Water Balance and Photosynthesis in Zea mays L. Seedlings Exposed to Drought and Flooding Stress

Biochem. Physiol. Pf1anlen 186, 145 - 152 (1990) VEB Gustav Fischer Verlag Jena

Water Balance and Photosynthesis in Zea mays L. Seedlings Exposed to Drought and Flooding Stress GIAN FRANCO SOLDATINI, ANNAMARIA RANIERI and ORESTE GERINI Istituto di Chimica Agraria, Universita di Pisa, Pisa Key Term Index: drought, flooding , photosynthesis; Zea mays

Summary The influence of drought and flooding on maize seedlings was compared by studying the water balance and photosynthetic activity in plants subjected to these stresses. In droughted plants the leaf water potential markedly decreased after seven days of drought but the reduction in water potential was not accompanied by a reduction in turgor pressure. In contrast, flooded maize seedlings did not show any changes in water potential or in its components. From the relationship between A and Cj it was concluded that photosynthesis is reduced both in flooded and droughted plants mainly due to non-stomatal effects.

Introduction Plant growth is influenced by environmental stresses, to which the plant may react using a variety of adaptive physiological mechanisms (GILL 1975; HENSON 1982; HSIAO 1973). The physiological responses to flooding have often been considered to be similar to those induced by drought (KOZLOWSKI 1976). Under drought conditions photosynthetic activity is limited not only by stomatal closure, which reduces the amount of CO 2 available for photosynthetic fixation , but also by a direct inhibition of the mesophyll photosynthetic reaction , i.e. by nonstomatal components (BOY ER 1976; CORNIC et al. 1983; HANSON and HITZ 1982; OSMOND et al. 1980; SOLDA TINI and GERINI 1988). Flooding conditions also reduce plant photosynthesis, but the mechanisms involved here have received little attention in comparison to those of droughted plants. Some authors (HIRON and WRIGHT 1973) have suggested that the reduction in photosynthetic activity seen after flooding is the result of changes in the leaf water potential and stomatal conductivity, this being an effect similar to that observed in droughted plants. In flooded plants stomatal closure would represent an adaptive reaction of the plant to the reduced water permeability of the roots, and the restriction of water losses by transpiration may avoid plant wilting. Opposing dala have been presented, however, which suggest that neither the leaf water potential nor the stomatal conductivity change in flooded plants (BRADFORD and HSIAO 1982; WAMPLE and THRONTON 1984) . That means the reduction in the photosynthetic rate in tomato plants subjected to flooding cannot be fully accounted for by stomatal closure (BRADFORD 1983) and effects at the mesophyll level must be considered. Abbreviations: A, assimilation rate; Cj , intercellular CO 2 ; Ww , water potential; W", osmotic potential; Wp , turgor potential; PPFD , photosynthetic photon flux density; RWC, relative water content BPP 186 (\990) 2

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This work was aimed at comparing the influence of drought and flooding on maize seedlings and to study their effects on water balance and photosynthesis. "Stomatal" and "nonstomatal" contributions to the reduction in photosynthesis were estimated by plotting the assimilation rate (A) against the intercellular CO 2 concentration (Ci)' Materials and Methods Plant material and stress conditions Maize seeds (Zea mays L. Fl Plenus) were genninated on moist filter paper in Petri dishes at 25 °C. The seedlings were transplanted after three days into pots containing sand and garden soil (2 : I) and were grown in a climate-controlled growth chamber with a 16 h light period and a light intensity of 500 Ilmol photons m- 2 s- 1 PPFD (photosynthetic photon flux density) at 25 and 22°C day/night and 75 % relative humidity . After two weeks the control plants continued to be watered daily to reach field capacity. A second group of plants were flooded by immersing the pots in water, maintaining the water level I cm above the soil surface. A third group of plants was subjected to drought by withholding water. These treatments were imposed for a period of seven days and the analyses of the water potential and gas exchanges of the leaves were performed at 2, 5 and 7 days from the imposition of the stress . All of the analyses were carried out using eight plants for each treatment.

Measurement of leaf water status The components of the leaf water potential ('I' w) were estimated using the pressure/volume method described by WILSON et al . (1979) . Measurements of 'I'w were carried out in a pressure chamber of the type originally introduced by SCHOLANDER et al. (1964) . The maize shoot, cut at the level of the frrst adventitious root, was enclosed in a plastic bag and placed in the pressure chamber with the cut end protruding from the chamber. The initial balance pressure, at that point where xylem fluid was first exuded, was recorded and taken to be the value of the leaf'l'w. The shoot was quickly weighed and replaced in the pressure chamber. Then a higher pressure was applied in the chamber, the new balance pressure was recorded and the shoot was weighed again. This procedure was repeated to obtain a series of values for 'I'w relative to the shoot weight. The fully turgid weight of each leaf was estimated by extrapolating the linear relation between balancing pressure and leaf fresh weight to obtain the potential leaf weight at 0 pressure according to LADIGES (1975). The RWC (Relative Water Content) was calculated from the turgid, fresh and dry weights according to RICHTER (1978) . The shoot dry weight was determined after drying for 48 h at 80°C. Estimates of the water potential components ('I'Jt and 'I'p) were obtained according to the method described in detail by WILSON et al. (1979). From the plot of lI'1'w against RWC, the parameters of osmotic potential at full turgor ('I'" 1(0), the water potential ('I' w0) and relative water content (RWCO) at zero turgor and the proportion of bound water (B) were derived. The 'I'"and 'I'p at each 'I'w was calculated from: '1'" = '1',,100 X (loo-B)/(RWC-B); 'I'p = 'I'w-'I'".

Gas exchange measurements Leaf gas exchanges were analyzed by an open system (Heinz Walz, Effeltrich, FRG) with an assimilation chamber thermostated by a Peltier battery . Both the CO2 and water vapour concentrations were measured by an infra-red gas analyser (BINOS, Leybold Heraeus , FRG) based on a differential system . The air circulating in the system was supplied from cylinders containing a gas mixture of known composition: O2 21 % and CO2 1,000 III 1- I, with N2 making up the remainder. Before entering the system, the gas was circulated by a temperature-controlled Peltier battery to establish the desired relative humidity. The variations in the concentration of CO 2 in the gas entering the system were obtained by reducing the CO 2 concentration of the gas in the cylinder (1,000 III I-I) with a G-600 gas diluting apparatus (ADC Limited, Hoddesdon, England). An incandescent lamp (OSRAM, 350 W) was used as the light source. The leaf temperature was monitored by a copper-constantan thermocouple pressed onto the abaxial leaf surface. Incident light was

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measured at the leaf level by a photosynthetic active radiation quantum sensor (LICOR , USA). The air flow in the system was 0. 7 I min I , and was continuously controlled by a mass flow meter in both the measuring and reference lines. The variations in the photosynthetic rate as a function of the CO 2 supply were measured by increasing stepwise the carbon dioxide concentration of the air entering the leaf chamber from to 1,000 !-tl I-I, maintaining constant the other environmental factors (R. H. 60 ± 2 % ; PPFD 1,500 !-tmol photons m - 2S - I;chamber temperature 25 ± 0.3 DC). The steady state in the gaseous exchanges of the leaves was considered to have been reached when for 60 measurements (at the rate of one measurement per second) the differences in concentration between the measuring and the reference gas streams entering the infrared gas analyser did not vary by more than 0.1 !-tl I-I for CO 2 and 20 !-t11-1 for H 2 0. The carbon dioxide intercellular concentration was calculated using the equation suggested by VON CAEMMERER and FARQUHAR (1981).

°

Results and Discussion The maize seedlings grown under well watered conditions showed a leaf water potential of -0.25 MPa, an osmotic potential of -1.60 MPa, a turgor potential of 1.34 MPa and a relative water content of 94 %. Fig. 1 shows the variations in 'If W' 'IfIt and 'If p during the seven-day period of the experiment, in response to limited or excess water in the soil. Under the conditions described above , flooding does not cause water stress in maize. The 'If w, 'If It and 'If p of the flooded plants in this experiment were similar to those of the control plants over the seven days of treatment time period. In addition, the RWC of the flooded plants remained at the same level as in the plants grown under normal conditions. Excess water and deficient water in soil have often been reported to have similar effects on photosynthesis and water balance (KOZLOWSKI 1976) . These results contradict the findings of KOZLOWSKI (1976) that flooding causes water stress due to root demage, but are consistent with reports which suggest that the leaf water potential is not reduced in sunflowers subjected to flooding (W AMPLE and THRONTON 1984). The lack of water stress in the flooded maize seedlings does indicate that plants under flooding can maintain a water balance at reduced water absorption by damaged roots . Apparently, the reduction in the transpiration rate of the flooded plants maintains the leaf 'If w at the same level as that of the control. This result is in agreement with other findings (BRADFORD 1983 ; HA NSON and HITz 1982 ; HANSON 1982; HEROLD 1980; W ENKERT et at. 1981). The maintenance of water balance happens at the expense of cell expansion and growth rate. Indeed the flooded plants were 25 % smaller at the end of the treatment time period. The decrease in 'If w in drought stressed plants was not accompanied by a corresponding reduction in turgor pressure, which remained at the same level as in the well watered plants (Fig. l , insert). It has been reported that variations in osmotic potential are a normal response when plants are subjected to water stress (CUTLER and RAINS 1978; HENSON 1982; TURNER et al. 1978) . The decrease in \fJ1t is dependent both on changes in solute concentration and on changes in water content. In our experiments, leaves of plants flooded for 2, 5 and 7 days, when subjected to pressure-induced water loss in a Scholander's chamber, showed curves of 'IfIt versus 'If w which were perfectly identical to those obtained with untreated plants . The leaves of the seedlings grown under stress, when subjected to pressure induced dehydration , underwent a reduction in 'If wand 'IfJt up to a point where 'If w equalled 'IfIt (the BPP 186 (1990) 2

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zero turgor point). The point at wich lJI w coincided with lJI rr became more negative as the treatment time period progressed from 2 days to 5 and 7 days (Fig. 2). The initial lJI rr declined as the stress developed. The decline in the initial '¥rr is a manifestation of active osmotic adjustment. These results agree with those reported by CUTLER et al. (1980) and RI CHTER (1988) , and indicate that an active osmotic adjustment is involved in maintaining a positive turgor pressure in maize seedlings subjected to drought when the water unbalance develops slowly.

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Fig. 3. Changes in transpiration (a) and photosynthesis (b) rates during the treatment time period, as determined at 1,5001,lmol photons m- 2 S-I and at an external CO 2 concentration of 3271,lIZ-I , in maize seedlings (e well watered; • droughted; ... flooded). The bars indicate the least significant difference at P=O.05. Fig. 2. Relationship between 'II Jt and 'II w for Zea mays seedlings subjected to pressure-induced dehydration during the development of drought stress. The numbers in the figure indicate: 0 = untreated; 2 = two days; 5 = five days and 7 = seven days of treatment. The arrows indicate the value of zero turgor point. BPP 186 (1990) 2

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Fig. 4. Net CO 2 uptake (A) as a function of the intercellular CO 2 concentration (C;) in Zea mays plants after seven days of treatment: (e) well watered; (_) droughted; (.) flooded. Ao represents the CO2 uptake of well watered plants at the external CO 2 concentration of 327!J,1 1-1; AF and AD are the corresponding values of net photosynthesis under flood and drought conditions, respectively; and AFm (flooding) and ADm (drought) are those theoretical values of photosynthesis which should be observed in flooded and droughted maize plants if the stress had no effect on stomatal conductivity. The last two parameters were determined by taking the points where the "demand functions" for the droughted (_) and flooded plants (.) intersected with the "supply function" for the well watered plants.

Both flood and drought conditions affected the assimilation rate (A) and the transpiration rate (E) (Fig. 3a, b). The reduction in E was very marked, not only in the plants subjected to drought, but also in those sUbjected to waterlogging. Both flood and drought stressed leaves reached the same rate of transpiration after seven days of treatment. The reduction in CO 2 assimilation was even greater in plants subjected to flooding than in those subjected to drought. An estimate of the contributions of stomatal and non-stomatal components to the reduction in CO 2 assimilation was achieved by analyzing the A vs. Cj curves. The A vs. Cj curves obtained for the control, droughted and flooded plants at the end of the experiment (seven days of treatment) are reported in Fig. 4. We apportioned the reduction in photosynthesis between the stomatal and non-stomatal components using the path-related method "mesophyllic processes changing first", as suggested by MOONEY et al. (1977) and by PRIOUL et al. (1984). This method considers the non-stomatal components to be the inhibition of photosynthesis which would remain if stress induced stomatal closure had not occurred and apply well to slowly developed water deficits. The percent of non-stomatal contribution to the decline in photosynthesis was calculated as follows: (Ao-ADm) X 100/(Ao-AD) for droughted plants and (Ao-AFm) X 100/(Ao-AF) for flooded plants. U sing this method we estimated that non-stomatal components accounted for 96 % of the total reduction in photosynthesis from Ao to AF in the flooded plants. For droughted plants we found a non-stomatal contribution of 93 % . 150

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It may be seen in Fig. 4 that opening the stomata of stressed plants (i.e., increasing C j ) would result only in a very small increase in photosynthetic CO 2 assimilation. At a partial pressure of 327 [11 1-1 CO 2 the plants subjected to flooding showed a much higher Cj than the droughted ones. This feature was not due to differences in stomatal conductance. This is in agreement with the nearly identical transpiration rates exhibited and again indicates that the differences in Cj between the treatments are do to differences in the CO 2 mesophyllic fixation capacity induced by the treatment.

Acknowledgements This work is supported by CNR, Italy. Special grant I.P.R.A. Subproject 1. Paper N. 1700. We thank Mr. RENATO PERINI for technical assistance.

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