Isolation and characterization of multiple forms of maize leaf sucrose-phosphate synthase

Sucrose-phosphate synthase SPS; (EC 2.4.1.14) from maize (Zea mays L. cv. Pioneer 3184) leaves was partially purified and kinetically characterized. Maize SPS was activated by glucose-6-phosphate (G-6-P) due to an increase in Vmax and a decrease in the Km for UDP-glucose. The UDP-glucose saturation profile was biphasic; thus two Km values for UDP-glucose were calculated. Inhibition by inorganic phosphate was observed only in the presence of G-6-P. Chromatography of partially purified maize leaf extracts on hydroxyapatite resolved two forms of SPS activity, which differed in their affinity for UDP-glucose and in the degree of activation by G-6-P. SPS was partially purified from maize leaves that were harvested in the light and in the dark. The light enzyme had a higher specific activity than the enzyme isolated from dark harvested leaves, and this difference persisted during enzyme purification. The apparent molecular weight (Stokes radius) of the light enzyme was 547 kDa, which was greater than that of the dark enzyme (457 kDa). Light and dark SPS differed in their affinities for UDP-glucose in the absence G-6-P. Both the light and the dark SPS were activated by G-6-P; the Km for UDP-glucose of the light enzyme was lowered by G-6-P, while the Km for UDP-glucose for the dark enzyme remained unchanged. These results suggest that light activation involves a conformational change that results in differences in maximum velocity, substrate affinities and regulation by metabolites. Chromatography of either the light or dark SPS on hydroxyapatite yielded two peaks of enzyme activity, suggesting that the occurrence of the two activity peaks was not due to an interconversion of the light and dark forms.     

Regulation of sucrose phosphate synthase by gibberellins in soybean and spinach plants

Exogenous applications of gibberellins (GAs) increased the extractable activity of leaf sucrose phosphate synthase (SPS) in soybean (Glycine max [L.]) and spinach (Spinacia oleracea [L.]). The response to GA applications was detectable within 2 h postapplication and was still observed 6 h, 24 h, and 7 d after treatment. When paclobutrazol, a GA biosynthesis inhibitor, was applied to intact soybean and spinach plants, decreased extractable SPS activity resulted within 24 h following the treatment. Different methods of GA application (spray, injection, capillary wick, and excised leaf systems) produced similar effects on SPS activity of soybean leaves. Protein synthesis in soybean leaves appeared to be necessary for GA-promoted SPS activity because gibberellic acid only partially reversed the inhibitory effect of pretreatment with cycloheximide. Levels of SPS protein from crude extracts of spinach plants were measured by a dot blot technique using monoclonal antibodies against SPS. Application of gibberellic acid to spinach leaves increased levels of SPS protein 2 h, 24 h, and 7 d after treatment. The results suggest that, in both soybean and spinach, GA is one of the endogenous hormonal factors that regulate the steady-state level of SPS protein and, hence, its activity.     

Changes of Sucrose-Phosphate Synthase Activity in Barley Primary Leaves during Light/Dark Transitions

The activity of sucrose-phosphate synthase (SPS) in 9-day-old barley (Hordeum vulgare L.) primary leaves was measured over a 24-hour period. Extractable enzyme activity was constant in the light, decreased 50 to 60% during the first one-half hour of darkness, and then returned to full activity before the start of the normal light period. Decreases of SPS activity in the dark were fully reversed by less than 10 minutes of illumination. In contrast to results with barley, the measurable activity of SPS in soybean, spinach, and pea leaves was unchanged during the first hour of darkness. Changes of SPS activity in barley primary leaves were stable upon gel filtration. The exact biochemical mechanism responsible for the enzyme activity changes in barley leaf extracts is unknown. The above findings support the suggestion by de Fekete (1973 Eur J Biochem, 10: 73-80) that SPS is controlled by posttranslational protein modification. These results are discussed in relation to the regulation of photosynthetic sucrose metabolism.     

Phytochrome mediated regulation of sucrose phosphate synthase activity in maize

The extractable activity of sucrose phosphate synthase was determined in etiolated seedlings of maize (Zea mays L.), soybean (Glycine max [L.] Merr.), and sugar beet (Beta vulgaris L.) following treatments of changing light quality. A 30-minute illumination of 30 microeinsteins per square meter per second white light produced a three-fold increase in sucrose phosphate synthase activity at 2 hours postillumination when compared to seedlings maintained in total darkness. Etiolated maize seedlings treated with 3.6 microeinsteins per square meter per second of red and far-red light showed a 50% increase and a 50% decrease in sucrose phosphate synthase activity, respectively, when compared to etiolated maize seedlings treated with white light. Maize seedlings exposed for 30 minutes to red followed by 30 minutes to far-red showed an initial increase in sucrose phosphate synthase activity followed by a rapid decrease to control level. Neither soybean or sugar beet sucrose phosphate synthase responded to the 30-minute illumination of white light. Phytochrome is involved in sucrose phosphate synthase regulation in maize, whereas it is not responsible for changes in sucrose phosphate synthase activity in soybean or sugar beet.     

Characterization of diurnal changes in activities of enzymes involved in sucrose biosynthesis

Experiments were conducted with vegetative soybean plants (Glycine max [L.] Merr., `Ransom') to determine whether the activities in leaf extracts of key enzymes in sucrose metabolism changed during the daily light/dark cycle. The activity of sucrose-phosphate synthase (SPS) exhibited a distinct diurnal rhythm, whereas the activities of UDP-glucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, and sucrose synthase did not. The changes in extractable SPS activity were not related directly to photosynthetic rates or light/dark changes. Hence, it was postulated that the oscillations were under the control of an endogenous clock. During the light period, the activity of SPS was similar to the estimated rate of sucrose formation. In the dark, however, SPS activity declined sharply and then increased even though degradation of starch was linear. The activity of SPS always exceeded the estimated maximum rate of sucrose formation in the dark. Transfer of plants into light during the normal dark period (when SPS activity was low) resulted in increased partitioning of photosynthate into starch compared to partitioning observed during the normal light period. These results were consistent with the hypothesis that SPS activity in situ was a factor regulating the rate of sucrose synthesis and partitioning of fixed carbon between starch and sucrose in the light.     

A comparative analysis of fructose 2,6-bisphosphate levels and photosynthate partitioning in the leaves of some agronomically important crop species

Starch, sucrose, and fructose 2,6-bisphosphate (F2, 6BP) levels were measured in pea (Pisum sativum L.), maize (Zea mays L.), onion (Allium cepa L.) and soybean (Glycine max L.) leaves throughout a light/dark cycle. Leaf starch accumulated in pea, maize, and soybean but not in onion. Sucrose was a major leaf storage reserve in pea, maize, and onion but was only found at low levels in soybean. In all species examined, the most dramatic changes in F2,6BP concentration coincided with light/dark transitions. During the light period F2,6BP levels were about 0.1 nanomole/milligram chlorophyll in soybean source leaves and there was a small increase in effector concentration in the dark. Levels of F2,6BP were also low in pea and maize leaves during the light period but then increased 10- or 20-fold in the dark. Dark onion leaf F2,6BP levels were about 1.1 to 1.3 nanomole/milligram chlorophyll and these values decreased by 20 to 30% in the light. Thus, three different patterns were identified that describe diurnal F2,6BP levels in source leaves. These results support the suggestion that F2,6BP is involved in the regulation of sucrose biosynthesis. However, it was not possible to demonstrate that high levels of F2,6BP are essential for starch synthesis in the chloroplast.     

Purification and preliminary characterization of sucrose-phosphate synthase using monoclonal antibodies

Monoclonal antibodies specific for sucrose phosphate synthase (SPS; EC 2.4.1.14) have been obtained for the first time. Three independent clones have been isolated which inhibited spinach (Spinacia oleracea L.) leaf SPS activity and facilitated the enzyme purification by immunoprecipitation. All three clones were specific for the spinach enzyme but neither inhibited nor precipitated the SPS present in tissue extracts of maize (Zea mays L.), barley (Hordeum vulgare L.), soybean (Glycine max L.), and sugar beet (Beta vulgaris L.). The inhibition of SPS activity by all three clones was reversible in the presence of UDPG, suggesting the presence of an epitope at the substrate-binding site. Immunoprecipitates of active enzyme preparations consistently revealed the presence of a 120 kilodalton polypeptide, indicating that the enzyme may be a homotetramer with a native molecular weight of about 480 kilodaltons. The occasional appearance of a 52 kilodalton polypeptide in the immunoprecipitates of some enzyme preparations was not the result of proteolysis, was not necessary for enzyme activity, and did not contain an antigenic site as revealed by Western blotting experiments. All three antibodies bind weakly to the SDS denatured 120 kilodalton subunit bound to nitrocellulose. The specific activity of the purified spinach enzyme was determined for the first time to be approximately 150 units per milligram SPS protein (pH 7.5 and 25°C) based on quantitative immunoprecipitation of the enzyme.     

Diurnal changes in sucrose phosphate synthase activity in leaves

Studies were conducted to identify and compare diurnal changes in sucrose phosphate synthase (EC 2.4.1.14) activity in leaves of different species, and the effect of nitrogen nutrition on the rhythm in soybean [ Glycine max (L). Merr] leaves. In recently expanded corn ( Zea mays L.) leaves, a single peak of enzyme activity was observed at the beginning of the photoperiod. A similar pattern was observed in older corn leaves, but activities (leaf fresh weight basis) were lower. In recently expanded pea ( Pisum sativum L.) and soybean leaves, two peaks of sucrose phosphate synthase activity were observed over a 24-h light:dark period, one at the beginning and one at the end of the photoperiod. A similar pattern was observed in older soybean leaves, but activities were generally lower and the amplitude of the changes was reduced. In a separate experiment, soybean plants were grown in the greenhouse with either 2 or 10 m M nitrate. The high-N plants had higher rates of photosynthesis and translocation, and greater activities of sucrose phosphate synthase in leaf extracts, compared to low-N plants. Over both experiments with soybeans, changes in sucrose phosphate synthase activity during the photoperiod were closely aligned with changes in translocation rate.     

Effects of Elevated Sucrose-Phosphate Synthase Activity on Photosynthesis,Assimilate Partitioning,and Growth in Tomato (Lycopersicon esculentum var UC82B)

The expression of a sucrose-phosphate synthase (SPS) gene from maize (Zea mays, a monocotyledon) in tomato (Lycopersicon esculentum, a dicotyledon) resulted in marked increases in extractable SPS activity in the light and the dark. Diurnal modulation of the native tomato SPS activity was found. However, when the maize enzyme was present the tomato leaf cells were unable to regulate its activation state. No detrimental effects were observed and total dry matter production was unchanged. However, carbon allocation within the plants was modified such that in shoots it increased, whereas in roots it decreased. There was, therefore, a change in the shoot:root dry weight ratio favoring the shoot. This was positively correlated with increased SPS activity in leaves. SPS was a major determinant of the amount of starch in leaves as well as sucrose. There was a strong positive correlation between the ratio of sucrose to starch and SPS activity in leaves. Therefore, SPS activity is a major determinant of the partitioning of photosynthetically fixed carbon in the leaf and in the whole plant. The photosynthetic rate in air was not significantly increased as a result of elevated leaf SPS activity. However, the light- and CO2-saturated rate of photosynthesis was increased by about 20% in leaves expressing high SPS. In addition, the temporary enhancement of the photosynthetic rate following brief exposures to low light was increased in the high SPS plants relative to controls. We conclude that the level of SPS in the leaves plays a pivotal role in carbon partitioning. Furthermore, high SPS levels have the potential to boost photosynthetic rates under favorable conditions.     

Effect of Photoperiod on Photosynthate Partitioning and Diurnal Rhythms in Sucrose Phosphate Synthase Activity in Leaves of Soybean (Glycine max L. [Merr.]) and Tobacco (Nicotiana tabacum L.)

Studies were conducted to identify the existence of diurnal rhythms in sucrose phosphate synthase (SPS) activity in leaves of three soybean (Glycine max L. [Merr.]) and two tobacco (Nicotiana tabacum L.) cultivars and the effect of photoperiod (15 versus 7 hours) on carbohydrate partitioning and the rhythm in enzyme activity. Acclimation of all the genotypes tested to a short day (7 hours) photoperiod resulted in increased rates of starch accumulation, whereas rates of translocation, foliar sucrose concentrations, and activities of SPS were decreased relative to plants acclimated to long days (15 hours). Under the long day photoperiod, two of the three soybean cultivars (`Ransom' and `Jupiter') and one of the two tobacco cultivars (`22NF') studied exhibited a significant diurnal rhythm in SPS activity. With the soybean cultivars, acclimation to short days reduced the activity of SPS (leaf fresh weight basis) and tended to dampen the amplitude of the rhythm. With the tobacco cultivars, photoperiod affected the shape of the SPS-activity rhythm. The mean values for SPS activity (calculated from observations made during the light period) were correlated positively with translocation rates and were correlated negatively with starch accumulation rates. Overall, the results support the postulate that SPS activity is closely associated with starch/sucrose levels in leaves, and that acclimation to changes in photoperiod may be associated with changes in the activity of SPS.     

Light and thiol activation of maize leaf glycerate kinase : the stimulating effect of reduced thioredoxins and ATP

Glycerate kinase (EC 2.7.1.31) from maize (Zea mays) leaves was shown to be regulated by light/dark transition. The enzyme more than doubled in activity after either the leaves or isolated mesophyll chloroplasts were illuminated with white light for 10 minutes. Rate of inactivation in the dark was faster in leaves than in the isolated chloroplast fraction. The stimulating effect of light could be mimicked in crude preparations by addition of 10 or 50 millimolar dithiothreitol or 100 millimolar 2-mercaptoethanol. The thiol treatment resulted in 8- to 10-fold activation of glycerate kinase, with the highest rates in the range of 27 to 30 micromoles per mg chlorophyll per hour. Activation was not accompanied by any changes in the apparent M_r value of glycerate kinase as determined by gel filtration (M_r = 47,000). In contrast to maize glycerate kinase, the enzyme from spinach was not affected by either light or thiol exposure.

Partially purified maize glycerate kinase was activated up to 3-fold upon incubation with a mixture of spinach thioredoxins m and f and 5 millimolar dithiothreitol. The thioredoxin and dithiothreitol-treated glycerate kinase could be further stimulated by addition of 2.5 millimolar ATP. The results suggest that glycerate kinase from maize leaves is capable of photoactivation by the ferredoxin/thioredoxin system. The synergistic effect of ATP and thioredoxins in activation of the enzyme supports the earlier expressed view that the ferredoxin/thioredoxin system functions jointly with effector metabolites in light-mediated regulation during photosynthesis.

     

Inactivation of highly activated spinach leaf sucrose-phosphate synthase by dephosphorylation

Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with [γ-³²P]ATP (JLA Huber, SC Huber, TH Nielsen [1989] Arch Biochem Biophys 270: 681-690). Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 25°C before assay. The “spontaneous” inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg²⁺ and was relatively insensitive to Ca²⁺ and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [³²P]Pi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 25°C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate. We postulate that highly activated SPS contains phosphorylated residue(s) that increase activation state, and that spontaneous inactivation occurs by removal of these phosphate group(s). Inactivation of SPS in vivo caused by feeding uncouplers to darkened leaf tissue that had previously been fed mannose in the dark, may occur by this mechanism. However, there is no evidence that this mechanism is involved in light-dark regulation of SPS in vivo.     

Variation among Species in Light Activation of Sucrose-Phosphate Synthase

Some species exhibit light activation of sucrose-phosphate synthase(SPS) in intact leaves. Twelve species which vary in extentof light modulation of SPS were studied to identify factorswhich regulate activation of the enzyme in situ. Leaves wereharvested in light and darkness, and SPS was assayed under highsubstrate (Vmax) or limiting substrate conditions (in the presenceof th inhibitor inorganic phosphate). The species tested fellinto three groups. In some species (Group I; barley and maize)light activation involved an increase in Vmax of the enzyme.In other species (Group II; spinach, swiss chard, sugarbeet,broad bean) light activation had no effect on Vmax but alteredcertain kinetic properties such that activation was only apparentwhen SPS was assayed with the limiting substration condition.Significantly, in some species (Group HI; soybean, pea, tobacco,Arabidopsis thaliana cucumber and melon) SPS activity was essentiallyunaffected by light/dark transitions. Phosphate sequesteringagents (mannose or gluco-samine) activated SPS in darkness inspecies which show light modulation (Groups I and II) but notspecies of Group III which do not light activate. Thus, changesin inorganic phosphate level may be one of the factors whichregulate the activation of SPS in situ. In addition, the catalyticactivity of SPS from species which exhibit light modulation(Groups I and II) is more strongly inhibited in vitro by inorganicphosphate (an allosteric effector) compared with the enzymeextracted from Group III species, which indicates that certainproperties of the enzyme itself may also vary among species.We conclude that species which do not exhibit light activationof SPS in intact leaves lack the mechanism responsible for thecovalent modification of enzyme activity (Walker and Huber 1989,Planta, in press) involved in light activation process. (Received October 14, 1988; Accepted January 5, 1989)     

Diurnal Changes in Maize Leaf Photosynthesis : II. Levels of Metabolic Intermediates of Sucrose Synthesis and the Regulatory Metabolite Fructose 2,6-Bisphosphate

Diurnal changes in the regulatory metabolite, fructose-2,6-bisphosphate (F26BP), and key metabolic intermediates of sucrose biosynthesis were studied in maize (Zea mays L. cv Pioneer 3184) during a day-night cycle. Whole leaf concentrations of dihydroxyacetonephosphate (DHAP) and fructose 1,6-bisphosphate changed markedly during the photoperiod. DHAP concentration was correlated positively with the rate of sucrose formation in vivo (assimilate export plus sucrose accumulation) and extractable activity of sucrose phosphate synthase (SPS). The changes closely followed net photosynthetic rate, which tracked irradiance. The other metabolic intermediates measured (glucose 6-phosphate, fructose 6-phosphate, and UDP-glucose) were either relatively constant over the 24 hour period or changed in a different pattern. Diurnal changes in leaf F26BP concentrations were pronounced, and fundamentally different than the pattern reported with other species. F26BP concentration decreased at the beginning of the day and remained low and constant; a 3- to 4-fold increase occurred with darkness, and slowly declined thereafter. In general, leaf F26BP concentration was negatively correlated with net photosynthetic rate, and also leaf DHAP concentration. Consequently, co-ordination of the regulation of cytosolic fructose 1,6-bisphosphatase and SPS was apparent. The results support the postulate that in maize leaves the activation state of SPS may be dependent on availability of DHAP and possibly other metabolites.     

Role of sucrose-phosphate synthase in partitioning of carbon in leaves

Variations in leaf starch accumulation were observed among four species (wheat [Triticum aestivum L.], soybean [Glycine max L. Merr.], tobacco [Nicotiana tabacum L.], and red beet [Beta vulgaris L.]), nine peanut (Arachis hypogea L.) cultivars, and two specific peanut genotypes grown under different nutritional regimes. Among the genotypes tested, the activity of sucrose phosphate synthase was correlated negatively with leaf sucrose content in seven of the nine peanut cultivars as well as the two peanut cultivars grown with different mineral nutrition. The peanut cultivars differed in the effect of 10 millimolar sucrose on sucrose phosphate synthase activity in leaf extracts. Enzyme activity in crude leaf extracts was inhibited by sucrose (10-42%) in four of the cultivars tested whereas five cultivars were not. Overall, the results suggest that a correlation exists between the activity of sucrose phosphate synthase and starch/sucrose levels in leaves.     

Comparative studies of the light modulation of nitrate reductase and sucrose-phosphate synthase activities in spinach leaves

We recently obtained evidence that the activity of spinach (Spinacia oleracea L.) leaf nitrate reductase (NR) responds rapidly and reversibly to light/dark transitions by a mechanism that is strongly correlated with protein phosphorylation. Phosphorylation of the NR protein appears to increase sensitivity to Mg²⁺ inhibition, without affecting activity in the absence of Mg²⁺. In the present study, we have compared the light/dark modulation of sucrose-phosphate synthase (SPS), also known to be regulated by protein phosphorylation, and NR activities (assayed with and without Mg²⁺) in spinach leaves. There appears to be a physiological role for both enzymes in mature source leaves (production of sucrose and amino acids for export), whereas NR is also present and activated by light in immature sink leaves. In mature leaves, there are significant diurnal changes in SPS and NR activities (assayed under selective conditions where phosphorylation status affects enzyme activity) during a normal day/night cycle. With both enzymes, activities are highest in the morning and decline as the photoperiod progresses. For SPS, diurnal changes are largely the result of phosphorylation/dephosphorylation, whereas with NR, the covalent modification is super-imposed on changes in the level of NR protein. Accumulation of end products of photosynthesis in excised illuminated leaves increased maximum NR activity, reduced its sensitivity of Mg²⁺ inhibition, and prevented the decline in activity with time in the light seen with attached leaves. In contrast, SPS was rapidly inactivated in excised leaves. Overall, NR and SPS share many common features of control but are not identical in terms of regulation in situ.     

Regulation of key enzymes of sucrose biosynthesis in soybean leaves : effect of dark and light conditions and role of gibberellins and abscisic Acid

An important part in the understanding of the regulation of carbon partitioning within the leaf is to investigate the endogenous variations of parameters related to carbon metabolism. This study of diurnal changes in the activities of sucrose-synthesizing enzymes and levels of nonstructural carbohydrates in intact leaves of field-grown soybean plants (Glycine max [L.]) showed pronounced diurnal fluctuations in sucrose phosphate synthase (SPS) activity. However, there was no distinct diurnal change in the activity of fructose-1,6-bisphosphatase (F1,6BPase). SPS activity in leaves from plants grown in controlled environments presented two peaks during the light period. In contrast to field-grown plants, F1,6BPase activity in leaves from growth chamber-grown plants manifested one peak during the first half of the light period. In plants grown under both conditions, sucrose and starch accumulation rates were highest during early hours of the light period. By the end of the dark period, most of the starch was depleted. A pattern of diurnal fluctuations of abscisic acid (ABA) levels in leaves was also observed under all growing conditions. Either imposition of water stress or exogenous applications of ABA inhibited F1,6BPase activity. However, SPS-extractable activity increased following water deficit but did not change in response to ABA treatment. Gibberellin application to intact soybean leaves increased levels of both starch and sucrose. Both gibberellic acid (10⁻⁶m) and gibberellins 4 and 7 (10⁻⁵m) increased the activity of SPS but had an inconsistent effect on F1,6BPase. Correlation studies between the activities of SPS and F1,6BPase suggest that these two enzymes are coordinated in their function, but the factors that regulate them may be distinct because they respond differently to certain environmental and physiological changes.     

Thiol-dependent regulation of glycerate metabolism in leaf extracts : the role of glycerate kinase in c(4) plants

We have recently reported that the activity of maize leaf glycerate kinase [EC 2.7.1.31] is regulated in vivo by the light/dark transition, possibly involving the ferredoxin/thioredoxin mechanism, and that the stimulating effect of light can be mimicked in vitro by incubation of crude leaf extract with reducing compounds (LA Kleczkowski, DD Randall 1985 Plant Physiol 79: 274-277). In the present study it was found that the time course of thiol activation of the enzyme was substantially dependent on the presence of some low molecular weight inhibitor(s) of activation found both in leaf extracts and mesophyll chloroplasts. Activity of glycerate kinase from maize as well as wheat leaves increased upon greening of etiolated plants and was correlated with the development of photosynthetic apparatus in these species. The maize enzyme was strongly activated by thiols at all stages of development from etiolated to green seedlings. Thiol activation of glycerate kinase was observed for a number of C₄ plants, notably of the nicotinamide adenine dinucleotide phosphate-malic enzyme type, with the strongest effect found for the enzyme from leaf extracts of maize and sorghum (10- and 8-fold activation, respectively). Among the C₃ species tested, only the enzyme from soybean leaves was affected under the same conditions (1.6-fold activation). This finding was reflected by an apparent lack of cross-reactivity between the enzyme from maize leaves and antibodies raised against purified spinach leaf glycerate kinase. We suggest that, in addition to its role as a final step of photorespiration in leaves, glycerate kinase from C₄ species may serve as a part of the facilitative diffusion system for the intercellular transport of 3-phosphoglycerate. Simultaneous operation of both the passive and the facilitative diffusion mechanisms of 3-phosphoglycerate transport in C₄ plants is postulated.     

Dependence of photosynthesis of sunflower and maize leaves on phosphate supply, ribulose-1,5-bisphosphate carboxylase/oxygenase activity, and ribulose-1,5-bisphosphate pool size

Sunflower (Helianthus annuus L. cv Asmer) and maize (Zea mays L. cv Eta) plants were grown under controlled environmental conditions with a nutrient solution containing 0, 0.5, or 10 millimolar inorganic phosphate. Phosphate-deficient leaves had lower photosynthetic rates at ambient and saturating CO₂ and much smaller carboxylation efficiencies than those of plants grown with ample phosphate. In addition, phosphate-deficient leaves contained smaller quantities of total soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit area, although the relative proportions of these components remained unchanged. The specific activity of Rubisco (estimated in the crude extracts of leaves) was significantly reduced by phosphate deficiency in sunflower but not in maize. Thus, there was a strong dependence of carboxylation efficiency and CO₂-saturated photosynthetic rate on Rubisco activity only in sunflower. Phosphate deficiency decreased the 3-phosphoglycerate and ribulose-1,5-bisphosphate (RuBP) contents of the leaf in both species. The ratio of 3-phosphoglycerate to RuBP decreased in sunflower but increased in maize with phosphate deficiency. The calculated concentrations of RuBP and RuBP-binding sites in the chloroplast stroma decreased markedly with phosphate deficiency. The ratio of the stromal concentration of RuBP to that of RuBP-binding sites decreased in sunflower but was not affected in maize with phosphate deficiency. We suggest that a decrease in this ratio made the RuBP-binding sites more vulnerable to blockage or inactivation by tight-binding metabolites/inhibitors, causing a decrease in the initial specific activity of Rubisco in the crude extract from phosphate-deficient sunflower leaves. However, the decrease in Rubisco specific activity was much less than the decrease in the RuBP content in the leaf and its concentration in the stroma. A large ratio of RuBP to RuBP-binding sites may have maintained the Rubisco-specific activity in phosphate-deficient maize leaves. We conclude that the effect of phosphate deficiency is more on RuBP regeneration than on Rubisco activity in both sunflower and maize.