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.     

Leaf Phosphate Status,Photosynthesis, and Carbon Partitioning in Sugar Beet (IV. Changes with Time Following Increased Supply of Phosphate to Low-Phosphate Plants)

Changes in photosynthesis, carbon partitioning, and growth following resupply of orthophosphate (Pi) to moderately P-deficient plants (low-P) were determined for sugar beets (Beta vulgaris L. cv F58-554H1) cultured hydroponically in growth chambers. One set of plants was supplied with 1.0 mM Pi in half-strength Hoagland solution (control plants), and a second set (low-P plants) was supplied with 0.05 mM Pi. At the end of 2 weeks, the low-P plants were resupplied with 1.0 mM Pi. Low-P plants rapidly accumulated large amounts of Pi, and the photosynthesis rate increased to control values within 4 to 6 h. The rate of photosynthesis appeared to be controlled by ribulose-1,5-bisphosphate (RuBP); low P reduced photosynthesis and RuBP levels, and P resupply increased photosynthesis and RuBP in a manner parallel with time. Low-P treatment reduced adenylate levels substantially but not nicotinamide nucleotides; adenylate levels recovered to control values over 3 to 6 h. With low P, more photosynthate is allocated to non-P carbon compounds (e.g. starch, sucrose) than to sugar phosphates. When P is resupplied, sugar phosphates increase as starch and sucrose pools decrease; this increase in leaf (chloroplast) sugar phosphates was most likely responsible for the increases in RuBP and photosynthesis and may have increased adenylate levels (through enhanced levels of ribose-5-phosphate).     

The photosynthetic induction response in wheat leaves: net CO2 uptake,enzyme activation,and leaf metabolites

The photosynthetic induction response was studied in whole leaves of wheat (Triticum aestivum L.) following 5-min, 30-min and 10-h dark periods. After the 5-min dark treatment there was a rapid burst in the rate of photosynthesis upon illumination (half of maximum after 30s), followed by a slight decrease after 1.5 more min and then a gradual rise to the maximum rate. During this initial burst in photosynthesis, there was a rapid rise in the level of 3-phosphoglycerate (PGA) and a high PGA/triose-phosphate (triose-P) ratio was obtained. In addition, after the 5-min dark treatment, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39), ribulose-5-phosphate kinase (EC 2.7.1.19) and chloroplastic fructose-1,6-bisphosphatase (EC 3.1.3.11) maintained a relatively high state of activation, and maximum activation occurred within 1 min of illumination. The results indicate there is a high capacity for CO₂ fixation in the cycle upon illumination but attaining maximum rates requires an increase in the ribulose-1,5-bisphosphate (RuBP) pool (adjustment in triose-P utilization for carbohydrate synthesis versus RuBP synthesis). With both the 30-min and 10-h dark pretreatments there was only a slight rise in photosynthesis upon illumination, followed by a lag, then a gradual increase to steady-state (half-maximum rate after 6 min). In contrast to the 5-min dark treatment, the level of PGA was low and actually decreased initially, whereas the level of RuBP increased and was high during induction, indicating that Rubisco is limiting. This regulation via the carboxylase was not reflected in the initial extractable activity, which reached a maximum by 1 min after illumination. The light activation of chloroplastic fructose-1,6-bisphosphatase in leaves darkened for 30 min and 10 h prior to illumination was relatively slow (reaching a maximum after 8 min). However, this was not considered to limit carbon flux through the carbon-fixation cycle during induction since RuBP was not limiting. When photosynthesis approached the maximum steady-state rate, a high PGA/triose-P ratio and a high PGA/RuBP ratio were obtained. This may allow a high rate of photosynthesis by producing a favorable mass-action ratio for the reductive phase (the conversion of PGA to triose phosphate) while stimulating starch and sucrose synthesis.Abbreviations Chl chlorophyll - FBP fructose-1,6-bisphosphate - FBPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA 3-phosphoglycerate - Pi inoganic phosphate - Rubisco RuBP carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - Ru5P ribulose-5-phosphate - triose-P triose phosphates (dihydroxyacetone phosphate+glyceraldehyde-3-phosphate)     

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.     

Phosphate Deficiency in Maize. I. Leaf Phosphate Status, Growth, Photosynthesis and Carbon Partitioning

Maize plants (Zea mays L.) were cultured with nutrient solutioncontaining 0.001 or 0.5 mM orthophosphate (Pi). Effects of lowphosphate (low-P) nutrition on growth, leaf phosphate status,photosynthesis, and carbon partitioning were investigated. Withlow-P treatment, the fresh weight of aerial parts decreasedby about 40% by 24 days after planting. Detailed studies ofthe effects of low-P treatment on the other characteristicsof maize leaves-were done using the middle part of the thirdleaf, counting from the base. Low-P treatment had almost noeffect on specific leaf weight or soluble protein content measured13 to 21 days after planting. Low-P treatment decreased Chicontent slightly (by 15% 19 days after planting). Twenty onedays after planting, low-P treatment had greatly decreased thelevels of leaf acid extractable Pi (by 77%) and photosynthesisrates (by 68%). The detrimental effects of low-P treatment onthe rates of photosynthesis and the amounts of acid extractablePi became progressively greater with time. There was a strongcorrelation between levels of leaf acid extractable Pi and ratesof photosynthesis. The minimum level of Pi necessary to sustainthe maximum photosynthesis rate was 0.6 mmol m^–2. Belowthis minimum content of Pi, the rate of photosynthesis decreasedsharply with decreasing Pi. To investigate the direct effectof Pi depletion on photosynthate partitioning at equivalentrates of photosynthesis, the rates in controls were reducedto almost the same as those in 18 or 19 day old low-P plants(about 50% of those in controls) by lowering light intensityand/ or ambient CO₂ concentration. The data clearly indicatesthat low-P treatment had a direct effect in lowering photosynthatepartitioning into starch. Starch mobilization during the nightwas also inhibited under low-P conditions. (Received January 7, 1991; Accepted March 5, 1991)     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

“Sink” regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin-cycle enzymes and an increase of glycolytic enzymes

Leaves on transgenic tobacco plants expressing yeast-derived invertase in the apoplast develop clearly demarcated green and bleached sectors when they mature. The green areas contain low levels of soluble sugars and starch which are turned over on a daily basis, and have high rates of photosynthesis and low rates of respiration. The pale areas accumulate carbohydrate, photosynthesis is inhibited, and respiration increases. This provides a model system to investigate the sink regulation of photosynthetic metabolism by accumulating carbohydrate. The inhibition of photosynthesis is accompanied by a decrease of ribulose-1,5-bisphosphate and glycerate-3-phosphate, and an increase of triosephosphate and fructose-1,6-bisphosphate. The extracted activities of ribulose-1,5-bisphosphate carboxylase, fructose-1, 6-bisphosphatase and NADP-glyeraldehyde-3-phosphate dehydrogenase decreased. The activity of sucrose-phosphate synthase remained high or increased, an increased portion of the photosynthate was partitioned into soluble sugars rather than starch, and the pale areas showed few or no oscillations during transitions between darkness and saturating light in saturating CO₂. The increased rate of respiration was accompanied by an increased level of hexose-phosphates, triose-phosphates and fructose-1,6-bisphosphate while glycerate-3-phosphate and phosphoenolpyruvate decreased and pyruvate increased. The activities of pyruvate kinase, phosphofructokinase and pyrophosphate: fructose-6-phosphate phosphotransferase increased two- to four-fold. We conclude that an increased level of carbohydrate leads to a decreased level of Calvin-cycle enzymes and, thence, to an inhibition of photosynthesis. It also leads to an increased level of glycolytic enzymes and, thence, to a stimulation of respiration. These changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.Abbreviations Fru 1,6bisP fructose-1,6-bisphosphate - Fru 1,6bisPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc 1P glucose-1-phosphate - Glc6P glucose-6-phosphate - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PFK phosphofructokinase - PEP phosphoenolpyruvate - PFP pyrophosphate:fructose-6-phosphate phosphotransferase - PGA glycerate-3-phosphate - PK pyruvate kinase - Pi inorganic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SPS sucrose-phosphate synthase - triose-P triose-phosphates     

Influences of leaf temperature on photosynthetic carbon metabolism in wheat

Net photosynthetic assimilation rate (A), extractable activities of three photosynthetic enzymes, and the concentrations of six metabolites were determined for wheat (Tricum aestivum L.) leaves as leaf temperature was varied under photorespiring (350 microliters per liter CO₂ and 21% O₂) and under nonphotorespiring conditions (800 microliters per liter CO₂ and 2% O₂). The extractable activity of ribulose-1,5-bisphosphate carboxylase (Rubisco) and fructose-1,6-bisphosphatase declined with increasing leaf temperature from 15 to 45°C. Leaf concentrations of ribulose-1,5-bisphosphate (RuBP) declined slightly between 15 and 25°C but increased to a level which is 4 to 5 times the binding site concentration of Rubisco at leaf temperatures of 35 and 45°C. Leaf concentrations of 3-phosphoglycerate, fructose-6-phosphate, and glucose-6-phosphate all declined with increasing leaf temperature. Outside of the limitations imposed by photorespiration, it is proposed that under high light and at suboptimal temperatures, A is limited by rate of utilization of triose phosphate; at optimal temperatures, by the availability of substrate (CO₂ and RuBP) under photorespiring conditions or utilization of triose phosphate under nonphotorespiring conditions; and at supraoptimal temperatures, by the activation state of Rubisco.     

The relationship between steady-state gas exchange of bean leaves and the levels of carbon-reduction-cycle intermediates

The relationship between the gas-exchange characteristics of attached leaves of Phaseolus vulgaris L. and the pool sizes of several carbon-reduction-cycle intermediates was examined. After determining the rate of CO₂ assimilation at known intercellular CO₂ pressure, O₂ pressure and light, the leaf was rapidly killed (<0.1 s) and the levels of ribulose-1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate were measured. In 210 mbar O₂, photosynthesis appeared RuBP-saturated at low CO₂ pressure and RuBP-limited at high CO₂ pressure. In 21 mbar (2%) O₂, the level of RuBP always appeared saturating. Very high levels of PGA and other phosphate-containing compounds were found with some conditions, especially under low oxygen.Abbreviations and symbols C₁ intercellular CO₂ pressure - PGA 3-phosphoglyceric acid - RuBP ribulose-1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

On the role of chloroplastic phosphoglucomutase in the regulation of starch turnover

When spinach (Spinacia oleracea L.) leaf disks were incubated in 10% polyethylene glycol to induce water stress, the ratio of glucose-1-phosphate to glucose-6-phosphate increased. This increase indicated an imbalance in the phosphoglucomutase (EC 2.7.5.1) reaction, which was earlier observed to be close to equilibrium, and was accompanied by higher fructose-1,6-bisphosphate and ribulose-1,5-bisphosphate concentrations. Because starch degradation was assumed to be the source of the glucose-1-phosphate accumulation, the kinetic properties of plastidic phosphoglucomutase were analysed. It was found that physiological concentrations of both sugar bisphosphates inhibited phosphoglucomutase by about 50%. From this observation it was concluded that under conditions in which fructose-1,6-bisphosphate and ribulose-1,5-bisphosphate accumulated, an inhibition of phosphoglucomutase activity restricted the carbon exchange between the Calvin cycle and starch turnover. Received: 23 March 1998 / Accepted: 26 August 1998     

亚适温弱光对黄瓜幼苗光合酶活性和基因表达的影响

以‘津优3号’为试材,研究亚适温弱光(18℃/12℃,100 μmol·m-2·s-1)下黄瓜幼苗叶片核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)、果糖-1,6-二磷酸酶(FBPase)、甘油醛-3-磷酸脱氢酶(GAPDH)、果糖-1,6-二磷酸醛缩酶(FBA)、转酮醇酶(TK) mRNA表达量及活性的变化.结果表明:亚适温弱光处理的单株叶面积和干物质量均明显减小.处理初期,Rubisco大亚基(rbcL)、小亚基(rbcS)、FBPase、GAPDH、FBA及TK的基因表达量大幅度下降,多数酶活性明显减弱(TK变化不明显),光合速率(Pn)快速降低;处理3d后,亚适温弱光处理的rbcL、rbcS基因表达量和Rubisco初始活性持续下降,但下降幅度明显减小,Rubisco总活性及FBPase、GAPDH、FBA和TK基因表达与活性均呈上升趋势,Pn同步回升;处理时间超过6d时,Rubisco和FBPase基因表达与活性趋于平稳,其他酶和Pn呈下降趋势.可见,亚适温弱光下黄瓜光合酶基因表达量和活性的降低是Pn降低的重要原因,光合机构对亚适温弱光的适应与光合酶的活化机制有关.     

Limiting Factors in Photosynthesis: VI. Regeneration of Ribulose 1,5-Bisphosphate Limits Photosynthesis at Low Photochemical Capacity

Earlier work (SE Taylor, N Terry [1984] Plant Physiol 75: 82-86) has shown that the rate of photosynthesis may be colimited by photosynthetic electron transport capacity, even at low intercellular CO₂ concentrations. Here we monitored leaf metabolites diurnally and the activities of key Calvin cycle enzymes in the leaves of three treatment groups of sugar beet (Beta vulgaris L.) plants representing three different in vivo photochemical capacities, i.e. Fe-sufficient (control) plants, moderately Fe-deficient, and severely Fe-deficient plants. The results show that the decrease in photosynthesis with Fe deficiency mediated reduction in photochemical capacity was through a reduction in ribulose 1,5-bisphosphate (RuBP) regeneration and not through a decrease in ribulose 1,5-bisphosphate carboxylase/oxygenase activity. Based on measurements of ATP and NADPH and triose phosphate/3-phosphoglycerate ratios in leaves, there was little evidence that photosynthesis and RuBP regeneration in Fe-deficient leaves were limited directly by the supply of ATP and NADPH. It appeared more likely that photochemical capacity influenced RuBP regeneration through modulation of enzymes in the photosynthetic carbon reduction cycle between fructose-6-phosphate and RuBP; in particular, the initial activity of ribulose-5-phosphate kinase was strongly diminished by Fe deficiency. Starch and sucrose levels changed independently of one another to some extent during the diurnal period (both increasing in the day and decreasing at night) but the average rates of starch or sucrose accumulation over the light period were each proportional to photochemical capacity and photosynthetic rate.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Ribulose-1,5-bisphosphate carboxylase-oxygenase,other Calvin-cycle enzymes,and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream

The inhibition of photosynthesis after supplying glucose to detached leaves of spinach (Spinacia oleracea L.) was used as a model system to search for mechanisms which potentially contribute to the sink regulation of photosynthesis. Detached leaves were supplied with 50 mM glucose or water for 7 d through the transpiration stream, holding the leaves in low irradiance (16 mol photons · m^–2 · s^–1) and a cycle of 9 h light/15 h darkness to prevent any endogenous accumulation of carbohydrate. Leaves supplied with water only showed marginal changes of photosynthesis, respiration, enzyme levels or metabolites. When leaves were supplied with 50 mM glucose, photosynthesis was gradually inhibited over several days. The inhibition was most marked when photosynthesis was measured in saturating irradiance and ambient CO₂, less marked in saturating irradiance and saturating CO₂, and least marked in limiting irradiance. There was a gradual loss of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein, fructose-1,6-bisphosphatase, NADP-glyceraldehyde-3-phosphate dehydrogenase and chlorophyll. The inhibition of photosynthesis was accompanied by a large decrease of glycerate-3-phosphate, an increase of triose-phosphates and fructose-1,6-bisphospate, and a small decrease of ribulose-1,5-bisphosphate. The stromal NADPH/NADP ratio increased (as indicated by increased activation of NADP-malate dehydrogenase), and the ATP/ADP ratio increased. Chlorophyll-fluorescence analysis indicated that thylakoid energisation was increased, and that the acceptor side of photosystem II was more reduced. Similar results were obtained when glucose was supplied by floating leaf discs in low irradiance on glucose solution, and when detached spinach leaves were held in high light to produce an endogenous accumulation of carbohydrate. Feeding glucose also led to an increased rate of respiration. This was not accompanied by any changes of pyruvate kinase, phosphofructokinase, or pyrophosphate: fructose-6-phosphate phosphotransferase activity. There was a decrease of phosphoenolpyruvate, glycerate-3-phosphate and glycerate-2-phosphate, an increase of pyruvate and triose-phosphates, and an increased ATP/ADP ratio. These results show (i) that accumulation of carbohydrate can inhibit photosynthesis via a long-term mechanism involving a decrease of Rubisco and other Calvin-cycle enzymes and (ii) that respiration is stimulated due to an unknown mechanism, which increases the utilisation of phosphoenolpyruvate.Abbreviations and Symbols C_i CO₂ concentration in the air space within the leaf - F_m fluorescence yield with a saturating pulse in dark-adapted material - F_o ground level of fluorescence using a weak non-actinic modulated beam in the dark - Fru1,6bisP fructose-1,6-bisphosphate - Fru1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - IRGA infrared gas analyser - NAD-MDH NAD-dependent malate dehydrogenase - NADP-MDH NADP-dependent malate dehydrogenase - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PEP phosphoenolpyruvate - PFK phospho-fructokinase - PFP pyrophospate: fructose-6-phosphate-phosphotransferase - 3-PGA glycerate-3-phospate - P_i inorganic phosphate - Ru1,5bisP ribulose 1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - triose-phosphates sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate This research was supported by the Deutsche Forschungsgemeinschaft (SFB 137).     

Increased activity of the tandem fructose-1,6-bisphosphate aldolase,triosephosphate isomerase and fructose-1,6-bisphosphatase enzymes in <Emphasis Type="Italic">Anabaena</Emphasis> sp. strain PCC 7120 stimulates photosynthetic yield

The regulation of photosynthetic yield at the genetic level has largely focused on manipulation of the catalytic enzymes in the Calvin cycle by genetic engineering. In order to investigate the contribution of increased enzymatic activity in the Calvin cycle on photosynthetic yield, the rice fructose-1,6-bisphosphate aldolase (FBA), spinach triosephosphate isomerase (TPI) and wheat fructose-1,6-bisphosphatase (FBPase) genes were cloned in tandem and co-overexpressed in cyanobacterium Anabaena sp. strain PCC 7120 cells. The enzymatic activities of FBA, TPI and FBPase, as well as sedoheptulose-1,7-bisphosphatase (SBPase), were remarkably increased in transgenic cells relative to the wild-type. The photosynthetic yield, as reflected by photosynthetic O₂ evolution and dry cellular weight, was also markedly increased in transgenic cells versus wide-type cells. The activity of SBPase is considered the most important factor for ribulose-1,5-bisphosphate (RuBP) regeneration in the Calvin cycle, and increased activity of TPI alone in transgenic cells does not stimulate photosynthetic yield. Thus, the increased activity of FBA and FBPase, but not TPI, significantly improved photosynthetic yield in transgenic cells by stimulating SBPase activity and consequently accelerating the RuBP regeneration rate.     

Short-term water stress leads to a stimulation of sucrose synthesis by activating sucrose-phosphate synthase

The aim of this work was to identify which aspects of photosynthetic metabolism respond most sensitively to leaf water deficit. Spinach (Spinacia oleracea L.) leaf discs were floated on sorbitol concentrations of increasing molarity and changes of the protoplast volume were estimated using [¹⁴C]sorbitol and ³H₂O penetration. Detached leaves were also wilted until 10% of their fresh weight was lost. Photosynthesis was studied at very high external CO₂ concentrations, to eliminate the effect of closing stomata. There was no large inhibition of CO₂ fixation after wilting leaves, or until the external water deficit was greater than-1.2 MPa. However, partitioning changed markedly at these moderate water deficits: more sucrose and less starch was made. When an inhibition of CO₂-saturated photosynthesis did appear at a water deficit of-2.0 MPa and above, measurements of chlorophyll-fluorescence quenching and metabolite levels showed the thylakoid reactions were not especially susceptible to short-term water stress. The inhibition was accompanied by a small increase of the triose phosphate: ribulose-1,5-bisphosphate ratio, showing regeneration of ribulose-1,5-bisphosphate was affected. However, there was also a general increase of the estimated concentrations of most metabolites, indicating that there is no specific site for the inhibition of photosynthesis. Increasing water deficit led to a large increase of fructose-2,6-bisphosphate. This is explained in terms of a simultaneous increase of fructose-6-phosphate and inorganic phosphate as the cell shrinks. The high fructose-2,6-bisphosphate led to the accumulation of triose phosphates, and the potential significance of this for protection against photoinhibition is discussed. There was an increase in the extractable activity of sucrose-phosphate synthase. This was only detected when the enzyme was assayed in conditions which distinguish between different kinetic forms which have previously been identified in spinach leaves. It is proposed that activation of sucrose-phosphate synthase is one of the first sites at which spinach leaves respond to a rising water deficit. This could be of importance for osmoregulation.Abbreviations Chl chlorophyll - Fru1,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA glycerate-3-phosphate - Pi inorgamic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - SPS sucrose-phosphate synthase - triose-P sum of glyceraldehyde-3-phosphate and dehydroxyacetone phosphate - UDPGlc uridine diphosphoglucose     

Limitation of CO(2) Assimilation and Regulation of Benson-Calvin Cycle Activity in Barley Leaves in Response to Changes in Irradiance, Photoinhibition, and Recovery

The response of the Benson-Calvin cycle to changes in irradiance and photoinhibition was measured in low-light grown barley (Hordeum vulgare) leaves. Upon the transition from the growth irradiance (280 micromoles per square meter per second) to a high photoinhibitory irradiance (1400 micromoles per square meter per second), the CO₂ assimilation rate of the leaves doubled within minutes but high irradiance rapidly caused a reduction in quantum efficiency. Following exposure to high light the activities of NADP-malate dehydrogenase and fructose-1,6-bisphosphatase obtained near maximum values and the activation state of ribulose-1,5-bisphosphate carboxylase increased. The activity of the latter remained constant throughout the period of photoinhibitory irradiance, but the increase in the activities of fructose-1,6-bisphosphatase and NADP-malate dehydrogenase was transient decreasing once more to much lower values. This suggests that immediately following the transition to high light reduction and activation of redox-modulated enzymes occurred, but then the stroma became relatively oxidized as a result of photoinhibition. The leaf contents of glucose 6-phosphate and fructose 6-phosphate increased following exposure to high light but subsequently decreased, suggesting that following photoinhibition sucrose synthesis exceeded the rate of carbon assimilation. The ATP content attained a constant value much higher than that in low light. During photoinhibition the glycerate 3-phosphate content greatly increased while ribulose-1,5-bisphosphate decreased. The fructose-1,6-bisphosphate and triose phosphate contents increased initially and then remained constant. During photoinhibition CO₂ assimilation was not limited by ribulose-1,5-bisphosphate carboxylase activity but rather by the regeneration of the substrate, ribulose-1,5-bisphosphate, related to a restriction on the supply of reducing equivalents.     

Perturbation of photosynthesis in spinach leaf discs by low concentrations of methyl viologen

Spinach leaf discs were floated on methyl-viologen solutions (5–200 nmol·l^-1) and the effect on photosynthetic metabolism was then investigated under conditions of saturating CO₂. Methyl viologen led to increased non-photochemical quenching, and the ATP/ADP ratio increased from <2 to >10. Comparison of the apparent quantum yield and non-photochemical quenching indicated that these concentrations of methyl viologen were only catalysing a marginal electron flux, and that the decrease in quantum yield was mainly the result of pH-triggered energy dissipation. Similar changes were also obtained after supplying tentoxin to inhibit the chloroplast ATP synthase and increase the energisation of the thylakoids. The photosystem-II acceptor, Q_A, was monitored by photochemical fluorescence quenching, and became more reduced. In contrast, the activation of NADP-malate dehydrogenase decreased, showing that the acceptor side of photosystem I becomes more oxidised. Similar changes were observed after supplying tentoxin. It is concluded that increased thylakoid energisation can lead to a substantial restriction of linear electron transport. Analysis of metabolite levels showed that glycerate-3-phosphate reduction was imporved, but that there was a large accumulation of triose phosphates and fructose-1,6-bisphosphate. This is the consequence of an inhibition of the regeneration of ribulose-1,5-bisphosphate, caused by inactivation of the stromal fructose-1,6-bisphosphatase and, to a lesser extent, phosphoribulokinase. Methyl viologen also led to inactivation of sucrose-phosphate synthase, and abolished the response of fructose-2,6-bisphosphate to rising rates of photosynthesis. This provides evidence for a primary role of glycerate-3-phosphate in controlling the activity of fructose-6-phosphate, 2-kinase and, thence, the fructose-2,6-bisphosphate concentration as the rate of photosynthesis increases. It is concluded that the very moderate ATP/ADP ratios found in chloroplasts are the results of constraints on the operation of ATP synthase. They can be increased if the thylakoid energisation is increased. However, the increased energisation acts directly or indirectly to disrupt many other aspects of photosynthetic metabolism including linear electron transport, activation of the Calvin cycle, and the control of sucrose and starch synthesis.Abbreviations and symbols Frul,6P₂ (Fru1,6Pase) fructose-1,6-bisphosphate(ase) - Fru2,6P, (Fru2,6Pase) fructose-2,6-bisphosphate(-ase) - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - Pi inorganic phosphate - PSI and PSII photosystems I and II - qE high energy' quenching of chlorophyll fluorescence - PGA glycerate-3-phosphate - Q_A primary stable acceptor of PSII - Ru5P (Ru1,5P₂) ribulose-5-phosphate (-1,5-bisphosphate) - SPS sucrose-phosphate synthase - triose P dihydroxyacetone phosphate plus glyceraldehyde-3-phosphate - _s apparent quantum yield Dedicated to Professor E. Latzko on the occasion of his 65th birthday