Norwegian University of Life

Sciences

Department of Plant and

Environmental Science

Thermoperiodic control of elongation growth and signaling

involved

by:

Deepak Mahat*

Department of Plant and Environmental Science

Norwegian University of Life Sciences

P.O. Box 5003, 1432 Ås, Norway

Ås, 2010

*Contact: mahatdeepak@gmail.com

Abstract

Production of short and compact plant in greenhouses is one of the major challenges. Due to high

demand of these types of plants by the customers, significant interest has been given in

greenhouse industries to produce such plants and reduce the energy cost. The hypocotyl

elongation depends upon temperature and light parameters. The aim of this study was to

investigate the effect of diurnal temperature drops and temperature light-interactions and the

signaling pathway. Arabidopsis and pea have been used as model plants to study the effect of

diurnal temperature drops and interaction between temperature drop treatment and irradiance. In

Arabidopsis, it was observed that a temperature drop (from 24 to 12°C) in the middle of the day

and night resulted in the largest and smallest reduction in hypocotyl growth. But, phyB and the

pif4 mutant as well as the plants mutated in four or all five DELLA proteins appeared less

sensitive to temperature than the wild type. Also, in pea, the combination of temperature drop

and increased irradiance showed the largest reduction in stem elongation. The phyB and the della

(la crys) mutant showed reduced response to temperature drop, and the long1 mutant was almost

temperature insensitive. Based on this research, a signaling pathway is proposed (with names of

components in Arabidopsis/pea): PhyB – (COP1/LIP1) – HY5/LONG1 – DELLAs – PIF4 and

HY5/LONG1 – GA2ox1/GA2ox2.

Contents

I. Abstract

II. Acknowledgements

III. Abbreviations

1. Introduction 1

1.1 Control of shoot elongation and its practical implications 1

1.2 Thermoperiodic control of shoot elongation 2

1.3 Gibberellins 3

1.4 Thermoperiodic control of gibberellin metabolism 6

1.5 Phytochromes 6

1.6 Involvement of phytochromes in temperature responses 8

1.7 Phytochrome interacting factors 8

1.8 Circadian Rhythms 9

1.9 Homologous genes: HY5 in Arabidopsis and LONG1 in pea 10

1.10 Arabidopsis thaliana 10

1.11 Pisum sativum 10

1.12 Objectives of the research 11

2. Materials and Methods 12

2.1 Experiments with Arabidopsis thaliana 12

2.2.1 Plant Materials 12

2.1.1.1 Seeds 12

2.1.1.2 Seed sterilization 12

2.1.1.3 Preparation of media 12

2.1.1.4 Growth conditions during germination 13

2.1.1.6 Light and temperature treatments 13

2.1.1.7 Hypocotyl length measurement 14

2.2 Experiments with Pisum sativum 15

2.2.1 Sowing and germination of pea seeds 15

2.2.2 Light and temperature treatments 15

2.2.3 Stem elongation rate recording through angular displacement

transducer 16

2.3 Statistical analyses 17

3. Results 18

3.1 Experiments with Arabidopsis thaliana 18

3.1.1 Temperature drop in the middle of the day resulted in the

largest reduction of hypocotyl length in Arabidopsis 18

3.1.2 Effect of gibberellin in hypocotyl growth 19

3.1.3 An apparent role of DELLA proteins in response to altered

temperature 19

i. Differential response to temperature in phy mutants 20

ii. PIF4 activity is involved in response to altered temperature 21

iii. Effect of toc1 mutation in response to altered temperature 22

b. Angular Displacement Transducer experiments with pea 22

i. Wild type 22

ii. The la crys mutant 23

iii. The long1 mutant 24

iv. The phyB mutant 25

c. Cumulative stem growth in pea 25

i. Wild type 26

ii. The la crys mutant 26

iii. The long1 mutant 27

iv. The phyB mutant 27

4. Discussion 28

4.1 Diurnal temperature drop reduces the hypocotyl length in Arabidopsis 28

4.2 Increased irradiance and temperature drop affect the stem elongation rate and

cumulative stem growth in pea 32

4.3 Proposed temperature signaling pathways 34

5. Conclusion 36

Suggestions for further research 36

References 37

ACKNOWLEDGEMENT

First and foremost, I would like to express my deep sense of honour and appreciation to my

supervisor Prof. Jorunn Elisabeth Olsen for her invaluable supervision, suggestions and regular

encouragements during the work. I am also grateful to my co-supervisor Micael Wendell for his

help, continuous encouragement and guidance during the lab work.

I am grateful to the Department of Plant Science (IPM) and Senter for Klimaregulert Forskning

(SKP) for providing necessary laboratory facilities and administrative support. Also, heartful

thanks to the project (Environmentally friendly development of Norwegian greenhouse industry)

for financial support for chemicals and seed expenses.

My sincere thanks goes to Ida Hagen, Marit Sirra, Tone Melbye and Dag Wenner for their kind

co-operation during lab work. Thanks to Mrs. Anju for her help during the scanning of the

plants. Finally, thanks to all for their direct and indirect help to complete this research.

ABBREVIATIONS

COP1 Constitutive Photomorphogenesis 1

GA gibberellic acid

GAI Gibberellic Acid Insensitive

DIF difference between DT and NT

DT day temperature

FR far-red light (generally 700-800nm)

HY5 Long HYpocotyl 5

Ler Landsberg erecta

LIP1 COP1 orthologous protein in pea

LONG1 HY5 orthologous protein in pea

NT night temperature

PhyA-E phytochromes A to E

PIF Phytochrome Interacting Factors

Pfr phytochrome in its far-red light absorbing form

R red light (generally 600-700nm)

R: FR ratio ratio of red light to far-red light

RGA Repressor of GA

RGL RGA-like

T1 constant temperature and constant irradiance

T2 constant temperature and increased irradiance

T3 temperature drop and constant irradiance

T4 temperature drop and increased irradiance

1

1. Introduction

1.1 Control of shoot elongation and its practical implications

Stem elongation is an important physiological process, which is central in deciding the

competitive success of plants in natural ecosystems. Rapid elongation may be beneficial in order

to maximize exposure to light in a situation with competition with neighbour plants. On the other

hand, there is no benefit for plants on being high if they are fragile because they then may easily

break when exposed to wind and heavy rains or during fruiting if the fruits are heavy. Thus,

plants need to control their shoot elongation.

Control of shoot elongation has important practical implications in horticulture and agriculture.

In production of cereals, control of shoot elongation is important to reduce lodging and thus

quality reduction and crop loss. Furthermore, for ornamentals and transplants, the customers

generally prefer short and compact plants. Tall plants are difficult to handle during transport and

in transplanting since they can easily break. Also, compact plants are commonly considered to

have a higher ornamental value. Due to lack of unlimited space in greenhouse for the production

of huge amounts of crops, high plant densities are desirable from an economical point of view.

However, in dense plant stands, light quality is shifted towards a reduced red: far-red ratio. This

elicits a shade-avoidance response with enhanced internode elongation (Smith, 1982).

Chemical growth retardants are widely used to reduce plant size. These retardants like onium

compounds (chlormequat chloride, chlorophonium, mepiquat chloride) and nitrogen containing

heterocyclic compounds (ancymidol, tetcyclacis, etc) affect gibberellins (GA) biosynthesis and

other metabolic pathways (Wilhelm, 2000). Such chemical growth retardants are harmful both

for human health and environment, and there are now quite strong limitations on their use. Thus,

it is highly desirable to use less growth retardants and find other ways to control shoot elongation

and obtain compact plants. One obvious way to achieve this in the greenhouse industry is by

exploiting the responses of plants to environmental factors, i.e. by manipulation with temperature

and light parameters such as irradiance and light quality.

In commercial greenhouse culture, the concept of temperature drop has been used and is still

used to control the height of plants in order to obtain compact plants in species such as poinsettia

(Euphorbia pulcherrima) and Begonia × hiemalis (Myster and Moe., 1995). In Norway,

poinsettia is by far the largest commercial pot plant culture with more than 6 million plants sold

yearly (Statistics of the Norwegian Gardeners Association (Gartnerforbundet)). In northern areas

such as Norway, diurnal temperature drops are used routinely in production of poinsettia and can

in the autumn easily be obtained by opening the hatches in the greenhouses in the early morning.

However, in warmer periods and in warmer parts of the world, cooling is necessary to obtain a

sufficient temperature drop. A lower day temperature (DT) than night temperature (NT) i.e.,

negative temperature difference (negative DIF) has been found to be most effective for the

reduction of plant height in many species and has been used in practice in a number of cases.

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However, in most part of the world, including northern areas, this is quite energy-demanding and

thus expensive since cooling during the day is normally needed.

Different types of energy sources are used in greenhouse to control the climate. Propane,

electricity and some other natural gases are widely used, but in some cases, alternative energy

sources such as solar and geothermal energy is used (Fowler et al., 2009). Since energy is

expensive, it is of great interest to reduce energy costs. The use of dynamic climate control has

been suggested as a way to save energy. This concept then is to let the temperature in the

greenhouse vary more in line with the outdoors temperature. There is ongoing research on this at

several places, including UMB. However, at least in areas with quite large differences between

DT and NT, this will lead to a situation with high DT and low NT (positive DIF), which

commonly results in promotion of stem elongation. This will again lead to a need for substantial

amounts of chemical growth retardants in order to obtain compact plants. By performing detailed

studies as to when in the diurnal cycle plants are most responsive to temperature drops, it might

be possible to control temperature preferentially at such time points and let the temperature

follow the outdoors temperature in other parts of the diurnal cycle. Also, increasing our

knowledge about environmental control of plant growth and its physiological and molecular

basis, will improve our possibilities for a sustainable manipulation of shoot elongation without

using high amount of harmful growth retardants.

1.2 Thermoperiodic control of shoot elongation

The stem elongation is influenced by the relationship between DT and NT, light parameters and

some portion of other environmental factors and physiological phenomena. Went (1944)

described thermoperiodism as a differential response of plants to temperature in the dark and

light period i.e. for physiological processes such as flowering, fruiting and growth. In poinsettia,

a temperature drop of 2 hours from 24°C to 8°C in the morning reduced the shoot elongation by

50% (Uber and Hendricks, 1992). Also, a short diurnal temperature drop of 4 hours in

Dendrathema grandiflorum at the beginning of the light period reduced elongation by 27%

(Nishijima et al., 1997). In pea (Pisum sativum) a diurnal temperature drop in the middle of the

light period reduced shoot elongation by 55% as compared to constant temperature, whereas a

temperature drop in the middle of the dark period reduced elongation by 27%. These facts also

indicate that the sensitivity of plants to temperature drop differs during the diurnal cycle.

Similarly, a temperature drop in the middle of the night had very little effect on stem elongation

in B.× hiemalis (Grindal and Moe., 1994). It has also been shown that different plant species

grown at a negative DIF had shorter internodes than plants grown at a positive DIF or constant

temperature (Erwin et al., 1989; Jensen et al., 1996: Grindal et al., 1998; Moe and Heins, 2000;

Stavang et al., 2005). Pea plants grown at a DT/NT combination of 13°C/21°C in a 12 hours

photoperiod reduced the stem elongation by 30% after 12 days as compared to 21°C/13°C

(Stavang et al., 2005). Similarly, reduction of stem elongation in negative DIF has also been

3

shown in a variety of other plant species (reviewed in Myster and Moe, 1995). Thus light and

temperature are interacting factors responsible for the control of stem elongation in plants.

1.3 Gibberellins

Gibberellins (GAs) are one of the naturally occurring plant hormones responsible for controlling

growth, differentiation and development (Davies, 2004). It has a key role in stem elongation.

Gibberellin stimulates the etiolated growth whereas light suppresses the elongation, growth,

expansion of true leaves and chloroplast development (Chory et al., 1996). Among 136

gibberellins, only few have biological activity. These are GA1, GA3, GA4 and GA7 (Hedden et

al., 2000). GA4 is the major bioactive GA in Arabidopsis thaliana and GA1 in pea (Ross et al.,

1989; Jianhong et al., 2008). GA is synthesized in three compartments of the cell i.e. the plastid,

the endoplasmic reticulum and the cytosol (Davies, 2004). A schematic description of GA

biosynthesis is shown in fig. 1. Three different classes of enzymes are involved in GA synthesis

from geranylgeranyl diphosphate (GGDP) i.e., terpene synthases (TPSs), the cytochrome P450

monooxygenase (P450) and the 2-oxaloglutarate dependent dioxygenase (2ODDs) (Yamaguchi,

2008).

The TPSs i.e., ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) located

in the plastid converts geranylgeranyl diphosphate (GGDP) to ent-kaurene in two steps. Ent-

kaurene is then converted to GA12 by two P450s. The ent-kaurene oxidase (KO) oxidizes C-19 to

produce ent-kaurenoic acid which is then converted to GA12 by a P450 i.e., ent-kaurenoic acid

oxidase (KAO). KO is located in the outer membrane of plastid, whereas, KAO is present in the

endoplasmic reticulum (Helliwell et al., 2001). GA12 is converted to the bioactive form, GA4 or

GA1 by sequential oxidations on C-20 (several steps) and C-3 by GA 20-oxidase (GA20ox) and

GA3-oxidase (GA3ox), respectively.

On the basis of substrates, pea contains two classes of GA2oxs i.e., a larger class which uses C19-

GAs as substrates and a smaller class which uses C20-GAs as substrates (Malcolm at al., 1991;

Lo et al., 2008). In pea, two C19-GA2oxs i.e., GA2ox1 and GA2ox2 have distinct overlapping

functions. GA2ox2 catalyzes the conversion of GA1 to GA8. Similarly, GA2ox1 predominantly

catalyzes the conversion of GA20 to GA29 (Reid at al., 1992). In Arabidopsis, the C19-GA2oxs

consist of AtGA2ox1, -2, -3, -4 and -6. These enzymes use C19-GAs as their substrate (GA4, GA1

and GA5). Similarly, AtGA2ox7 and -8 of C20-GA2oxs, hydroxylate C20-GAs (GA12 and GA53)

and convert these inactive precursors to bioactive GAs (Achard et al., 2008).

The activity of GA20oxs is enhanced when Arabidopsis plants are transferred from short days to

long days. This results in increase in the level of GA20 or GA9 (Xu et al., 1997). GA3oxs in turn

converts GA20 and GA9 to bioactive GA1 and GA4, respectively. The GA5 gene, which encodes

GA20ox, is highly expressed in elongated stem while GA4, which encodes GA3ox, is not

expressed as much as that of GA5, in this tissue. The content of bioactive GA1 in pea was found

4

to be reduced upon transfer of etiolated seedlings to light (O’Neill et al., 2000; Garcia-Martinez

and Gil, 2001). Thus, light is essential for the regulation of GA biosynthesis. Weller et al. (2009)

reported that reduced GA1 levels under de-etiolation are linked largely to increased expression of

GA2ox2. The apical stem portion of 7 days old dark grown pea seedlings of the

photomorphogenesis mutant, lip1, contained 3 to 4 fold less GA1 as compared to the wild type.

This was associated with a 4 fold increase in transcript level of GA2ox2 as compared to the wild

type.

Figure1. Biosynthesis of gibberellins (GAs) from geranylgeranyl pyrophosphate (GGPP) in

plants (source: www.4eplantphys.net/article.php?ch=2&id=366). KS stands for ent-kaurene

5

synthase; CPS for ent-copalyl diphosphate synthase; KO for ent-kaurene oxidase; KAO for ent-

kaurenoic acid oxidase and ox stands for oxidase enzyme. These above mentioned enzymes are

involved in the conversion of GA12 to the bioactive GA4 and GA1, as well as their inactivation.

Treatment with GA stimulates the stem growth by promoting cell division and cell elongation in

the subapical meristem (Yang et al., 1996; Hansen et al., 1999). Also, some previous studies

revealed that some enzymes like xyloglucan endotransglucosylase/ hydrolase are involved in GA

promoted stem growth (Xu et al., 1996). GA stimulates the expression of the cell cycle

controlling genes encoding cyclin-dependent kinase and cyclins and thus promotes the mitotic

activity (Fabian et al., 2000). It is also well known that GA affects the orientation of

microtubululi and accordingly cellolose microfibriles, which get more parallel and perpendicular

to the axis of elongation upon the influence of GA.

The GA signaling pathway consists of several positive and negative regulators including soluble

GA receptors in Arabidopsis. The DELLA protein repressors turn off the pathway in the absence

of GAs. This DELLA protein belongs to the GRAS family of transcription factors. The DELLA

proteins of Arabidopsis are Repressor of GA (RGA), Gibberellic Acid Insensitive (GAI), RGL1

(RGA- like), RGL2 and RGL3 (Zentella et al., 2007; Hussain and Peng, 2003; Cao et al., 2006).

RGA and GAI repress vegetative growth and floral initiation which is promoted by GA. RGL1

encodes a negative regulator of GA responses (Wen and Chang, 2002). RGL2 regulates seed

germination induced by GA. The role of RGL3 is not well known, but the transcript level of

RGL3 is increased in response to low temperature (Achard et al., 2009). RGL1 along with RGA

and RGL2 take part in fruit flower and fruit development (Zentella et al., 2007). In pea, the

existence of two different DELLA genes denoted LA and CRY, was recently shown (Weston et

al., 2008). Mutations in these genes results in an elongated slender phenotype. DELLA proteins

have also been found to promote the biosynthesis of active GAs. In the Arabidopsis della mutant

rga, the expression of the GA biosynthesis gene GA4 is reduced indicating, that the DELLA

protein levels are associated with an up-regulation of GA synthesis genes (Silverstone et al.,

2001).

In Arabidopsis, the DELLA proteins are known to interact with the DNA- binding domain of

PIF3 and PIF4 (phytochrome interacting factors 3 and 4), two transcription factors that promote

cell expansion in response to GA and light (Spartz et al., 2008). By degrading the DELLA

protein repressor, the GA signaling pathway is continued and GA induced genes are expressed.

Simply, Arabidopsis without GA are found to be short and late flowering whereas the plants

treated with GA has normal growth responses (Richard et al., 2001). Thus, GA increases the

hypocotyl length by decreasing the effects of DELLA growth repressor proteins (Peng et al.,

1997; Silverstone et al., 1998; Dill and Sun, 2001; King et al., 2001).

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1.4 Thermoperiodic control of GA metabolism

Pea plants grown at negative DIF have reduced length and reduced levels of GA as compared to

plants grown at positive DIF and constant temperature (Grindal et al., 1998; Stavang et al., 2005;

2010). The decreased GA1 level under negative DIF was explained by a strong stimulation up to

19-fold of the expression of GA2ox2 (Stavang et al., 2005). It has also been found that a negative

DIF treatment affects the stem, leaf and root mass ratio (Stavang et al., 2010). Reduced levels of

active GA under negative DIF have also been found in other species such as Campanula

isophylla, tomato (Lycopersicum esculentum) and Dendrathema grandiflorum (Jensen et al.,

1996; Langton et al., 1997; Nishijima et al., 1997). Also in pea, a 2 hour temperature drop from

21°C to 13°C in the middle of the day reduced the stem elongation rate temporarily by 55% and

increased the mRNA levels of the GA-deactivation gene PsGA2ox2 by 2-fold within 30 min and

up to 4-fold after 1.5 hour (Stavang et al., 2007). A 36% reduction in the GA1 levels was

recorded after 3-4 hour time lag. Similarly, when the temperature was dropped in the night, the

stem elongation rate was reduced by 27% but this had no effect on transcript levels of PsGA2ox2

or GA1 levels (Stavang et al., 2007). However, there was a slight stimulation on the expression of

GA biosynthesis genes i.e. NA, PsGA20ox1 and PsGA3ox, indicating that the effect of

temperature drop on GA metabolism in pea is qualitatively different in light and dark. In D.

grandiflorum the level of GA1 in stem tissue was also reduced after 4 hours diurnal temperature

drop (Nishijima et al., 1997). Also, in an experiment with a temperature increase from 20-29°C,

the expression of genes in both the GA and auxin metabolism in Arabidopsis was shown to be

regulated by temperature (Stavang et al., 2009).

Like della mutants in Arabidopsis, the la crys della double mutant of pea does not respond to

applied GAs but shows a saturated GA response independently of growth conditions (Potts et al.,

1985; Ingram and Reid, 1987). The elongation of la crys mutant is similar to that of the wild type

when the wild type is treated with a saturating dose of active GA (Weston et al., 2008). It was

recently shown that a negative DIF treatment does not influence shoot elongation or stem, leaf or

root mass ratio in pea mutated in its two DELLA genes, LA and CRY in contrast to the wild type

(Stavang et al., 2010). Furthermore, Arabidopsis plants mutated in four or all five DELLA genes

(quadruple and pentuple mutant) showed a partial response only to a temperature increase from

20-29°C (Stavang et al., 2009). This indicates that DELLA proteins play a role in temperature

signaling. In the present work, the effects of mutation in four or all five DELLA genes in

Arabidopsis or the two DELLA genes in pea were investigated further as related to temperature

drop-light interactions. Also, the effect of GA application was investigated in this respect to

further evaluate the importance of the GA pathway.

1.5 Phytochromes

Plants contain several light absorbing pigments, including chlorophyll and carotenoids, which

are active in light harvest in photosynthesis, as well as pigments which play important roles in

developmental processes. Of these, phytochromes are involved in sensing of red (R) and far red

7

(FR) light, cryptochromes and phototropins in blue light sensing. Phytochromes are a family of

soluble proteins consisting of a light absorbing chromophore pigment and a polypeptide chain

i.e., apoprotein. They are involved in a variety of processes such as chloroplast development,

initiation of seed germination in response to light, control of flowering and inhibition of

elongation growth through inhibition of cell elongation (Reed et al., 1994; Heschel et al., 2007).

The two forms of phytochromes interconvert upon light absorption (fig. 2). These are Pr

(absorbing light of 670 nm) and Pfr (absorbing light of 730 nm). When Pr absorbs R light, it is

converted to the active form Pfr and this converts back to Pr by absorbing FR light. The effects

of phytochromes depend upon the amount of light harvested. These pigments are encoded by a

multigene family and classified on the basis of light exposure. Type I phytochrome is stable to

light whereas type II phytochrome is light labile.

Red light (670 nm)

No response Pr Pfr Response

Far red light (730 nm)

Figure 2. Two forms of phytochromes differ in their absorption spectra.

In Arabidopsis thaliana, the genes PHYA, PHYB, PHYC, PHYD and PHYE encode five distinct

phytochromes (Dehesh et al., 1991; Basu et al., 2000). Of these, the PHYA gene encodes type I

phytochrome and PHYB and other PHY genes encode type II phytochrome. Different

phytochromes, of which PhyA and PhyB are best studied, have specific roles to control several

diverse phases of plant growth and development during all phases of life cycle (Casal et al.,

1998). In Arabidopsis, PhyB plays a role in de-etiolation of seedlings under high irradiance of

red light. However, both, PhyA and PhyB act together under low irradiance or continuous light.

PhyB is known to interact with FR light to increases the expression of AtGA20ox2. This in turn

results in increased GA levels and enhanced elongation (Hisamatsu et al., 2005).

The phyA/phyB double mutant has slightly longer hypocotyl length than the phyB mutant in

white light; and longer hypocotyl length in R light (Yang et al., 1995). It has the same length as

that of the phyA mutant in FR light. 97% of phyA/phyB mutant seeds germinated when they were

exposed to white light. They also found that when R light was given, there was a slight effect on

seed germination. Devlin et al., (1996) observed that when FR light was given at the end of the

day, the phyA/phyB double mutant of A. thaliana showed increased internode length and early

flowering.

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1.6 Involvement of phytochromes in temperature responses

A long hypocotyl phyB deficient mutant (lh) and its isogenic wild type of cucumber (Cucumis

sativus L.) were studied for the interaction effects between thermo- and photomorphogenetic

stem elongation by growing plants under 12 hours photoperiod with DT/NT of 25/19°C and

19/25°C with 30 min end-of-day (EOD)-R or FR light (Xiong et al., 2002). 30 min EOD-FR

compared to EOD-R, increased length of stem, hypocotyl and internodes in WT while the lh

mutants showed less response to the positive and negative DIF. This indicates that phyB plays a

key role in thermo- and photomorphogenetic responses. In Arabidopsis, decrease in petiole

elongation in response to a negative DIF treatment was also shown to be reduced in a phyB

mutant as compared to the wild type (Thingnæs et al., 2008). However, mutation in phyD had no

influence and a phyE mutant showed a slightly positive influence on the temperature effect.

Thus, these results indicate that phyB is needed for a complete thermoperiodic control of

elongation growth in Arabidopsis. Also, Halliday et al., (2003) showed that the early flowering

phenotype of a phyB mutant in Arabidopsis is temperature dependent. In Populus, which has one

PHYA and two PHYB genes, effect of changed expression of PHYA suggests an activity of phyA

in photoperiodic control of shoot elongation (Olsen et al., 1997; Ingvarsson et al., 2006). The

PHYA overexpressors did not show growth cessation and apical bud formation in response to

short days (Olsen et al., 1997). However, in low NT these plants ceased their growth and formed

a terminal bud under short days, indicating that temperature altered phytochrome action

(Mølmann et al., 2005). The wild type even showed growth cessation and apical bud formation

under long days when NT was low. Also, in seed germination, phytochrome action appears to be

dependent on temperature (Heschel et al., 2007). Taken together, these studies suggest a role of

phytochromes in temperature responses. In the present work, this was investigated further as

related to responses to light-temperature interactions.

1.7 Phytochrome interacting factors

Phytochrome interacting factors (PIFs) are a group of basic helix-loop-helix transcription factors

which have been identified by genetic, molecular and photobiological techniques. When

phytochrome is exposed to R light, it gets converted to Pfr and translocated to the nucleus

(Castillon et al., 2007). The Pfr there induces the phosphorylation of PIFs, either directly or

indirectly. The PIF gene family in Arabidopsis consists of 7 members which are PIF1, PIF2,

PIF3, PIF4, PIF5, PIF6 and PIF7, that controls the light regulated gene expression directly or

indirectly (Castillon et al., 2007).

The involvement of PIF4 and PIF5 were found to control de-etiolation in FR light in phyA

signaling (Huq and Quail, 2002). Among the PIFs, PIF4 appears to play a significant role in

temperature responses (Koini et al., 2009; Stavang et al., 2009). The expression of PIF4

increased both in cotyledons and hypocotyls when plants were transferred from 20°C to 29°C,

whereas, the expression of PIF5 increased slightly only in hypocotyls (Stavang et al., 2009).

There was no alteration in the transcript level of PIF1 and PIF3 in any organ. Also, a pif4 mutant

9

showed little response to the transfer of plants from 20°C to 29°C (Stavang et al., 2009). In this

thesis work, the role of PIF4 and PIF5 in temperature responses was further evaluated as related

to temperature-light interactions.

1.8 Circadian Rhythms

Circadian clocks, which depend on light and generate circadian rhythms, synchronize the

flowering, growth and development of a plant with day length and thus regulate plant fitness.

The circadian clocks also confer daily rhythms in growth and metabolism and interact with

signaling pathways involved in plant response to the environment. The circadian rhythms can be

seen at cellular levels i.e., changes in gene expression, and at organism level i.e., activity

changes. This can be seen in the form of sinusoidal waves i.e., period, phase and amplitude.

Small changes in light also affect the rhythmic amplitude of clock outputs (Harmer, 2009).

The exposure to high intensity of continuous light shortens the free running period of the rhythm

(Aschoff, 1960). Some observations from the metabolic process in leaves have shown a coupling

of circadian rhythms between plant cells (Rascher, 2001). This verifies that a single cell is

capable to perform the circadian rhythms whether it is unicellular or multicellular organisms, and

helps to anticipate the changes in the environment and synchronize different physiological

processes with each other.

Arabidopsis thaliana has been a model plant for analyzing and genetic dissection of complex

processes such as circadian rhythms (Redei, 1975; Hubbard et al., 2009). When the short period

mutant toc1 (timing of cab expression) is grown along with the long period mutant ztl (Zeitlupe)

under short day/night cycle, the toc1 produces more chlorophyll, accumulate more biomass and

fix more carbon. This process is reverse for ztl when these plants were grown under long

day/night cycles (Dodd et al., 2005). This is due to the entraining capacity of Arabidopsis

towards day lengths different from 24 hours (Roden et al., 2002; Somers et al., 1998; Yanovsky

et al., 2002). The toc1 mutant grown in artificial short days has the same output as the wild type

grown in 24 hours day. Similarly, the process is reversed with ztl mutants when they were grown

under artificial long days.

The plant circadian clock consists of three interlocked transcriptional feedback loops. These are

TOC1, CCA and LHY1. The TOC1, also known as pseudo response regulator (PRR1), is an

evening phased and clock regulated gene of unknown molecular function. The two proteins CCA

and LHY1 bind with the TOC1 promoter and inhibit its expression (Alabadi et al., 2001). This

shortens the period of two bioluminescence rhythms i.e., the expression of chlorophyll a/b-

binding protein (CAB) genes and the movement of primary leaves (www.arabidopsis.

org/servlets/tairobject). Since, according to earlier and present results, there appears to be a

diurnal rhythm in response to temperature-drops, the effect of mutation in TOC1 in response to

light-temperature interactions was evaluated in this present work

10

1.9 Homologous genes: HY5 in Arabidopsis and LONG1 in pea

In pea, two transcription factors i.e., LONG1 and LIP1 have been discovered, which are

orthologous to HY5 and COP1 of Arabidopsis (Weller et al., 2009). GA together with COP1

(constitutive photomorphogenic 1, a very strong repressor of photomorphogenesis) prevent the

accumulation of HY5 in darkness. hy5 mutants have impaired ability to de-etiolate when GA

biosynthesis is obstructed through inhibitors (Alabadi et al., 2008). Although very similar to

HY5 in the C-terminal end, LONG1 contains an additional N-terminal domain which is different

from HY5, but these two genes have similar functions on photomorphogenesis (Weller et al.,

2009). LONG1 plays important role in de-etiolation under R, blue and FR light. During de-

etiolation in pea, GA1 content is dropped due to increased expression of GA2ox2 and this is

recovered during the darkness. The long1 mutant fails to down-regulate GA1 production upon

light exposure in the initial phase. The response of long1 mutants to light exhibits weaker

transient down-regulation of GA, thus the elongation of stem is slightly inhibited. This also

shows that in addition to LONG1, other genes are also involved in this process that regulated the

process of de-etiolation (Weller et al., 2009).

1.10 Arabidopsis thaliana

Arabidopsis thaliana (common name: thale cress), which belongs to Brassicaceae, has for a few

decades been an important model plant for study of molecular genetics and gene functions

(Borevitz and Ecker, 2004). It has a rapid life cycle of 6-8 weeks i.e., from germination to mature

seeds. Landsberg erecta (Ler) and Columbia (Col) are the most commonly used background

lines of this species. In addition to a small size and rapid life cycle, a small genome and the fact

that it is easy to transform and mutate, have made it the best studied plant model species in the

recent few decades.

The Landsberg ecotype is obtained from the Liabach seed stock. Landsberg erecta (Ler-0) was

obtained by irradiation of Landsberg seeds so they are not considered the true breeding line as

that of Landsberg. The Columbia ecotypes were also selected from the Liabach seed population.

These ecotypes are fertile and also respond well to a changed photoperiod. They have different

accessions i.e., Col-0 to Col-7 (www.arabidopsis.org/portal/education/aboutarabidopsis). The

ecotype C24 is very much similar to ecotype Columbia, but some differences can be obtained,

such as flowering time. SNPs (Single Nucleotide Polymorphisms) of C24 versus Col have also

been recorded (Torjek et al., 2003).

1.11 Pisum Sativum

Pisum sativum, an annual plant usually cultivated for vegetable, has a rapid life cycle of about

two-three months. Although not easily amenable to transformation, a variety of mutations have

been obtained. The pea plant appears to contain two DELLA proteins i.e., LA and CRY, which

are important regulators of GA synthesis and root growth. The elongated la crys mutant plant

behaves like being GA saturated and was recently reported to be mutated in two DELLA genes

11

LA and CRY, which appear to be the only DELLA genes operating in the shoot. Different

phytochrome mutants in pea have been studied. These are the phyB-1 to phyB-6 mutants which

vary in their modification extent of the phyB molecule (Weller et al., 2001). The phyB-5 mutants

are characterized by being long, slender and pale. This mutant exhibits clear photoperiod

responsiveness for vegetative treats, showing that the mechanism which detects photoperiod is

still functional (Weller et al., 2001). LONG1 and LIP1 are two transcription factors that recently

have been discovered in pea. The long1 mutants do not fully responsd to light, which results in

slight inhibition of stem elongation only in light. The partial response suggests involvement of

other genes along with LONG1 in regulating the process of de-etiolation (Weller et al., 2009).

1.12 Objectives of the research

The research in this thesis is concerned with control of elongation growth by different

temperature-light interaction regimes. Increasing the knowledge on this is of significant interest

with respect to dynamic temperature control in greenhouses in order to reduce energy cost and

produce short and compact plants. Earlier research concerned with stem elongation in poinsettia,

Begonia and pea (as mentioned earlier) has revealed that there is a differential diurnal sensitivity

to temperature drop in these species. However, the different studies have been conducted under

different day lengths and different light conditions and generalizations are thus difficult. No

studies through the entire diurnal cycle have been reported and only little information is available

about the background of differential sensitivity to temperature changes during the diurnal cycle.

In this thesis, the main aim was to investigate further thermoperiodic control of elongation

growth and signaling involved in sensing of light-temperature alteration (drop) at different time

points in the day and in the night. In these experiments, the temperature was dropped (changed)

on different time points in the diurnal cycle, using hypocotyls of Arabidopsis as a model system.

This enables rapid screening of large numbers of plants and a variety of relevant mutants are

available. Since control of hypocotyl elongation might differ from control of elongation of the

proper (internodes), the effect of diurnal temperature drops on stem elongation and growth rates

was also studied, using pea as a model system. In pea, various relevant mutants are also

available. Another specific aim in this respect was to study effects of the interaction between

temperature drop and irradiance.

12

2. Materials and methods

2.1 Experiments with Arabidopsis thaliana

2.1.1 Plant Materials

2.1.1.1 Seeds

In this experiment, Landsberg erecta i.e., Ler-0 (ecotype of Arabidopsis thaliana) was the

genetic background for the following mutants of Arabidopsis thaliana (L.) Heyhn: phyA-201,

phyB-5, phyA-201/phyB-5, della quadruple (rga, gai, rgl1,

rgl2) and della pentuple mutants (rga, gai, rgl1, rgl2, rgl3).

Similarly, Columbia (Col-0) was the genetic background for

the pif4, pif5 and pif4/pif5 double mutants. Finally, C24 was

the genetic background for toc1 mutant. Seed stock was

supplied by the Nottingham Arabidopsis Stock Centre (UK),

and grown for seed harvest in Centre for Climatically

regulated plant research at Ås (SKP; fig. 3). In SKP, the seeds

were harvested from the plants grown at 21°C in 18 hours

photoperiod (06.00-00.00) consisting of natural light and

supplement light intensity of 150 µmol m-2

s-1

from fluorescent

tube (Gavita, Andebu, Norway; www.gavita.com).

Figure 3. Arabidopsis thaliana grown to harvest seeds.

2.1.1.2 Seed Sterilization.

All seeds of Arabidopsis were surface sterilized for 3 min in 70% (v/v) ethanol and 0.1% (v/v)

SDS using vortex. The seeds were rinsed with sterile water for 6 times.

2.1.1.3 Preparation of Media

½ strength Murashige-Skoog (MS) medium (Murashige and Skoog, 1962) was prepared using

0.8% (w/v) agar (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and 2.15 g MS Medium

(Duchefa Biochemie, Netherlands) .The pH was adjusted to 5.6 and the medium was autoclaved.

40 ml media was poured in each petriplate. 0.1% (w/v) agar was also prepared for the seed

sowing purpose. Furthermore, a filter-sterilized stock solution of GA3 was prepared to make the

final concentration (50 µM) of media for studies of effect of GA on shoot elongation.

2.1.1.4 Seed Sowing

The sterilized seeds of Arabidopsis mixed with 0.1% (w/v) agar were sown on plates of ½ MS

medium with the help of a pipette. Together with each mutant genotype, its wild type

13

background was sown on the same plate separated by a line (indicator). This process was carried

out in a laminar airflow cabinet (Medianor Asa, Norway).

2.1.1.5 Growth conditions during germination

The Arabidopsis seeds were stratified at 4°C for 4 days in the dark

to synchronize germination. Germination was induced by placing

the plates for 2 days (12 hours light; light period from 07.00 –

19.00) under white fluorescent light (F96T12/CW/1500, General

Electrics, USA) of 100 µmol m-2

sec-1

at 20° C in a germination

Chamber (Conviron, Controlled environments Ltd., Canada; fig.4).

Figure 4. Agar plates with ½ MS-medium with seeds of Arabidopsis thaliana in the germination

chamber.

2.1.1.6 Light and temperature treatments

Six growth cabinets (locally made by SKP, UMB along with Micro-Matic Norge As, Nesbru,

Norway) were used for the experimental setup (fig. 5). The photoperiod was 12 hours and the

red:far red light ratio and the irradiance were adjusted to 1.7 and a photon flux density of 50

µmol m-2

s-1

respectively. This light was supplied by white fluorescent tubes (F96T12/CW/1500,

General Electrics, USA,) combined with incandescent lamps (60W, Osram, Munich, Germany).

The humidity could not be precisely controlled in these

chambers, but varied between 45±12% relative humidity

corresponding to 1.4-0.9 kPa water vapour defecit. The

temperature was 24°C except for 4 hours where temperature

was reduced from 24°C to 12°C. Such a temperature drop

was given at different time points in the diurnal cycle

according to table 1. To ensure a temperature of 12°C at the

onset of light in the morning, the temperature drop treatment

started 30 min before the light was turned on. In another

experiment, seedlings were exposed to different constant

temperatures, which correspond to the lowest (12°C) and

highest (24°C) temperatures as well as the diurnal average

temperature (22°C) in the drop experiments. All experiments

were carried out for 8 days before the hypocotyl lengths were

measured.

Figure 5. The experimental set up with seedlings of Arabidopsis thaliana in growth cabinet.

14

Table 1. Temperature drops on different chambers. The photoperiod was 12 hours, from 09:00 to

21:00.

Chamber

number

Temperature reduction time

From To

1 08.30 12.30

2 12.30 16.30

3 16.30 20.30

4 20.30 00.30

5 00.30 04.30

6 04.30 08.30

2.1.1.7 Hypocotyl length measurement

To measure the hypocotyl lengths, seedlings were scanned (fig. 6) and length was measured

using Adobe Photoshop (1990 – 2005 Adobe systems Incorporated; Version 9.0.2) and image

tool software i.e., UTHSCSA Image tool (Version 3.0) (http://ddsdx.uthscsa.edu/dig/downloa

.html).

Transfer of plants from

plates to transparent sheet

Transparent sheet

ready to scan

Scanned (pdf) form ready for measurement

Figure 6. Measurement procedure of hypocotyl length of seedlings of Arabidopsis thaliana.

15

2.2 Experiments with Pisum sativum

2.2.1. Sowing and germination of pea seeds.

Seeds of the pea mutant la crys, phyB, long 1, (Weller et al., 2001, 2009; Weston2008) and of the

wild type variety of pea, line 107 (Pisum

sativum L. cv. Torsdag) were sown in plastic

pots (base diameter 8 cm and top diameter 11

cm) containing fertilized peat (Floralux; Nittedal

Torvindustrier, Norway) and grown in

environmentally controlled chambers (cabinet)

(fig. 7). The cabinets and the light conditions

were same as in the experiment with

Arabidopsis thaliana (section 2.1.1.6), i.e., a 12

h photoperiod, an R: FR ratio of 1.7 and a

photon flux density of 50 µmol m-2

s-1

. The

seedlings were watered daily with a nutrient

solution of EC= 1.5 mS cm-1

for the proper

growth (Stavang et al., 2005). The temperature

for the day and night was constant for all the

plants, i.e., 21°C until the epicotyls had emerged.

Figure 7. Pea plants in a growth chamber to measure the stem elongation rate through an angular

displacement transducer.

2.2.2 Light and temperature treatments

Six days after sowing, the plants were transferred to different growth chambers for further

experiments regarding irradiance and temperature drop effects on shoot elongation during a

period of 10 days. Each chamber containing 12-15 plants of mutant plants and 12-15 plants of

the wild type were set for the experiment. The treatments in each chamber are shown in Table 2.

Table 2. Light and temperature details for 12 h photoperiod in experiment with pea.

T1. Time schedule for treatment 1: Constant irradiance of 50 µmol m-2

s-1

and constant

temperature 21°C.

Time Temperature Light in µmol m-2

s-1

07.00 – 19.00 21°C 50

19.00 – 07.00 21°C 0 (Darkness)

16

T2. Time schedule for treatment 2: Increased irradiance to 150 µmol m-2

s-1

for 4 hours in the

middle of the light period and constant temperature 21°C.

Time Temperature Light in µmol m-2

s-1

07.00 – 11.00 21°C 50

11.00 – 15.00 21°C 150

15.00 – 19.00 21°C 50

19.00 – 07.00 21°C 0 (Darkness)

T3. Time schedule for treatment 3: Constant irradiance of 50 µmol m-2

s-1

and temperature

drop from 21°C to 13°C for 4 hours in the middle of the light period.

Time Temperature Light in µmol m-2

s-1

07.00 – 11.00 21°C 50

11.00 – 15.00 13°C 50

15.00 – 19.00 21°C 50

19.00 – 07.00 21°C 0 (Darkness)

T4. Increased irradiance to 150 µmol m-2

s-1

for4 hours in the middle of the light period and

temperature drop from 21°C to 13°C

for 4 hours in the middle of the light period.

Time Temperature Light in µmol m-2

s-1

07.00 – 11.00 21°C 50

11.00 – 15.00 13°C 150

15.00 – 19.00 21°C 50

19.00 – 07.00 21°C 0 (Darkness)

2.2.3 Stem elongation rate recording through angular displacement transducer

Eleven days after sowing plants, the growth rates of plants exposed to the four different

temperature and light treatments described above (Table 2) were monitored. Each chamber

contained 2 mutant (la crys, phyB or long1) and 2 wild type plants. The average recording from

these two plants from each of 3-7 replicate experiments was calculated. For all treatments, fine

scale measurement of stem elongation was conducted every 10 seconds for up to 2 days using an

angular displacement transducer, series 604 (Trans-Tec. Ellington, Connecticut, USA), which

was connected to a data logger, type CR10-AM416 (Campbell Scientific Inc., Shepshed,

Loughborough, England ) as described by Torre and Moe (1998). The values obtained from the

experiment were averaged over a 10 minute period (Stavang et al., 2007).

The curves for growth rates were not always at the same level before the start of the treatments

possibly due to variation between the transducer devices and their adjustments, as well as

17

inherent differences in growth rates between plants. As absolute comparison of the reduction in

growth rate between the control with constant conditions (T1) and other treatments could then

not be done. Thus within each ecotype, we have normalized the values of all curves to the initial

values (before the light and temperature treatments started) for the curve giving the largest

reduction in growth rate during the treatments (T4 i.e., temperature drop and increased

irradiance)

2.3 Statistical Analyses

The effects of the temperature treatments in different genotypes were subjected to statistical

analyses using the general linear model (GLM) analysis of variance procedure in Minitab

(15.1.0.0) program (Minitab Inc., State College, PA, USA). For the Arabidopsis experiments,

effects of the different temperature treatments and relevant genotypes as well as the interaction

between these factors were included in the model: genotype · temperature · genotype x

temperature. For the experiments with pea where the cumulative growth was recorded, the

effects of treatments and genotype as well as the interaction between these factors were analysed

after 10 days of growth. Thus, this model was the following genotype · temperature · genotype x

temperature. In case of statistically significant effects of the different factors, Tukey’s post hoc

test was used to identify specific statistical differences between treatments and genotypes as well

as for interaction genotype x temperature (where the genotypes respond different to the different

treatments).

18

3. Results

3.1 Experiments with Arabidopsis thaliana

3.1.1 Temperature drop in the middle of the day resulted in the largest reduction of

hypocotyl length in Arabidopsis.

In experiments with Arabidopsis, temperature was dropped from 24°C to 12°C in different

diurnal time periods under constant irradiance of 50 µmol m-2

s-1

and 12 h photoperiod (Table 1).

The overall results showed that the hypocotyl length exhibited the strongest reduction when the

temperature was reduced in the middle of the day (from 12:30 to 16:30; fig. 8). On the other

hand, the hypocotyl lengths were least affected when temperature was dropped in the middle of

the night (from 00:30 to 04:30). Both wild type genotypes Ler and Col respond similarly to all

temperature drop treatments. For these genotypes i.e., Ler and Col, the hypocotyl length after a

temperature drop in the middle of the day were 64 and 82% (fig. 8a) lower than after a

temperature drop in the middle of the night. Compared to the middle of the day, the hypocotyl

length was less responsive to a temperature drop during the first and last treatment time in light

(temperature reduction time from 08:30 - 12:30 and 16:30 - 20:30, respectively). After the strong

decline in the growth of hypocotyls in response to a temperature drop in the middle of the day,

the hypocotyl elongation was less sensitive to a temperature drop at all points. When Ler and Col

seedlings were exposed to different constant temperature treatments, the hypocotyl length

increased significantly (P ≤ 0.0001) with increase in temperature with an increase of about 98

and 113%, respectively from 12°C to 22°C and 111 and 168%, respectively from 12 to 24°C (fig

8b). The two genotypes showed a deviation when grown at 24°C. The ecotype Col showed a

greater increase in length from 22 to 24°C as compared to Ler.

Figure 8. (8a) Effect on hypocotyl length of Arabidopsis thaliana (Ler and Col) exposed to 8

days of 4 h diurnal temperature drops from 24°C to 12°C at different times during the diurnal

cycle (denoted 1-6, for time points see table 1) of a 12 h photoperiod. (8b) Hypocotyl length after

8 days in different constant temperatures (12°C, 22°C

and 24°C). Results are mean of 20 plants

in each of 3 replicate plates in 2 and 4 experiments for Ler and Col exposed to diurnal

temperature drops and one experiment for constant temperatures.

19

3.1.2 Effect of gibberellin in hypocotyl growth

To study whether the involvement of the GA pathway is necessary for an effect of diurnal

temperature drops or different constant temperatures on the hypocotyl length in Arabidopsis,

GA3 (50 µM) was included in the growth medium. Although GA3 application stimulated

hypocotyl elongation significantly (P ≤ 0.0001), similar to the untreated seedlings (fig. 8a), the

response to a temperature drop in the middle of the day was strongest and the response to a drop

in the middle of the night was weakest in the GA3 treated plants (fig. 9a). For Col and Ler, the

difference in hypocotyl length between these two treatments was 91% and 80%, respectively.

The response of Ler and Col was similar although Col appeared to elongate less than Ler in

response to GA3 upon a temperature drop in the end of night. Arabidopsis Col plants applied

with GA3 (50µM) under constant temperatures of 12°C, 22°C and 24°C (fig. 9b) also elongated

significantly 32% more than untreated Col plants (P ≤ 0.0001). The GA3 treated Col plants

showed a hypocotyl length difference between 12°C and 22°C of 118%. Similarly, the difference

between 12°C and 24°C

was found to be 190%.

Figure 9. (9a) Effect of application of GA3 (50µM) on hypocotyl length of Arabidopsis thaliana

(Ler and Col) exposed for 8 days to 4 h diurnal temperature drops from 24°C to 12°C at different

times during the diurnal cycle (denoted 1-6, for time points see table 1) of a 12 h photoperiod.

(9b) Effect of GA3 on Col hypocotyl length in different constant temperatures (12°C, 22°C and

24°C) after 8 days. Results are mean of 20 plants in each of 3 replicate plates ± SE.

3.1.3 An apparent role of DELLA proteins in response to altered temperature

The role of DELLA proteins in response to altered temperature was investigated by studies of

della quadruple and pentuple mutants in Arabidopsis. Both della mutants followed the same

pattern as the wild type with highest and lowest sensitivity to a temperature drop in the middle of

the day and night, respectively. However, the overall results suggest that the della mutants

showed slightly-less difference in hypocotyl length than the wild type in response to the

temperature drop during the middle of day time as compared to the middle of night. The

hypocotyl length difference between the mid-day and mid-night drops for della quadruple, della

pentuple and their background i.e., Ler were 51%, 58% and 65%, respectively (fig. 10a). In the

wild type, the effect of drop in the middle and end of the day was significantly different from all

other drop treatments (P ≤ 0.0001). In contrast, there was no significant differences in effect of a

temperature drop in the middle and end of the day for the two della mutants (drop time 2 and 3

20

(P=1.0). Also, both the wild type and the della quadruple mutant differed from the della pentuple

mutant in that they showed no significant difference in effect of a temperature drop in the

beginning of the day (1) and the beginning of the night (4) (P=1.0). These differences indicate

that the responses to the different diurnal drop treatments differ between these genotypes. The

hypocotyl growth pattern also increased with increased constant temperatures. The hypocotyl

length difference of della quadruple, della pentuple and Ler between constant temperatures 12°C

and 22°C were 58%, 36% and 98% respectively. Similarly, the hypocotyl length difference of

the above mentioned mutants and their background between 12°C and 24°C were 85%, 46% and

111% respectively (fig. 10b). Thus, it appeared that the pentuple della mutant was the least

sensitive to different constant temperatures.

Figure 10. (10a) Effect on hypocotyl length of della quadruple and pentuple mutants and wild

type Ler of Arabidopsis thaliana exposed for 8 days to 4 h diurnal temperature drops from 24°C

to 12°C at different times during the diurnal cycle (denoted 1-6, for time points see table 1) of a

12 h photoperiod. (10b) Hypocotyl length of the della mutants and wild type (Ler) after 8 days in

different constant temperatures (12°C, 22°C

and 24°C). Results are mean ± SE of 20 plants in

each of 3 replicate plates in one and two experiments for constant temperature and diurnal

temperature drops, respectively.

3.1.4 Differential response to temperature in phy mutants

To investigate further whether phytochromes are somehow involved in temperature signaling,

the response to diurnal temperature drops and different constant temperatures were investigated.

Decreased temperature of 4 hours in the middle of the day reduced the hypocotyl length of phy

mutants. However, the phyB and phyA/phyB double mutant showed a smaller difference in

response to temperature drop treatment in the middle of the day than in the middle of night as

compared to the phyA mutant and its background wild type grown along with it (fig. 11a). The

hypocotyl length difference between the mid-day and mid-night drops for the phyA, phyB,

phyA/phyB double mutant and Ler were 70%, 23%, 18%, and 74%, respectively. Similarly, the

phyB and the phyA/phyB double mutant showed less response to differences in constant

temperatures as compared to other genotypes (fig. 11b). The difference in hypocotyl length of

phyA, phyB, phyA/phyB double mutant and Ler between constant temperatures 12°C and 22°C

were 66%, 18%, 18% and 98%, respectively. Similarly, the hypocotyl length differences of the

above mentioned mutants and their background between the temperatures 12°C and 24°C were

73%, 25%, 33% and 111%, respectively.

21

Figure 11. (11a) Effect on hypocotyl length of different phy mutants and the wild type Ler of

Arabidopsis thaliana exposed for 8 days to 4 h diurnal temperature drops from 24°C to 12°C at

different times during the diurnal cycle (denoted 1-6, for time points see table 1) of a 12 h

photoperiod. (11b) Hypocotyl length of the phy mutants after 8 days in different constant

temperatures (12°C, 22°C

and 24°C). Results are mean ± SE of 20 plants in each of 3 replicate

plates in each of one, two and three experiments for the double mutant, the individual phy

mutants and the wild type Ler exposed to diurnal temperature drops and one experiment for

constant temperatures.

3.1.5 PIF4 activity is involved in response to altered temperature

To investigate further the involvement of PIF genes in control of hypocotyl growth by

temperatures, pif4, pif5 and pif4/pif5 double mutants were compared with wild type plants with

the same genetic background i.e., Col-0. The hypocotyl length difference between temperature

drop treatments in the middle of the day and night for pif4, pif5, pif4/pif5 double mutant and Col

were 37%, 93%, 38% and 82%, respectively. This indicates that the pif4 and the pif4/pif5 double

mutant were less sensitive to the temperature drops than Col and the pif5 mutant (fig. 12a). The

response of the pif mutants to constant temperatures was somewhat similar to the response to

reduced temperature of 4 hour periods. The hypocotyl length difference of pif4, pif5, pif4/pif5

double mutant and Col between the constant temperatures 12°C and 22°C

were 38%, 71%, 42%,

and 118% respectively. Similarly, the differences in hypocotyl length of these mutants between

12°C and 24°C were 62%, 99%, 98% and 190% respectively (P ≤ 0.0001) (fig.12b).

Figure 12. (12a) Effect on hypocotyl length of pif mutants of Arabidopsis thaliana exposed for 8

days to 4 h diurnal temperature drops from 24°C to 12°C at different times during the diurnal

22

cycle (denoted 1-6, for time points see table 1) of a 12 h photoperiod. (12b) Hypocotyl length of

the pif mutants after 8 days in different constant temperatures (12°C, 22°C

and 24°C). Results are

mean ± SE of 20 plants in each of 3 replicate plates in one and three experiments for pif mutants

and the wild type (Col) exposed to diurnal temperature drops, respectively and one experiment

for constant temperatures.

3.1.6 Effect of toc1 mutation in response to altered temperature

In order to try to evaluate whether the rhythm in temperature sensitivity is due to an underlying

circadian mechanism, the sensitivity of the toc1 mutant to diurnal temperature drops was

analyzed. The hypocotyl length suggested a difference between the mid-day and mid-night drops

for toc1 and its background i.e., C24 were 76% and 118%, respectively (fig. 13a). Similarly, the

growth pattern was increased with increased constant temperatures (12°C, 22°C and 24°C). The

hypocotyl length differences of toc1 and C24 mutants between constant temperatures 12°C and

22°C were 86% and 103%, respectively. Similarly, the hypocotyl length differences of the toc1

mutant and its background between the temperatures 12°C and 24°C were 118% and 151%,

respectively (fig. 13b).

Figure 13. Effect on hypocotyl length of the toc1 mutant of Arabidopsis thaliana exposed for 8

days to 4 h diurnal temperature drops from 24°C to 12°C at different times during the diurnal

cycle (denoted 1-6, for time points see table 1) of a 12 h photoperiod. (13b) Hypocotyl length of

the toc1 mutants after 8 days in different constant temperatures (12°C, 22°C

and 24°C). Results

are mean of 20 plants in each of 3 replicate plates ± SE.

3.2. Angular displacement transducer experiments with pea

The elongation rates of different mutants compared to the wild types were evaluated after 4 hour

temperature drop from 21 to 13°C or increased irradiance from 50–150 µmol m-2

s-1

in the

middle of the light period, either given separately (T3 and T2 respectively) or in combination

(T4). These treatments were compared with exposure to constant irradiance at 50 µmol m-2

s-1

and constant temperature of 21°C (T1).

3.2.1 Wild type

Compared to T1, the wild type showed a reduction in growth rate in all treatments (fig 14). T4

treated plants showed the largest reduction in growth by 41% (averaged over the treatment

23

period), whereas T2 and T3 resulted in reduction of growth by 27 and 34%, respectively, all as

compared to T1 (fig. 14). After the treatment period, there was a gradual increase in growth.

Figure 14. Effect of different irradiance and temperature combinations on rate of shoot

elongation in the wild type of pea. The treatment period of 4 h in the middle of the light period of

12 h (07:00-19:00) is indicated by blue arrows. 1 CT (referred to as T1 in the thesis text) =

constant irradiance of 50 µmol m-2

s-1

at 21°C. 2 CT Light (T2) = increased irradiance from 50-

150 µmol m-2

s-1

at 21C. 3 Drop (T3) = temperature drop from 21°C to 13°C at irradiance of 50

µmol m-2

s-1

. 4 Drop light (T4) = Temperature drop from 21°C to 13°C and increased irradiance

from 50-150 µmol m-2

s-1

. Results are mean of 6 and 5 experiments with 2 plants in each for T1

and T2, and 7 and 6 experiments with 2 plants in each for T3 and T4, respectively.

3.2.2 The la crys mutant

From the angular displacement transducer experiment, it appeared that in contrast to the wild

type, there were no differences in the growth rate of the la crys della mutant plants exposed to

different irradiances at the same temperature, i.e., T1 and T2 on the one hand and T3 and T4 on

the other. However, the temperature drop treatments, T3 and T4 were both found to be effective

for reduction of the growth rate (fig.15). The T4 (constant irradiance and temperature drop)

showed a sharp decline in growth rate by 53% (averaged over the treatment period), whereas the

growth rate in plants treated with T3 were reduced by a similar value (50 %) after a lag-time of

about 4 h, as compared to T1 and T2.

24

Figure 15. Effect of different irradiances and temperature combinations on the rate of shoot

elongation in the la crys mutant in pea. The treatment period of 4 h in the middle of the light

period of 12 h (07:00-19:00) is indicated by blue arrows. For treatment details see fig. 14.

Results are mean of 3 experiments with 2 plants in each.

3.2.3 The long1mutant

The long1 mutant did not show any clear difference in growth rate in response to the different

temperature and light combinations (fig. 16), except for a possible slight tendency of decreased

growth rate (by 9%) after a temperature drop either given separately (T3) or in combination with

increased irradiance (T4). Like the wild type, the elongation followed a normal pattern with

increased growth rate after the light was turned off in the evening (at 19:00).

Figure 16. Effect of different irradiance and temperature combinations on rate of shoot

elongation in the long1 mutant of pea. The treatment period of 4 h in the middle of the light

25

period of 12 h (07:00-19:00) is indicated by blue arrows. The treatments were as described in

figure 14. Results are the mean of 3 and 4 experiments with two plants in each for treatment 1

and 3, and 2 and 4, respectively.

3.2.4 The phyB mutant

As expected, the phyB mutant showed a higher growth rate than the wild type. Interestingly,

upon transition from darkness to light (at 07:00), it showed an inhibition of elongation growth

like wild type plants, but as opposed to the wild type this transient only and a growth rate similar

to in darkness was restored after about 2 hours in light (fig. 17). Furthermore, the phyB mutant

showed similar gradual response patterns to the treatments as wild type plants. T4 showed a

gradual decrease in stem elongation rate during the treatment period. T4 was reduced by 56%,

whereas T3 was declined by 45%. Similarly, T2 was decreased by 32%, as compared to T1.

Figure 17. Effect of different irradiance and temperature combinations on rate of shoot

elongation in phyB mutant of pea. The treatment period of 4 h in the middle of the light period of

12 h (07.00-19:00) is indicated by blue arrows. Growth conditions as in figure 14. Results are the

mean of 4 replicate experiments with 2 plants in each.

3.3 Cumulative stem growth in pea

To examine the effect of irradiance and temperature drop on cumulative shoot elongation, plants

were exposed to the same four diurnal temperature-light treatments as described above (Table 2)

for 10 days.

26

3.3.1 Wild type

Among the 4 treatments, T4 was responsible for the most prominent reduction of shoot

elongation in the wild type with a significant 29% reduction (P ≤ 0.0001) compared to T1 after

10 days of growth (fig. 18). Plants treated with T2 and T3 remained in an intermediary position

and were not clearly different from each

other, although T2 significantly differed

from T1 (P ≤ 0.0001) where as T3 was not

different from T1 (P = 0.1655). Similarly,

T4 was not significantly different from T3

(P ≥ 0.4130). Looking isolated at the

situation after 10 days, there appeared to be

about 6-9% reduction only in shoot

elongation, for these T2 and T3 as

compared to T1.

Figure 18. Average cumulative growths of the wild type of pea exposed to different light and

temperature combinations for 4 h in the middle of the 12 h photoperiod. 1 CT (referred to as T1

in the thesis text) = constant irradiance (50 µmol m-2

s-1

) and temperature (21oC). 2 CT Light =

increased irradiance (from 50 µmol m-2

s-1

to 150 µmol m-2

s-1

) and constant temperature as in

T1. 3 Drop = constant irradiance as in T1 and a 4 h temperature drop in the middle of the light

period (from 21 to 13oC). 4 Drop Light= increased irradiance as in T2 and temperature drop as in

T3. Results are mean ± SE of 4 experiments with 51 plants in each experiment for each genotype

3.3.2 The La crys mutant

The differences were small among the four treatments. Two of them, T3 and T4 i.e., constant

irradiance for 4 hours in day time and a temperature drop at the same time, and the application of

increased irradiance with temperature drop appeared the most effective for the reduction of shoot

elongation (fig. 19). After 10 days these treatments appeared to result in a 10% decrease in

elongation growth compared to T1 where constant irradiance and temperature was given. There

was no significant effect of increased

irradiance (T2 and T3) (P=0.1655). As

expected, the la crys mutant was more

elongated than the wild type. Whereas, the

mutant had grown about 45 mm during 10

days period, the wild type had grown about

30 mm.

Figure 19. Average cumulative growth of la

crys mutant of pea exposed to different light

and temperature combinations for 4 h in the

27

middle of the 12 h photoperiod. Growth conditions were as described in figure 18. Results are

mean ± SE of 12 plants for each treatments.

3.3.3 The long1 mutant

The average cumulative growths of long1

mutants after 10 days growth were similar

in all treatments (P ≤ 0.0001) (fig. 20).

Figure 20. Average cumulative growth of

long1 mutant of pea exposed to different

light and temperature combinations for 4 h

in the middle of the 12 h photoperiod.

Growth conditions were as described in

figure 18. Results are mean of 15 plants in

each treatment ± SE.

3.3.4 The phyB mutant

Treatments 1, 2 and 3 appeared to affect

shoot elongation in the phyB mutant

almost equally (fig. 21). However, a

temperature drop in combination with

increased irradiance (T4) resulted in

significantly shorter plants (22%)

compared to control plants (P ≤ 0.0001)

(fig. 21).

Figure 21. Average cumulative growth of

phyB mutant of pea exposed to different

light and temperature combinations for 4 h in the middle of the 12 h photoperiod. Growth

conditions were as described in figure 18. Results are mean ± of 2 experiments with 12 and 15

plants in each for each treatment.

28

4. Discussion

4.1 Diurnal temperature reduction reduces the hypocotyl length in Arabidopsis

In order to investigate the effect of temperature drops in reduction of elongation growth at

different time points during the diurnal cycle, Arabidopsis seedlings were exposed to 4 hours

diurnal temperature drops from 24 to 12°C at 6 different time points (table 1). Plants were also

grown at different constant temperatures of 12 and 24°C as well as 22°C, which correspond to

the average temperature in each diurnal drop treatment. The hypocotyl length of different wild

types of Arabidopsis showed the greatest reduction when the temperature was reduced during the

mid-day (fig. 8a). The lowest sensitivity to a temperature drop was observed in the middle of the

night. The different wild types Ler and Col showed very similar responses. A few earlier

experiments have also showed decrease in stem length in response to temperature reductions in

light. In pea, a 2 hours temperature drop in the middle of the day reduced shoot elongation more

than a temperature drop in the middle of the night (Stavang et al., 2007). Also, a diurnal

temperature drop of 2 hours at the end of the night and the beginning of the day reduced plant

height, diameter and other parameters in B.×hiemalis and poinsettia (Moe et al., 1992). However,

in these studies, no systematic studies of effect of temperature changes through the entire diurnal

cycle were performed. The different sensitivity to temperature drop in the middle of the day and

in the middle of the night, but also different sensitivity within the light period and within the dark

period, as observed in the present work, might suggest the involvement of a circadian clock.

Indeed, several hormone related-genes and also levels of some growth-controlling hormones

such as indole-3-acetic acid (IAA) have been shown to exhibit circadian rhythms (Stavang et al.,

2005; Michaels et al., 2008; Mizuno and Yamashino, 2008; Alabadi and Blazquez, 2008).

Accordingly, this might at least partly explain the differential temperature sensitivity through the

diurnal cycle. During constant temperature treatments (12, 22 and 24°C), the two ecotypes Ler

and Col responded similarity, i.e., hypocotyls elongation increased with increasing temperature

(fig. 8b). Effects of different temperatures on elongation growth are well documented (Penfield,

2008). However, the hypocotyl length of Col appeared shorter as compared to Ler, except for at

24°C where the heights of these ecotypes were similar (fig. 8b). It can thus be speculated

whether Col responds more strongly to higher temperatures. However, the results of Stavang et

al. (2009) where Ler and Col plants were transferred from 20 to 29°C did not indicate any

systematic difference in temperature response between the genotypes, only variation between

experiments. Thus, although the plant number was quite high in the present experiment (60

plants altogether), it would be advisable to repeat it since it was performed once only. Since we

often observed curling of plants, it was necessary to sow in excess and select straight plants to be

able to measure hypocotyl lengths properly. This selection might have influenced the results to a

certain extent, and underline the importance of including high numbers of plants in such

experiments.

In pea, it has been shown that a larger effect of a diurnal temperature drop on shoot elongation in

the middle of the day than in the middle of the night is linked to reduced levels of active GA1 and

29

increased expression of the GA-inactivation gene GA2ox2 (Stavang et al., 2007). Thus, we

hypothesized that GA-saturation might reduce the difference in response to temperature drops in

the day and in the night. As expected, application of GA3 (50µM) in the medium was effective

for stimulation of hypocotyl length in all temperature treatments (fig. 9). Both genotypes Ler and

Col responded similarly, although the curve for effect of diurnal temperature drops in Col was

more irregular for some reason or another, possibly due to experimental error since many of the

plants showed non-straight hypocotyl growth. However, the GA3-concentration used was also

applied in a study of effect of elevated temperature on hypocotyl elongation (increase from 20-

29°C), and then such apparently stress-related curling was generally not observed (Stavang et al.,

2009). This might be due to that sucrose was included in the medium in the experiment by

Stavang et al. (2009). In our experiment this was aimed to avoid any interaction between sucrose

and the treatments and sucrose was accordingly not added. It might also be that simultaneous

GA3-application and temperature drop treatments was perceived as more stressful than GA3-

application together with elevated temperature. Somewhat surprisingly, no effect of GA

application on the response to temperature drop at different time points during the diurnal cycle

was observed (fig. 9a). This might suggest that the level of active GA in temperature response is

not as important as hypothesized earlier (Grindal et al., 2008; Stavang et al., 2005, 2007, 2009).

Thus, in spite of that a different response to temperature drop in the middle of the day and in the

middle of the night was correlated with lower and higher GA1 levels and higher and lower

expression of GA2ox2 (Stavang et al., 2007), the possibility exists that there is not a causal

relationship in this respect. Alternatively, the GA application might have induced the feed-

forward mechanism which exists under high levels of active GA, resulting in increased

expression of GA2ox2-genes (Yamaguchi, 2008). However, since GA3 in contrast to the active

GAs, GA4 and GA1 has a double bound between carbon 1 and 2, it is less prone to 2β-oxidation

and thus more stable than GA1 and GA4 (Yamaguchi, 2008). It was nicely demonstrated by

comparing effects of GA1 and GA3 application on pea exposed to negative DIF treatment

(Grindal et al., 1998). The GA1 application reduced the inhibitory effect of negative DIF on

shoot elongation more than GA3 application. On the other hand, in pea exposed to different DIF

treatments for 12 days, active GA1 levels were reduced under negative DIF, but there were no

diurnal differences in GA1 levels for any of the DIF treatments (Stavang et al., 2005). In that

experiment, it might be that the GA1 levels had somehow stabilized since analyses were done

only after long-term DIF exposure. Taken together, why a smaller difference between a

temperature drop in the middle of the day as compared to the middle of the night was not

observed in the GA3 applied plants, appears somewhat surprising and a good explanation cannot

as yet be provided.

We had studied the role of della mutants in response to altered temperature. The result showed

that stem elongation in the della quadruple and della pentuple mutant is sensitive to altered

temperature particularly at the mid-day and to different constant temperatures. The della

quadruple mutant showed a reduced hypocotyl length as compared to the della pentuple mutant,

30

but higher length than its background (Ler).These della mutants appear somewhat less sensitive

than the wild type to diurnal temperature drops in the middle of the day as compared to the

middle of the night. The della quadruple and della pentuple were 40 and 50% less sensitive as

compared to Ler. Furthermore, the della mutants, particularly the pentuple della mutant appeared

less sensitive to different constant temperatures than the wild type (fig. 10b). At least in constant

temperatures, this might suggest a particular temperature-response related to role of RGL3,

which is mutated in the pentuple but not in the quadruple mutant. A role of RGL3 in response to

low, above-zero temperature with respect to cold acclimatization has been suggested (Achard et

al., 2009). Two of the DELLA proteins of Arabidopsis i.e., RGA and GAI are mainly responsible

for inhibition of stem growth in being high when GA levels are low (Dilli et al., 2001; King et

al., 2001). Stavang et al., (2009) showed that many DELLA proteins in conjugation contribute to

the regulation of growth responses at different temperatures. Plants bearing semi-dominant

DELLA alleles (gai-1 and rga-17) exhibit the same relative growth response at two temperatures

i.e., 20oC and 29

oC, as wild type plants. This indicates that the obstruction in the GA signaling

pathway caused by single dominant DELLA alleles can be overcome by temperature. The

seedlings deficient in four (rga-t2, gai-t6, rgl1-1 and rgl2-1; Achard et al., 2006) or in all five

DELLA genes (rga-t2, gai-t6, rgl1-1, rgl2-1 and rgl3-1; Feng et al., 2008) also showed a partial

response in fully active GA pathway. A larger group of DELLA proteins is needed to regulate

growth responses at stressful temperatures (Stavang et al., 2009). That the della mutants showed

a partial response only to temperature change as compared to wild type indicates that the activity

of an intact GA signaling pathway is necessary to fully promote hypocotyl growth when the

temperature changes (fig. 10; Stavang et al., 2009).

To examine the role of phytochromes in the temperature control of stem growth in Arabidopsis,

we conducted experiments with phyA, phyB and phyA/phyB double mutants. The phyA, phyB

and phyA/phyB double mutant showed higher elongation rates, i.e. 32, 92 and 111%, respectively

than the wild type (Ler). As expected on the basis of earlier studies, the phyB lacking plants were

slender and longer than the wild types and the phyA mutants (fig. 11; Weller et al., 1995). Both

the phyA/phyB double mutant and the phyB mutant showed less response to altered temperature

as compared to the phyA mutant and its wild type (Ler), which had more reduced hypocotyl

length upon a temperature reduction during mid-day as compared to the middle of the night

(fig.11). The wild type appeared to be almost one and half-fold more affected by the treatment as

compared to plants mutated in phyB. It might be that a temperature drop in the middle of the

light period is perceived by the plant as more stressful to the growth than a temperature drop in

the dark. Thingnaes et al., (2008) also observed that a negative DIF reduced stem and petiole

elongation in Arabidopsis genotypes lacking phyB, while phyD and phyE mutation had very little

influence on temperature effect. These results as well as the result from the present study suggest

that phyB is needed for a complete thermoperiodic control of elongation growth in A. thaliana.

Similarly, during constant temperatures at 24oC, the phyB mutant showed 102% elongation as

31

compared to its wild type. Thus, phyB presence is also a prerequisite for a complete response to

different constant temperatures.

Stavang et al., (2009) and Koini et al., (2009) showed that PIF4 is a critical factor for regulating

growth in response to increased temperature. Also, they found that the PIF4 deficient mutants did

not show an elongation response or leaf hyponasty upon transfer to high temperature. To

investigate the involvement of the PIF4 and PIF5 genes in control of hypocotyl growth by

temperature drops at different diurnal time points as well as different constant temperatures, the

pif4, pif5 and pif4/pif5 double mutants were compared with their genetic background (Col) in the

present study. The results showed that pif4 is less sensitive to altered temperature generally. The

pif4, pif5 and pif4/pif5 double mutant subjected to the treatment exhibited 41, 18 and 44%,

respectively, decrease in growth as compared to its wild type (Col). A similar pattern was

followed during the constant temperature treatments also (fig. 12). The induction of PIF4

expression after transfer from 20 to 29oC was detected in cotyledons and hypocotyls where as

PIF5 expression was detected only in hypocotyls (Stavang et al., 2009) i.e., at higher

temperatures the level of PIF4 and PIF5 increase. The role of PIF4 in temperature signaling does

not, however, appear to operate through interaction with either phytochrome or DELLA proteins,

suggesting the existence of a novel regulatory mechanism (Koini et al., 2009). Taken together,

the earlier studies related to increased temperature (Stavang et al., 2009, Koini et al., 2009) as

well as the present study where the effect of diurnal temperature drops and different constant

temperatures show that PIF4 is an important component of plant high temperature signaling and

integrates multiple environmental cues during plant development.

If the circadian clocks are affected in Arabidopsis, the mutant shows increased hypocotyl length

(Zagotta et al., 1996). In the present research, we aimed to study whether the rhythmicity in

temperature sensitivity is due to an underlying circadian mechanism. Similar to its C24 wild type

background, the toc1 mutant showed the largest reduction in hypocotyl length during the 4 h

temperature drop in the mid-day and rhythmicity through the diurnal cycle. However, it appeared

to exhibit a larger difference in hypocotyl length between a temperature drop in the middle of the

day and the middle of the night. Many genes in Arabidopsis has been identified, whose

expression oscillates robustly and diurnally during day/night cycle (Harmer et al., 2000; Schaffer

et al., 2001; Mizuno and Yamashino, 2008). Mizuno and Yamashino, (2008) used Arabidopsis

public microarray database with Affymetrix Genechip Arabidopsis ATH1 22K arrays,

representing almost all protein-coding transcripts of the reference plant. Finally, Blasing et al.

(2005) used these databases and identified certain number of genes (classified into two groups)

whose expression was inferred to oscillate diurnally and robustly with high amplitudes. Group A

(CCA1, LHY, TOC1/PRR1, PRR3/5/7/9, G1 and ELF4) indicating high amplitude gene family

whose expression oscillates diurnally and robustly. Similarly, Group B representing moderate

gene family. The involvement of the circadian clock in the different response to temperature

drop through the diurnal cycle is highly probable, but need further investigation. In the present

32

study we aimed to include also mutants in other central components of the circadian clock, i.e.,

the cca1 and lhy-mutants, but unfortunately the seeds did not germinate.

4.2 Increased irradiance and temperature drop affect the stem elongation rate and

cumulative stem growth in pea

The effects of the temperature and light responses on elongation growth in pea were studied in

different mutants. The elongation rates of the different mutants were analyzed in 4 different

treatments by using angular displacement transducer in short-time experiments and cumulative

growths were studied in a 10 days period. Both these experimental approaches showed similar

results in each genotype.

In the short-term transducer experiments, the wild type, pea plants subjected to a temperature

drop and increased irradiance in the middle of the day (T4) showed a reduction in growth rate by

41% (fig. 14) compared to treatment 1 (T1) where irradiance and temperature was kept constant.

Similarly, wild type plants subjected to 10 days treatment exhibited 29% (fig. 18) decrease in the

stem elongation in response to T4 as compared to T1. Results were similar to previous

experiments conducted by Stavang et al., (2007), where the temperature was dropped from 21 to

13°C for two hours in the middle of the light period. In this experiment, the irradiance was

constant i.e., about 180 µmolm-2

s-1

. Then stem elongation was then reduced by 55% within 30

min. Also, Begonia hiemalis plants exposed to a temperature drop of 3-6°C showed reduced

growth as compared to the plants grown at constant temperature (Grindal et al., 1994). Similarly,

Petunia×hybrida grown at an equivalent temperature increase of 2 hours light period resulted in

higher plants (Sysoyeva et al., 2001). In the present experiments, the application of only

temperature drop (T3) had less effect as compared to when irradiance was increased

simultaneously (T4). These experiments demonstrate than an increase and decrease of

temperature in the photoperiod affects the growth of the plants.

The la crys mutants subjected to 4 h diurnal temperature drop treatment showed similar growth

reduction pattern like the wild type. From the transducer experiment, T4 was found as the most

effective treatment for the reduction of the stem growth rate. There was 49% reduction in growth

as compared to T1 (fig. 15). This is similar to the wild type, which must suggest that the la and

crys DELLA proteins are not essential for response to a temperature drop in the middle of the

light phase. On the other hand, cumulative growth at the end of a 10 days period was less

different between T4 and T1, i.e., T4 resulted in a reduced stem elongation by 11% only after 10

days (fig. 19). This might suggest that the DELLA proteins may be more important in long-term

than short-term responses to diurnal temperature changes. A role in more long-term responses to

temperature changes is supported by that the la crys mutant showed no or very poor DIF

response on stem elongation (Stavang et al., 2010). Stavang et al. (2009), showed that many

DELLA proteins in conjunction contribute to the regulation of hypocotyl growth responses at

different temperatures (20 and 29°C). Also, a role of DELLA in more long-term temperature

33

responses is supported by the present Arabidopsis experiments discussed above. These plants

were exposed to different temperature treatments in an 8 days period. Plants mutated in four or

all five DELLA proteins appeared somewhat less sensitive to temperature than the wild type (fig.

10). Particularly the pentuple della mutant appeared less sensitive to different constant

temperatures, suggesting a role of RGL3 in the temperature responses. On the other hand, the la

crys mutant showed little response to changes irradiance in both types of experiments in the

present study, indicating an important role of these DELLA proteins in response to increased

irradiance. The della mutant are generally elongated and slender due to lack of functional

DELLA proteins, i.e., la and crys (Weston et al., 2008). In Arabidopsis, the DELLA proteins i.e.,

RGA and GAI are known to be responsible for the growth responses of hypocotyl (Dill et al.,

2001).

The phyB mutant also showed similar response to the different treatments as the wild type. In

this genotype also, T4 was the most effective treatment for the short-term reduction of the stem

elongation rate, which was reduced by 39% as compared to T1 (fig. 17). The effect of T4 on

cumulative elongation growth after 10 days of treatment was similar, although smaller, with a

22% reduction of stem growth (fig. 21). The smaller response of the phyB pea mutant in the

longer term experiment compared to the short-term registration of elongation rate, might suggest

that PHYB might play a role in more long-term temperature responses. This is supported by the

experiments in Arabidopsis where hypocotyl lengths were recorded after a period of 8 days in

different temperature treatments (fig. 11). In these experiments phyB mutants were less sensitive

to temperature than the wild type and the phyA mutant. However, since the short term transducer

experiments with the pea phyB mutant suggested that the mutant was responding quite similarly

to the wild type to the different treatments. It would be interesting to investigate also the

involvement of PhyA in response to temperature in pea. Seeds of this mutant were unfortunately

not available when the experimental work of the thesis was done. Stavang et al., (2009) observed

PIF4 as a critical factor in Arabidopsis for regulating growth in response to temperature. The

expression of PIF4 and PIF5 is enhanced at higher temperature in hypocotyls. PIF4 is known to

act downstream of phyB (Lorrain et al., 2008), but the results of Stavang et al. (2009) also

suggest the existence of a PhyB-independent pathway for effects of temperature on PIF4.

Unfortunately, no pif-mutants are as yet available in pea, so this connection cannot be tested in

this species. Taken together, our results with Arabidopsis and to a certain although smaller

extent, also with pea, might suggest a role of PhyB in temperature responses with respect to

elongation growth. In control of flowering by ambient temperature PhyB was shown to play a

role (Hallidat et al., 2003), but also other phytochromes or the interaction between them have

been suggested to be involved (Halliday and Whitelam, 2003). Furthermore, Mølmann et al.

(2005) showed an effect of temperature on PhyA action in Populus. Also, a study of seed

germination in Arabidopsis suggests a similar situation for different phytochromes in this respect

(Heschel et al., 2007). Taken together, it might be hypothesized that phytochromes might, in

some way or another play a role in temperature sensing (Olsen, 2010). Furthermore, there might

34

be other associated photoreceptors that are involved in the regulation of growth under changed

light and temperature conditions. Cry2 has been suggested to play a role in control of flowering

by ambient temperature since a cry2-mutant exhibit changes flowering time in 23°C as compared

to the wild type (Blazquez et al., 2003). Interestingly, the phyB mutant showed an inhibition of

the elongation growth rate upon transfer from darkness to light, but within about 2 hours the

growth rate was again similar to in darkness. Thus, although the plants apparently sense changes

in light, it appears that the lack of PhyB make the plants unable to sense an extended period of

light.

The LONG1, which is a transcription factor in pea, is orthologous to HY5 of Arabidopsis. Seeds

of a hy5 mutant were not yet available when the work of this thesis was done. The long1 mutant

of pea was introduced in this experiment to study its response towards the light and temperature.

The long1 mutant seedling showed an immediate relationship in the light regulation of active GA

levels and the expression of several GA biosynthetic genes, e.g. GA2ox2 (Weller et al., 2009).

Since a negative DIF treatment and a temperature drop in the light have been shown to stimulate

expression of GA2ox2 and thus reduction of the active GA1, we hypothesized that LONG1 is also

involved in temperature responses. This was observed in the present study. This genotype

appeared to be virtually insensitive to temperature drop and increased irradiance. Only a very

small 9% reduction (fig. 16) in growth was observed when treated with T4 (as compared to T1).

Similarly, there was hardly any reduction in shoot elongation after 10 days of treatment (fig. 20).

This might be due to the condition that LONG1 has a central role in mediating the effects of both

light and temperature on GA biosynthesis and in photomorphogenic development. Stavang et al.

(2007) and Gonnet (2009; master thesis) has analysed the transcript levels of different genes of

the GA metabolism in wild type of pea and phyB mutant. Also, the analysis of transcript level of

long1 mutant is on progress (Olsen JE, personal communication). These analyses showed that

while expression of GA2ox2 is rapidly up-regulated in the wild type by increased irradiance and

temperature drop as well as their interaction (T4), the expression of GA2ox2 is similar in all

treatments for the long1 mutants. Thus, in addition to being important in response to increased

irradiance (Weller et al., 2009; fig 20), LONG1 apparently plays a role also in response to

temperature (fig. 16 and fig. 20).

4.3 Proposed temperature signaling pathways

On the basis of our results, it can be proposed that the temperature signaling network is at least

partly analogous to the signaling network in light, i.e., light and low temperature acts at least

partly through a common signaling network. The proposed signaling pathways are (with the

names of components in Arabidopsis/pea) PhyB – (COP1/LIP1) – HY5/LONG1 – DELLAs/LA

CRYs – (PIF4) and HY5/LONG1 –GA2ox1/GA2ox2.

In pea, two transcription factors i.e., LONG1 and LIP1 are orthologous to HY5 and COP1 of

Arabidopsis, respectively (Weller et al., 2009). Although, very similar in the C-terminal, LONG1

35

contains an additional N-terminal domain, which is different from HY5. HY5/LONG1 acts a

downstream of the photoreceptors PhyA and phyB as well as Cry(s) (Weller at al., 2009). The

HY5/LONG1 protein is present in light. In darkness, it is degraded due to action of COP1/LIP1,

which is then present in the cell nucleus (Bae et al., 2008). After transfer to light, COP1/LIP1 is

translocated to the cytoplasm. Thus, in light HY5/LONG1 builds up and contributes to the

photomorphogenesis due to its interaction with GA2ox1/GA2ox2 and DELLAs. DELLAs in turn

interact with PIFs. In light, a functional PHYB protein targets PIF4 to degradation by the 26S-

proteasome (De Lucas et al., 2008), and also contribute positively to the light-promoted

accumulation of DELLA proteins (Achard et al., 2007). Although, several of the factors shown

to be involved in light signaling (transition dark-light) have not been studied yet with respect to

effects of temperature in both Arabidopsis and pea, the results obtained so far (Stavang et al.,

2005; 2007; 2009, 2010, present study) suggest that we can propose a similar model for

temperature signaling with PhyB, LONG1, DELLAs, PIF4, GA2ox1/GA2ox2 and active GA as

central players. However, it might be that there is also a DELLA-dependent pathway for effect of

PhyB on PIF4 at least in Arabidopsis, since the della mutants in this species appear somewhat

less sensitive to a temperature drop than the phyB and pif4 (this study) mutants. This was also

suggested by Stavang et al. (2009).

36

5. Conclusions

From the present investigation, it was observed that the temperature drop of 4 hours in the

middle of the day and night resulted in the largest and smallest reduction of hypocotyl length,

respectively in Arabidopsis. Although, involvement of GA pathway (i.e., changed GA-levels)

seem necessary for a temperature control of shoot elongation in plants (Stavang et al., 2005;

2007; 2009; 2010). The application GA3 (50 µm) in our research did not, for unknown reasons,

seem effective to reduce the response to temperature. Mutations in della were effective to a

certain degree for reduction of hypocotyl length by temperature. Thus, DELLAs appear

important in temperature responses. The phyB mutant responded less to temperature than its

background wild type genotype and the phyA mutant. Similarly, the pif4 mutant showed less

response to temperature than the wild type and the pif5 mutant. From the experiments with the

toc1 mutant, grown along with its wild type C24 it is unclear whether TOC1 is involved in

response to temperature. Furthermore, it was observed that the combination of increased

irradiance and temperature drop (T4) had largest reduction effect in stem elongation in pea. The

treatment (T4) might be stressful to the pants, which might reduce the stem growth i.e., due to

the induced expression of GA2ox2 (Gonnet, 2009 master thesis). The la crys della mutant

appeared slightly less sensitive to temperature drop than the wild type. Also, the phyB mutant

appeared slightly less inhibited in shoot elongation than wild type after 10 days of treatment. The

long1-mutant was virtually insensitive to temperature, indicating an important role of LONG1 in

effect of temperature on stem elongation. Although, not all components have been tested yet in

temperature experiments with Arabidopsis and pea, based on the findings in this thesis and

earlier results by Stavang and co-authors (Stavang et al., 2005; 2007; 2009; 2010), a signaling

pathway is proposed (with names of components in Arabidopsis/pea): PhyB – (COP1/LIP1) –

HY5/LONG1 – DELLAs/LA CRYs – PIF4 and HY5/LONG1 – GA2ox1/GA2ox2. Thus, it

appears that temperature signaling shares strong similarities with signaling involved in dark-light

transition.

Suggestions for further research

Experiments with the hy5 mutants could be done to verify a role in temperature signaling, similar

as suggested in the long1 pea experiments. It would be interesting to investigate the link between

COP1 – HY5 and LIP1 – LONG1 in temperature signaling by studying the response to the

relevant temperature drop treatments also in the cop1 and lip1-mutants. Also, since DELLA

proteins appear to play a role in temperature signaling, it would also be interesting to investigate

the roles of individual DELLA proteins. Furthermore, other circadian clock mutants i.e., cca1

and lhy could be studied to investigate if the rhythmicity in temperature sensitivity is due to an

underlying circadian mechanism.

37

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