Sodium and chloride exclusion and retention by non-grafted and grafted melon and Cucurbita plants.

The effects of grafting on Na and Cl(-) uptake and distribution in plant tissues were quantified in a greenhouse experiment using six combinations of melon (Cucumis melo L. cv. Arava) and pumpkin (Cucurbita maxima Duchesne×Cucurbita moschata Duchesne cv. TZ-148): non-grafted, self-grafted, melons grafted on pumpkins, and pumpkins grafted on melons. Total Na concentration in shoots of plants with pumpkin or melon rootstocks was <60 mmol kg(-1) and >400 mmol kg(-1), respectively, regardless of the scion. In contrast, shoot Cl(-) concentrations were quite similar among the different scion-rootstock combinations. Na concentrations in exudates from cut stems of plants with a pumpkin rootstock were very low (<0.18 mM), whereas those in the exudates of plants with melon rootstocks ranged from 4.7 mM to 6.2 mM, and were quite similar to the Na concentration in the irrigation water. Root Na concentrations averaged 11.7 times those in the shoots of plants with pumpkin rootstocks, while in plants with melon rootstocks, values were similar. Two mechanisms could explain the decrease in shoot Na concentrations in plants with pumpkin rootstocks: (i) Na exclusion by the pumpkin roots; and (ii) Na retention and accumulation within the pumpkin rootstock. Quantitative analysis indicated that the pumpkin roots excluded ∼74% of available Na, while there was nearly no Na exclusion by melon roots. Na retention by the pumpkin rootstocks decreased its amount in the shoot by an average 46.9% compared with uniform Na distribution throughout the plant. In contrast, no retention of Na could be found in plants grafted on melons.

Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate.

One of the major protein kinases (PK(III)) that phosphorylates serine-158 of spinach sucrose-phosphate synthase (SPS), which is responsible for light/dark modulation of activity, is known to be a member of the SNF1-related family of protein kinases. In the present study, we have developed a fluorescence-based continuous assay for measurement of PK(III) activity. Using the continuous assay, along with the fixed-time-point (32)P-incorporation assay, we demonstrate that PK(III) activity is inhibited by glucose-6-phosphate (Glc-6-P). Relative inhibition by Glc-6-P was increased by decreasing pH from 8. 5 to 5.5 and by reducing the concentration of Mg(2+) in the assay from 10 to 2 mM. Under likely physiological conditions (pH 7.0 and 2 mM Mg(2+)), 10 mM Glc-6-P inhibited kinase activity approximately 70%. Inhibition by Glc-6-P could not be ascribed to contaminants in the commercial preparations. Other metabolites inhibited PK(III) in the following order: Glc-6-P > mannose-6-P, fructose-1,6P(2) > ribose-5-P, 3-PGA, fructose-6-P. Inorganic phosphate, Glc, and AMP were not inhibitory, and free Glc did not reverse the inhibition by Glc-6-P. Because SNF1-related protein kinases are thought to function broadly in the regulation of enzyme activity and gene expression, Glc-6-P inhibition of PK(III) activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.

Carbon Isotope Discrimination, Gas Exchange, and Growth of Sugarcane Cultivars under Salinity.

Physiological features associated with differential resistance to salinity were evaluated in two sugarcane (Saccharum spp. hybrid) cultivars over an 8-week period during which greenhouse-grown plants were drip-irrigated with water or with NaCI solutions of 2, 4, 8, or 12 decisiemens (dS) m-1 electrical conductivity (EC). The CO2 assimilation rate (A), stomatal conductance (g), and shoot growth rate (SGR) began to decline as EC of the irrigation solution increased above 2 dS m-1. A, g, and SGR of a salinity-resistant cultivar (H69-8235) were consistently higher than those of a salinity-susceptible cultivar (H65-7052) at all levels of salinity and declined less sharply with increasing salinity. Carbon isotope discrimination ([delta]) in tissue obtained from the uppermost fully expanded leaf increased with salinity and with time elapsed from the beginning of the experiment, but [delta] was consistently lower in the resistant than in the susceptible cultivar at all levels of salinity. Gas-exchange measurements suggested that variation in [delta] was attributable largely to variation in bundle sheath leakiness to CO2 ([phi]). Salinity-induced increases in [phi] appeared to be caused by a reduction in C3 pathway activity relative to C4 pathway activity rather than by physical changes in the permeability of the bundle sheath to CO2. A strong correlation between [delta] and A, g, and SGR permitted these to be predicted from [delta] regardless of the cultivar and salinity level. [delta] thus provided an integrated measure of several components of physiological performance and response.

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. CO(2) assimilation was then determined on acclimated and nonacclimated plants. The decrease of CO(2) 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 CO(2) partial pressure. The slopes of net CO(2) assimilation versus intercellular CO(2) 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 CO(2) partial pressure, the declined inhibition in CO(2) assimilation of stress-acclimated plants was found for both salinity and water stress.

Salinity effects on photosynthesis in isolated mesophyll cells of cowpea leaves.

Mesophyll cells from leaves of cowpea (Vigna unquiculata [L.] Walp.) plants grown under saline conditions were isolated and used for the determination of photosynthetic CO(2) fixation. Maximal CO(2) fixation rate was obtained when the osmotic potential of both cell isolation and CO(2) fixation assay media were close to leaf osmotic potential, yielding a zero turgor pressure. Hypotonic and hypertonic media decreased the rate of photosynthesis regardless of the salinity level during plant growth. No decrease in photosynthesis was obtained for NaCl concentrations up to 87 moles per cubic meter in the plant growing media and only a 30% decrease was found at 130 moles per cubic meter when the osmotic potential of cell isolation and CO(2) fixation media were optimal. The inhibition was reversible when stress was relieved. At 173 moles per cubic meter NaCl, photosynthesis was severely and irreversibly inhibited. This inhibition was attributed to toxic effects caused by high Cl(-) and Na(+) accumulation in the leaves. Uptake of sorbitol by intact cells was insignificant, and therefore not associated with cell volume changes. The light response curve of cells from low salinity grown plants was similar to the controls. Cells from plants grown at 173 moles per cubic meter NaCl were light saturated at a lower radiant flux density than were cells from lower salinity levels.

Effect of altered sink: source ratio on photosynthetic metabolism of source leaves.

When seven crop species were grown under identical environmental conditions, decreased sink:source ratio led to a decreased photosynthetic rate within 1 to 3 days in Cucumis sativus L., Gossypium hirsutum L., and Raphanus sativus L., but not in Capsicum annuum L., Solanum melongena L., Phaseolus vulgaris L., or Ricinus communis L. The decrease was not associated with stomatal closure. In cotton and cucumber, sink removal led to an increase in starch and sugar content, in glucose 6-phosphate and fructose 6-phosphate pools, and in the proportion of (14)C detected in sugar phosphates and UDPglucose following (14)CO(2) supply. When mannose was supplied to leaf discs to sequester cytoplasmic inorganic phosphate, promotion of starch synthesis, and inhibition of CO(2) fixation, were observed in control discs, but not in discs from treated plants. Phosphate buffer reduced starch synthesis in the latter, but not the former discs. The findings suggest that sink removal led to a decreased ratio inorganic phosphate:phosphorylated compounds. In beans (14)C in sugar phosphates increased following sink removal, but without sucrose accumulation, suggesting tighter feedback control of sugar level. Starch accumulated to higher levels than in the other plants, but CO(2) fixation rate was constant for several days.

A simple procedure to overcome polyethelene glycol toxicity on whole plants.

A procedure is described that can be used to minimize toxic effects of polyethylene glycol (PEG) to plants. The procedure is based on recycling nutrient solutions containing PEG-6000 through two plant cultures. Tomato plants grown in -0.3 megapascals PEG solutions used after two growth cycles exhibited minimal toxic effects. Long-term responses like dry matter production and chlorophyll content as well as short-term responses like CO(2) fixation rates and leaf conductance were severely inhibited by fresh PEG-6000 and only slightly reduced by recycled PEG-6000. Complete osmotic adjustment was obtained with tomatoes grown in recycled but not in fresh PEG solutions.

Effect of translocation-hindering procedures on source leaf photosynthesis in cucumber.

THREE TREATMENTS WHICH ALTERED TRANSLOCATION RATE WERE APPLIED TO CUCUMBER PLANTS: Girdling of source leaf petiole; removal of all aerial sinks; removal of all source leaves except one. Two different effects were observed, one short-term (during the initial 6 hours), and one long-term (detected after several days).The short-term effect was observed exclusively in girdled leaves and involved a reduction in (14)CO(2) fixation rate paralleled by an increase in stomatal resistance. The effects were maximal after 3 hours with subsequent recovery. Stomatal closure apparently resulted from the 5 to 10% water deficit temporarily detected in girdled leaves which probably induced the observed temporary increases in abscisic acid content. Kinetin counteracted the effects of girdling.The long-term effect was detected 3 days after girdling and 3 to 5 days after sink manipulation. An increase or decrease in (14)CO(2) fixation rate was observed when the sink-source ratio was increased or decreased respectively, accompanied by a respective decrease or increase in starch content. Changes in the relative amount of (14)CO(2) incorporated into various photosynthetic products were also observed. Stomatal closure was not involved, and the decrease in CO(2) fixation was not counteracted by kinetin.

Steady-state photosynthesis in alfalfa leaflets: effects of carbon dioxide concentration.

When the CO(2) concentration to which Medicago sativa L. var. El Unico leaflets were exposed was increased from half-saturation to saturation (doubled rate of photosynthesis), glycolate and glycine production apparently decreased due to inhibition of a portion of the glycolate pathway. Serine and glycerate production was not inhibited. We conclude that serine and glycerate were made from 3-phosphoglycerate and not from glycolate and that the conversion of glycine to serine may not be the major source of photorespiratory CO(2) in alfalfa. In investigations of glycolate and photorespiratory metabolism, separate labeling data should be obtained for glycine and serine as those two amino acids may be produced from different precursors and respond differently to environmental perturbations. The increased photosynthetic rate (at saturating CO(2)) resulted in greater labeling of both soluble and insoluble products. Sucrose labeling increased sharply, but there was no major shift of tracer carbon flow into sucrose relative to other metabolites. The flow of carbon from the reductive pentose phosphate cycle into the production of tricarboxylic acid cycle intermediates and amino acids increased. Only small absolute increases occurred in steady-state pool sizes of metabolites of the reductive pentose phosphate cycle at elevated CO(2), providing further evidence that the cycle is well regulated.

Nitrite Reduction in Reconstituted and Whole Spinach Chloroplasts during Carbon Dioxide Reduction.

Nitrite reduction in either whole, isolated spinach chloroplasts (Spinacia oleracea L.) or in reconstituted spinach chloroplasts is stimulated by a short period of photosynthetic CO(2) fixation in the light prior to nitrite addition. With reconstituted chloroplasts, a similar stimulation can be obtained in nitrite reduction without CO(2) fixation by the addition of dihydroxyacetone phosphate or fructose 6-phosphate. Specific intermediate metabolites of the photosynthetic carbon reduction cycle may have a regulatory role in nitrite reduction in chloroplasts in the light.

Analysis of steady state photosynthesis in alfalfa leaves.

A method for carrying out kinetic tracer studies of steady state photosynthesis in whole leaves has been developed. An apparatus that exposes whole leaves to (14)CO(2) under steady state conditions, while allowing individual leaf samples to be removed as a function of time, has been constructed. Labeling data on the incorporation of (14)C into Medicago sativa L. metabolite pools are reported. A carbon dioxide uptake rate of 79 micromoles (14)CO(2) per milligram chlorophyll per hour was observed at a CO(2) level slightly below that of air. Several actively turning over pools of early and intermediate metabolites, including 3-phosphoglyceric acid, glycerate, citrate, and uridine diphosphoglucose, showed label saturation after approximately 10 to 20 minutes of photosynthesis with (14)CO(2) under steady state conditions. Alanine labeling increased more rapidly at first, and then at a lower rate as saturation was approached. Sucrose was a major product of photosynthesis and label saturation of the sucrose pool was not observed. Labeled carbon appeared rapidly in secondary metabolites. The steady state apparatus used has numerous advantages, including leaf temperature control, protection against leaf dehydration, high illumination, known (14)CO(2) specific radioactivity, and provision for control and adjustment of (14)CO(2) concentration. The apparatus allows for experiments of long duration and for sufficient sample points to define clearly the metabolic steady state.

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 CO(2) fixation media below -6 to -8 bars resulted in decreased rates of fixation regardless of solute composition. A decrease in CO(2) fixation rate of isolated chloroplasts was also found when leaves were dehydrated in air prior to chloroplast isolation. An inverse response of CO(2) 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.Low leaf water potentials were found to inhibit CO(2) fixation of intact leaf discs to almost the same degree as they did CO(2) fixation by chloroplasts isolated from those leaves. CO(2) fixation by intact leaves was decreased by 50 and 80% when water potentials were reduced from -7.1 to -9.6 and from -7.1 to -17.6 bars, respectively. Transpiration was decreased by only 40 and 60%, under the same conditions. However, correction for the increase in leaf temperature indicated transpiration decreases of 57 and 80%, similar to the relative decreases in CO(2) fixation.Despite the 4-fold increase in leaf resistance to CO(2) diffusion in the gas phase when the water potential of leaves was reduced from -6.5 to -14.0 bars, an additional increase of about 50% in mesophyll resistance was obtained. CO(2) concentration at compensation also increased when leaf water potential was reduced.

Inhibition of photosynthetic carbon dioxide fixation in isolated spinach chloroplasts exposed to reduced osmotic potentials.

Reduced osmotic potentials inhibited the rate of CO(2) fixation by isolated intact spinach (Spinacia oleracea) chloroplasts. This inhibition was observed immediately after transfer of chloroplasts from a solution containing 0.33 m sorbitol to higher sorbitol concentrations, and the depressed rate remained constant. The inhibited CO(2) fixation could not be attributed to a decreased rate of photosynthetic electron transport, since NADP reduction was unaffected by subjecting the chloroplasts to low potentials. It could also not result from restricted permeability to CO(2), as CO(2) concentrations had no effect on the relative inhibition induced by the lowered potential.A procedure was developed for the determination of several enzymes of the photosynthetic carbon reduction cycle in the intact chloroplast without their being extracted. The activities of the combined three enzymes: ribose-5-phosphate isomerase, ribulose-5-phosphate kinase, and ribulose-1,5-diphosphate carboxylase and of ribulose-1,5-diphosphate carboxylase alone were found to be inhibited at low osmotic potentials. Analysis of the photosynthetic products showed that the formation of glycerate-3-phosphate was inhibited to a greater extent than the formation of all other products.CO(2) fixation was partly resumed when chloroplasts were returned from a 0.67 m sorbitol to a 0.33 m sorbitol solution, regardless whether the transfer occurred in the light or in the dark.

Glycolate formation in intact spinach chloroplasts.

Photosynthetic (14)CO(2) fixation and the accumulation of photosynthetic products and the response of each process to both 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) and ascorbate were investigated in the intact spinach chloroplast.Ascorbate increased the rate of CO(2) uptake with an increase in all photosynthetic products, but, proportionally, there was a much larger increase in glycolate formation. CO(2) fixation inhibited by DCMU was partially restored on addition of ascorbate. Under conditions not optimal for glycolate formation, such as saturating levels of CO(2) and an anaerobic atmosphere, ascorbate in the presence of DCMU restored the formation of all photosynthetic products excluding glycolate. This effect of ascorbate on glycolate synthesis in the presence of DCMU was diminished under conditions which favored glycolate formation. Externally added glycerate 3-phosphate and fructose 1,6-diphosphate depressed the appearance of radioactivity in glycolate.The data are interpreted to indicate that glycolate is produced during photosynthesis as a result of a reaction between a 2-carbon piece derived from a sugar phosphate and an oxidant generated by the photochemical act. The oxidant may be an intermediate of photosystem 2 or a peroxide generated by a mechanism of the Mehler type involving molecular oxygen.