Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : II. Partitioning between Sucrose and Starch

The role of fructose 2,6 bisphosphate in partitioning of photosynthate between sucrose and starch has been studied in spinach (Spinacia oleracea U.S. hybrid 424). Spinach leaf material was pretreated to alter the sucrose content, so that the rate of starch synthesis could be varied. The level of fructose 2,6-bisphosphate and other metabolites was then related to the accumulation of sucrose and the rate of starch synthesis. The results show that fructose 2,6-bisphosphate is involved in a sequence of events which provide a fine control of sucrose synthesis so that more photosynthate is diverted into starch in conditions when sucrose has accumulated to high levels in the leaf tissue. (a) As sucrose levels in the leaf rise, there is an accumulation of triose phosphates and hexose phosphates, implying an inhibition of sucrose phosphate synthase and cytosolic fructose 1,6-bisphosphatase. (b) In these conditions, fructose 2,6-bisphosphate increases. (c) The increased fructose 2,6-bisphosphate can be accounted for by the increased fructose 6-phosphate in the leaf. (d) Fructose 2,6-bisphosphate inhibits the cytosolic fructose 1,6-bisphosphatase so more photosynthate is retained in the chloroplast, and converted to starch.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : III. Properties of the Cytosolic Fructose 1,6-Bisphosphatase

The cytosolic fructose 1,6-bisphosphatase from spinach (Spinacia oleracea U.S. hybrid 424) leaves has been partially purified and its response to fructose 2,6-bisphosphate, AMP, and fructose 1,6-bisphosphate studied, using concentrations present in the cytosol during photosynthesis. In the presence of fructose 2,6-bisphosphate, the substrate saturation kinetics for fructose 1,6-bisphosphate are sigmoidal, with half-maximal activity being attained in 0.1 to 1 millimolar concentration range. The inhibition is enhanced by AMP. Using these results, and information published elsewhere on metabolite concentrations, it is discussed how fructose 1,6-bisphosphatase activity will vary in vivo in response to alterations in the availability of triose phosphate and AMP, and the accumulation of the product, fructose 6-phosphate.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : I. Coordination of CO(2) Fixation and Sucrose Synthesis

A mechanism is proposed for a feed-forward control of photosynthetic sucrose synthesis, which allows withdrawal of carbon from the chloroplast for sucrose synthesis to be coordinated with the rate of carbon fixation. (a) Decreasing the rate of photosynthesis of spinach (Spinacia oleracea, U.S. hybrid 424) leaf discs by limiting light intensities or CO₂ concentrations leads to a 2-to 4-fold increase in fructose 2,6-bisphosphate. (b) This increase can be accounted for by lower concentrations of metabolites which inhibit the synthesis of fructose 2,6-bisphosphate, such as dihydroxyacetone phosphate and 3-phosphoglycerate. (c) Thus, as photosynthesis decreases, lower levels of dihydroxyacetone phosphate should inhibit the cytosolic fructose bisphosphatase via simultaneously lowering the concentration of the substrate fructose 1,6-bisphosphate, and raising the concentration of the inhibitor fructose 2,6-bisphosphate.     

Metabolic Regulation in the Senescing Tobacco Leaf: II. Changes in Glycolytic Metabolite Levels in the Detached Leaf

Yellowing of detached mature tobacco leaves standing in water in the dark was accompanied by a strong “climacteric rise” in respiration rate. During this period the ATP level and energy charge of the adenylate system also rose. The levels of glycolytic intermediates between glucose 1-phosphate and triose phosphates rose, those between 3-phosphoglycerate and phosphoenolpyruvate fell, and pyruvate rose. On the assumption of a drop in NAD/NADH ratio, as found by other workers in wheat leaves, the reverse crossover between triose phosphates and 3-phospholglycerate was attributed to inhibition of glyceraldehyde 3-phosphate dehydrogenase. The forward crossover between phosphoenolpyruvate and pyruvate was taken to indicate activation of pyruvate kinase, possibly by fructose diphosphate. Secondary large rises in pyruvate and fructose diphosphate occurred well after the climacteric peak had been passed. No evidence was found for participation of phosphofructokinase in metabolic control in the yellowing leaf. Possible limitations to the use of the crossover theorem in the present situation, such as changes in compartmentation and in flux through branch points, are emphasized.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : V. Modulation of the Spinach Leaf Cytosolic Fructose 1,6-Bisphosphatase Activity in Vitro by Substrate, Products, pH, Magnesium, Fructose 2,6-Bisphosphate, Adenosine Monophosphate, and Dihydroxyacetone Phosphate

How fructose 2,6-bisphosphate and metabolic intermediates interact to regulate the activity of the cytosolic fructose 1,6-bisphosphatase in vitro has been investigated. Mg²⁺ is required as an activator. There is a wide pH optimum, especially at high Mg²⁺. The substrate dependence is not markedly pH dependent. High concentrations of Mg²⁺ and fructose 1,6-bisphosphate are inhibitory, especially at higher pH. Fructose 2,6-bisphosphate inhibits over a wide range of pH values. It acts by lowering the maximal activity and lowering the affinity for fructose 1,6-bisphosphate, for which sigmoidal saturation kinetics are induced, but the Mg²⁺ dependence is not markedly altered. On its own, adenosine monophosphate inhibits competitively to Mg²⁺ and noncompetitively to fructose 1,6-bisphosphate. In the presence of fructose 2,6-bisphosphate, adenosine monophosphate inhibits in a fructose 1,6-bisphosphate-dependent manner. In the presence of adenosine monophosphate, fructose 2,6-bisphosphate inhibits in Mg²⁺-dependent manner. Fructose 6-phosphate and phosphate both inhibit competitively to fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate does not affect the inhibition by phosphate, but weakens inhibition by fructose 6-phosphate. Dihydroxyacetone phosphate and hydroxypyruvate inhibit noncompetitively to fructose 1,6-bisphosphate and to Mg²⁺, but both act as activators in the presence of fructose 2,6-bisphosphate by decreasing the S_0.5 for fructose 1,6-bisphosphate. A model is proposed to account for the interaction between these effectors.     

Diurnal Changes in Maize Leaf Photosynthesis : III. Leaf Elongation Rate in Relation to Carbohydrates and Activities of Sucrose Metabolizing Enzymes in Elongating Leaf Tissue

Maize (Zea mays L. cv. Pioneer 3184) leaf elongation rate was measured diurnally and was related to diurnal changes in the activities of sucrose metabolizing enzymes and carbohydrate content in the elongating portion of the leaf. The rate of leaf elongation was greatest at midday (1300 hours) and was coincident with the maximum assimilate export rate from the distal portion of the leaf. Leaf elongation during the light period accounted for 70% of the total observed increase in leaf length per 24 hour period. Pronounced diurnal fluctuations were observed in the activities of acid and neutral invertase and sucrose phosphate synthase. Maximum activities of sucrose phosphate synthase and acid invertase were observed at 0900 hours, after which activity declined rapidly. The activity of sucrose phosphate synthase was substantially lower than that observed in maize leaf source tissue. Neutral invertase activity was greatest at midday (1200 hours) and was correlated positively with diurnal changes in leaf elongation rate. There was no significant change in the activity of sucrose synthase over the light/dark cycle. Sucrose accumulation rate increased during a period when leaf elongation rate was maximal and beginning to decline. Maximum sucrose concentration was observed at 1500 hours, when the activities of sucrose metabolizing enzymes were low. At no time was there a significant accumulation of hexose sugars. The rate of starch accumulation increased after the maximum sucrose concentration was observed, continuing until the end of the light period. There was no delay in the onset of starch mobilization at the beginning of the dark period, and essentially all of the starch was depleted by the end of the night. Mobilization of starch in the elongating tissue at night could account for a significant proportion of the calculated increase in the tissue dry weight due to growth. Collectively, the results suggested that leaf growth may be controlled by the activities of certain sucrose metabolizing enzymes and may be coordinated with assimilate export from the distal, source portion of the leaf. Results are discussed with reference to diurnal photoassimilation and export in the distal, source portion of the leaf.     

Subcellular Metabolite Levels in Spinach Leaves : Regulation of Sucrose Synthesis during Diurnal Alterations in Photosynthetic Partitioning

The alterations of subcellular metabolite levels during the day in spinach leaves have been investigated using nonaqueous density gradient centrifugation to separate chloroplasts, cytosol, and vacuole. The results provide direct evidence for the role of sucrose phosphate synthase and cytosolic fructose 1,6-bisphosphatase in regulating sucrose synthesis in leaves and also show that the phosphate translocator is kinetically limiting in vivo.     

Changes in Fructose 2,6-Bisphosphate Levels in Green Pepper (Capsicum annuum L.) Fruit in Response to Temperature

The regulatory metabolite, fructose 2,6-bisphosphate (Fru 2,6-P₂) was found in green pepper (Capsicum annuum L.). The Fru 2,6-P₂ level was found to: (a) rise rapidly in response to heat; (b) drop rapidly, followed by recovery, in response to cold storage of fruit and, (c) oscillate during cold storage of fruit. The possible existence of a relationship between chilling injury and Fru 2,6-P₂ is considered.     

Modulation of Sucrose Content by Fructose 2,6-bisphosphate during Photosynthesis in Rice Leaves Growing at Different Light Intensities

Reddy, A. R. and Das, V. S. R. 1987. Modulation of sucrose contentby fructose 2,6-bisphosphate during photosynthesis in rice leavesgrowing at different light intensities.—J. exp. Bot. 38:828–833. The relationship between the rate of CO₂ fixation and sucroseconcentration in the leaves of rice (Oryza sativa L.) grownat different light intensities was investigated. Maximum sucrosecontent coincided with maximum rates of CO₂ fixation, achievedat a photon flux density of 1600 µmol m^–2 s^–1.The levels of sucrose and fructose 2,6-bisphosphate were alsocompared in the leaves under different light intensities. Fructose2,6-Msphosphate accumulated during growth at low light. Theactivity of fructose-6-phosphate 2-kinase was high in the leavesgrown at low light while that of fructose-2,6-bisphosphatasewas low. The activities of phosphoglucose isomerase and phospho-glucomutasewere slightly increased by growth at low light The activitiesof UDP glucose pyrophosphorylase were adversely affected invitro with increased concentrations of fructose 2,6-bisphosphatewhile those of sucrose phosphate synthase were moderately affected.Phosphoglucose isomerase and phosphoglucomutase were activatedby fructose 2,6-bisphosphate (8-0 mmol m^–3) by 12-15%.The results suggested that low light intensities during growthresult in an accumulation of fructose 2,6-bisphosphate whichmodulates the key enzymes of sucrose biosynthesis thus regulatingcarbon flow under conditions of limited photosynthesis. Key words: Oryza sativa, photosynthesis, sucrose synthesis, fructose 2,6-bisphosphate, light     

Responses of Ribulose-1,5-Bisphosphate Carboxylase,Cytochrome f,and Sucrose Synthesis Enzymes in Rice Leaves to Leaf Nitrogen and Their Relationships to Photosynthesis

The photosynthetic gas-exchange rates and various biochemical components of photosynthesis, including ribulose-1,5-bisphosphate carboxylase (Rubisco) content, cytochrome (Cyt) f content, and the activities of two sucrose synthesis enzymes, were examined in young, fully expanded leaves of rice (Oryza sativa L.) grown hydroponically in different nitrogen concentrations. The light-saturated rate of photosynthesis at an intercellular CO2 pressure of 20 Pa (CO2-limited photosynthesis) was linearly dependent on leaf nitrogen content, but curvilinearly correlated with Rubisco content. This difference was due to a greater than proportional increase in Rubisco content relative to leaf nitrogen content and the presence of a CO2 transfer resistance between the intercellular air spaces and the carboxylation sites. CO2-limited photosynthesis was proportional to Cyt f content, one of the key components of electron transport, but was not proportional to the activities of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, the two regulatory enzymes of sucrose synthesis. Light-saturated photosynthesis above an intercellular CO2 pressure of 60 Pa (CO2-saturated photosynthesis) was curvilinearly dependent on leaf nitrogen content. This CO2-saturated photosynthesis was proportional to Cyt f content in the low- and normal-nitrogen leaves, and correlated better with the activities of cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase in the high-nitrogen leaves. The increase in the activities of these two enzymes with increasing leaf nitrogen was not as great as the increase in Cyt f content. Thus, as leaf nitrogen increased, the limitation caused by the activities of sucrose synthesis enzymes came into play, which resulted in the curvilinear relationship. However, this limitation by sucrose synthesis enzymes did not affect photosynthesis under normal ambient air.     

Synergistic Inhibition of Fructose 1,6-Bisphosphatase by Fructose 2,6-Bisphosphate and Adenosine Monophosphate in Round Spermatids of Rats

The effects of adenosine monophosphate (AMP) and fructose 2, 6-bisphosphate (fruc-2, 6-P₂) on the key-enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (fruc-P₂ase; D-fructose 1, 6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) in spermatid extract from rat testes were studied. The fruc-P₂ase activity in the spermatids of rats was suppressed by AMP and fruc-2, 6-P₂. The inhibition of fruc-2, 6-P₂ was much stronger at low than at high substrate concentrations, and enhanced synergistically with AMP. The substrate saturation curve was changed by fruc-2, 6-P₂ hyperbolic to sigmoidal. Furthermore, the concentration of AMP that decreased the activity to 50% was much lower in the presence than in the absence of fruc-2, 6-P₂. These results indicate the possibility that gluconeogenesis in spermatids of rats is controlled by AMP and fruc-2, 6-P₂.     

Photosynthesis and Ribulose 1,5-Bisphosphate Carboxylase in Rice Leaves: Changes in Photosynthesis and Enzymes Involved in Carbon Assimilation from Leaf Development through Senescence

Changes in photosynthesis and the ribulose 1,5-bisphosphate (RuBP) carboxylase level were examined in the 12th leaf blades of rice (Oryza sativa L.) grown under different N levels. Photosynthesis was determined using an open infrared gas analysis system. The level of RuBP carboxylase was measured by rocket immunoelectrophoresis. These changes were followed with respect to changes in the activities of RuBP carboxylase, ribulose 5-phosphate kinase, NADP-glyceraldehyde 3-phosphate dehydrogenase, and 3-phosphoglyceric acid kinase.

RuBP carboxylase activity was highly correlated with the net rate of photosynthesis (r = 0.968). Although high correlations between the activities of other enzymes and photosynthesis were also found, the activity per leaf of RuBP carboxylase was much lower than those of other enzymes throughout the leaf life. The specific activity of RuBP carboxylase on a milligram of the enzyme protein basis remained fairly constant (1.16 ± 0.07 micromoles of CO₂ per minute per milligram at 25°C) throughout the experimental period.

Kinetic parameters related to CO₂ fixation were examined using the purified carboxylase. The K_m(CO₂) and V_max values were 12 micromolar and 1.45 micromoles of CO₂ per minute per milligram, respectively (pH 8.2 and 25°C). The in vitro specific activity calculated at the atomospheric CO₂ level from the parameters was comparable to the in situ true photosynthetic rate per milligram of the carboxylase throughout the leaf life.

The results indicated that the level of RuBP carboxylase protein can be a limiting factor in photosynthesis throughout the life span of the leaf.

     

Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway during Induction of Photosynthesis in Maize Leaves

Changes in the level of metabolites of the C₄ cycle and reductive pentose phosphate (RPP) pathway were measured simultaneously with induction of photosynthesis in maize (Zea mays L.) to evaluate what may limit carbon assimilation during induction in a C₄ plant.

After 20 minutes in the dark, there was an immediate rise in photosynthesis during the first 30 seconds of illumination, followed by a gradual rise approaching steady-state rate after 20 minutes of illumination. Among metabolites of the C₄ cycle, there was a net increase in the level of C₃ compounds (the sum of pyruvate, alanine, and phosphoenolpyruvate) during the first 30 seconds of illumination, while there was a net decrease in the level of C₄ acids (malate plus aspartate). The total level of metabolites of the C₄ cycle underwent a sharp increase during this period. At the same time, there was a sharp rise in the level of intermediates of the RPP pathway (ribulose-1,5-bis-phosphate, 3-phosphoglycerate, dihydroxyacetonephosphate, and fructose-1,6-bisphosphate) during the first minute of illumination. The net increase of carbon among intermediates of the C₄ cycle and RPP pathway was far above that of carbon input from CO₂ fixation, and the increase in intermediates of the RPP pathway could not be accounted for by decarboxylation of C₄ acids, suggesting that an endogenous source of carbon supplies the cycles. After 3 minutes of illumination there was a gradual rise in the levels of intermediates of the C₄ cycle and in the total level of metabolites measured in the RPP pathway. This rise in metabolite levels occurs as photosynthesis gradually increases and may be required for carbon assimilation to reach maximum rates in C₄ plants. This latter stage of inductive autocatalysis through the RPP pathway may contribute to the final buildup of these intermediates.

     

Leaf Phosphate Status, Photosynthesis and Carbon Partitioning in Sugar Beet: II. Diurnal Changes in Sugar Phosphates, Adenylates, and Nicotinamide Nucleotides

Sugar Beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers. Leaf orthophosphate (Pi) levels were varied nutritionally. The effect of decreased leaf phosphate (low-P) status was determined on the diurnal changes in the pool sizes of leaf ribulose 1,5-bisphosphate (RuBP), 3-phosphoglycerate (PGA), triose phosphate, fructose 1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, adenylates, nicotinamide nucleotides, and Pi. Except for triose phosphate, low-P treatment caused a marked reduction in the levels of leaf sugar phosphates (on a leaf area basis) throughout the diurnal cycle. Low-P treatment decreased the average leaf RuBP levels by 60 to 69% of control values during the light period. Low-P increased NADPH levels and NADPH/NADP⁺ ratio but decreased ATP; the ATP/ADP ratio was unaffected. Low P treatment caused a marked reduction in RuBP regeneration (RuBP levels were half the RuBP carboxylase binding site concentration) but did not depress PGA reduction to triose phosphate. These results indicate that photosynthesis in low-P leaves was limited by RuBP regeneration and that RuBP formation in low-P leaves was not limited by the supply of ATP and NADPH. We suggest that RuBP regeneration was limited by the supply of fixed carbon, an increased proportion of which was diverted to starch synthesis.     

Regulation of Carbon Partitioning in Source and Sink Leaf Parts in Sweet Pepper (Capsicum annuum L.) Plants : Role of Fructose 2,6-Bisphosphate

Area expansion rate, partitioning of photosynthetically fixed carbon, and levels of fructose 2,6-bisphosphate (fru-2,6-P₂) were determined in individual parts of developing leaves of sweet pepper (Capsicum annuum L.). The base was rapidly expanding and allocated less carbon to sucrose synthesis in comparison to the leaf tip, where expansion had almost stopped. The change in leaf expansion rate and carbon partitioning happened gradually. During day time levels of fru-2,6-P₂ were consistently higher in the leaf base than in the leaf tip. Leaf expansion rate and carbon partitioning were closely related to day time levels of fru-2,6-P₂, suggesting that fru-2,6-P₂ is an important factor in adjustment of metabolism during sink-to-source transition of leaf tissue. The levels of fru-2,6-P₂ changed markedly after a dark-to-light transition in the leaf base, but not in the leaf tip, suggesting that regulatory systems based on fru-2,6-P₂ are different in sink and source leaf tissue. During the period upon dark-to-light transition the variations in level of fru-2,6-P₂ did not show a close correlation to changes in the carbon partitioning, until the metabolism had reached a steady state.     

秦艽与小秦艽光合日变化的研究

运用光合作用测定系统,对野生秦艽(Gentiana macrophylla Pall．)和小秦艽(G．dahurica Fisch．)的开花期的光合特性进行研究。结果表明,2种秦艽的净光合速率、蒸腾速率、气孔导度日变化曲线都呈双峰型,有明显的光合“午休”现象。小秦艽的光合速率、蒸腾速率、气孔导度照著高于秦艽,分别高出2．7 μmolCO2·m-2·s-1、1．5 mmol·m-2·s-1和140．7 mmol．m-2·s-1而叶温则低于秦艽2．8C。相关分析表明,开花期的温度、光照对2种秦艽的光合都有一定影响,蒸腾速率、气孔导度调节光合作用,而引起2种秦艽光合速率降低的主要因素为非气孔因素。     

Leaf Phosphate Status, Photosynthesis, and Carbon Partitioning in Sugar Beet: III. Diurnal Changes in Carbon Partitioning and Carbon Export

The effect of low phosphate supply (low P) was determined on the diurnal changes in the rate of carbon export, and on the contents of starch, sucrose, glucose, and fructose 2,6-bisphosphate (F2,6BP) in leaves. Low-P effects on the activities of a number of enzymes involved in starch and sucrose metabolism were also measured. Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers and the low-P treatment induced nutritionally. Low-P treatment decreased carbon export from the leaf much more than it decreased photosynthesis. At growth chamber photon flux density, low P decreased carbon export by 34% in light; in darkness, export rates fell but more so in the control so that the average rate in darkness was higher in low-P leaves. Low P increased starch, sucrose, and glucose contents per leaf area, and decreased F2, 6BP. The total extractable activities of enzymes involved in starch and sucrose synthesis were increased markedly by low P, e.g. adenosine 5-diphosphoglucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, uridine 5-diphosphoglucose pyrophosphorylase, and sucrose-phosphate synthase. The activities of some enzymes involved in starch and sucrose breakdown were also increased by low P. We propose that plants adapt to low-P environments by increasing the total activities of several phosphatases and by increasing the concentrations of phosphate-free carbon compounds at the expense of sugar phosphates, thereby conserving Pi. The partitioning of carbon among the various carbon pools in low-P adapted leaves appears to be determined in part by the relative capacities of the enzymes for starch and sucrose metabolism.