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Photosynthetic Recovery of a Perennial Grass Leymus chinensis after Different Periods of Soil Drought

Abstract The effects of rewatering after different periods of soil drought stress on the photosynthetic capacity of Leymus chinensis in pots were investigated. The plants were subjected to short-term (10-d), moderate-term (20-day) and long-term (30-d) drought each followed by rewatering. Control plants were well watered during the experimental periods. The long-term water stress without rewatering decreased the chlorophyll content, Chl a/b ratio, carbonic anhydrase (CA) activity, net photosynthetic rate (A), and leaf area compared with the control. Rewatering increased the ratio of Chl a/b, CA activity and A, but decreased the leaf area and ion leakage from the cut leaf pieces. The long-term water stress without rewatering reduced the maximal efficiency of PSII (Fv/Fm), the actual quantum yield (Φp), and photochemical quenching (qp), but these values were increased by rewatering to more than the control level, though non-photochemical quenching (qN) was decreased as compared with the control. This implied that long-term drought aggravated PSII, but rewatering improved it. The net CO2-exchange rate showed similar diurnal changes in all treatments, but the rate in the morning was lower in long-term drought (before rewatering) than in the other treatments. These results suggest the photosynthesis of Leymus chinensis may be well adapted to episodical soil drought.

Water defi cit is one of the key environmental factors limiting grassland productivity (Carlen et al., 1999;Bai et al., 2004). Severe water defi cit results in the decreases in leaf photosynthetic capacity and the key enzyme activity (Hsiao, 1973;Kaiser, 1987;Inamullah and Isoda, 2005;Xu and Zhou, 2006b), and leads to oxidative damage (García and Lamattina, 2001;Xu and Zhou, 2006a). However, a moderate water defi cit improves photosynthetic capacity and regulates carbon allocation due to acclimation to environmental stresses (Hsiao, 1973;Havaux, 1992;Yang et al., 2001;Xu and Zhou, 2005b). Thus, how grassland plants respond to water stress is a critical issue.
Grasslands dominated by Leymus chinensis, are widespread from the loess plateau of China (the southeast edge) to Bajkal of Russia (the northeast edge), and from Sanjiang plain of northeastern China (the east edge) to Ulaanbaatar of Mongolia (the west edge). Leymus chinensis is a rhizomatous and native perennial plant with good palatability and high forage value. However, the distribution region of Leymusdominated grassland is severely constrained due to climatic change and land-use practices (for example, overgrazing, reclamation, etc.) in the recent decades (Zhou et al., 2002;Bai et al., 2004;Zhou et al., 2006). Many researches indicated that drought and the seasonal precipitation seriously affect the growth and the community composition of grasslands (Distel et al., 1996;O'connor et al., 2001;Kröpfl et al., 2002;Wang and Gao, 2003), and drought is particularly a problem, occurring episodically in many regions (Kang et al., 2002;Shah and Paulsen, 2003). The global climatic change may result in not only the change of total precipitation but also the occurrence of discontinuous drought. How the discontinuous drought induced by the global climatic change will affect plant growth and plant community composition remains unknown, especially for grassland plants that are more sensitive to drought.
In this research, comprehensive experiments with different soil drought and rewatering treatments were conducted in the greenhouse at the Institute of Botany, the Chinese Academic of Sciences in order to elucidate how discontinuous drought affects the photosynthetic capacity of grassland plants. The chlorophyll content, net photosynthetic rate, PSII capacity, a key photosynthetic enzyme activity (carbonic anhydrase), soluble protein and sugar concentrations, as well as ion leakage from the leaf (as a damage indicator) were examined to elucidate the adaptive mechanisms of Leymus chinensis plants to soil drought and rewatering. We propose a hypothesis that rewatering promotes recovery the photosynthetic capacity, but not the leaf growth of the plants, from the suppression by soil-water stress.

Plant culture and water treatments
Seeds of Leymus chinensis (Trin.) Tzvel. were collected from a grassland fi eld in Xilinhot, Inner Mongolia in 2001. They were sterilized with 5% potassium permanganate solution for 5 min, rinsed, and immersed in water for 2 d, and then kept below 0ºC in a refrigerator. They were sown in plastic seedling trays (50 cm in length, 30 cm in width and 20cm in height) on 6 May 2002, and transplanted into plastic pots (5.4 L) wrapped with plastic fi lm and grown there until the fi fth leaf appeared. Each pot was fi lled with 4.16 kg dry soil obtained from the fi eld and planted with 6 plants per pot. The soil contained organic C, total N, available N and P at the concentrations of 10.18 ±0.52 g kg -1 , 1.68 ±0.05 g kg -1 , 89.46 ±2.37 mg kg -1 and 126 ±5.21 mg kg -1 , respectively. The soil composition is similar to that of the fi eld soil. The experiment was conducted in a greenhouse, day/night temperature was 28-30/20-25ºC, and maximal photosynthetic photon fl ux density (PPFD) was around 1100 µmol m -2 s -1 .
Water was withheld at the plant tillering stage (about 50 d after sowing) when the plant height was ca. 25 cm. Then, the plants were subjected to severe drought with less than 45% soil water content (Hsiao, 1973), which was set to simulate natural drought conditions in this area, for 0, 10, 20 and 30 days, each followed by rewatering (Fig. 1). These treatments are called the control, Std (short-term drought), Mtd (moderate-term drought) and Ltd (long-term drought), respectively, in this paper. Each treatment had 12 pots as replicates. Water was supplied to each pot by weighing at 17:00 every day. Water addition was recorded for all treatments. Most of the parameters were measured twice: on 12 August and on 22 August (see Fig. 1).

Leaf area
Leaf fresh samples from whole plants in a pot were dried in an oven at 80ºC to a constant weight to measure dry weight. Leaf area was estimated from the relationship between leaf area and dry weight (Kolb and Sperry, 1999).

Net CO 2 -exchange rate
On the youngest fully-expanded leaves, the net CO 2exchange rate was measured by an open system (ADC BioScientifi c, Hoddeson, UK) at a CO 2 concentration of 350-360 µmol mol -1 , 65-80% relative humidity, and light intensity of 900 µmol m -2 s -1 .

Chlorophyll fl uorescence measurements
The youngest expanded leaves were dark-adapted for 30 min prior to fl uorescence measurements; and the chlorophyll fl uorescence was measured at room temperature (25ºC) with a portable fl uorometer (PAM-2000, Walz, German). The fl uorescence parameters were calculated according to the formulae (van Kooten and Snel, 1990;Lu and Zhang, 1999): The maximal effi ciency of PSII was shown by F v / F m =(F m −F 0 )/F m , the actual quantum yield of PSII electron centers by Φ p = (F m '−F S )/F m ', photochemical quenching by q P =(F m '−F S )/(Fm'−F 0 '), and non-photochemical quenching by q N =1−(F m '−F 0 ' )/(F m −F 0 ).

Leaf relative water content
The relative water content (RWC) was determined according to the method of , which was modifi ed by Wilkinson et al. (2001). The detached leaves (about 1g fresh mass) were cut, fresh weight (FW) was measured, and then placed in a beaker (25 mL) fi lled with water overnight in the dark. They were reweighed the next morning to obtain the fully turgid weight (TW) and dry weight (DW) was determined after drying at 80ºC for 24 h in an oven. Leaf relative water content (RWC) of the leaves was expressed as (FW -DW)/(TW−DW) ×100.

Carbonic anhydrase activity assay
The fresh samples of leaves were frozen in liquid N for 1 min and then stored at -80ºC for carbonic anhydrase activity assay. About 0.5 g leaves were vigorously ground in a mortar on an ice plate with 40 mmol l -1 Hepes-KOH (pH 8.2), containing 10 mmol L -1 dithiothreitol (DTT). After adding additional 1 mL of the buffer mixture, the homogenate was fi ltered through four-layer gauze cloths. The remainder was diluted in the blending buffer as required (diluted 10 times) and stored at 0ºC prior to assay. The rate of CO 2 hydration was measured at 0ºC at different pHs. The reaction mixture contained 25 mmol L -1 Hepes-KOH buffer (pH 8.2) in a fi nal 1 mL. The reaction was started by adding a CO 2 solution (distilled water saturated with CO 2 at 0ºC). The mixtures were stirred by up and down agitation of the precooled pH electrode probe. Control assays contained boiled extracts. The observed rate of pH was converted into equivalent mol H +1 generated by titrating the reaction mixture buffer in the range from 8.3 to 7.6 of pH with a standard solution of H 2 SO 4 (Dai et al., 2000).

Chlorophyll, soluble sugar, and soluble protein concentrations
Chlorophyll (Chl) in leaves was extracted with 10 mL 80% acetone and their compositions were spectrophotometrically calculated according to the equations reported by Lichtenthaler and Wellburn (1983). The Chl concentration was expressed as the percentage of fresh weight (FW) of viable leaf.
Soluble sugar was exhaustively extracted from leaf samples by hot ethanol extraction. Five mL of 70% (v/v) ethanol was mixed by shaking with 100 mg of ground leaf material in a test tube. The sample was then incubated at 80ºC for 20 min with shaking, and centrifuged for 5 min at 1,500 g. The freshly prepared anthrone reagent (0.2% anthrone in concentrated H 2 SO 4 ) was pipetted into test tube and chilled in ice water. The extract was thoroughly mixed with the anthrone reagent. The tubes were heated in boiling water for 15 min and then rapidly cooled. The absorbance was read at 630 nm (Yemm and Willis, 1954;Güitman et al., 1991). Total soluble sugar content was calculated as the percentage of dry matter of viable leaf.
About 1 g leaves were homogenized with 10 mL of 50 mmol L -1 sodium phosphate, pH 7.8, containing 2 mmol L -1 EDTA and 80 mmol L -1 L-ascorbic acid. After centrifugation at 15,000 g for 20 min, the supernatants were used to determine soluble protein (Cruz et al., 1970). Soluble protein concentration was measured according to the method of Bradford (1976).

Ion leakage from leaves
Ion leakage was determined according to García and Lamattina (2001). Leaves were cut into about 20 mm 2 pieces (1g fresh mass) and placed in a beaker (25 mL) with 15 mL de-ionized water, and exhausted by rinsing with an air extractor for 10 minutes. After the incubation at 25ºC for 2 h, the conductivity of the bathing solution (Xi) was determined with a conductivity meter (HI9033 multi-range, Hanna Instruments, Sigma, Singapore). Then, the samples were heated at 85ºC for 2 h, and the conductivity (Xt) was read again in the bathing solution. Electrolyte leakage was expressed as a percentage of the total conductivity after heating at 85ºC (Xi / Xt).

Statistical analyses
All statistical analyses were performed using SPSS 10.0 (SPSS, Chicago, Illinois, USA). Differences in photosynthetic rate, RWC, Chl content, and CA activity were tested using one-way analysis of variance (ANOVA) (p = 0.05 or p = 0.01) followed by Duncan's multiple range test. Interactive effects of treatments and daily times were analyzed using a two-way ANOVA (UNIANOVA) (p = 0.05).

Relative water content of leaves
On 12 August, the plants in Ltd were still in drought, and their leaf relative water content (RWC) was signifi cantly lower than that in the control ( Fig. 2A). However, RWC in Std and Mtd, which were rewatered, was slightly higher than that in the control. On 22 August, RWC was the highest in Ltd and lowest in the control. Generally, RWC in all treatments on 22 August was lower than that on 12 August, implying that RWC decreased gradually with plant growth.

Leaf area
As shown in Fig. 2B, leaf area was largest in Std among the treatments including the control, on both 12 August and 22 August, indicating that rewatering following short-term drought stress stimulated leaf growth but that following long-term drought did not; i.e., leaf growth was not recovered by rewatering. Table 1 shows the effects of different soil drought treatments on Chlorophyll (Chl) concentration at two growth stages. These data showed signifi cant differences among different soil drought treatments (p < 0.01 for Chl a, a / b, but p < 0.05 for Chl b). The concentrations of Chl a, Chl b and Chl a / b ratio in Ltd were 43.4, 28.6 and 21.2%, respectively, lower than those in the control, and 78.6, 29.3 and37.1% respectively, higher than those in Mtd on 12 August. The concentrations of leaf Chl in both Ltd and Mtd treatments on 22 August were higher than those in the other treatment (p < 0.05). The difference in the Chl concentrations in leaves between Mrd and Ltd and that between the control and Std treatments were not signifi cant. As compared with the control, the concentrations of Chl a and b and a / b ratio in Ltd were 193.0, 54.8 and 97.6%, respectively, higher than those in the control. Table 2 shows the effects of drought and rewatering on chlorophyll fl uorescence. The maximal effi ciency of PSII photochemistry (F v / F m ) represents the most photochemical effi ciency or primary effi ciency of light energy transitions, the actual quantum yield (Φ p ) indicates the effi ciency of transfer of absorbed photons to the reaction center of PSII, photochemical quenching coeffi cient (q P ) represents the portion of light energy absorbed by PSII, and non-photochemical quenching coeffi cient (q N ) is the portion of absorbed light energy by antenna pigment but not used in electron transport and dissipate as thermal energy. On 12 August, F v / F m , Φ p , and q p in Ltd were 9.6, 48.4 and 33.3%, respectively lower, and q N in Ltd was 4.5% higher than those in the control, suggesting that longterm soil drought reduces PSII capacity. However, F v / F m , Φ p , and q p were 4.2, 52.1 and 43.1%, respectively, higher and q N in Mtd was 14.2% lower than those in the control. These data showed signifi cant differences among different soil moisture treatments (p < 0.01 for F v / F m , Φ p , and q p , but p < 0.05 for q N ). On 22 August, F v / F m , Φ p , and q p in Mtd were 4.4, 27.0 and 15.9%, respectively, higher, and those in Ltd were 10.4, 101.2 and 40.8%, respectively, higher than in the control. However, those in Mtd and q N in Ltd were 9.9 and 8.9%, respectively, lower than those in the control. These fi ndings implied that rewatering, especially Ltd enhances PSII capacity.

Ion leakage from leaves
The liberation of ions from the cell to the extracellular space can be used as the index of membrane injury of the plant cell. As shown in Fig.  3A, ion leakage expressed as the percentage of total conductivity (Xi / Xt) in Std and control was 25.5% on the average, but that in Mtd and Ltd was 17.0% on the average in upper leaves, which was signifi cantly lower than in the control and Std. In the lower leaves, similar results were observed but the difference was not signifi cant. These results suggest a higher ability to adapt to the change in soil water moisture in young leaves than in old leaves.

Carbonic anhydrase activity
The carbonic anhydrase (CA) activity in Ltd on 12 August was markedly affected by soil water stress, but that on 22 August was signifi cantly stimulated by rewatering (Fig. 3B). The leaf CA activity on 12 August was highest in Mtd followed by Std, control and Ltd treatments in this order, but that on 22 August was highest in Ltd followed by Mtd, Std and control treatments.

Soluble protein and soluble sugar concentrations
On 22 August, Mtd treatment had the highest concentration of leaf soluble protein followed by Ltd, Std and the control (Fig. 4A). Compared with the control, soluble protein concentrations in leaves in Mtd and Ltd were 2.4-and 2.0-fold higher, respectively.
The concentration of soluble sugar was also highest in Mtd followed by Ltd, Std and the control in this order (Fig. 4B). The concentration in Mtd and Ltd was 94.9 and 81.5%, respectively, higher than that in the control. Fig. 5A shows the diurnal patterns of net CO 2exchange rate in leaves of Leymus chinensis measured on 8 August. Daily courses were similar for all treatment; there was a gradual increase from about 7:00 with a peak at about 8:00. Then it decreased and was lowest between 11:00 and 12:00, followed by an increase again with a peak at 14:00. Net photosynthetic rate was reduced to almost zero at 18:00, indicating that the respiratory rate of the leaves might be equal to CO 2 -exchange rate without sunlight. The leaf respiration showed little fl uctuation at night, and the net photosynthetic rate started to increase again at 4:00

Diurnal change of leaf CO 2 -exchange rate
The maximal effi ciency of PSII photochemistry; Φ p , the actual quantum yield of PSII electron centers; q P , photochemical; q N , non-photochemical quenching. -5:00. Fig. 5A also demonstrates the effects of soil drought and rewartering on net CO 2 -exchange rate in leaves. There were signifi cant differences in the CO 2 exchange rate among the treatments. Mtd treatment showed the highest rate before 12:00, particularly between 8:00 and 11:00, but not after 13:00, indicating that rewatering stimulated leaf photosynthesis. Under soil drought conditions, the photosynthetic rate was greatly decreased. However, leaf respiratory rate at night was not signifi cantly different among the treatments. Fig. 5B presents the diurnal changes of leaf net CO 2exchange rate in different soil drought treatments measured on 18 August. The leaf photosynthetic rate increased in the morning in all treatments. The leaf photosynthesis in Ltd treatment showed a higher rate, and the rate in the control treatment the lowest. After midday, the net CO 2 -exchange rate decreased in all treatments although there was a minor peak in the afternoon. The net CO 2 -exchange rate in leaves was close to zero at 7:00 p.m.
Statistical analysis by Univariate Analysis of Variance (UAV) indicated that the net photosynthetic rate signifi cantly varied with the time of day, and the ratio in the upper and lower leaves were signifi cantly different (Table 3). Ltd treatment increased the net CO 2 -exchange rate from 7:00 to 11:00 by 35% and 4.38% in upper and lower leaves, respectively, as compared with those in the control treatment. There were signifi cant differences among different times, and treatments, and interaction between time and treatment, in the upper leaves (p < 0.01), but no signifi cant differences were observed in the lower leaves except at noon. Leaf respiration rates at night, were not greatly infl uenced by rewatering.

Discussion
Two measurement days (Aug 12 and 22) refl ected different plant water conditions among different drought treatments. On 12 August, plants in Std and Mtd treatments were under rewatering, while those in Ltd treatment were still under drought stress. On 22 August, all plants were under rewatering, although they differed in duration of rewatering. Thus, Ltd plants on 12 August could be used to assess the effects of long-term drought, while Ltd plants on 22 August to assess the effect of rewatering after the stress. Chl a and Chl b contents and Chl a / b ratios could be used as indices of plant photosynthetic capacity (Johnston et al., 1989). The present results showed that long-term water stress reduced the Chl contents and Chl a/b ratio (Table 1), that represents leaf senescence and a decrease in energy available for the photosystems (Johnston et al., 1989). Carbonic anhydrase (CA) is involved in the fi rst step of CO 2 fi xation in leaf mesophyll cells (O'Leary, 1982;Hatch and Burnell, 1990). The present result showed that CA activity decreased when plants were subjected to long-term soil water stress, which is consistent with the result for the mature leaves of rice (Oryza sativa L) (Dai et al., 2000). However, Hatch and Burnell (1990) reported that the activity of the enzyme might be just for the conversion of CO 2 to HCO 3 in mesophyll cells, and does not limit photosynthesis. Thus, the role of CA in drought acclimation needs to be assessed in the future. Under fi eld conditions, plants often suffer from damage including photoinhibition and photooxidation due to soil drought stress (Chaves et al., 2003). Rainfall occurs after various durations of dry periods in natural fi elds. The drought treatments in this study simulated drought conditions in natural fi elds, and the results may represent the plant responses to drought stress in natural fi elds.
The chlorophyll fl uorescence parameters measured on plant leaves may be used to assess of PSII capacity (van Kooten and Snel, 1990;Taub et al., 2000;. This experiment showed that rewatering increased PSII capacity. Severe soil drought resulted in cell-membrane damage (Benhassaine-Kesri et al., 2002), and a decrease in leaf photosynthetic capacity (García and Lamattina, 2001;Xu and Zhou, 2006b). However, rewatering after long-term drought may repair the damage and stimulate the well photosynthesis. The other studies indicated that elevated CO 2 could protect photosynthesis against high-temperature and drought damage (Hamerlynck et al., 2000;Taub et al., 2000), and water stress or salinity increased the ability to protect plant from high temperatures (Lu and Zhang, 1999;Lu et al., 2003;Qiu and Lu, 2003). However, extreme drought weakens the photosynthetic adaptation to a high temperature (Xu and Zhou, 2005a;Xu and Zhou, 2006a), and the leaf photoprotective mechanism is associated with leaf temperature change, depending on plant species (Inamullah and Isoda, 2005). The adaptive mechanisms may be involved in the stress history of the plant. There is evidence that plants, which have been previously exposed to water stress, show an improved tolerance to water stress through increase in solute levels (Takami et al., 1981;Morgan, 1984). In the present study, the concentrations of soluble protein and sugars in the leaves were higher in the plants previously subjected to soil drought than in those grown in ample soil moisture. The drought treatment followed by rewatering also increased Chl a / b ratio, CA activity and PSII capacity. The osmoregulation under drought treatment may have increased the tolerance to drought.
The present results showed that a decrease in leaf physiological capacity (activity) by long-term drought might be overcome by rewatering. The capacity (activity) can be increased over that in well-watered treatment (control). Investigations on the stimulating effect of drought treatment followed by rewatering in wheat by Liu et al. (2001) indicated that severe water stress increased the leaf area more greatly than the moderate water stress. Recently, Siopongco et al. (2005Siopongco et al. ( , 2006 reported that the stimulation of rice plant growth by drought followed by rewatering depended on the genotype. In the present study, although rewatering stimulated Chl content, Chl a / b ratio, and CA activity, the leaf growth of the plants subjected to rewatering following long-term water stress was inferior to that of the control. It is suggested that leaf expansion is more sensitive to water stress than many other physiological processes in Leymus chinensis. This point highlights the difference between the responses of plant growth and some physiological processes to soil moisture. Note that we reported here only the experiment, the physiological responses of the grassland plant at juvenile stage. The responses at adult stage with rhizomes and extensive root system may be largely different. On the other hand, the plant growth in pots is different from that in the fi eld, because of different environmental conditions, such as temperature, light. The limited volume of soil in pots might also be responsible (Kang et al., 2002). However, the data may be comparable among different soil water treatments because all plants were grown in the same size pots and under same environmental conditions. Thus, the present results may provide insight into underlying mechanism of the grassland plant in response to soil drought and rewatering at the juvenile stage. However, more investigation is required in the fi eld at different plant growth stages in the future.