Effect of Preparations Exhibiting Cytokinin-like Activity on the Specific Density of Leaf in Grasses

The effects of synthetic preparations exhibiting cytokinin-like activity (6-benzylaminopurine, Thidiazuron, and kartolin-2) on the specific leaf area (SLA) were studied in plants of the family Gramineae (wheat, Triticum aestivum L.; meadow fescue, Festuca pratensis Huds.; and reed fescue, F. arindinacea Schreb.). At the early stages of ontogeny (until the leaf area reached 50–60% of the maximum value), treatment of plants of the three species with cytokinin-like preparations caused an increase in SLA. The SLA value in these plants was correlated with the rate of photosynthetic assimilation of carbon dioxide and activities of carbon metabolism enzymes: ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), NAD-malate dehydrogenase (EC 1.1.1.37), and NADP-glyceraldehydephosphate dehydrogenase complex, which includes phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehydephosphate dehydrogenase (EC 1.2.1.13). However, there was no correlation of SLA with the activity of phospho(enol)pyruvate carboxylase (EC 4.1.1.31), an anaplerotic carboxylation enzyme of grasses. SLA is suggested to reflect the state and activity of the photosynthetic apparatus and can be recommended as a characteristic of photosynthesis variability (e.g., caused by cytokinin-like preparations).     

A Chlamydomonas reinhardii mutant with catalytically and structurally altered ribulose-5-phosphate kinase

The biochemical lesion in a light-sensitive, acetate-requiring Chlamydomonas mutant was identified. This strain, designated rpk, exhibited photosynthetic rates less than 3% of the wild-type. Analysis of photosynthetic products by high-performance liquid chromatography demonstrated an accumulation of ¹⁴C label in pentose and hexose monophosphates. After 1 min of photosynthesis in ¹⁴CO₂ these intermediates comprised 27.5% of the label in the mutant compared with 8% in the wild-type. The mutant pheno-type was caused by a 20-fold reduction in ribulose-5-phosphate (Ru5P)-kinase (EC 2.7.1.19) activity. The mutant exhibited wild-type levels of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) and transketolase (EC 2.2.1.1) indicating that the mutation specifically affected Ru5P kinase. In a cross of the mutant with the wild-type, tetrad progeny segregated in a Mendelian fashion (1:1) and light-sensitivity cosegregated with reduced Ru5P-kinase activity and an acetate requirement for growth. Almost normal levels of Ru5P-kinase protein were detected in the mutant by probing nitrocellulose replicas of sodium dodecylsulfate-polyacrylamide gels with anti-Ru5P-kinase antibody. The subunit size of the mutant enzyme, 42 kDa, was identical to that of the wild-type. Isoelectric focusing of the native protein determined that the mutant protein was altered, exhibiting a more acidic isoelectric point than the wild-type protein. Thus, the molecular basis for the lesion affecting Ru5P-kinase activity in mutant rpk is a charge alteration which results in a partially impaired enzyme.Abbreviations Chl chlorophyll - Da dalton - FCCP carbonylcyanide-p-trifluorophenylhydrazone - RuBP ribulose-1,5-bisphosphate - Ru5P ribulose-5-phosphate     

Effect of preparations exhibiting cytokinin-like activity on the specific density of leaf in grasses

The effects of synthetic preparations exhibiting cytokinin-like activity (6-benzylaminopurine, Thidiazuron, and kartolin-2) on the specific leaf area (SLA) were studied in plants of the family Gramineae (wheat, Triticum aestivum L.; meadow fescue, Festuca pratensis Huds.; and reed fescue, F. arindinacea Schreb.). At the early stages of ontogeny (until the leaf area reached 50-60% of the maximum value), treatment of plants of the three species with cytokinin-like preparations caused an increase in SLA. The SLA value in these plants was correlated with the rate of photosynthetic assimilation of carbon dioxide and activities of carbon metabolism enzymes: ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), NAD-malate dehydrogenase (EC 1.1.1.37), and NADP-glyceraldehydrophosphate dehydrogenase complex, which includes phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehydrophosphate dehydrogenase (EC 1.2.1.13). However, there was no correlation of SLA with the activity of phospho(enol)pyruvate carboxylase (EC 4.1.1.31), an anaplerotic carboxylation enzyme of grasses. SLA is suggested to reflect the state and activity of the photosynthetic apparatus and can be recommended as a characteristic of photosynthesis variability (e.g., caused by cytokinin-like preparations).     

Carbon assimilation by different developmental stages of Laminaria saccharina

Various stages of the life cycle of the marine brown alga Laminaria saccharina (L.) Lamour. (Laminariales, Phaeophyta) including male and female gametophytes, female gametes, zygotes and young sporophytes of different age were investigated for their potentials of carbon dioxide (¹⁴CO₂) fixation. Rates of photosynthesis attain the same order of magnitude in all stages. Photosynthetic ¹⁴CO₂-fixation is accompanied by a substantial light independent carbon assimilation. This is confirmed by rate determinations of the equivalent carboxylating enzymes present in the plants, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxokinase (EC 4.1.1.32) as well as by chromatographic analyses of the appropriate [¹⁴C]-assimilate patterns.Abbreviations RuBP-C ribulose-1,5-bisphosphate carboxylase - PEP-CK phosphoenolpyruvate carboxykinase - PEP phosphoenolpyruvate - PS photosynthesis - DF dark fixation     

“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     

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     

Antisense RNA Inhibition of RbcS Gene Expression Reduces Rubisco Level and Photosynthesis in the C4 Plant Flaveria bidentis

The C4 dicot Flaveria bidentis was genetically transformed with an antisense RNA construct targeted to the nuclear-encoded gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; RbcS). RbcS mRNA levels in leaves of transformants were reduced by as much as 80% compared to wild-type levels, and extractable enzyme activity was reduced by up to 85%. There was no significant effect of transformation with the gene construct on levels of other photosynthetic enzymes. Antisense transformants with reduced Rubisco activity exhibited a stunted phenotype. Rates of photosynthesis were reduced in air at high light and over a range of CO2 concentrations but were unaffected at low light. From these results we conclude that, as is the case in C3 plants, Rubisco activity is a major determinant of photosynthetic flux in C4 plants under high light intensities and air levels of CO2.     

Ribulose-1,5-bisphosphate carboxylase-deficient plastome mutants of Oenothera

In spite of only slightly subnormal pigment contents, two plastome mutants of Oenothera (Vα, Iσ) were practically incapable of photosynthetic CO₂ fixation and another one exhibited considerably reduced photosynthesis (IVβ). While other photosynthetic enzymes were present as far as investigated, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity was very low or missing altogether. As shown by gel electrophoresis, mutant IVβ contained some, though little, fraction I protein. In the other two mutants fraction I protein could not be detected. Also, neither the small nor the large subunit of ribulose-1,5-bisphosphate carboxylase could be found in these mutants. In immunodiffusion experiments with a monospecific antiserum against rye ribulose-1,5-bisphosphate carboxylase, only extracts from wild-type Oenothera produced visible precipitation lines. Still, the presence of very low levels of immunochemically reactive antigen was indicated for all three mutants. The highest level was observed in mutant IVβ. The behaviour of the mutant extracts suggested that the antigens of mutant and wild type leaves reacting with the antiserum were not identical. All mutants appeared to have a coupled electron transport system as shown by ATP measurements, light scattering and 515 nm absorption changes. Linear electron transport was possible in the mutants. Still, the photoresponse of cytochrome f and fluorescence measurements suggested altered electron transport properties in the mutants. These are interpreted to be secondary lesions of the photosynthetic apparatus caused by primary deficiency in ribulose-1,5-bisphosphate carboxylase activity. From the absence in two mutants (Vα, Iσ) of the small subunit of ribulose-1,5-bisphosphate carboxylase, which is known to be coded for by nuclear DNA and to be synthesized on cytoplasmic ribosomes, it appears that the genetic system of the plastids is capable of interfering with the genome-controlled synthesis of plastid components.     

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)     

Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase efficiency by the presence of sucrose during the tissue culture of strawberry plantlets

Summary In order to identify the physiological and biochemical events leading to the negative effects of the presence of sucrose in culture medium on the photosynthetic capacity of plantlets cultivated in vitro, time course in photosynthesis, metabolite pool sizes, and ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity were investigated in strawberry (Fragaria x ananassa Duch. cv. Kent) plantlets following their transfer to medium with or without sucrose. When the plantlets grown in medium without sucrose were transferred to a similar medium with 30 g liter⁻¹ sucrose, their net photosynthesis decreased and their level of phosphorylated compounds increased with time. In addition, initial catalytic turnover, total catalytic turnover, and the activation state of ribulose-1,5-bisphosphate carboxylase decreased in these plantlets. Conversely, when the plantlets grown in medium with 30 g liter⁻¹ sucrose were transferred to a similar medium without sucrose, their net photosynthesis slowly increased with time and their level of phosphorylated compounds slowly decreased. A slow increase with time of initial catalytic turnover, total catalytic turnover, and the activation state of ribulose-1,5-bisphosphate carboxylase was also observed in these plantlets. The results of the present paper suggest that the reduced photosynthetic capacity of strawberry plantlets cultivated in vitro in the presence of sucrose is the consequence of a reduction in the efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase due to its deactivation and the possible presence of putative inhibitors of carboxylation sites.     

C4 Photosynthesis (The Effects of Leaf Development on the CO2-Concentrating Mechanism and Photorespiration in Maize)

The effect of O2 on photosynthesis was determined in maize (Zea mays) leaves at different developmental stages. The optimum level of O2 for maximum photosynthetic rates was lower in young and senescing tissues (2-5 kPa) than in mature tissue (9 kPa). Inhibition of photosynthesis by suboptimal levels of O2 may be due to a requirement for functional mitochondria or to cyclic/pseudocyclic photophosphorylation in chloroplasts; inhibition by supraoptimal levels of O2 is considered to be due to photorespiration. Analysis of a range of developmental stages (along the leaf blade and at different leaf ages and positions) showed that the degree of inhibition of photosynthesis by supraoptimal levels of O2 increased rapidly once the ribulose-1,5-bisphosphate carboxylase/oxygenase and chlorophyll contents were below a critical level and was similar to that of C3 plants. Tissue having a high sensitivity of photosynthesis to O2 may be less effective in concentrating CO2 in the bundle sheath cells due either to limited function of the C4 cycle or to higher bundle sheath conductance to CO2. An analysis based on the kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase was used to predict the maximum CO2 level concentrated in bundle sheath cells at a given degree of inhibition of photosynthesis by supraoptimal levels of O2.     

Ribulose-1,5-biphosphate carboxylase-deficient plastome mutants of Oenothera.

In spite of only slightly subnormal pigment contents, two plastome mutants of Oenothera (Valpha, Isigma) were practically incapable of photosynthetic CO2 fixation and another one exhibited considerably reduced photosynthesis (IVbeta). While other photosynthetic enzymes were present as far as investigated, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity was very low or missing altogether. As shown by gel electrophoresis, mutant IVbeta contained some, though little, fraction I protein. In the other two mutants fraction I protein could not be detected. Also, neither the small nor the large subunit of ribulose-1,5-biphosphate carboxylase could be found in these mutants. In immunodiffusion experiments with a monospecific antiserum against rye ribulose-1,5-bisphosphate carboxylase, only extracts from wild-type Oenothera produced visible precipitation lines. Still, the presence of very low levels of immunochemically reactive antigen was indicated for all three mutants. The highest level was observed in mutant IVbeta. The behaviour of the mutant extracts suggested that the antigens of mutant and wild type leaves reacting with the antiserum were not identical. All mutants appeared to have a coupled electron transport system as shown by ATP measurements, light scattering and 515 nm absorption changes. Linear electron transport was possible in the mutants. Still, the photoresponse of cytochrome f and fluorescence measurements suggested altered electron transport properties in the mutants. These are interpreted to be secondary lesions of the photosynthetic apparatus caused by primary deficiency in ribulose-1,5-bisphosphate carboxylase activity. From the absence in two mutants (Valpha, Isigna) of the small subunit of ribulose-1,5-bisphosphate carboxylase, which is known to be coded for by nuclear DNA and to be synthesized on cytoplasmic ribosomes, it appears that the genetic system of the plastids is capable of interfering with the genome-controlled synthesis of plastid components.     

Effect of inorganic carbon supply on the photosynthetic physiology of Gracilaria tenuistipitata

Gracilaria tenuistipitata Zhang et Xia was cultured for 15 d at low, normal and high inorganic carbon concentrations under constant light, temperature and nutrient conditons. Carbonic anhydrase (CA; EC 4.2.1.1.) activity, ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco; EC 4.1.1.39) content, pigment content and C/N ratio were measured, and also the photosynthesis and growth rates. Both Rubisco content and CA activity increased under conditions of low inorganic carbon (C_i) but decreased at high C_i with respect to the control. The amount of pigments declined considerably at high C_i and was slightly higher at low C_i. The maximum rate of photosynthesis and the photosynthetic efficiency increased in low C_i and the opposite was found at high C_i concentration. The effects of C_i concentration on maximum rate of photosynthesis and photosynthetic efficiency are discussed in relation to the variation in pigment and Rubisco contents and CA activity. The data indicate that C_i may be an important factor controlling the photosynthetic physiology of G. tenuistipitata with regard, not only to the enzymes of C_i metabolism, but also to the pigment content.Abbreviations APS_max maximum apparent photosynthetic rate - CA carbonic anhydrase - Chl chlorophyll - C_i inorganic carbon - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work has been supported by grants No. PB91-0962 and No. MAR90-0365 from Spanish Direction for Science and Technology (DIGICYT). M.J. G-S holds a fellowship from the DIGICYT.     

Differences between maize and rice in N-use efficiency for photosynthesis and protein allocation

The N-use efficiency for photosynthesis was higher in a C(4) plant, maize, than in a C(3) plant, rice, including rbcS antisense rice with optimal ribulose-1,5-bisphosphate carboxylase (Rubisco) content for CO(2)-saturated photosynthesis, even when photosynthesis was measured under saturating CO(2) conditions. The N cost for the C(4) cycle enzymes in maize was not large, and the lower amount of Rubisco allowed a greater N investment in the thylakoid components. This greater content of the thylakoid components as well as the CO(2) concentrating mechanism may support higher N-use efficiency for photosynthesis in maize.     

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).     

Effects of a Short-Term Shift to Low Temperature and of Long-Term Cold Hardening on Photosynthesis and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Sucrose Phosphate Synthase Activity in Leaves of Winter Rye (Secale cereale L.)

The effect of a short-term (hours) shift to low temperature (5[deg]C) and long-term (months) cold hardening on photosynthesis and carbon metabolism was studied in winter rye (Secale cereale L. cv Musketeer). Cold-hardened plants grown at 5[deg]C exhibited 25% higher in situ CO2 exchange rates than nonhardened plants grown at 24[deg]C. Cold-hardened plants maintained these high rates throughout the day, in contrast to nonhardened plants, which showed a gradual decline in photosynthesis after 3 h. Associated with the increase in photosynthetic capacity following cold hardening was an increase in ribulose-1,5-bisphosphate carboxylase/oxygenase and sucrose phosphate synthase activity and 3- to 4-fold increases in the pools of associated metabolites. Leaves of nonhardened plants shifted overnight to 5[deg]C required 9 h in the light at 5[deg]C before maximum rates of photosynthesis were reached. The gradual increase in photosynthesis in leaves shifted to 5[deg]C was correlated with a sharp decline in the 3-phosphoglycerate/triose phosphate ratio and by an increase in the ribulose bisphosphate/3-phosphoglycerate ratio, indicating the gradual easing of aninorganic phosphate-mediated feedback inhibition on photo-synthesis. We suggest that the strong recovery of photosynthesis in winter rye following cold hardening indicates that the buildup of photosynthetic enzymes, as well as those involved in sucrose synthesis, is an adaptive response that enables these plants to maximize the production of sugars that have both cryoprotective and storage functions that are critical to the performance of these cultivars during over-wintering.     

Phosphorylation in vitro of the large subunit of the ribulose-1,5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase

A protein kinase activity responsible for the in vitro phosphorylation of at least six endogenous polypeptides including the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is present in the stroma (3000 X g supernatant, S30) of spinach chloroplasts. The phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is strongly enhanced when sodium fluorure is used as a protein phosphatase inhibitor. Phosphorylation occurs on threonine and serine residues. The protein kinase involved is not Ca2+-dependent. There is also evidence for a protein phosphatase activity which suggests a coupled regulation by a phosphorylation-dephosphorylation process. The phosphorylating activity is drastically reduced when S30 is prepared from leaves harvested after a dark period. Phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is not related to its own synthesis. The in vitro phosphorylation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) is also demonstrated.     

The role of enzyme activation state in limiting carbon assimilation under variable light conditions

The mechanisms regulating transient photosynthesis by soybean (Glycine max) leaves were examined by comparing photosynthetic rates and carbon reduction cycle enzyme activities under flashing (saturating 1 s lightflecks separated by low photon flux density (PFD) periods of different durations) and continuous PFD. At the same mean PFD, the mean photosynthetic rates were reduced under flashing as compared to continuous light. However, as the duration of the low PFD period lengthened, the CO₂ assimilation attributable to a lightfleck increased. This enhanced lightfleck CO₂ assimilation was accounted for by a greater postillumination CO₂ fixation occurring after the lightfleck. The induction state of photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fructose 1,6-bisphosphatase (FBPase) and ribulose 5-phosphate kinase (Ru5P kinase) activities all responded similarly and were all lower under flashing as compared to constant PFD of the same integrated mean value. However, the fast phase of induction and FBPase and Ru5P kinase activities were reduced more than were the slow phase of induction and rubisco activity. This was consistent with the role of the former enzymes in the fast induction component that limited RuBP regeneration. Competition for reducing power between carbon metabolism and thioredoxin-mediated enzyme activation may have resulted in lower enzyme activation states and hence lower induction states under flashing than continuous PFD, especially at low lightfleck frequencies (low mean PFD).Abbreviations FBPase fructose 1,6-bisphosphatase (EC 3.1.3.11) - LUE lightfleck use efficiency - P-glycerate 3-phosphoglycerate - PICF post-illumination CO₂ fixation - Ru5P kinase ribulose 5-phosphate kinase (EC 2.7.1.19) - RuBP ribulose 1,5-bisphosphate - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - SBpase sedoheptulose 1,7-bisphosphatase (EC 3.1.3.37)     

Differential sensitivity of stomatal and non-stomatal components to NaCl or Na2SO4 salinity in horsegram, Macrotyloma uniflorum (Lam.)

14CO2 assimilation rate (P), leaf diffusive conductance (gs), photosynthetic electron flow, and activities of enzymes of Calvin cycle were studied in a horsegram [Macrotyloma uniflorum (Lam.)] in response to salinity induced by NaCl or Na2SO4. A significant reduction in P and gs by both salt treatments was registered. Na2SO4 caused a greater reduction in gs than the NaCl salinity. Studies with isolated chloroplasts confirmed a greater sensitivity to NaCl than to Na2SO4. Salinity inhibited the photosynthetic electron transport. The activity of ribulose-1,5-bisphosphate carboxylase (E.C.4.1.1.39) was under salinity inhibited more than the activities of other three enzymes of the Calvin cycle, ribulose-5-phosphate kinase (E.C.2.7.1.19), ribose-5-phosphate isomerase (E.C.5.3.16), and NADP-glyceraldehyde-3-phosphate dehydrogenase (E.C.1.2.13). These inhibitions lead to a reduced capacity for ribulose-1,5-bisphosphate regeneration. Isolated chloroplasts extracted from salt stressed plants and supplemented with the substrates of Calvin cycle could elevate P, but the P was always lower than in the controls. Decreased P in horsegram exposed to high salinity can be attributed to both stomatal and non-stomatal components, however, the sensitivity to the salt source, NaCl or Na2SO4, was different.     

Activity of Carbon Metabolism Enzymes in Wheat Plants Treated with Kartolin-4 and Exposed to Water Stress

Enzymatic activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39), phospho(enol)pyruvate carboxylase (EC 4.1.1.31), NAD malate dehydrogenase (EC 1.1.1.37), and NADP glyceraldehyde phosphate dehydrogenase complex including phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.13) were comparatively assayed in wheat seedlings of the cultivar Lyutestsens 758 grown under normal conditions, water deficiency conditions, and subsequent rehydration. Water stress was found to decrease the activity of all enzymes tested, the effect being most pronounced in the case of Rubisco. The content of Rubisco in wheat plants exposed to water deficiency was reduced less significantly than the activity of the enzyme. Pretreatment of plant seeds with kartolin-4 (o-isopropyl-N-2-hydroxyethyl carbamate), a preparation with cytokinin activity, reduced the dehydration-induced inhibition of enzymatic activity. Upon a subsequent rehydration, kartolin-4 facilitated rapid recovery of the photosynthetic activity, the process being based on the kartolin-induced stimulation of reparation reactions. Under conditions of water stress, a partial decrease in the activity of carbon metabolism enzymes in vitrowas accompanied by complete inhibition of photosynthesis in vivo, perhaps, as a result of an abrupt increase in the stomatal resistance.