ФИЗИОЛОГИЯ РАСТЕНИИ, 2011, том 58, № 3, с. 476-480
КРАТКИЕ ^^^^^^^^^^^^^^^^ СООБЩЕНИЯ
УДК 581.1
Effect of Water Deficit on Biomass Production and Accumulation of Secondary Metabolites in Roots of Glycyrrhiza uralensis
© 2011 W. D. Li***, J. L. Hou***, W. Q. Wang***, X. M. Tang***, C. L. Liu***, D. Xing***
* School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing, China ** Engineering Research Center of Good Agricultural Practice for Chinese Crude Drugs, Beijing, China *** School of Traditional Chinese Medicine, Capital Medical University, Beijing, China
Received October 27, 2009
Two-year-old seedlings of licorice plant (Glycyrrhiza uralensis Fisch) were exposed to three degrees of water deficit, namely weak (60—70%), moderate (40—50%), and strong (20—30%) relative water content in soil, whereas control plants were grown in soil with 80—90% water content. Moderate and strong water deficit decreased the net photosynthetic rate, stomatal conductance, and biomass production. Water use efficiency and the root-to-shoot ratio increased significantly in response to water deficit, indicating a high tolerance to drought. Weak water deficit did not decrease root biomass production, but significantly increased the production of glycyrrhizic acid (by 89%) and liquiritin (by 125%) in the roots. Therefore, a weak water deficit can increase the yield of root medical compounds without negative effect on root growth.
Keywords: Glycyrrhiza uralensis — biomass production — gas exchange — glycyrrhizic acid — liquiritin — water deficit
INTRODUCTION
Licorice (Glycyrrhiza uralensis Fisch) is a very popular medicinal plant, which roots contain glycyrrhizic acid and liquiritin mainly accumulated in the root and rhizome tissues [1, 2]. Recently, glycyrrhizic acid has been found to be highly active in inhibiting the replication of the severe acute respiratory syndrome (SARS)-associated virus and has been suggested as a potential therapeutic agent for chronic hepatitis and acquired immunodeficiency syndrome (AIDS) [3]. Licorice plants appear to be highly drought-tolerant, being a favorable plant to restore degraded desert, arid and semiarid ecosystems of northwest China [4]. However, data on physiological processes, such as biomass production and secondary metabolite yield, in response to environmental conditions are lacking [5].
Water deficit usually inhibits plant growth and productivity by affecting gas exchange and especially photosynthesis [6, 7]. Water use efficiency (WUE) can be traditionally defined as the ratio of net photosynthesis to transpiration over a period of seconds or minutes [8]. The higher WUE has been mentioned as a strategy to improve crop performance under water-limited conditions [9]. However, in licorice plants photosyn-
Abbreviations: WC — water content; WUE — water use efficiency. Corresponding author: Wenquan Wang. School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing, 100102 China. E-mail: wwq57@126.com
thesis and biomass production as well as WUE in response to water deficit were not studied.
Water deficit can induce the biosynthesis of some secondary metabolites [10—12], resulting in their accumulation in medicinal plants [10, 13, 14]. For example, the concentration of rutin and chlorogenic acid increased with drought severity in tomato plants [14]. Although the responses of the metabolites to drought have been investigated in some medicinal plants [4, 10], no reference concerning the effect of various water deficit levels on their production by licorice roots is available.
The present study aims to determine the effect of water deficit on gas exchange, biomass and secondary metabolites production in licorice plants. It was hypothesized that a suitable water deficit, in addition to saving water, can also increase the amount of root secondary metabolites without negative effect on root growth.
MATERIALS AND METHODS
Plants and experimental design. The experiment was performed in a greenhouse at Beijing University of Chinese Medicine. Seeds were collected from one population in Hangjinqi, Inner Mongolia. Seeds were soaked in concentrated H2SO4 for 20 min, washed several times with tap water, and then sown immediately in small plastic pots filled with 450 ml of sand mixture
Table 1. Net photosynthetic rate, stomatal conductance, transpiration rate, and water use efficiency in Glycyrrhiza uralensis under different soil relative water contents
Soil relative water contentf % Net photosynthetic rate, p.mol/(m2 s) Stomatal conductance, mol/(m2 s) Transpiration rate, mmol/(m2 s) Water use efficiency, ^mol/mmol
80-90 15.0 ± 0.3a 0.24 ± 0.01a 5.8 ± 0.1a 2.6
60-70 14.3 ± 0.4a 0.23 ± 0.01a 3.6 ± 0.1b 4.0
40-50 12.3 ± 0.5b 0.22 ± 0.02a 3.5 ± 0.1b 3.5
20-30 10.3 ± 0.4c 0.17 ± 0.01b 3.4 ± 0.1b 3.0
Notes: Measurements were made at a sunny day on July; 2007 after more than two-month-long
treatment. Water use efficiency was calculated as the ratio of net photosynthetic rate to transpiration rate. Means followed by different letters indicate significant differences at P < 0.05. n = 4.
on May 6, 2006. After germination, four seedlings per pot were selected and cultivated for a year. On April 15, 2007, the seedlings were transplanted to bigger plastic pots filled with sandy soil with pH 7.06, containing nitrogen (223 mg/kg), organic mass (3.1 g/kg), and the available P and K contents of13.7 and 28.0 mg/kg, respectively. Each pot contained four seedlings.
Water treatments were carried out from May 15 until the end of October in 2007. Four levels of soil relative water content (WC), 80-90, 60-70, 40-50, and 20-30%, represented control plants, weak, moderate, and strong water deficit, respectively. Each pot was weighed and water was added to reach the target level
at 6:00 p.m. every day. There were four replications per treatment arranged in a completely randomized block design.
Leaf gas exchange. The newly developed leaves from the middle part of the shoot were chosen for gas exchange measurement using a Li-6400 portable photosynthesis system ("Li-Cor", United States) at a sunny day on July, 2007. The measurements were made from 8:00 a.m. to 9:30 a.m., under approximate photosynthetic photon flux density of 1200-1400 ^mol/(m2 s), and ambient CO2 concentration of 380 ^mol/mol. The net photosynthetic rate, stomatal conductance, and transpiration rate were simultaneously measured. Water
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Contents of glycyrrhizic acid and liquiritin in the roots of Glycyrrhiza uralensis expressed per gram dry weight (a, b) and per plant roots (c, d) under different soil relative water contents on the end of October in 2007 after more than five-month-long treatment. Different letters indicate significant differences at P < 0.05. n = 4.
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Table 2. Dry weights of whole plant, roots, and shoots, decreases in the root and shoot dry weights, and root-to-shoot ratio in Glycyrrhiza uralensis under different soil relative water contents
Soil relative water content, % Whole plant dry wt, g Root dry wt, g Root dry wt decrease, % Shoot dry wt, g Shoot dry wt decrease, % Root-to-shoot ratio
80-90 5.6 ± 0.2a 3.1 ± 0.1a 0 2.5 ± 0.2a 0 1.26 ± 0.10
60-70 5.2 ± 0.2a 3.0 ± 0.2ab -2.3 2.2 ± 0.2ab -11.9 1.40 ± 0.12
40-50 4.6 ± 0.2b 2.7 ± 0.1b -13.1 1.9 ± 0.2b -24.6 1.48 ± 0.15
20-30 3.3 ± 0.2c 1.9 ± 0.1c -37.6 1.4 ± 0.2c -46.0 1.53 ± 0.18
Notes: Measurements were made at the end of October in 2007 after more than five-month-long treatment. Dry weight of root or shoot decrease percent (%) = (treatment — control)/control x 100%. Means followed by different letters indicate significant differences at P < 0.05. n = 10.
use efficiency (WUE) was calculated as the ratio of the net photosynthetic rate to transpiration rate.
Biomass determination. Biomass determination was carried out by the end of October. Licorice plants were separated into the roots and shoots. Dry weights were determined after drying for 72 h at 50°C in an oven. Root-to-shoot ratio = root dry weight/shoot dry weight. Then the dried roots were used for glycyrrhizic acid and liquiritin analyses.
Glycyrrhizic acid and liquiritin analyses. Glycyrrhizic acid and liquiritin were extracted as described in [15]. Dry roots were extracted with a tenfold volume of 0.3% ammonia for 30 min under ultrasonication (250 W 20 KHz). Glycyrrhizic acid and liquiritin concentrations were determined with a HP1100 high performance liquid chromatography system ("Agilent Technologies", United States) consisting of a G1311A pump, a G1379A degasser, and a G1313A autoinjector connected to a G1315B diode array detector (DAD). The separation was performed on a DIKMA Diamon-silTM-C 18 column (250 mm x 4.6 mm x 5 ^m) with a mobile phase consisting of 0.1% H3PO4 (solvent A) and acetonitrile (solvent B). The sample (10 ^l) was eluted with a gradient profile, and the column was maintained at 25°C [5, 15].
Statistical analysis. Statistical treatment was performed using a SPSS statistical package (version 13, SPSS, Chicago, United States). The difference between the mean values of each treatment was determined using Duncan's multiple range test and considered significant at P < 0.05.
RESULTS
Gas exchange
The net photosynthetic rate, stomatal conductance, and transpiration rate decreased with increasing water deficit (table 1). Compared to the control,
60—70% WC had no effect on the net photosynthetic rate and stomatal conductance, but at 40—50 and 20— 30% WC, photosynthesis and transpiration were significantly reduced. However, water deficit increased WUE and
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