PHYSIOLOGIA PLANTARUM 81: 37^14, Copenhagen 199] Water stress, carbon dioxide, and light effects on sucrosephosphate synthase activity in Phaseolus vulgaris Terry L. Vassey, W, Paul Quick, Thomas D. Sharkey and Mark Stitt Vassey, T. L., Quick, W. P., Sharkey, T. D. and Stitt, M. 1991. Water stress, carbon dioxide, and light effects on sucrose-phosphate synthase activity in Fhaseolus vulgar«. - Physiol. Plant. 8!: 37-44. The characteristics of sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity in leaves of Phaseolus vulgaris L. cv. Linden was studied in plants subjected to water stress and various C d and light treatments. When water was withheld for 3 days causing mild water stress (-0.9 MPa), the activity of SPS measured in crude extracts was reduced ca 50%. The effect of water stress was most evident when the enzyme was assayed with saturating amounts of its substrates fructose 6-phosphate and UDP glucose. Placing a water-stressed plant in an atmosphere containing 1% CO, reversed the effect of water stress on SPS activity over 5 h even though the water stress was not relieved. Holding unstressed leaves in low CO, partial pressure reduced the extractabie activity of SPS. After 1 h of low CO, treatment the effect of low CO, could be reversed by 20 min of 5% COj. However, after 24 h of low COj treatment', less SPS activity was recovered by the 20 min treatment. The cytosolic protein synthesis inhibitor cycloheximide prevented the stow recovery of SPS activity, but did not affect the rapid recovery of SPS. We conclude that the effect of water stress on SPS activity was a consequence of the inhibition of photosynthesis caused by stomatal closure. Responses of Phaseolus vulgaris SPS to light were similar to the response to low COT in that the effects were most pronounced under V.^^^ assay conditions. This is the first report of this type of light response of SPS in a dicotyledonous species. Key words - Bea.n, carbon dioxide, light activation (of enzymes), Fhaseolus vulgaris, sucrose-phosphate synthase, water stress. r. L. Vassey and T. D. Sharkey, Dept of Botany, Univ. of Wisconsin, Madison, WI 53706, VSA; W. P. Quick and M. Slitl (corresponding author), Lehrsluhl fur Fflanzenphysiologie, Univ. Bayreulh, D-85H0 Bayreuth, Germany. Introduction In many plants, the last important regulatory step in sucrose synthesis during photosynthesis is catalyzed by sucrose-phosphate synthase (SPS) (Stitt et al. 1987). When sucrose synthesis is insufficietit to use all of the triose phosphate tnade available by photosynthesis, photosynthesis becomes feedback iimited (Sharkey 1985b, 1990), This feedback limitation is characterized by a loss of sensitivity of photosynthesis to CO, and O, partial pressure (Sharkey 1985a), When water stress develops over several days, photosynthesis becomes feedback limited (Sharkey 1985a), We found that in P, vulgaris, feedback-limited photosynthesis after water stress was highly cori-elated with a loss of extractabie SPS activity (Vassey and Sharkey 1989). The recovery ^j photosynthesis after rewatering stressed bean plants paralleled the recovery of SPS activity. On the other hand. Quick et al. (1989) found a stimulation in the activity of SPS in rapidly stressed spinach leaves. We undertook these experiments to determine whether this discrepancy could be explained and to understand how mild water stress which develops over several days reduces the activity of SPS in P, vulgaris, Enzyme activity can be controlled by the concentration of effector molecules, by stable modification of the enzyme molecule, atid by changes in the amount of enzyme present, SPS activity is affected by glucose 6phosphate (Glc 6-P) and phosphate (Doehlert and Huber 1984), but this fine control of SPS activity will Received 29 May, 1990; revised 24 September, 1990 Physiol. Planl. 81, 1«I 37 not affect the activity assayed in crude plant extracts because the concentration of effector molecules is diluted before the assay. Therefore, changes in extractabie activity of SPS likely are caused by stable modifications of the protein present, or changes in the amount of protein present. Changes in extractabie activity of SPS have been demonstrated and is called coarse control (Stitt et al, 1988), Changes in extractabie activity have been seen in response to light (Sicher and Kremer 1984, 1985, Pollock and Housley 1985, Ohsugi and Huber 1987, Stitt and Grosse 1988), endogenous rhythms (Kerr et al, 1985), sucrose accumulation (Stitt et al, 1988), and phytochrome control (Vassey 1988). The activation of SPS upon illumination is slower in N, than in air in maize, and this can be interpreted as a CO, effect on extractabie SPS activity (Huber et al. 1987). The effect of light on the extractabie activity of SPS varies between species (Huber et al, 1989), In spinach and several other species, the increase in extractabie SPS activity is only observed when the enzyme is assayed with limiting levels of substrate. In Zea mays and several other monocotyledons, the increase in extractabie activity of SPS is observed when the enzyme is assayed with saturating amounts of substrate (Huber et al, 1989), Other plant species, including Glycine max, the species most closely related to P. vulgaris of those tested, do not exhibit light activation of SPS. SPS is difficult to study because crtide extracts contain hexose phosphate isomerase which will convert the substrate fructose 6-phosphate (Fru 6-P), to the activator, Glc 6-P. Therefore, it is not possible to change the substrate concentration without also changing the concentration of an activator (Doehlert and Huber 1984). In purified extracts it is equally difficult to relate the observed properties to the situation inside the leaf (Stitt et al. 1988). Because of these problems, various methods for standardizing the assay of SPS have been developed (Stitt et al, 1988, Huber et al. 1989, Quick et al. 1989). These methods usually assess SPS activity in the presence of saturating levels of substrates and no inhibitors, and also under limiting levels of substrates in the presence of the inhibitor phosphate (called the selective assay by Quick et al, [1989]), We have investigated the extractabie activity of SPS in crude extracts of P, vulgaris leaves to determine how water stress affects its activity. We tested the hypothesis that the effect of water stress on SPS activity is indirect and caused by low CQ, partial pressure inside leaves resulting from stomatal closure (Vassey and Sharkey 1989) and whether high CQj, which can overcome the effect of stomatal closure, can reverse the decline in SPS activity during water stress. To characterize the CQj response of SFS activity we compared it to the light responses of SPS activity in P, vulgaris. phate; Glc 6-P, glucose 6-phosphate; SPS, sucrose-phosphate synlhase; UDP-Glc, UDP glucose. Materials antl methods Plant culture All experiments were carried out with Phaseolus vulgaris L. cv. Linden. Plants were grown in a growth chamber in 4 1 pots containing a 3;3;3;2 (v;v) mixture of soil;peat;perlite;rice hulls. Plants were grown under a 12 h photoperiod with 24/17°C day/night temperature, 60% RH with a photon flux density of 600 (.imol m'- s'' (cool white, very high output, Sylvania F96TI2 lamps). Mild water stress was obtained by withholding water until leaves had reached a water potential of ca -0.9 MPa, Water potential was measured with a pressure bomb (Soil Moisture Equipment Corp., Santa Barbara, CA) on primary leaves. All the leaves of a given plant were assumed to have the same water potential. Measurement of gas exchange Photosynthetic CO, assimilation and partial pressure of CO2 inside the leaf was measured in a small circular cuvette made of aluminium. The cuvette had Saran windows to admit light. Water from a constant temperature bath flowed through the aluminum chamber to control the leaf temperature at 25°C. Air was mixed from NT, O2, and 5% CO2 in air using mass flow controllers (Datametrics type 825, Edwards High Vacuum, Wilmington, MA). The air was humidified, then the water was condensed in a copper coil held at 18.5°C. The amount of air going to the chamber was measured with a Datametrics mass flow meter (type 831), Humidity was measured with a Dew 10 dew point hygrometer from General Eastern, Depletion of CQ2 was measured with a LiCor 6250 infrared gas analyzer. The air flow was ca 1 1 min~' to the top and bottom of 11 cm" leaf area. All experiments were carried out at 25°C. Measurements carried out in a leaf disc electrode were performed as described in Quick et al. (1989). Extraction and assay of enzymes SPS (EC 2,4,1,14) was assayed as described by Vassey (1989) with slight modification. Briefly, fresh leaf material (ca 100 mg) was ground in a 7 ml Ten-Broeck ground glass tissue homogenizer in 2 ml extraction buffer containing 50 mM HEPES-NaQH (pH 7,2), 5 mM MgCl,, 1 mJW EDTA, 15 mM KCI, 2 mg ml"' polyvinylpolypyrolidone (insoluble), 2 mM sodium diethyldithiocarbamate, 10% glycerol (v/v), 0,1% Triton X-100 (w/v), and 5 mM freshly added dithiothreitol. The extract was immediately transferred to a 2 ml microfuge tube and centrifuged (Model E, Beckman Inst,, Fullerton, CA) for 1 min. The supernatant was then Abbreviations - CH, cycloheximide; Fru 6-P, fructose 6-phos- desalted through a 5 ml bed of Sephadex G25 in a 5 ml 38 Physiol. Plant. 81,1»1 syringe and centrifuged for 30 s. The effluent was used for enzyme assay, SPS was assayed by measuring the sucrose and sucrose phosphate produced from Fru 6-P and UDP-Glc. The reaction mixture contained 150 yd of leaf extract and 50 yd of a Fru 6-P, Glc 6-P, and UDP-Glc mixture, in 100 niM HEPES-NaOH (pH 8.0) containing 10 mM MgCl,, Estimated final concentrations of substrates are given in the figure legends. Since we used relatively unpurified extracts to minimize the effects of the purification process on the results, the final Fru 6-P/Glc 6-P ratio was determined by the equilibrium established by hexose phosphate isomerase. Mixtures were incubated at 2S°C and al! reactions were terminated by mixing a 50 (il satnple aliquot with 50 [i\ of 30% NaOH. Samples were taken 5 min and 15 min after introduction of substrate. Hexoses were destroyed by placing the reaction tubes in boiUng water for 10 min. The tubes were cooled, 1 ml of anthrone reagent (76 ml H2SO4, 36 ml HjO, and 150 mg anthrone) was added, the mixture incubated at 38°C for 20 min, and absorbance at 620 nm was measured. The units we report are the same as the units for photosynthesis since SPS activity is required for photosynthesis. In addition, these units are ftilly consistent with SI guidelines, unlike the standard units for enzyme activity of |imol (mg protein)"' min"'. During our measurements of kinetic parameters we discovered that after incubation with the crude extract for several minutes, our assay buffers contained ca 1 mM phosphate. Therefore, the phosphate concentrations we report are added phosphate concentrations and may not be absolute phosphate concentrations. It is unclear whether this phosphate measured by the molybdate assay is. available (Sharkey and Vanderveer 1989). Because we were unable to remove this phosphate using an enzymatic sequestering system, this phosphate may not be available to influence reactions as has been found in phosphate starved chloroplasts (Robinson and Giersch 1987), We believe that sucrose synthase activity was negligible in these assays because the low level of phosphate found in the assay mixtures indicates that the potential conversion of Fru 6-P to Fru was limited and sucrose synthase levels are often low in mature leaves (Vassey 1989), saturated with Fru 6-P. Total protein levels were 1.75 ±0,05 (n = 20) g m~^ in controls leaves and 1,67 ±0.06 (n = 20) g m"- in water-stressed leaves (these protein levels can be used to convert the units of enzyme activity). At present, there is no way to determine how much SPS protein is present in leaves of P. vulgaris, since the only reported antibodies for SPS do not crossreact with SPS from other species (Walker and Huber 1989), The effect of water stress on SPS was strongest in the absence of added phosphate in the assay medium (Fig. 2), Added phosphate reduced SPS activity of both water-stressed and control leaf extracts until both had the same low activity. The effect of phosphate was competitive with Fru 6-P but not with UDP-Glc (data not shown). How does water stress reduce the activity of SPS? Water stress could reduce the activity of SPS as a consequence of the reduced CO, inside the leaf caused by stomatal closure. To test this, we put a water-stressed (-0,9 MPa water potential) plant in a growth chamber with 1 kPa (1%) CO, in an attempt to overcome the effect of stomatal closure on the CO, ievel inside the leaf. The plants were not rewatered and, because the roots had explored the total soil volume, there was no way for the plant to recover from the water stress in the high CO, treatment. The activity of SPS in leaves from 2,0 o 1,5 "5 1,0 E O control • water-stressed 0- Resuits Characteristics of SPS reduction caused by water stress We first characterized SPS from water-stressed and control leaves of Phaseoltts vulgaris. The maximutn activity of SPS was found at 7 mM Fru 6-P (Fig, 1), In these assays Glc 6-P was added to equal 4 times the concentration of Fru 6-P, since in these crude extracts hexose phosphate isomerase would have converted much of the added Fru 6-P to Glc 6-P (Doehlert and Huber 1984). The effect of water stress was still evident when SPS was Physiol. Pianl. 81, i99i " 0,0 12 [Fru 6-P], mM Fig. 1. Effect of Fru 6-P concentration on SPS activity extraeted from control and water-stressed bean leaves. Water stress was imposed by withholding water for 3 days until the water potential of adjacent leaves was -0.9 MPa. Bars indicate SEM of 4 experiments, if no bar is present, it was smaller than the symbol. In addition to the Fru 6-P, Glc 6-P was added to give 4 times the eoncentration of Fru 6-P. The concentration of UDP-Glc was 10 mM, The first data point, at 0,3 mAf Fm6-P appears higher than expected but is correct. We did not eonsistently observe such a pattern and attach no significance to it. 39 O control • water-stressed E CD 2 o C/3 "o E -5 1 0 0 I Phosphate concentration, mM Fig. 2. Effect of phosphate concentration on extractable SPS activity of control and water-stressed bean leaves. Water stress was imposed as described for Fig. 1. Bars indicate SEM of 4 experiments. The substrate concentrations in these assays were: Fru 6-P, 4 mM; Glc 6-P, 20 mM; UDP-Glc, 10 mAf. the water-stressed plant was ca 50% of the control level when the plant was first put into high CO, (Fig. 3). Over 5 h, the activity of SPS recovered to the control level (shown as a triangle in Fig. 3). During this treatment, photosynthesis increased from 3,3 to 10.2 [imol 2,0 We next tested whether control plants held in low CO, would lose SPS activity. After 1 h in 7,5 Pa CO,, the activity of SPS was reduced to the level found in the water-stressed plant (Fig. 4). The low CO, had no significant effect on SPS activity from water-stressed leaves. We also tested for rapid reversibility of the effect of low CO, on SPS activity. Leaves which had been in low CO, for 1 h were put into a leaf disk electrode and held in 5 kPa (5%) CO, for 20 min. This 20 min high COj treatment reversed the effect of low CO, on SPS activity in control leaves. When SPS activity was reduced by water stress, a 20 min high CO, treatment did not cause recovery of the SPS activity. Water stress developed over 2-3 days, while the low CO, treatment lasted only 1 h. We decided to test whether a longer period of time caused the reduction in SPS activity that could not be reversed in 20 min. The results of this experiment are shown in Fig. 5. After 1 h in low CO2, most of the reduction in SPS activity could be reversed by 20 min in high CO,, However, after 24 h in low CO,, less SPS activity could be restored by the 20 min high CO, treatment. The response of SPS to CO, was strongest when both substrates were saturating (labelled V^gj conditions. Fig, 6). At low Fru 6-P concentration the effect of CO, was reduced while in limiting UDP-Glc there was no effect of CO,. The data in Fig. 6 indicate that in many cases, SPS activity can be saturated by 3 mM Fru 6-P even though that level of Fru 6-P is often called limiting. - E CD U3 1,5 - 2 o U) "o 1,0 1 0,5 0- cn 0,0 1 h low Time in high Fig. 3. Extractable SPS activity of water-stressed bean leaves held in 1 kPa CO, and 300 nmol photons m"- s"' over 5 h. Water stress was imposed as described for Fig. 1 and the plant was not rewatered when put into the high CO, chamber. Bars indicate SEM of 3 experiments. The triangle is the activity of SPS in a crude leaf extract from unstressed plants. Substrate concentrations were as given for Fig. 2. 40 1 hlow4 20 min high COg levels l-ig. 4. Activity of SPS in crude bean leaf extracts of control and water-stressed plants held in normal CO, partial pressure (35 Pa), 1 h low CO, (7,5 Pa), and 1 h low CO, followed by 20 min high CO2 (5 kPa', 5%, n = 4). The photon flux density was 600 (xtnbl m"^ s~' during the treatment. Water stress was imposed as described for Fig. 1, and substrate concentrations were as given for Fig. 2, Physioi. Piant. 81. IWi •I 0-3 iI CO CO CL ^ 0.0 1 1 1 0,6 1— finguish SPS light responses. We found a marked light response at substrate saturation but not at low substrate eoncentration in the presence of 10 mM phosphate (Fig. 7). We did find, however, that when phosphate was left out, the lighf response was apparent when Fru 6-F was low (Fig. 7). This amount of Fru 6-P (3 miW) was limiting in the presence of phosphate and for the enzyme from leaves held in darkness,, but not for enzyme from leaves taken in the light. When UDP-Glc was limiting, the light freafment had no effect oti extractabie SPS activity regardless of the presence or absence of phosphate. I 5 a. I "-'•^ I en o 1 ! 1 11 I 1,2 24 2,5 Time in low COj, h I light I dark Fig. 5. Activity of SPS in crude bean leaf extracts before, and after 1 and 24 h exposure to low CO, (7.5 Pa). The open bar represents data from leaf extracts taken without further treatment; the hatched bar, those from leaves exposed to 5 kPa CO, for an additional 20 min to test for rapid reversibility. The leaves were exposed to 600 (imol photons m~' s"' during the treatment. The data are all determined from one trifoliolate to reduce the effects of leaf to leaf variability, and so no error bars shown. 2,0 1,5 1.0 Light activation of SPS The extractabie activity of SPS increased with light (Fig. 7). We tested the light response of P. vulgaris using the protocol used by Huber et al. (1989) to dis- 2,5 - 1 1 low CO2 - 0,5 I 0,0 I I I .•& 2,0 ^ 1 normal CO2 O 2,0 - 1,5 - ' 1,5 b yy\, 1,0 - y/, /y CO D. ± 11 i Limiting Limiting Fru 6-P UDP-Glc Fig. 6. Activity of SPS extracted from bean leaves treated with normal (35 Pa) or low (7.5 Pa) CO, for 1 h in 600 |imol photons m"^ s"' (n = 4). The limiting Fru 6-P assay had 3 mM Fru 6-P, 12 mM Glc 6-P, and 10 mM UDP-Glc. There was no phosphate added to the assay medium. The limiting UDP-Glc assay had 10 mM Fru 6-P, 40 miW Glc 6-P, and only 3 mM UDP-Glc. The V^.,. assay had 10 mM Fru 6-P, 40 mM Glc 6-P, and lOmM UDP-Glc. Physiol. Planl. 81, 1991 I 1.0 0,5 0,0 Limiting Limiting V,max Fru 6-P UDP-Glu Fig. 7. Activity of SPS from bean leaves kept in darkness or in 600 [imol photons m"^ s"' for 1 h (n - 4). Assay conditions are as described for Fig. 6 except that additional experiments were carried out with 10 mM phosphate added to the assay medium (lower panel). Phosphate was not present during the V„„ assays. Different plants were used for the data in the top panel and the bottom panel but within a panel, all data were obtained from the same leaf extracts. 41 control 1,5 - detached from plants, fed water or 1 mM CH, and exposed to high CO2. We used 1 mM CH because preliminary experiments indicated that this concentration would inhibit protein synthesis without inhibiting photosynthesis under the conditions we used. The SPS activity of the water stressed leaf fed water recovered rapidly in high CO, (Fig, 8). The recovery was probably speeded by the recovery from water stress caused when the detached leaf quickly rehydrated in the beaker of water. This accounts for the faster recovery than observed in Fig. 3. When CH was fed to the leaf, the recovery of SPS activity was blocked. After 5 h, the SPS activity of CH-fed leaves was substantially lower than that of the leaf fed water (Fig. 8). CH had no significant effect on the control plants, indicating that CH did not inhibit metabolism or SPS activity. To test whether CH affected the rapid recovery of SPS following a 1 h low CO, treatment, we exposed a leaf to 7.5 Pa CO, for 1 h. During the second 0.5 h, 3 mM CH was fed as above. At the end of the 1 h in low CO2, SPS activity was 50% of that in control leaves. However, a 20 min treatment at 100 Pa (ca 1000 ppm) caused the complete recovery of SPS activity (data not shown). Discussion Water stress The inhibition of SPS caused by water stress can be reversed by high CO, treatment of water-stressed plants without relief of the water stress. A reduction in extractabie SPS activity can be induced in healthy plants by incubation in low CO,. We therefore conclude that the effect of mild water stress on SPS activity is a secondary 0 1 5 effect mediated by reduced CO, partial pressure inside Recovery time,, h the leaf caused by stomatal closure. Because it can lake up to 5 h for recovery of the SPS activity, photosyntheFig. 8. Recovery of SPS activity after water stress in control and CH fed bean leaves. F. vulgaris leaves were detached and sis can be Hmited by SPS activity during short-term gas the petioles held under water while the leaf blade was exposed exchange measurements at enhanced COj partial presto 1 kPa CO,. One mM CH was fed through the transpiration sure. In other words, photosynthesis affects SPS activity stream for ] or 5 h. Bars = SEM of 3 experiments. Water stress was imposed as described for Fig. 1 and substrate concentra- and if photosynthesis is low for a long time, the recovery of SPS activity can be slow enough that a transient SPS tions were as described for Fig. 2. limitation of photosynthesis is observed in gas exchange data. When photosynthesis is limited this way, it should Cyetoheximide treatment be insensitive to COj and Oj (Sharkey 1985a). This We fed cycloheximide (CH) to water-stressed and con- behavior has been reported often for water stressed trol leaves held in high CO, to get some indication leaves (Forseth and Ehlringer 1983, Morgan 1984,, von whether protein synthesis was required for the recovery Caemmerer and Farquhar 1984, Sharkey 1985a, Vassey of SPS activity following water stress. CH affects a and Sharkey 1989). number of cellular processes in addition to cytosolic Apparent water stress effects on the biochemistry of protein synthesis, so this experirnent is only an indicator photosynthesis have also been attributed to uneven of the requirement for protein synthesis, not conclusive (patchy) stomatalclosure.(Downton etal. 1988). Patchproof (Ellis and MacDonald 1970). In addition, CH iness does occur in this variety of P, vulgaris (Sharkey causes stomatal closure (data not shown), but the exper- and Seemann 1989). However, when photosynthesises iments we report were done with 10 times greater COj limited by patchy stomatal closure,, it should be sensitive concentration than was required to completely over- to CO2 and especially O^, This was true for the plants in come the stomatal closure effect of CH. Leaves were the experiments of Downton et al. (1988), but in many 42 Piiysiol. Plant. 8!, 1991 water-stress-sensitive species, COj-sensitive photosynthesis is unaffected by water stress (von Caemmerer and Farquhar 1984, Sharkey 1985a, Sharkey and Seeman 1989). Both patchiness and the decline in SPS activity change the response of photosynthesis to CO, partial pressure inside the leaf following water stress. This change has contributed to the idea that water stress adversely affects the biochemical reactions of photosynthesis. However, under mild water stress (—0.9 MPa water potential in bean) both of these effects are consequences of stomatal closure. We conclude that stomatai closure is the primary effect of mild water stress on photosynthesis. Apparent changes in the biochemical capacity for photosynthesis induced by water stress are either artifactuai (patchiness) or a consequence of the stomatal closure (reduced SPS activity). The conclusion that the reduction in SPS activity is caused by the reduction in CO, partial pressure inside the leaf is supported by the findings of Quick et al. (1989) with spinach. They showed that detached spinach leaves wilted to 90% of their fresh weight exhibited increased SPS activity when placed in very high external CO, concentrations. Such leaves retained complete photosynthetic capacity and did not lose SPS activity. In P, vutgaris under water stress as imposed here, the rate of photosynthesis declined (Sharkey and Seeman 1989, Vassey and Sharkey 1989) and SPS activity was lost. Therefore, SPS activity is sensitive to the rate of photosynthesis, not to water stress per se. When th^ data are interpreted this way, what appears to be opposite results of Vassey and Sharkey (1989) and Quick et al. (1989) are fully consistent. The hypothesis put forward by Vassey and Sharkey (1989) that the effect of water stress is indirect and mediated by stomatal closure has withstood detailed testing. This is of fundamental importance since the hypothesis explains away the last support from gas exchange data for direct effects of water stress on photosynthesis. Studies of chloroplast functioning have often found no effect of mild water stress on photosynthetic electron transport and the carbon reduction cycle (Dietz and Heber 1983, Kaiser 1987, Downton et al. 1988, Stuhlfauth et al. 1988, Quick et al. 1989, Sharkey and Seemann 1989). The data from intact leaf studies is now in complete agreement; there is no effect of water stress on photosynthesis except by way of stomatal closure until relatively severe water stress occurs. The direct effect of more severe water stress on the biochemistry of photosynthesis appears to result from changes in the volume of chloroplasts (Kaiser 1987) or protoplasts (Santakumari and Berkowitz 1990). Kaiser and Forster (1989) report that the capacity for nitrate reduction is sensitive to CO,, as we report here for SPS activity. It may be that a number of enzymes are affected by the rate of photosynthesis. The mechanism by which photosynthesis is sensed for this regulation is unknown at present. Physiol. Plant. 81, 1991 Regulation of SPS in P, vulgaris The study of light activation of SPS in P, vulgaris was undertaken to help characterize the regulation by CO, seen during water stress. SPS of P, vulgaris exhibits light activation which was most apparent at saturating levels of substrate. In this respect P. vulgaris responds most like Zea mays and barley (Huber et al. 1989). However, unlike these monocotyledons, light activation was not evident when UDP-Glc was limiting in the assay mixture. When Fru 6-P was low, light activation could be suppressed by adding phosphate to the assay medium. The SPS of P, vulgaris is unlike any SPS reported by Huber et al. (1989), adding to the evidence that there are large species differences in regulation of this important photosynthetic enzyme (Huber et al. 1989). Like the SPS response to darkness, inhibition of SPS activity by low CO, treatment was most apparent under V^,^ conditions. Some response was observed when Fru 6-P was low but not when UDF-Glc was limiting. We suggest that a similar mechanism underlies the light response and the CO, response of SPS in P, vulgaris. The rapid reversibility of the light and CO2 responses is consistent with protein modification rather than changes in protein amount. The observation of Huber et al. (1989) of protein modification of SPS in spinach leaves provides a potential mechanism for the rapid changes in SPS activity. However, the data presented here are from studies conducted with crude extracts and cannot be taken as evidence for or against this hypothesis. In addition to the short-term regulation of SPS, we found evidence for a slowly reversible regulation which could involve protein turnover (Figs 3 and 5). The CH data in Fig. 8 is an additional indicator that protein turnover might be involved in SPS^ regulation in response to water stress. We have no indication whether similar or different signals are involved in the shortterm, quickly reversible regulation, and the longerterm, CH sensitive regulation. Acknowledgements - Supported by U.S. Department of Energy contracts DE-FG02-87ER13785 and DE-FG0287ER60568 and an OECD grant to W.P.Q. References Dietz, K.-J. & Heber, U. 1983. Carbon dioxide gas exchange and the energy status of leaves of Primula palinuri under water stress. - Planta 158: 349-356. Doehlert, D. C. & Huber, S. C. 1984. Phosphate inhibition of spinach leaf sucrose phosphate synthase as affected by glucose-6-phosphate and phosphoglucoisomerase. - Plant Physiol 76: 250-253. Downton, W. J. S., Loveys, B. R. & Grant, W. 1. R. 1988. Non-uniform stomatal closure induced by water stress causes putative non-stomatal inhibition of photosynthesis. — New Phytol. 110: 503-509. Ellis, R. J. & MacDonald, 1. R. 1970. Specificity of cyeloheximidc in higher plant systems. - Plant Physiol. 46: 227-232. Forseth, I. N. & Ehlringer, J. R. 1983. Ecophysiolcgy of two solar tracking desert winter annuals. III. Gas exehange 43 responses to light, CO, and VPD in relation to long-term drought. - Oeeologia 57: 344-351. Huber, ]., Huber, S. C. & Nielson, T. H. 1989. Protein phosphoryiation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity. - Arch. Biochem. Biophys. 270: 681-690. Huber, S. C , Ohsugi, R., Usuda, H. & Kalt-Torres, W. 1987. Light modulation of maize leaf sucrose phosphate synthase. - Plant Physiol. Biochem. 25: 515-523. - . Nielsoo, T. H., Huber, J. L. A. & Pharr, D. M. 1989. Variation among species in light activation of sucrose-phosphate synthase. - Plant Cell Physiol. 30: 277-285. Kaiser, W. M. 1987. Effects of water deficit on photosynthetic capacity. - Physiol. Plant. 71: 142-149. - & Forster,, 1. 1989. Low COj prevents nitrate reduction in leaves. - Plant Physiol. 91: 970-974. Kerr, P. S., Rufty, T. W. & Huber, S. C. 1985. Endogeoous rhythms in photosynthesis, sucrose-phosphate synthase aetivity and stomatal resistance in leaves of soybean {Gtycine max (L) Merr.). - Plant Physiol. 77: 275-280. Morgan, J. A. 1984. Interaction of water supply and N in wheat. - Plant Physioi. 76: 112-117. Obsugi, R. & Huber, S. C. 1987. Light modulation and localization of sucrose phosphate synthase activity between mesophyll cells and bundle sheath cells in C4 species. Plant Physio!. 84: 109^1101. Pollock, C. 1. & Housley, T. L. 1985. Light-induced increase in sucrose phosphate synthetase activity in leaves of Lolium temulentum. - Ann. Bot. 55: 593-596. Quick, W. P., Siegl, G., Neuhaus, E., Feil, R. & Stitt, M. 19B9. Short term water stress leads to a stimulation of sucrose synthesis by activ,ating sucrose phosphate synthase. - Planta 177: 535-546. Robinson, S. P & Giersch, C. 1987. Inorganic phosphate concentration in the stroma of isolated chloroplasts and its influence on photosynthesis. - Aust. 1. Plant Physiol. 14: 451-462. Santakumari, M. & Berkowitz, G. A. 1990. Correlation between the maintenance of photosynthesis and in situ protoplast volume at low water potentials in droughted wheat. - Plant Physiol. 92: 733-739. Sharkey, T. D. 1985a. O,-insensitive photosynthesis in C3 plants. Its occurrence and a possible explanation. - Plant Physiol. 78: 71-75. - 1985b. Photosynthesis in intact leaves of Cj plants: Physics, physiology and rate limitations. — Bot. Rev. 51: 53-105. - 1990. Feedback limitation of photosynthesis and the physiological role of ribulose bisphosphate carboxylase carbamyiation. - Bot. Mag. Tokyo Special Issue 2 (in press). - & Seemann, J. R. 1989. Mild water stress effects on carbon- reduction-cycle intermediates, RuBP carboxylase activity, and spatial homogeneity of photosynthesis in leaves. Plant Physiol. 89: 1060-1065. - & Vanderveer,, P. J. 1989. Stromal phosphate concentration is low during feedbaek limited photosynthesis. - Plant Physiol. 91: 679-684. Sicher, R. C. & Kremer, D. F. 1984. Changes in sucrosephosphate synthase activity in barley primary leaves during hght/dark transitions. - Plant Physiol. 76: 910-912. - & Kremer, D. F. 1985. Possible control of maize leaf sucrose-phosphate synthase activity by light modulation. Plant Physiol. 79: 695-698. Stitt, M. & Grosse, H. 1988. Interaction between sucrose synthesis and CO, fixation. I. Secondary kinetics during photosynthetic induction are related to a delay in activation of sucrose synthesis. - 1. Plant Physiol. 133: 129-137. - , Huber, S. & Kerr, P. 1987. Control of photosynthetic sucrose formation. - In The Biochemistry of Plants. A Comprehensive Treatise (P. K. Stumpf and E. E. Conn, eds), pp. 327-409. Academic Press, New York. ISBN 0-12675410-1. - , Wilke, 1, Feil, R. & Heldt, H. W. 1988. Coarse control of sucrose-phosphate synthase in leaves: alterations of kinetic properties in response to the rate of photosynthesis and accumulation of sucrose. - Planta 174: 217-230. Stuhlfauth, T., Sultemeyer, D. E, Wemz, S. & Fock, H. P. 1988. Fluorescence quenching and gas exchange in a water stressed C3 plant. Digitalis lanata. - Plant Physiol. 86: 246250. Vassey, T. L. 1988. Phytochrome mediated regulation of sucrose phosphate synthase activity in maize. - Plant Physiol. 88: 540-542. - 1989. Light/dark profiles of sucrose phosphate synthase, sucrose synthase, and acid invertase in leaves of sugar beet. - Plant Physiol. 89: 347-351. - cfe Sharkey, T. D. 1989. Mild water stress of Phaseolus vulgaris plants leads to reduced starch synthesis and extractabie sucrose phosphate synthase activity. - Plant Physiol. 89: 1066-1070. von Caemmerer, S. & Farquhar, G. D. 1984. Effects of partial defoliation, changes in irradiance during growth, shortterm water stress, and growth at enhanced p(CO;) on the photosynthetic capacity of leaves of Phaseolus vulgaris L. Planta 160: 320-329. Walker, J. L. & Huber, S. C. 1989. Regulation of suerosephosphate-syEthase activity in spinach leaves by protein level and covalent modification. - Planta 177: 116-120. Edited by C. Larsson 44 Physiol. Plant. 81, 1991