Reduced osmotic potential effects on photosynthesis : identification of stromal acidification as a mediating factor

Addition of sorbitol, which facilitated reductions in reaction medium osmotic potential from standard (0.33 molar sorbitol, −10 bars) isotonic conditions to a stress level of 0.67 molar sorbitol (−20 bars), inhibited the photosynthetic capacity of isolated spinach (Spinacia oleracea) chloroplasts. This inhibition, which ranged from 64 to 74% under otherwise standard reaction conditions, was dependent on reaction medium inorganic phosphate concentration, with the phosphate optimum for photosynthesis reduced to 0.05 millimolar at the low osmotic potential stress treatment from a value of 0.25 millimolar under control conditions.

Stromal alkalating agents such as NH₄Cl (0.75 millimolar) and KCl (35 millimolar) were also found to affect the degree of low osmotic potential inhibition of photosynthesis. Both agents doubled the rate of NaHCO₃-supported O₂ evolution under the stress treatment, while hardly affecting the control rate at optimal concentrations. These agents also reduced the length of the lag phase of photosynthetic O₂ evolution under the stress treatment to a much greater degree. The rate-enhancement effect of these agents under the stress treatment was reversed by sodium acetate, which is known to facilitate stromal acidification.

The reaction medium pH optimum for photosynthesis under the stress treatment was higher than under control conditions. In the presence of optimal NH₄Cl, this shift was no longer evident.

Internal pH measurements indicated that the stress treatment caused a 0.43 and 0.24 unit reduction in the stromal and intrathylakoid pH, respectively, under illumination. This osmotically induced acidification was not evident in the dark. The presence of 0.75 millimolar NH₄Cl partially reversed the osmotically induced reduction in the illuminated stromal pH. It was concluded that stromal acidification is a mediating mechanism of the most severe site of low osmotic potential inhibition of the photosynthetic process.

     

Effect of water stress on the reduction of nitrate and nitrite by soybean nodules

The effects of water stress on nitrate reductase and nitrite reductase activities in symbiotic nodules were examined in field-grown soybean plants (Glycine max L Merr. cv Clark). The in vitro assays of enzyme activity indicated that the nodule cytosol and bacteroids contained both nitrate reductase and nitrite reductase activities. The reduction of nitrate in bacteroids increased significantly as nodule water potential declined from −0.6 to −1.4 megapascals, and then decreased when −1.8 megapascals water potential was reached. On the contrary, the reduction of nitrate in nodule cytosol was inhibited as water stress progressed. Increases in water stress intensity also caused a significant inhibition in nitrite reductase activities of bacteroids and nodule cytosol within soybean nodules. The results show that nitrate reduction occurred both in the cytosol and bacteroids of water-stressed soybean nodules. The reduction of nitrate functioned at different physiological modes in these two fractions.     

Nitrite photoreduction in vivo is inhibited by oxygen

It was hypothesized previously that an O₂ inhibition of NO₂⁻ photoreduction would reflect a competition between O₂ and NO₂⁻ for electrons from ferredoxin at the site of plastid nitrite reductase. In order to test this in vivo, intact spinach (Spinacia oleracea L.) leaf chloroplast and mesophyll cell isolates held in high light were aerated with streams of 20% O₂/80% N₂ (250 micromolar O₂ in aqueous solution) or, alternatively, streams of 100% N₂. Bicarbonate plus CO₂ and NO₂⁻ were supplied to reaction mixtures at levels just sufficient to promote maximal assimilations of CO₂ and NO₂⁻. In chloroplast isolates, there was a 9 to 30% O₂ inhibition of NO₂⁻ reduction while there were high rates of CO₂ fixation. In spinach and soybean (Glycine max) leaf cell isolates, NO₂⁻ photoreduction rates were 10 to 55% inhibited by O₂ at near ambient levels. It is possible that O₂ may compete, albeit weakly, with NO₂⁻ (nitrite reductase) for equivalents derived from reduced ferredoxin. Also, O₂ may oxidize sulfhydryl groups on nitrite reductase which are involved in substrate binding and/or activation.     

Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : site-specific inhibition of the photosynthetic carbon reduction cycle

The effects of reduced osmotic potential on the photosynthetic carbon reduction cycle were investigated by monitoring photosynthetic processes of spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts exposed to increased assay medium sorbitol concentrations. CO₂ assimilation was found to be inhibited at 0.67 molar sorbitol by about 60% from control rates at 0.33 molar sorbitol. This level of stress inhibition was greater than that affecting the reductive phase of the cycle; glycerate 3-phosphate reduction was inhibited at 0.67 molar by 27 to 40%. Sorbitol (0.67 molar) inhibited the rate of O₂ evolution at saturating and limiting concentrations of NaHCO₃, and extended the lag phase of O₂ evolution. This indicated that factors which are rate-limiting to the photosynthetic process are adversely affected by reduced osmotic potential.

Analysis of photosynthetic products following CO₂ fixation in 0.33 molar sorbitol and 0.67 molar sorbitol indicated that reduced osmotic potential facilitated increases in the levels of fructose 1,6-bisphosphate and triose phosphates with reductions in glucose 6-phosphate and fructose 6-phosphate, implicating fructose 1,6-bisphosphatase as a site of osmotic stress. Osmotic inhibition of the reductive portion (glycerate 3-phosphate to triose phosphate) of the photosynthetic carbon reduction cycle was partially attributed to feedback inhibition by the product, triose phosphate, on glycerate 3-phosphate reduction. A saturating concentration of ribose 5-phosphate partially overcame osmotic inhibition of CO₂-supported O₂ evolution, indicating another but apparently less severe site of stress inhibition in the sequence of ribose 5-phosphate to glycerate 3-phosphate.

     

Response of carbon dioxide fixation to water stress: parallel measurements on isolated chloroplasts and intact spinach leaves

Application of water stress to isolated spinach (Spinacia oleracea) chloroplasts by redutcion of the osmotic potentials of CO₂ fixation media below −6 to −8 bars resulted in decreased rates of fixation regardless of solute composition. A decrease in CO₂ fixation rate of isolated chloroplasts was also found when leaves were dehydrated in air prior to chloroplast isolation. An inverse response of CO₂ fixation to osmotic potential of the fixation medium was found with chloroplasts isolated from dehydrated leaves—namely, fixation rate was inhibited at −8 bars, compared with −16 or −24 bars.     

Chloroplast osmotic adjustment and water stress effects on photosynthesis

Previous studies have suggested that chloroplast stromal volume reduction may mediate the inhibition of photosynthesis under water stress. In this study, the effects of spinach (Spinacia oleracea, var `Winter Bloomsdale') plant water deficits on chloroplast photosynthetic capacity, solute concentrations in chloroplasts, and chloroplast volume were studied. In situ (gas exchange) and in vitro measurements indicated that chloroplast photosynthetic capacity was maintained during initial leaf water potential (Ψ_w) and relative water content (RWC) decline. During the latter part of the stress period, photosynthesis dropped precipitously. Chloroplast stromal volume apparently remained constant during the initial period of decline in RWC, but as leaf Ψ_w reached −1.2 megapascals, stromal volume began to decline. The apparent maintenance of stromal volume over the initial RWC decline during a stress cycle suggested that chloroplasts are capable of osmotic adjustment in response to leaf water deficits. This hypothesis was confirmed by measuring chloroplast solute levels, which increased during stress. The results of these experiments suggest that stromal volume reduction in situ may be associated with loss of photosynthetic capacity and that one mechanism of photosynthetic acclimation to low Ψ_w may involve stromal volume maintenance.     

Leaf water potential, stomatal resistance, and photosynthetic response to water stress in peach seedlings

Individual groups of peach (Prunus persica [L.] Batsch) seedlings stressed to −17, −26 and −36 bars recovered to control levels within 1, 3, and 4 days, respectively. Stomatal resistance was significantly correlated with both leaf water potential and net photosynthesis. In seedlings stressed to −52 bars, leaf water potential and stomatal resistance recovered sooner than net photosynthesis, despite recovery of 0₂ evolution at a rate similar to leaf water potential. Therefore, some nonstomatal factor other than reduction in photochemical activity must be responsible for the lag in recovery of CO₂ assimilation following irrigation.     

Evaluation of water stress control with polyethylene glycols by analysis of guttation

The water relations of pepper plants (Capsicum frutescens L.) under conditions conducive to guttation were studied to evaluate the control of plant water stress with polyethylene glycols. The addition of polyethylene glycol 6000 to the nutrient solution resulted in water relations similar to those expected in soil at the same water potentials. Specifically, xylem pressure potential in the root and leaf became more negative during a 24-hour treatment period, while osmotic potential of the root xylem sap remained constant. The decrease in pressure potential was closely correlated with the decrease in osmotic potential of the nutrient solution. In contrast, the addition of polyethylene glycol 400 to the nutrient medium resulted in a reduction of osmotic potential in the root xylem sap; this osmotic adjustment in the xylem was large enough to establish an osmotic gradient for entry of water and cause guttation at a nutrient solution osmotic potential of −4.8 bars. Pressure potential in the root and leaf xylem became negative only at nutrient solution osmotic potentials lower than −4.8 bars. About half of the xylem osmotic adjustment in the presence of polyethylene glycol 400 was caused by increased accumulation of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the root xylem. These studies indicate that larger polyethylene glycol molecules such as polyethylene glycol 6000 are more useful for simulating soil water stress than smaller molecules such as polyethylene glycol 400.     

Water Relations of Cotton Plants under Nitrogen Deficiency: II. Environmental Interactions on Stomata

Nitrogen deficiency in cotton plants (Gossypium hirsutum L.) considerably increased the sensitivity of stomata to water stress. At air temperatures of 27, 35, and ≥40 C, threshold potentials for complete stomatal closure were −10, −15, and −26 bars in N-deficient plants and −20, −20, and −30 bars in high-N plants, respectively. This three-way interaction among N supply, water potential, and air temperature was similar to that exerted on leaf expansion. The effects of N supply on stomatal behavior could not be explained on the basis of either osmotic or structural considerations. Rather, effects of N deficiency on mesophyll and stomata were independent and divergent. Stomatal behavior may impart a stress avoidance type of drought resistance to N-deficient plants.     

Stomatal and Nonstomatal Components to Inhibition of Photosynthesis in Leaves of Capsicum annuum during Progressive Exposure to NaCl Salinity

Young bell pepper (Capsicum annuum L.) plants grown in nutrient solution were gradually acclimated to 50, 100, or 150 moles per cubic meter NaCl, and photosynthetic rates of individual attached leaves were measured on several occasions during the salinization period at external CO₂ concentrations ranging from approximately 70 to 1900 micromoles per mole air. Net CO₂ assimilation (A) was plotted against computed leaf internal CO₂ concentration (C_i), and the initial slope of this A-C_i curve was used as a measure of photosynthetic ability. During the 10 to 14 days after salinization began, leaves from plants exposed to 50 moles per cubic meter NaCl showed little change in photosynthetic ability, whereas those treated to 100 or 150 moles per cubic meter NaCl had up to 85% inhibition, with increase in CO₂ compensation point. Leaves appeared healthy, and leaf chlorophyll content showed only a 14% reduction at the highest salinity levels. Partial stomatal closure occurred with salinization, but reductions in photosynthesis were primarily nonstomatal in origin. Photosynthetic ability was inversely related to the concentration of either Na⁺ or Cl⁻ in the leaf laminas sampled at the end of the experimental period. However, the concentration of Cl⁻ expressed on a tissue water basis was greater, exceeding 300 moles per cubic meter, and Cl⁻ was more closely associated (R² = 0.926) with the inhibition of photosynthetic ability. Leaf turgor was not reduced by salinization and leaf osmotic potential decreased to a slightly greater extent than the osmotic potential decreases of the nutrient solutions. Concentration of accumulated Na⁺ and Cl⁻ (on a tissue water basis) accounted quantitatively for maintenance of leaf osmotic balance, assuming that these ions were sequestered in the vacuoles.     

Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : generation and use of reducing power

The effect of increasing assay medium sorbitol concentration from 0.33 to 1.0 molar on the photosynthetic reactions of intact and broken spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts was investigated by monitoring O₂ evolution supported by the addition of glyceric acid 3-phosphate (PGA), oxaloacetic acid (OAA), 2,5-dimethyl-p-benzoquinone, and 2,6-dichlorophenolindophenol or as O₂ uptake with methyl viologen as acceptor.

Uncoupled 2,6-dichlorophenolindophenol-supported whole chain electron transport (photosystems I and II) was inhibited from the 0.33 molar rate by 14% and 48.6% at 0.67 and 1.0 molar sorbitol in the intact chloroplast and by only 0.4% and 25.0% in the broken chloroplast preparation. Whole chain electron flow from water to other oxidants (OAA, methyl viologen) was also inhibited at increased osmoticum in intact preparations while electron flow from water to methyl viologen, ferricyanide, and NADP in broken preparations did not demonstrate the osmotic response. Electron transport to 2,5-dimethyl-p-benzoquinone (photosystem II) from H₂O and to methyl viologen (photosystem I) from 3,3′-diaminobenzidine were found to be unaffected by osmolarity in both intact and broken preparations.

The stress response was more pronounced (26-38%) with PGA as substrate in the presence of 0.67 molar sorbitol than the inhibition found with uncoupled and coupled linear electron flow. In addition, substrate availability and ATP generated by cyclic photophosphorylation evaluated by addition of Antimycin A were found not to be mediating the full osmotic inhibition of PGA-supported O₂ evolution. In a reconstituted (thylakoids plus stromal protein) chloroplast system to which a substrate level of PGA was added, O₂ evolution was only slightly (7.8%) inhibited by increased osmolarity (0.33-0.67 molar sorbitol) indicating that the level of osmotic inhibition above that contributed by adverse effects on electron flow can be attributed to the functioning of the photosynthetic carbon reduction cycle within the intact chloroplasts.

     

Nitrate and Nitrite Reduction in Wheat Leaves as Affected by Different Types of Water Stress

The effect of different types of water stress on nitrate and nitrite reductases of wheat (Triticum vulgare L. cv. Mivhor) leaves was investigated. Water stress was applied either to leaf tissue by its incubation in mannitol or various salt solutions, or to intact plants by exposure of the root system to low temperatures or to salinity. Nitrite reductase was much less sensitive to water stress than nitrate reductase, and was not sensitive to salinity up to osmotic potentials of about — 13 bars. The decrease in nitrite reductase activity by water stress was attributed to a direct inhibition of the enzyme rather than to a repression of enzyme synthesis. This was based on the fast response of the enzyme after exposure of leaf tissue to reduced osmotic potential, on the lack of a continuous decrease in enzyme activity during a prolonged stress, and on the fact that light activation of reductase was unaffected by water stress. The inhibition of nitrate reductase under water stress was attributed to both a direct inhibition and a reduced rate in enzyme synthesis. This is concluded from the fact that a decrease in its activity was obtained already within 1 h after stress application and from the fact that light induction of the enzyme was inhibited by stress.     

Mild Water Stress of Phaseolus vulgaris Plants Leads to Reduced Starch Synthesis and Extractable Sucrose Phosphate Synthase Activity

Mild water stress, on the order of −1.0 megapascals xylem water potential, can reduce the rate of photosynthesis and eliminate the inhibition of photosynthesis caused by O₂ in water-stress-sensitive plants such as Phaseolus vulgaris. To investigate the lack of O₂ inhibition of photosynthesis, we measured stromal and cytosolic fructose-1,6-bisphosphatase, sucrose phosphate synthase, and partitioning of newly fixed carbon between starch and sucrose before, during, and after mild water stress. The extractable activity of the fructose bisphosphatases was unaffected by mild water stress. The extractable activity of SPS was inhibited by more than 60% in plants stressed to water potentials of −0.9 megapascals. Water stress caused a decline in the starch/sucrose partitioning ratio indicating that starch synthesis was inhibited more than sucrose synthesis. We conclude that the reduced rate of photosynthesis during water stress is caused by stomatal closure, and that the restriction of CO₂ supply caused by stomatal closure leads to a reduction in the capacity for both starch and sucrose synthesis. This causes the reduced O₂ inhibition and abrupt CO₂ saturation of photosynthesis.     

In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite

An NADPH-dependent NO₂⁻-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP⁺ oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO₂⁻ reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO₂⁻ reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP⁺. NADP⁺ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO₂⁻ reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO₂⁻ reduction and (b) consume NADP⁺, releasing FNR from NADP⁺ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO₂⁻ reduction observed in vitro is capable of accounting for the observed rates of dark NO₃⁻ assimilation by C. reinhardtii.     

Correlation between the Maintenance of Photosynthesis and in Situ Protoplast Volume at Low Water Potentials in Droughted Wheat

Studies were undertaken to examine the relationship between water deficit effects on photosynthesis and the extent of protoplast volume reduction which occurs in leaves at low water potential (Ψ_w). This relationship was monitored in two cultivars (`Condor' and `Capelle Desprez') of cultivated wheat (Triticum aestivum) that differed in sensitivity to drought, and in a wild relative of cultivated wheat (Triticum kotschyi) that has been previously found to be `drought resistant.' When subjected to periods of water stress, Condor and T. kotschyi plants underwent osmotic adjustment; Capelle plants did not. Photosynthetic capacity was maintained to different extents in the three genotypes as leaf Ψ_w declined during stress; Capelle plants were most severely affected. Calculations of internal leaf [CO₂] and stomatal conductance from gas exchange measurements indicated that differences in photosynthetic inhibition at low Ψ_w among the genotypes were primarily due to nonstomatal effects. The extent of protoplast volume reduction that occurred in leaves at low Ψ_w was also found to be different in the three genotypes; maintenance of protoplast volume and photosynthetic capacity in stressed plants of the genotypes appeared to be correlated. When the extent of water stress-induced inhibition of photosynthesis was plotted as a function of declining protoplast volume, this relationship appeared identical for the three genotypes. It was concluded that there is a correlative association between protoplast volume and photosynthetic capacity in leaves of wheat plants subjected to periods of water stress.     

Acclimation of CO(2) Assimilation in Cotton Leaves to Water Stress and Salinity

Cotton (Gossypium hirsutum L. cv Acala SJ2) plants were exposed to three levels of osmotic or matric potentials. The first was obtained by salt and the latter by withholding irrigation water. Plants were acclimated to the two stress types by reducing the rate of stress development by a factor of 4 to 7. CO₂ assimilation was then determined on acclimated and nonacclimated plants. The decrease of CO₂ assimilation in salinity-exposed plants was significantly less in acclimated as compared with nonacclimated plants. Such a difference was not found under water stress at ambient CO₂ partial pressure. The slopes of net CO₂ assimilation versus intercellular CO₂ partial pressure, for the initial linear portion of this relationship, were increased in plants acclimated to salinity of −0.3 and −0.6 megapascal but not in nonacclimated plants. In plants acclimated to water stress, this change in slopes was not significant. Leaf osmotic potential was reduced much more in acclimated than in nonacclimated plants, resulting in turgor maintenance even at −0.9 megapascal. In nonacclimated plants, turgor pressure reached zero at approximately −0.5 megapascal. The accumulation of Cl⁻ and Na⁺ in the salinity-acclimated plants fully accounted for the decrease in leaf osmotic potential. The rise in concentration of organic solutes comprised only 5% of the total increase in solutes in salinity-acclimated and 10 to 20% in water-stress-acclimated plants. This acclimation was interpreted in light of the higher protein content per unit leaf area and the enhanced ribulose bisphosphate carboxylase activity. At saturating CO₂ partial pressure, the declined inhibition in CO₂ assimilation of stress-acclimated plants was found for both salinity and water stress.     

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR⁻ 108, NR⁻ 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N₂-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR⁻ 108 and NR⁻ 303 contained neither nitrate reductase nor nitrite reductase activities.

Addition of 20 millimolar KNO₃ to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO₃ treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.

The addition of either KNO₃ or KNO₂ to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.

     

Effect of photosynthetic inhibitors and uncouplers of oxidative phosphorylation on nitrate and nitrite reduction in barley leaves

The effects of several photosynthetic inhibitors and uncouplers of oxidative phosphorylation on NO₃⁻ and NO₂⁻ assimilation were studied using detached barley (Hordeum vulgare L. cv Numar) leaves in which only endogenous NO₃⁻ or NO₂⁻ were available for reduction. Uncouplers of oxidative phosphorylation greatly increased NO₃⁻ reduction in both light and darkness, while photosynthetic inhibitors did not.

The NO₂⁻ concentration in the control leaves was very low in both light and darkness; 98% or more of the NO₂⁻ formed from NO₃⁻ was further assimilated in control leaves. More NO₂⁻ accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO₂⁻, 94% or more was further assimilated. It appears that metabolites, either external or internal to the chloroplast, capable of reducing NADP (which, in turn, could reduce ferredoxin via NADP reductase) might support NO₂⁻ reduction in darkness and light when photosynthetic electron flow is inhibited by photosynthetic inhibitors.

Nitrite assimilation was much more sensitive to uncouplers in darkness than in light: in darkness, 74% or more of NO₂⁻ formed from NO₃⁻ was further assimilated, whereas in light, 95% or more of the NO₂⁻ was further assimilated.

     

Osmotic adjustment, symplast volume, and nonstomatally mediated water stress inhibition of photosynthesis in wheat

At low water potential (ψ_w), dehydration reduces the symplast volume of leaf tissue. The effect of this reduction on photosynthetic capacity was investigated. The influence of osmotic adjustment on this relationship was also examined. To examine these relationships, comparative studies were undertaken on two wheat cultivars, one that osmotically adjusts in response to water deficits (`Condor'), and one that lacks this capacity (`Capelle Desprez'). During a 9-day stress cycle, when water was withheld from plants grown in a growth chamber, the relative water content of leaves declined by 30% in both cultivars. Leaf osmotic potential (ψ_s) declined to a greater degree in Condor plants. Measuring ψ_s at full turgor indicated that osmotic adjustment occurred in stressed Condor, but not in Capelle plants. Two methods were used to examine the degree of symplast (i.e. protoplast) volume reduction in tissue rapidly equilibrated to increasingly low ψ_w. Both techniques gave similar results. With well-watered plants, symplast volume reduction from the maximum (found at high ψ_w for each cultivar) was the same for Condor and Capelle. After a stress cycle, volume was maintained to a greater degree at low ψ_w in Condor leaf tissue than in Capelle. Nonstomatally controlled photosynthesis was inhibited to the same degree at low ψ_w in leaf tissue prepared from well-watered Condor and Capelle plants. However, photosynthetic capacity was maintained to a greater degree at low ψ_w in tissue prepared from stressed Condor plants than in tissue from stressed Capelle plants. Net CO₂ uptake in attached leaves was monitored using an infrared gas analyzer. These studies indicated that in water stressed plants, photosynthesis was 106.5% higher in Condor than Capelle at ambient [CO₂] and 21.8% higher at elevated external [CO₂]. The results presented in this report were interpreted as consistent with the hypothesis that there is a causal association between protoplast (and presumably chloroplast) volume reduction at low ψ_w and low ψ_w inhibition of photosynthesis. Also, the data indicate that osmotic adjustment allows for maintenance of relatively greater volume at low ψ_w, thus reducing low ψ_w inhibition of chloroplast photosynthetic potential.     

Oxidation of proline by mitochondria isolated from water-stressed maize shoots

Proline oxidation and coupled phosphorylation were measured in mitochondria after isolation from shoots of water-stressed, etiolated maize (Zea mays L.) seedlings. Both state III and state IV rates of proline oxidation decreased as a logarithmic function of increased seedling water stress between −5 and −10 bars. Proline oxidation rates decreased 62% (state III) and 58% (state IV) as seedling water potentials were decreased from −5 to −10 bars. By comparison, oxidation of succinate, exogenous NADH, or malate + pyruvate decreased only 10 to 15% in this stress range. These decreases were a linear function of increased stress and were comparable to oxidation rates of mitochondria subjected to varying in vitro osmotic potentials. Osmotically induced in vitro stress reduced proline oxidation rates linearly with more negative osmotic potentials, a decrease that was similar to the responses of the other substrates to more negative osmotic potentials. Some decrease in coupling, with all substrates as determined by ADP/O ratios, was observed under osmotic stress. Mitochondria were also isolated from shoot tissue that had been stressed and then rewatered. On a percentage basis, the recovery of proline oxidation was greater than that of the other substrates.