ULTRAVIOLET LIGHT AND ITS EFFECT ON GERMINATION, GROWTH,

PHYSIOLOGY AND RESPONSES OF COOL SEASON GRASSES.

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Edward John Nangle, M.S.

Graduate Program in Horticulture and Crop Science

The Ohio State University

2012

Dissertation Committee:

Dr. David Gardner, Adviser

Dr. T. Karl Danneberger

Dr. James D. Metzger

Dr. Luis E. Rodriguez-Saona

Copyrighted by

Edward John Nangle

2012

ii

ABSTRACT

The increase of ultraviolet (UV) light levels in the northern hemisphere raises the spectre

of possible problems for turfgrass plants due to long term exposure. Cool season

turfgrasses which are susceptible to photoinhibition may suffer from a loss of

productivity and growth, reducing their ability to sequester carbon. The effect on

germination, plant responses in relation to UV absorbing compounds and how turfgrasses

were evaluated for loss of quality with regards to linking new technology to visual ratings

have had no or limited research.

Enhancing the percentage of Kentucky bluegrass (Poa pratensis L.) seed

germination and speed could benefit establishment of the grass in a greater range of

environmental and geographical conditions. Potential for the use of ultraviolet light to

enhance Kentucky bluegrass seed germination exists through exposure to UV light. The

effect of ultraviolet light may be lost with seed age. In altering wavelength exposure,

there may be an opportunity to enhance the effect.

In measuring turf quality, traditionally human measurement has been the standard

method for both color and cover. Color, in particular, is thought to be controlled by

pigment changes. In evaluating a total of 51 cultivars of tall fescue (Schedenorus phoenix

Scop. Holub), perennial ryegrass (Lolium perenne L.) creeping bentgrass (Agrostis

stolonifera L.) cv. ‘Penncross’ and ‘L-93’ it was found that nitrogen content is

iii

most crucial in color measurements. Reflective measurements did not correlate with

nitrogen or chlorophyll content. Extract measurements had stronger correlation with

nitrogen content than pigmentation concentration. Current reflective measurement

equipment may not be closely linked to visual rating of turfgrass color possibly due to

variation in leaf surfaces.

There is a difference in response to UV light among grass species. Creeping

bentgrass ‘L-93’ produced increasing anthocyanin in response to UV light. The

characterization of Cyanidin – 3 – O – glucoside was the first reported in the literature in

creeping bentgrass. Carotenoids, zeaxanthin and β-carotene decrease in creeping

bentgrass after exposure to UV-B and turfgrass quality and vegetative production in

bentgrasses decreased to a greater extent than tall fescue or perennial ryegrass. All

grasses have the ability to initially accumulate phenolic compounds and flavonoids in the

tissue most exposed to light, although this still doesn’t mean a prevention in damage to

photosynthetic machinery. Turfgrass recovery and maintenance of optimal photosynthetic

rates will be crucial as breeders try to develop new cultivars that are adapted to higher

levels of UV light.

iv

DEDICATION

To Eddie and Catherine Nangle

v

ACKNOWLEDGEMENTS

I would like to thank Dr. Dave Gardner for giving me a chance at a PhD, it has been a

wonderful life changing experience here and I feel that I have achieved more than I could

ever have imagined. The friendship and comments, which were sharp, incredibly witty

and sometimes intelligent, were more than I could have asked for. I hope to carry on the

good name of the program wherever I may end up in a happy and successful career.

Thank you Dr. Metzger, for your invaluable advice and brevity. Dr. Danneberger, your

insight and always excellent stories kept me smiling and motivated. Thank you, Dr.

Rodriguez-Saona for your patience and willingness to teach and interact with me.

To the people in the lab, Dominic Petrella – I have learned as much from you as

you did from me; Aneta Studzinska, your determination to finish drove me on. To Pam

Sherratt, you took the first step and I am sure many more will faithfully follow your life

changing decision – I did! Dr. John Street, Emily, Matt, Dave Snodgrass, Jim Vent,

Andrew Muntz, Arly Drake and Phil Young, your help and input was greatly appreciated.

To Suemi, I couldn’t have gotten this far and further without you. To Mike O’Keeffe –

what you have done for me cannot be overestimated and I will be forever indebted.

Finally to my parents, it’s been tough and arduous, but this has been something

for which your support never wavered. For that, I will be eternally grateful and cannot

ever pay you back. I just hope you’re as proud as I am thankful.

vi

VITA

October 7, 1980 .............................................Born Enniscorthy, Wexford, Ireland

2008................................................................M.S. Horticulture and Crop Science,

The Ohio State University.

2008 to present ..............................................Graduate Research Associate, The Ohio

State University

PUBLICATIONS

Nangle, E.J., D.S. Gardner, J.D. Metzger, J. R. Street, and T.K. Danneberger. 2012. The effect of Nitrogen source and Trinexapac-ethyl on Creeping bentgrass (Agrostis stolonifera L.) physiology under neutral shade, deciduous tree shade and full sunlit conditions. Hortscience 47(7):1-7. Nangle, E., D. Gardner, A.S. Studzinska, T.K. Danneberger, and J.R. Street, 2010. Nitrogen absorption and movement in creeping bentgrass Agrostis stolonifera L. as affected by nitrogen source and shade. Proc. of 2nd Annual European Turfgrass Conference, Angers, France, 2010. Studzinska, A.K., D. S. Gardner, J. Yan, E. Nangle, and T. K. Danneberger. 2009. Development and Characterization of Transgenic Creeping Bentgrass Transformed with Arabidopsis BAS1 Gene. Int. Turf Soc. Res. J. 11:859-870.

FIELDS OF STUDY Major Field: Horticulture and Crop Science

vii

TABLE OF CONTENTS

ABSTRACT ........................................................................................................................ ii

DEDICATION ................................................................................................................... iv

ACKNOWLEDGEMENTS ................................................................................................ v

VITA .................................................................................................................................. vi

PUBLICATIONS ............................................................................................................... vi

FIELDS OF STUDY ......................................................................................................... vi

LIST OF TABLES ............................................................................................................. xi

LIST OF FIGURES ......................................................................................................... xiii

CHAPTER 1: LITERATURE REVIEW ............................................................................ 1

LIGHT .................................................................................................................... 1

ULTRAVIOLET LIGHT ........................................................................................ 2

CAROTENOIDS: LIGHT ABSORBING COMPOUNDS .................................... 6

FLAVONOIDS: UV ABSORBING COMPOUNDS WITH SPECIFIC

INTEREST IN ANTHOCYANINS ...................................................................... 11

PHENOLIC COMPOUNDS................................................................................. 18

viii

MEASUREMENT TECHNIQUES ...................................................................... 19

REFERENCE ........................................................................................................ 24

CHAPTER 2: SUBJECTIVE AND OBJECTIVE COLOR RATINGS OF

TURFGRASSES AND THEIR LINKS TO PIGMENTATION AND NITROGEN

CONCENTRATIONS. ..................................................................................................... 38

ABSTRACT .......................................................................................................... 38

INTRODUCTION ................................................................................................ 39

MATERIALS AND METHODS .......................................................................... 44

RESULTS ............................................................................................................. 49

DISCUSSION AND CONCLUSION .................................................................. 52

REFERENCE ........................................................................................................ 64

CHAPTER 3: INFLUENCE OF ULTRAVIOLET LIGHT ON GERMINATION RATE

AND SPEED OF KENTUCKY BLUEGRASS (Poa pratensis L.) ................................. 70

ABSTRACT .......................................................................................................... 70

INTRODUCTION ................................................................................................ 71

MATERIALS AND METHODS .......................................................................... 73

RESULTS AND DISCUSSION ........................................................................... 74

CONCLUSIONS .................................................................................................. 76

REFERENCE ........................................................................................................ 81

ix

CHAPTER 4: ULTRAVIOLET-B LIGHT INFLUENCE ON COOL SEASON

TURFGRASS GROWTH AND MORPHOLOGY .......................................................... 84

ABSTRACT .......................................................................................................... 84

INTRODUCTION ................................................................................................ 85

MATERIALS AND METHODS .......................................................................... 88

RESULTS ............................................................................................................. 91

DISCUSSION ....................................................................................................... 93

REFERENCE ...................................................................................................... 101

CHAPTER 5: PIGMENT CHANGES IN COOL SEASON TURFGRASSES IN

RESPONSE TO ULTRAVIOLET-B LIGHT IRRADIANCE ....................................... 106

ABSTRACT ........................................................................................................ 106

INTRODUCTION .............................................................................................. 107

MATERIALS AND METHODS ........................................................................ 113

RESULTS ........................................................................................................... 122

DISCUSSION ..................................................................................................... 125

CONCLUSION ................................................................................................... 129

REFERENCE ...................................................................................................... 141

CHAPTER 6: OVERALL CONCLUSIONS ON UV-B INFLUENCE ON TURFGRASS

PIGMENTS AND GROWTH ........................................................................................ 149

x

ABSTRACT ........................................................................................................ 149

DISCUSSION ..................................................................................................... 150

APPENDIX A: HANDHELD REFELCTANCE MEASUREMENTS OF TURFGRASS

TISSUE ........................................................................................................................... 154

APPENDIX B: COLOR OF LEAVES OF CREEPING BENTGRASS (Agrostis

stolonifera L.) TISUE AFTER EXPOSURE TO UV-B LIGHT .................................... 156

APPENDIX C: CHROMATOGRAM CAROTENOID STANDARDS ........................ 158

APPENDIX D: CHROMATOGRAM CHOKEBERRY ARONIA ................................ 160

APPENDIX E: CHLOROPHYLL A CONTENT AFTER D7 UV TREATMENT OF

CREEPING BENTGRASS ‘L-93’ ................................................................................. 162

APPENDIX F: CHROMATOGRAM BENTGRASS CAROTENOIDS ....................... 164

APPENDIX G: TALL FESCUE CAROTENOID CHROMATOGRAM ...................... 166

COMPLETE REFERENCE ........................................................................................... 168

xi

LIST OF TABLES

Table 2.1: Normality test results and data processing results for six cool season grasses

evaluated for color using transmission and reflection type measurements post

ROBUSTREG analysis. .................................................................................................... 56

Table 2.2: Correlation values for chlorophyll pigmentation and transmission

characteristics obtained using a Hunter XE colorquest colorimeter on extracts of six cool

season turfgrasses.............................................................................................................. 57

Table 2.3: Correlation values for chlorophyll pigmentation and tissue nitrogen levels with

visual ratings of six cool season turfgrasses. .................................................................... 58

Table 2.4: Correlation of visual ratings to transmission and reflection color characteristics

of six cool season turfgrasses. ........................................................................................... 59

Table 2.5: Correlation values for chlorophyll pigmentation and reflection characteristics

obtained using a Konica CR310 handheld colorimeter on surfaces of six cool season

turfgrasses. ........................................................................................................................ 60

Table 2.6: Correlations between reflectance measurements and transmission

measurements with tissue nitrogen for 6 cool season turfgrasses. .................................... 61

xii

Table 2.7: Human color rankings for cultivars of six cool grasses. (1= brown / 6 =

acceptable / 9 = optimum)................................................................................................. 62

Table 3.1: Germination percentages of Kentucky bluegrass (Poa pratensis L.) seed lots

treated with PAR (400 – 700 nm) and ultraviolet light (300 – 400 nm). .......................... 78

Table 4.1: Lighting conditions in control and ultraviolet treated light environments for

turfgrasses grown at The Ohio State University March – April 2011. ............................. 96

Table 5.1: Light intensity and light distribution for Conviron E15 growth chambers

illuminated using high output F12T12-CW.HO fluorescent tubes and QUV UV-B 313 nm

UV-B lights. .................................................................................................................... 131

Table 5.2: Anthocyanin pigmentation detected in creeping bentgrass ‘L-93’ after 7 days

of enhanced UV-B treatments. ........................................................................................ 132

xiii

LIST OF FIGURES

Figure 1.1: Carotenoid biosynthesis pathway to non-photochemical (NPQ) quenching

xantophyll cycle. ................................................................................................................. 8

Figure 1.2: Anthocyanin biosynthesis pathway. ............................................................... 14

Figure 2.1: Principal component analysis for turfgrass samples analyzed for color using

reflectance and transmission measurements. .................................................................... 63

Figure 3.1: Percentage germination of Kentucky bluegrass (Poa pratensis L.) seed treated

with photosynthetically active radiation (400 – 700 nm) and ultraviolet light (280 – 480

nm) in seed lot one. ........................................................................................................... 79

Figure 3.2: Percentage germination of Kentucky bluegrass (Poa pratensis L.) seed treated

with photosynthetically active radiation (400 – 700 nm) and ultraviolet light (280 – 480

nm) in seed lot two. ........................................................................................................... 80

Figure 4.1: Relative growth rate for four cool season grasses grown in control and

enhanced ultraviolet-B light conditions in repeated experiments. .................................... 97

Figure 4.2: Color ratings for four cool season grasses grown in control and enhanced

ultraviolet-B light conditions in repeated experiments. (1-9 scale, 1 = brown/dead, 9 =

dark green optimal color, 6 = acceptable). ........................................................................ 98

xiv

Figure 4.3: Number of tillers per plant for four cool season grasses grown in control and

enhanced ultraviolet-B light conditions in experiment one. ............................................. 99

Figure 4.4: Number of tillers per plant for four cool season grasses grown in control and

enhanced ultraviolet-B light conditions in experiment two. ........................................... 100

Figure 5.1: Fv / Fm values for Perennial Ryegrass and Creeping Bentgrass ‘Penncross’

treated with UV-B light (280 – 315 nm) and PAR light (400 – 700 nm) conditions over

seven days. ...................................................................................................................... 133

Figure 5.2: Total chlorophyll content for Tall Fescue and Creeping Bentgrass ‘L-93’

treated with UV-B light (280 – 315 nm) and PAR light (400 – 700 nm) conditions over

seven days. ...................................................................................................................... 134

Figure 5.3: Distribution of phenolics in creeping bentgrass ‘L-93’ after treatment with

both UV-B light (280 – 315 nm) and PAR light (400 – 700 nm). Data was pooled as all

grasses had the same response. ....................................................................................... 135

Figure 5.4: Distribution of total flavonoids in perennial ryegrass tissue after treatment

with both UV-B light (280 – 315 nm) and PAR light (400 – 700 nm). Data was pooled as

all grasses had the same response. .................................................................................. 136

Figure 5.5: Turfgrass flavonoid content in Creeping bentgrass, Tall Fescue and Perennial

Ryegrass grown in UV-B light (280 – 315 nm) and PAR light (400 – 700 nm) D0 to D7.

......................................................................................................................................... 137

Figure 5.6: Anthocyanin profile of creeping bentgrass ‘L-93’ exposed for 7 days to UV-B

light. Produced from HPLC analysis using C18 column and light absorbance measured at

520 nm using a PDA. ...................................................................................................... 138

xv

Figure 5.7: Chromatogram of Perennial ryegrass carotenoids with absorbance at 453 nm.

Data produce using C30 column and PDA connected to Chemstation software. ............ 139

Figure 5.8. β-carotene (top) and zeaxanthin (bottom) content in creeping bentgrass, tall

fescue and perennial ryegrass exposed to UV-B light D0 – D7. .................................... 140

1

CHAPTER 1

LITERATURE REVIEW

LIGHT

Plant growth and development depends mainly on energy derived from light

(Smith, 1982). The habitat and ecosystem of plants can vary greatly even when there are

minor changes in light (Schmitt and Wulff, 1993). There are two properties of light that

relate to plant growth and development: light quality and quantity. Light quality can refer

to proportions of visible light spread across the 400-700 nm spectra which influences

plant growth responses, e.g. changes in the ratio of red light to far red light can influence

biomass allocation (Smith, 1994; Maliakal et al., 1999) and light quantity is the actual

amount of energy associated with radiation hitting the earth’s surface (Smith, 1994).

There has been an increase in research concerning the effects of ultraviolet light (UV)

100-400 nm on plant growth and development (Caldwell et al., 1989; Mendez et al.,

1999; Rowland 2006; Sarkar et al., 2011). This includes UV-C (100-280nm), UV-B (315-

280nm) and UV-A (400-315nm). The ozone (O3) layer in the stratosphere between 15

km and 60 km above the earth filters UV wavelengths to hit the earth’s surface

selectively (Rind et al., 1990; Rowland, 2006). Ozone can absorb ultraviolet-B light,

while oxygen (O2) absorbs UV-C light (Caldwell, 1979; Björn et al., 1998). Exposure

UV-B is increasing due to the decline of ozone linked to chlorofluorocarbons and the

breakage of the O3 structure by chlorine (Rowland, 2006).

2

The levels of atmospheric ozone, however, have varied and declined since 1950 with

20% decreases in content in some locations (Björn et al., 1998; Frederick et al., 1989;

Kerr and McElroy, 1993; Rowland, 2006). Ozone is also created by UV striking O2 and

splitting it in two followed by the attachment to an unbroken O2 (Rozema et al., 1997).

The incidence of UV radiation varies by season with winter time increases as high 35%

per year while there is a 7% increase in summer due to greater ozone loss in colder

temperatures catalyzing chlorine depletion of the gas (Kerr and McElroy, 1993; Solomon,

1990). Cloud cover decreases UV radiation by as much as 20% compared to a clear day

due mainly to scattering (Lubin and Frederick, 1991).

ULTRAVIOLET LIGHT

The discovery of ultraviolet light as a wavelength occurred in 1800 with Ritter

(Berg, 2008). The issue of UV radiation hitting the earth’s atmosphere was first noted in

1881 by Hartley when he was able to measure ultraviolet energy hitting the earth’s

surface and found it varied depending on altitude (Hartley, 1881). The description of the

wavelengths occurred not long after with Hertz who developed a method for measuring

microwaves.

The difference in light energy associated with each photon in each UV light

portion has led to developments of action spectrums (Caldwell, 1971; Flint and Caldwell,

2003). The energy associated with each portion of the UV range elicits different

responses, creating the need for the spectrum. Currently, regions on earth between 40°N

and 40°S latitude receive 2-11 kJ m-2 d-1 of UV radiation and this has the potential to

increase due to ozone loss (Taalas et al., 2000; Kakani et al, 2003; Rowland, 2006).

3

Exposure to UV-B levels along different lines of latitudes vary due to ozone depletion,

notably at the poles. Increases in UV levels since 1970 are as great as 130% in the

Antarctic in the spring as compared to a 7% increase in northern hemisphere mid-

latitudes over the same time period (Madronich et al., 1998). Levels of ozone depleted by

40% in the Antarctic while negligible increases occurred in the mid-latitudes since the

1970s (McKenzie et al., 2003).

Ultraviolet-C and B, impacts DNA through the production of cyclobutane

pyrimidine dimers leading to mutations (McLennan, 1987) while UV-B has a greater

impact on plant physiology (Stapleton, 1992) and UV-A is considered to have little

impact (Flint and Caldwell, 2003). Exposure to UV-B also tends to promote dimerization

while UV-A exposure tends to promote repair enzymes such as photolyase which

photoreactivate DNA strands by dimer breakage (Strid et al., 1994; Britt, 1996). The

increased amount of UV-B is considered most important as it has physiological impact

(Caldwell, 1979; Stapleton, 1992). Ultraviolet-B levels are projected to increase by 50-

60% from current levels in the northern hemisphere with peak irradiance levels predicted

to occur between 2010 and 2019 in the spring time (Taalas et al., 2000). There is

variation in the possible increases with seasonal and geographical variations. Alpine

regions already have experienced an increase of UV radiation by 1.1% per anum

(Blumenthaler and Ambach, 1990) with higher elevations having higher levels of UV-B

due to a thinner atmosphere (Caldwell et al., 1980). UV light initially damages the

photosynthetic apparatus within the plant. The majority of research indicates that damage

4

is mainly in photosystem II of the light harvesting complex (Tevini et al., 1988;

Bornman, 1989).

The impact of ultraviolet-A light on plants had been noted in various ways

including phototrophic responses in the coleoptile tip in wheat (Triticum spp.) (Castle,

1935), and control of phytochrome via UV-A light - near blue was proposed by Galston

(1949). Pfeiffer (1928) reported that without UV irradiance, plants had longer shoots,

thinner leaves and poor root development. Popp and Brown found that there were only

negative attributes associated with UV light, (1936). They also disputed the results of

Pfeiffer based on lack of replication and poor lighting control. Other studies have

demonstrated a decrease in pollen production (Flint and Caldwell, 1984), declination in

competitive ability of plants against weeds (Barnes et al., 1990), and reductions in

biomass in a range of plants including pines (Pinaceae L.), (Tevini et al., 1981; Sullivan

and Teramura, 1988). Thinning of the leaf surface and increased levels of flavonoids and

phenolics are also found in plants (Tevini et al., 1981; Sarkar et al., 2011). The potential

for long term reduction in the ozone layer has driven interest in how long term adaptation

of plants will occur in response to increasing UV levels.

There are many causes for the variation of the impact of UV light on plants

observed in the literature. Plants with sensitivity to UV-B light produce increased

epicuticular wax on the adaxial surface while there is a reduction in leaf thickness in

cotton (Gossypium hirstum L.). Decreases in mesophyll and palisade tissue also occurs

(Kakani et al., 2003). Stomatal densities may also decrease but this is crop dependent and

other factors play a role. In saturated CO2 conditions, treatments of UV-B were more

5

effective in decreasing stomatal density (Ziska et al., 1992). Altitude changes increase the

UV effect on growth habits of plant material. Plant tissue found above 3000 m has

increased dark respiration, an earlier effort at reproduction and maintained photosynthetic

efficiency compared to plant material from 0 m above sea level when treated with

supplemental UV light due to adaptation to the conditions (Ziska et al., 1992). There can

be a greater than 40% increase in UV irradiance between the elevations (Caldwell et al.,

1980; Sullivan et al., 1992). Variations in levels of ozone with elevation change are

considered possible due to regional aerosol conditions solar elevation and also albedo

(Pfeifer et al., 2006).

Dilation of thylakoid membranes in (Pisium sativum L.) and damage to

chloroplast membranes occurred after UV-B irradiation (Brandle et al., 1977).

Herbaceous dicots cannot attenuate UV-B in the epidermis with 18-41% of UV-B light

reaching the mesophyll layer leaving increased chances for damage to photosynthetic

apparatus. Conifer needles, regardless of age, absorb essentially all UV-B light. Grasses

and woody dicots are considered intermediate in their ability to protect the mesophyll

layer from damage with attenuation in the epidermis ranging from 3-12% (Day et al.,

1992). In contrast to the negative effects, tomato ripening can be faster with treatments of

UV-B as colored pigments build up in the plant which can absorb the excess energy,

(Bacci et al., 1999). Flowering color can be enhanced with UV-B treatments in smoke

tree (Cotinus coggygria ‘Royal purple’). The increase in anthocyanin content in the leaf

in response to the light treatment resulted in more favorable performance ratings in color

(Oren-Shamir and Nissim, 1997).

6

CAROTENOIDS: LIGHT ABSORBING COMPOUNDS

The potential long term effects are considered to be both anatomical and physiological

and plants do possess response mechanisms for dealing with UV-B light. These are

mainly antioxidants such as photolyase and super oxide dismutase (Zhang et al., 2005;

Sarkar et al., 2011). These systems repair damage caused by excited radicals due to

excess energy from UV-B radiation. There are compounds however, that may absorb the

light directly and aid the reduction of UV-B light attenuation and energy dissipation in

the plant such as flavonoids and carotenoids (Gould, 2004; Rowland, 2006).

Carotenoids are lipid soluble isoprenoid compounds containing C40 chains

(tetraprenoids) which are ubiquitous in nature and have been investigated in relation to

human health benefits. Interest exists in looking at the capabilities they can have in plant

protection (Bartley and Scolnik, 1995; Müller et al., 2001). Biosynthesis of carotenoids

occurs in cellular plastids where they are associated with light harvesting complexes

(Kopsell and Kopsell, 2006). The basic C5 isoprenoid structures are converted to

isopentyl diphosphate (IPP) and into dimethylallyl diphosphate (DMAPP). These

compounds are substrates for the enzyme geranylgeranyl diphosphate synthase (GGPP)

(Cunningham and Gantt, 1998). The enzyme catalyzes GGPP formation which is paired

with another GGPP in a head to head condensation reaction creating phytoene (Gross,

1991). The pathway then branches after the conversion of phytoene to lycopene via

phytoene desaturase and ξ carotene desaturase; the following cyclization of the lycopene

rings can form carotenoids with two β rings (Fig. 1.1), (zeaxanthin, antheraxanthin,

7

violaxnathin) or one β ring and one ε ring (lutein, α carotene) on the C40 structures (Fig.

1.1), (Kopsell and Kopsell, 2006).

The reversibility of accumulation of lycopene with increased FR light sources

following red light exposure indicates that initial responses in carotenoid synthesis are

due in part to phytochrome (Alba et al., 2000). This hypothesis was first thought of early

in the 1950’s (Piringer and Heinze, 1954) when accumulations of certain pigments were

noted in response to the red light wavelengths. The inability to clarify this result further

inhibited work past the 1970’s. The general thought was that this physiological trait

occurred which was recently confirmed (Alba et al., 2000).

8

Geranylgeranyl diphosphate

Phytoene

ζ-carotene

Lycopene

β – carotene α - carotene

zeaxanthin lutein

NPQ antheraxanthin loroxanthin

violaxanthin

Figure 1.1: Carotenoid biosynthesis pathway to non-photochemical (NPQ) quenching xantophyll cycle.

9

The variations in the carotenoids impact various functions in the plant. Lutein is

believed to have a greater impact on light quenching than β carotene (Cuttriss and

Pogson, 2004). The xantophyll cycle has received a lot of attention due to its antioxidant

capacity and photoprotection element (Bartley and Scolnik, 1995; McElroy et al., 2006).

Carotenoids are essential in reducing light photooxidative stress to chlorophyll and the

light harvesting complex (Foyer et al., 1994, Moradas-Fereira et al., 1996). The

downward energy transfer of singlet oxygen (1O2) to zeaxanthin reduces the damage it

can cause to lipids and cell walls (Owens et al., 1992). In carotenoids with no rings at the

end of the structure, such as lycopene, the large amount of double bonds allows for a

similar effect (Kopsell and Kopsell, 2006). Light harvesting complexes have the -trans

configuration providing efficient singlet energy transfer to chlorophyll molecules. The -

cis forms tend to isomerize towards the -trans configuration upon photo excitation. The

carotenoids attached to reaction centers are also found to be in –cis conformation and are

unaffected by temperature change. This may make them preferred carotenoids for energy

dissipation (Lutz et al., 1978; Koyama and Fujii, 1999).

Creeping bentgrass xantophylls in high light conditions cumulatively increase as a

percentage of the total carotenoids in high irradiance but decrease in low irradiance.

Neoxanthin and β-carotene decrease in high irradiance but increase in low irradiance

(Bell and Danneberger, 1999; McElroy et al., 2006). Testing of creeping bentgrass

showed that after up to 24 hours of exposure there was an increase of carotenoids in

higher irradiance but a resultant decrease and probable degradation after 168hrs

(McElroy et al., 2006). Maximum buildup of lutein, β-carotene and chlorophyll B

10

occurred at 24 hours in Kale (Brassica oleracea L.) but an overall increase in

photoperiod also increased total pigment accumulations (Lesfrud et al., 2006). Seeds of

certain maize hybrids had lower carotenoid content grown at cooler temperatures (16 °C)

(Massacci et al., 1995).

The application of nutrient elements to plants increases carotenoid content.

Increasing nitrogen applications to spinach (Spinacia oleracea L.) almost doubled the

quantity of lutein on a fresh weight basis (Lesfrud et al. 2007). This is a benefit that may

already have been realized with current applications of nitrogen but also may allow for

improvements in areas which are prone to excess levels of sunlight. The addition of

nitrogen to plants increases production of many pigments (Greenwood et al., 1990) until

a level of nitrogen toxicity is reached (Carrow et al., 2001). In periods of reduced

nitrogen content rice plants may increase their content in an attempt to increase thermal

dissipation abilities in responses following decreases in chlorophyll (Huang et al., 2004).

The management of turfgrasses on sand based golf greens is dependent on sufficient

fertility in a growing medium that is classed as having poor nutrient retention capabilities.

Thus poor fertility management may lead to photoinhibition and loss of productivity.

Control or inhibition of carotenoid biosynthesis is a mode of action that has been

exploited in the recent development of herbicides. Mesotrione (2-[4-(methylsulfonyl)-2-

nitrobenzoyl]-1,3-cyclohexanedione) has proved useful in controlling common turfgrass

weeds (McCurdy et al., 2008; Beam et. al., 2006). Inhibition of certain carotenoids leads

to bleaching of the leaf and damage to the photosystems of weeds (McCurdy et al., 2008).

The damage to the photosystems then allows for improved turfgrass competition although

11

there is also potential for damage to turfgrass systems in certain situations (Beam et al.,

2006). This form of chemical control is relatively new to the turfgrass industry and

suggests the potential for more useful information related to the development and control

of secondary metabolites.

There have been efforts to enhance levels of carotenoids in plants. This has been

carried out for a variety of reasons including ecological fitness due to reduced

photoinhibition and increased human health benefits. Variations occur in tomato in

particular providing a wide range of varieties with different carotenoid profiles (Fraser

and Bramley, 2004; Ronen et al., 1999; Ronen et al., 2000). Genetic engineering of

carotenoids in plants has occurred at an increased rate as our understanding has

developed about the pathways. Control is offered with genetic manipulation and allows

for a greater understanding and possibility for greater change in compounds (Fraser and

Bramley, 2004). Research on the effects of UV-B impact on carotenoid profiles of

turfgrass species has not been carried out.

FLAVONOIDS: UV ABSORBING COMPOUNDS WITH SPECIFIC INTEREST IN

ANTHOCYANINS

Pigments such as flavonoids have specific absorption characteristics in the UV

region (Ziska et al., 1992). Flavonoids are compounds defined as having a 15 carbon

structure in a form of a C6 – C3 –C6 backbone (Anonymous, 1997). They can be broken

down into different pigments which have UV-B absorbing characteristics such as

anthocyanins (Gould, 2004). Anthocyanins are a pigment in plants which are associated

with various functions. They are derived from shikimic acid via flavonoid formation

12

(Bennett and Wallsgrove, 2006). There have been at least 4,000 flavonoids described and

many are found in higher plants (Seigler, 1998). Plants use them as a color source for

attraction of pollinators which can see in the UV wavelengths and also as a defense

mechanism for disease and herbivory (Briscoe and Chittka, 2001; Irani and Grotewold,

2005). They are primarily known for their bright red colors and are used in the food

industry as a colorant. In plants, anthocyanin response is related to environmental stresses

including drought stress, excessive UV-B conditions, heavy metal toxicity, cold

hardiness, insect and fungal attack (Chalker-Scott, 1999; Gould, 2004).

Anthocyanins are naturally found in higher levels in plants such as berries and red

fruits (Lohachoompol et. al, 2004). Anthocyanins are found in shoots of plants but in

general are predominantly found in the upper epidermis or just beneath it in the

mesophyll layer in leaves (Chalker-Scott, 1999). The areas in which they are most often

found tend to have greater levels of light exposure (McLure 1975, Chalker-Scott 1999). It

has been shown that the synthesis is autonomous to cells (Lancaster et al., 1994). The

localization of production would indicate that a specific screen for cell ultra-structure

may occur versus whole plant changes.

Anthocyanin synthesis involves approximately seven steps and the pigments are

assembled similarly to other flavonoids from two different streams (Bennett and

Wallsgrove, 2006). One stream involves the shikimate pathway to produce the amino

acid phenylalanine. The other stream produces malonyl-CoA, a C3 unit from a C2 unit

(acetyl-CoA). These streams meet and are coupled together by the enzyme chalcone

synthase (CHS), which forms an intermediate chalcone (Fig. 1.2). The chalcone synthase

13

is considered to be the first enzyme in all direct flavonoid biosynthesis. The chalcone is

subsequently isomerized by the enzyme chalcone isomerase (CHI) to the prototype

pigment naringenin (Fig 1.2), (Heller and Forkmann, 1988). Naringenin is oxidized and

then further reduced by the enzyme dihydroflavonol 4-reductase (DFR) to the

corresponding leucoanthocyanidins. The resulting, unstable anthocyanidins are further

coupled to sugar molecules by enzymes like UDP-3-O-glucosyl transferase to yield the

final relatively stable anthocyanins (Seigler, 1998). One of the final steps is light

controlled and in particular it seems that UV-B light induces increased synthesis

(Kubasek et al., 1992).

The initiation of synthesis is thought to be controlled by the light receptors

phytochrome or cryptochrome (Mancinelli et al., 1991; Sponga et al 1986).

Cryptochrome, a blue light receptor, is inferred to elicit anthocyanin synthesis in tomato

seedlings. Blue light applications significantly increased levels of anthocyanins in

tomato. The phytochrome response was more significant in cabbage, however, with about

7x efficiency in anthocyanin production with red light (650 – 800nm) applications

(Sponga et al., 1986). The initiation response of chalcone synthase to UV-A is mediated

by cryptochrome 1 (CRY1) but there is no response to UV-B light via either CRY1 or

CRY2 to enhance chalcone synthase, the key flavonoid biosynthesis enzyme (Wade et

al., 2001). The interaction between the two light receptors is also possibly important as

both phytochrome and cryptochrome converge in a single pool to initiate light responses

(Ang et al., 1998). It has been proposed that PHYB, in particular, equilibrates the flux

through CRY1 and UV-B signaling pathways (Wade et al., 2001).

14

Phenylalanine

4 – Coumaryl CoA

Malonyl CoA Chalcone synthase

Chalcone

Chalcone isomerase

Flavanone

Flavones, isoflavones Flavanone 3-hyrdoxylase (F3H) Flavan-4-ols (colorless)

Dihydroflavanol

Anthocyanins Flavanol glycosides (red pigmentation) (colorless)

Figure 1.2: Anthocyanin biosynthesis pathway.

15

Plant age affects response to changes in the light and induction of chalcone

synthase activity via phytochrome. In 6 days old mustard (Sinapis hirta L.) seedlings far

red, UV-B and UV-A / blue light induced the CHS transcripts (Kaiser el al., 1995). In

older mustard seedlings and mature Arabidopsis leaf tissue, however, there is no

phytochrome induction but there was a response due to UV-B and UV-A / blue light

treatments (Jackson et al., 1995).

There are 6 main anthocyanins and many others that occur less frequently. The six

aglycones (pelargonidin, cyanidin, delphinidin, petunidin, malvidin and peonidin) are

widely distributed among angiosperms and gymnosperms (Seigler, 1998). The

differences between the various anthocyanins are mainly due to the nature and number of

sugars attached to the ‘3 or ‘5 position on the C ring of the structure. The addition of

sugars and acids at either of these positions leads to a more stable structure and alters the

absorption abilities of the pigments (Garćia-Viguera and Bridle, 1999). Variations can be

caused by the nature and number of aliphatic or aromatic acids attached to the sugars

(Kong et al., 2003). The variations lead to color changes which can be seen across a

whole range of plants. The leaves of red cabbage contain eight acylated cyanidin

glucosides while red onion contains four major anthocyanins; cyanidin 3-glucoside,

cyanidin 3-laminaribioside, cyanidin 3-malonylglucoside, cyanidin 3-

malonyllaminaribioside and four minor ones (Brouillard, 1988; Donner et al., 1997).

There is some argument as to whether anthocyanins are directly related to general

stress tolerance or if they are produced as a direct result of the stress under which the

plant has been placed. The impact of cold hardiness, changes in light intensity and

16

wavelengths seems to enhance responses in particular. Photoprotection has been offered

as a specific anthocyanin function and hazelnut seeds lacking anthocyanins have died in

field conditions versus anthocyanin producing progeny (Mehlenbacher and Thompson,

1991). The anthocyanins also favor absorption of UV-B and green light over blue light

with only small quantities of red light being absorbed (McClure, 1975). The absorption of

the blue-green light means that there is only red light available to chloroplasts. This is

important as it reduces the potential for excessive energy build up (Pietrini and Massacci,

1998). An increased tolerance for photoinhibition in cold acclimated jack pine seed /

needles was a result of light attenuation by anthocyanins and increased utilization of light

energy (Krol et al., 1995).

Temperature changes elicit many anthocyanin responses. In fall, color changes in

leaves lead to increased quantities of carotenoids and anthocyanins giving a range of red,

orange and yellow colors. The increase in content has been related to masking of

chlorophylls as leaves are senescing thus decreasing potential for photo oxidation damage

reducing nutrient retrieval prior to winter-like conditions (Field et. al., 2001). The red

leaves are seen when there is de novo synthesis of anthocyanins unlike carotenoids which

are revealed with chlorophyll breakdown and there is no increase in levels (Matile et al.,

1992). Increased absorption in green yellow light by anthocyanins (Neill and Gould,

1999) may counteract the loss of chlorophyll and attenuate sufficient light to prevent

damage as leaves senesce (Field et al., 2001). The reflection of red light may be

independent of anthocyanin content and concentration of the pigments is more important

than their location (Neill and Gould, 1999).

17

Temperature also plays a role in the stability of anthocyanins, and they are

susceptible to degradation if exposed to prolonged warmer temperatures when used in

food colorants (Giusti, personal comm. 2008, Ahmed et al., 2004). In the natural

environment the anthocyanins are seen in varying temperatures depending on the plant

and the functionality of the anthocyanin. The alterations in temperature are seen as

leading to a response also. In cabbage (Brassica oleracea L.) colder temperatures

increase light dependent anthocyanin production; however, there is disagreement as to

whether the development is a result of temperature effects on the phytochrome system or

on chalcone synthase activity related to photoinhibition (Rabino and Mancinelli, 1986).

The pH plays a major role in coloration. Lower pH levels (<2) tend to produce a

red color while a pH greater than 4 results in predominantly colorless anthocyanins

(Seigler, 1998). The change in plant pH can be caused by a variety of environmental

effects including sunlight (Zu-Hua et al., 1990). The pH also affects anthocyanin bases

which are stabilized by forming very colorful complexes with metals. The complexes

with such minerals as aluminum, iron or magnesium which are extremely susceptible to

pH change and have narrow limitations (Seigler, 1998). The extraction and quantification

of anthocyanins tends to occur from plant material using a solvent containing a low

percentage of hydrochloric or formic acid. This allows samples to be kept at lower pH

and reducing the chances of degradation of non-acylated pigments (Kong et al., 2003).

Anthocyanins may also have potential to reduce aluminum toxicity. Formation of

complexes between anthocyanins and Al3+ allows for pigment stabilization and reduces

18

degradation due to oxygen reactions as well as internal Al3+ levels (Moncada et al.,

2003).

PHENOLIC COMPOUNDS The class of phenolic compounds is widespread and has been identified in large

numbers. Approximately 40% of the organic carbon is allocated to phenolic compounds

in the biosphere (Croteau et al., 2000). Their production is predominantly linked to the

shikimate and acetate pathways (Floss, 1997; Hare and Cress, 1997). They are largely

derived from phenylalanine and are formed usually in cells close to locations of stress.

The enzyme phenylalanine ammonia-lyase (PAL) is used to direct carbons from amino

acids into the synthesis of the metabolites. They can be constitutively present (e.g.

lignins) or they can be induced (Langebartels et al., 2002). Their production is highly

complex and variable as there are so many compounds with multi step reactions required

for their synthesis (Mustafa and Verpoorte, 2007). The structures have a minimum of 6

carbons and their structure has an aromatic phenyl ring. There are generally one or more

acidic hydroxyl groups attached to the aromatic ring (Croteau et al., 2000).

The action of UV within the photosynthetic apparatus is predominantly involved

in the formation of highly excited compounds such as singlet oxygen (Foyer et al., 1994).

A majority of phenolic compounds act as scavengers of singlet oxygen and have an

ability to donate hydrogen from hydroxyl groups around their aromatic ring to free

radicals. This then reduces the impact of oxidation of lipids and other structures within

the cell (Foti et al., 1994). This can be used to reduce protein denaturation in the

photosynthetic apparatus which undergoes continuous cycles of damage and repair and

19

can become overwhelmed in high light or enhanced UV-B conditions (Takashi and

Murata, 2008). The production of these compounds is stimulated by UV radiation as well

as flavonols (Tevini, 1993). The range of compounds means that they can be induced in

many situations. High concentrations of phenolic compounds are believed to reduce

herbivore attack (Dicke et al., 2003). In Brassicaceae, sinapate esters of phenolics

provide protection against UV irradiation due to specificity in UV light wavelength

absorption (Sheahan, 1996). Phenolics in epidermal cells have the ability to screen and

absorb UV irradiation (Bornman and Teramura, 1993).

Plants do also possess the ability to distribute protective compounds based on

where light exposure is highest. In Jersey cudweed (Gnaphalium luteo-album L.), surface

concentrations of flavonols increased in response to UV-B radiation (Cuadra et al., 1997).

Regular removal of tissue may reduce a plant’s ability to respond to these conditions and

enhance the impact of UV-B light stress. Phenolic compounds found in the epidermal

layers of leaves are believed to offer screening of elevated light levels to the

photosynthetic apparatus (Gould, 2004). The upper part of the plant may alter its

pigment composition in response to the light location also (Ju et al., 1999).

MEASUREMENT TECHNIQUES

The ability to separate and identify the compounds has increased as equipment

sensitivity and improved structural elucidation developed. This happens via use of

spectrophotometry, mass spectrometry, high performance liquid chromatography and

nuclear magnetic resonance spectrometry has created better separation techniques which

do not damage the compounds during analysis. This has allowed for the creation of a vast

20

amount of information on these materials and in food sources there is a lot of information

known about their structure and molecular weight (Wrolstad and Giusti, 2001). A range

of these systems are used to characterize and quantify pigmentation changes.

Spectrophotometry is dependent on variations in reflective or transmission properties in

compounds based on their reaction to excitation via light energy. The absorption of light

leads to a change in energy in bonding electrons (electronic, vibrational, and rotational).

The spectra arise when the molecules of interest absorb photons of a specific energy.

They remit the energy at a slightly longer wavelength which is then divided out by a

monochromator and absorbed by a light sensor. The values are then compared against a

set of standards (Hardy, 1938). Spectrophotometers can be used in a pH differential

method to quantify specific anthocyanin levels depending on the standards available.

There is a very high correlation with the standards (R >0.9), (Lee et al., 2005). Analysis

of all pigments in this study could be carried out with spectrophotometry (Wellburn,

1994).

More in-depth analysis can be carried out using high pressure liquid

chromatography (HPLC). The process is the separation of chemical substances by

partitioning them between two media. The most common form is via a stationary phase

and a mobile phase. The first may be a solid or liquid while the second maybe a liquid or

gas. The separation of substances depends on a range of situations – compound size,

polarity and volatility are some of the common methods. It provides a precise method

which can separate compounds which are very similar thus difficult to analyze using

other methods. Chromatography itself has many fields and methods such as gas

21

chromatography, thin layer chromatography, paper chromatography, and gel permeation

chromatography (Shackleton, 2010). Specific known wavelengths are again used with a

photodiode array along with retention time, order of elution, standards, solvents used in

phases and also the extraction methods to characterize and quantify the pigments.

Anthocyanins and carotenoids can be separated within the chemical classes using this

system and it has become consistent and reliable over time (Hong and Wrolstad, 1990;

McCurdy et al., 2008).

Enhancing the separation and developing greater precision requires mass analysis

of compounds. This can be carried out using mass spectrometry. It analyzes compounds

based on a mass to charge ratio of an analyte. This mass to charge is used in two ways;

the charge on the compound and its mass affect its flight through a charged field and

depending on this its structure can be determined using time of flight and distance flown.

The compounds are ionized and have a charge added and then analyzed. This

occurs simply through a five step process. The loading of samples is carried out usually

with an automatic injector or it can be done manually if single samples are being used.

The samples are then vaporized undergoing a phase transition from liquid to gas phase.

Ionization then occurs via a range of ways – including electrospray and lasers, and this

leaves a charged particle which is then accelerated through a larger column which is

surrounded by electromagnetic fields. The ions are then deflected by a magnetic field

according to their masses. This method has been used successfully to elucidate

anthocyanins (McEwen et al., 2005; Pascual-Teresa et al., 2002). These systems allow for

further information to be produced on turfgrass changes in response to both biotic and

22

abiotic stresses. These methods will allow for deeper insight into producing quality

turfgrasses with more specific topographical, geological and geographical variances

catered for. It will give turfgrass breeders new tools in selecting potential turfgrass

cultivars.

The changes that are currently ongoing in the earth’s atmosphere and potential

future issues with higher light intensities and alterations in spectral radiation mean that

understanding the protective pigment capacities of turfgrass in response to this stress is

important. In turfgrass there has been very little work on these pigments. There is a

known increase in total anthocyanin concentration in Kentucky bluegrass (Poa pratensis

L.) in response to UV-B light (Zhang et al., 2005), but there has been no characterization

of the pigments. Identification of changes within turfgrass plants based on possible

attainable levels of UV-B is important and could aid potential breeding efforts in trying to

combat the problem. Their influence on turfgrass color and productivity may not be

understood in this situation and this warrants further investigation. The physiological

aspect of the plant and the response location is also of importance. It can be expected that

tissue receiving more sunlight at the top of the canopy may have a dual purpose. It

creates shaded conditions for the understory, reducing potential for light damage while

also it could be expected that there is a concentration of protective light pigmentation

where the light intensity is greatest. This also may influence carbohydrate allocation and

force a change in plant growth habit. A reduction, for instance, in biomass accumulation

or tillering would lead to a reduction in recovery capability and thus limiting turfgrass use

in certain situations. These questions do pose a learning moment for physiologists and

23

breeders and the objective of this study is to give insight into what protective

pigmentation cool season turfgrasses possess and how plants may or may not use them in

relation to a stress response.

24

REFERENCE Ahmed, J., U.S. Shivhare, and G.S.V. Raghavan. 2004. Thermal degradation kinetics of

anthocyanin and visual colour of plum puree. Euro. Food Res. Tech. 218:525-528. Alba, R., M.M. Cordonnier-Pratt, and L.H. Pratt. 2000. Fruit-localized phytochromes

regulate lycopene accumulation independently of ethylene production in tomato. Plant Physiol. 123:363-370.

Ang, L.-H., S. Chattopadhyay, N. Wei, T. Oyama, K. Okada, A. Batschauer, and X.-W

Deng. 1998. Molecular interaction between COP1 and HY5 defines a regulatory switch for light control of Arabidopsis development. Mol. Cell. 1:213–222.

Anonymous, 1997. Flavonoids (isoflavonoids and neoflavonoids). In McNaught A.D. and

A.Wilkinson (ed.) IUPAC Compendium of Chemical Terminology, 2nd ed. Available at http://old.iupac.org/goldbook/F02424.pdf (verified 28 Jun. 2012).

Bacci, L., D. Grifoni, F. Sabatini, and G. Zipoli. 1999. UV-B radiation causes early

ripening and reduction in size of fruits in two lines of tomato (Lycopersicon esculentum Mill.). Glob. Change Bio. 5:635-646.

Barnes, P.W., S.D. Flint, and M.M. Caldwell. 1990. Morphological responses of crop and weed species of different growth forms to ultraviolet-B radiation. Am. J. Bot. 77:1354-1360.

Bartley, G.E., and P.A. Scolnik. 1995. Plant carotenoids: Pigments for photoprotection, visual attraction and human health. Plant Cell. 7:1027-1038.

Beam, J.B., W.L. Barker, and S.D. Askew. 2006. Selective creeping bentgrass (Agrostis

stolonifera L.) control in cool season turfgrass. Weed Tech. 20:3430-344.

25

Bell, G.E., and T.K. Danneberger. 1999. Temporal shade on creeping bentgrass turf. Crop Sci. 39:1142-1146.

Bennett, R.N., and R.M. Wallsgrove. 2006. Secondary metabolites in plant defense

mechanisms. New Phytol. 127:617-633. Berg, H. 2008. Johann Wilhelm Ritter – The founder of scientific electrochemistry. Rev.

Polarography. 54:99-103.

Björn, L.O., T.V. Callaghan, C. Gehrke, U. Johanson, M. Sonesson, and D. Gwynn-

Jones. 1998. The problem of ozone depletion in northern Europe. Ambio 27:275-279.

Blumenthaler, M., and W. Ambach. 1990. Indication of increasing solar ultraviolet-B radiation flux in Alpine regions. Science 248:206-208.

Bornman, J.F. 1989. Target sites of UV-B radiation in photosynthesis of higher plants. J.

Photochem. Photobiol. 4:145-158.

Bornman, J.F., and A.H. Teramura. 1993. Effects of ultraviolet-B radiation on terrestrial plants. p. 427-471. In A.R. Young et al. (ed.) Environmental UV Photobiology. Plenum Press, New York.

Brandle, J.R., W.F. Campbell, W.B. Sisson, and M.M. Caldwell. 1977. Net photosynthesis, electron transport capacity and ultrastructure of Pisium sativum L. exposed to ultraviolet-B radiation. Plant Physiol. 60:165-169.

Briscoe, A.D., and L. Chittka. 2001. The evolution of colour vision in insects. Ann. Rev.

Entomol. 46:471-510 Britt, A.B. 1996. DNA damage and repair in plants. Annu. Rev. Plant Physiol. Plant Mol.

Biol. 47.75-100.

26

Brouillard, R. 1988. Flavonoids and flower color. p. 525-538. In J.B. Harborne (ed.) The flavonoids: Advances in research since 1980. Chapman Hall, London, United Kingdom.

Caldwell, M.M. 1971. Solar UV irradiation and the growth and development of higher

plants. Photophysiology 6:131-177.

Caldwell, M.M. 1979. Plant life and ultraviolet radiation: Some perspective in the history of the earth's UV climate. BioScience 29:520-525.

Caldwell, M.M., R. Robrecht, and W.D. Billings. 1980. A steep latitudinal gradient of

solar ultraviolet-B radiation in the arctic-alpine life zone. Ecology 6:600-611. Caldwell, M.M., A.H. Teramura, and M. Tevini. 1989. The changing solar ultraviolet

climate and the ecological consequences for higher plants. Trends Ecol. Evol. 4:363-367.

Carrow, R.N., D.V. Waddington, and P.E. Reike. 2001. Turfgrass soil fertility and

chemical problems. John Wiley & Sons, Inc., Hoboken, NJ. Castle, E.S. 1935. Photic excitation and phototropism in single plant cells. Cold Spring

Harbor Symp. Quant. Biol. 3:224-229. Chalker-Scott, L. 1999. Environmental significance of anthocyanins in plant stress

responses. Photochem. Photobiol. 70:1-9.

Croteau, R., T.M. Kutchan, and N.G. Lewis. 2000. Natural products (secondary

metabolites). p. 1250-1318. In B. Buchanan et al. (ed.) Biochem. Molec. Biol. Plts. Am. Soc. Plant. Phys.

Cuadra, P., J.B. Harborne, and P.G. Waterman. 1997. Increases in surface flavonols and

photosynthetic pigments in Gnaphalium luteo-album in response to UV-B radiation. Phytochemistry 45:1377-1383.

27

Cunningham, F.X., and E. Gantt. 2002. Genes and enzymes of carotenoid biosynthesis in plants. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49:577-583.

Cuttriss, A., and B. Pogson. 2004. Carotenoids. In: Plant pigments and their

manipulation. Ann. Plant Rev. 14:57-91.

Day, T.A., T.C. Vogelmann, and E.H. DeLucia. 1992. Are some plant life forms more

effective than others in screening out ultraviolet-B radiation? Oecologia 92:513-519.

Dicke, M., A.A. Agrawal, and J. Bruin. 2003. Plants talk, but are they deaf? Trends

Plant Sci. 8:403-405.

Donner, H., L. Gao, and G. Mazza. 1997. Separation and characterization of simple and

malonylated anthocyanins in red onions, Allium cepa L. Food Res. Int. 30:637-643.

Field, T.S., D.W. Lee, and N.M. Holbrook. 2001. Why leaves turn red in autumn. The

role of anthocyanins in senescing leaves of red osier dogwood. Plant Physiol. 127:566-574.

Flint, S.D., and M.M. Caldwell. 1984. Partial inhibition of in vitro pollen germination by

simulated solar ultraviolet‑B radiation. Ecology 65:792-795.

Flint, S.D., and M.M. Caldwell. 2003. A biological spectral weighting function for ozone

depletion research with higher plants. Physiol. Planta. 117:137-144. Floss, H.G. 1997. Natural products derived from unusual variants of the shikimate

pathway. Natural Prod. Rep. 14:433-452.

Foti, M., M. Piattelli, V. Amico, and G. Ruberto. 1994. Antioxidant activity of phenolic

meroditerpenoids from marine algae. Photochem. Photobiol. 26:159-164.

28

Foyer, C.H., M. Lelandais, and K.J. Kunert. 1994. Photooxidative stress in plants. Physiol. Planta. 92:696-717.

Foyer, C.H., P. Descourvieres, and K.J. Kunert. 1994. Protection against oxygen radicals:

an important defence mechanism studied in transgenic plants. Plant Cell Environ. 17:507-523.

Fraser, P.D., and P.M. Bramley. 2004. The biosynthesis and nutritional uses of

carotenoids. Prog. Lipid Res. 43:228-265.

Frederick, J.E., H.E. Snell, and E.K. Haywood. 1989 Solar ultraviolet radiation at the

earths surface. Photochem. Photobio. 50:443-450.

Galston, A.W., and R.S. Baker. 1949. Studies on the physiology of light action II. The

photodynamic action of riboflavin. Am. J. Bot. 36:773-780.

Garcı́a-Viguera, C., and P. Bridle. 1999. Influence of structure on colour stability of

anthocyanins and flavylium salts with ascorbic acid. Food Chem. 64:21-26.

Gould, K.S. 2004. Nature's swiss army knife: The diverse protective roles of

anthocyanins in leaves. J. Biomed. Biotech. 5:314-320.

Greenwood, D.J., G. LeMaire, G. Gosse, P. Cruz, A. Draycott, and J.J. Neeteson. 1990.

Decline in percentage N of C3 and C4 crops with increasing plant mass. Ann. Bot. 66:425-436.

Gross, J., 1991. Pigments in vegetables: chlorophylls and carotenoids. Van Nostrand

Reinhold, NY. Hardy, A.C. 1938. History of the design of the recording spectrophotometer. J. Opt.

Soc. Am. 28:360-363.

Hare, P.D., and W.A., Cress. 1997. Metabolic implications of stress-induced proline

accumulation in plants. Plant Growth Reg. 21:79-102.

29

Hartley, W.N. 1881. On the absorption of solar rays by atmospheric ozone. J. Chem. Soc. 39:111-128.

Heller, W., and G. Forkmann. 1988. Biosynthesis in the flavonoids: p. 319-425. In J.B.

Harborne (ed.) The flavonoids: Advances in research since 1980. Chapman Hall, London, United Kingdom.

Hong, V., and R.E. Wrolstad. 1990. Use of HPLC separation/photodiode array detection

for characterization of anthocyanins. J. Agric. Food Chem. 38:708-71. Huang, Z.-A., D.-A. Jiang, Y. Yang, J.-W. Sun, and S.-H. Jin. 2004. Effects of nitrogen

deficiency on gas exchange, chlorophyll fluorescence, and antioxidant enzymes in leaves of rice plants. Photosynthetica. 42:357-364.

Irani, N.G., and E. Grotewold. 2005. Light-induced morphological alteration in

anthocyanin-accumulating vacuoles of maize cells. BMC Plant Bio. 5:7. Jackson, J.A., G. Fuglevand, B.A. Brown, M.J. Shaw, and G.I. Jenkins. 1995. Isolation of

Arabidopsis mutants altered in the light regulation of chalcone synthase gene expression using a transgenic screening approach. Plant J. 8:369-380.

Ju, Z., Y. Duan, and Z. Ju. 1999. Effects of covering the orchard floor with reflecting

films on pigment accumulation and fruit coloration in `Fuji' apples. Sci. Hortic. 82:47-56.

Kaiser, T., K. Emmler, B. Weisshaar, E. Schafer, and A. Batschauer. 1995. Promoter

elements of the mustard CHS1 gene are sufficient for light regulation in transgenic plants. Plant Mol. Biol. 28:219-229.

Kakani, V.G., K.R. Reddy, D. Zhao, and A.R. Mohammed. 2003. Effects of ultraviolet‐B

radiation on cotton (Gossypium hirsutum L.). Morph. Anat. Ann. Bot. 91:817-826.

Kerr, J.B., and C.T. McElroy. 1993. Evidence for large upward trends of ultraviolet-B

radiation linked to ozone depletion. Science 262:1032-1034.

30

Kong. J.M., C. Lian-Sai, G. Ngoh-Khang, C. Tet-Fatt, and R. Brouillard. 2003. Analysis and biological activities of anthocyanins. Phytochemistry 64:923-933.

Kopsell, D.A., and D.E. Kopsell. 2006. Accumulation and bioavailability of dietary

carotenoids in vegetable crops. Trends Plant Sci. 11:1360-1385. Koyama, Y., and R. Fujii, 1999. Cis-trans carotenoids in photosynthesis: configurations,

excited–state properties and physiological functions. p. 161-188. In H.A. Frank (ed.) The photochemistry of carotenoids. Kluwer Publishers, Dordrecht, Netherlands.

Krol, M., G.R. Gray, V.M. Hurry, G. Oquist, L. Malek, and N.P.A. Huner, 1995. Low

temperature stress and photoperiod effect on increased tolerance to photoinhibition in Pinus banksia seedlings. Canadian J. Bot. 73:1119-1127.

Kubasek, W.L., B.W. Shirley, A. McKillop, H.M. Goodman, W. Briggs, and F.M.

Ausubel. 1992. Regulation of flavonoid biosynthetic genes in germinating Arabidopsis seedlings. Plant Cell. 4:1229-1236.

Lancaster, J.E., J.E. Grant, C.E. Lister, and M.C. Taylor. 1994. Skin color in apples –

Influence of copigmentation and plastid pigments on shade and darkness of red color in five genotypes. J. Am. Soc. Hort. Sci. 119:63-69.

Langebartels, C., M. Schraudner, W. Heller, D. Ernst, and H. Sanderman Jr. 2002.

Oxidative stress and defense reactions in plants exposed to air pollutants and UV-B radiation. p. 105-137. In D. Inzé and M. Van Montagu (ed.) Oxidative Stress in Plants. Taylor and Francis New York, NY.

Lee, J., R.W. Durst, and R.E. Wrolstad. 2005. Determination of total monomeric

anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. J. of Ass. Analyt. Com. Int. 88:1269-1278.

Lesfrud, M.G., D.A. Kopsell, R.M. Auge, and A.J. Both. 2006. Biomass production and

pigment accumulation in kale grown under increasing photoperiods. Hortscience 41:603-606.

31

Lesfrud, M.G., D.A. Kopsell, and D.E. Kopsell. 2007. Nitrogen levels influence biomass, elemental accumulations, and pigment concentrations in spinach. J. Plant Nut. 30:171-185.

Lohachoompol, V., G. Srzednicki, and J. Craske. 2006. The change of total anthocyanins

in blueberries and their antioxidant effect after drying and freezing. J. Biomed. Biotechnol. 5:248-252.

Lubin, D., and J.E. Frederick. 1991. The ultraviolet radiation environment of the antarctic

peninsula: the roles of ozone and cloud cover. J. Appl. Meteorol. 30:478-493. Lutz, M., I. Agalidis, G. Hervo, R.J. Cogdell, and F. Reiss-Husson. 1978. On the state of

carotenoids bound to reaction centers of photosynthetic bacteria: A resonance raman study. Biochim. Biophys. Acta (BBA) – Bioenergetics. 503:287-303.

Madronich, S., R.L. McKenzie, L.O. Björn, M.M. Caldwell. 1998. Changes in

biologically active ultraviolet radiation reaching the earth's surface. J. Photochem. Photobiol. B: Biol. 46:5-19.

Maliakal, S.K, K. McDonnell, S.A. Dudley, and J. Schmitt. 1999. Effects of red to far-red

ratio and plant density on biomass allocation. Int. J. Plant Sci. 160:723-733. Mancinelli, A.L., F. Rossi, and A. Moroni. 1991. Cryptochrome, phytochrome and

anthocyanin production. Plant physiol. 96:1079-1085. Massacci, A., M. Adelaide-lannelli, F. Pietrini, and F. Loreto. 1995. The effect of growth

at low temperature on photosynthetic characteristics and mechanisms of photoprotection of maize leaves. J. Exp. Bot. 46:119-127.

Matile, P., B.M.P. Flach, and B.M. Eller. 1992. Autumn leaves of Ginkgo biloba L.:

optical properties, pigments, and optical brighteners. Bot. Acta. 105:13-17. McClure, J.W. 1975. Physiology and functions of flavonoids. p. 970-1055. In. J.

Harborne, and T. Mabry (ed.) The flavonoids. Chapman and Hall Ltd., United Kingdom.

32

McCurdy, J.D., J.S. McElroy, D.A. Kopsell, C.E. Sams, and J.C. Sorochan. 2008. Effects of mesotrione on perennial ryegrass (Lolium perenne L.) carotenoid concentrations under varying environmental conditions. J. Ag. Food Chem. 56:9133-9139.

McElroy, J.S., D.A. Kopsell, J.C. Sorochan, and C.E. Sams. 2006. Response of creeping

bentgrass carotenoid composition to high and low irradiance. Crop Sci. 46:2606-2612.

McEwen, C.N., R.G. McKay, and B.S. Larsen. 2005. Analysis of solids, liquids, and

biological tissues using solids probe introduction at atmospheric pressure on commercial LC/MS instruments. Anal. Chem. 77:7826-7831.

McKenzie, R.L., L.O. Björn , A. Bais, and M. Ilyas. 2003. Changes in biologically

active ultraviolet radiation reaching the earth's surface. Photochem. Photobiol. Sci. 2:5-1.

McLennan, A.G. 1987. DNA damage, repair, and mutagenesis. p. 135-186. In J.A.

Bryant and V.L. Dunham (ed.) DNA replication in Plants. CRC Press, Boca Raton, FL.

Mehlenbacher, S.A., and M.M. Thompson. 1991. Inheritance of a chlorophyll deficiency

in hazelnut. Hortscience 26:1414-1416. Mendez, M., D.G. Jones, and Y. Manetas. 1999. Enhanced UV-B radiation under field

conditions increases anthocyanin and reduces the risk of photoinhibition but does not affect growth in the carnivorous plant Pinguicula vulgaris. New Phytol. 144:275-282.

Moncada, M.C., S. Moura, M. João Melo, A. Roque, C. Lodeiro, and F. Pina. 2003.

Complexation of aluminum (III) by anthocyanins and synthetic flavylium salts: A source for blue and purple color. Inorganica Chimica Acta. 356:51-61.

Moradas-Fereira, P.V., P.P. Costa, and W. Mager. 1996. The molecular defenses against

reactive oxygen species in yeast. Mol. Microbiol. 19:651-658.

33

Müller P., L. Xiao-Ping, and K.K. Niyogi. 2001. Non-photochemical quenching. A response to excess light energy. Plant Physiol. 125:1558-1566.

Mustafa, N.R., and R.Verpoorte. 2007. Phenolic compounds in Catharanthus

roseus. Phytochem. Rev. 6:243-258. Neill, S., and K.S. Gould. 1999. Optical properties of leaves in relation to anthocyanin

concentration and distribution. Can J. Bot. 77:1777-1782. Oren-Shamir, M., and A. Levi-Nissim. 1997. UV-light effect on the leaf pigmentation of

Cotinus coggygria ‘Royal Purple’. Sci. Hortic. 71:59-66. Owens, T.G., A.P. Shreve, and A.C. Atbrecht. 1992. Dynamics and mechanisms of

singlet energy transfer between carotenoids and chlorophylls: light harvesting and nonphotochemical fluorescence quenching. p. 170-186. In N. Murata (ed.) In Research in Photosynthesis 4. Kluwer, Dordrecht, Netherlands.

Pascual-Teresa, S. de., C. Santos-Buelga, and J.C. Rivas-Gonzalo. 2002. LC–MS analysis

of anthocyanins from purple corn cob. J. Sci. Food and Agric. 82:1003-1006. Pfeiffer, N.E. 1928. Anatomical study of plants grown under glasses transmitting light of

various ranges of wavelength. Bot. Gaz. 85:427-436. Pfeifer, M.T., P. Koepke, and J. Reuder. 2006. Effects of altitude and aerosol on UV

radiation. J. Geophys. Res. 111. D01203. doi:10.1029/2005JD006444. Pietrini, F., and A. Massacci. 1998. Leaf anthocyanin content changes in Zea mays grown

at low temperature: Significance for the relationship between quantum yield of PS II and the apparent quantum yield of CO2 assimilation. Photosynth. Res. 58:213-219.

Piringer, A.A., and P.H. Heinze. 1954. Effect of light on the formation of a pigment in

the tomato fruit cuticle. Plant Physiol. 29:467-472.

34

Popp, H.W., and F. Brown. 1936. The effect of ultra- violet radiation upon seed plants. p. 853-887. In B.M. Dugger (ed.) Biological effects of radiation. Vol. 2. McGraw-Hill Book Co., New York.

Rabino, I., and A.L. Mancinelli. 1986. Light, temperature and anthocyanin production.

Plant Physiol. 81:922-924. Rind, D., R. Suozzo, N.K. Balachandran, and M.J. Prather. 1990. Climate change and the

middle atmosphere. I: The doubled CO2 climate. J. Atmos. Sci. 47:475-494. Ronen, G., M. Cohen, D. Zamir, and J. Hirschberg. 1999. Regulation of carotenoid

biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon-cyclase is down-regulated during ripening and is elevated in the mutant delta. Plant J. 17:341-351.

Ronen, G., M. Cohen, D. Zamir, and J. Hirschberg. 2000. An alternative pathway to β-

carotene formation in plant chromoplasts discovered by map-based cloning of beta and old-gold color mutations in tomato. Proc. Natl. Acad. Sci. U.S.A. 97:11102-11107.

Rowland, F.S. 2006. Stratospheric ozone depletion. Philos. Trans. R. Soc. London Ser. B.

361:769-790. Rozema, J., J. van de Staaij, L.O. Björn, and M. Caldwell. 1997. Review. UV-B as an

environmental factor in plant life: stress and regulation. Trends in Ecol. 12:22–28. Sarkar, D., P.C. Bhowmik, Y-I. Kwon, and K. Shetty. 2011. The role of proline-

associated pentose phosphate pathway in cool-season turfgrasses after UV-B exposure. Environ. Exp. Bot. 70:251-258.

Schmidt, R.E., and X. Zhang. 2001. Alleviation of photochemical activity decline of

turfgrasses exposed to soil moisture stress or UV radiation. Int. Turfgrass Soc. Res. J. 9:340-346.

35

Schmitt, J., and R.D. Wulff. 1993. Light spectral quality, phytochrome and plant competition. Trends in Ecol. Evol. 8:47-51.

Seigler, D.S. 1998. Plant secondary metabolism. Kluwer Publishers, Norwell, MA. Shackleton, C. 2010. Clinical steroid mass spectrometry: A 45-year history culminating

in HPLC–MS/MS becoming an essential tool for patient diagnosis. J. Ster. Biochem. Mol. Biol. 121:481-490.

Sheahan, J.J. 1996. Sinapate esters provide greater UV-B attenuation than flavonoids in

Arabidopsis thaliana (Brassicaceae). Am J. Bot. 83:679-686. Smith, H. 1982. Light quality, photo perception and plant strategy. Ann. Rev. Plant

Physiol. 33:481-518. Smith, H. 1994. The light environment. p. 377-416. In R.E. Kendrick and G.H.

Kronenberg. (ed.) Photomorphogenesis in plants. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Solomon, S. 1990. Progress towards a quantitative understanding of Antarctic ozone

depletion. Nature 347:347-354. Sponga, F., G.F. Deitzer, and A.L. Mancinelli. 1986. Cryptochorme, Phytochrome and

the photoregualtion of anthocyanin production under blue light. Plant Physiol. 82:952-955.

Stapleton, A.E. 1992. Ultraviolet radiation and plants: Burning questions. Plant Cell. 4:1353-1358.

Strid, Å., and R.J. Porra. 1992. Alterations in pigment content in leaves of Pisium sativum after exposure to supplementary UV-B. Plant Cell Physiol. 33:1015-1023.

Strid, Å., W.S. Chow, and J.M. Anderson. 1994. UV-B damage and protection at the

molecular level in plants. Photosynth. Res. 39:475-489.

36

Sullivan, J.H., and A.H. Teramura. 1988. Effects of ultraviolet-B irradiation on seedling growth in the Pinaceae. Am. J. Bot. 75:225-230.

Taalas, P., J. Kaurola, and A. Kylling. 2000. The impact of greenhouse gases and

halogenated species on the future solar UV radiation doses. Geog. Res. Lett. 27:1127-1130.

Takashi, S., and N. Murata. 2008. How do environmental stresses accelerate

photoinhibition? Trends Plant. Sci. 13:178-182. Tevini, M., W. Iwanzik, and U. Thoma. 1981. Some effects of enhanced UV-B

irradiation on the growth and composition of plants. Planta 153:388-394. Tevini, M. 1993. Effects of enhanced UV-B radiation on terrestrial plants. p. 125-153. In

Tevini M. (ed.) UV-B Radiation and Ozone Depletion. Lewis Publ., Boca Raton, FL.

Wade, H.K., T.N. Bibikova, W.J. Valentine, and G.I. Jenkins. 2001. Interactions within a

network of phytochrome, cryptochrome and UV-B phototransduction pathways regulate chalcone synthase gene expression in Arabidopsis leaf tissue. Plant J. 25:675-685.

Wellburn, A.R. 1994. The spectral determination of chlorophylls a and b, as well as total

carotenoids using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 144:307-313.

Wrolstad, R.E., and M.M. Giusti. 2001. Characterization and measurement of

anthocyanins by UV-visible spectroscopy. Unit F1.2. p. F1.2.1-F1.2.13. In R.E. Wrolstad and S.J. Schwartz (ed.) Current Protocols in Food Analytical Chemistry. John Wiley & Sons, Inc. New York, NY.

Zhang, X., E.H. Ervin, and R.E. Schmidt. 2005. The role of leaf pigment and antioxidant

levels in UV-B resistance of dark - and light - green Kentucky bluegrass cultivars. J. Am Soc. Hort. Sci. 130:836-841.

37

Ziska, L.H, A.H. Teramura, and J.H. Sullivan. 1992. Physiological sensitivity to plants along an elevation gradient to UV-B radiation. Am. J. Bot. 79:863-871.

Zu-Hua, Y., K. Siebke, and U. Heber. 1990. Light dependent changes in leaves of C3

plants. III. Effect of inhibitors of photosynthesis and of developmental state of photosynthetic apparatus on cystolic and vacuolar pH changes. Planta 182:262-269.

38

CHAPTER 2

SUBJECTIVE AND OBJECTIVE COLOR RATINGS OF TURFGRASSES AND THEIR LINKS TO PIGMENTATION AND NITROGEN CONCENTRATIONS.

ABSTRACT

Turfgrass color is used to correlate turfgrass responses to management inputs such as

fertility treatments and response to environmental stresses. Visual perception of color is

considered a crucial measurement of turfgrass quality as it is the quickest method of

judging turfgrass performance. The measurement can be subjective; therefore the digital

analysis of color has been added as a means to add objectivity to standardize color

measurements. The purpose of this study was to test for a correlation between subjective

and objective turfgrass color rankings and chlorophyll pigment concentrations in several

cool season grasses on an area and volume basis. Fifty one cultivars of six different cool

season grasses were used; perennial ryegrass (Lolium perenne L.), Kentucky bluegrass

(Poa pratensis L.), creeping bentgrass (Agrostis stolonifera L.), tall fescue (Schedenorus

phoenix (Scop.) Holub, fine fescues (Festuca spp.), and colonial bentgrass (Agrostis

capillaris L.). Turfgrasses were germinated and maintained in fifteen cm pots for two

months. Subjective analysis was then carried out using a visual rating for greenness on a

1-9 scale.

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Objective analysis of chlorophyll pigmentation on a mass and area basis was carried out

using a handheld colorimeter on the grass surface and a bench top colorimeter on pigment

extract. Both systems used the CIELab color measurement system. Tissue nitrogen and

chlorophyll content by area and mass were measured. The handheld system measuring

reflectance did not correlate to pigmentation concentration (P=0.05). Visual rating was

closely linked to nitrogen levels and the benchtop system was influenced by pigment

concentrations (P=0.05). Hand held systems using reflectance type measurements may

suffer from excess error due to characteristics such as surface texture and leaf thickness.

INTRODUCTION

Turfgrass color is traditionally ranked on a 1-9 scale with a ranking of one equal

to dead brown turf and nine equal to optimal green turf color in the eyes of a rater.

However, this is a subjective measurement. In trying to develop a rapid method of

measuring turfgrass responses to either stresses or management inputs, researchers have

found success with using handheld reflectance systems in field situations to measure

plant color. The measurement of plant disease damage and plant nutrient deficiencies

have been successfully tested in creeping bentgrass (Agrostis stolonifera L.), and corn

(Zea mays L.), (Nutter et al., 1993; Bausch and Duke, 1996). These systems provided

highly correlated data and were shown to have positively correlated (R) values to visual

ratings and even tended to be less biased (Nutter et al., 1993). There has been success

using systems which are wavelength dependent to indicate biomass status and

chlorophyll (680 – 780 nm) content (Filella and Penuelas, 1994). Wavelengths in the red

40

range 680-780nm are considered the best estimator of leaf area index in (Capsicum

annuum L.) and (Phaseolous vulgaris L.). Water stress can be measured using this

technology also, however this was seen only after the stress was well developed (Nutter

et al., 1993).

The digital color measurement systems in use have different functions. Digital

imaging has been found to be extremely successful (r2=0.99) in correlating Munsell color

chips to nitrogen treatments in creeping bentgrass (Karcher and Richardson, 2003). The

digital system used is also more precise when compared to traditional methods

(Richardson et al., 2001). The digital analysis uses photography and software to develop

ranking systems and has the ability to obtain large volumes of data from one picture, and

separate out colors with excellent reproducibility. Colorimeters, which depend on a

reflective light wavelength and intensity measurement have been used to separate

creeping bentgrass cultivars and different nitrogen treatments (Landschoot and Mancino,

2000; Mangiafico and Guillard, 2005). The method used in our trials is offered as an

alternative method for turfgrass color analysis which can use either handheld reflective

measurements or spectral transmission benchtop measurements and link them to nitrogen

measurement in turfgrass.

A majority of the color-based systems in use are dependent on a reflective

measurement of turfgrass leaf surfaces. However, leaf surfaces vary in many ways due to

environmental conditions and varietal differences. Tall fescue (Schedenorus phoenix

(Scop.) Holub.) cultivars have a leaf thickness of between 0.31 and 0.36mm under

drought stress and a drought free leaf width of 0.44 cm to 0.52 cm (Fu and Huang, 2004).

41

Nitrogen content causes variation in tall fescue leaf width from 0.31 cm to 0.75 cm

(Rademacher and Nelson, 2001). Colorimeter systems do not have the capability to

measure such narrow sizes creating the potential for inaccurate measurements due to

different leaf age and different nitrogen concentrations. Upper and lower leaf surfaces

also vary in thickness. The upper epidermal surface of Kentucky bluegrass can be 0.58

µm and 1.23 µm on the lower epidermal surface. This thickness variation in the leaf

surface plays a role in light absorption, reflection and transmission (Cameron, 1970;

Clark and Lister, 1975). Penetration and reflection of light varies by leaf thickness.

Treatment by UV-B of alfalfa (Brassica campestris L.) resulted in an increase of leaf

thickness by 45% - causing an increase in the amount of light back scattered (Bornman

and Vogelmann, 1991). Variations across and within turfgrass species leaf thickness

could influence reflective type measurements.

Human eyesight uses rods and cones in the retina. The cones are sensitive to

short, medium and long wavelengths and work to absorb opposing colors of red, green

and blue (Brown and Wald, 1964; Roorda and Williams, 1999). The methods of color

measurement by different handheld reflectance systems vary. The systems can use color

rankings from the Munsell system (Escadafal et al., 1989), Hunter color systems or CIE

Lab scales (Commission Internationale de l'éclairage), (Pattee et al., 1991). The use of the

Munsell color system (Munsell, 1915) was designed to rank color by Albert Munsell

(Nickerson, 1940). This is considered the initial color system, but it did not transfer

consistently into digital reproduction and so more recent systems were developed such as

CIELab and Hunter scales (Sharma and Trussell, 1997). These two systems attempt to

42

replicate how the rods and cones work in the retina and are based on a fixed scale so as

not to become device dependent and lacking reproducibility (Lemaire et al., 2005).

A handheld colorimeter system uses measurements in different color spaces and

L* is considered a measure of lightness while a* measures green (-) or red, (+) and b* is

a measure of blue (-) and yellow (+) colors on the wheel. Hue or the color attribute is also

measureable in a degree system to fit the color wheel while chromaticity is used to

measure the lightness (100 = white) or darkness (0 = black) of the color. This represents a

similar system to human eyesight and can be converted into a 3-D image for digital

analysis (American Society for Testing and Materials, 1989). The system emits a diffuse

uniform light source over a set measuring area that is reflected and collected by an optical

fiber cable which enables analysis of color (Minolta, Chroma Meter CR-300 Instruction

manual). The light source can be manipulated to equal daylight conditions D65 or C

which have similar light circumstances but D65 is preferred by CIE. There are other

illuminant types which can replicate indoor lighting. The handheld systems are fast,

simple and field usable (Shimada et al., 2004).

Previous work investigated correlating colorimeter values to creeping bentgrass

cultivars using the CIELab system and the Minolta CR-310 handheld colorimeter. The

results indicated that the hue angle – the component of the system that describes the true

color which is then further influenced by the lightness or chroma of the color – was the

most consistent part of the data and correlated favorably with data collected from visual

rankings (Landschoot and Mancino, 2000). The data, however, were not linked to any

plant characteristic thus offering no ability to replicate the study. CIELab system data can

43

be transferred to tristimulus based values such as XYZ values and so it offers a possible

system that is transferrable between multiple pieces of equipment. This then allows for

comparison of the same characteristics across grass types (Reinhard et al., 2001). Trials

on creeping bentgrass using the normalized difference vegetation indices (NDVI) system

compared to turfgrass pigmentation content indicated an influence on color ratings when

nitrogen, magnesium and iron fertilization rates were varied. The variation in the color

was only explained 36% of the time by chlorophyll pigmentation and that another factor

or combination of factors was responsible for the result (Stiegler et al., 2005).

Extract measurements of chlorophyllous pigments using benchtop colorimeters

can also give color measurements based on either transmittance or reflectance dimensions

(Ahmed et al., 2000). Transmitted or reflected light beams are collected through a lens

which is 8° from perpendicular to the sample surface. The beams are then integrated

using a sphere and flash beams. The flash reduces the noise and allows for higher

intensity light which normally reduces sensitivity of the pigments due to degradation

when exposed to light for an extended period (Joilot and Joilot, 1984).

Spectrophotometers have been used to test color differences between grasses extracts

(Kavanagh et al., 1985). The transmitted light is then passed through a diffraction grating

which separates and measures the wavelengths of light. These systems also possess the

ability to measure in L*a*b* color space (Yudin et al., 1987). The use of extracts requires

lab time however; this can be a downside to the use of benchtop color measurement due

to cost and time while field measurements are easily and quickly carried out.

44

These systems offer opportunities to standardize color system management and

take out subjectivity that can occur between trained and untrained raters of color

(Gooding and Gamble, 1990; Landschoot and Mancino, 1997). There, however, needs to

be a larger bank of color data compiled with these systems. There are limited data in

relation to pigmentation influence on color rating and whether or not surface reflectance

measurements or transmission type measurements are more appropriate for color

analysis. The objective of this study was to discover how chlorophyll pigments influence

color systems analysis and link pigment concentration to a particular trait within the color

system to create a baseline of data for breeders and other end users. The study aims to

determine what influences human color perception which could be related to stress

responses during periods of pigment decline such as exposure to UV-B. It further aims to

discover whether there is a correlation between visual rating and color system ratings.

MATERIALS AND METHODS Plant material

Two separate Turfgrass trials were run from March, 1st 2010 until June, 11th 2010,

and June 15th 2010 until September 30th 2010 at The Ohio State University, Howlett Hall

greenhouse, 680 Vernon Tharp St., Columbus OH, 43210. Six turfgrass varieties were

seeded in a sand based rootzone, of 80/20 volume/volume sand / peat mix. There were 9

cultivars of perennial ryegrass, Kentucky bluegrass, creeping bentgrass, tall fescue, fine

fescues, sheeps fescue (Festuca ovina L.), creeping red fescue (Festuca rubra L.), hard

fescue (Fesutca brevapila Tracey.). Six cultivars of colonial bentgrass were evaluated.

45

The grasses were chosen based on their color rankings from national turfgrass

evaluation program (NTEP) trial results for 2003 – 2007 for all grasses and based on

commercial availability. Cultivars were randomly separated by the rankings of high /

intermediate / low (Top 1/3, Middle 1/3 Lowest 1/3) of grasses on the NTEP evaluation

results.

Turfgrasses were watered with a mist system initially for 30 seconds every 10

minutes during germination (21 days). Once established, grasses were watered twice

daily for 3 minutes. All turfgrass samples were treated with 20-20-20 liquid fertilizer

(Scotts Co. Marysville, OH) at a rate of 4.06 kg ha-1 on a bi weekly basis. Each grass was

then grown out to a height of 10 cm and maintained at this height for two months in a

greenhouse with temperatures maintained at ~20° Celsius.

Data collection

Turfgrass samples were placed in a completely randomized design in full sun

conditions. A total of 44 untrained (not trained in evaluating turfgrass color) and trained

(turfgrass researchers with training and experience in ranking turfgrass color) evaluators

ranked grasses based on their color. Ranking was set with 1 = brown 6 = satisfactory and

9 = optimal dark green. Turfgrasses were irrigated twice on both days to prevent wilt and

plant material was unranked for 1 hour periods post irrigation to prevent any impact of

water remaining on leaf surfaces on rankings. On both occasions grasses were in full sun

for the total period of time during ranking. A total of forty two surveys were collected

between both experiments. Visual ratings by humans were carried out with permission of

46

institutional review board (IRB project #2010E0291) on June 12th and again on

September 22nd 2010.

Turfgrass samples were taken immediately following the visual rating and leaves

were collected by area (40 mm-2) and on a mass basis (0.05 g) with three subsamples

taken from each cultivar and placed in N,N-dimethylformamide (Sigma-Aldrich, St

Louis, MO) to extract chlorophyll, (Moran and Porath, 1980). Samples were stored in

dark conditions for a period of 48–72 hours after this. Extract was subjected to absorption

analysis using a UV-Vis spectrophotometer (Shimadzu Scientific Instruments Inc.,

Columbia, MD) at wavelengths of 664, 647, 625 and 603 nm. Data were calculated using

formulas from Moran, (1982) and chlorophyll totals, chlorophylls A and B were

quantified on an area and mass basis. The premise for this was that the hand-held system

measures directly on a set area on leaf tissue while benchtop systems require tissue

extract for analysis. Correlating color with chlorophyll content based on the area and

mass measurements were designed to fit this variation between color measurement

systems.

Freshly harvested samples were used for reflectance analysis. Turfgrass leaves

were layered four times to create an optically dense stack (Mangiafico and Guillard,

2005), then three light measurements were taken on the top layer (Appendix A), using a

Konica Minolta CRM 310 (Konica Minolta Sensing, Ramsey, NJ). The stacking and

multiple readings were done to reduce variability due to turfgrass surface irregularity.

The layers were then randomly re-ordered and measurements repeated. Samples were re-

configured three times for a total of nine color measurements per pot and thirty six

47

measurements per cultivar. Data were taken for chroma, hue lightness (L*) red/green

(±a*) yellow/blue (±b*). Prior to analysis the system was calibrated with a white plate

provided by the manufacturer.

Color analysis of extract was carried out using the Hunter Colorquest XE system

(Hunter Associates Laboratory Inc., Reston, VA). Turfgrasses were initially tested for

spectral transmission properties to assess which volume of turfgrass was suitable to

reduce high viscosity or high reflectance levels which could distort data. Turfgrass

samples weighing 0.5 g were determined to be most consistent across all grasses used.

Turfgrass samples were then placed in 15 mL glass tubes with 10mL of N,N-

dimethylformamide. N,N-dimethylformamaide was used to extract all color related to

chlorophyll pigments due to its high efficiency (Stiegler et al., 2004). Samples were

placed in reagent for 48hrs in darkness. Extract was then placed in a 20 mL cuvette with

10 mm path length. Each sample was then tested at illuminant settings of D65 and

transmittance settings with data acquired for chroma, hue lightness (L*) green/red (±a*)

yellow/blue (±b*). Prior to analysis, calibration was performed with a white plate

provided by manufacturer and dimethylformamide was used as a blank.

Tissue samples from each grass were taken and dried for 48 hrs at 60 °C prior to

tissue analysis for percentage nitrogen content at this time (A&L Eastern Laboratories,

Richmond, VA).

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Data analysis and experimental design

Four replications of each cultivar were used in two repeated trials. The pots were

ranked by humans in a completely randomized design in an outdoor setting with full sun

on turfgrasses throughout the day. During the second experiment a precipitation event

forced the stoppage of ranking, this resumed once water was removed from leaf surface

and cloud cover was gone.

Data transformation of a*b* values is required to create the hue value which is in

a degree format to fit onto the 3-D color circle associated with the system. The

conversion formula is:

degrees ((Atan2(b*,a*))

Following this conversion data was tested using robust regression ROBUSTREG

analysis for data influence and outliers (Chen, 2002). Normality tests were carried out

using the Kolmogorov – Smirnov test while skew and kurtosis were tested also. Data was

then analyzed using multivariate principal components analysis with the R program (R

Development core team, 2011; Jaisingh et al., 2006) to gain an in initial understanding on

variable influence on color. The results of the multivariate analysis were then used to

develop regression and correlation data using PROCREG and PROCCORR with SAS

(SAS Institute, Cary, NC). Differences between cultivar color, nitrogen content and

chlorophyll content were analyzed using PROCGLM. All grasses were analyzed

separately and as a completely randomized design.

49

RESULTS

There was some influence from raw values in the data and outliers were discarded

accordingly (P=0.05). Normality tests (Kolmogorov-Smirnov) found the data to be non-

normal (P=0.05) and so data was tested for outliers using PROC ROBUSTREG. Data

with leverage due to errors in entry or large variability were removed. Kurtosis and skew

varied between grasses and also between transmission and reflectance measurements

(Table 2.1). Transmission data was distributed normally for fine fescue, colonial

bentgrass and Kentucky bluegrass. The data for other three grasses were also normally

distributed with tall fescue (0.04) (P=0.05), creeping bentgrass (0.03) and perennial

ryegrass (0.04) according to Kolmogorov-Smirnov tests. All grasses, with the exception

of fine fescue (0.03) and Kentucky bluegrass (0.03), were found to have normal data for

reflectance measurements (Table 2.1). Skew was similar for both transmission and

reflectance though no grass had a normal distribution based on kurtosis and skew analysis

(Table 2.1).

There was some correlation between hue and chlorophyll B in Kentucky

bluegrass (-0.6), colonial bentgrass (-0.6) and creeping bentgrass (-0.3) cultivars for

transmission analysis with the bench system (Table 2.2). The negative value obtained

correlation is due to the values obtained for the -A* measurements in the green region.

Perennial ryegrass, tall fescue and fine fescue had limited to poor correlation with hue,

chroma A* and B* values (Table 2.2). The strongest correlation across all pigments and

transmission values was -0.6 (Chl B – Hue : Colonial bentgrass / Kentucky bluegrass)

50

with all grasses except colonial bentgrass having no correlation between a certain

pigment and measurement value at least once (Table 2.2).

No, or limited, relationships existed between chlorophyll levels or variations in

chlorophyll pigments and visual rating (Table 2.3). Visual rating was closely linked in

particular with nitrogen levels in perennial ryegrass (0.7) but there was limited linkage

overall, either on an area basis or chlorophyll volume basis between visual rating and

chlorophyll content (Table 2.3). Visual analysis and correlation with the reflectance and

transmission data was also very limited with data having a correlation of no more than -

0.5 which occurred when correlating reflectance characteristics to the visual rating of fine

fescue (Table 2.4).

There was a correlation between nitrogen levels and visual rating of green color

(Figure 2.1). Lower levels of tissue nitrogen content led to lower rankings within species.

Neither pigment concentrations nor nitrogen content influenced CIElab measured

reflectance. The transmission analysis was influenced by all of the pigments. Visual

rating was independent of transmission and reflectance while N and grass variety had

positive correlations with human color rankings (Figure 2.1).

There was a very poor relationship found between reflectance measurements for

color and turfgrass pigmentation (Table 2.5). The strongest noted relationship existed

between the red/green (A*) component and blue yellow component (B*) of colonial

bentgrass with a value of 0.5 and -0.5 for both. Grasses such as tall fescue, perennial

ryegrass and fine fescue had an even more limited relationship between pigments and

color measurements (Table 2.5). Hue values were not seen to be linked closely to total

51

chlorophyll in all grasses while reflective analysis did have stronger correlation with Chl

b concentrations (Table 2.5).

The a* and b* values were influenced minimally on their own by chlorophyll

content in perennial ryegrass, creeping bentgrass and Kentucky bluegrass using both

transmission and reflectance methods (Tables 2.2, 2.5). Correlation between turfgrasses

fine fescue, tall fescue and colonial bentgrass and chlorophyll was comparatively stronger

but the strongest relationship was only found to be 0.5 between chlorophyll B content and

A* and B* reflectance measurement (Table 2.5). A correlation of 0.6 and -0.7 was found

between nitrogen levels and reflectance measurements for colonial bentgrass and A* and

B* (Table 2.6).

The reflectance measurements were linked closest to nitrogen content for A* and

B* with the strongest relationships occurring between nitrogen fine fescue, tall fescue

and colonial bentgrass (Table 2.6). Nitrogen levels had the strongest relationship in

reflectance measured hue for fine fescue, tall fescue and colonial bentgrass (-0.4) and a

higher correlation with chroma for the same three grasses (-0.6, -0.5, -0.6). Nitrogen did

correlate with colonial bentgrass in transmission measurements for hue (0.8) but

transmission analysis in general did not link as closely to nitrogen content in fine fescue

or tall fescue. Colonial bentgrass, creeping bentgrass and Kentucky blue grass had

stronger links between color characteristics and tissue nitrogen content when using

transmission analysis (Table 2.6).

Cultivar differences were noted within grasses. They did not follow visual rating

for reflection analysis and there was limited similarity in the rankings from the

52

transmission measurements (Table 2.4). The visual rating did separate cultivars though

not following the same ranking as NTEP evaluations to a large extent. Perennial ryegrass

had similar rankings but not values, while tall fescue had limited separation (P=0.05)

using human data (Table 2.7).

DISCUSSION AND CONCLUSION

The correlation between key components of turfgrass color and objective

measurements using refelectance and transmission systems in this study was limited.

Pigment concentrations in our study had a poor relationship with the handheld measuring

system in particular. This is consistent with Stiegler at al., (2005) who stated that pigment

concentration could only account for 36% of the variability in turfgrass color and that

other factors play a role when using reflective measuring devices. The studies that had

success with the reflectance system included variations in nitrogen rates as part of the

studies (Landschoot and Mancino, 1997; Mangiafico and Guillard, 2005). The variation

in nitrogen rates would presumably enhance differences in turfgrass colors making it

more likely that reflection analysis could distinguish differences similar to human

eyesight. Forcing of differences was not the aim of this trial so nitrogen rates were not

altered.

There was some what more correlation between pigment concentrations in our

study and transmission measurements. The use of the extract and use of the

spectrophotometer equipment may have been more beneficial for this relationship. In

using reflectance systems there also could have been an increase in light scattering from

53

the leaf surfaces, possibly reducing the precision of the results. The isolation of the

pigments through extraction would also have helped the transmission results and offered

a clear influence on the results. There may be a greater opportunity to more strongly

correlate the reflectance measurements if scattering from the leaf surfaces can be

reduced.

Other studies have concentrated on using smooth surface turfgrass leaves such as

Kentucky bluegrass and creeping bentgrass. Turfgrass surfaces which are known for

varied veination and narrower leaves such as the tall fescue, perennial ryegrass and fine

fescues used here offer a problem for attempting to obtain a consistent reflectance type

measurement. In tall fescue for instance with a change in ploidy from 4X to 8X, which

could have played a role in this study, there was a decrease in vein number on the leaf

surface of 14.2 to 10.2 in major veins. Minor veins on average increase from 4.2 to 6.6

with ploidy changes (Byrne et al., 1981). These surface differences and leaf angle play a

role in light reflectance properties; abaxial surfaces reflect greater levels of light in

grasses than adaxial surfaces. The structure of the epidermal layer (Stiff and Powell,

1974; Thomas and Barber, 1974) which varies in grasses plays a large role in their optical

properties (Sheehy, 1975). Glaucous leaves of eucalyptus (Eucalyptus obliqua L’Hér.)

absorbed only 37% of total radiation while green leaves absorbed 50% of total radiation

(Thomas and Barber 1974). These are all possible areas that would affect the use of a

handheld system in our study which could reduce its reproducibility.

For a more accurate assessment of the ability of these handheld systems other

pigments may need to be taken into account when working with reflectance

54

measurements. Flavonoids absorb light up to 430 nm (Saxby, 1964) and thus reach into

the visible light range; variations in their concentrations can occur with leaf age and also

location in the leaf (Morales et al., 2011). The carotenoids also absorb blue light in the

visible spectrum (Zscheile et al., 1942). The pools of carotenoids found in turfgrass

including lutein and the xantophylls can change based on irradiance levels (McElroy et

al., 2006). Anthocyanins also absorb light in the 500-530 nm region (Gould, 2004), and

may play a role in light wavelengths reflected in that region of PAR. We did not measure

these pigments and they may have played a role in creating the variability we found using

the handheld system in this study.

Leaf texture may also play a role in the lack of strong data correlation. Grasses

which possess leaf hairs and trichomes such as buffalograss (Buchloe dactyloides L.) also

offer a problem for this equipment. There may be increased scattering of radiation on

turfgrasses thus increasing diffuse reflectance (Gausman and Cardenas, 1969; Chien and

Sussex, 1996). Texture influences not only turfgrasses as it has been noted in the food

industry that these issues do exist when analyzing color (Giusti and Wrolstad, 2005.)

It could be expected that visual ratings by untrained evaluators in our study would

not follow NTEP rankings due to a lack of training and possible differences in light and

locations. There can also be variation between trained observers and their color rankings

(Landschoot and Mancino, 1997). The value of having trained observers may be lost in

this instance with greater argument for use of objective standardized rankings of color.

Variability in human eyesight and perspective of color allows for variation in comparison

to NTEP trials and also as compared to trained evaluators. Due to location the potential

55

for visual ratings varying is increased with disparities in light intensity and light angle

playing a role in color analysis (Giusti and Wrolstad, 2005). This issue is part of the

reason that there is interest in using a standardized system to analyze turfgrass color. The

transmission and reflectance methods which offer a standardized color system with

CIELab did not follow the visual ratings with NTEP or local visual ratings. This is

despite the fact the two systems are designed to replicate human eyesight in standard

daylight illumination. This is the first time such a broad scale measurement of turfgrass

species and turfgrass cultivars has been tested however, and previous studies did not aim

to link results to NTEP trial results.

Visual analysis is linked closest to nitrogen content, which has been noted before.

Higher nitrogen rates lead to higher color rankings (Guertal and Evans, 2006; Frank et

al., 2004). Transmission and reflection based analysis also linked most strongly with

nitrogen although in this research the correlation values of 0.4 are not considered strong.

Altering nitrogen rates could lead to a more favorable result (Landschoot and Mancino,

1997). The measurement of nitrogen rather than color may prove to be a better option for

future research as a broader database is created for turfgrasses and reflectance-based

measurements using the same field equipment or, at minimum, the same scale.

56

*Indicates significance in data different from normal at P=0.05. § NS, Non significant at P=0.05. † Data considered to have skew of 0 are considered normal. ‡ Data considered to have kurtosis of 0 are considered normal. Table 2.1: Normality test results and data processing results for six cool season grasses evaluated for color using transmission and reflection type measurements post ROBUSTREG analysis.

Transmission Reflectance Grass Skew† Kurtosis‡ Kolmogorov - Smirnov Skew Kurtosis Kolmogorov - Smirnov

Fine fescue 0.6 2.4 0.05 NS§ 0.6 1.5 0.03 * Tall fescue 0.7 1.5 0.04 * 0.5 0.6 0.05 NS

Colonial Bentgrass 0.3 0.8 0.05 NS 0.4 0.5 0.07 NS Creeping bentgrass 0.6 0.8 0.03 * 0.6 1.0 0.07 NS Perennial ryegrass 0.5 1.4 0.04 * 0.4 0.5 0.09 NS

Kentucky bluegrass 0.7 1.3 0.05 NS 0.6 0.7 0.03 *

57

† A measure of color using CIELab system – 3-d color system using lightness / red-green / blue-yellow components. ‡ A measure of intensity of color using CIElab. § The red-green component of the color system. ¶ The blue yellow component of the color system. Correlation values below 0.7 are considered to have limited relationship between components analyzed Table 2.2: Pearsons correlation values for chlorophyll pigmentation and transmission characteristics obtained using a Hunter XE colorquest colorimeter on extracts of six cool season turfgrasses.

Chl Total Chl a Chl b Grass Hue† Chroma‡ A*§ B*¶ Hue Chroma A* B* Hue Chroma A* B*

Fine fescue 0.1 0.2 -0.1 0.1 0.1 0.2 -0.1 0.1 0.1 0.0 0.0 0.1 Tall fescue 0.0 -0.4 -0.3 0.3 0.0 -0.3 -0.3 0.3 0.0 -0.3 -0.3 -0.3

Colonial bentgrass -0.5 0.2 0.3 -0.4 -0.4 0.2 0.2 -0.3 -0.6 0.1 0.4 -0.5 Creeping bentgrass -0.2 0.1 0.1 -0.1 -0.1 0.2 0.2 -0.2 -0.3 -0.2 -0.1 0.0 Perennial ryegrass 0.0 -0.4 -0.2 0.1 0.0 -0.4 -0.2 0.1 -0.0 -0.4 -0.2 0.1

Kentucky bluegrass -0.5 -0.1 0.1 -0.3 -0.4 -0.0 0.1 -0.2 -0.6 -0.4 0.1 -0.4

58

† Correlation values below 0.7 are considered to have limited relationship between components analyzed. Table 2.3: Pearsons correlation values for chlorophyll pigmentation and tissue nitrogen levels visual rating of six cool season turfgrasses.

Pigment / g-1 Pigment / m-2 Grass Chl total Chl a Chl b N Chl total Chl a Chl b N

Fine fescue -0.2† -0.2 -0.2 0.2 -0.4 -0.4 -0.2 0.2 Tall fescue -0.1 -0.0 -0.1 0.1 0.1 0.1 -0.0 0.0

Colonial bentgrass -0.2 -0.2 -0.1 0.2 -0.0 -0.0 0.0 0.1 Creeping bentgrass -0.2 -0.1 -0.2 -0.1 0.1 0.1 0.1 -0.1 Perennial ryegrass 0.1 0.1 0.1 0.7 0.1 0.1 0.1 0.7

Kentucky bluegrass -0.3 -0.3 -0.1 -0.3 -0.1 -0.1 -0.1 -0.3

59

† A measure of color using CIELab system – 3-d color system using lightness / red-green / blue-yellow components. ‡ A measure of intensity of color using CIElab. § The red-green component of the color system. ¶ The blue yellow component of the color system. Correlation values below 0.7 are considered to have limited relationship between components analyzed Table 2.4: Pearsons correlation of visual rating to transmission and reflection color characteristics of six cool season turfgrasses.

Transmission Reflectance Grass Hue† Chroma‡ A§ B¶ Hue Chroma A B

Fine fescue 0.1 0.0 -0.1 0.1 0.1 -0.5 0.3 -0.1 Tall fescue 0.0 0.2 0.0 0.0 0.1 -0.1 0.1 -0.1

Colonial bentgrass -0.1 -0.1 0.1 -0.1 -0.1 -0.0 -0.1 -0.0 Creeping bentgrass 0.1 -0.0 0.2 -0.1 0.1 -0.0 0.0 0.1 Perennial ryegrass -0.1 -0.0 -0.2 0.2 0.0 -0.1 -0.0 -0.0

Kentucky bluegrass -0.1 0.0 0.1 -0.1 0.0 0.1 -0.1 0.1

60

† A measure of color using CIELab system – 3-d color system using lightness / red-green / blue-yellow components. ‡ A measure of intensity of color using CIElab. § The red-green component of the color system. ¶ The blue yellow component of the color system. Correlation values below 0.7 are considered to have limited relationship between components analyzed Table 2.5: Pearsons correlation values for chlorophyll pigmentation and reflection characteristics obtained using a Konica CR310 handheld colorimeter on surfaces of six cool season turfgrasses.

Chl Total Chl a Chl b Grass Hue† Chroma‡ A§ B¶ Hue Chroma A B Hue Chroma A B

Fine fescue -0.4 0.2 0.0 -0.4 -0.4 0.2 0.0 -0.4 -0.4 0.1 0.1 -0.3 Tall fescue -0.1 -0.1 0.2 -0.1 -0.1 -0.1 0.2 -0.1 -0.0 -0.1 0.1 -0.1

Colonial bentgrass -0.2 -0.3 0.4 -0.4 -0.2 -0.3 0.4 -0.4 -0.3 -0.4 0.5 -0.5 Creeping bentgrass 0.0 -0.0 0.0 -0.0 0.0 -0.0 0.0 -0.0 0.0 -0.0 0.0 0.0 Perennial ryegrass 0.1 0.1 -0.1 0.1 0.1 0.1 -0.1 0.1 0.1 0.1 -0.1 0.1

Kentucky bluegrass 0.1 0.0 -0.0 0.1 0.1 0.0 -0.0 0.1 0.0 0.1 -0.1 0.0

61

† A measure of color using CIELab system – 3-d color system using lightness / red-green / blue-yellow components. ‡ A measure of intensity of color using CIElab. § The red-green component of the color system. ¶ The blue yellow component of the color system. Correlation values below 0.7 are considered to have limited relationship between components analyzed Table 2.6: Pearsons correlations between reflectance measurements and transmission measurements with tissue nitrogen for 6 cool season turfgrasses.

Reflectance Transmission Grass Hue† Chroma‡ A§ B¶ Hue Chroma A B

Fine fescue -0.4 -0.6 0.6 -0.7 0.1 0.3 -0.4 0.3 Tall fescue -0.4 -0.5 0.5 -0.5 -0.0 -0.4 -0.2 0.1

Colonial bentgrass -0.4 -0.6 0.6 -0.7 0.8 -0.2 -0.6 0.7 Creeping bentgrass 0.1 -0.0 -0.1 0.1 0.2 0.5 0.3 -0.2 Perennial ryegrass -0.1 -0.1 -0.1 0.1 -0.2 -0.0 -0.4 0.3

Kentucky bluegrass -0.1 -0.2 0.2 -0.2 0.4 0.5 -0.3 0.4

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Grass Cultivar Colonial bentgrass Fine fescue Kentucky bluegrass Creeping bentgrass Perennial ryegrass Tall fescue

1 6.4 a† 7.7 ab 7.2 ab 6.6 a 6.9 a 8.2 a 2 5.4 c 6.4 c 6.2 def 4.9 b 7.1 a 7.4 b 3 6.1 ab 8.0 a 6.0 ef 5.3 b 6.5 ab 7.2 b 4 6.4 a 8.0 a 6.7 bcd 6.5 a 6.6 ab 8.1 a 5 5.2 c 6.1 c 6.0 ef 5.2 b 5.8 cd 7.0 b 6 5.6 ab 6.1 c 7.4 a 6.6 a 6.2 bcd 7.3 b 7 n/a‡ 5.5 d 7.0 abc 5.1 b 6.4 abc 8.0 a 8 n/a 7.3 b 5.9 f 5.0 b 5.1 e 7.0 b 9 n/a 7.3 b 6.5 cde 5.5 b 5.6 de 7.3 b

LSD§ (P=0.05) 0.5 0.6 0.6 0.7 0.7 0.5 † Data followed by a different letter in each column represent significant differences between cultivars (P=0.05). ‡ Only six cultivars could be found for colonial bentgrass. § Means separation carried out using Fishers least protected means (P=0.05). Table 2.7: Human color rankings for cultivars of six cool grasses. (1= brown / 6 = acceptable / 9 = optimum).

63

† Directional line indicates influence of component on either transmission or reflection analysis. PC = principal component. PC1 = Influence of chlorophyll total, chlorophyll a and b, hue, chroma l* a* b* on transmission color analysis. PC 2 = Influence of chlorophyll total, chlorophyll a and b, hue, chroma l* a* b* on reflection color analysis. Figure 2.1: Principal component analysis for turfgrass samples analyzed for color using reflectance and transmission measurements.

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REFERENCE Ahmed, J., U.S. Shivhare, and G.D. Singh. 2000. Chlorophyll and color of green chilli

puree as affected by mesh size and temperature. Int. J. Food Propog. 3:305-315. American Society for Testing and Materials. 1989. Standard test method for specifying

color by the munsell system. ASTM standard D1535-89. ASTM, Philadelphia, PA.

Bausch, W.C., and H.R. Duke. 1996. Remote sensing of plant nitrogen status in corn.

Trans. ASAE. 39:1869-1875. Bornmann, J.F., and T.C. Vogelmann. 1991. Effect of UV-B radiation on leaf optical

properties measured with fiber optics. J. Exp. Bot. 42:547-554. Brown, P.K., and G. Wald. 1964. Visual pigments in single rods and cones of the human

retina. Science 1964. 144:45-52. Byrne, M.C., C.J. Nelson, and D.D. Randall. 1981. Ploidy effects on anatomy and gas

exchange of tall fescue leaves. Plant Physiol. 68:891-893. Cameron, R.J. 1970. Light intensity and the growth of Eucalyptus seedlings: II. The

effect of cuticular waxes on light absorption in leaves of Eucalyptus species. Aus. J. Bot. 18:275-284.

Chen, C. 2002. Robust regression and outlier detection with the ROBUSTREG

procedure. Paper 265–27. SAS Institute Inc., Cary, NC, Proceedings SUGI 27, Orlando FL Apr 14–17. Available at http://www2.sas.com/proceedings/sugi27/p265-27.pdf (verified, 28 June, 2012).

65

Chien, J.C., and I.M. Sussex. 1996. Differential regulation of trichome formation on the adaxial and abaxial leaf surfaces by gibberellins and photoperiod in Arabidopsis thaliana (L.) Heynh. Plant Physiol. 111:1321-1328.

Clark, J.B, and G.R. Lister. 1975. Photosynthetic action spectra of trees: II. The

relationship of cuticle structure to the visible and ultraviolet spectral properties of needles from four coniferous species. Plant Physiol. 55:407-413.

Escadafal, R., M-C. Girard, and D. Courault. 1989. Munsell soil color and soil

reflectance in the visible spectral bands of landsat MSS and TM data. Remote Sens. Env. 27:37-46.

Filella, I., and J. Penuelas. 1994. The red edge and shape as indicators of plant

chlorophyll, biomass and hydric status. Int. J. Remote Sensing. 15:1459-1470.

Frank, K.W., R.E. Gaussoin, T.P. Riordan, R.C. Shearman, J.D. Fry, E.D. Miltner, and

P.G. Johnson. 2004. Nitrogen rate and mowing height effects on turf-type buffalograss. Crop Sci. 44:1615-1621.

Fu, J., and B. Huang. 2004. Leaf characteristics associated with drought resistance in tall

fescue cultivars. Acta Hortic. 661:233-239. Gausman, H.W., and R. Cardenas. 1969. Effect of leaf pubescence of Gynura aurantiaca

on light reflectance. Bot. Gaz. 130:158-162. Giusti, M.M., and R.E. Wrolstad. 2005. Characterization and measurement of

anthocyanins by UV-visible spectroscopy. p. 19–32. Unit F1.2. In Wrolstad R.E. (ed.) Handbook of food analytical chemistry—pigments, colorants, flavors, texture, and bioactive food components. John Wiley & Sons Inc., New York.

Gooding, M.J., and L.J. Gamble. 1990. Color evaluation of Poa pratensis cultivars. J.

Sports Turf Res. Inst. 66:134-140. Gould, K.S. 2004. Nature's swiss army knife: The diverse protective roles of

anthocyanins in leaves. J. Biomed. Biotech. 5:314-320.

66

Guertal, E.A., and D.L. Evans. 2006. Nitrogen rate and mowing height effects on TifEagle bermudagrass establishment. Crop Sci. 46:1772-1778.

Jaisingh, J., E.W.K. See-To, and K.Y. Tamin. 2006. The impact of open source software

on the strategic choices of firms developing proprietary software. J. Manage. Info. Sys. 25:241-275.

Joliot, P., and A. Joliot. 1984. Electron transfer between the two photosystems. I. flash excitation under oxidizing conditions. Biochim. Biophys. Acta. 765:210-218.

Karcher, D.E., and M.D. Richardson. 2003. Quantifying turfgrass color using digital

image analysis. Crop Sci. 43:943-951.

Kavanagh, T., D. O’Beirne, and T.P. Cormican. 1985. Use of the hunter colour difference

meter for colour evaluation in turfgrass experiments. p. 255-261. In: F. Lemaire (ed.) Proc. 5th Intl. Turfgrass Res. Conf., Avignon, France. 1-5 Jul.

Landschoot, P.J., and C.F. Mancino. 1997. Assessment of the minolta CR-310 chroma

meter for predicting nitrogen status of Agrostis stolonifera L. Intl. Turfgrass Soc. Res. J. 8:711-718.

Landschoot, P.J., and C.F. Mancino. 2000. A comparison of visual vs. instrumental

measurement of color differences in bentgrass turf. HortScience 35:914-916. Lemaire, G., G. Escadeillas, and E. Ringot. 2005. Evaluating concrete surfaces using an

image analysis process. Cons. Build. Mat. 19:604-611. Mangiafico, S.S., and K. Guillard. 2005. Turfgrass reflectance measurements,

chlorophyll, and soil nitrate desorbed from anion exchange membranes. Crop Sci. 45:259-265.

McElroy, J.S., D.A. Kopsell, J.C. Sorochan, and C.E. Sams. 2006. Response of creeping

bentgrass carotenoid composition to high and low irradiance. Crop Sci. 46:2606-2612.

67

Morales, L.O., R. Tegelberg, M. Brosché, A. Lindfors, S. Siipola, P.J. Aphalo. 2011. Temporal variation in epidermal flavonoids due to altered solar UV radiation is moderated by the leaf position in Betula pendula. Physiol. Planta. 143:1399-3054.

Moran, R. 1982. Formulae for determination of chlorophyllous pigments extracted with

N,N- dimethylformamide. Plant Physiol. 69:1376-1381. Moran, R., and D. Porath. 1980. Chlorophyll determination in intact tissues using N,N –

dimethylformamide. Plant Physiol. 65:478-479. Munsell, A.H. 1915. Atlas of the munsell color system. Wadsworth-Howland and

Company, Malden, MA. Nickerson, D. 1940. History of the munsell color system and its scientific application. J.

Optic. Soc. Am. 30:575-586. Nutter, F.W., Jr., M.L. Gleason, J.H. Jenco, and N.C. Christians. 1993. Assessing the

accuracy, intra-rater repeatability and inter-rate repeatability of disease assessment systems. Phytopathology 83:806-812.

Pattee, H.E., F.G. Giesbrecht, and C.T. Young. 1991. Comparison of peanut butter

color determination by CIELAB L*,a*,b* and Hunter color-difference methods and the relationship of roasted peanut color to roasted peanut flavor response. J. Agric. Food Chem. 39:519-523.

R Development Core Team. (2011). R: A language and environment for statistical

computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0. Available at http://www.R-project.org/ (Verified 29 Jun. 2012).

Rademacher, I.F., and C.J. Nelson. 2001. Nitrogen effects on leaf anatomy within

intercalary meristems of tall fescue leaf blades. Ann. Bot. 88:893-903. Reinhard, E., M. Adhikkmin, B. Gooch, and P. Shirley. 2001. Color transfer between

images. Comp. Graphic. App. 21:34-41.

68

Richardson, M.D., D.E. Karcher, and L.C. Purcell. 2001. Quantifying turfgrass cover using digital image analysis. Crop Sci. 41:1884–1888.

Roorda, A., and D.R. Williams. 1999. The arrangement of the three cone classes in the

living human eye. Nature 397:520-522. SAS Institute. 2011. Release 9.2 SAS Inst. Cary, NC. Saxby, M.J. 1964. Characterization of flavonoids and coumarins by their absorption

spectra on paper chromatograms. Anal. Chem. 36:1145-1146. Sharma, G., and H.J. Trussell. 1997. Digital color imaging. Trans. Image Proc. 6:901-

932. Sheehy, J.E. 1975. Some optical properties of leaves of eight temperate forage grasses.

Ann. Bot. 39:377-386. Shimada. K., Y. Kakehashi, H. Matsumura and N. Tanoue. 2004. In vivo quantitative

evaluation of tooth color with hand-held colorimeter and custom template. J. Prosth. Dent. 91:389-391.

Stiegler, J.C., G. Bell and N. Maness. 2004. Comparison of acetone and N,N-

dimethylformamide for pigment extraction in turfgrass. Comm. Soil Sci. Plant Anal. 35:1801-1813.

Stiegler, J.C., G.E. Bell, N.O. Maness, and M.W. Smith. 2005. Spectral detection of

pigment concentrations in creeping bentgrass golf greens. Int. Turf. Res. J. 10:818-825.

Stiff, M.L., and J.B. Powell. 1974. Stem anatomy of turfgrass. Crop Sci. 14:181-186. Thomas, D.A., and H.N. Barber. 1974. Studies on leaf characteristics of a cline of

Eucalyptus urnigera from Mount Wellington, Tasmania. II. Reflection, transmission and absorption of radiation. Aust. J. Bot. 22:701-707.

69

Wang, Y., M. Zhang, X.Y. Zhang, and H.D. Dong. 2002. Comparative studies on elasticity and scrape tolerance characters of Zoysia japonica and Poa pratensis. Prataculture Sci. 19:56-59.

Yudin, L.S., C.W. Mitchell, and J.J Cho. 1987. Color preference of thrips (Thysanoptera: Thripidae) with reference to aphids (Homoptera: Aphididae) and leafminers in Hawaiian lettuce farms. J. Econo. Entomol. 80:51-55.

Zscheile, F.P., J.W. White, Jr., B.W. Beadle, and J.R. Roach. 1942. The preparation and

absorption spectra of five pure carotenoid pigments. Plant Physiol. 17:331-346.

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CHAPTER 3

INFLUENCE OF ULTRAVIOLET LIGHT ON GERMINATION RATE AND SPEED OF KENTUCKY BLUEGRASS (Poa pratensis L.)

ABSTRACT

Kentucky bluegrass (Poa pratensis L.) is a commonly used turfgrass in the

temperate climates of the United States. The grass is predominantly established from sod,

because of its slow time to germinate (21 d). Ultraviolet light has been noted to enhance

germination speed of other crops and it may similarly enhance Kentucky Bluegrass

germination. Turfgrass seeds from two seed lots were placed in two separate germination

chambers and set on an 8hr / day 16hr / night schedule. Supplemental ultraviolet light

(11.2 kJ / m-2 / d-1) was added on a treatment of two 1 hr time periods split over a 3 hour

period versus no UV light. The treatments were applied for 21 days. Seed germination

was counted at 7, 14 and 21 days. Ultraviolet light increased the germination

completeness and speed of germination in the newer but not older seed lot. Seed

germination was greater (P=0.05) at day 7 and after 21 days there was a greater

germination percentage or completeness in ultraviolet light conditions than in control in

seed lot one but not lot two. These results suggest that treatment of bluegrass seed with a

UV light may enhance germination; however seed age may override the effect.

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INTRODUCTION

Turfgrass seed germination is dependent on a number of environmental factors.

Manipulating the light environment may be a method to enhance germination speed and

completeness. Kentucky bluegrass (Poa pratensis L.) may reach only 60% germination

after 28 days and 90% germination after 4 months (Maguire and Steen, 1971). This is a

low rate of germination and leads to difficulties in establishment of the turfgrass.

Treatments to enhance this have included the use of hydrogen peroxide to increase fresh

weight of seed and germination by 20% and 30% respectively (Takabe, 2004). There are

limited methods to improve its germination completeness and hence the establishment of

Kentucky bluegrass lawns is limited by speed of seed germination. Kentucky bluegrass is

considered a favorable turfgrass in the northern United States variety due to its spreading

growth habit and desirable color especially when nitrogen rates are increased (Skogley

and Ledeboer, 1968).

Methods to enhance germination speed and vigor of grass seedlings include seed

placement in favorable moisture conditions prior to seeding. This resulted in a 40hr

decrease in germination time of crested wheatgrass (Agropyron cristatum L.) (Keller and

Bleak, 1968). The aim of this method is to have the seed active prior to seeding and thus

naturally increase the germination rate. Priming seed with initial hydration followed by

air drying has proven to increase germination speed and seedling fresh mass (Pill et al.,

1997) in Kentucky bluegrass. Osmoconditioning or pre-treating with water, of perennial

ryegrass (Lolium perenne L.) seeds increased germination rate and root growth even

under suboptimum temperatures (Danneberger et al., 1992). Sonication for 4 minutes

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significantly increased germination in orchids (Calanthe discolor L.) by 50% (Miyoshi

and Mii, 1988). This was due to a perceived requirement for scarification of a thicker

seed coat. Different energy treatments using microwaves removes fungal propagules thus

reducing the need for fungicide applications and this had no effect on germination rates

(Cavalcante and Muchovej, 1993). Gamma radiation of tall fescue (Schedenorus phoenix

(Scop.) Holub) reduces endophyte levels by ~90% while not affecting germination

(Bagegni et al., 1990).

The potential for the use of ultraviolet-B (UV-B) light in promoting germination

has received no attention in turfgrass seedling studies. Light is crucial in controlling plant

germination in Kentucky bluegrass (Toole and Borthwick, 1971), as well as plant growth

and development (Frankhauser and Chory, 1997). Ultraviolet light, (200-400nm) has

higher energy compared to photosynthetically active radiation (400-700nm), (PAR). The

impact of ultraviolet-C (UV-C) has been found to be inhibitory in sunflower (Helianthus

annuus L.). This effect, however, can be reversed during dark storage of seeds after the

UV-C treatment (Torres et al., 1991). Kale, cabbage, (Brassica oleracea), radish

(Raphanus sativus L.) and agave (Agave L) treated with UV-B light germinated more

rapidly but subsequent growth was markedly inhibited (Noble, 2002). The number of

seeds and dry weight of seeds in Bromus catharticus receiving 90% UV-B almost

doubled compared to the control (Deckmyn and Impens, 1998). There was, however, not

an effect on germination. The objective of this study was to investigate if ultraviolet light

could be used as a treatment to speed germination of Kentucky bluegrass and to increase

average germination completeness.

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MATERIALS AND METHODS

Plant material

Kentucky bluegrass seeds from two different seed lots based on age of the same

cultivar ‘Bariris’ were used in this trial (Barenbrug USA, Tangent, OR). Fifty turfgrass

seeds were placed in each of 9 petri-dishes (9 cm diam.) for a total 450 seeds per light

treatment. Two repeated studies for each lot were carried out in each of two growth

chambers with a day / night regime of 8 / 16 hours and temperatures of 25 / 15° C. The

visible light (400 nm – 700 nm) intensity was measured using cosine corrected light

meters (Spectrum Technologies, Plainfield, IL). Ultraviolet light was provided by four

Reptisun 10.0 UV overhead fluorescent tubes (Zoo Med Laboratories, San Luis Obispo,

CA) in one chamber. The ultraviolet light was measured using a portable IL1350

radiometer with UV-B sensor (International Light, Newburyport, MA). The UV light was

then reported as kJ / m-2 / d-1. Each study was conducted for twenty-one days. The lights

were switched between chambers for each experiment. The petri dishes were covered in

both chambers for 21 hours per day for moisture retention. Seeds were placed on standard

blue blotters (Anchor Paper Co., Plymouth, MN), to ensure seed stability and consistent

moisture levels. Prior to daily UV treatment initiation, covers were removed to prevent

any reflection of UV rays from seed surfaces.

Data collection and analysis

Initial treatment of seed with distilled H2O at 5 mL per day occurred and this then

decreased to 3 mL per day after 14 d as seed volume and water requirements decreased.

74

Radicle emergence was used to determine germination and seed count data were

collected at 7, 14 and 21 days. Germination rate based on radicle emergence against total

seeds used and germination vigor based on seed counts were analyzed. The petri dishes

were set up in a randomized complete block design to reduce the impact of lighting

variability. Data were analyzed using the general linear models (GLM) procedure of the

SAS program (SAS Institute, 2008). The data from both seed lots were pooled from each

repetition. Fisher’s protected least significant difference testing (LSD) was used to test

for differences in the average germination values and total germination percentages. Data

were combined to check for differences between seed lots.

RESULTS AND DISCUSSION

Treatments of UV totaled 11.2 kJ m-2 d-1 in the UV chamber, and daily PAR was

4.3 mol m-2 d-1. The control chamber had a total PAR of 4.2 mol m-2 d-1 but no detectable

level of UV light. There was no supply of UV light in the UV-A region (320 – 400 nm) in

either chamber. Due to significant differences (P=0.05) in overall percentage germination

between the two seed lots data were analyzed separately. The newer seed lot which was

one year old (Lot 1), had significantly higher (P=0.05) germination rates compared to the

older seed lot (>2 years old) in UV (Table 3.1). There was lower germination rates in

control light environment in Lot 1 compared to Lot 2 (Table 3.1). The age of the seed

may play a role in the germination percentage change in the UV chamber as has been

noted previously where older seeds had reduced germination percentages (Shaidaee et al.,

1969).

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Overall total germination percentage was significantly higher (Figure 3.1) in lot 1

for UV treated seeds compared to control seeds. In tests on lot two UV germination

decreased from lot one. The control total germination percentage rates in lot two

increased from tests on lot one. This meant there was no overall separation between the

germination rates due to the light treatments (Figure 3.2).

Days to germination of lot one was affected by light treatments. Applications of

ultraviolet light significantly (P=0.05) enhanced seed germination on day 7 compared to

the control treatment. The cumulative germination percentage was still higher at day 14

for the UV chamber in lot one but not lot two (Figure 3.1 and 3.2). On day 21 both sets of

light treatments were significantly different in lot one. The observation of higher

germination on day 7 resulted in significantly faster germination times in the UV treated

grass chambers (Figure 3.1). The pattern of greater seed germination (P=0.05) on day 7

in the first experiment was also repeated in the second experiment for seed lot one

(Figure 1). Seed lot one almost reached germination completeness after 21 days in UV,

however data was not collected past this date.

The same results were not observed with the 2nd seed lot. UV treatment did not

result in increased germination by day 7 in seed lot 2 (Figure 3.2). The initial difference

at day 7 from seed lot one was not noticed in the second seed lot. The second seed lot

demonstrated the greatest percentage of germination on day 21 in both experiments and

there was no significant difference between light treatments (Figure 3.2). Overall

germination decreased in UV conditions in lot 2 compared to lot 1 while there was an

increase in germination from lot one to seed lot two in control conditions (Table 3.1).

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CONCLUSIONS

The variation between the seed lots means that further work is required in order to

conclusively determine if a clear benefit due to UV irradiance exists. The initial results

were very promising and agree with data produced from lettuce and wheat seed (Noble,

2002, Wagné, 1966). The amount of published work done with ultraviolet light on

turfgrass germination is limited and variability in seed lots due to age and weight has

been noted previously regardless of light treatment (Larsen and Andreasen, 2004,

Shaidaee et al., 1969). This may be a large part of the reason for the difference in results

and a specific cultivar which has more readily available seed lots of similar age would be

desirable.

The use of specific wavelengths also requires investigation. The experimental

system provided elevated UV-B levels but it also had UV-A wavelengths. This was due

to a size restriction with UV-B specific lights. If the study focused solely around the main

UV-B wavelengths it may enhance the biologically activity spectrum (Caldwell, 1971). It

has been noted in that UV-C (100 nm - 290 nm) does not provide potential for

germination enhancement, with inhibition of sunflower germination (Helianthus annus

L.), (Torres et al., 1991). The wavelengths and energy associated with ultraviolet light

may have triggered response from the light receptor phytochrome – A, especially at low

fluence rates which irreversibly trigger germination (Shinomura et al., 1996).

Field testing is also a step that has to be considered as well as combing hydration

and temperature cycles with the UV irradiation in future studies. It does offer a potential

option however to produce enhanced Kentucky bluegrass germination if consistency can

77

be found with the seed lots. This would allow for greater seasonal use of the grass in

home lawn establishment.

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Light Ultraviolet Control

Lot Df Germination # --------------------------- % --------------------------- 1 1 81.6 a† 60.3 b 2 1 71.6 b 70.7 a

LSD (0.05) 5.8 8.9 † Means followed by different letter were significantly different (P=0.05)

Table 3.1: Germination percentages of Kentucky bluegrass (Poa pratensis L.) seed lots treated with PAR (400 – 700 nm) and ultraviolet light (300 – 400 nm).

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Figure 3.1: Percentage germination of Kentucky bluegrass (Poa pratensis L.) seed treated with photosynthetically active radiation (400 – 700 nm) and ultraviolet light (280 – 480 nm) in newer seed lot one.

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Figure 3.2: Percentage germination of Kentucky bluegrass (Poa pratensis L.) seed treated with photosynthetically active radiation (400 – 700 nm) and ultraviolet light (280 – 480 nm) in older seed lot two.

81

REFERENCE Bagegni, A.M., D.A. Sleper, H.D. Kerr, and J.S. Morris. 1990. Viability of Acremonium

coenophialum in tall fescue seed after ionizing radiation treatments. Crop Sci. 30:1272-1275.

Caldwell, M.M. 1971. Solar ultraviolet radiation and the growth and development of

higher plants. p. 131-174. In: A.C. Giese (ed.) Photophysiology. Academic Press, New York.

Cavalcante, M.J.B., and J.J. Muchovej. 1993. Microwave irradiation of seeds and

selected fungal spores. Seed Sci. Technol. 21:247-253. Danneberger, T.K., M.B. McDonald Jr., C.A. Geron, and P. Kumari. 1992. Rate of

germination and seedling growth of perennial ryegrass seed following osmoconditioning. HortScicience 27:28-30.

Deckmyn, G., and I. Impens. 1998. Effects of solar UV-B irradiation on vegetative and

generative growth of Bromus catharticus. Environ. Exp. Bot. 40:179-185. Frankhauser, S., and J. Chory. 1997. Light control of plant development. Ann. Rev. Cell

Dev. Biol. 13:203-29. Keller, W., and A.T. Bleak. 1968. Preplanting treatment to hasten germination and

emergence of grass seed. J. Range Manage. 21:213-216. Larsen, S.U., and C. Andreasen. 2004. Light and heavy turfgrass seeds differ in

germination percentage and mean germination thermal time. Crop Sci. 44:1710-1720.

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Larsen, S.U., and B.M. Bibby. 2004. Use of germination curves to describe variation in germination characteristics in three turfgrass species. Crop Sci. 44:891-899.

Maguire, J.D., and K.M. Steen. 1971. Effects of potassium nitrate on germination and

respiration of dormant and non dormant Kentucky bluegrass (Poa pratensis L.) seed. Crop Sci. 11:48-50.

Miyoshi, K., and M. Mii. 1985. Ultrasonic treatment for enhancing seed germination of

terrestrial orchid Calanthe discolor in a symbiotic culture. Sci. Hortic. 35:127-130.

Noble, R.E. 2002. Effects of UV-irradiation on seed germination. Sci. Total Environ.

299:173-176. Pill, W.G., J.J. Frett, and I.H. Williams. 1997. Matric priming of Kentucky bluegrass and

tall fescue seeds benefits seedling emergence. HortScience 32:1061-1063. Shaidaee, G, B.E. Dahl, and R.M. Hansen. 1969. Germination and emergence of different

age seeds of six grasses. J. of Range Manage. 22:240-243. Shinomura, T., A. Nagatani, H. Hanzawa, M. Kubota, M. Watanabe, and M. Furuya,

1996. Action spectra for phytochrome A and B-specific photoinduction of seed germination in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 15:8129-8133.

Skogley, C.R., and F.B. Ledeboer. 1968. Evaluation of several Kentucky bluegrass and

red fescue strains maintained as lawn turf under three levels of fertility. Agron. J. 60:47-49.

Takabe, T., A. Uchida, and T. Tetsuko. 2004. Effects of hydrogen peroxide on germination of Kentucky bluegrass. In Abstracts, Abs # 709. Plant Biology 2004: July 24 – July 28, 2004. Lake Buena Vista, FL.

Toole, V.K., and H.A. Borthwick. 1971. Effect of light, temperature and their interactions

on seeds of Kentucky bluegrass (Poa pratensis L.). J Am. Soc. Hort. Sci. 96:301-304.

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Torres, M., G. Frutos, and J.M. Duran. 1991.Sunflower seed deterioration from exposure to UV-C radiation. Environ. Exp. Bot. 31:201-207.

Wagné, C. 1966. Effect of UV light on lettuce seed germination and on the unfolding of

grass leaves. Physiol. Planta. 19:128-133.

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CHAPTER 4

ULTRAVIOLET-B LIGHT INFLUENCE ON COOL SEASON TURFGRASS GROWTH AND MORPHOLOGY

ABSTRACT

Ultraviolet light is a potential issue for plant growth and development due to its

effect on photosynthesis and plant productivity. Turfgrass plants may be affected by

ultraviolet light causing loss of quality and ability to recover from injury. The objective

of this study was to investigate the effects of ultraviolet light on turfgrass productivity

and morphology in three cool season grasses. Tall fescue, (Schedonorus phoenix (Scop.)

Holub.), creeping bentgrass (Agrostis stolonifera L.), cultivars ‘Penncross’, ‘L-93’ and

perennial ryegrass (Lolium perenne L.), were selected due to limited or no information on

their growth and development in elevated ultraviolet light conditions at lawn height of

10-12 cm. The impact of ultraviolet light treatment on color, root and shoot biomass and

tillering was measured over a four week period of exposure in repeated experiments.

Ultraviolet-B light was tested in particular and light levels were measured at 16 kJ m-2 d-1

biologically effective light in growth chambers programmed for a day/night regime of

14/10 hours. Ultraviolet-B light significantly (P=0.05) reduced shoot and root production

in all grasses and inhibited tiller production (P=0.05). Turfgrasses exposed to this level of

ultraviolet-B light at lawn height of cut showed visual damage and significantly lower

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color rankings after 2 weeks. The experiment demonstrated that after extended exposure

to ultraviolet-B light there is a loss of productivity and color in cool season turfgrasses.

INTRODUCTION

Light plays a crucial role in turfgrass growth and development. It is normally

considered most important in the wavelengths of 400-700 nm; photosynthetically active

radiation (PAR), (Pons et al., 1993). Light wavelengths can often vary and spectral

distribution can fluctuate with environmental conditions (Bell et al., 2000). There is

interest in how the degradation of the ozone layer will influence spectral distribution on

the earth’s surface (Manney et al., 2011). The decrease in ozone is predicted to lead to a

significant increase in exposure to wavelengths in the ultraviolet (UV) range (Kerr and

McElroy, 1993). Ultraviolet light is found in the range of 10 – 400 nm and the energy

associated with these wavelengths is considered damaging when compared to PAR

(Fiscus and Booker, 1995). The UV-B wavelengths which hit the surface of the earth are

predicted to increase in springtime radiation by 50-60% from 2010-2020 (Taalas et al.,

2000) in the northern hemisphere. The reduction in ozone and a resulting increase in UV

irradiance has been linked to chlorofluorocarbons in the atmosphere (Rowland, 1990).

It has been shown that leaf area indices of some Poaceae species can change in

response to different levels of biologically effective UV-B irradiance (Caldwell, 1971;

Deckmyn and Impens, 1999). Increases in leaf and axillary shoot production in response

to UV-B exposure are noted in both monocot crops such as wheat (Triticum aestivum L.),

and dicots such as beans (Phaseolus vulgaris L.), (Barnes et al., 1990). Red fescue

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(Festuca rubra L.) growth was positively affected by UV-B radiation in all seasons

(Deckmyn and Impens, 1999), showing potential for differences in response to exposure.

Epidermal thickness alters leaf absorbance properties so much that it is considered an

excellent predictor of the effectiveness of a plant’s ability to screen UV-B light (Day,

1993). Root length was inhibited at UV-B irradiances of 2.5 – 5 kJ m-2 d-1 in Antarctic

hairgrass (Deschampsia antartica L.), while there was an increase in tillering but no

overall impact in the relative growth rate due to the compensation in tiller production

compared to root loss (Rozema et al., 2001). Reductions in stem elongation and leaf

expansion in young plants of long spined thorn apple (Datura ferox L.) occur in UV-B

light (Ballare et al., 1996). Smaller leaf sizes in white clover (Trifolium repens L.) also

have been influential in inferring tolerance to UV-B exposure (Hoffman, 2000). In bean

plants, low levels of PAR, combined with UV-B treatments, led to a decrease in leaf area

of 47% and a decrease in leaf dry weight of ~25% (Cen and Bornman, 1990). Ultraviolet

light exposure is linked to reduced photosynthesis due to direct damage to photosystem II

(Brandle et al., 1977), which results in decreased leaf blade and internode lengths and

plant productivity.

Varietal differences in rice (Oryza sativa L.) following exposure to UV have been

observed. Some cultivars had smaller leaf areas, stunted plant height and a reduction in

relative growth rate whereas others showed little response. Plants with rapid growth rates

such as squash (Cucurbita pepo L.) tend to be more sensitive to damage while slower

growing plants such as peppers (Capsicum frutescens L.) are tolerant of the UV-B

irradiance (Smith et al., 2000). These effects were not noted until after 2 weeks of UV-B

87

treatment (Dai et al., 1992). Perennial ryegrass and orchardgrass (Dactylis glomerata L.)

biomass production was reduced in response to comparable spring and summer

treatments of elevated UV levels (Deckmyn and Impens, 1999). In poplars (Populus

cathayana Rehd.), however, there was no effect on biomass following exposure to

different levels of UV-B (Zhang et al., 2011). Variability among plant species in response

to UV-B has been noted in review papers and could be related to other issues such as

temperature, or concentrations of CO2 (Krupa and Nickert, 1989). Limited work has been

conducted to determine the impact of UV-B on vegetative productivity of turfgrasses that

would be used in homelawn situations covering potentially 163,800 km-2 ± 35,850 km-2

in the United States (Milesi et al., 2005).

Ultraviolet light has varying effects on quality ratings among the turfgrasses.

Chewings fescue (Festuca rubra L. ssp. commutata Gaud.), is considered the most UV-B

tolerant turfgrass with Kentucky bluegrass being considered the least tolerant (Zhang and

Ervin, 2009). Quality ratings were reduced by as much as 74% for Kentucky bluegrass

after 10 days of exposure. There was also cultivar variation in the tolerance of UV-B in

this trial. Darker green Kentucky bluegrass cultivars are thought to be also more tolerant

of UV-B conditions (Ervin et al., 2004c). In other plants such as apples (Malus

domestica) ‘Royal Gala’, increasing the red color post-harvest with the use of UV-B

treatments has been seen as a benefit to production (Dong et al., 1995). This response was

due to increased production of flavonoid-type pigments. Combined treatments of UV-B

and visible light were most effective in enhancing the color in other apple cultivars such

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as (Malus domestica Borkh.), cv. ‘Aroma’, (Hagen et al., 2007). The resulting increase in

red color on the skin in both cases added value to the apple crop.

Limited research has been conducted to test the effect of UV-B exposure on

turfgrasses at higher heights of cut such as home lawns and the estimated area covered by

irrigated turfgrass crop covers potentially 163,800 km-2 (Milesi et al., 2005). The

objective of this study was to test four cool season grasses for their growth rate, tillering

and color responses to elevated UV-B conditions predicted to occur in this decade

(Taalas et al., 2000). It was hypothesized that thicker-leaved species, such as perennial

ryegrass and tall fescue would have greater tolerance to UV-B than thin leaved species

like Kentucky bluegrass and creeping bentgrass.

MATERIALS AND METHODS

Sample preparation

Experiments were initiated in March, 2011 and May, 2011 with no noticeable

differences in turfgrasses following establishment in Columbus, Ohio. Turfgrasses were

initially seeded into a mason sand rootzone containing >97% sand (Hummel & Co,

Trumansburg, NY) as a single seed with structured timing to match germination speeds.

The rootzone met United States Golf Association (USGA) classification and had a pH of

7.4 with 2.7% clay silt fraction. Creeping bentgrasses were seeded first into 3.8 cm

diameter ‘conetainers’ (Steuwe & Sons, Tangent, OR) and 4 days later perennial ryegrass

and tall fescue were seeded. Each conetainer contained a single plant. The plants were

placed in a mist house in a ten minute misting cycle of 15 seconds per water application

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to maintain moisture. Turfgrasses were grown for 6 weeks and initially treated with

Peters 20-20-20 (Scotts-Miracle Gro, Marysville, OH) at a rate of 11.8 kg ha-1 every two

weeks. Six weeks after germination all plants were removed and placed in a greenhouse

maintained at ~20°C for four more weeks to allow for maturation. Imidacloprid N-[1-[(6-

Chloro-3-pyridyl) methyl]-4,5-dihydroimidazol-2-yl]nitramide (Bayer Environmental

Science, Research Triangle PK, NC) was applied to prevent insect damage. Mefenoxam

(R,S)-2-[(2,6-dimethylphenyl)-methoxyacetylamino]-propionic acid methyl ester

(Syngenta AG, Greensboro, NC) and thiophanate-methyl (dimethyl 4,4’-o-

phenylenebis[3-thioallophanate]) (Cleary Chemicals, Dayton, NJ) were applied for

preventative disease control. Water was applied for a 5 minute period daily to prevent

wilt, with automated heads using an ESP modular irrigation system (Rain Bird, Azusa,

CA). Ten weeks after turfgrass emergence, plants were placed in Conviron E15 growth

chambers (Controlled Environment Ltd., Winnipeg, Canada). Plants were left in complete

darkness at 20 °C for 24 hours.

Ultraviolet-B treatment was implemented using specific UV-B lamps that have

peak light emission at 313 nm, QUV UV-B 313 nm, (Q-Lab Corp., Westlake, OH). The

control treatment consisted of only light from fluorescent lamps. The UV-B lamps did not

produce any extra visible radiation. Treatment exposure was calculated based on the

predicted time of exposure and measured lamp irradiance. Radiation from ultraviolet

sources were measured using both UV-B and UV-A sensors attached to an IL1350

photometer (International Light, Newburyport, MA). The radiation is reported in kJ m-2

d-1. Photosynthetic active radiation was measured every 15 minutes for the period of the

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study using a cosine corrected light sensor (Spectrum Technologies, Plainfield, Il) and

reported as mol m-2 d-1. The chambers were programmed for a day/night length of 14/10

hours. Photosynthetic active radiation (PAR) in the range of 400 – 700 nm was provided

in all chambers using 85 watt s-1 high output F12T12-CW.HO fluorescent tubes (General

Electric, Fairfield, CT). Water was applied twice daily to prevent wilting. Temperature in

each chamber was set to 20°C during light exposure and 17 °C in darkness. Fertility

treatments continued for the duration of each study. Turfgrass plants were placed 0.4 m

below the lighting in an even spacing.

Data collection and analysis

The experiment was designed as a randomized complete block with weeks being

randomized in each block. Under each light regime, there were three blocks with four

replications per block of each grass for a total of 12 samples of each grass per

measurement. Data were analyzed using the linear model (GLM) method in SAS Institute

9.2 (2008). Means separation was carried out using Fishers protected least significant

difference test (P=0.05) and data were pooled. At the end of each week samples were

harvested and separated into roots and shoots, then dried at 70 °C for two days. The

shoot: root ratio was then calculated based on dry weight (Gwynn-Jones and Johanson,

1996). Tissue was dried in a VWR 1350F (Univar USA, Redmond, WA) oven in 8.9 x

16.5 cm coin envelopes (Officemax, Naperville, IL). Relative growth rate was calculated

based on the formula of Gardner et al., (1985):

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RGR = Relative growth weight; lnW2 = natural log weight 2; lnW1 = natural log weight

1; T1 = Time 1; T2 = Time 2.

RGR = (lnW2 – lnW1) / (T1 – T2)

Tillering was measured by counting daughter plants extending from the crown of

each seeded conetainer. Tillers were counted when samples were harvested for each week

of treatment at weeks 1, 2, 3 and 4. Tillers were defined as daughter plants arising

directly from the crown individual plants. Color rankings were carried out on a 1-9

ranking scale with 1 = dead/brown 9 = dark green 6 = acceptable. The color data were

collected prior to trial initiation and weekly during the trial under the same light

conditions.

RESULTS

Turfgrasses in the UV-B treated environment received a total of 24 kJ m-2 d-1.

Once this was adjusted for UV-B biologically effective radiation the total effective UV-B

light seen by turfgrasses (Caldwell, 1971) was decreased to 16 kJ m-2 d-1 (Table 4.1). The

daily photosynthetic integral totaled 18.5 mol m-2 d-1 (Table 4.1).

After 3 weeks, growth of all grasses in ultraviolet light was significantly inhibited

(P=0.05) compared to grasses grown in the control light environments. There was,

however, a decrease in growth rate in the control chambers after four weeks and so no

significant differences were observed. The growth rate in UV conditions, however, had

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become negative as turfgrasses began to completely fail (Figure 4.1). There was

variability noted within the data, which occurred despite using twelve different samples

from each grass at each sampling date.

There was a significant decrease (P=0.05) in color quality ratings for all grasses

exposed to UV-B (Figure 4.2). There was a 22% decrease in the ‘L-93’ color and a 17%

decrease in ‘Penncross’ color. Tall fescue and perennial ryegrass had a loss of 18% and

15% in color quality respectively. After four weeks, both creeping bentgrass cultivars

were ranked below acceptable levels and visible damage was clearly seen with browning

and yellowing of the leaf surfaces (Figure 4.2), (Appendix B). Dark lesions similar to

burn spots were also noted on the UV-treated bentgrass plants in the UV-B treatment.

The loss of color was not observed until week two of the experiments and was consistent

in both trials (Appendix B). The color differences were consistent after week two until

the end of each experiment. There was some reddening of stems and tissue material but

this was not consistently observed.

Under UV-B conditions in experiment one there was a clear inhibition (P=0.05)

of plant tillering in three of the grasses (Figure 4.3). There was a 40% inhibition of

tillering in creeping bentgrass ‘L-93’ and 39% decrease in the creeping bentgrass

‘Penncross’ cultivar. Perennial ryegrass was not inhibited significantly in either

experiment. In experiment one, tall fescue tillering was significantly inhibited (P=0.05)

when it was treated with UV-B light. There was a 30% decrease of tall fescue tillering by

30% in experiment one but this did not occur in experiment two. In experiment two, there

was an overall decrease in tillering regardless of light environment. The bentgrasses

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exposed to UV-B had lower tiller numbers in experiment one but they were not

significantly different from the control treatment in experiment two (Figure 4.4). This

was possibly due to greater light intensities in the maturation of plants in experiment 2

which occurred two months later in the year. Perennial ryegrass tiller number was not

significantly different in response to light regime, between the light treatments. The root :

shoot ratio did not change significantly in response to light regime, but the data were

highly variable.

DISCUSSION

The decrease in growth rate in the bentgrasses after exposure to UV light has been

reported previously (Schmidt and Zhang, 2001). Reducing the efficiency of the

photosynthetic machinery due to photolytic damage would effectively reduce plant

productivity and explain previous findings. Biological variation and seasonal light

changes affect plant maturation and may have affected the turfgrass plants in experiment

two. This had an effect on the control plants in experiment two, thus reducing the

potential for separation of treatment effects. The potential exists for differences in the leaf

surface characteristics of each of the turfgrasses. In particular, differences in leaf

thickness are known to alter light absorption and reflection (Cameron, 1970; Clark and

Lister, 1975). Exposure to UV-B light can also lead to thickening of the leaf surfaces

(Bornman and Vogelmann, 1991), thus affecting plant productivity though not

necessarily in a beneficial manner. The difference in productivity between the grasses

was also of interest. The two thicker-leafed grasses - tall fescue and perennial ryegrass -

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tended to produce greater biomass levels than the thinner-leafed creeping bentgrass. The

issue of leaf thickness may be crucial in this situation as a plant tries to maintain normal

growth rates in high UV-B conditions.

The inhibition of tillering by UV-B could be considered problematic for turfgrass

wear-recovery from daily use as well as regular growth and development. This reduction

has been noted before in rice but there were also cultivar differences (Teramura at al.,

1991). In that study, 30% of the rice cultivars showed a significant sensitivity to UV-B

irradiance. This was also shown in other rice cultivars at ambient levels of UV-B of 8 kJ

m-2 d-1, where tillering was reduced occurred in cultivar ‘CL161’ but not in ‘Cocodrie’.

The results were not linked to hormonal or carbohydrate changes in the plants

(Mohammed et al., 2007). In Antarctic hairgrass (Deschampsia antarctica), tillering was

increased by UV-B; however, the levels of UV-B were not as high as in this study

(Rozema et al., 2001). The lack of separation in the root and shoot ratio was somewhat

surprising; however, the light levels may have suppressed the growth and allocation of

carbohydrate equally. This does not agree with previous results (Gwynn-Jones, 2001), but

the light levels were higher in this study and it was conducted over a shorter period of 35

days compared to 45 days.

Light intensity variation plays a role in the overall responses of plants to both UV-

B exposure and standard PAR irradiance. Its effect varies regarding the amount of

exposure that is received by plants. In lapland reedgrass, (Calamagrostis lapponica) and

(Calamgrostis pupurea), levels of UV-B simulating 15% loss of ozone reduced tillering.

In light levels simulating 25% ozone depletion, there was an increase in tillering (Gwynn-

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Jones and Johanson, 1996). The exposure value equated to 6.5 kJ m-2 d-1, which is lower

than turfgrasses were subjected to in this study. Visible light intensities in this study were

also lower than normal daily integrals. The effect of UV-B could have been increased due

to lower levels of light protecting pigments in the lower visible light conditions

(Teramura et al., 1980). In an attempt to gain a consistent turfgrass response, UV-B levels

in our study were elevated above normal and this allowed for a clearer insight into the

turfgrass response. Another goal of our approach was to subject turfgrasses to equivalents

of extended exposures to UV-B irradiation which could simulate a season-long exposure.

The visual evaluation of color and differences between ratings agrees with

previous studies (Millington and King, 2010). Turfgrasses may have suffered from

probable chlorophyll degradation and this has been connected to a decrease in

photochemical efficiency (Ervin et al., 2004c). The loss of color is a principal factor for

evaluating turfgrass quality and while the thicker-leaved plants had significant color loss,

they still maintained acceptable color levels compared to the two creeping bentgrass

cultivars. This measurement can be construed as subjective and future potential use of

objective color measurements could offer more accurate and consistent visual

representations of the changes. This may be an important factor to consider regarding

tolerance of cool season grasses to UV light stress. If the predicted changes in UV light

levels occur over time, further research into leaf cuticle thickness and changes in resins

and waxes in the cuticular layers will be required. The use of synthetic, reflective type

pigments as plant coatings may be an alternative to breeding for increased UV-B

tolerance in turfgrasses.

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Light Chamber UV-B (280-320nm) UV-A (320-400nm) PAR (400-700nm)

______________ kJ m-2 d-1 ______________ mol / m-2 / d-1 Control 0.0 0.1 18.6

UV- Chamber 24.0 0.4 18.4

Table 4.1: Lighting conditions in control and ultraviolet treated light environments for turfgrasses grown at The Ohio State University March – April 2011.

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† Significant difference between grasses grown in ultraviolet and control conditions on this date (P=0.05). Figure 4.1: Relative growth rate for four cool season grasses grown in control and enhanced ultraviolet-B light conditions in repeated experiments

98

† Significant difference between grasses grown in ultraviolet and control conditions on this date (P=0.05).

Figure 4.2: Color ratings for four cool season grasses grown in control and enhanced ultraviolet-B light conditions in repeated experiments. (1-9 scale, 1 = brown/dead, 9 = dark green optimal color, 6 = acceptable).

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† Bars signify least significant difference between treatments (P=0.05). ‡ Histograms with different letters for each grass signify differences at (P=0.05).

Figure 4.3: Number of tillers per plant for four cool season grasses grown in control and enhanced ultraviolet-B light conditions in experiment one.

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† Bars signify least significant difference between treatments (P=0.05). ‡ Histograms with different letters for each grass signify differences at (P=0.05).

Figure 4.4: Number of tillers per plant for four cool season grasses grown in control and enhanced ultraviolet-B light conditions in experiment two.

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REFERENCE

Ballare, C.L., A.L. Scopel, A.E. Stapleton, and M.J. Yanovsky. 1996. Solar ultraviolet-B radiation affects seedling emergence, DNA integrity, plant morphology, growth rate, and attractiveness to herbivore insects in Datura ferox. Plant Physiol. 112:161-170. Barnes, P.W., S.D. Flint, and M.M. Caldwell. 1990. Morphological responses of crop and weed species of different growth forms to ultraviolet-B radiation. Am. J. Bot. 77:1354-1360. Bell, G.E., T.K. Danneberger, and M.J. McMahon. 2000. Spectral irradiance available for turfgrass growth in sun and shade. Crop Sci. 40:189-195. Bornman, J.F., and T.C. Vogelmann. 1991. Effect of UV-B radiation on leaf optical properties measured with fiber optics. J. Exp. Bot. 42:547-554. Brandle, J.R, W.F. Campbell, W.B Sisson, and M.M. Caldwell. 1977. Net photosynthesis, electron transport capacity, and ultrastructure of Pisium sativum

L. exposed to ultraviolet-B radiation. Plant Physiol. 60:165-169. Caldwell, M.M. 1971. Solar ultraviolet radiation and the growth and development of higher plants. p. 131-177. In A.C. Giese (ed.) Photophysiology: Academic Press, NY. Cameron, R.J. 1970. Light intensity and the growth of Eucalyptus seedlings: II. The effect of cuticular waxes on light absorption in leaves of Eucalyptus species. Aus. J. Bot. 18:275-284.

102

Clark, J.B., and G.R. Lister. 1975. Photosynthetic action spectra of trees: II. The relationship of cuticle structure to the visible and ultraviolet spectral properties of needles from four coniferous species. Plant Physiol. 55:407-413. Cen, Y.-P., and J.F. Bornman. 1990. The response of bean plants to UV-B radiation

under different irradiances of background visible light. J. Exp. Bot.41:1489-1495. Dai, Q., V.P. Coronel, B.S. Vergara, P.W. Barnes, and A.T. Quintos. 1992. Ultraviolet-B radiation effects on growth and physiology of four rice cultivars. Crop Sci. 32:1269-1274. Day, T.A. 1993. Relating UV-B radiation screening effectiveness of foliage to absorbing- compound concentration and anatomical characteristics in a diverse group of plants. Oecologia 95:542-550. Deckmyn, G., and I. Impens. 1999. Seasonal responses of six Poaceae to differential levels of solar UV-B radiation. Environ. Exp. Bot. 41:177-184. Dong, Y. hu, D. Mitra, A. Kootstra, C. Lister, and J. Lancaster. 1995. Postharvest stimulation of skin color in royal gala apple. J. Am. Soc. Hortic. Sci. 120:95-100. Ervin, E.H., X. Zhang, and J.H. Fike. 2004a. Ultraviolet-B radiation damage on Kentucky bluegrass I: antioxidant and colorant effects. HortScience 39:1465-1470. Ervin, E.H., X. Zhang, and J.H. Fike. 2004c. Ultraviolet-B radiation damage on Kentucky bluegrass. III. Cultivar effects. HortScience 39:1475-1477. Fiscus, E.L., and F.L. Booker 1995. Is increased UV-B a threat to crop photosynthesis

and productivity? Photosynth. Res. 43:81-92. Gardner, F.P., R.B. Pearce, and R.L. Mitchell (ed.) 1985. Physiology of crop plants. Iowa State Univ. Press, Ames IA.

103

Gwynn-Jones, D., and U. Johanson. 1996. Growth and pigment production in two subarctic grass species under four different UV-B irradiation levels. Physiol. Planta. 97:701-707. Gwynn-Jones, D. 2001. Short-term impacts of enhanced UV-B radiation on photo- allocation and metabolism: a possible interpretation for time-dependent inhibition of growth. Plant Ecol. 154:67-73. Hagen, S.F., G.I.A. Borge, G.B. Bengtsson, W. Bilger, A. Berge, K. Haffner, and K.A. Solhaug. 2007. Phenolic contents and other health and sensory related properties of apple fruit (Malus domestica Borkh., cv. Aroma): Effect of postharvest UV-B irradiation. Postharvest Bio. Technol. 45:1-10. Hofmann, R.W. 2000. Population studies of ultraviolet-B radiation responses in white clover (Trifolium repens L.). Ph.D. Thesis, Massey University, N.Z. http://muir.massey.ac.nz/bitstream/handle/10179/2248/02_whole.pdf?sequence=1

(Verified 30 Jun. 2012). Kerr, J.B., and C.T. McElroy. 1993. Evidence for large upward trends of ultraviolet-B radiation linked to ozone depletion. Science 262:1032-1034. Krupa, S.V., and R.N. Kickert. 1989. The Greenhouse effect: Impacts of ultraviolet-B (UV-B) radiation, carbon dioxide (CO2), and Ozone (O3) on vegetation. Environ. Pollut. 61:263-393. Manney, G.L., M.L. Santee, M. Rex, N.J. Livesey, M.C. Pitts, P. Veefkind, E.R. Nash, I. Wohltmann, R. Lehmann, L. Froidevaux, L.R. Poole, M.R. Schoeberl, D.P. Haffner, J. Davies, V. Dorokhov, H. Gernandt, B. Johnson, R. Kivi, E. Kyrö, N. Larsen, P.F. Levelt, A. Makshtas, C.T. McElroy, H. Nakajima, M.C. Parrondo, D.W. Tarasick, P. von der Gathen, K.A. Walker, and N.S. Zinoviev. 2011. Unprecedented Arctic ozone loss in 2011. Nature 478:469-475. Milesi, C., S.W. Running, C.D. Elvidge, J.B. Dietz, and B.T. Tuttle. 2005. Mapping and modeling the biogeochemical cycling of turf grasses in the United States. Environ. Manage. 36:426-438.

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Millington, K.R., and A.L. King. 2010. Measuring colour and photostability of small fleece wool samples. Anim. Prod. Sci. 50:589-592. Mohammed, A.R., E.W. Rounds, and L. Tarpley. 2007. Response of rice (Oryza sativa L.) tillering to sub-ambient levels of ultraviolet-B radiation. J. Agron. Crop Sci. 193:324-335. Pons, T.L., H. van Rijnberk, I. Scheurwater, and A. van der Werf. 1993. Importance of the gradient in photosynthetically active radiation in a vegetation stand for leaf nitrogen allocation in two monocotyledons. Oecologia 95:416-424. Rowland, F.S. 1990. Stratospheric ozone depletion by chlorofluorocarbons. Ambio 19:281-292. Rozema, J., R. Broekman, D. Lud, H.J. Huiskes, T. Moerdijk, N. de Bakker, B. Meijkamp, and A. van Beem. 2001. Consequences of depletion of stratospheric ozone for terrestrial antarctic ecosystems: the response of Deschampsia antarctica to enhanced UV-B radiation in a controlled environment. Plant Ecol. 154:103- 115. Smith, J.L., D.J. Burritt, and P. Bannister. 2000. Shoot dry weight, chlorophyll and UV- B-absorbing compounds as indicators of a plant's sensitivity to UV-B radiation. Annal. Bot. 86:1057-1063. SAS Version 9.2. SAS Copyright, 2002-2008 by SAS Institute Inc., Cary, NC, USA. Schmidt, R.E., and X. Zhang. 2001. Alleviation of photochemical activity decline of turfgrasses exposed to soil moisture stress or UV radiation. Int. Turfgrass Res. J. 9:340-346. Teramura, A.H., R. Hilton Biggs, and S. Kossuth. 1980. Effects of ultraviolet-B

irradiances on Soybean II. Interaction between ultraviolet-B and photosynthetically active radiation on net photosynthesis, dark respiration, and transpiration. Plant Physiol. 65:483-488.

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Teramura, A.H., L.H. Ziska, and A.E. Sztein. 1991. Changes in growth and photosynthetic capacity of rice with increased UV-B radiation. Physiol. Planta. 83:373-380. Zhang, S., H. Jiang, S. Peng, H. Korpelainen, and C. Li. 2011. Sex-related differences in morphological, physiological, and ultrastructural responses of Populus cathayana to chilling. J. Exp. Bot. 62:675-686. Zhang, X., and E.H. Ervin. 2009. Physiological assessment of cool-season turfgrasses under ultraviolet-B stress. HortScience 44:1785-1789.

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CHAPTER 5

PIGMENT CHANGES IN COOL SEASON TURFGRASSES IN RESPONSE TO ULTRAVIOLET-B LIGHT IRRADIANCE

ABSTRACT

Pigments involved in protection of photosynthetic apparatus in high light

conditions have potential to aid turfgrasses in periods of enhanced ultraviolet-B (UV-B)

radiation. These pigments have light absorbing capacities in the UV-B range (280-320

nm) and also may aid in energy dissipation as an initial protective mechanism from

photosystem damage. These pigments and their function in elevated UV-B conditions

have received little attention. This study aimed to characterize protective pigment

responses to enhanced UV-B conditions in cool-season turfgrass. Three cool season

grasses were used - tall fescue (Schedenorus phoenix (scop.) Holub), perennial ryegrass

(Lolium perenne L.) and creeping bentgrass (Agrostis stolonifera L.) cultivars ‘L93’ and

‘Penncross’. Turfgrasses were treated for pigment responses over a one week period and

subjected to 16 kJ m-2 d-1 of UV-B in growth chambers. The chambers were run on a day

/ night regime of 14 / 10 hours and plants were subjected to 18.5 mol m-2 d-1

photosynthetically active radiation (PAR) and 23 / 20 °C. Samples were collected at day

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0, 1, 3, 5 and 7, and chlorophyll fluorescence was measured and chlorophyll

pigmentation, flavonoids, phenolics, anthocyanins and carotenoids content were

determined. Fluorescence measurements and chlorophyll levels decreased significantly

(P=0.05) in UV-B conditions compared to the control environment. All species possessed

significantly (P=0.05) higher quantities of total phenolics and flavonoids in the top of the

tissue canopy compared to roots or tissue at the soil surface. After day one all of these

compounds decreased but there were more significant decreases in UV-B conditions.

Anthocyanins were only characterized in creeping bentgrass ‘L93’ with Cyanidin – 3 – O

– glucoside making up 60% of the anthocyanins identified. The carotenoids, zeaxanthin

and β-carotene declined in total content in UV-B treatments in creeping bentgrass ‘L-93’

and ‘Penncross’ after seven days, but not in perennial ryegrass or tall fescue.

INTRODUCTION

Light drives many photosynthetic responses in plants. High irradiance can lead to

photoinhibition and chlorophyll degradation in plants. This may cause a decrease in

productivity (Curran et al., 1990; Anderson and Aro, 1994; Gitelson et al., 2006) and

leaves plants susceptible to poor recovery from wear and biotic stress. The irradiance that

plants receive on a daily basis varies in both quantity and spectral distribution. The light

distribution refers to the photosynthetically active radiation (PAR) that occurs in the 400-

700 nm wavelengths. It can be measured instantaneously in μmoles m-2 s-1 (Smith, 1994).

The direct effect of wavelength variations differs however from high levels of irradiance.

Light low in red light leads to alterations in photoreceptors signaling whole plant and

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biochemical changes (Wherley et al., 2005). In Italian ryegrass (Lolium multiflorum

Lam.), there was reduced tillering while increasing length of leaf blades and reproductive

shoots due to increased red light (655 nm – 665 nm) (Casal et al., 1985). On the other end

of the spectrum, concentrations of blue light enhanced activity of cryptochrome leading

to chalcone synthase induction in Arabaidposis thaliana, (Lin et al., 1996). Outside of

PAR, other wavelengths such as ultraviolet light (UV) impact plant growth and

physiology. Ultraviolet light wavelengths are found between 100 nm and above 400 nm

(Rosario et al., 1979).

The effects of these wavelengths vary on plant physiology and growth.

Ultraviolet-A increases biomass by 22% in cucumber compared to UV-B wavelengths

but standard PAR additionally increased biomass by 14% above UV-A (Krizek et al.,

1997). The influence of UV-B in particular is considered to be negative due to

development of reactive oxygen species, inhibition of photosynthesis and effects on

genetic expression (Mackerness, 1999; Teramura and Sullivan, 1994). Lower

regeneration of ribulose bisphosphate, decreases in fluorescence, increased D1

polypeptide turnover within photosystem II have also been linked to UV-B exposure

(Greenburg et al., 1989; Strid et al., 1990; Ziska et al., 1993). Pea plants (Pisium sativum

L.) and cotton (Gossypium hirsutum L.) suffer decreases in chlorophyll content when

exposed to UV-B (Ziska et al., 1993; Gao et al., 2003). These losses occur within plants

particularly from UV-B radiation (Caldwell, 1971). The potential for further damage

could increase from 2010-2020. Peak irradiance of UV-B wavelengths are predicted to

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occur in this time period prior to degradation of chlorine and bromine containing species

(Taalas et al., 2000).

The protective mechanisms that plants possess vary in their action and also in

location of response. Plant pigments such as anthocyanins, a class of UV-B absorbing

flavonoids are produced in Kentucky bluegrass (Poa pratensis L.), in response to

elevated UV-B conditions (Zhang et al., 2005). Anthocyanins are flavonoids which

absorb in the UV-B region (Gould, 2004). Polyacylated anthocyanins completely reduced

DNA lesions in blue morning glory (Ipomoea tricolor cv. ‘Heavenly Blue’), (Mori et al.,

2005). The anthocyanins themselves can vary in their absorption spectrum due to

changes in methylation or acylation (Gitz et al., 1998; Do and Cormier, 1991). These

changes are affected predominantly by pH alterations within the plant which are light

driven (Yin et al., 1990). The actual location of the pigments in the adaxial epidermis of

leaf surfaces is also considered crucial for UV protection in coleus (Solenostemon L.)

(Burger and Edwards, 1996). There are also other pigments which are used by plants with

the aim of reducing light damage to photosynthetic apparatus. The light absorption

characteristics are different from anthocyanins but offer light protection.

Carotenoids are another set of compounds which have another role in light

protection in high intensity light. A pool of the 40 carbon compounds known as

xantophylls alters its composition in creeping bentgrass (Agrostis stolonifera L.) in

response to high and low light conditions (Bell and Danneberger, 1999; McElroy et al.,

2006). Zeaxanthin is found in larger proportion to violaxanthin in high light conditions

but reverts to equal or lower levels in low light conditions. Lutein has also been shown to

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be the carotenoid of greatest concentration regardless of light conditions (McElroy et al.,

2006). In Kentucky bluegrass treated with UV-B, total carotenoid content decreased over

10 days with recovery occurring after 15 days of treatment completion. There was also a

positive correlation (>0.7) between these compounds and turfgrass quality in enhanced

UV-B conditions (Zhang et al., 2005). The ability of carotenoids to quench singlet

oxygen and other excited free radicals after UV-B treatment (Mathews-Roth, 1996), may

be responsible for reduction in damage to LHC in plants and limit the loss of plant

quality. The damage to photosynthetic apparatus is used to infer possible reductions in

photosynthetic efficiency and this damage has also a positive correlation to total

carotenoid content (Zhang et al., 2005).

The repair and defense mechanisms can also include more than pigments.

Enzymes which act as catalysts for energy-quenching reactions such as super oxide

dismutase build up in response to UV-B exposure in Kentucky bluegrass (Zhang et al.,

2005). Glucose-6-phosphate dehydrogenase activity and higher levels of proline occur in

creeping bentgrass, Kentucky bluegrass, perennial ryegrass and tall fescue treated with

UV-B. A possible repair mechanism allocating resources from glycolysis to the pentose

phosphate pathway leads to a buildup in enzymatic activity in turfgrasses exposed to UV-

B light (Sarkar et al., 2011). These repair systems stabilize the D1 protein within PSII and

offer a method of scavenging excess levels of energy. The systems also help to split up

cyclobutane dimers in DNA between two adjacent pyrimidines which are probably

formed during UV exposure. Photolyase uses excision repair or recombination repair to

complete restoration of DNA damaged from UV-B exposure (Sancar and Sancar, 1988).

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The damage to photosynthetic apparatus linked to high energy light within PAR is

known as photoinhibition and UV-B light is known to cause similar problems for plants

(Friso et al., 1994). The enhanced rate of degradation of both the D1 and D2 proteins

inhibits the rate of photosynthesis. The damage to the D2 protein is increased in high UV-

B conditions which are not considered normal. In general the D1 protein degrades at a

more rapid rate than D2 with the exception where UV-B treatments are imposed in

combination with visible light (Jansen et al., 1993). This is due to the UV absorption by

the manganese cluster which lies in the oxygen evolving complex. This may act as a

component magnifying the protein degradation (Babu et al., 1999; Tyystjärvi, 2008). The

extent of the damage may initially be expressed in PSII and then damage will begin to

occur in the more robust PSI. The damage in PSII may be construed as an enhanced rate

of protein degradation which can be repaired. This damage, however, then leads to

damage of PSI which requires complete breakdown and reconstruction and long term

damage to PSI (Takahashi and Murata, 2008). The degradation of PSII leads to a buildup

of reactive oxygen species and peroxyl radicals with increased lipid peroxidation (Hideg

and Vass, 1996; He and Häder, 2002).

In measuring damage to photosynthetic apparatus within plants, damage to

photosystem II is most prevalent in relation to UV-B damage (Vass et al., 1996). This

damage can be measured indirectly via fluorescence of reaction centers of chlorophyll A

(Maxwell and Johnson, 2000). The degradation of the centers leads to a decrease in

fluorescence. Chlorophyll content within tissue, which also degrades due to

photoinhibition, can also be measured (Strid and Porra, 1992). This degradation in UV-B

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conditions is at a higher rate than normal PAR and leads to a decrease in productivity

from plants and can occur on a time scale of minutes to hours (Aro et al., 1993).

Turfgrass photoinhibition occurs under elevated UV-B light conditions with Kentucky

bluegrass suffering significant decreases in the ratio of variable fluorescence to maximal

fluorescence (Fv/Fm) (Zhang et al., 2005). This results in a decrease in photosynthetic

activity due to increased levels of fluorescence within the photosynthetic apparatus.

Under elevated UV-B light conditions it has been shown that photoinhibition also affects

the PSII apparatus in tall fescue and perennial ryegrass but not as badly as Kentucky

bluegrass (Zhang and Ervin, 2009).

There is a need to elucidate the variations that occur within the plant canopy while

also characterizing pigment changes across a range of cool season turfgrasses exposed to

UV-B irradiation. The influence of UV-B may create different responses within cool

season turfgrasses and how they protect their photosynthetic apparatus in an attempt to

retain productivity. The objective of this study was to characterize pigmentation

alteration in UV-B conditions and investigate if these changes offer potential for

protection from UV-B damage to the photosynthetic apparatus in cool season turfgrasses

via energy dissipation and direct absorption of excess light. We hypothesized that

turfgrasses would produce higher quantities of protective pigments in response to the

UV-B stress. These higher pigment quantities would then allow turfgrasses to retain their

photosynthetic capacity during the period of stress. This will aid in reducing summer

stress and allow turfgrasses to maintain photosynthetic activity close to normal levels.

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MATERIALS AND METHODS

Sample preparation

The studies were conducted in October, 2010 and March, 2011 in Columbus,

Ohio. Turfgrasses were initially seeded into a mason sand rootzone >97% sand (Hummel

& Co, Trumansburg, NY). The rootzone met USGA requirements and had a pH of 7.4

with a clay silt fraction of less than 5%. The seeding was timed to match germination

speeds between the grasses. Creeping bentgrasses were seeded first, and 4 days later

perennial ryegrass and tall fescue were seeded into 3.8 cm diameter ‘conetainers’

(Steuwe & Sons, Tangent, OR). Moisture was maintained by placing turfgrasses on a 30

second irrigation cycle every 10 minutes in a mist house. Turfgrasses were allowed to

establish for six weeks in the mist house.

Grasses were treated with Peters 20-20-20 (Scotts-Miracle Gro, Marysville, OH)

at a rate of 11.8 kg ha-1 every two weeks during this period. After germination, plants

were removed and placed in standard greenhouse conditions for a further four weeks to

allow for maturation. Turfgrasses were maintained at a height of ~10cm with clippings

removed 3 times weekly. Fertility treatments were continued and imidacloprid N-[1-[(6-

chloro-3-pyridyl) methyl]-4,5-dihydroimidazol-2-yl]nitramide [Bayer Environmental

Science, Research Triangle PK, NC] was applied to prevent insect damage. Mefenoxam

(R,S)-2-[(2,6-dimethylphenyl)-methoxyacetylamino]-propionic acid methyl ester

[Syngenta AG, Greensboro, NC] and thiophanate-methyl (dimethyl 4,4’-o-

phenylenebis[3-thioallophanate]) [Cleary Chemicals, Dayton, NJ] were applied for

preventative disease control. Water was applied to prevent wilt, with automated heads

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using an ESP modular irrigation system (Rain Bird, Azusa, CA) once daily. Ten weeks

after initial turfgrass emergence, plants were placed in Conviron E15 growth chambers

(Controlled Environment Ltd., Winnipeg, Canada). To equalize plants they were placed

in dark growth chambers at 20 °C. This happened for 24 hours for all plants in both

experiments.

Ultraviolet-B treatment commenced using UV-B lights having peak emission at

313 nm, QUV UV-B 313 nm, (Q-Lab Corp., Westlake, OH). Treatment exposure was

calculated using time of exposure multiplied by measured irradiance energy per second.

Wavelengths within UV-B and UV-A were measured using an IL1350 photometer

(International Light, Newburyport, MA) with specific sensors for each portion of the UV

light spectrum. Biologically effective radiation was calculated from spectrum provided

by light producers. Ultraviolet radiation is reported in kJ m-2 d-1. Photosynthetically

active radiation was instantaneously measured every 15 minutes during the study and

reported as mol m-2 d-1 using cosine corrected light sensors (Spectrum Technologies,

Plainfield, Il). Light treatments of 14 hours daytime and night time periods of 10 hours

were controlled by the chamber. The lamps were pre-lit for 48 hours to prevent any

fluctuation in energy output during trials. Photosynthetic active radiation (PAR) which

lies in the range of 400 – 700 nm was provided using fluorescent lamps. The same lamps

were used in the control and UV chambers, 85 w s-1 high output F12T12-CW.HO

fluorescent tubes (General Electric, Fairfield, CT). The turfgrasses were irrigated twice

daily to prevent wilt in the chambers. Each chamber was fixed at 20°C during light

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periods and 17 °C in darkness. In each chamber, plants were placed 0.4 m below the

lighting in an even spacing.

Chlorophyll content and fluorescence

The experiment was set up in a randomized complete block design with four

blocks and three replications per block for each grass. Analysis was carried out at Day0

(D0), Day1 (D1), Day3 (D3), Day5 (D5) and Day7 (D7) for all data. After each treatment

turfgrasses were placed in darkness for one hour. Fluorescence measurements were then

taken with 12 total replications for each grass per block to reduce variability. The Fv/Fm

values were obtained using a handheld chlorophyll fluorometer OS-30p (Opti-Sciences,

Hudson, NH). Leaf tissue from the top 5 cm was used and measured in-vivo with initial

660 nm pulses from an integral probe fluoresced to a photodiode with a 700 – 750 nm

bandpass filter. Values were averaged for each block and analyzed separately due to the

fact that different turfgrass plants were used for each sample date.

Three turfgrass tissue samples from the top of each grass of 40 mm-2 were taken

and placed into 5 mL of N,N-dimethylformamide (Sigma-Aldrich, St. Louis, MO) for 48

hours in the dark at 4°C. The extract was then placed in 4 mL cuvettes and absorbances

were measured at 664, 647, 625, 603 nm. This was carried out using a UV1800

spectrophotometer (Shimadzu Corp., Colombia, MD). Total chlorophyll, chlorophyll A

and chlorophyll B were calculated using pre-determined formulae (Moran, 1982).

Chlorophyll content was reported in µg m-2. The rest of the tissue material was separated

into the top 5 cm (T5) of tissue, all tissue between the root / shoot interface and the top

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cm (T) and roots (R) (Appendix C), and frozen in liquid nitrogen at -210 °C. All samples

were then freeze dried using a Virtis ultra 35 LE lyophilizer (The Virtis Co., Gardiner,

NY) under a vacuum of 125 mTorr. This ran on an air temperature of -40°C to 10°C over

a 20 hour period and maintained at 10°C for a further four hours while tray temperatures

were maintained at -20°C. Samples were then stored at -20°C while analysis was

completed.

Total flavonoids

Soluble flavonoids were measured using the method described by Pourmorad et

al., (2006). Samples of 0.035 g were added to 10 mL of methanol and vortexed in 15 mL

conical centrifuge tubes and allowed to stand for 10 minutes at room temperature. The

samples were centrifuged at 3,400 rpm for 5 minutes. The material stood with an

occasional vortex for at least 10 minutes before centrifuging. A standard curve was

prepared using quercitin solutions at concentrations of 0, 10, 20, 40 and 50 μg mL in

methanol. The extract was then placed into a tube with 1.5 mL CH3OH, 0.1 mL of 10%

AlCl3, 0.1 mL potassium acetate and 2.8 mL distilled H2O. The material was vortexed

and incubated at room temperature for 30 minutes. The supernatant absorbance was

measured at 415 nm with a UV1800U spectrophotometer. Linear regression analysis was

used to determine concentrations of flavonoids in each sample. This was carried out for

R, T and T5 of all samples to determine concentrations of pigments based on location.

This test was performed for each day in both experiments.

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Total phenolics

Total phenolics were measured in accordance McDonald et al., (2001). Gallic acid

standard curves were made using concentrations of 0, 5, 10, 20, 40 and 80 mg L in

MeOH:H2O, 50:50 v/v. Plant tissue (0.035 g) was placed in 15 mL of methanol and

vortexed and let sit for 10 minutes. Tissue sample was then centrifuged for 3 minutes at

3600 rpm. Supernatant (0.5 mL) was collected and placed in a 15 mL tube with 5 mL of

10% folin ciocalteu reagent and 4 mL of 1M Na2CO3 and let sit at room temperature for

15 minutes. Total phenol concentration was then determined at absorbance of 765 nm

using a UV1800U spectrophotometer. Linear regression analysis was then used to

estimate phenol concentrations in each sample on a µg g-1 basis. This was carried out for

R, T and T5 of all samples to determine concentrations of pigments based on location.

This was carried out for each day in both experiments.

Carotenoid extraction and analysis

Carotenoid extraction was carried out with slight modifications from a method

described by Ferruzzi et al., (1998). All experiments were carried out under caution of

low light to prevent carotenoid degradation. Tissue sample (0.5 g) was placed in 10 mL

of MeOH in a 15 mL centrifuge tube and vortexed for 30 seconds. Samples were then

homogenized using a polytron PT10 (Brinkman Institute, Westbury, NY), and then

centrifuged for 7 minutes with an Accuspin 3R (Fisher Scientific, Pittsburgh, PA).

Supernatant was removed and the pellet was extracted 3 times in 10 mL acetone/hexane

1:1 (v/v). All extracts were pooled in a 50 mL tube, a small volume of NaCl crystals were

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added, and the extract was brought up to 50 mL with water and vortexed. The upper

hexane layer was brought to 10 mL in a volumetric flask with hexane and transferred to a

25 mL conical flask.

Samples were run through glass wool and Na2SO4 crystals and then saponified to

remove the phytol tail from the chlorophyll pigments to allow them to partition into the

aqueous phase for removal. Samples (10 mL) were placed in 50 mL conical flasks with

10% KOH (Sigma Aldrich), in a 1:2 v/v with the extract. Flasks were then placed on a

benchtop shaker for 2 hours covered in foil. The saponified mixture was then transferred

to a 125 mL separatory funnel. The green chlorophyll separated into the lower phase

which was removed. The remaining upper phase was washed with 40% Na2SO4 three

times. The remaining sample was then dried under nitrogen at less than 2 PSI using a

reactive-vap 18780 (Pierce, Rockford, IL). Samples were then resuspended in 2 mL of

methyl tert-butyl ether: MeOH 1:1 (v/v) and passed through a 0.22 µm nylon syringe

filter (Fisher Scientific, Pittsburgh PA) into 2 mL amber HPLC vials (Waters, Milford,

MA).

Sample volumes of 20 µL were injected into an HP1050 series HPLC, with a

diode array detector (DAD), were used to quantitate the carotenoids. Samples (20 µL)

were injected onto a YMC C30 carotenoid column 4.6 x 250 mm, 3 µm particle size,

(Waters, Milford, MA). A gradient system was set up and a tertiary solvent system was

used. The gradient was used based on a modified method of Moros et al., (2002), with a

flow rate of mL/min. Solvent A consisted of methanol/MTBE/water (81:15:4), solvent B

consisted of methanol/MTBE (9:91) and solvent C consisted of MTBE alone. The initial

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conditions were 100% A, with a linear increase to 45% A and 55% B over 21 min,

changing to 100% C and holding for 2 minutes and returning to 100% A and holding for

5 minutes. Data was collected using HP Chemstation 3D and 453 nm wavelength was

used for quantitation of the compounds. External calibration curves of zeaxanthin

(CaroteNature, Lupsingen, Switzerland), lutein, β-carotene (Sigma Aldrich),

antheraxanthin and violaxanthin (Chromadex, Irving, CA) were used to quantitate the

respective carotenoids. Turfgrass samples were used from D0 and D7 of the trials to look

at overall changes in concentrations of carotenoids. Samples were taken from the T5

portion of turfgrasses

Anthocyanins

Anthocyanins were extracted using a method modified from Giusti et al., (1999).

Freeze dried tissue (0.5g) was crushed into a fine powder using a mortar and pestle while

suspended in liquid nitrogen. Samples were then placed in 10 mL of 100% acetone in 15

mL tubes, vortexed and left to sit for 5 minutes. Samples were then centrifuged at 3600

rpm for 5 minutes. Supernatant was collected and samples were re-extracted with 0.1%

HCL acidified 70:30% acetone:H2O (V:V). Samples were re-extracted two more times

until supernatant was clear. Extract was combined in a 50 mL centrifuge tube and

brought to 30 mL. Supernatant and tissue were then combined and passed through a No.1

filter (Whatman, Kent, United Kingdom) to maximize extract collection in a Büchner

funnel. This was carried out under vacuum using a Rocker 300 pump (Rocker,

Kaohsiung, Taiwan), and samples were collected in a conical flask. To separate water

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soluble from lipid soluble compounds, 2:1 volume chloroform was added and samples

were sat overnight in dark cool conditions in 125 ml separating funnels. Chloroform and

acetone (now mixed together) were removed and the top aqueous portion was collected

and retained. To purify the anthocyanins, C-18 cartridges (Biotage, Charlotte, NC) were

used. To prepare the cartridges 100% MeOH x2 volumes of cartridge were flushed

through under an Alltech 210351 12-port vacuum manifold (Alltech, Deerfield, IL) and

washed with 3 volumes of acidified water (0.1% v/v HCL). Samples were then loaded

into the cartridge and anthocyanins, sugars and other phenolics were adsorbed onto the C-

18 column under vacuum. To remove water soluble compounds (0.01%) acidified water

was then passed through the cartridge followed by ethyl acetate to elute non-anthocyanin

phenolics. Anthocyanins were finally recovered using v/v 0.01% HCL acidified MeOH in

concentrated format. In all stages, the pH of extract was sustained at 2 or lower to retain

stability. Samples were collected in 2.5 mL microfuge tubes and spun at 5000 rpm x 5

minutes to remove any debris. Samples were then concentrated using a Büchi R114

rotoevaporator and re suspended in 1 mL distilled deionized water which was acidified

with 0.01% HCL.

Samples were passed through a .22 µ filter using 3 mL luer lock syringes and

injected into 2 mL amber LCMS vials with 300 µL inserts with springs. Samples were

then injected into a LCMS apparatus set up as follows. A CTO-20A (Shimadzu Scientific

Inst., Columbia, MD) oven was used to maintain temperatures at 35°C, flow of 0.8 mL

min was controlled with LC-20AD – LC pumps, while 50 µL sample injection was

managed with a SIL-20AC autosampler. Samples were injected onto a Varian C18-A,

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150 x 4.6 mm i.d. column (Varian Inc., Lake Forest CA). Samples were eluted through a

SPD-M20A PDA (Shimadzu) with wavelengths 280, 320 and 520 nm simultaneously

selected. Spectral analysis of wavelengths between 250 and 700 nm were recorded for all

peaks. Solvents for mobile phase were first filtered through .22 µm filters. A binary flow

mobile phase consisting of (A) 4.5% formic acid:H2O v/v and (B) acetonitrile with a flow

rate of 0.8 mL min was used. Anthocyanins were separated using a solvent gradient from

5 % B to 15 % in 5 minutes followed by an increase in B to 30 % at 20 minutes. From 20

minutes to 23 minutes solvent B increased to 100 % and was maintained there until 25

minutes. Concentration of B then decreased to 5 % at 28 minutes and sample injection

completed after 35 minutes. Samples were then injected into an electron spray mass

spectrometer and fragmentation analysis carried out. Due to limited samples only

qualitative data was obtained. Known anthocyanin pigments (Chandra et al., 2001) from

(Aronia melanocarpa) were injected under the same conditions and used as a reference

material (Appendix D). Compound mass, elution order, spectral absorbance and retention

time were used to characterize anthocyanins found in turfgrass samples.

Data analysis

Data analysis for chlorophyll, fluorescence, total phenolics and flavonoids was

carried out using the general linear model from the SAS program (SAS Inst., 2008).

Analysis was carried on block averages for comparison of data from each light

environment collected on each specific date. Comparisons between light environments

were carried out for each variable on each date because different samples were used for

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each date. Carotenoid content for each light environment was calculated as a difference

between D0 and D7 to compare overall changes during treatments. The lack of samples

due to growth chamber size constrictions for anthocyanin analysis meant that only

characterization of these pigments could occur at this time.

RESULTS

Light intensities for the two chambers were very similar within the PAR region of

light (400-700 nm). The main difference between chambers was measured in the UV-B

region with turfgrasses subjected to 16.9 KJ m-2 d-1 of UV-Bbe light in the UV chamber

compared to the 0 in the control chamber (Table 5.1).

Fluorescence measurements in all grasses followed the same pattern in both

experiments and data was pooled after testing for differences between experiments. In

both chambers there was a relative decrease in photosynthetic activity from D0 in all

grasses. However, significantly lower fluorescence measurements occurred in chambers

that had elevated UV-B conditions (P=0.05) after D3 and this continued through D7. All

grasses suffered greatest decline in Fv / Fm up to D5. Perennial ryegrass and “Penncross’

creeping bentgrasses had declines of Fv / Fm greater than 10% (Figure 5.1). No grass

exhibited complete loss of photosynthetic activity.

Chlorophyll pigmentation exhibited a similar pattern in all grasses in both

experiments. Treatment of UV-B resulted in significant declines in total chlorophyll

content (P=0.05). The decreases were noted after D1 in both perennial ryegrass and tall

fescue. This separation between control and UV treated grasses did not occur until D3 in

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the creeping bentgrass cultivars. There were also declines in the control chamber in all

grasses; however, the decline in chlorophyll content in the control chambers was faster in

the bentgrass cultivars (Figure 5.2). Overall, chlorophyll levels were higher on an area

basis in perennial ryegrass and tall fescue compared to the creeping bentgrass cultivars.

The change in chlorophyll A and B also followed the same pattern. Significant declines

(P=0.05) occurred in both of these chlorophyll pigments in UV-B conditions (Appendix

E).

The distribution of both phenolics and flavonoids were similar across all grasses

regardless of light environment. This occurred in both experiments. Turfgrasses exhibited

the ability to accumulate significantly higher (P=0.05) quantities of both phenolics and

flavonoids in the T5 portion of the tissue sample. There was almost a 50% difference in

total phenolics between the T5 and T in creeping bentgrass ‘L-93’ in control conditions at

the end of the trial. On that same date there was an 85% difference between T5 and R

tissues in ‘L-93’ (Figure 5.3). There was an initial increase in phenolic content in both

portions of tissue on D1 and this was then followed by a decline. Flavonoids also

exhibited similar significant separation with T5 tissue containing significantly greater

concentrations. In perennial ryegrass, concentrations of total flavonoids varied between

T5 and T tissue was as much as 50% after D7 (Figure 5.4). There was for the most part a

spike in response after day 1 but this was followed by a decline in concentration in both

parts of the plant tissue.

The overall concentration of flavonoids increased on D1 in both light

environments. This response was then followed by a decline in all grasses until D5 when

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quantities then leveled off. There was significant decreases in all grasses in UV-B

irradiated conditions at D7. There was on average a 27.5% decrease in all grasses at D7

(Figure 5.5). A similar decrease was observed in both light environments in total phenolic

concentrations (Data not shown). The difference at D7 between control and UV-B

chambers was only significantly different in the two creeping bentgrass cultivars (Data

not shown).

Anthocyanin responses to UV-B exposure were very limited. Extracts of tissue

from D1, D3 and D5 yielded no data when passed through HPLC conditions at 520 – 530

nm. This occurred in all grasses for both sets of light environments and in both

experiments. In D7 tissue from creeping bentgrass ‘L-93’, HPLC analysis indicated that

the presence of anthocyanins sufficiently strong for detection at 520 nm (Figure 5.6) and

320 nm. Electrospray ionization of compounds resulted in masses for four main pigments

in the ‘L-93’ tissue. The elution order and approximate retention time was similar to

Aronia samples which were also injected and checked in previous literature (Chandra et

al., 2001). This information was then used to characterizing the anthocyanins found in

creeping bentgrass ‘L-93’ (Table 5.2). First eluted compound at 8.4 minutes was cyanidin

3 – O – glucoside which was followed by peonidin 3 – O – galactoside at 9.95 minutes.

At 11.05 minutes cyanidin 3 – O – malonyl glycoside was detected and this was followed

by cyanidin 3 – O – succinyl glucoside at 13.25 minutes (Figure 5.6). No other grasses

displayed detectable anthocyanin increases at D7 in either light environment.

Using internal spikes to quantify potential pigment degradation resulted in noted

loss of antheraxanthin, lutein and violaxanthin due to saponification. Zeaxanthin and β-

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carotene were identified based on absorbance, peak shape, elution order and retention

time linked to standards (Figure 5.7). Zeaxanthin eluted at 11.1 minutes while β-carotene

eluted at 20.1 minutes. Peaks that did fit any of standards available eluted at 16, 19 and

24 minutes (Figure 5.7). Degradation of β-carotene was ~10% through saponification and

zeaxanthin had ~15% degradation. Saponification was required to separate chlorophyll

pigments from carotenoids prior to HPLC analysis. There was no difference in carotenoid

content between light environments at D0 or D7. There was however greater degradation

in the UV environment from D0 to D7 with significant decreases in β-carotene content in

both of the bentgrasses (Appendix F) in the UV chamber. Perennial ryegrass and tall

fescue (Appendix G) did not have such a significant loss of β-carotene in UV conditions

(Figure 5.8a). Creeping bentgrass ‘Penncross’ also had significant β-carotene loss

between samples from D0 to D7 in control light conditions (Data not shown). Creeping

bentgrass ‘L-93’ had significant losses (P=0.05) of zeaxanthin in both UV and control

chambers. No other grass exhibited this loss of zeaxanthin and this did not happen in the

control chamber (Figure 5.8b). There was no difference in zeaxanthin content between

UV and control treated turfgrasses at D7.

DISCUSSION

The decrease in photosynthetic activity in our study occurred regardless of grass

and agreed with previous data in cool season turfgrass (Zhang et al., 2005; Sarkar et al.,

2011). This loss of activity in our study could be attributed to the decrease in phenolics or

also loss of enzymes used for repair of the photosynthetic apparatus. The decrease in

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carotenoid pigments may also have played a role in the decrease in photosynthetic

activity in this study. The loss after D1 of UV the absorbing compounds would indicate

that the photosynthetic apparatus would have been suffering damage at the same time.

This was born out in the results show using the flouresence measurements.

The loss of chlorophyll pigmentation in this study was despite taller turfgrasses

having a dense canopy. The loss of chlorophyll coincided with the decrease in

fluorescence also and this would then lead to a loss of biomass productivity as has been

noted in wheat (Triticum aestivum L.) which had a decline of 18% (Correia et al., 1999)

in response to elevated UV-B levels. Degradation of chlorophyll is a common response to

UV-B radiation in plants. Kentucky bluegrass has exhibited this response before as have

sunflower cotyledons and other plants (Zhang et al., 2005; Costa et al., 2002; Tevini et

al;, 1981). The energy associated with the photons in the UV-B wavelengths (3.94 – 4.43

electronvolts) is considered strong enough to destroy many chemical bonds and it may

have contributed directly to the chlorophyll degredation (Kovács and Keresztes, 2002).

The variation in distribution of phenolics and flavonoids in our study could be

attributed to two causes. Turfgrass plants in such a dense canopy may have led to self-

shading thus the elevated levels in the upper canopy of these UV absorbing compounds is

a response to the light conditions in the grasses tested. This argument is further

strengthened by the lack of response and much lower levels of each set of compounds in

the root tissue (Buer and Muday, 2004). Similar distributions of flavonols have been

noted (Cortell and Kennedy, 2006) and also in phenolic compounds in roots and leaf

tissue of cow lily (Nuphar advena) and large leaf pondweed (Potamogeton amplifolius

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L.) (Cronin and Lodge, 2003). The second cause could relate to age of the leaf tissue

which can be more susceptible to damage from UV-B exposure when it is younger (Lois,

1994). We feel this distribution of compounds in our study is due to a light response as

material was clipped 3 days before trial initiation to reduce the influence of leaf age.

The actual quantities of phenolics and flavonoids had an initial increase from D0

to D1 in both light environments in our study. This type of response is considered a plant

protective response in an attempt by the plant to reduce the impact of the UV-B

wavelengths on the photosynthetic apparatus. Phenolics have antioxidant capacity and

offer a method to reduce oxidized radicals and reduce damage to the photosynthetic

apparatus (Duval et al., 1999). Mutant Arabidopsis plants unable to accumulate

flavonoids are highly susceptible to UV damage (Landry et al., 1995). There was then a

decline in flavonoid and phenolic levels in all grasses, with greater declines for both in

UV-B. This loss may be related to an inability of turfgrass plants to allocate enough

energy to retain initial response levels due to lower levels of visible light in the chambers

(Teramura et al., 1980). The UV-B exposure may have overwhelemed the plants

capability to repair itself and recover from free radical damage in this study.

The potential benefit of anthocyanins was only seen after seven days of exposure

only in the ‘L-93’ cultivar of the creeping bentgrass in our study. This delay in

production was after a decline in photosynthetic activity and so the benefit was both

delayed and limited. Increased anthocyanins have been found in Kentucky bluegrass in

response to UV-B exposure (Zhang et al., 2005). The UV-B absorption capabilities of

anthocyanins are argued as a benefit and that plants possessing this response are

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acclimated to UV-B stress (Gould, 2004). There is some argument to the photoprotective

capabilities of these compounds as only marginally higher photosynthetic efficiency was

found in red Kermes oak (Quercus coccifera L.) leaves compared to greener ones

(Karageorgou and Manetas, 2006). Also limited or no photoprotective value was found in

myrtle plants with anthocyanins in the epidermis (Woodall and Stewart, 1998).

In our study the creeping bentgrass cultivar ‘L-93’ only exhibited such a response

while ‘Penncross’ did not. The variety differences in anthocyanin response noted in our

study have also been noted before though not in turfgrass. In maize (Zea mays L.), of

seven varieties tested for UV-B response, only two varieties responded with anthocyanin

production while one other showed a strong anthocyanin development response to far red

light (Beggs and Wellmann, 1985). The other reason that both the perennial ryegrass and

tall fescue may have had limited response in this study was that epidermal thickness plays

a large role in determining the transmission of light into the epidermis. Tall fescue has

large variations in its thickness and it can significantly alter its leaf thickness from 0.31

cm to 0.36 cm in response to drought (Fu and Huang, 2004). This may require much

larger consideration for UV-B screening effectiveness (Day, 1993) combined with

pigment concentrations.

The concentration of β-carotene decreased in both light environmentsin our study

but the decrease was greatest in UV conditions and also only in the bentgrass species.

The loss in β-carotene content has also been linked to a loss of photosynthetic activity in

Dunlaliella cells exposed to UV-B (White and Jahnke, 2002). This loss of an important

pigment may relate to the loss of quality and also the greater loss of photosynthetic

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activity in the UV chamber-treated bentgrasses. Zeaxanthin decreases have occurred over

extended periods of UV- B exposure in maize (Carletti et al., 2003). There have been

initial increases in content also but possible changes in energy allocation may reduce

their levels after prolonged exposure. This degradation of both carotenoids reduces the

turfgrass capabilities to moderate damage from reactive oxygen species. The impact of

carotenoids may be crucial in high UV conditions due to their capabilities of energy

dissipation and is particularly important for the thinner leaf bentgrasses. Further work

completing the xantophyll cycle response and also quantifying and characterizing these

compounds is required to gain a fuller understanding of their value.

The loss of xantophylls due to saponification has been observed in prior studies

(Kimura et al., 1990). It is the first time that the problem has been reported in turfgrass

and may reduce the capability to cleanly separate carotenoid and chlorophyllous

pigments when using HPLC analysis. The alkalinity of the methanolic KOH leads to

hydrolysis of the compounds; however, it was thought that the lower percentage of KOH

would reduce the problem (de Sá and Rodriguez-Amaya, 2003).

CONCLUSION The loss of photosynthetic activity in turfgrasses observed in this experiment due

to exposure to UV-B was expected. It was interesting to note a decrease in quantity of

UV-B absorbing pigments over extended exposure. However, the combination of high

UV and low visible light levels may have played a role in this. The loss of carotenoids in

the creeping bentgrasses is of interest as is the production of anthocyanins in ‘L-93’

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creeping bentgrass due to their UV absorbing abilities. The reduction in carotenoids

would lead to an increase in highly energized products thus requiring some form of

antioxidant response and also a compound that absorbs the excess light. Immediate

breeding responses for a crop that covering potentially 163,800 km-2 (Milesi et al., 2005),

of cultivated land are not possible. In an attempt to address the problem rapidly, the use

of synthetic coatings may offer a practical response and tool for turfgrass managers. This

first characterization and extraction of turfgrass anthocyanins also offers some potential

for further research in turfgrass stress physiology. Further work on carotenoid

characterization and chromatogram publication is also required due to their importance in

light stress and new herbicide research.

131

Light _______________ Chambers _______________ Control Ultraviolet-B

PAR (400-700 nm) 26.2 mol m-2 d-1 25.9 mol m-2 d-1

UV-A (400 – 320 nm) 1.0 KJ m-2 d-1 0.8 KJ m-2 d-1 UV-B be† ( 320 – 280 nm) 0.0 KJ m-2 d-1 16.9 KJ m-2 d-1

†Biologically effective radiation within UV-B wavelengths based on Caldwell, (1971).

Table 5.1: Light intensity and light distribution for Conviron E15 growth chambers illuminated using high output F12T12-CW.HO fluorescent tubes and QUV UV-B 313 nm UV-B lights.

132

Compound Tr (min) † [M] +m / z‡ Fragments m / z§ UV – vis nm¶ Area % (520 nm) # Identity 1 8.42 449 287 280, 516 60.5 Cyanidin 3 – O – glucoside 2 9.95 463 301 324, 516 4.3 Peonidin 3 – O – galactoside 3 11.05 535 287 281, 519 27.5 Cyanidin 3 – O – malonyl glycoside 4 13.25 549 287 329, 516 7.4 Cyanidin 3 – O – succinyl glucoside

† Retention time. ‡ Mass of whole compound. § Breakdown of compound sizes after passage through mass spectrometer. ¶ Peak absorption of compounds in PAR and UV regions. # Percentage of total compounds identified. Table 5.2: Anthocyanin pigmentation detected in creeping bentgrass ‘L-93’ after 7 days of enhanced UV-B treatments.

133

*† Significantly different at (P=0.05)

Figure 5.1: Fv / Fm values for Perennial Ryegrass and Creeping Bentgrass ‘Penncross’ treated with UV-B light (280 – 315 nm) and PAR light (400 – 700 nm) conditions over seven days.

134

*† Significantly different at (P=0.05)

Figure 5.2: Total chlorophyll content for Tall Fescue and Creeping Bentgrass ‘L-93’ treated with UV-B light (280 – 315 nm) and PAR light (400 – 700 nm) conditions over seven days.

135

*† Significantly different at (P=0.05) between Roots (r), Top (T) and Top 5 cm (T5) tissue.

Figure 5.3: Distribution of phenolics in creeping bentgrass ‘L-93’ after treatment with both UV-B light (280 – 315 nm) and PAR light (400 – 700 nm). Data was pooled as all grasses had the same response.

136

*† Significantly different at (P=0.05) between Top (T) and Top 5 cm (T5) tissue Figure 5.4: Distribution of total flavonoids in perennial ryegrass tissue after treatment with both UV-B light (280 – 315 nm) and PAR light (400 – 700 nm). Data was pooled as all grasses had the same response.

.

137

()† Significant difference occurred in flavonoid content for each grass on each day where letters are different. Significance at (P=0.05). Figure 5.5: Turfgrass flavonoid content in Creeping bentgrass, Tall Fescue and Perennial Ryegrass grown in UV-B light (280 – 315 nm) and PAR light (400 – 700 nm) D0 to D7.

138

† Retention times, mass, spectral information and order of elution provided in Table 5.2.

Figure 5.6: Anthocyanin profile of creeping bentgrass ‘L-93’ exposed for 7 days to UV-B light. Produced from HPLC analysis using C18 column and light absorbance measured at 520 nm using a PDA.

139

† 1 – Zeaxanthin; 2 – β – carotene. Figure 5.7: Chromatogram of Perennial ryegrass carotenoids with absorbance at 453 nm. Data produce using C30 column and PDA connected to Chemstation software.

140

†

Means followed by different letters significantly different (P=0.0).

Figure 5.8. β-carotene (top) and zeaxanthin (bottom) content in creeping bentgrass, tall fescue and perennial ryegrass exposed to UV-B light D0 – D7.

141

REFERENCE

Anderson, J.M., and E-M. Aro. 1994. Grana stacking and protection of Photosystem II in

thylakoid membranes of higher plant leaves under sustained high irradiance: Ann. Hypoth. Photosynth. Res. 41:315-326.

Aro, E-M., I. Virgin, and B. Andersson. 1993. Photoinhibition of photosystem II.

Inactivation, protein damage and turnover. Biochim. Biophys. Acta. 1143:113-34. Babu, T.S., M.A.K. Jansen, B.M. Greenberg, V. Gaba, S. Malkin, A.K. Mattoo, and M.

Edelman. 1999. Amplified degradation of photosystem II D1 and D2 proteins under a mixture of photosynthetically active radiation and UV-B radiation: Dependence on redox status of photosystem II. Photochem. Photobiol. 69:553-559.

Beggs, C.J., and E. Wellmann. 1985. Analysis of light-controlled anthocyanin formation

in coleoptiles of Zea mays L.: The role of UV-B, blue, red and far-red light. Photochem. Photobiol. 41:481-486.

Bell, G.E., and T.K. Danneberger, 1999.Temporal shade on creeping bentgrass turf. Crop

Sci. 39:1142-1146. Britton, G., S. Liaaen-Jensen, and H. Pfander. 2004. Carotenoids handbook. Birkhäuser

Verlag, Boston, MA.

Buer, C.S., and G.K. Muday. 2004. The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light. Plant Cell. 16:1191-1205.

142

Burger, J., and G. E. Edwards. 1996. Photosynthetic efficiency, and photodamage by UV and visible radiation, in red versus green leaf coleus varieties. Plant Cell Physiol. 37:395-399.

Caldwell, M.M. 1971. Solar UV irradiation and the growth and development of higher

plants. p. 131-177. In A.C. Giese (ed.) Photophys. Academic Press, NY. Caldwell, M.M., J.H. Richards, D.A. Johnson, R.S. Nowak, and R. S. Dzurec. 1981.

Coping with herbivory: Photosynthetic capacity and resource allocation in two semiarid Agropyron bunchgrasses. Oecologia 50:14-24.

Carletti, P., A. Masi, A. Wonisch, D. Grill, M. Tausz, and M. Ferretti. 2003. Changes in

antioxidant and pigment pool dimensions in UV-B irradiation maize seedlings. Environ. Exp. Bot. 50:149-157.

Casal, J.J., V.A. Deregibus, and R.A. Sánchez. 1985. Variations in tiller dynamics and

morphology in Lolium multiflorum Lam. vegetative and reproductive plants as affected by differences in red/far-red irradiation. Ann. Bot. 56:553-559.

Chandra, A., J. Rana, and Y. Li. 2001. Separation, identification, quantification, and

method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS. J. Agric. Food Chem. 49:3515-3521.

Correia, C.M., M.S. Torres-Pereira, and J.M.G. Torres-Pereira. 1999. Growth,

photosynthesis and UV-B absorbing compounds of Portuguese Barbela wheat exposed to ultraviolet-B radiation. Environ. Pollut. 104:383-388.

Cortell, J.M., and J.A. Kennedy. 2006. Effect of shading on accumulation of flavonoid

compounds in (Vitis vinifera L.) pinot noir fruit and extraction in a model system. J. Agric. Food Chem. 49:3515-3521.

Costa, H., S.M. Gallego, and M.L. Tomaro. 2002. Effect of UV-B radiation on

antioxidant defense system in sunflower cotyledons. Plant Sci. 162:939-945.

143

Cronin, G., and D.M. Lodge. 2003. Effects of light and nutrient availability on the growth allocation, carbon/nitrogen balance, phenolic chemistry and resistance to herbivory of two freshwater macrophytes. Oecologia 137:31-41.

Curran, P.J., J.L. Dungan, and H.L. Gholz. 1990. Exploring the relationship between

reflectance red edge and chlorophyll content in slash pine. Tree Physiol. 7:33-48. Day, T.A. 1993. Relating UV-B radiation screening effectiveness of foliage to absorbing-

compound concentration and anatomical characteristics in a diverse group of plants. Oecologia 95:542-550.

de Sá, M.C. and D.B. Rodriguez-Amaya. 2003. Carotenoid composition of cooked green

vegetables from restaurants. Food Chem. 83:595-600. Do, C.B., and F. Cormier. 1991. Accumulation of peonidin 3-glucoside enhanced by

osmotic stress in grape (Vitis vinifera L.) cell suspension. Plant Cell Tiss. Organ Cult. 24:49-54.

Duval, B., K. Shetty, and W.H. Thomas. 1999. Phenolic compounds and antioxidant

properties in the snow alga Chlamydomonas nivalis after exposure to UV light. J. App. Phycol. 11:559-566.

Ferruzzi, M.G., L.C. Sander, C.L. Rock, and S.J. Schwartz. 1998. Carotenoid

determination in biological microsamples using liquid chromatography with a coulometric electrochemical array detector. Anal. Biochem. 256:74-81.

Friso, G., R. Barbato, G.M. Giacometti, and J. Barber. 1994. Degradation of D2 protein

due to UV-B irradiation of the reaction center of photosystem II. FEES Lett. 339: 217-221.

Fu, J., and B. Huang. 2004. Leaf characteristics associated with drought resistance in tall

fescue cultivars. Acta Hortic. 661:233-239.

144

Gao, W., Y. Zheng, J.R. Slusser, and G.M. Heisler. 2003. Impact of enhanced ultraviolet- B irradiance on cotton growth, development, yield, and qualities under field conditions. Agric. For. Meteorol. 120:241-248.

Gitelson, A.A., A. Vina, S.B. Verma, D.C. Rundquist, T.J. Arkebauer, G. Keydan, B.

Leavitt, V. Ciganda, G.G. Burba, and A.E. Suyker. 2006. Relationship between gross primary production and chlorophyll content in crops: Implications for the synoptic monitoring of vegetation productivity. J. Geophys. Res. 111: D08S11 Available at http://dx.doi.org/10.1029/2005JD006017 (Verified 30 Jun. 2012).

Gitz, D., D.L. Liu, and J.W. McClure. 1998. Phenolic metabolism, growth, and UV-B

tolerance in phenylalanine ammonia-lyase inhibited red cabbage seedlings. Photochemistry 49:377-386.

Giusti, M.M., L.E. Rodríguez-Saona, D. Griffin, and R.E. Wrolstad. 1999. Electrospray

and tandem mass spectroscopy as tools for anthocyanin characterization. J. Agric. Food Chem. 47:4657-4664.

Gould, K.S.2004. Nature's swiss army knife: The diverse protective roles of anthocyanins

in leaves. J Biomed. Biotechnol. 5:314-320. Greenburg, B.M., V. Gaba, A.K. Mattoo, and M. Edelman. 1989. Degradation of the 32-

KDa Photosystem II reaction center protein in UV, visible and far red light occurs through a common 23.5 KDa intermediate. Z Naturforsch. C:Biosci. 44:450-452.

He, Y-Y. D-P. Häder. 2002. UV-B-induced formation of reactive oxygen species and

oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine. J. Photochem. Photobiol. B. 66: 115-124.

Hideg, É. and I. Vas. 1996. UV-B induced free radical production in plant leaves and

isolated thylakoid membranes. Plant Sci. 115:251-260. Jansen, M.A.K, B. Depka, A. Trebst, and M. Edelman. 1993. Engagement of specific

sites in the plastoquinone niche regulates degradation of the D1 protein in Photosystem II. J. Biol. Chem. 268:21246-21252.

145

Karageorgou, P. and Y. Manetas. 2006. The importance of being red when young: anthocyanins and the protection of young leaves of Quercus coccifera from insect herbivory and excess light. Tree Physiol. 26:613-621.

Kimura, M., D.B. Rodriguez-Amaya, and H.T. Godoy. 1990. Assessment of the

saponification step in the quantitative determination of carotenoids and provitamins Ann. Food Chem. 35:187-195.

Kovács, E., and Á. Keresztes. 2002. Effect of gamma and UV-B/C radiation on plant

cells. Micron 33:199-210. Krizek, D.T., R.M. Mirecki, and S.J. Britz. 1997. Inhibitory effects of ambient levels of

solar UV-A and UV-B radiation on growth of cucumber. Physiol. Plant. 100:886-893.

Landry, L.G., C.C. Chapple, and R.L. Last. 1995. Arabidopsis mutants lacking phenolic

sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant Physiol. 109:1159-1166.

Lin, C., M. Ahmad, and A.R. Cashmore. 1996. Arabidopsis cryptochrome 1 is a soluble

protein mediating blue light-dependent regulation of plant growth and development. Plant J. 5:893-902.

Lois, R. 1994. Accumulation of UV-absorbing flavonoids induced by in Arabidopsis

thaliana L. I. Mechanisms of UV - resistance in Arabidopsis. Planta 194:498- 503.

Mackerness, S.A-H. 2000. Plant responses to ultraviolet-B (UV-B: 280–320 nm) stress:

What are the key regulators? Plant Growth. Reg. 32:27-39. Mathews-Roth, M.M. 1986. Carotenoids quench evolution of excited species in

epidermis exposed to UV-B (290-320 nm) light. Photochem. Photobiol. 43:91–93. Maxwell, K., and G.N. Johnson. 2000. Chlorophyll fluorescence – a practical guide. J.

Exp. Bot. 51:659-668.

146

McDonald, S., P.D. Prenzler, M. Autolovich, and K. Robards. 2001. Phenolic content and antioxidant activity of olive extracts. Food Chem. 73:73-84.

McElroy, J.S., D.A. Kopsell, J.C. Sorochan, and C.E. Sams. 2006. Response of creeping

bentgrass carotenoid composition to high and low irradiance. Crop Sci. 46:2606-2612.

Milesi, C., S.W. Running, C.D. Elvidge, J.B. Dietz, and B.T. Tuttle. 2005. Mapping and

modeling the biogeochemical cycling of turf grasses in the United States. Environ. Manage. 36:426-438.

Moran, R. 1982. Formulae for determination of chlorophyllous pigments extracted with

N,N-dimethylformamide. Plant Physiol. 69:1376-381. Mori, M., K. Yoshida, Y. Ishigaki, T. Matsunaga, O. Nikaido, K. Kameda, and T. Kondo.

2005. UV-B protective effect of a polyacylated anthocyanin, HBA, in flower petals of the blue morning glory, Ipomoea tricolor cv. Heavenly Blue. Bioorg. Med. Chem. 13:2015-2020.

Moros, E.E., D. Dsrnoko, M. Cheryan, E.G. Perkins, and J. Jerrell. 2002. Analysis of

xantophylls in corn by HPLC. J. Agric. Food Chem. 50:5787-5790. Pourmorad, F., S.J. Hosseinimehr, and N. Shahabimajd. 2006. Antioxidant activity,

phenol and flavonoid contents of some selected Iranian medicinal plants. Afric. J. Biotechnol. 5:1142-1145.

Rosario, R., G.J. Mark, J.A. Parrish, and M.C. Mihm Jr. 1979. Histological changes

produced in skin by equally erythemogenic doses of UV-A, UV-B, UV-C and UV-A with psoralens. Brit. J. Derm. 101:299-308.

Sancar, A. and G.B. Sancar. 1988. DNA repair enzymes. Ann. Rev. Biochem. 57:29-67. Sarkar, D., P.C. Bhowmik, Y.I. Kwon, and K. Shetty. 2011. The role of proline-

associated pentose phosphate pathway in cool-season turfgrasses after UV-B exposure. Environ. Exp. Bot. 70:251-258.

147

Smith, H. 1994. Sensing the light environment: the functions of the phytochrome family. p. 377-416. In J.R.E. Kendrick and G.H Kronenberg (ed.) Photomorphogenesis in Plants. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Strid, A., W.S. Chow, and J.M. Anderson. 1990. Effects of supplementary ultraviolet – B

radiation on photosynthesis in Pisium sativum. Biophys. Biochim. Acta. 1020:260-268.

Taalas, P., J. Kaurola, A. Kylling, D. Shindell, R. Sausen, M. Dameris, V. Grewe, J.

Herman, J. Damski, and B. Steil. 2000. The impact of greenhouse gases and halogenated species on future solar UV radiation doses. Geophys. Res. Lett. 27:1127-1130.

Takahashi, S. and N. Murata. 2008. How do environmental stresses accelerate

photoinhibition? Trends Plant Sci. 13:178-182. Teramura, A.H., R. Hilton Biggs, and S. Kossuth. 1980. Effects of ultraviolet-B

irradiances on Soybean II. Interaction between ultraviolet-B and photosynthetically active radiation on net photosynthesis , dark respiration, and transpiration. Plant Physiol. 65:483-488

Teramura, A.H., and J.H. Sullivan. 1994. Effects of UV-B radiation on photosynthesis

and growth of terrestrial plants. Photosynth. Res. 39:463-473. Tevini, M., M.W. Iwanzik, and U. Thoma. 1981. Some effects of enhanced UV-B

irradiation on the growth and composition of plants. Planta 153:388-394. Tyystjärvi, E. 2008. Photoinhibition of photosystem II and photodamage of the oxygen

evolving manganese cluster. Coordination Chemistry Reviews. The Role of Manganese in Photosystem II. 252:361-376.

Vass, I., L. Sass, C. Spetea, A. Bakou, D.F. Ghanotakis, and V. Petroulea. 1996. UV-B-

induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence. Impairment of donor and acceptor side components. Biochem. J. 35:8964-8973.

148

Wherley, B.G., D.S. Gardner, and J.D. Metzger. 2005. Tall fescue photomorphogenesis as influenced by changes in the spectral composition and light intensity. Crop Sci. 45:562-568.

White, A.L., and L.S. Jahnke. 2002. Contrasting effects of UV-A and UV-B on

photosynthesis and photoprotection of β-carotene in two Dunaliella spp. Plant Cell Physiol. 43:877-884.

Woodall, G.S., and G.R. Stewart. 1998. Do anthocyanins play a role in UV protection of

the red juvenile leaves of Syzygium? J. Exp. Bot. 49:1447-1450. Yin, Z-H., S. Neimanis, U. Wagner, and U. Hebe. 1990. Light-dependent pH changes in

leaves of C3 plants I. Recording pH changes in various cellular compartments by fluorescent probes. Planta 182:244-252.

Zhang, X., E.H. Ervin, and R.E. Schmidt. 2005. The role of leaf pigment and antioxidant

levels in UV-B resistance of dark- and light-green Kentucky bluegrass cultivars. J. Am Soc. Hortic. Sci. 130:836-841.

Zhang, X., and E.H. Ervin. 2009. Physiological assessment of cool-season turfgrasses

under ultraviolet-B stress. HortScience 44:1785-1789. Ziska, L.H., A.H. Teramura, J.H. Sullivan, and A. McCoy. 1993. Influence of ultraviolet-

B (UV-B) radiation on photo-synthetic and growth characteristics in field-grown cassava (Manihot esculentum Crantz). Plt. Cell Environ. 16:73-79.

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CHAPTER 6

OVERALL CONCLUSIONS ON UV-B INFLUENCE ON TURFGRASS PIGMENTS AND GROWTH

ABSTRACT

The studies conducted for this project have yielded a range of insights as to the

effects of UV-B on different cool season turfgrasses. In looking at growth and

germination there is an applied element to the turfgrass knowledge which adds to the

expanding base of information in place. Investigating the changes in pigmentation and

developing new information and areas of research has created potential for new turfgrass

cultivar breeding and also offers insight into physiological changes that previously were

undocumented in turfgrass. Overall conclusions and ideas for future work are offered.

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DISCUSSION

The influence of UV-B light from these studies on turfgrass is varied. From the

initial growth and germination stage to maturity and canopy effects, there were some

benefits and some negative impacts. The use of UV to enhance Kentucky bluegrass seed

germination offers the potential to increase chances of successful establishment in a

broader range of environmental conditions. Further studies, including field research,

should be conducted in this area as this was the first trial using UV light and Kentucky

bluegrass seed. There were also some issues with both the chambers and the lighting.

Seed age seemed to reduce the effectiveness of UV light in seed germination overriding

any benefit. The exposure of Kentucky bluegrass seedlings to UV did not seem to

damage seed coats prior to germination and further work on long term exposure of

Kentucky bluegrass seedlings to UV is warranted. Enzymatic responses and breakdown

of starch reserves undoubtedly will paint a clearer picture for the method’s potential use.

Investigating what affects turfgrass color raised some interesting questions.

Previous research suggests that adding nitrogen darkens turfgrass. The pigment

correlation or lack ofit in cultivars which were not forced by different rates of nitrogen

applications was somewhat surprising as plants were not chlorotic and seemed dark green

in the trial. Nitrogen was supplied at a single rate and thus this would explain the green

color on its own. The data analysis indicated that the hand-held system which depended

on light reflection failed to be influenced by pigment content and did not correlate to

human visual quality rankings. This is somewhat of a concern as handheld systems are

becoming prevalent and may mean that a whole new ranking database will need to be

151

built up if they are going to become standard. There was a relatively strong relationship

found between both systems and leaf tissue nitrogen content, thus handheld systems

measuring nitrogen might be more appropriate. The extract analysis undoubtedly offered

more information and links to pigment changes; however its cost and time delay is

problematic. Other pigments could also play a role in color which we did not account for

of course. Pigments such as carotenoids absorb light and may completely reduce

reflection of light in the blue region. This would account for the fact that the systems

could not separate out green colors. Trained evaluators, using the long standard NTEP +9

scale, may still be the most consistent and precise method for color rankings.

Increasing UV-B light in this situation on mature stands of cool season turfgrasses

produced some interesting and exciting responses. Characterizing anthocyanins for the

first time in turfgrass is a mile stone and offers a whole new area of research in turfgrass.

Potential lies for pigments in a range of areas and if turfgrasses can be controlled in a

way that naturally enhances pigment production, then a potential for improved stress

tolerance exists. The result here was not overly consistent; however, by varying light

intensity and possibly temperature then opportunity exists for quantification and greater

characterization of the pigments. The perennial habit of turf also offers a possibility to be

harvested with the aim of using the pigments possibly as natural food colorants or light

absorbing dyes. The characterization of the impact of UV-B on specific carotenoid

content is also a first in turfgrass management and shows how important these pigments

are in grass. Reductions in carotenoid content and the publication of chromatograms are a

first within the turfgrass research area on carotenoids. As herbicide modes of action

152

continue to diversify into bleaching and photoinhibition due to damage to photosynthetic

apparatus, knowledge of these pigments is required within grasses to offer potential for

selective control.

The turfgrass decline in both the UV-B and control chambers is of interest. In

summer periods PAR light intensity is far higher than was found in these studies and so

turfgrass undergoes a daily stress of photo-inhibition. This low light however is a

problem which occurs when using growth chambers, depending on the grass, turfgrasses

may still be susceptible to photoinhibition in chambers. The photoinhibition can vary

between rapidly repaired dynamic photoinhibition to chronic photoinhibition leaving

turfgrass with a reduced capacity for optimal productivity. This is particularly true for C3

turfgrasses, and during the period of the year when opportunities for carbon sequestration

are greatest due to higher light levels. This undoubtedly plays into a loss of quality during

stressful periods of the year. It also may reduce turfgrass plant’s competitive ability with

weedy grasses and further work is currently ongoing looking into this acclimation during

seasonal changes in light.

The difference between the quality of grasses that have a shiny leaf surface and

thicker leaves compared to the thin-leaved bentgrasses is also of interest. Perennial

ryegrass and tall fescue which are considered to have shiny leaf surfaces were not as

affected as the bentgrasses and so reflective characteristics of the leaf surface may be

important. Limited attention has been paid to turfgrass leaf epidermal content changes in

high light conditions. This may become more of an interest going forward as new

herbicides aim for selectivity with greater potential for damage to target species due to an

153

ability to penetrate leaf surfaces. It is also of concern for herbicide use in summer periods

where potential damage to non-target species can occur due to changes in epidermal

surfaces. Reducing the impact of higher UV-B levels becomes more important from a

plant health standpoint with the likely increase in levels penetrating the ozone in the next

ten years (2010-2020).

154

APPENDIX A: HANDHELD REFELCTANCE MEASUREMENTS OF TURFGRASS TISSUE

155

156

APPENDIX B: COLOR OF LEAVES OF CREEPING BENTGRASS (Agrostis stolonifera L.) TISUE AFTER EXPOSURE TO UV-B LIGHT

157

158

APPENDIX C: CHROMATOGRAM CAROTENOID STANDARDS

159

# Standard Time (min) Area Area % 1 Violaxanthin 6.5 78.9 2.6 2 Antheraxanthin 8.3 1482.6 50.4 3 Lutein 10.0 510.6 17.3 4 Zeaxanthin 11.2 267.2 9.1 5 Β-carotene 23.0 603.6 20.5

Time (min)

160

APPENDIX D: CHROMATOGRAM CHOKEBERRY ARONIA

161

162

APPENDIX E: CHLOROPHYLL A CONTENT AFTER D7 UV TREATMENT CREEPING BENTGRASS ‘L-93’

163

0

100

200

300

400

0 2 4 6 8

Chl

orop

hyll

A c

onte

nt (µ

g m

-2)

Day

Chlorophyll A Creeping bentgrass 'Penncross'

UVCTRL

164

APPENDIX F: CHROMATOGRAM BENTGRASS CAROTENOIDS

165

# Time (min) Area Area% Zeaxanthin 10.4 43.1 3.2 2 17.5 72.4 5.4 3 19.5 31.1 2.3 4 20.2 50.3 3.8 β-carotene 22.5 891.4 66.7 6 23.6 168.6 12.6

Time (min)

166

APPENDIX G: TALL FESCUE CAROTENOID CHROMATOGRAM

167

# Time (min) Area Area% Zeaxanthin 10.5 54.6 1.6 2 17.5 32.9 0.8 3 19.4 66.9 2.0 4 20.0 143.3 4.2 β-carotene 22.53 2686.3 80.4 6 23.3 339.3 10.16

Time (min)

168

COMPLETE REFERENCE Ahmed, J., U.S. Shivhare, and G.D. Singh. 2000. Chlorophyll and color of green chili

puree as affected by mesh size and temperature. Int. J. Food Prop. 3:305-315. Ahmed, J., U.S. Shivhare, and G.S.V. Raghavan. 2004. Thermal degradation kinetics of

anthocyanin and visual colour of plum puree. Euro. Food Res. Tech. 218:525-528 Alba, R., M.M. Cordonnier-Pratt, and L.H. Pratt. 2000. Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato.

Plant Physiol. 123:363-370. American Society for Testing and Materials. 1989. Standard test method for specifying color by the munsell system. ASTM standard D1535–89. ASTM, Philadelphia,

PA. Anderson, J.M., and E.-M. Aro. 1994. Grana stacking and protection of Photosystem II in thylakoid membranes of higher plant leaves under sustained high irradiance: Ann.

Hypoth. Photosynth. Res. 41:315-326. Ang, L.-H., S. Chattopadhyay, N. Wei, T. Oyama, K. Okada, A. Batschauer, and X.-W

Deng. 1998. Molecular interaction between COP1 and HY5 defines a regulatory switch for light control of Arabidopsis development. Mol. Cell. 1:213–222.

Anonymous, 1997. Flavonoids (isoflavonoids and neoflavonoids). McNaught A.D. and

A.Wilkinson (ed.) IUPAC Compendium of Chemical Terminology, 2nd ed. Available at http://old.iupac.org/goldbook/F02424.pdf (verified 28 Jun. 2012).

Aro, E-M., I. Virgin, and B. Andersson. 1993. Photoinhibition of photosystem II. Inactivation, protein damage and turnover. Biochim. Biophys. Acta. 1143:113-34.

169

Babu, T.S., M.A.K. Jansen, B.M. Greenberg, V. Gaba, S. Malkin, A.K. Mattoo, and M. Edelman. 1999. Amplified degradation of photosystem II D1 and D2 proteins

under a mixture of photosynthetically active radiation and UV-B radiation: Dependence on redox status of photosystem II. Photochem. Photobio. 69:553-559.

Bacci, L., D. Grifoni, F. Sabatini, and G. Zipoli. 1999. UV-B radiation causes early ripening and reduction in size of fruits in two lines of tomato (Lycopersicon

esculentum Mill.). Glob. Change Bio. 5:635-646. Bagegni, A.M., D.A. Sleper, H.D. Kerr, and J.S. Morris. 1990. Viability of Acremonium coenophialum in tall fescue seed after ionizing radiation treatments. Crop Sci.

30:1272-1275. Ballare, C.L., A.L. Scopel, A.E. Stapleton, and M.J. Yanovsky. 1996. Solar ultraviolet-B radiation affects seedling emergence, DNA integrity, plant morphology, growth

rate, and attractiveness to herbivore insects in Datura ferox. Plant Physiol. 112:161-170.

Barnes, P.W., S.D. Flint, and M.M. Caldwell. 1990. Morphological responses of crop and weed species of different growth forms to ultraviolet-B radiation. American J. of

Bot. 77:1354-1360. Bartley, G.E., and P.A. Scolnik. 1995. Plant carotenoids: Pigments for photoprotection, visual attraction and human health. Plant Cell. 7:1027-1038. Bausch, W.C., and H.R. Duke. 1996. Remote sensing of plant nitrogen status in corn. Trans. ASAE. 39: 1869-1875. Beam, J.B., W.L. Barker, and S.D. Askew. 2006. Selective creeping bentgrass (Agrostis stolonifera L.) control in cool season turfgrass. Weed Tech. 20:3430-344. Beggs, C.J., and E. Wellmann. 1985. Analysis of light-controlled anthocyanin formation in coleoptiles of Zea mays L.: The role of UV-B, blue, red and far-red light.

Photochem. Photobio. 41:481-486.

170

Bell, G.E., and T.K. Danneberger. 1999. Temporal shade on creeping bentgrass turf. Crop Sci. 39:1142-1146. Bell, G.E., T.K. Danneberger, and M.J. McMahon. 2000. Spectral irradiance available for growth in sun and shade. Crop Sci. 40:189-195. Bennett, R.N., and R.M. Wallsgrove. 2006. Secondary metabolites in plant defense mechanisms. New Phytol. 127:617-633. Berg, H. 2008. Johann Wilhelm Ritter – The founder of scientific electrochemistry. Rev. Polarography. 54:99-103. Bilger, W., M. Veit, L. Schreiber, and U. Schreiber. 1997. Measurement of leaf epidermal transmittance of UV radiation by chlorophyll fluorescence. Physiolog.

Planta. 101:754-763. Björn, L.O., T.V. Callaghan, C. Gehrke, U. Johanson, M. Sonesson, and D. Gwynn- Jones. 1998. The problem of ozone depletion in northern Europe. Ambio 27:275-

279. Blumenthaler, M., and W. Ambach. 1990. Indication of increasing solar ultraviolet-B radiation flux in Alpine regions. Science 248:206-208. Bornman, J.F., and T.C. Vogelmann. 1988. Penetration of blue and UV radiation measured by fiber optics in spruce and fir needles. Physiolog. Planta. 72:699-705. Bornman, J.F. 1989. Target sites of UV-B radiation in photosynthesis of higher plants. J. Photochem. Photobiol. 4:145-158. Bornman, J.F., and T.C. Vogelmann. 1991. Effect of UV-B radiation on leaf optical properties measured with fiber optics. J. Exp. Bot. 42:547-554.

171

Bornman, J.F., and A.H. Teramura. 1993. Effects of ultraviolet-B radiation on terrestrial plants. p. 427-471. In A.R. Young et al. (ed.) Environmental UV Photobiology.

Plenum Press, New York. Brandle, J.R., W.F. Campbell, W.B. Sisson, and M.M. Caldwell. 1977. Net photosynthesis, electron transport capacity and ultrastructure of Pisium sativum L.

exposed to ultraviolet-B radiation. Plant Physiol. 60:165-169. Briscoe, A.D., and L. Chittka. 2001. The evolution of colour vision in insects. Ann. Rev.

Entomol. 46:471-510 Britt, A.B. 1996. DNA damage and repair in plants. Annu. Rev. Plant Physiol. Plant Mol.

Biol. 47.75-100. Britton, G., S. Liaaen-Jensen, and H. Pfander. 2004. Carotenoids handbook. Birkhäuser Verlag, Boston, MA. Brouillard, R. 1988. Flavonoids and flower color. P. 525–538. In J.B. Harborne (ed.) The flavonoids: Advances in research since 1980. Chapman Hall, London, United

Kingdom. Brown, P.K., and G. Wald. 1964. Visual pigments in single rods and cones of the human retina. Science 1964. 144:45-52. Buer, C.S., and G.K. Muday. 2004. The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to

gravity and light. Plant Cell. 16:1191-1205. Burger, J., and G. E. Edwards. 1996. Photosynthetic efficiency, and photodamage by UV and visible radiation, in red versus green leaf coleus varieties. Plant Cell Physiol.

37:395-399. Byrne, M.C., C.J. Nelson, and D.D. Randall. 1981. Ploidy effects on anatomy and gas exchange of tall fescue leaves. Plant Physiol. 68:891-893.

172

Caldwell, M.M. 1971. Solar ultraviolet radiation and the growth and development of higher plants. p. 131-174. In: A.C. Giese (ed.) Photophysiology. Academic Press,

New York. Caldwell, M.M. 1979. Plant life and ultraviolet radiation: Some perspective in the history of the earth's UV climate. BioScience 29:520-525. Caldwell, M.M., R. Robrecht, and W.D. Billings. 1980. A steep latitudinal gradient of solar ultraviolet-B radiation in the arctic-alpine life zone. Ecology 6:600-611. Caldwell, M.M., J.H. Richards, D.A. Johnson, R.S. Nowak, and R. S. Dzurec. 1981. Coping with herbivory: Photosynthetic capacity and resource allocation in two

semiarid Agropyron bunchgrasses. Oecologia 50:14-24. Caldwell, M.M., A.H. Teramura, and M. Tevini. 1989. The changing solar ultraviolet climate and the ecological consequences for higher plants. Trends Ecol. Evol.

4:363-367. Cameron, R.J. 1970. Light intensity and the growth of Eucalyptus seedlings: II. The effect of cuticular waxes on light absorption in leaves of Eucalyptus species. Aus.

J. Bot. 18:275-284. Carletti, P., A. Masi, A. Wonisch, D. Grill, M. Tausz, and M. Ferretti. 2003. Changes in antioxidant and pigment pool dimensions in UV-B irradiation maize seedlings.

Environ. Exp. Bot. 50:149-157. Carrow, R.N., D.V. Waddington, and P.E. Reike. 2001. Turfgrass soil fertility and

chemical problems. John Wiley & Sons, Inc., Hoboken, NJ. Casal, J.J., V.A. Deregibus, and R.A. Sánchez. 1985. Variations in tiller dynamics and morphology in Lolium multiflorum Lam. vegetative and reproductive plants as

affected by differences in red/far-red irradiation. Ann. Bot. 56:553-559. Castle, E.S. 1935. Photic excitation and phototropism in single plant cells. Cold Spring Harbor Symp. Quant. Biol. 3:224-229.

173

Cavalcante, M.J.B., and J.J. Muchovej. 1993. Microwave irradiation of seeds and selected fungal spores. Seed Sci. Technol. 21:247-253. Cen, Y.-P., and J.F. Bornman. 1990. The response of bean plants to UV-B radiation

under different irradiances of background visible light. J. Exp. Bot. 41:1489-1495. Chalker-Scott, L. 1999. Environmental significance of anthocyanins in plant stress responses. Photochem. Photobiol. 70:1-9. Chandra, A., J. Rana, and Y. Li. 2001. Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by

HPLC and HPLC-MS. J. Agric. Food Chem. 49:3515-3521. Chen, C. 2002. Robust regression and outlier detection with the ROBUSTREG procedure. Paper 265-27. SAS Institute Inc., Cary, NC, Proceedings SUGI 27,

Orlando FL Apr 14–17. Available at http://www2.sas.com/proceedings/sugi27/p265-27.pdf (verified, 28 June, 2012). Chien, J.C., and I.M. Sussex. 1996. Differential regulation of trichome formation on the adaxial and abaxial leaf surfaces by gibberellins and photoperiod in Arabidopsis

thaliana (L.) Heynh. Plant Physiol. 111:1321-1328. Clark, J.B, and G.R. Lister. 1975. Photosynthetic action spectra of trees: II. The relationship of cuticle structure to the visible and ultraviolet spectral properties of

needles from four coniferous species. Plant Physiol. 55:407-413. Correia, C.M., M.S. Torres-Pereira, and J.M.G. Torres-Pereira. 1999. Growth, photosynthesis and UV-B absorbing compounds of Portuguese Barbela wheat

exposed to ultraviolet-B radiation. Environ. Pollut. 104:383-388. Cortell, J.M., and J.A. Kennedy. 2006. Effect of shading on accumulation of flavonoid compounds in (Vitis vinifera L.) pinot noir fruit and extraction in a model system.

J. Agric. Food Chem. 49:3515-3521.

174

Costa, H., S.M. Gallego, and M.L. Tomaro. 2002. Effect of UV-B radiation on antioxidant defense system in sunflower cotyledons. Plant Sci. 162:939-945. Cronin, G., and D.M. Lodge. 2003. Effects of light and nutrient availability on the growth allocation, carbon/nitrogen balance, phenolic chemistry and resistance to

herbivory of two freshwater macrophytes. Oecologia 137:31-41. Croteau, R., T.M. Kutchan, and N.G. Lewis. 2000. Natural products (secondary metabolites). p. 1250-1318. In B. Buchanan et al. (ed.) Biochem. Molec. Biol.

Plants Am. Soc. Plant Physiol. Cuadra, P., J.B. Harborne, and P.G. Waterman. 1997. Increases in surface flavonols and photosynthetic pigments in Gnaphalium luteo-album in response to UV-B

radiation. Phytochemistry 45:1377-1383. Cunningham, F.X., and E. Gantt. 2002. Genes and enzymes of carotenoid biosynthesis in plants. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49:577-583. Curran, P.J., J.L. Dungan, and H.L. Gholz. 1990. Exploring the relationship between reflectance red edge and chlorophyll content in slash pine. Tree Physiol. 7:33-48. Cuttriss, A., and B. Pogson. 2004. Carotenoids. In: Plant pigments and their manipulation. Ann. Plant Rev. 14:57-91. Dai, Q., V.P. Coronel, B.S. Vergara, P.W. Barnes, and A.T. Quintos. 1992. Ultraviolet-B radiation effects on growth and physiology of four rice cultivars. Crop Sci.

32:1269-1274. Danneberger, T.K., M.B. McDonald Jr., C.A. Geron, and P. Kumari. 1992. Rate of germination and seedling growth of perennial ryegrass seed following

osmoconditioning. HortScience 27:28-30. Day, T.A., T.C. Vogelmann, and E.H. DeLucia. 1992. Are some plant life forms more effective than others in screening out ultraviolet-B radiation? Oecologia 92:513-

519.

175

Day, T.A. 1993. Relating UV-B radiation screening effectiveness of foliage to absorbing- compound concentration and anatomical characteristics in a diverse group of

plants. Oecologia 95:542-550. Deckmyn, G., and I. Impens. 1999. Seasonal responses of six Poaceae to differential levels of solar UV-B radiation. Environ. Exp. Bot. 41:177-184. Deckmyn, G., and I. Impens. 1998. UV-B and PAR in a grass (Lolium perenne L.) canopy. Plant Ecol. 137:13-19. Deckmyn, G., and I. Impens. 1998. Effects of solar UV-B irradiation on vegetative and generative growth of Bromus catharticus. Env. Exp. Bot. 40:179-185. de Sá, M.C. and D.B. Rodriguez-Amaya. 2003. Carotenoid composition of cooked green vegetables from restaurants. Food Chem. 83:595-600. Dicke, M., A.A. Agrawal, and J. Bruin. 2003. Plants talk, but are they deaf? Trends Plant Sci. 8:403-405. Do, C.B., and F. Cormier. 1991. Accumulation of peonidin 3-glucoside enhanced by osmotic stress in grape (Vitis vinifera L.) cell suspension. Plant Cell Tiss. Organ

Cult. 24:49-54. Dong, Y. hu, D. Mitra, A. Kootstra, C. Lister, and J. Lancaster. 1995. Postharvest stimulation of skin color in royal gala apple. J. Am. Soc. Hortic Sci. 120:95-100. Donner, H., L. Gao, and G. Mazza. 1997. Separation and characterization of simple and malonylated anthocyanins in red onions, Allium cepa L. Food Res. Int. 30:637-

643. Duval, B., K. Shetty, and W.H. Thomas. 1999. Phenolic compounds and antioxidant properties in the snow alga Chlamydomonas nivalis after exposure to UV light. J.

Appl. Phycol. 11:559-566.

176

Ervin, E.H., X. Zhang, and J.H. Fike. 2004a. Ultravolet-B radiation damage on Kentucky bluegrass I: antioxidant and colorant effects. HortScience 39:1465-1470. Ervin, E.H., X. Zhang, and J.H. Fike. 2004c. Ultraviolet-B radiation damage on Kentucky bluegrass. III. Cultivar effects. HortScience 39:1475-1477. Escadafal, R., M-C. Girard, and D. Courault. 1989. Munsell soil color and soil reflectance in the visible spectral bands of landsat MSS and TM data. Remote

Sens. Environ. 27:37-46. Ferruzzi, M.G., L.C. Sander, C.L. Rock, and S.J. Schwartz. 1998. Carotenoid determination in biological microsamples using liquid chromatography with a

coulometric electrochemical array detector. Anal. Biochem. 256:74-81. Field, T.S., D.W. Lee, and N.M. Holbrook. 2001. Why leaves turn red in autumn. The role of anthocyanins in senescing leaves of red osier dogwood. Plant Physiol.

127:566-574. Filella, I., and J. Penuelas. 1994. The red edge and shape as indicators of plant chlorophyll, biomass and hydric status. Int. J. Remote Sensing. 15:1459-1470. Fiscus E.L., and F.L. Booker. 1995. Is increased UV-B a threat to crop photosynthesis and productivity? Photosynth. Res. 43:81-92. Flint, S.D., and M.M. Caldwell. 1984. Partial inhibition of in vitro pollen germination by simulated solar ultraviolet‑B radiation. Ecology. 65:792-795. Flint, S.D., and M.M. Caldwell. 2003. A biological spectral weighting function for ozone depletion research with higher plants. Physiol. Planta. 117:137-144. Floss, H.G. 1997. Natural products derived from unusual variants of the shikimate pathway. Natural Prod. Rep. 14:433-452.

177

Foti, M., M. Piattelli, V. Amico, and G. Ruberto. 1994. Antioxidant activity of phenolic meroditerpenoids from marine algae. J. Photochem. Photobiol. 26:159-164. Foyer, C.H., M. Lelandais, and K.J. Kunert. 1994. Photooxidative stress in plants. Physiol. planta. 92:696-717. Foyer, C.H., P. Descourvieres, and K.J. Kunert. 1994. Protection against oxygen radicals: an important defence mechanism studied in transgenic plants. Plant Cell and Env.

17:507-523. Frank, K.W., R.E. Gaussoin, T.P. Riordan, R.C. Shearman, J.D. Fry, E.D. Miltner, and P.G. Johnson. 2004. Nitrogen rate and mowing height effects on turf-type

buffalograss. Crop Sci. 44:1615–1621. Frankhauser, S., and J. Chory. 1997. Light control of plant development. Ann. Rev. Cell Dev. Biol. 13:203-29. Fraser, P.D., and P.M. Bramley. 2004. The biosynthesis and nutritional uses of carotenoids. Prog. Lipid Res. 43:228-265. Frederick, J.E., H.E. Snell, and E.K. Haywood. Solar ultraviolet radiation at the earths surface. Photochem. Photobio. 50:443-450. Friso, G., R. Barbato, G.M. Giacometti, and J. Barber. 1994. Degradation of D2 protein due to UV-B irradiation of the reaction center of photosystem II. FEES Lett. 339:

217-221. Fu, J., and B. Huang. 2004. Leaf characteristics associated with drought resistance in tall fescue cultivars. Acta Hortic. 661:233-239. Galston, A.W., and R.S. Baker. 1949. Studies on the physiology of light action II. The photodynamic action of riboflavin. Am. J. Bot. 36:773-780.

178

Gao, W., Y. Zheng, J.R. Slusser, and G.M. Heisler. 2003. Impact of enhanced ultraviolet- B irradiance on cotton growth, development, yield, and qualities under field

conditions. Agric. For. Meteorol. 120:241-248. Garcı́a-Viguera, C., and P. Bridle. 1999. Influence of structure on colour stability of anthocyanins and flavylium salts with ascorbic acid. Food Chem. 64:21-26. Gardner, F.P., R.B. Pearce, and R.L. Mitchell (ed.) 1985. Physiology of crop plants. Iowa State Univ. Press, Ames IA. Gausman, H.W., and R. Cardenas. 1969. Effect of leaf pubescence of Gynura aurantiaca on light reflectance. Bot. Gaz. 130:158-162. Gitelson, A.A., A. Vina, S.B. Verma, D.C. Rundquist, T.J. Arkebauer, G. Keydan, B. Leavitt, V. Ciganda, G.G. Burba, and A.E. Suyker. 2006. Relationship between

gross primary production and chlorophyll content in crops: Implications for the synoptic monitoring of vegetation productivity. J. Geophys. Res. 111: D08S11 Available at http://dx.doi.org/10.1029/2005JD006017 (Verified 30 Jun. 2012).

Gitz, D., D.L. Liu, and J.W. McClure. 1998. Phenolic metabolism, growth, and UV-B

Tolerance in phenylalanine ammonia-lyase inhibited red cabbage seedlings. Photochemistry 49:377-386.

Giusti, M.M., L.E. Rodríguez-Saona, D. Griffin, and R.E. Wrolstad. 1999. Electrospray and tandem mass spectroscopy as tools for anthocyanin characterization. J. Agric.

Food Chem. 47:4657-4664. Giusti, M.M., and R.E. Wrolstad. 2005. Characterization and measurement of anthocyanins by UV-visible spectroscopy. p. 19-32. Unit F1.2. In Wrolstad R.E.

(ed.) Handbook of food analytical chemistry—pigments, colorants, flavors, texture, and bioactive food components. John Wiley & Sons Inc., New York.

Gooding, M.J., and L.J. Gamble. 1990. Color evaluation of Poa pratensis cultivars. J. Sports Turf Res. Inst. 66:134-140.

179

Gould, K.S. 2004. Nature's swiss army knife: The diverse protective roles of anthocyanins in leaves. J. Biomed. Biotechnol. 5:314-320. Greenburg, B.M., V. Gaba, A.K. Mattoo, and M. Edelman. 1989. Degradation of the 32- KDa Photosystem II reaction center protein in UV, visible and far red light occurs

through a common 23.5 KDa intermediate. Z Naturforsch. C:Biosci. 44:450-452. Greenwood, D.J., G. LeMaire, G. Gosse, P. Cruz, A. Draycott, and J.J. Neeteson. 1990. Decline in percentage N of C3 and C4 crops with increasing plant mass. Ann. Bot.

66:425-436. Gross, J., 1991. Pigments in vegetables: chlorophylls and carotenoids. Van Nostrand Reinhold, NY. Guertal, E.A., and D.L. Evans. 2006. Nitrogen rate and mowing height effects on TifEagle bermudagrass establishment. Crop Sci. 46:1772-1778. Gwynn-Jones, D., and U. Johanson. 1996. Growth and pigment production in two subarctic grass species under four different UV-B irradiation levels. Physiol.

Planta. 97:701-707. Gwynn-Jones, D. 2001. Short-term impacts of enhanced UV-B radiation on photo- assimilate, allocation and metabolism: a possible interpretation for time-

dependent inhibition of growth. Plant Ecol. 154:67-73. Hagen, S.F., G.I.A. Borge, G.B. Bengtsson, W. Bilger, A. Berge, K. Haffner, and K.A. Solhaug. 2007. Phenolic contents and other health and sensory related properties

of apple fruit (Malus domestica Borkh., cv. Aroma): Effect of postharvest UV-B irradiation. Postharvest Bio. Technol. 45:1-10.

Hardy, A.C. 1938. History of the design of the recording spectrophotometer. J. Optic. Soc. Am. 28:360-363. Hare, P.D., and W.A., Cress. 1997. Metabolic implications of stress-induced proline accumulation in plants. Plant Growth Reg. 21:79-102.

180

Hartley, W.N. 1881. On the absorption of solar rays by atmospheric ozone. J. Chem. Soc. 39:111-128. He, Y-Y. D-P. Häder. 2002. UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of

ascorbic acid and N-acetyl-L-cysteine. J. Photochem. Photobio. B: Biology. 66: 115-124.

Heller, W., and G. Forkmann. 1988. Biosynthesis in the flavonoids: p. 319-425. In J.B. Harborne (ed.) The flavonoids: Advances in research since 1980. Chapman Hall,

London, United Kingdom. Hideg, É. and I. Vas. 1996. UV-B induced free radical production in plant leaves and isolated thylakoid membranes. Plant Sci. 115:251-260. Hofmann, R.W. 2000. Population studies of ultraviolet-B radiation responses in white clover (Trifolium repens L.). Ph.D. Thesis Massey University, N.Z.

http://muir.massey.ac.nz/bitstream/handle/10179/2248/02_whole.pdf?sequence=1 (Verified 30 Jun. 2012).

Hong, V., and R.E. Wrolstad. 1990. Use of HPLC separation/photodiode array detection for characterization of anthocyanins. J. Agric. Food Chem. 38:708-71 Huang, Z.-A., D.-A. Jiang, Y. Yang, J.-W. Sun, and S.-H. Jin. 2004. Effects of nitrogen

deficiency on gas exchange, chlorophyll fluorescence, and antioxidant enzymes in leaves of rice plants. Photosynthetica. 42:357-364.

Irani, N.G., and E. Grotewold. 2005. Light-induced morphological alteration in anthocyanin-accumulating vacuoles of maize cells. BMC Plant Bio. 5:7. Jackson, J.A., G. Fuglevand, B.A. Brown, M.J. Shaw, and G.I. Jenkins. 1995. Isolation of Arabidopsis mutants altered in the light regulation of chalcone synthase gene

expression using a transgenic screening approach. Plant J. 8:369-380.

181

Jaisingh, J., E.W.K. See-To, and K.Y. Tamin. 2006. The impact of open source software on the strategic choices of firms developing proprietary software. J. of Manage.

Info. Sys. 25:241-275. Jansen, M.A.K, B. Depka, A. Trebst, and M. Edelman. 1993. Engagement of specific

sites in the plastoquinone niche regulates degradation of the D1 protein in Photosystem II. J. Biol. Chem. 268:21246-21252.

Joliot, P., and A. Joliot. 1984. Electron transfer between the two photosystems. I. flash excitation under oxidizing conditions. Biochim. Biophys. Acta. 765:210-218. Ju, Z., Y. Duan, and Z Ju. 1999. Effects of covering the orchard floor with reflecting films on pigment accumulation and fruit coloration in `Fuji' apples. Sci. Hortic.

82:47-56. Kaiser, T., K. Emmler, B. Weisshaar, E. Schafer, and A. Batschauer. 1995. Promoter elements of the mustard CHS1 gene are sufficient for light regulation in

transgenic plants. Plant Mol. Biol. 28:219-229. Kakani, V.G., K.R. Reddy, D. Zhao, and A.R. Mohammed. 2003. Effects of ultraviolet‐B radiation on cotton (Gossypium hirsutum L.). Morph. Anat. Ann. Bot. 91:817-

826. Karageorgou, P., and Y. Manetas. 2006. The importance of being red when young: anthocyanins and the protection of young leaves of Quercus coccifera from insect

herbivory and excess light. Tree Physiol. 26:613-621. Karcher, D.E., and M.D. Richardson. 2003. Quantifying turfgrass color using digital image analysis. Crop Sci. 43:943-951. Kavanagh, T., D. O’Beirne, and T.P. Cormican. 1985. Use of the hunter colour difference meter for colour evaluation in turfgrass experiments. p. 255-261. In: F. Lemaire

(ed.) Proc. 5th Intl. Turfgrass Res. Conf., Avignon, France. 1-5 Jul.

182

Keller, W., and A.T. Bleak. 1968. Preplanting treatment to hasten germination and emergence of grass seed. J. Range Manage. 21:213-216. Kerr, J.B., and C.T. McElroy. 1993. Evidence for large upward trends of ultraviolet-B radiation linked to ozone depletion. Science 262:1032-1034. Kimura, M., D.B. Rodriguez-Amaya, and H.T. Godoy. 1990. Assessment of the saponification step in the quantitative determination of carotenoids and

provitamins A. Food Chem. 35:187-195. Kong, J.M., C. Lian-Sai, G. Ngoh-Khang, C. Tet-Fatt, and R. Brouillard. 2003. Analysis and biological activities of anthocyanins. Phytochemistry 64:923-933. Kopsell, D.A., and D.E. Kopsell. 2006. Accumulation and bioavailability of dietary carotenoids in vegetable crops. Trends Plant Sci. 11:1360-1385. Kovács, E., and Á. Keresztes. 2002. Effect of gamma and UV-B/C radiation on plant cells. Micron 33:199-210. Koyama, Y., and R. Fujii, 1999. Cis-trans carotenoids in photosynthesis: configurations, Excited–state properties and physiological functions. p.161-188. In H.A. Frank (ed) The photochemistry of carotenoids. Kluwer Publishers, Dordrecht, Netherlands. Krizek, D.T., R.M. Mirecki, and S.J. Britz. 1997. Inhibitory effects of ambient levels of solar UV-A and UV-B radiation on growth of cucumber. Phys. Planta. 100:886-

893. Krol, M., G.R. Gray, V.M. Hurry, G. Oquist, L. Malek, and N.P.A. Huner, 1995. Low Temperature stress and photoperiod effect on increased tolerance to

photoinhibition in Pinus banksia seedlings. Canadian J. Bot. 73:1119-1127. Krupa, S.V., and R.N Kickert. 1989. The Greenhouse effect: Impacts of ultraviolet-B (UV-B) radiation, carbon dioxide (CO2), and Ozone (O3) on vegetation. Environ.

Pollut. 61:263-393.

183

Kubasek, W.L., B.W. Shirley, A. McKillop, H.M. Goodman, W. Briggs, and F.M. Ausubel. 1992. Regulation of flavonoid biosynthetic genes in germinating

Arabidopsis seedlings. Plant Cell. 4:1229-1236. Lancaster, J.E., J.E. Grant, C.E. Lister, and M.C. Taylor. 1994. Skin color in apples – Influence of copigmentation and plastid pigments on shade and darkness of red

color in five genotypes. J. Am. Soc. Hortic. Sci. 119:63-69. Landry, L.G., C.C. Chapple, and R.L. Last. 1995. Arabidopsis mutants lacking phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant

Physiol. 109:1159-1166. Landschoot, P.J., and C.F. Mancino. 1997. Assessment of the minolta CR-310 chroma meter for predicting nitrogen status of Agrostis stolonifera L. Intl. Turfgrass Soc.

Res. J. 8:711-718. Landschoot, P.J., and C.F. Mancino. 2000. A comparison of visual vs. instrumental measurement of color differences in bentgrass turf. HortScience 35:914-916. Langebartels, C., M. Schraudner, W. Heller, D. Ernst, and H. Sanderman Jr. 2002. Oxidative stress and defense reactions in plants exposed to air pollutants and UV-

B radiation. p.105-137. In D. Inzé and M. Van Montagu (ed.) Oxidative Stress in Plants. Taylor and Francis New York, NY.

Larsen, S.U., and C. Andreasen. 2004. Light and heavy turfgrass seeds differ in germination percentage and mean germination thermal time. Crop Sci. 44:1710-

1720. Larsen, S.U., and B.M. Bibby. 2004. Use of germination curves to describe variation in germination characteristics in three turfgrass species. Crop Sci. 44:891-899. Lee, J., R.W. Durst, and R.E. Wrolstad. 2005. Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and

wines by the pH differential method: collaborative study. J. Ass. Analyt. Com. Int. 88:1269-1278.

184

Lemaire, G., G. Escadeillas, and E. Ringot. 2005. Evaluating concrete surfaces using an image analysis process. Cons. Build. Mat. 19:604-611. Lesfrud, M.G., D.A. Kopsell, R.M. Auge, and A.J. Both. 2006. Biomass production and pigment accumulation in kale grown under increasing photoperiods. Hortscience

41:603-606. Lesfrud, M.G., D.A. Kopsell, and D.E. Kopsell. 2007. Nitrogen levels influence biomass, elemental accumulations, and pigment concentrations in spinach. J. Plant Nut.

30:171-185. Lin, C., M. Ahmad, and A.R. Cashmore. 1996. Arabidopsis cryptochrome 1 is a soluble protein mediating blue light-dependent regulation of plant growth and

development. Plant J. 5:893-902. Lohachoompol, V., G. Srzednicki, and J. Craske. 2006. The change of total anthocyanins in blueberries and their antioxidant effect after drying and freezing. J. Biomed.

Biotechnol. 5:248-252. Lois, R. 1994. Accumulation of UV-absorbing flavonoids induced by in Arabidopsis thaliana L. I. Mechanisms of UV - resistance in Arabidopsis. Planta 194:498- 503. Lubin, D., and J.E. Frederick. 1991. The ultraviolet radiation environment of the antarctic peninsula: the roles of ozone and cloud cover. J. Appl. Meteorol. 30:478-493. Lutz, M., I. Agalidis, G. Hervo, R.J. Cogdell, and F. Reiss-Husson. 1978. On the state of

carotenoids bound to reaction centers of photosynthetic bacteria: A resonance raman study. Biochim. Biophys. Acta (BBA) – Bioenergetics. 503:287-303.

Mackerness S.A-H. 2000. Plant responses to ultraviolet-B (UV-B: 280–320 nm) stress: What are the key regulators? Plant Growth Reg. 32:27-39.

185

Madronich, S., R.L. McKenzie, L.O. Björn, M.M. Caldwell. 1998. Changes in biologically active ultraviolet radiation reaching the earth's surface. J. Photochem. Photobiol. B: Biol. 46:5-19.

Maguire, J.D., and K.M. Steen. 1971. Effects of potassium nitrate on germination and respiration of dormant and non dormant Kentucky bluegrass (Poa pratensis L.)

seed. Crop Sci. 11:48-50. Maliakal, S.K, K. McDonnell, S.A. Dudley, and J. Schmitt. 1999. Effects of red to far-red ratio and plant density on biomass allocation. Int. J. Plant Sci. 160:723-733. Mancinelli, A.L., F. Rossi, and A. Moroni. 1991. Cryptochrome, phytochrome and anthocyanin production. Plant Physiol. 96:1079-1085. Mangiafico, S.S., and K. Guillard. 2005. Turfgrass reflectance measurements, chlorophyll, and soil nitrate desorbed from anion exchange membranes. Crop Sci.

45:259-265. Manney, G.L., M.L. Santee, M. Rex, N.J. Livesey, M.C. Pitts, P. Veefkind, E.R. Nash, I. Wohltmann, R. Lehmann, L. Froidevaux, L.R. Poole, M.R. Schoeberl, D.P. , J.

Davies, V. Dorokhov, H. Gernandt, B. Johnson, R. Kivi, E. Kyrö, N. Larsen, P.F. Levelt, A. Makshtas, C.T. McElroy, H. Nakajima, M.C. Parrondo, D.W. Tarasick, P. von der Gathen, K.A. Walker, and N.S. Zinoviev. 2011. Unprecedented Arctic ozone loss in 2011. Nature 478:469-475.

Massacci, A., M. Adelaide-lannelli, F. Pietrini, and F. Loreto. 1995. The effect of growth at low temperature on photosynthetic characteristics and mechanisms of

photoprotection of maize leaves. J. Expt. Bot. 46:119-127. Mathews-Roth, M.M. 1986. Carotenoids quench evolution of excited species in epidermis exposed to UV-B (290-320 nm) light. Photochem. Photobiol. 43:91-93 Matile, P., B.M.P. Flach and B.M. Eller. 1992. Autumn leaves of Ginkgo biloba L.: optical properties, pigments, and optical brighteners. Bot. Acta. 105:13-17.

186

Maxwell, K., and G.N. Johnson. 2000. Chlorophyll fluorescence – a practical guide. J. Exp. Bot. 51:659-668. McClure, J.W. 1975. Physiology and functions of flavonoids. p. 970-1055. In. J. Harborne, and T. Mabry (ed.) The flavonoids. Chapman and Hall Ltd., United

Kingdom. McCurdy, J.D., J.S. McElroy, D.A. Kopsell, C.E. Sams, and J.C. Sorochan. 2008. Effects of mesotrione on perennial ryegrass (Lolium perenne L.) carotenoid

concentrations under varying environmental conditions. J. Ag. Food Chem. 56:9133-9139.

McDonald, S., P.D. Prenzler, M. Autolovich, and K. Robards. 2001. Phenolic content and antioxidant activity of olive extracts. Food Chem. 73:73-84. McElroy, J.S., D.A. Kopsell, J.C. Sorochan, and C.E. Sams. 2006. Response of creeping bentgrass carotenoid composition to high and low irradiance. Crop Sci. 46:2606-

2612. McEwen, C.N., R.G. McKay, and B.S. Larsen. 2005. Analysis of solids, liquids, and biological tissues using solids probe introduction at atmospheric pressure on

commercial LC/MS instruments. Anal. Chem. 77:7826-7831. McKenzie, R.L. L.O. Björn, A. Bais, and M. Ilyas. 2003. Changes in biologically

active ultraviolet radiation reaching the earth's surface. Photochem. Photobiol. Sci. 2:5-1.

McLennan, A.G. 1987. DNA damage, repair, and mutagenesis. p. 135-186. In J.A. Bryant and V.L. Dunham (ed.) DNA replication in Plants. CRC Press, Boca

Raton, FL. Mehlenbacher, S.A., and M.M. Thompson. 1991. Inheritance of a chlorophyll deficiency in hazelnut. Hortscience 26:1414-1416.

187

Mendez, M., D.G. Jones, and Y. Manetas. 1999. Enhanced UV-B radiation under field conditions increases anthocyanin and reduces the risk of photoinhibition but does

not affect growth in the carnivorous plant Pinguicula vulgaris. New Phytol. 144:275-282.

Milesi, C., S.W. Running, C.D. Elvidge, J.B. Dietz, and B.T. Tuttle. 2005. Mapping and modeling the biogeochemical cycling of turf grasses in the United States.

Environ. Manage. 36:426-438. Millington, K.R., and A.L. King. 2010. Measuring colour and photostability of small fleece wool samples. Anim. Prod. Sci. 50:589-592. Miyoshi, K., and M. Mii. 1985. Ultrasonic treatment for enhancing seed germination of terrestrial orchid Calanthe discolor in a symbiotic culture. Sci. Hortic. 35:127-

130. Mohammed, A.R., E.W. Rounds, and L. Tarpley. 2007. Response of rice (Oryza sativa L.) tillering to sub-ambient levels of ultraviolet-B radiation. J. Agron. Crop

Sci.193:324-335. Moncada, M.C., S. Moura, M. João Melo, A. Roque, C. Lodeiro, and F. Pina. 2003.

Complexation of aluminum (III) by anthocyanins and synthetic flavylium salts: A source for blue and purple color. Inorganica Chimica Acta. 356:51-61.

Moradas-Fereira, P.V., P.P. Costa, and W. Mager. 1996. The molecular defenses against reactive oxygen species in yeast. Mol. Microbiol. 19:651-658. Morales, L.O., R. Tegelberg, M. Brosché, A. Lindfors, S. Siipola, and P.J. Aphalo. 2011. Temporal variation in epidermal flavonoids due to altered solar UV radiation is

moderated by the leaf position in Betula pendula. Phys. Planta. 143:1399-3054. Moran, R. 1982. Formulae for determination of chlorophyllous pigments extracted with N,N- dimethylformamide. Plant Physiol. 69:1376-1381.

188

Moran, R., and D. Porath. 1980. Chlorophyll determination in intact tissues using N,N – dimethylformamide. Plant Physiol. 65:478-479. Mori, M., K. Yoshida, Y. Ishigaki, T. Matsunaga, O. Nikaido, K. Kameda, and T. Kondo. 2005. UV-B protective effect of a polyacylated anthocyanin, HBA, in flower

petals of the blue morning glory, Ipomoea tricolor cv. Heavenly Blue. Bioorg. Med. Chem. 13:2015-2020.

Moros, E.E., D. Dsrnoko, M. Cheryan, E.G. Perkins, and J. Jerrell. 2002. Analysis of xantophylls in corn by HPLC. J. Agric. Food Chem. 50:5787-5790. Müller, P., L. Xiao-Ping, and K.K. Niyogi. 2001. Non-photochemical quenching. A response to excess light energy. Plant Physiol. 125:1558-1566. Munsell, A.H. 1915. Atlas of the munsell color system. Wadsworth-Howland and Company, Malden, MA. Mustafa, N.R., and R.Verpoorte. 2007. Phenolic compounds in Catharanthus roseus. Phytochem. Rev. 6:243-258. Neill, S., and K.S. Gould. 1999. Optical properties of leaves in relation to anthocyanin concentration and distribution. Can J. Bot. 77:1777-1782. Nickerson, D. 1940. History of the munsell color system and its scientific application. J. Optic. Soc. Am. 30:575-586. Noble, R.E. 2002. Effects of UV-irradiation on seed germination. Sci. Total Environ. 299:173-176. Nutter, Jr., F.W., M.L. Gleason, J.H. Jenco, and N.C. Christians. 1993. Assessing the accuracy, intra-rater repeatability and inter-rate repeatability of disease

assessment systems. Phytopatholgy 83:806-812.

189

Oren-Shamir, M., and A. Levi-Nissim. 1997. UV-light effect on the leaf pigmentation of Cotinus coggygria ‘Royal Purple’. Sci. Hortic. 71:59-66. Owens, T.G., A.P. Shreve, and A.C. Atbrecht. 1992. Dynamics and mechanisms of

singlet energy transfer between carotenoids and chlorophylls: light harvesting and nonphotochemical fluorescence quenching. p. 170-186. In N. Murata (ed.) In Research in Photosynthesis 4. Kluwer, Dordrecht, Netherlands.

Pascual-Teresa, S. de., C. Santos-Buelga, and J.C. Rivas-Gonzalo. 2002. LC–MS analysis of anthocyanins from purple corn cob. J. Sci. Food and Agric. 82:1003-1006. Pattee, H.E., F.G. Giesbrecht, and C.T. Young. 1991. Comparison of peanut butter color determination by CIELAB L*, a*, b* and Hunter color-difference methods

and the relationship of roasted peanut color to roasted peanut flavor response. J. Agric. Food Chem. 39:519-523.

Pfeiffer, N.E. 1928. Anatomical study of plants grown under glasses transmitting light of various ranges of wavelength. Bot. Gaz. 85:427-436. Pfeifer, M.T. P. Koepke, and J. Reuder. 2006. Effects of altitude and aerosol on UV

radiation. J. Geophys. Res. 111. D01203. doi:10.1029/2005JD006444. Pietrini, F., and A. Massacci. 1998. Leaf anthocyanin content changes in Zea mays grown at low temperature: Significance for the relationship between quantum yield of PS

II and the apparent quantum yield of CO2 assimilation. Photosynth. Res. 58:213-219.

Pill, W.G., J.J. Frett, and I.H. Williams. 1997. Matric priming of Kentucky bluegrass and tall fescue seeds benefits seedling emergence. HortScience 32:1061-1063. Piringer, A.A., and P.H. Heinze. 1954. Effect of light on the formation of a pigment in the tomato fruit cuticle. Plant Physiol. 29:467-472.

190

Pons, T.L., H. van Rijnberk, I. Scheurwater, and A. van der Werf. 1993. Importance of the gradient in photosynthetically active radiation in a vegetation stand for leaf

nitrogen allocation in two monocotyledons. Oecologia 95:416-424. Popp, H.W., and F. Brown. 1936. The effect of ultra- violet radiation upon seed plants. p. 853-887. In B.M. Dugger (ed.) Biological effects of radiation. Vol. 2. McGraw-

Hill Book Co., New York. Pourmorad, F., S.J. Hosseinimehr, and N. Shahabimajd. 2006. Antioxidant activity, phenol and flavonoid contents of some selected Iranian medicinal plants. Afric. J.

Biotechnol. 5:1142-1145. R Development Core Team. (2011). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-

900051-07-0. Available at http://www.R-project.org/ (Verified 29 Jun. 2012). Rabino, I., and A.L. Mancinelli. 1986. Light, temperature and anthocyanin production. Plant Physiol. 81:922-924. Rademacher, I.F., and C.J. Nelson. 2001. Nitrogen effects on leaf anatomy within intercalary meristems of tall fescue leaf blades. Ann. Bot. 88:893-903. Reinhard, E., M. Adhikkmin, B. Gooch, and P. Shirley. 2001. Color transfer between images. Comp. Graphic App. 21:34-41. Richardson, M.D., D.E. Karcher, and L.C. Purcell. 2001. Quantifying turfgrass cover using digital image analysis. Crop Sci. 41:1884-1888. Rind, D., R. Suozzo, N.K. Balachandran, and M.J. Prather. 1990. Climate change and the middle atmosphere. I: The doubled CO2 climate. J. of Atmos. Sci. 47:475-494. Ronen, G., M. Cohen, D. Zamir, and J. Hirschberg. 1999. Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon-cyclase is down-regulated during ripening and is elevated in the mutant

delta. Plant J. 17:341-351.

191

Ronen, G., M. Cohen, D. Zamir, and J. Hirschberg. 2000. An alternative pathway to β- carotene formation in plant chromoplasts discovered by map-based cloning of beta and old-gold color mutations in tomato. Proc. Natl. Acad. Sci. U.S.A.

97:11102-11107. Roorda, A., and D.R. Williams. 1999. The arrangement of the three cone classes in the living human eye. Nature 397:520-522. Rosario, R., G.J. Mark, J.A. Parrish, and M.C. Mihm Jr. 1979. Histological changes produced in skin by equally erythemogenic doses of UV-A, UV-B, UV-C and

UV-A with psoralens. Brit. J. Derm. 101:299-308. Rowland, F.S. 1990. Stratospheric ozone depletion by chlorofluorocarbons. Ambio 19:281-292. Rowland, F.S. 2006. Stratospheric ozone depletion. Philos. Trans. R. Soc. London, Ser. B. 361:769-790. Rozema, J., J. van de Staaij, L.O. Björn, and M. Caldwell. 1997. Review. UV-B as an

environmental factor in plant life: stress and regulation. Trends in Ecol. 12:22–28. Rozema, J., R. Broekman, D. Lud, H.J. Huiskes, T. Moerdijk, N. de Bakker, B. Meijkamp, and A. van Beem. 2001. Consequences of depletion of stratospheric

ozone for terrestrial Antarctic ecosystems: the response of Deschampsia antarctica to enhanced UV-B radiation in a controlled environment. Plant Ecol. 154:103-115.

Sancar, A., and G.B. Sancar. 1988. DNA repair enzymes. Ann. Rev. Biochem. 57:29-67. Sarkar, D., P.C. Bhowmik, Y-I. Kwon, and K. Shetty. 2011. The role of proline- associated pentose phosphate pathway in cool-season turfgrasses after UV-B

exposure. Environ. Exp. Bot. 70:251-258. SAS Institute. 2011. Version 9.2 SAS Inst. Cary, NC.

192

Saxby, M.J. 1964. Characterization of flavonoids and coumarins by their absorption spectra on paper chromatograms. Anal. Chem. 36:1145-1146. Schmidt, R.E., and X. Zhang. 2001. Alleviation of photochemical activity decline of turfgrasses exposed to soil moisture stress or UV radiation. Int. Turfgrass Soc. Res. J. 9:340-346. Schmitt, J., and R.D. Wulff. 1993. Light spectral quality, phytochrome and plant competition. Trends in Ecol. Evol. 8:47-51. Seigler, D.S. 1998. Plant secondary metabolism. Kluwer Publishers, Norwell, MA. Shackleton, C. 2010. Clinical steroid mass spectrometry: A 45-year history culminating in HPLC–MS/MS becoming an essential tool for patient diagnosis. J. Ster.

Biochem. Mol. Biol. 121:481-490. Shaidaee, G, B.E. Dahl, and R.M. Hansen. 1969. Germination and emergence of different age seeds of six grasses. J. of Range Manage. 22:240-243. Sharma, G., and H.J. Trussell. 1997. Digital color imaging. Trans. Image Proc. 6:901- 932. Sheahan, J.J. 1996. Sinapate esters provide greater UV-B attenuation than flavonoids in Arabidopsis thaliana (Brassicaceae). Am. J. Bot. 83:679-686. Sheehy, J.E. 1975. Some optical properties of leaves of eight temperate forage grasses. Ann. Bot. 39:377-386. Shimada, K., Y. Kakehashi, H. Matsumura, and N. Tanoue. 2004. In vivo quantitative evaluation of tooth color with hand-held colorimeter and custom template. J.

Prosth. Dent. 91:389-391. Shinomura, T., A. Nagatani, H. Hanzawa, M. Kubota, M. Watanabe, and M. Furuya. 1996. Action spectra for phytochrome A and B-specific photoinduction of seed

germination in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 15:8129-8133.

193

Skogley, C.R., and F.B. Ledeboer. 1968. Evaluation of several Kentucky bluegrass and red fescue strains maintained as lawn turf under three levels of fertility. Agron. J.

60:47-49. Smith, H. 1982. Light quality, photo perception and plant strategy. Ann. Rev. Plant Physiol. 33:481-518. Smith, H. 1994. Sensing the light environment: the functions of the phytochrome family. p. 377-416. In J.R.E. Kendrick and G.H Kronenberg (ed.) Photomorphogenesis in

Plants. Kluwer Academic Publishers, Dordrecht, The Netherlands. Smith, J.L., D.J. Burritt, and P. Bannister. 2000. Shoot dry weight, chlorophyll and UV-

B-absorbing compounds as indicators of a plant's sensitivity to UV-B radiation. Annal. Bot. 86:1057-1063.

Solomon, S. 1990. Progress towards a quantitative understanding of Antarctic ozone

depletion. Nature 347:347-354 Sponga, F., G.F. Deitzer, and A.L. Mancinelli. 1986. Cryptochorme, phytochrome and the photoregualtion of anthocyanin production under blue light. Plant Physiol.

82:952-955. Stapleton, A.E. 1992. Ultraviolet radiation and plants: Burning questions. Plant Cell. 4:1353-1358. Stiegler, J.C., G. Bell and N. Maness. 2004. Comparison of acetone and N,N- dimethylformamide for pigment extraction in turfgrass. Comm. Soil Sci. Plant

Anal. 35:1801-1813. Stiegler, J.C., G.E. Bell, N.O. Maness, and M.W. Smith. 2005. Spectral detection of pigment concentrations in creeping bentgrass golf greens. Int. Turf. Res. J.

10:818-825.

194

Stiff, M.L., and J.B. Powell. 1974. Stem anatomy of turfgrass. Crop Sci. 14:181-186. Strid, Å., W.S. Chow, and J.M. Anderson. 1990. Effects of supplementary ultraviolet – B radiation on photosynthesis in Pisium sativum. Biophys. Biochim. Acta.

1020:260-268. Strid, Å., and R.J. Porra. 1992. Alterations in pigment content in leaves of Pisium

sativum after exposure to supplementary UV-B. Plant Cell Physiol. 33:1015-1023. Strid, Å., W.S. Chow, and J.M Anderson. 1994. UV-B damage and protection at the

molecular level in plants. Photosynth. Res. 39:475-489. Sullivan, J.H., and A.H. Teramura. 1988. Effects of ultraviolet-B irradiation on seedling growth in the Pinaceae. Am. J. Bot. 75:225-230. Taalas, P., J. Kaurola, A. Kylling, D. Shindell, R. Sausen, M. Dameris, V. Grewe, J. Herman, J. Damski, and B. Steil. 2000. The impact of greenhouse gases and

halogenated species on future solar UV radiation doses. Geophys. Res. Lett. 27:1127-1130.

Takabe, T., A. Uchida, and T. Tetsuko. 2004. Effects of hydrogen peroxide on germination of Kentucky bluegrass. In Abstracts, Abs # 709. Plant Biology 2004:

July 24 – July 28, 2004. Lake Buena Vista, FL. Takashi, S., and N. Murata. 2008. How do environmental stresses accelerate photoinhibition? Trends Plant. Sci. 13:178-182. Teramura, A.H., R. Hilton Biggs, and S. Kossuth. 1980. Effects of ultraviolet-B

irradiances on Soybean II. Interaction between ultraviolet-B and photosynthetically active radiation on net photosynthesis, dark respiration, and transpiration. Plant Physiol. 65:483-488.

Teramura, A.H., L.H. Ziska, and A.E. Sztein. 1991. Changes in growth and photosynthetic capacity of rice with increased UV-B radiation. Physiol. Planta.

83:373-380.

195

Teramura, A.H., and J.H. Sullivan. 1994. Effects of UV-B radiation on photosynthesis and growth of terrestrial plants. Photosynth. Res. 39:463-473. Tevini, M., W. Iwanzik, and U. Thoma. 1981. Some effects of enhanced UV-B irradiation on the growth and composition of plants. Planta 153:388-394. Tevini, M. 1993. Effects of enhanced UV-B radiation on terrestrial plants. p. 125-153. In Tevini M. (ed.) UV-B Radiation and Ozone Depletion. Lewis Publ., Boca Raton,

FL. Thomas, D.A., and H.N. Barber. 1974. Studies on leaf characteristics of a cline of Eucalyptus urnigera from Mount Wellington, Tasmania. II. Reflection,

transmission and absorption of radiation. Aust. J. Bot. 22:701-707. Toole, V.K., and H.A. Borthwick. 1971. Effect of light, temperature and their interactions on seeds of Kentucky bluegrass (Poa pratensis L.). J Am. Soc. Hortic. Sci.

96:301-304. Torres, M., G. Frutos, and J.M. Duran. 1991.Sunflower seed deterioration from exposure to UV-C radiation. Environ. Exp. Bot. 31:201-207. Tyystjärvi, E. 2008. Photoinhibition of photosystem II and photodamage of the oxygen evolving manganese cluster. Coordination Chemistry Reviews. The Role of

Manganese in Photosystem II. 252:361-376. Vass, I., L. Sass, C. Spetea, A. Bakou, D.F. Ghanotakis, and V. Petroulea. 1996. UV-B- induced inhibition of photosystem II electron transport studied by EPR and

chlorophyll fluorescence. Impairment of donor and acceptor side components. Biochemistry 35:8964-8973.

Wade, H.K., T.N. Bibikova, W.J. Valentine, and G.I. Jenkins. 2001. Interactions within a network of phytochrome, cryptochrome and UV-B phototransduction pathways

regulate chalcone synthase gene expression in Arabidopsis leaf tissue. Plant J. 25:675-685.

196

Wagné, C. 1966. Effect of UV light on lettuce seed germination and on the unfolding of grass leaves. Physiol. Planta. 19:128-133. Wang, Y., M. Zhang, X.Y. Zhang, and H.D. Dong. 2002. Comparative studies on elasticity and scrape tolerance characters of Zoysia japonica and Poa pratensis.

Prataculture Sci. 19:56-59. Wellburn, A.R. 1994. The spectral determination of chlorophylls a and b, as well as total carotenoids using various solvents with spectrophotometers of different

resolution. J. Plant Physiol. 144:307-313. Wherley, B.G., D.S. Gardner, and J.D. Metzger. 2005. Tall fescue photomorphogenesis as influenced by changes in the spectral composition and light intensity. Crop

Sci. 45:562-568. White, A.L., and L.S. Jahnke. 2002. Contrasting effects of UV-A and UV-B on photosynthesis and photoprotection of β-carotene in two Dunaliella spp. Plant

Cell Physiol. 43:877-884. Woodall, G.S., and G.R. Stewart. 1998. Do anthocyanins play a role in UV protection of the red juvenile leaves of Syzygium? J. Exp. Bot. 49:1447-1450. Wrolstad, R.E., and M.M. Giusti. 2001. Characterization and measurement of anthocyanins by UV-visible spectroscopy. Unit F1.2. p. F1.2.1-F1.2.13. In R.E.

Wrolstad and S.J. Schwartz (ed.) Current Protocols in Food Analytical Chemistry. John Wiley & Sons, Inc. New York, NY.

Yin, Z-H., S. Neimanis, U. Wagner, and U. Hebe. 1990. Light-dependent pH changes in leaves of C3 plants I. Recording pH changes in various cellular compartments by

fluorescent probes. Planta 182:244-252. Yudin, L.S., C.W. Mitchell, and J.J Cho. 1987. Color preference of thrips (Thysanoptera: Thripidae) with reference to aphids (Homoptera: Aphididae) and leafminers in

Hawaiian lettuce farms. J. Econo. Entomol. 80:51-55.

197

Zhang, S., H. Jiang, S. Peng, H. Korpelainen, and C. Li. 2011. Sex-related differences in morphological, physiological, and ultrastructural responses of Populus cathayana

to chilling. J. Exp. Bot. 62:675-686. Zhang, X., E.H. Ervin, and R.E. Schmidt. 2005. The role of leaf pigment and antioxidant levels in UV-B resistance of dark- and light-green Kentucky bluegrass cultivars.

J. Am Soc. HortScience 130:836-841. Zhang, X., and E.H. Ervin. 2005. Exogenous salicylic acid and jasmonic acid effects on Kentucky bluegrass UV-B tolerance. Int. Turfgrass Soc. Res. J. 10:910-915. Zhang, X., and E.H. Ervin. 2009. Physiological assessment of cool-season turfgrasses under ultraviolet-B stress. HortScience. 44:1785-1789. Ziska, L.H, A.H. Teramura, and J.H. Sullivan. 1992. Physiological sensitivity to plants along an elevation gradient to UV-B radiation. Am. J. Bot. 79:863-871. Ziska, L.H., A.H. Teramura, J.H. Sullivan, and A. McCoy. 1993. Influence of ultraviolet- B (UV-B) radiation on photo-synthetic and growth characteristics in field-grown

cassava (Manihot esculentum Crantz). Plant Cell Environ. 16:73-79. Zscheile, F.P., J.W. White, Jr., B.W. Beadle, and J.R. Roach. 1942. The preparation and

absorption spectra of five pure carotenoid pigments. Plant Physiol. 17:331-346. Zu-Hua, Y., K. Siebke, and U. Heber. 1990. Light dependent changes in leaves of C3 plants. III. Effect of inhibitors of photosynthesis and of developmental state of

photosynthetic apparatus on cystolic and vacuolar pH changes. Planta 182:262-26.