Transgenic Arabidopsis plants with decreased activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase have altered carbon partitioning

The role of fructose-2,6-bisphosphate (Fru-2,6-P(2)) as a regulatory metabolite in photosynthetic carbohydrate metabolism was studied in transgenic Arabidopsis plants with reduced activity of Fru-6-phosphate,2-kinase/Fru-2,6-bisphosphatase. A positive correlation was observed between the Fru-6-phosphate,2-kinase activity and the level of Fru-2,6-P(2) in the leaves. The partitioning of carbon was studied by (14)CO(2) labeling of photosynthetic products. Plant lines with Fru-2,6-P(2) levels down to 5% of the levels observed in wild-type (WT) plants had significantly altered partitioning of carbon between sucrose (Suc) versus starch. The ratio of (14)C incorporated into Suc and starch increased 2- to 3-fold in the plants with low levels of Fru-2,6-P(2) compared with WT. Transgenic plant lines with intermediate levels of Fru-2,6-P(2) compared with WT had a Suc-to-starch labeling ratio similar to the WT. Levels of sugars, starch, and phosphorylated intermediates in leaves were followed during the diurnal cycle. Plants with low levels of Fru-2,6-P(2) in leaves had high levels of Suc, glucose, and Fru and low levels of triose phosphates and glucose-1-P during the light period compared with WT. During the dark period these differences were eliminated. Our data provide direct evidence that Fru-2,6-P(2) affects photosynthetic carbon partitioning in Arabidopsis. Opposed to this, Fru-2,6-P(2) does not contribute significantly to regulation of metabolite levels in darkness.     

RNA interference-mediated repression of sucrose-phosphatase in transgenic potato tubers (Solanum tuberosum) strongly affects the hexose-to-sucrose ratio upon cold storage with only minor effects on total soluble carbohydrate accumulation

Storage of potato tubers at low temperatures leads to the accumulation of glucose and fructose in a process called 'cold sweetening'. The aim of this work was to investigate the role of sucrose-phosphatase (SPP) in potato tuber carbohydrate metabolism at low temperature (4 degrees C). To this end, RNA interference (RNAi) was used to reduce SPP expression in transgenic potato tubers. Analysis of SPP specific small interfering RNAs (siRNAs), SPP protein accumulation and enzyme activity indicated that SPP silencing in transgenic tubers was stable during the cold treatment. Analysis of soluble carbohydrates showed that in transgenic tubers, cold-induced hexogenesis was inhibited while, despite strongly reduced SPP activity, sucrose levels exceeded wild-type (WT) values four- to fivefold after 34 d of cold treatment. This led to a drastic change in the hexose-to-sucrose ratio from 1.9 in WT tubers to 0.15 to 0.11 in transgenic tubers, while the total amount of soluble sugars was largely unchanged in both genotypes. Sucrose-6(F)-phosphate (Suc6P), the substrate of SPP, accumulated in transgenic tubers in the cold which most likely enables the residual enzyme to operate with maximal catalytic activity in vivo and thus, in the long term, counterbalances reduced SPP activity in the transformants. Northern analysis revealed that cold-induced expression of vacuolar invertase (VI) was blocked in SPP-silenced tubers explaining a reduced sucrose-to-hexose conversion. Suc6P levels were found to negatively correlate with VI expression. A possible role of Suc6P in regulating VI expression is discussed.     

Expression of Arabidopsis plastidial phosphoglucomutase in tobacco stimulates photosynthetic carbon flow into starch synthesis

Phosphoglucomutase (PGM, EC 2.7.5.1) is one of the enzymes constituting the carbohydrate synthesis pathway in higher plants. It catalyzes the reversible conversion of glucose 6-phosphate (Glc6P) to glucose 1-phosphate (Glc1P). Previously, metabolic turnover analysis using (13)CO(2) in tobacco leaves demonstrated that conversion of Glc6P to Glc1P may limit carbon flow into carbohydrate synthesis. In order to assess the effects of PGM, Arabidopsis thaliana cytosolic or plastidial PGM was expressed under the control of cauliflower mosaic virus 35S promoter in tobacco plants (Nicotiana tabacum cv. Xanthi) and phenotypic analysis was performed. The transgenic plants expressing Arabidopsis plastidial PGM showed 3.5-8.2-fold higher PGM activity than that of wild-type, and leaf starch and sucrose contents increased 2.3-3.2-fold and 1.3-1.4-fold, respectively over wild-type levels. In vivo(13)C-labeling experiments indicated that photosynthetically fixed carbon in the transgenic plants could be converted faster to Glc1P and adenosine 5'-diphosphate glucose than in wild-type, suggesting that elevation of plastidial PGM activity should accelerate conversion of Glc6P to Glc1P in chloroplasts and increase carbon flow into starch. On the other hand, transgenic plants expressing Arabidopsis cytosolic PGM showed a 2.1-3.4-fold increase in PGM activity over wild-type and a decrease of leaf starch content, but no change in sucrose content. These results suggest that plastidial PGM limits photosynthetic carbon flow into starch.     

Cell wall-bound invertase limits sucrose export and is involved in symptom development and inhibition of photosynthesis during compatible interaction between tomato and Xanthomonas campestris pv vesicatoria

Cell wall-bound invertase (cw-Inv) plays an important role in carbohydrate partitioning and regulation of sink-source interaction. There is increasing evidence that pathogens interfere with sink-source interaction, and induction of cw-Inv activity has frequently been shown in response to pathogen infection. To investigate the role of cw-Inv, transgenic tomato (Solanum lycopersicum) plants silenced for the major leaf cw-Inv isoforms were generated and analyzed during normal growth and during the compatible interaction with Xanthomonas campestris pv vesicatoria. Under normal growth conditions, activities of sucrolytic enzymes as well as photosynthesis and respiration were unaltered in the transgenic plants compared with wild-type plants. However, starch levels of source leaves were strongly reduced, which was most likely caused by an enhanced sucrose exudation rate. Following X. campestris pv vesicatoria infection, cw-Inv-silenced plants showed an increased sucrose to hexose ratio in the apoplast of leaves. Symptom development, inhibition of photosynthesis, and expression of photosynthetic genes were clearly delayed in transgenic plants compared with wild-type plants. In addition, induction of senescence-associated and pathogenesis-related genes observed in infected wild-type plants was abolished in cw-Inv-silenced tomato lines. These changes were not associated with decreased bacterial growth. In conclusion, cw-Inv restricts carbon export from source leaves and regulates the sucrose to hexose ratio in the apoplast. Furthermore, an increased apoplastic hexose to sucrose ratio can be linked to inhibition of photosynthesis and induction of pathogenesis-related gene expression but does not significantly influence bacterial growth. Indirectly, bacteria may benefit from low invertase activity, since the longevity of host cells is raised and basal defense might be dampened.     

Leaflet photosynthesis rate and carbon metabolite accumulation patterns in nitrogen-limited, vegetative soybean plants

Prolonged inorganic nitrogen (NO₃ ^–+NH₄ ⁺) limitation of non-N₂-fixing soybean plants affected leaflet photosynthesis rates, photosynthate accumulation rates and levels, and anaplerotic carbon metabolite levels. Leaflets of nitrogen-limited (N-Lim), 27–31-day-old plants displayed 15 to 23% lower photosynthesis rates than leaflets of nitrogen-sufficient (N-Suff) plants. In contrast, N-Lim plant leaflets displayed higher sucrose and starch levels and rates of accumulation, as well as higher levels of carbon metabolites associated with sucrose and starch synthesis, e. g., glycerate-3-phosphate and glucose phosphates, than N-Suff plant leaflets. Concurrently, levels of soluble protein, chlorophyll, and anaplerotic metabolites, e.g., malate and phosphoenolpyruvate, were lower in leaflets of N-Lim plants than N-Suff plants, suggesting that the enzymes of the anaplerotic carbon metabolite pathway were lower in activity in N-Lim plant leaflets. Malate net accumulation rates in the earliest part of the illumination period were lower in N-Lim than in N-Suff plant leaflets; however, by the midday period, malate accumulation rate in N-Lim plant leaflets exceeded that in leaflets of N-Suff plants. Further, soluble protein accumulation rates in leaflets of N-Suff and N-Lim plants were similar, and the rate of dark respiration, measured in the early part of the dark period, was higher in N-Lim plant leaflets than in N-Suff plant leaflets. It was concluded that during prolonged N-limitation, foliar metabolite conditions favored the channelling of a large proportion of the carbon assimilate into sucrose and starch, while assimilate flow through the anaplerotic pathway was diminished. However, in some daytime periods, there was a normal level of carbon assimilate channelled through the anaplerotic pathway for ultimate use in amino acid and protein synthesis.Abbreviations ADPG-PPiase ADPglucose pyrophosphorylase - Ce CO₂ in the leaf photosynthesis measuring cuvette - Ci leaf internal CO₂ during photosynthesis measurement - Chl chlorophyll - DHAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - G_sw stomatal conductance with units as mmol H₂O m^–2 s^–1 - G1P glucose-1-phosphate - G6P glucose-6-phosphate - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphate - FBPase-pH 8.1 chloroplastic fructose-1,6-bisP (C-1) phosphatase (pH 8.1) - MAL malate - N inorganic nitrogen, i.e. NO₃ ^–+NH₄ ⁺ (at levels and molar ratios indicated) - PE post-emergence - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PGA 3-phosphoglycerate - PYR pyruvate - PYR kinase pyruvate kinase - Pn net CO₂ photoassimilation in leaves - PPFD photosynthetic photon flux density - PPRC pentose phosphate reductive cycle - RuBP ribulose-1,5-bisphosphate; rubisco-ribulose-1,5-bisphosphate carboxylase/oxygenase - SLW specific leaf mass - SPS sucrose-6-phosphate synthase - TCA cycle tricarboxylic acid cycle; triose-P-DAP+GAP     

The impact of reduced vacuolar invertase activity on the photosynthetic and carbohydrate metabolism of tomato

The impact of reduced vacuolar invertase activity on photosynthetic and carbohydrate metabolism was examined in tomato (Solanum lycopersicon L.). The introduction of a co-suppression construct (derived from tomato vacuolar invertase cDNA) produced plants containing a range of vacuolar invertase activities. In the leaves of most transgenic plants from line INV-B, vacuolar invertase activity was below the level of detection, whereas leaves from line INV-A and untransformed wild-type plants showed considerable variation. Apoplasmic invertase activity was not affected by the co-suppression construct. It has been suggested that, in leaves, vacuolar invertase activity regulates sucrose content and its availability for export, such that in plants with high vacuolar invertase activity a futile cycle of sucrose synthesis and degradation takes place. In INV-B plants with no detectable leaf vacuolar invertase activity, sucrose accumulated to much higher levels than in wild-type plants, and hexoses were barely detectable. There was a clear threshold relationship between invertase activity and sucrose content, and a linear relationship with hexose content. From these data the following conclusions can be drawn. (i) In INV-B plants sucrose enters the vacuole where it accumulates as hydrolysis cannot take place. (ii) There was not an excess of vacuolar invertase activity in the vacuole; the rate of sucrose hydrolysis depended upon the concentration of the enzyme. (iii) The rate of import of sucrose into the vacuole is also important in determining the rate of sucrose hydrolysis. The starch content of leaves was not significantly different in any of the plants examined. In tomato plants grown at high irradiance there was no impact of vacuolar invertase activity on the rate of photosynthesis or growth. The impact of the cosuppression construct on root vacuolar invertase activity and carbohydrate metabolism was less marked.Abbreviations CaMV Cauliflower Mosaic Virus - WT wild type     

Increase in the activity of fructose-1,6-bisphosphatase in cytosol affects sugar partitioning and increases the lateral shoots in tobacco plants at elevated CO<Subscript>2</Subscript> levels

We generated transgenic tobacco plants with high levels of fructose-1,6-bisphosphatase expressing cyanobacterialfructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol. At ambient CO₂ levels (360 ppm), growth, photosynthetic activity, and fresh weight were unchanged but the sucrose/hexose/starch ratio was slightly altered in the transgenic plants compared with wild-type plants. At elevated CO₂ levels (1200 ppm), lateral shoot, leaf number, and fresh weight were significantly increased in the transgenic plants. Photosynthetic activity was also increased. Hexose accumulated in the upper leaves in the wild-type plants, while sucrose and starch accumulated in the lower leaves and lateral shoots in the transgenic plants. These findings suggest that cytosolic fructose-1,6-bisphosphatase contributes to the efficient conversion of hexose into sucrose, and that the change in carbon partitioning affects photosynthetic capacity and morphogenesis at elevated CO₂ levels.     

Gas Exchange and Carbon Partitioning in the Leaves of Celery (Apium graveolens L.) at Various Levels of Root Zone Salinity

Both mannitol and sucrose (Suc) are primary photosynthetic products in celery (Apium graveolens L.). In other biological systems mannitol has been shown to serve as a compatible solute or osmoprotectant involved in stress tolerance. Although mannitol, like Suc, is translocated and serves as a reserve carbohydrate in celery, its role in stress tolerance has yet to be resolved. Mature celery plants exposed to low (25 mM NaCl), intermediate (100 mM NaCl), and high (300 mM NaCl) salinities displayed substantial salt tolerance. Shoot fresh weight was increased at low NaCl concentrations when compared with controls, and growth continued, although at slower rates, even after prolonged exposure to high salinities. Gas-exchange analyses showed that low NaCl levels had little or no effect on photosynthetic carbon assimilation (A), but at intermediate levels decreases in stomatal conductance limited A, and at the highest NaCl levels carboxylation capacity (as measured by analyses of the CO2 assimilation response to changing internal CO2 partial pressures) and electron transport (as indicated by fluorescence measurements) were the apparent prevailing limits to A. Increasing salinities up to 300 mM, however, increased mannitol accumulation and decreased Suc and starch pools in leaf tissues, e.g. the ratio of mannitol to Suc increased almost 10-fold. These changes were due in part to shifts in photosynthetic carbon partitioning (as measured by 14C labeling) from Suc into mannitol. Salt treatments increased the activity of mannose-6-phosphate reductase (M6PR), a key enzyme in mannitol biosynthesis, 6-fold in young leaves and 2-fold in fully expanded, mature leaves, but increases in M6PR protein were not apparent in the older leaves. Mannitol biosynthetic capacity (as measured by labeling rates) was maintained despite salt treatment, and relative partitioning into mannitol consequently increased despite decreased photosynthetic capacity. The results support a suggested role for mannitol accumulation in adaptation to and tolerance of salinity stress.     

Transgenic Arabidopsis plants expressing Escherichia coli pyrophosphatase display both altered carbon partitioning in their source leaves and reduced photosynthetic activity

The effects of the cytosolic expression of Escherichia coli pyrophosphatase (ppa) were investigated in the rosette leaves of transgenic Arabidopsis plants. During the daytime, glucose and fructose were found to accumulate at levels that were approximately two- to threefold higher in these plants than in the wild type. Interestingly, however, neither sucrose nor starch levels showed any distinctive build up in transgenic plants except under continuous white light growth conditions, during which they accumulated at high levels. Additionally, the leaves of transgenic Arabidopsis plants contain two- to threefold higher levels of inorganic phosphate (Pi) and two- to sixfold higher levels of uridine diphosphate-glucose than wild type plants during the diurnal cycle. In contrast, triose phosphate contents in the leaves of E. coli ppa transformants were either similar or slightly decreased when compared with wild type leaves. Furthermore, the photosynthetic activity of these transgenic plants was found to be reduced by 20–40% compared to normal levels. These results indicate that induction of ppa activity in the cytosol affects carbon partitioning between source and sink organs and also that the concomitant increase in Pi caused the accumulation of carbon metabolites and reduced photosynthetic activity.     

Carbon partitioning in leaves and tubers of transgenic potato plants with reduced activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase

The role of fructose-2,6-bisphosphate (Fru-2,6-P₂) in regulation of carbon metabolism was investigated in transgenic potato plants ( Solanum tuberosum L. cv Dianella) transformed with a vector containing a cDNA-sequence encoding fructose-6-phosphate,2-kinase (F6P,2-K, EC 2.7.1.105)/fructose-2,6-bisphosphatase (F26BPase, EC 3.1.3.46) in sense or antisense direction behind a CaMV 35S promoter. The activity of F6P,2-K in leaves was reduced to 5% of wild-type (WT) activity, and the level of Fru-2,6-P₂ was reduced both in leaves (10% of the WT level) and in tubers (40% of the WT level). Analysis of photosynthetic ¹⁴CO₂ metabolism, showed that in plant lines with reduced Fru-2,6-P₂ level the carbon partitioning in the leaves was changed in favour of sucrose biosynthesis, and the soluble sugars-to-starch labelling ratio was doubled. The levels of soluble sugars and hexose phosphates also increased in leaves of the transgenic plants. Most notably, the levels of hexoses were four- to six-fold increased in the transgenic plants. In tubers with reduced levels of Fru-2,6-P₂ only minor effects on carbohydrate levels were observed. Furthermore, carbon assimilation in tuber discs supplied with [U-¹⁴C]-sucrose showed only a moderate increase in labelling of hexoses and a decreased labelling of starch. Similar results were obtained using [U-¹⁴C]-glucose. No differences in growth of the transgenic lines and the WT were observed. Our data provide evidences that Fru-2,6-P₂ is an important factor in the regulation of photosynthetic carbon metabolism in potato leaves, whereas the direct influence of Fru-2,6-P₂ on tuber metabolism was limited.     

Photosynthetic Carbon Metabolism and Translocation in Wild-Type and Starch-Deficient Mutant Nicotiana sylvestris L

A high rate of daytime export of assimilated carbon from leaves of a starch-deficient mutant tobacco (Nicotiana sylvestris L.) was found to be a key factor that enabled shoots to grow at rates comparable to those in wild-type plants under a 14-h light period. Much of the newly fixed carbon that would be used for starch synthesis in leaves of wild-type plants was used instead for sucrose synthesis in the mutant. As a result, export doubled and accumulation of sucrose and hexoses increased markedly during the day in leaves of the mutant plants. The increased rate of export to sink leaves appeared to be responsible for the increase in the proportion of their growth that occurred during the day compared to wild-type plants. Daytime growth of source leaves also increased, presumably as a result of the increased accumulation of recently assimilated soluble carbon in the leaves. Even though starch accumulation did not occur in the leaves of mutant plants, nearly all the sugar that accumulated during the day was exported in the period of decreasing irradiance at the end of the diurnal light period. Changes in carbon allocation that occurred in leaves of wild-type and mutant plants near the end of the light period appeared to result from endogenous diurnal regulation associated with the day-night transition.     

Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants

By using barley seeds, developmental changes of ADPglucose (ADPG)-producing sucrose synthase (SS) and ADPG pyrophosphorylase (AGPase) have been compared with those of UDPglucose (UDPG), ADPG, sucrose (Suc) and starch contents. Both ADPG-synthesizing SS and AGPase activity patterns were found to correlate well with those of ADPG and starch contents. Remarkably, however, maximal activities of ADPG-synthesizing SS were found to be several fold higher than those of AGPase throughout seed development, the highest rate of starch accumulation being well accounted for by SS. Kinetic analyses of SS from barley endosperms and potato tubers in the Suc cleavage direction showed similar K(m) values for ADP and UDP, whereas apparent affinity for Suc was shown to be higher in the presence of UDP than with ADP. Moreover, measurements of transglucosylation activities in starch granules incubated with purified SS, ADP and [U-(14)C]Suc revealed a low inhibitory effect of UDP. The ADPG and UDPG contents in the transgenic S-112 SS and starch deficient potato mutant [Zrenner et al. (1995) Plant J. 7: 97] were found to be 35% and 30% of those measured in wild-type plants, whereas both glucose-1-phosphate and glucose-6-phosphate contents were found to be normal as compared with those of wild-type plants. The overall results thus strongly support a novel gluconeogenic mechanism reported previously [Pozueta-Romero et al. (1999) CRIT: Rev. Plant Sci. 18: 489] wherein SS catalyses directly the de novo production of ADPG linked to starch biosynthesis in heterotrophic tissues of plants.     

Reduction of the cytosolic fructose-1,6-bisphosphatase in transgenic potato plants limits photosynthetic sucrose biosynthesis with no impact on plant growth and tuber yield

Sucrose produced in source leaves is the predominant carbon source for developing sink tissues in most higher plants. Consequently the rate of sucrose synthesis is likely to be important for sink development and final crop yield. Two sucrose biosynthetic enzymes are believed to possess regulatory properties with respect to the rate of sucrose synthesis: (i) cytosolic FBPase and (ii) sucrose phosphate synthase. To study the impact of reduced photosynthetic sucrose biosynthesis on plant growth and crop yield a cDNA clone encoding cytosolic FBPase was isolated from a potato leaf cDNA library and used for antisense experiments in transgenic potato plants. The cDNA clone cy-F1, containing an open reading frame of 1020 bp highly homologous (85%) to other known sequences of plant cytosolic FBPases, was cloned in reversed orientation between the 35S CaMV promoter and the octopine synthase polyadenylation signal. Out of 75 independent transformants five transgenic lines having 9 to 55% of the wild-type FBPase activity were chosen for further analysis. A 45% reduction of the cytosolic FBPase activity did not cause any measurable change in metabolite concentrations, growth behaviour or photosynthetic parameters of the transgenic plants. Inhibition of cytosolic FBPase activity below 20% of the wild-type activity led to an accumulation of 3-PGA, triose-phosphates and fructose-1,6bisphosphate in source leaves. This resulted in a reduced light-saturated rate of assimilation measured via gas exchange and a decreased photosynthetic rate under conditions of the leaf disc electrode with saturating light and CO₂. Measuring photosynthetic carbon fluxes by labelling leaf discs with ¹⁴CO₂ revealed a 53–65% reduction of sucrose synthesis whereas starch synthesis decreased only by 18–24%. The flux into the anionic and cationic fraction was not altered. Despite these changes steadystate sucrose concentrations were not effected in source leaves from transgenic plants. Starch accumulated by more than a factor of 3 compared with wild-type leaves and was degraded during the night. This provides strong evidence for the hypothesis that hexoses and/or hexosephosphates are exported out of the chloroplasts, thereby circumventing the limitation of sucrose biosynthesis caused by the inhibition of cytosolic FBPase in the dark. Accordingly, plant growth and potato tuber yield remained unaltered. From these data it can be concluded that a reduced photosynthetic sucrose biosynthetic capacity can be efficiently compensated without any reduction in crop yield under greenhouse or growth chamber conditions by changing carbon export strategy. Whether the same holds true for field conditions remains to be elucidated.     

Effect of High Temperature on Plant Growth and Carbohydrate Metabolism in Potato

This study was undertaken to determine the role of sucrose-metabolizing enzymes in altered carbohydrate partitioning caused by heat stress. Potato (Solanum tuberosum L.) genotypes characterized as susceptible and tolerant to heat stress were grown at 19/17[deg]C, and a subset was transferred to 31/29[deg]C. Data were obtained for plant growth and photosynthesis. Enzyme activity was determined for sucrose-6-phosphate synthase (SPS) in mature leaves and for sucrose synthase, ADP-glucose pyrophosphorylase, and UDP-glucose pyrophosphorylase in developing tubers of plants. High temperatures reduced growth of tubers more than of shoots. Photosynthetic rates were unaffected or increased slightly at the higher temperature. Heat stress increased accumulation of foliar sucrose and decreased starch accumulation in mature leaves but did not affect glucose. SPS activity increased significantly in mature leaves of plants subjected to high temperature. Changes in SPS activity were probably not due to altered enzyme kinetics. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was reduced in tubers, albeit less quickly than leaf SPS activity. There was no interaction of temperature and genotype with regard to the enzymes examined; therefore, observed differences do not account for differences between genotypes in heat susceptibility.     

Sources of Carbon for Export from Spinach Leaves throughout the Day

Rates of net carbon exchange, export, starch, and sucrose synthesis were measured in leaves of spinach (Spinacia oleracea L.) throughout a 14-hour period of sinusoidal light to determine the sources of carbon contributing to export. Net carbon exchange rate closely followed light level, but export remained relatively constant throughout the day. In the morning when photosynthesis was low, starch degradation provided most of the carbon for export, while accumulated sucrose was exported during the evening. At high photosynthesis rate, the regulatory metabolite fructose 2,6-bisphosphate was low, allowing more of the newly fixed carbon to flow to sucrose through cytosolic fructose bisphosphatase. When the rate of sucrose synthesis exceeded the rate of export from the leaf, sucrose accumulated and soon thereafter sucrose synthesis declined. A decreasing sucrose synthesis rate resulted in additional carbon moving to the synthesis of starch, which was maintained throughout the remainder of the day. The declining sucrose synthesis rate coincided with decreasing activity of sucrose phosphate synthase present in gel-filtered leaf extracts. A rise in the leaf levels of uridine diphosphoglucose and fructose 6-phosphate throughout the day was consistent with this declining activity.     

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.     

Increased sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development

Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase) was increased by overexpression of an Arabidopsis (Arabidopsis thaliana) cDNA in tobacco (Nicotiana tabacum) plants. In plants with increased SBPase activity, photosynthetic rates were increased, higher levels of Suc and starch accumulated during the photoperiod, and an increase in leaf area and biomass of up to 30% was also evident. Light saturated photosynthesis increased with increasing SBPase activity and analysis of CO2 response curves revealed that this increase in photosynthesis could be attributed to an increase in ribulose 1,5-bisphosphate regenerative capacity. Seedlings with increased SBPase activity had an increased leaf area at the 4 to 5 leaf stage when compared to wild-type plants, and chlorophyll fluorescence imaging of these young plants revealed a higher photosynthetic capacity at the whole plant level. Measurements of photosynthesis, made under growth conditions integrated over the day, showed that mature plants with increased SBPase activity fixed 6% to 12% more carbon than equivalent wild-type leaves, with the young leaves having the highest rates. In this paper, we have shown that photosynthetic capacity per unit area and plant yield can be increased by overexpressing a single native plant enzyme, SBPase, and that this gives an advantage to the growth of these plants from an early phase of vegetative growth. This work has also shown that it is not necessary to bypass the normal regulatory control of SBPase, exerted by conditions in the stroma, to achieve improvements in carbon fixation.     

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol

Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose 6-phosphate - FW fresh weight - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - 3PGA glycerate-3-phosphate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - Pi inorganic phosphate - PPi inorganic pyrophosphate - UDPGlc UDP-glucose This research was supported by the Deutsche Forschungsgemein-Schaft (SFB 137) and Sandoz AG (T.J., M.H., M.S.) and by the Bundesminister für Forschung und Technologie (U.S., L.W.).     

Diurnal fluctuations in cotton leaf carbon export, carbohydrate content, and sucrose synthesizing enzymes

In fully expanded leaves of greenhouse-grown cotton (Gossypium hirsutum L., cv Coker 100) plants, carbon export, starch accumulation rate, and carbon exchange rate exhibited different behavior during the light period. Starch accumulation rates were relatively constant during the light period, whereas carbon export rate was greater in the afternoon than in the morning even though the carbon exchange rate peaked about noon. Sucrose levels increased throughout the light period and dropped sharply with the onset of darkness; hexose levels were relatively constant except for a slight peak in the early morning. Sucrose synthase, usually thought to be a degradative enzyme, was found in unusually high activities in cotton leaf. Both sucrose synthase and sucrose phosphate synthetase activities were found to fluctuate diurnally in cotton leaves but with different rhythms. Diurnal fluctuations in the rate of sucrose export were generally aligned with sucrose phosphate synthase activity during the light period but not with sucrose synthase activity; neither enzyme activity correlated with carbon export during the dark. Cotton leaf sucrose phosphate synthase activity was sufficient to account for the observed carbon export rates; there is no need to invoke sucrose synthase as a synthetic enzyme in mature cotton leaves. During the dark a significant correlation was found between starch degradation rate and leaf carbon export. These results indicate that carbon partitioning in cotton leaf is somewhat independent of the carbon exchange rate and that leaf carbon export rate may be linked to sucrose formation and content during the light period and to starch breakdown in the dark.