Environmental and Experimental Botany 44 (2000) 133–139

Growth, soluble carbohydrates, and aloin concentration ofAloe 6era plants exposed to three irradiance levels

Alejandra Paez a, G. Michael Gebre b, Maria E. Gonzalez a,Timothy J. Tschaplinski c,*

a Laboratorio de Ecofisiologia. Dept. Biologia, Facultad de Ciencias, Uni6ersidad del Zulia, Maracaibo, Venezuelab Department of Bioagricultural Sciences and Pest Management, Colorado State Uni6ersity, Fort Collins, CO 80523-1177, USA

c En6ironmental Sciences Di6ision, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6422, USA

Received 17 June 1999; received in revised form 6 June 2000; accepted 8 June 2000

Abstract

Research was conducted on Aloe 6era, a traditional medicinal plant, to investigate the effects of light on growth,carbon allocation, and the concentrations of organic solutes, including soluble carbohydrates and aloin. The plantswere vegetatively propagated and grown under three irradiances: full sunlight, partial (30% full sunlight), and deepshade (10% full sunlight) for 12–18 months. After 1 year of growth, five plants from each treatment were harvestedto determine total above- and below ground dry mass. Four plants from the full sunlight and the partial shadetreatments were harvested after 18 months to assess the soluble carbohydrate, organic acid and aloin concentrationsof the clear parenchyma gel and the yellow leaf exudate, separately. Plants grown under full sunlight produced morenumerous and larger axillary shoots, resulting in twice the total dry mass than those grown under partial shade. Thedry mass of the plants grown under deep shade was 8.6% that of plants grown under full sunlight. Partial shadeincreased the number and length of leaves produced on the primary shoot, but leaf dry mass was still reduced to 66%of that in full sunlight. In contrast, partial and deep shade reduced root dry mass to 28 and 13%, respectively, of thatunder full sunlight, indicating that carbon allocation to roots was restricted under low light conditions. When plantswere sampled 6 months later, there were only minor treatment effects on the concentration of soluble carbohydratesand aloin in the leaf exudate and gel. Soluble carbohydrate concentrations were greater in the gel than in the exudate,with glucose the most abundant soluble carbohydrate. Aloin was present only in the leaf exudate and higherirradiance did not induce a higher concentration. Limitation in light availability primarily affected total dry massproduction and allocation, without substantial effects on either primary or secondary carbon metabolites. © 2000Elsevier Science B.V. All rights reserved.

Keywords: Aloe 6era ; Aloin; Carbon allocation; Partitioning; Irradiance; Carbohydrates

www.elsevier.com/locate/envexpbot

* Corresponding author. Tel.: +1-865-5744597; fax: +1-865-5769939.

E-mail address: t2t@ornl.gov (T.J. Tschaplinski).

S0098-8472/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

PII: S0 098 -8472 (00 )00062 -9

A. Paez et al. / En6ironmental and Experimental Botany 44 (2000) 133–139134

1. Introduction

Aloe 6era, a member of the Liliaceae plantfamily, is a common crop on Margarita Island,Venezuela, and a wild species in other xerophyticregions of Venezuela (Hoyos, 1985). A. 6era gelhas been used as a traditional medicine to inducewound healing, and as an anti-cancer, and anti-vi-ral agent (Maze et al., 1997). Some of the medici-nal properties of aloe have been attributed toaloin, also known as barbaloin, a C-glycosidederivative of anthraquinone (Reynolds, 1985). Al-though aloe is widely cultivated and valued for itsmedicinal properties (Grindlay and Reynolds,1986; Maze et al., 1997), few studies have beenconducted to determine the effects of variousgrowing conditions on plant dry mass and theproduction of aloin.

An increase in leaf thickness of aloe plantswith moisture and a corresponding increase ingel production have been reported (Genet andvan Schooten, 1992). McCarthy and van Rheedevan Oudtshoorn (1966) reported seasonal varia-tion in the concentration of aloin from leaf exu-date of two aloe species in South Africa. Theconcentration of aloin increased from winter tosummer and the authors suggested that thischange may have been due to temperature-in-duced changes in metabolic processes. Sincelight regimes influence growth and physiologicalresponses of all plants (Nobel, 1976; Givnish,1988), the growth response of A. 6era and itsaloin production may also be influenced bylight.

The present study was undertaken to deter-mine the effect of irradiance on growth, carbonallocation (distribution), and carbon partitioning(chemical fractionation) of assimilated carboninto soluble carbohydrates, organic acids andaloin in tissues of aloe. Leaves acclimated tohigh irradiance levels generally have higher pho-tosynthetic rates (Givnish 1988). It has also beensuggested that high irradiation can lead tohigher concentrations of phenolic compounds asa result of the increased carbon production(Shure and Wilson 1993). We hypothesized thatthe highest concentrations of aloin would beachieved under the high irradiance given the in-

creased availability of carbon precursors andstimulation of secondary carbon metabolism.The study specifically addressed the main effectsof shading to evaluate the growth potential ofA. 6era in response to light and its potential toproduce aloin in relation to soluble carbohy-drate availability.

2. Material and methods

The research was conducted under field condi-tions in an area adjacent to the Facultad deCiencias, Zulia University, Maracaibo,Venezuela. A total of 60 A. 6era L. plants werevegetatively propagated in 30 kg plastic potsand grown under three irradiance regimes: fullsunlight, partial (30% full sunlight) and deepshade (10% full sunlight). The irradiance re-ceived by the plants on a typical sunny day wasdetermined with a LiCor Li-188b radiometer(LiCor, Lincoln, NE). The range of readings av-eraged between 1340 and 1860 mmol m−2 s−1

(full sunlight), 420–670 mmol m−2 s−1 (partialshade), and 130–190 mmol m−2 s−1 (deepshade). Plants were irrigated every morning withtap water. Nutrients were applied at a rate of100 kg N, 50 kg P, and 50 kg K ha−1 every 3months.

After 1 year of growth, destructive and non-destructive measurements were conducted duringthe summer (July). The non-destructive measure-ments included the number, width and length ofleaves; number, width and length of lateralbranches; and total shoot length. Five plants pertreatment were harvested to assess dry massproduction. The plants were cut at the root col-lar and separated into leaves, stems and roots.The roots were washed and all tissues dried inan oven at 60°C for 2 weeks for dry mass deter-mination.

Leaf samples from four plants growing underfull sunlight and partial shade treatments werecollected midday in January after 18 months.The leaves consist of the outer green (chloro-phyll-containing) leathery margin (including theepidermis and palisade cell layers) and an inter-nal clear gelatinous (gel) matrix. A yellowish

A. Paez et al. / En6ironmental and Experimental Botany 44 (2000) 133–139136

liquid exudate, produced by the bundle sheathcells of outer margin of the leaf, has a characteris-tic smell and bitter taste (Grindlay and Reynolds,1986). In contrast, the gel is the inner, colorlessand tasteless parenchyma tissue. The exudate wasseparated from the clear parenchyma gel by al-lowing it to drip into a container and the gel wasexcised with a scalpel as a filet from the core ofthe leaf lamina. All samples were prepared forsoluble carbohydrate, organic acid and aloinanalyses, as follows. Samples were freeze dried for48 h, weighed, and placed directly into the deriva-tizing reagent. Approximately 10–20 mg of driedexudate and 10–25 mg of gel were subjected toanalysis. Soluble carbohydrates, organic acids andaloin were analyzed as trimethylsilyl derivativesby dissolving and heating samples with 2 ml ofTri-Sil ‘Z’ (Pierce Chemical, Rockford, IL) for 45min with samples left overnight before analysisthe next day (Tschaplinski et al., 1993). Sampleswere then analyzed using capillary gas chromatog-raphy–mass spectrometry (GC–MS), which wasalso used to confirm the identity of solutes mea-sured. A total of 1 ml of each sample was injectedinto a HP 5972 GC–MS (Hewlett-Packard,Avondale, PA). Operating conditions of the GC–MS were as described elsewhere (Gebre et al.,1998). External standards of known carbohy-drates were injected to determine the concentra-tion in plant samples. The data were subjected toanalysis of variance, and treatment means werecompared using Student’s t-tests at a significancelevel of P50.05. However, probabilities ofPB0.10 are reported, and those of P\0.10 aredesignated not significant (ns).

3. Results

3.1. Carbon production and allocation

At the end of 1 year of growth, A. 6era plantsthat were exposed to partial shade (30% full sun-light) produced 27% more leaves that were 21%longer relative to the leaves of plants under fullsunlight. Overall, partial shading increased shootlength (Table 1). However, plants grown underfull sunlight produced wider leaves and had moreand larger axillary shoots than partially shadedplants (Table 1), resulting in twice as much totaldry mass as those plants grown under partialshade and more than 11× that of plants grownunder deep shade (Table 2). The allocation ofcarbon within plants grown under full sunlightwas 53% to leaves and 28% to roots. Partial shadeincreased allocation to leaves to 70%, but de-creased allocation to roots to 13%. A similartrend was observed under deep shade with thecorresponding values being 70% for leaves and21% for roots. Root dry mass of plants in the fullsunlight treatment was 4× and 15× that ofplants under partial shade and deep shade,respectively.

3.2. Carbon partitioning

3.2.1. Soluble carbohydrates and organic acidsAfter 18 months of growth, partial shade re-

duced the myoinositol concentration of the gel to47% of that of plants grown in full sun (Table 3).Otherwise, there was no effect of shading on theconcentration of the major solutes measured in

Table 2Dry biomass of Aloe 6era plants grown under three irradiances: full sunlight, partial and deep shade (30 and 10% of full sunlight,respectively) after 1 yeara

Leaf DW (g) Stem DW (g)Irradiance Axillary shoot DW (g) Root DW (g) Total DW (g)

12.791.3a12.191.1a 36.293.4a 130.395.7a69.3094.1aFull sunlightPartial shade 46.092.8b 65.593.8b6.990.3b 4.190.4b 8.690.7b

11.491.1c2.490.2c1.090.c 0c8.090.7cDeep shade

a Treatment means (9SE) followed by different letters are significantly different at P50.05 (n=5).

A. Paez et al. / En6ironmental and Experimental Botany 44 (2000) 133–139 135

Tab

le1

Gro

wth

ofA

loe6e

rapl

ants

unde

rth

ree

irra

dian

ces:

full

sunl

ight

,pa

rtia

lan

dde

epsh

ade

(30

and

10%

offu

llsu

nlig

ht,

resp

ecti

vely

)af

ter

1ye

ar*

Irra

dian

ceL

engt

hof

leav

esN

umbe

rof

Wid

thof

leav

esP

lant

leng

thN

umbe

rof

axill

ary

Len

gth

ofax

illar

ysh

oots

(cm

)sh

oots

(cm

)le

aves

(cm

)(c

m)

4.49

0.1a

5.59

0.1a

10.69

0.8a

16.59

0.4b

Ful

lsu

nlig

ht26

.39

0.1b

349

3c

7.79

0.3b

Par

tial

shad

e3.

09

0.1b

479

1a3.

89

0.1b

21.09

0.2a

31.99

1.2a

Dee

psh

ade

0c419

1b14

.89

0.1c

26.89

0.1b

2.39

0.1c

0c

*T

reat

men

tm

eans

(9SE

)fo

llow

edby

diff

eren

tle

tter

sar

esi

gnifi

cant

lydi

ffer

ent

atP5

0.05

(n=

5).

A. Paez et al. / En6ironmental and Experimental Botany 44 (2000) 133–139 137

Table 3Mean (9SE) concentrations of soluble carbohydrates, organic acids, and aloin of Aloe 6era plants grown under full sunlight andpartial shade (30% full sunlight) treatments after 18 months (n=4 except n=3 for full sunlight treatment of gel)a

Exudate (mmol gDW−1)Organic solutes Gel (mmol gDW−1)

Partial shade Probability Full sunlightFull sunlight Partial shade Probability

Glucose 77.394.6 100.6910.3 0.084 655958 730993 ns7.990.7Fructose 12.292.9 ns 288922 275947 ns

8.791.7 0.056 1629174.390.7 179922Galactose ns1.190.1Arabinose 1.890.3 0.049 nd nd0.590.3Sucrose 0.390 ns nd nd

0.490 0.007 12.391.20.790.1 5.790.7Myoinositol 0.00411.693.5 0.066 20.495.0 34.398.2Quinic acid ns3.690.9

nd 409964nd 6569224Malic acid ns20079543Aloin ns20969129 nd nd

a nd, solute was not detected in the tissue analyzed.

the gel. Partial shade tended to increase the con-centrations of some of the major solutes includingglucose, galactose and quinic acid. However, thesechanges were only significant at PB0.10. Partialshade reduced the myoinositol concentration ofleaf exudate to 54% of that observed in full sun.Partial shade also increased the arabinose concen-tration of the exudate by 1.66× , but both ofthese changes involved minor constituents. Glu-cose was the major soluble carbohydrate mea-sured in both the exudate and the gel samples(Table 3), constituting 81–84% of the total solu-ble carbohydrates (excluding phenolic glucosides)in the exudate and 59–61% of the total in the gel.In contrast, the sucrose concentration was low,which is not surprising given the nature of thetissues analyzed (i.e. neither chlorophyll-contain-ing tissues nor conducting tissues were analyzed).

With the exception of aloin, most of the solutesmeasured were at higher concentrations in the gelthan in the exudate (Table 3). For example, theconcentrations of fructose and galactose weremuch higher (20–40× ) in the gel than the exu-date. The main exudate constituents were aloinand glucose, followed by low concentrations offructose, galactose, arabinose, myoinositol, andsucrose. Quinic acid was the major organic acid inthe exudate, but it was detected at low concentra-tions ranging from 4 to 12 mmol gDW−1, incontrast with the higher concentrations of 20–34mmol gDW−1 in the gel. Although malic acid was

not detected in the exudate, it was the majororganic acid in the gel.

3.2.2. AloinAloin was only detected in the exudate with no

differences between sun and shade plants (Table3). Any limitation in carbon availability due tolower light levels was largely evident in dry massdifferences, rather than in the concentrations ofsoluble carbohydrates or secondary carbon com-pounds. The concentration of aloin in the exudateranged from 2007 to 2096 mmol gDW−1, account-ing for 93.7–95.6% of the major organic solutesdetermined in the exudate. Although there was nosignificant effect of treatment, our samplesshowed high variability in the aloin concentrationof partially shaded plants (Table 3).

4. Discussion

Although partial shading increased the numberand length of leaves produced, the greater produc-tion of axillary shoots under full sun, coupledwith greater root dry mass, resulted in the greatertotal plant dry mass. The number of leaves perplant in all treatments was within the values re-ported in the literature for A. 6era (Grindlay andReynolds, 1986; Genet and van Schooten, 1992).The width and length of leaves was slightly lowerthan the average. Partial shading favored

A. Paez et al. / En6ironmental and Experimental Botany 44 (2000) 133–139138

retention of carbon in shoots at the expense ofroots. A decrease in the allocation of carbon toroots in plants grown under shade has also beenreported in other species such as cogongrass (Im-perata cylindrica) (Patterson, 1980).

Whereas glucose was the major soluble carbo-hydrate in aloe leaves, shading did not affect itsconcentration in the leaf gel. Christopher andHoltum (1996) also reported that glucose was themajor soluble storage carbohydrate in A. 6era,peaking at �350 mmol gDW−1 at 1500 h, fol-lowed by sucrose at �275 mmol gDW−1, andfructose at �125 mmol gDW−1. The somewhathigher concentrations reported for the gel in ourstudy, relative to the concentrations reported forthe whole leaf, is not unexpected because theparenchymatous gel tissue has less inactive dryresidue that would dilute the concentrations ob-served. In addition to glucose accumulation, aloeis known to accumulate starch (Christopher andHoltum, 1996) and acetylated mannans (aceman-nan) as storage polysaccharides in protoplasts ofparenchymatous cells (Femenia et al., 1999).

Many factors contribute to the fluctuation inthe concentrations of the major organic solutesobserved. Given that leaves were sampled in thelate morning, the relatively high concentrations ofmalate were expected. Malate concentrations inaloe leaves typically peak at 06:00 h at �250mmol gDW−1 and reach their minimum, negligi-ble concentrations at 15:00 h (Christopher andHoltum, 1996). Again, the low amount of inactiveresidue of the parenchymatous tissue produce thesomewhat higher concentrations in our studycompared to studies where the whole leaf is ex-tracted. Our sampling occurred in the winter (dryseason, daytime temperature 28–30°C) withleaves older than when growth measurementswere taken. However, Yaron (1993) reported thatalthough irrigation of A. 6era affected the concen-tration of carbohydrates, leaf age had no signifi-cant effect. Season of harvest also had no effecton dry matter content. The extract in that studycontained �1% dry matter with soluble carbohy-drates constituting 0.2–0.3% and polysaccharides0.1–0.2%.

There are few reports on the effect of shadingon aloin concentration, but Chauser-Volfson and

Gutterman (1998) noted that the concentrationsof aloin (barbaloin), homonataloin, and nataloinwere higher in A. mutabilis plants growing in theshade than in the direct sun light. Such a reportcoupled with our findings suggest reduced lightavailability does not restrict the production ofaloin or glucose, its conjugated moiety. Aloin wasonly detected in the exudate, which was similar tothat reported by Grindlay and Reynolds (1986),with no differences between sun and shade plants.In Aloe ferox, van Wyk et al. (1995) found thecontribution of aloin, aloeresin A and aloesin was70–97% of total dry weight of leaf exudate with ageographical variation in the aloin content aloneranging from 9.5 to 31.2%. A difference in aloinconcentration between leaves within the sameplant has also been reported with the highestconcentration just below the apex of the plant(younger leaves) and lowest at the base (Okamuraet al., 1996; Chauser-Volfson and Gutterman,1998). All leaves in our study were collected fromthe same position on each plant. The concentra-tion of aloin and other closely-related anthroneC-glycosides have also been shown to be highestin the top third of a leaf and lowest at its base inAloe mutabilis and A. hereroensis (Chauser-Volfson and Gutterman, 1997, 1998). There aremany potential sources of variability reported inthe literature. McCarthy and van Rheede vanOudtshoorn (1966) found seasonal variation inaloin concentration with an increase during thesummer corresponding to an increase in tempera-ture. They also suggested that wind affects aloinproduction by shrinking the leaf. Park et al.(1998) reported that aloin content determinedfrom total leaf weight (gel and exudate combined)was variable throughout the season.

5. Conclusions

Although the dry mass of all plant componentswere reduced by shading, leaf dry mass was theleast affected due to an offsetting increase in thenumber and length of leaves. In contrast, root drymass was reduced significantly under partial shad-ing, suggesting that carbon allocation to roots isrestricted under low light conditions. Limitation

A. Paez et al. / En6ironmental and Experimental Botany 44 (2000) 133–139 139

in light availability primarily affected total drymass production and carbon allocation, withoutsubstantial effects on soluble carbohydrates.There were only minimal treatment effects in or-ganic solute concentrations of exudate and the gelafter 18 months growth. Soluble carbohydrateswere more abundant in the gel than in the exu-date. Aloin was present only in the exudate andshading did not affect its concentration. The hy-pothesis that the higher irradiance would inducehigher glucose concentrations in tissues, and con-sequently, higher aloin concentrations was notsubstantiated.

Acknowledgements

The authors wish to express their gratitude toCONDES (Universidad del Zulia) and CONICITfor supporting part of this research conducted inVenezuela. The research was also funded, in part,by the Bioenergy Feedstock Development Pro-gram, US Department of Energy, at Oak RidgeNational Laboratory, managed by UT-Battelle,LLC, for the US Department Energy under con-tract DE-AC05-00OR22725. Publication No.5006, Environmental Sciences Division, OakRidge National Laboratory. The second authorwas also supported by an appointment to the OakRidge National Laboratory Postdoctoral Re-search Associates Program administered jointlyby the Oak Ridge National Laboratory and theOak Ridge Institute for Science and Education.

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