Water Potential, Stomatal Dimension and Leaf Gas Exchange in Soybean Plants under Long-term Moisture Deficit

Jpn. J. Trop. Agr. 44 (1) :30-37, 2000 Water Potential, Stomatal Dimension and Leaf Gas Exchange in Soybean Plants under Long-term Moisture Deficit Ashok K. GHOSH, K. ISHIJIKI, M. TOYOTA, A. KUSUTANI and K. ASANUMA Faculty of Agriculture, Kagawa University Ikenobe 2393, Miki-cho, Kagawa-ken 761-0795, Japan Abstract Frequency of stomata, area of stomatal aperture and their relationship to leaf gas exchange in soybean [Glycine max (L.) Merr.] leaves grown under different soil moisture levels are not well documented. In this experiment, attempts were made to investigate the effects of soil moisture deficit imposed from the early growth stage until maturity on the area of stomatal aperture, photosynthesis, dark respiration and transpiration. Determinate cultivar 'Akiyoshi' was grown in a greenhouse at optimal (80% of field capacity) and three sub-optimal soil moisture levels (65%, 50% and 40% of field capacity). Stomatal frequency was increased, but the total area of stomatal aperture per unit leaf area decreased (P<0.05) with decreasing plant available water. Photosynthetic rate was markedly reduced (P<0.01) as the soil moisture deficit increased. Dark respiration rate of the leaves also decreased by moisture deficit throughout the growth stages of the plants. The net carbon assimilation efficiency of soybean leaf drastically reduced by water limitation. Increasing moisture deficit decreased the transpiration rate. Photosynthetic and dark respiration rates were correlated with the leaf water potential and the area of stomatal aperture per unit leaf area. Plants with larger stomatal apertures transpired significantly more water than the plants with a smaller aperture area. Increasing moisture stress significantly reduced the water use efficiency throughout the growth stages. Moisture deficit decreased the area of stomatal aperture per unit area which reduced the leaf gas exchange rate along with a higher stomatal resistance. Key words Dark respiration, Moisture deficit, Photosynthesis, Soybean, Stomata, Transpiration Introduction It is well known that the stomatal density in leaves varies depending on the environment in which the plants grow6,10,14,17,20,21). It is also well known that the degree of stomatal opening depends on the surrounding environment. However, detailed studies on the area of stomatal aperture of soybean leaves for varying intensities of a particular stress are still lacking. Soybean is often cultivated under non-irrigated conditions. There are considerable variations in the soil moisture status in relation to the types of soils in the soybean producing areas in the world. Sometimes the soils contain sub-optimal levels of plant available water throughout the growing Received Feb. 12, 1999 Accepted Aug. 27. 1999 season, in particular when precipitation is insufficient or erratic, or under continuous drought. In the tropical soybean-growing soils, this kind of moisture status is frequently observed which limits growth and yield. Stomatal closure is the initial response of plants to soil moisture deficit, presumably initiated by root signals8). The photosynthetic rate is reduced if the soil moisture deficiency occurred at earlier stages of growth19). High carbon exchange rate (CER) is effective in increasing the yield of indeterminate cotton and soybean18). We assume that the area of stomatal aperture per unit leaf area may differ in leaves grown at different moisture levels which should be related to the CER. Thus, biomass accumulation and yield may be indirectly associated with the area of stomatal aperture. These results are derived

GHosx et al.: Moisture Stress and Gas Exchange in Soybean 31 from experiments where water stress was applied only at certain stage (s) of plant growth. However, it remains to be determined how soybean leaves respond in terms of leaf water potential, photosynthetic rate, dark respiration and transpiration when soil moisture remains far below the optimum level throughout the growth. The respiratory loss of photoassimilated carbon is likely to be altered with the progression of water stress within the plant body. Data are also lacking on the seasonal trend of the dark respiration rate of soybean leaves which develop under different degrees of moisture stress. It is essential to determine how the rate of respiration varies with the changes in the leaf water potential due to differences in the soil moisture deficit. There is also inadequate information regarding the area of stomatal aperture in relation to soil moisture deficit, and the relationship of stomatal aperture to leaf gas exchange characteristics. In a companion paper13), we reported the growth and yield of soybean plants cultivated at different soil moisture levels throughout the growing season. In this paper, we investigated the relationship between the stomatal opening and leaf gas exchange, and the influence on other factors in plants grown under continuous optimum and sub-optimal soil moisture conditions. Materials and Methods Plant culture Four soil moisture levels (80%, 65%, 50% and 40% of FC) were simulated in clay pots, 30 cm in diameter and 24 cm in depth. Seeds of the latematuring determinate cultivar 'Akiyoshi' were sown on 26th June, 1996. After emergence, two healthy plants were allowed to grow in each pot. Soil moisture levels were maintained by irrigating every pot two times a day. The whole experiment was conducted inside a glasshouse under natural sun light and atmospheric humidity. The detailed description of the materials and plant culture methods was given elsewhere13). Leaf water potential At predawn (from 4:00 to 5:00 am), the leaf water potential (ƒõ1) was estimated using a Scholander-type pressure chamber (PMS Instrument Corporation, Oregon, USA) at the V11, R1, R3 and R5 stages11). The terminal leaflet of the trifoliate of 4th uppermost node of the main stem was used. Data were collected in six replications, i, e., using six leaves from six different plants in each treatment. Stomatal frequency and size For the stomatal measurements, fully expanded leaves of the 10th or 11th nodes on the main stem were used at the R1 stage of the plants. On a sunny clear day, the stomata were considered to be fully open between 9:30 to 10:30 am. Stomatal impressions were taken during that period using clear nail varnish (from Shiseido Cosmenity, Japan) coated on both leaf surfaces on either side of the midrib in the central part of the terminal leaflets. Impressions of six leaves from six different plants were collected in each treatment. Twenty five randomly selected microscope fields were studied for stomatal counts under 100x magnification from each leaf impression. Guard cell length and the area of aperture were measured by imageanalyzing optical micrography using an image analyzer (Luzex III-U: Nireco Co. Ltd.). Area of the same leaves was measured to calculate the number of stomata on both surfaces. After tracing on black sheets of paper through the outer edge of the leaflets, the papers were cut to the exact shape of the leaflets. Then the leaf shaped black papers were passed through an automatic area meter (Hayashi Denko Co. Ltd., Japan) to obtain the area of individual leaflets. Six replications of the data were obtained for subsequent mean calculation and statistical analysis. Leaf gas exchange measurements The fully expanded leaves of the 4th uppermost node of the main stem were used for the gas exchange measurements, i, e., net photosynthesis, stomatal conductance, stomatal resistance, transpiration and dark respiration. The same leaves were used to estimate the water potential. A portable photosynthesis system, the LI-COR model 6200 (LiCor Inc., Lincoln NB, USA) was used. Under natural sunlight the measurements were performed between 9:30 am and 11:30 am. In these sunny days, the photon flux density within the PAR wave band (400 `700nm) ranged between 1300 `1500,ƒÊmolm-2s-1. The leaf temperature ranged from 33 Ž to 36 Ž during most of the measurements,

32 Jpn. J. Trop. Agr. 44 (1) 2000 which corresponds to the ambient summer temperature at the site of the experiment. Glass walls were replaced with steel net and the exhaust fans were continuously running to avoid excessive heat inside the glasshouse. The data were collected at about 15-day intervals. By covering the leaf chamber with an opaque polyethylene sheet, it was possible to measure the dark respiration rate of the same leaves, using the same machine at the same temperatures. Instantaneous water use efficiency (WUE) was calculated by dividing the net photosynthetic rates by the transpiration rates. Effect of moisture deficit on water potential, stomatal dimension and gas exchange parameters was examined by analysis of variance according to STEEL and TORRIE24) and using statistical software MSTATC (Michigan State University, USA). Mean separation was performed through the computation of least significant differences (LSD). Results Leaf water potential and Discussion The seasonal trends of the leaf water potential (ƒõ1) of the plants grown at four levels of soil moisture are presented in Table 1. There was a significant (P<0.01) consistent decrease in ƒõ1 with the decrease of the soil moisture level throughout the vegetative and reproductive stages of growth. ƒõ1 decreased by about 55% due to the 50% decrease of available soil moisture at all the four critical stages of plant growth (V11, R1, R3 and R5). With the increase in plant age, the ƒõ1 did not decrease significantly in the plants grown on higher moisture deficit soils, while minimal changes were also observed in the nonstressed plants without showing any clear trend. Table 1. Leaf water potential (in MPa) at four critical growth stages of soybean plants cv. Akiyoshi cultivated at four soil moisture levels. FC denotes the field capacity These results indicate a very significant effect of soil moisture levels on the xylem water potential of leaves at all stages of growth. These changes in the water potential can be considered to be the primary indicators of many morphological and physiological changes. Decrease of ƒõ1 in soybean and other crops by soil moisture deficit was reported by many authors,4,5,15,22,23). In all of these experiments, water stress was applied for a short period of time and mainly at the early reproductive stage of the plants. Our data clearly depict the seasonal changes of ƒõ1 in soybean plants grown at different levels of moisture deficit uniformly throughout the growing season. ƒõ1 changed at different stages of plant growth. Irrespective of the size of the plants, the visible symptoms of water stress (temporary wilting) were prominent in plants with increasing moisture deficiency as the day temperature increased until noon, while in the early morning and late afternoon the symptoms of wilting were not evident. Although this visual observation was not analyzed in detail, it appears that the soybean plants were subjected to a severe stress when the water deficit was combined with a high temperature. Stomatal frequency and aperture size Guard cell length decreased with the progression of the soil moisture deficit on both the abaxial and abaxial surfaces (Table 2). Compared to the optimum level of soil moisture (80%FC), the stomatal length on the abaxial surface was 30% smaller in the plants grown at 40% FC while 25% on the abaxial surface. Growth of the stomata was restricted by inadequate supply of moisture. Under water stress conditions, the stomata are only partially opened, while the guard cell length was still shorter compared to that in the non-stressed plants. Stomata! frequency increased significantly (P<0.01) on both the abaxial and abaxial surfaces of leaves, when the plants were subjected to increasing levels of moisture deficit. Hence, the frequency of the total stomata including both surfaces was higher in the leaves of stressed plants than in those of non-stressed plants. During the microscopic studies for the stomatal measurements, it was commonly observed that the size of the epidermal cells of the water-stressed leaves was smaller than that of the non-stressed leaves. Although data on the

GHosH et al.: Moisture Stress and Gas Exchange in Soybean 33 Table 2. Stomatal frequency and aperture area in leaves of soybean plants grown under optimum and sub-optimum soil moisture conditions. FC denotes the field capacity cell size were not collected in this experiment, higher stomatal frequency in the stressed leaves could be due to the smaller cell size. CIHA and BRUN6) and PENFOUND20) reported a lower frequency of stomata in plants grown at adequate soil moisture levels compared to the frequency in moisture-stressed plants. The area of the single leaflet was significantly smaller (P<0.01) in the plants grown under increased moisture stress than in the non-stressed plants. Water is an essential material for the growth and development of plants. Inadequate supply of moisture restricts cell division and cell growth and eventually induces a limited enlargement of leaves. Since the leaf area is highly correlated with the average leaf water potential (P<0.01), the volume of plant available moisture in soil is an important factor for the growth of leaves as well as the whole plant. BOYER3) and CIHA and BRUN6) reported that leaf enlargement was restricted when soybean plants were subjected to water stress. The number of stomata per leaflet consistently decreased on both leaf surfaces with the increasing moisture deficit (P 0.05). ƒõ1 was correlated with the stomatal frequency (r=0.986*) and the number of stomata per leaflet (r=0.979*). These relationships indicate that moisture limitation also inhibits the formation of stomata. Area of the stomatal aperture decreased significantly (P<0.01) with the increase of the soil moisture deficit on both abaxial and abaxial surfaces. As the soil moisture level decreased from 80% to 40% of FC, the area of stomatal aperture decreased by 51% on the abaxial surface and 55% on the abaxial surface with consistent reduction at every step of moisture differences. The total area of stomatal aperture per unit leaf area similarly decreased with increasing soil moisture deficit on both leaf surfaces. The total area of stomatal aperture per unit area of both abaxial and abaxial surfaces showed a highly positive correlation with the carbon exchange rate, as discussed later (Table 4). Therefore, the moisture deficit led to a decrease in the number and growth of stomata per leaflet and also resulted in a smaller area of aperture per unit leaf area. JUNG and SCOTT16) observed that the stomata of non-irrigated soybean plants were partially closed during the

34 Jpn. J. Trop. Agr. 44 (1) 2000 daylight hours and closed earlier in the afternoon than the stomata of irrigated plants. In their experiment, moisture stress was imposed on plants grown under adequate soil moisture levels. Although their observations are not exactly similar to our investigation, one of their important findings that agrees with our results is that under drought conditions stomatal aperture is smaller. Since water is an important factor of plant growth, the data of this experiment showed that the soil moisture deficit caused an inhibition of the formation and growth of the stomata as well as their pore size during the active gas exchange period. Since stomata are the gateway for the exchange of CO2, O2 and water vapor between the plant system and the atmosphere, the aperture area might be related to the photosynthesis, respiration and transpiration Leaf gas exchange rates. Net photosynthesis was greatly reduced as Table 3. Dynamics of leaf gas exchange characteristics of 4th uppemost leaves of soybean plants grown at four soil moisture levels. DAE, days after emergence; FC, field capacity; LSD (0.05), least significant difference at 5% probability level.

GHOsH et al.: Moisture Stress and Gas Exchange in Soybean 35 the soil moisture decreased (Table 3). These differences among the treatments were significant (P<0.001) and consistent. A 50% decrease of soil moisture from the optimum level resulted in a 86% reduction of net photosynthetic rates at V11. This difference was narrowed to 70% at the R2 stage and then widened again to 80% at the R4 stage. This observation indicates that the decrease in net photosynthesis with increasing water deficit was less appreciable during the flowering stage than during the pod filling stage. These results agree with the report of GHORASITY et al.12) TROEDSON et al.25) reported that the photosynthetic rate increased with increasing soil moisture up to the saturation level. In indeterminate cultivars, CORTES and SINCLAIR7) observed that as a result of drought during reproductive growth, the carbon exchange rate was reduced by approximately 25% of the remainder of the season. The plants grown at higher soil moisture levels showed higher dark respiration rates throughout the growing season. However, when the moisture supply was adequate, the increase in the rate of photosynthesis was several times larger than the increase in the dark respiration rates of the same leaves compared to those of moisturedeficient leaves. These results were reflected in the higher total dry matter accumulation per plant13). Under each treatment, the changes of the dark respiration rates at different stages of growth did not show any significant difference. The ratio of the photosynthetic rate to the rate of dark respiration decreased significantly (P<0.05) with the increasing moisture deficit in the plants at all the six stages of growth when the data were taken. At the V11 stage, nonstressed leaves had a ratio of 11:1 which gradually decreased to 3:1 in the leaves of the plants in the highest moisture stress treatment. Again, this ratio changed at different stages of growth. With the increase of the plant age, this ratio became smaller until the R4 stage and then increased slightly at the R5 stage in all the treatments, with a decreasing trend in the stressed plants. Thus the ratio of carbon fixation to carbon release in the leaf tissues decreased due to the decreasing supply of moisture. These ratios indicate that under water stress conditions, the photosynthetic rate decreased more sharply than the rate of respiration at all the stages of plant growth. Thereby, the net carbon assimilation efficiency was drastically reduced. These are the main reasons for the decrease in dry matter production and seed production of soybean plants under soil moisture deficit which were reported in the companion paper13). Increasing moisture deficit resulted in a significantly higher (P<0.05) stomatal resistance throughout the vegetative and reproductive stages of the plants. Stomatal resistance also increased with the increasing age of the plants. At every stage of plant growth, the rate of transpiration decreased significantly (P<0.05) as the water supply decreased. The transpiration rates decreased with the increasing age of the plants for all the four moisture levels. Data from the stomatal resistance and transpiration indicated that the moisture deficiency increased the stomatal resistance, which eventually resulted in a decrease in the loss of moisture from the plant body. WUE declined (P<0.05) with increasing soil moisture deficit at all the stages of growth. Compared to the optimum soil moisture (80% FC), WUE under the highest moisture deficit conditions (40% FC) decreased by 55% to 72% at different stages of growth. This observation agrees with the data reported by ALLEN et al.1), who found that the WUE decreased by 3050% after irrigation was withheld for 11 days. WUE was correlated with the area of stomatal aperture per unit area of the leaf (Table 4), suggesting that the plants with a larger stomatal aperture per unit leaf area had a higher WUE. Significant correlations were observed between (ƒõ1 and the total area of stomatal aperture per unit leaf area, photosynthetic rate and stomatal conductance, photosynthetic rate and the area of stomatal aperture per unit leaf area, ƒõ1 and photosynthetic rate. These relationships indicate that increasing water stress affected the leaf water potential and stomatal dimensions, and that these two characteristics brought about significant negative changes in the leaf gas exchange rates. Stomatal regulation of gas exchange showed a direct relationship with the amount of moisture available in soil. Smaller aperture with higher stomatal resistance may cause a slower rate of gas exchange between leaf and the atmosphere in moisturestressed plants. Photosynthesis and respiration, the two primary events in a plant system are thus impaired at the time of water deficiency. Therefore, stomatal behavior reflects the soil

36 Jpn. J. Trop. Agr. 44 (1) 2000 Table 4. Correlations among area of stomatal aperture, leaf water potential and leaf gas exchange characteristics at selected growth stages. Sa, area of stomatal aperture per unit leaf area; ƒõ1, leaf water potential; Cs, stomatal conductance; Pn, net photosynthesis; Rd, dark respiration; Rs, stomatal resistance; WUE, water use efficiency; * and **, levels of significance at 5% and 1%, respectively. moisture status which is an important determinant of carbon assimilation efficiency of the plant. GHORASHY et al.12) reported that the photosynthetic rate decreased linearly as ƒõ1 decreased. DJEKOUN and PLANCHON9) reported that the decrease of ƒõ1 was accompanied by a reduction in photosynthesis and stomatal conductance. According to BOYER3), the differences in the photosynthetic behavior could be attributed solely to the differences in the stomatal behavior down to the ƒõ1 value of-16 bars in the soybean cultivar 'Harosoy'. These results are derived from experiments in which water stress was applied at only one or two stages of growth. Our data showed that the leaf area was drastically decreased over long-term water stress resulting in a higher stomatal density and smaller aperture area. For a short period of water stress, the effect might be different depending on the stage of growth when the water deficit occurred. If the moisture deficit occurs at the early vegetative stage and is removed afterwards, plants may resume growth to some extent and the new leaves may show different characteristics. Water shortage periods generally vary from short to long in different regions. Developing cultivars suitable for shortterm and others for long-term water stress could be useful. Therefore, it is necessary to monitor the soil water status together with stomatal conditions, including the frequency and area of opening per unit of leaf area. Breeding cultivars adaptable to short or long periods of water stress may be a better approach to address the problem since regions differ in the water shortage period. This experiment involved one late maturing determinate cultivar. It is thus necessary to conduct similar experiments with indeterminate soybeans to confirm the effects. The other part of the experiment dealing with mineral nutrition under variable soil moisture deficits, will be reported References later. 1. ALLEN, L. H. Jr., R. R. VALLE, J. W. MISHOE and J. W. JONES 1994 Soybean leaf gas exchange responses to carbon dioxide and water stress. Agron. J. 86: 625-636. 2. ASHRAF, M. and J. W. O'LEARY 1996 Effect of drought

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