Growth, soluble carbohydrates, and aloin concentration of Aloevera plants exposed to three irradiance levels

Environmental and Experimental Botany 44 (2000) 133 – 139 www.elsevier.com/locate/envexpbot

Growth, soluble carbohydrates, and aloin concentration of Aloe 6era plants exposed to three irradiance levels Alejandra Paez a, G. Michael Gebre b, Maria E. Gonzalez a, Timothy J. Tschaplinski c,* b

a Laboratorio de Ecofisiologia. Dept. Biologia, Facultad de Ciencias, Uni6ersidad del Zulia, Maracaibo, Venezuela 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 plants were vegetatively propagated and grown under three irradiances: full sunlight, partial (30% full sunlight), and deep shade (10% full sunlight) for 12–18 months. After 1 year of growth, five plants from each treatment were harvested to determine total above- and below ground dry mass. Four plants from the full sunlight and the partial shade treatments were harvested after 18 months to assess the soluble carbohydrate, organic acid and aloin concentrations of the clear parenchyma gel and the yellow leaf exudate, separately. Plants grown under full sunlight produced more numerous and larger axillary shoots, resulting in twice the total dry mass than those grown under partial shade. The dry mass of the plants grown under deep shade was 8.6% that of plants grown under full sunlight. Partial shade increased 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 that under full sunlight, indicating that carbon allocation to roots was restricted under low light conditions. When plants were sampled 6 months later, there were only minor treatment effects on the concentration of soluble carbohydrates and 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 higher irradiance did not induce a higher concentration. Limitation in light availability primarily affected total dry mass production and allocation, without substantial effects on either primary or secondary carbon metabolites. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Aloe 6era; Aloin; Carbon allocation; Partitioning; Irradiance; Carbohydrates

* Corresponding author. Tel.: + 1-865-5744597; fax: +1865-5769939. E-mail address: [email protected] (T.J. Tschaplinski). S0098-8472/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 9 8 - 8 4 7 2 ( 0 0 ) 0 0 0 6 2 - 9

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1. Introduction Aloe 6era, a member of the Liliaceae plant family, is a common crop on Margarita Island, Venezuela, and a wild species in other xerophytic regions of Venezuela (Hoyos, 1985). A. 6era gel has been used as a traditional medicine to induce wound healing, and as an anti-cancer, and anti-viral agent (Maze et al., 1997). Some of the medicinal properties of aloe have been attributed to aloin, also known as barbaloin, a C-glycoside derivative of anthraquinone (Reynolds, 1985). Although aloe is widely cultivated and valued for its medicinal properties (Grindlay and Reynolds, 1986; Maze et al., 1997), few studies have been conducted to determine the effects of various growing conditions on plant dry mass and the production of aloin. An increase in leaf thickness of aloe plants with moisture and a corresponding increase in gel production have been reported (Genet and van Schooten, 1992). McCarthy and van Rheede van Oudtshoorn (1966) reported seasonal variation in the concentration of aloin from leaf exudate of two aloe species in South Africa. The concentration of aloin increased from winter to summer and the authors suggested that this change may have been due to temperature-induced changes in metabolic processes. Since light regimes influence growth and physiological responses of all plants (Nobel, 1976; Givnish, 1988), the growth response of A. 6era and its aloin production may also be influenced by light. The present study was undertaken to determine the effect of irradiance on growth, carbon allocation (distribution), and carbon partitioning (chemical fractionation) of assimilated carbon into soluble carbohydrates, organic acids and aloin in tissues of aloe. Leaves acclimated to high irradiance levels generally have higher photosynthetic rates (Givnish 1988). It has also been suggested that high irradiation can lead to higher concentrations of phenolic compounds as a result of the increased carbon production (Shure and Wilson 1993). We hypothesized that the highest concentrations of aloin would be achieved under the high irradiance given the in-

creased availability of carbon precursors and stimulation of secondary carbon metabolism. The study specifically addressed the main effects of shading to evaluate the growth potential of A. 6era in response to light and its potential to produce aloin in relation to soluble carbohydrate availability.

2. Material and methods The research was conducted under field conditions in an area adjacent to the Facultad de Ciencias, Zulia University, Maracaibo, Venezuela. A total of 60 A. 6era L. plants were vegetatively propagated in 30 kg plastic pots and grown under three irradiance regimes: full sunlight, partial (30% full sunlight) and deep shade (10% full sunlight). The irradiance received by the plants on a typical sunny day was determined with a LiCor Li-188b radiometer (LiCor, Lincoln, NE). The range of readings averaged between 1340 and 1860 mmol m − 2 s − 1 (full sunlight), 420–670 mmol m − 2 s − 1 (partial shade), and 130–190 mmol m − 2 s − 1 (deep shade). Plants were irrigated every morning with tap water. Nutrients were applied at a rate of 100 kg N, 50 kg P, and 50 kg K ha − 1 every 3 months. After 1 year of growth, destructive and nondestructive measurements were conducted during the summer (July). The non-destructive measurements included the number, width and length of leaves; number, width and length of lateral branches; and total shoot length. Five plants per treatment were harvested to assess dry mass production. The plants were cut at the root collar and separated into leaves, stems and roots. The roots were washed and all tissues dried in an oven at 60°C for 2 weeks for dry mass determination. Leaf samples from four plants growing under full sunlight and partial shade treatments were collected midday in January after 18 months. The leaves consist of the outer green (chlorophyll-containing) leathery margin (including the epidermis and palisade cell layers) and an internal clear gelatinous (gel) matrix. A yellowish

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liquid exudate, produced by the bundle sheath cells of outer margin of the leaf, has a characteristic smell and bitter taste (Grindlay and Reynolds, 1986). In contrast, the gel is the inner, colorless and tasteless parenchyma tissue. The exudate was separated from the clear parenchyma gel by allowing it to drip into a container and the gel was excised with a scalpel as a filet from the core of the leaf lamina. All samples were prepared for soluble carbohydrate, organic acid and aloin analyses, as follows. Samples were freeze dried for 48 h, weighed, and placed directly into the derivatizing reagent. Approximately 10 – 20 mg of dried exudate and 10 – 25 mg of gel were subjected to analysis. Soluble carbohydrates, organic acids and aloin were analyzed as trimethylsilyl derivatives by dissolving and heating samples with 2 ml of Tri-Sil ‘Z’ (Pierce Chemical, Rockford, IL) for 45 min with samples left overnight before analysis the next day (Tschaplinski et al., 1993). Samples were then analyzed using capillary gas chromatography–mass spectrometry (GC – MS), which was also used to confirm the identity of solutes measured. A total of 1 ml of each sample was injected into 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 carbohydrates were injected to determine the concentration in plant samples. The data were subjected to analysis of variance, and treatment means were compared using Student’s t-tests at a significance level of P 5 0.05. However, probabilities of PB0.10 are reported, and those of P \ 0.10 are designated not significant (ns).

3. Results

3.1. Carbon production and allocation At the end of 1 year of growth, A. 6era plants that were exposed to partial shade (30% full sunlight) produced 27% more leaves that were 21% longer relative to the leaves of plants under full sunlight. Overall, partial shading increased shoot length (Table 1). However, plants grown under full sunlight produced wider leaves and had more and larger axillary shoots than partially shaded plants (Table 1), resulting in twice as much total dry mass as those plants grown under partial shade and more than 11 × that of plants grown under deep shade (Table 2). The allocation of carbon within plants grown under full sunlight was 53% to leaves and 28% to roots. Partial shade increased allocation to leaves to 70%, but decreased allocation to roots to 13%. A similar trend was observed under deep shade with the corresponding values being 70% for leaves and 21% for roots. Root dry mass of plants in the full sunlight treatment was 4× and 15× that of plants under partial shade and deep shade, respectively.

3.2. Carbon partitioning 3.2.1. Soluble carbohydrates and organic acids After 18 months of growth, partial shade reduced the myoinositol concentration of the gel to 47% of that of plants grown in full sun (Table 3). Otherwise, there was no effect of shading on the concentration of the major solutes measured in

Table 2 Dry 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 Irradiance

Leaf DW (g)

Stem DW (g)

Axillary shoot DW (g)

Root DW (g)

Total DW (g)

Full sunlight Partial shade Deep shade

69.309 4.1a 46.092.8b 8.09 0.7c

12.191.1a 6.99 0.3b 1.09 0.c

12.7 91.3a 4.1 90.4b 0c

36.2 9 3.4a 8.6 90.7b 2.4 90.2c

130.3 95.7a 65.5 9 3.8b 11.4 9 1.1c

a

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

26.3 9 0.1b 31.9 9 1.2a 26.8 9 0.1b

16.5 9 0.4b 21.0 9 0.2a 14.8 9 0.1c

349 3c 479 1a 419 1b

Full sunlight Partial shade Deep shade

Number of axillary shoots 5.5 9 0.1a 3.0 9 0.1b 0c

Width of leaves (cm) 4.4 9 0.1a 3.8 90.1b 2.3 90.1c

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

Length of leaves (cm)

Number of leaves

Plant length (cm)

Irradiance

10.690.8a 7.79 0.3b 0c

Length of axillary shoots (cm)

Table 1 Growth of Aloe 6era plants under three irradiances: full sunlight, partial and deep shade (30 and 10% of full sunlight, respectively) after 1 year*

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

Glucose Fructose Galactose Arabinose Sucrose Myoinositol Quinic acid Malic acid Aloin a

Exudate (mmol gDW−1)

Gel (mmol gDW−1)

Full sunlight

Partial shade

Probability

Full sunlight

Partial shade

Probability

77.39 4.6 7.99 0.7 4.39 0.7 1.19 0.1 0.59 0.3 0.79 0.1 3.69 0.9 nd 20969 129

100.69 10.3 12.29 2.9 8.79 1.7 1.89 0.3 0.39 0 0.49 0 11.69 3.5 nd 20079 543

0.084 ns 0.056 0.049 ns 0.007 0.066

655 958 288 922 162 917 nd nd 12.3 91.2 20.4 9 5.0 409 9 64 nd

730 993 275 947 179 9 22 nd nd 5.790.7 34.39 8.2 656 9 224 nd

ns ns ns

ns

0.004 ns ns

nd, solute was not detected in the tissue analyzed.

the gel. Partial shade tended to increase the concentrations of some of the major solutes including glucose, galactose and quinic acid. However, these changes were only significant at P B 0.10. Partial shade reduced the myoinositol concentration of leaf exudate to 54% of that observed in full sun. Partial shade also increased the arabinose concentration of the exudate by 1.66×, but both of these changes involved minor constituents. Glucose was the major soluble carbohydrate measured in both the exudate and the gel samples (Table 3), constituting 81 – 84% of the total soluble 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 the tissues analyzed (i.e. neither chlorophyll-containing tissues nor conducting tissues were analyzed). With the exception of aloin, most of the solutes measured were at higher concentrations in the gel than in the exudate (Table 3). For example, the concentrations of fructose and galactose were much higher (20 – 40×) in the gel than the exudate. The main exudate constituents were aloin and glucose, followed by low concentrations of fructose, galactose, arabinose, myoinositol, and sucrose. Quinic acid was the major organic acid in the exudate, but it was detected at low concentrations ranging from 4 to 12 mmol gDW − 1, in contrast with the higher concentrations of 20–34 mmol gDW − 1 in the gel. Although malic acid was

not detected in the exudate, it was the major organic acid in the gel.

3.2.2. Aloin Aloin was only detected in the exudate with no differences between sun and shade plants (Table 3). Any limitation in carbon availability due to lower light levels was largely evident in dry mass differences, rather than in the concentrations of soluble carbohydrates or secondary carbon compounds. The concentration of aloin in the exudate ranged from 2007 to 2096 mmol gDW − 1, accounting for 93.7–95.6% of the major organic solutes determined in the exudate. Although there was no significant effect of treatment, our samples showed high variability in the aloin concentration of partially shaded plants (Table 3).

4. Discussion Although partial shading increased the number and length of leaves produced, the greater production of axillary shoots under full sun, coupled with greater root dry mass, resulted in the greater total plant dry mass. The number of leaves per plant in all treatments was within the values reported in the literature for A. 6era (Grindlay and Reynolds, 1986; Genet and van Schooten, 1992). The width and length of leaves was slightly lower than the average. Partial shading favored

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retention of carbon in shoots at the expense of roots. A decrease in the allocation of carbon to roots in plants grown under shade has also been reported in other species such as cogongrass (Imperata cylindrica) (Patterson, 1980). Whereas glucose was the major soluble carbohydrate in aloe leaves, shading did not affect its concentration in the leaf gel. Christopher and Holtum (1996) also reported that glucose was the major soluble storage carbohydrate in A. 6era, peaking at 350 mmol gDW − 1 at 1500 h, followed by sucrose at 275 mmol gDW − 1, and fructose at 125 mmol gDW − 1. The somewhat higher concentrations reported for the gel in our study, relative to the concentrations reported for the whole leaf, is not unexpected because the parenchymatous gel tissue has less inactive dry residue that would dilute the concentrations observed. In addition to glucose accumulation, aloe is known to accumulate starch (Christopher and Holtum, 1996) and acetylated mannans (acemannan) as storage polysaccharides in protoplasts of parenchymatous cells (Femenia et al., 1999). Many factors contribute to the fluctuation in the concentrations of the major organic solutes observed. Given that leaves were sampled in the late morning, the relatively high concentrations of malate were expected. Malate concentrations in aloe leaves typically peak at 06:00 h at 250 mmol gDW − 1 and reach their minimum, negligible concentrations at 15:00 h (Christopher and Holtum, 1996). Again, the low amount of inactive residue of the parenchymatous tissue produce the somewhat higher concentrations in our study compared to studies where the whole leaf is extracted. Our sampling occurred in the winter (dry season, daytime temperature 28 – 30°C) with leaves older than when growth measurements were taken. However, Yaron (1993) reported that although irrigation of A. 6era affected the concentration of carbohydrates, leaf age had no significant effect. Season of harvest also had no effect on dry matter content. The extract in that study contained 1% dry matter with soluble carbohydrates constituting 0.2 – 0.3% and polysaccharides 0.1–0.2%. There are few reports on the effect of shading on aloin concentration, but Chauser-Volfson and

Gutterman (1998) noted that the concentrations of aloin (barbaloin), homonataloin, and nataloin were higher in A. mutabilis plants growing in the shade than in the direct sun light. Such a report coupled with our findings suggest reduced light availability does not restrict the production of aloin or glucose, its conjugated moiety. Aloin was only detected in the exudate, which was similar to that reported by Grindlay and Reynolds (1986), with no differences between sun and shade plants. In Aloe ferox, van Wyk et al. (1995) found the contribution of aloin, aloeresin A and aloesin was 70–97% of total dry weight of leaf exudate with a geographical variation in the aloin content alone ranging from 9.5 to 31.2%. A difference in aloin concentration between leaves within the same plant has also been reported with the highest concentration just below the apex of the plant (younger leaves) and lowest at the base (Okamura et al., 1996; Chauser-Volfson and Gutterman, 1998). All leaves in our study were collected from the same position on each plant. The concentration of aloin and other closely-related anthrone C-glycosides have also been shown to be highest in the top third of a leaf and lowest at its base in Aloe mutabilis and A. hereroensis (ChauserVolfson and Gutterman, 1997, 1998). There are many potential sources of variability reported in the literature. McCarthy and van Rheede van Oudtshoorn (1966) found seasonal variation in aloin concentration with an increase during the summer corresponding to an increase in temperature. They also suggested that wind affects aloin production by shrinking the leaf. Park et al. (1998) reported that aloin content determined from total leaf weight (gel and exudate combined) was variable throughout the season.

5. Conclusions Although the dry mass of all plant components were reduced by shading, leaf dry mass was the least affected due to an offsetting increase in the number and length of leaves. In contrast, root dry mass was reduced significantly under partial shading, suggesting that carbon allocation to roots is restricted under low light conditions. Limitation

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in light availability primarily affected total dry mass production and carbon allocation, without substantial effects on soluble carbohydrates. There were only minimal treatment effects in organic solute concentrations of exudate and the gel after 18 months growth. Soluble carbohydrates were more abundant in the gel than in the exudate. Aloin was present only in the exudate and shading did not affect its concentration. The hypothesis that the higher irradiance would induce higher glucose concentrations in tissues, and consequently, higher aloin concentrations was not substantiated.

Acknowledgements The authors wish to express their gratitude to CONDES (Universidad del Zulia) and CONICIT for supporting part of this research conducted in Venezuela. The research was also funded, in part, by the Bioenergy Feedstock Development Program, US Department of Energy, at Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the US Department Energy under contract DE-AC05-00OR22725. Publication No. 5006, Environmental Sciences Division, Oak Ridge National Laboratory. The second author was also supported by an appointment to the Oak Ridge National Laboratory Postdoctoral Research Associates Program administered jointly by the Oak Ridge National Laboratory and the Oak Ridge Institute for Science and Education.

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