Reduction of the Cytosolic Phosphoglucomutase in Arabidopsis Reveals Impact on Plant Growth,Seed and Root Development,and Carbohydrate Partitioning

Phosphoglucomutase (PGM) catalyses the interconversion of glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P) and exists as plastidial (pPGM) and cytosolic (cPGM) isoforms. The plastidial isoform is essential for transitory starch synthesis in chloroplasts of leaves, whereas the cytosolic counterpart is essential for glucose phosphate partitioning and, therefore, for syntheses of sucrose and cell wall components. In Arabidopsis two cytosolic isoforms (PGM2 and PGM3) exist. Both PGM2 and PGM3 are redundant in function as single mutants reveal only small or no alterations compared to wild type with respect to plant primary metabolism. So far, there are no reports of Arabidopsis plants lacking the entire cPGM or total PGM activity, respectively. Therefore, amiRNA transgenic plants were generated and used for analyses of various parameters such as growth, development, and starch metabolism. The lack of the entire cPGM activity resulted in a strongly reduced growth revealed by decreased rosette fresh weight, shorter roots, and reduced seed production compared to wild type. By contrast content of starch, sucrose, maltose and cell wall components were significantly increased. The lack of both cPGM and pPGM activities in Arabidopsis resulted in dwarf growth, prematurely die off, and inability to develop a functional inflorescence. The combined results are discussed in comparison to potato, the only described mutant with lack of total PGM activity.     

The contribution of plastidial phosphoglucomutase to the control of starch synthesis within the potato tuber

The aim of this work was to evaluate the extent to which plastidial phosphoglucomutase (PGM) activity controls starch synthesis within potato (Solanum tuberosum L. cv. Desirée) tubers. The reduction in the activity of plastidial PGM led to both a correlative reduction in starch accumulation and an increased sucrose accumulation. The control coefficient of plastidial PGM on the accumulation of starch was estimated to approximate 0.24. The fluxes of carbohydrate metabolism were measured by investigating the metabolism of [U-14C]glucose in tuber discs from wild-type and transgenic plants. In tuber discs the control coefficient of plastidial PGM over starch synthesis was estimated as 0.36, indicating that this enzyme exerts considerable control over starch synthesis within the potato tuber.     

Antisense inhibition of plastidial phosphoglucomutase provides compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose-6-phosphate

The aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole-plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose-6-phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.     

Metabolite pools during starch synthesis and carbohydrate oxidation in amyloplasts isolated from wheat endosperm

Starch synthesis and CO₂ evolution were determined after incubating intact and lysed wheat (Triticum aestivum L. cv. Axona) endosperm amyloplasts with ¹⁴C-labelled hexose-phosphates. Amyloplasts converted [U-¹⁴C]glucose 1-phosphate (Glc1P) but not [U-¹⁴C]glucose 6-phosphate (Glc6P) into starch in the presence of ATP. When the oxidative pentose-phosphate pathway (OPPP) was stimulated, both [U-¹⁴C]Glc1P and [U-¹⁴C]Glc6P were metabolized to CO₂, but Glc6P was the better precursor for the OPPP, and Glc1P-mediated starch synthesis was reduced by 75%. In order to understand the basis for the partitioning of carbon between the two potentially competing metabolic pathways, metabolite pools were measured in purified amyloplasts under conditions which promote both starch synthesis and carbohydrate oxidation via the OPPP. Amyloplasts incubated with Glc1P or Glc6P alone showed little or no interconversion of these hexose-phosphates inside the organelle. When amyloplasts were synthesizing starch, the stromal concentrations of Glc1P and ADP-glucose were high. By contrast, when flux through the OPPP was highest, Glc1P and ADP-glucose inside the organelle were undetectable, and there was an increase in metabolites involved in carbohydrate oxidation. Measurements of the plastidial hexose-monophosphate pool during starch synthesis and carbohydrate oxidation indicate that the phosphoglucose isomerase reaction is at equilibrium whereas the reaction catalysed by phosphoglucomutase is significantly displaced from equilibrium. Received: 29 March 1997 / Accepted: 5 June 1997     

Alteration of photosynthate partitioning by high-level expression of phosphoglucomutase in tobacco chloroplasts

Plastidial phosphoglucomutase (PGM) plays an important role in starch synthesis and degradation. Nonetheless, the impact of enhanced plastidial PGM activity on metabolism in photosynthetic tissue is yet to be elucidated. In this study, we generated transplastomic tobacco plants overproducing Arabidopsis thaliana plastidial PGM (AtptPGM) in chloroplasts and analyzed the consequent metabolic and physiological parameters in the transplastomic plants. AtptPGM accumulated in the chloroplasts to up to 16% of total soluble protein in the leaves. PGM activity in leaves increased 100-fold relative to that of wild-type plants. The transplastomic plants were phenotypically indistinguishable in their growth rates, photosynthetic activities, and starch synthesis from wild-type plants, but hexose partitioning in the light period was dramatically different. Furthermore, alteration of extracellular invertase activity was observed in the lower leaves of the transplastomic plants. These observations suggest that high-level expression of plastidial PGM alters hexose partitioning in light periods via modification of extracellular invertase activity.     

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.     

Glucose-1-phosphate transport into protoplasts and chloroplasts from leaves of Arabidopsis

Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-(14)C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less (14)C into starch when unlabeled bicarbonate is supplied in addition to the (14)C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-(14)C]Glc-1-P incorporate (14)C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate (14)C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.     

Altered activity of the P2 isoform of plastidic glucose 6-phosphate dehydrogenase in tobacco (Nicotiana tabacum cv. Samsun) causes changes in carbohydrate metabolism and response to oxidative stress in leaves

Expression of one specific isoform of plastidic glucose 6-phosphate dehydrogenase (G6PDH) was manipulated in transgenic tobacco. Antisense and sense constructs of the endogenous P2 form of G6PDH were used to transform plants under the control of the cauliflower mosaic virus (CaMV) 35S promotor. Recombinant plants with altered expression were taken through to homozygosity by selective screening. Northern analyses revealed substantial changes in the expression of the P2 form of G6PDH, with no apparent impact on the activity of the cytosolic isoenzyme. Analysis of G6PDH activity in chloroplasts showed that despite the large changes in expression of P2-G6PDH, the range of enzyme activity varied only from approximately 50 to 200% of the wild type, reflecting the presence of a second G6PDH chloroplastic isoform (P1). Although none of the transgenic plants showed any visible phenotype, there were marked differences in metabolism of both sense and antisense lines when compared with wild-type/control lines. Sucrose, glucose and fructose contents of leaves were higher in antisense lines, whereas in overexpressing lines, the soluble sugar content was reduced below that of control plants. Even more striking was the observation that contents of glucose 6-phosphate (Glc6P) and 6-phosphogluconate (6PG) changed, such that the ratio of Glc6P:6PG was some 2.5-fold greater in the most severe antisense lines, compared with those with the highest levels of overexpression. Because of the distinctive biochemical properties of P2-G6PDH, we investigated the impact of altered expression on the contents of antioxidants and the response of plants to oxidative stress induced by methyl viologen (MV). Plants with decreased expression of P2-G6PDH showed increased content of reduced glutathione (GSH) compared to other lines. They also possessed elevated contents of ascorbate and exhibited a much higher ratio of reduced:oxidised ascorbate. When exposed to MV, leaf discs of wild-type and overexpressing lines demonstrated increased oxidative damage as measured by lipid peroxidation. Remarkably, leaf discs from plants with decreased P2-G6PDH did not show any change in lipid peroxidation in response to increasing concentrations of up to 15 micro m MV. The results are discussed from the perspective of the role of G6PDH in carbohydrate metabolism and oxidative stress. It is suggested that the activity of P2-G6PDH may be crucial in balancing the redox poise in chloroplasts.     

Molecular and biochemical characterization of cytosolic phosphoglucomutase in wheat endosperm (Triticum aestivum L. cv. Axona)

Evidence from a number of plant tissues suggests that phosphoglucomutase (PGM) is present in both the cytosol and the plastid. The cytosolic and plastidic isoforms of PGM have been partially purified from wheat endosperm (Triticum aestivum L. cv. Axona). Both isoforms required glucose 1,6-bisphosphate for their activity with K(a) values of 4.5 micro M and 3.8 micro M for cytosolic and plastidic isoforms, respectively, and followed normal Michaelis-Menten kinetics with glucose 1-phosphate as the substrate with K(m) values of 0.1 mM and 0.12 mM for the cytosolic and plastidic isoforms, respectively. A cDNA clone was isolated from wheat endosperm that encodes the cytosolic isoform of PGM. The deduced amino acid sequence shows significant homology to PGMs from eukaryotic and prokaryotic sources. PGM activity was measured in whole cell extracts and in amyloplasts isolated during the development of wheat endosperm. Results indicate an approximate 80% reduction in measurable activity of plastidial and cytosolic PGM between 8 d and 30 d post-anthesis. Northern analysis showed a reduction in cytosolic PGM mRNA accumulation during the same period of development. The implications of the changes in PGM activity during the synthesis of starch in developing endosperm are discussed.     

Antisense repression of cytosolic phosphoglucomutase in potato (Solanum tuberosum) results in severe growth retardation, reduction in tuber number and altered carbon metabolism

The aim of this work was to investigate the role of cytosolic phosphoglucomutase (PGM; EC 5.4.2.2) in the regulation of carbohydrate metabolism. Many in vitro studies have indicated that PGM plays a central role in carbohydrate metabolism; however, until now the importance of this enzyme in plants has not been subject to reverse-genetics investigations. With this intention we cloned the cytosolic isoform of potato PGM (StcPGM) and expressed this in the antisense orientation under the control of the CaMV 35 S promoter in potato plants. We confirmed that these plants contained reduced total PGM activity and that loss in activity was due specifically to a reduction in cytosolic PGM activity. These plants were characterised by a severe phenotype: stunted aerial growth combined with limited root growth and a reduced tuber yield. Analysis of the metabolism of these lines revealed that leaves of these plants were inhibited in sucrose synthesis whereas the tubers exhibited decreased levels of sucrose and starch as well as decreased levels of glycolytic intermediates but possessed unaltered levels of adenylates. Furthermore, a broader metabolite screen utilising GC-MS profiling revealed that these lines contained altered levels of several intermediates of the TCA cycle and of amino acids. In summary, we conclude that cytosolic PGM plays a crucial role in the sucrose synthetic pathway within the leaf and in starch accumulation within the tuber, and as such is important in the maintenance of sink-source relationships.     

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.     

Starch metabolism in developing embryos of oilseed rape

The aim of this work was to characterise the metabolism of starch in developing embryos of oilseed rape (Brassica napus L. cv. Topaz). The accumulation of starch in embryos in siliques which were darkened or had been exposed to the light was similar, suggesting that the starch is synthesised from imported sucrose rather than via photosynthesis in the embryo. Starch content and the activities of plastidial enzymes required for synthesis of starch from glucose 6-phosphate (Glc6P) both peaked during the early-mid stage of cotyledon development (i.e. during the early part of oil accumulation) and then declined. The mature embryo contained almost no starch. The starch-degrading enzymes α-(EC 3.2.1.1) and β-amylase (EC 3.2.1.2) and phosphorylase (EC 2.4.1.1) were present throughout development. Most of the activity of these three enzymes was extraplastidial and therefore unlikely to be involved in starch degradation, but there were distinct plastidial and extraplastidial isoforms of all three enzymes. Activity gels indicated that distinct plastidial isoforms increase during the change from net synthesis to net degradation of starch. Plastids isolated from embryos at stages both before and after the maximum starch content could convert Glc6P to starch although the rate was lower at the later stage. The results are consistent with the idea that starch synthesis and degradation occur simultaneously during embryo development. The possible roles of transient starch accumulation during embryo development are discussed. Received: 15 May 1997 / Accepted: 30 May 1997     

Antisense inhibition of cytosolic phosphorylase in potato plants (Solanum tuberosum L.) affects tuber sprouting and flower formation with only little impact on carbohydrate metabolism

To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants were created in which the expression of the enzyme was inhibited by introducing a chimeric gene containing part of the coding region for cytosolic phosphorylase linked in antisense orientation to the 35S CaMV promotor. As revealed by Northern blot analysis and native polyacrylamide gel electrophoresis, the expression of cytosolic phosphorylase was strongly inhibited in both leaves and tubers of the transgenic plants. The transgenic plants propagated from stem cuttings were morphologically indiscernible from the wild-type. However, sprouting of the transgenic potato tubers was significantly altered: compared with the wild-type, transgenic tubers produced 2.4 to 8.1 times more sprouts. When cultivated in the greenhouse, transgenic seed tubers produced two to three times more shoots than the wild-type. Inflorescences appeared earlier in the resulting plants. Many of the transgenic plants flowered two or three times successively. Transgenic plants derived from seed tubers formed 1.6 to 2.4 times as many tubers per plant as untransformed controls. The size and dry matter content of the individual tubers was not noticeably altered. Tuber yield was significantly higher in the transgenic plants. As revealed by carbohydrate determination of freshly harvested and stored tubers, starch and sucrose pools were not noticeably affected by the antisense inhibition of cytosolic phosphorylase; however, glucose and fructose levels were markedly reduced after prolonged storage. These results favour the view that cytosolic phosphorylase does not participate in starch degradation. The possible links between the reduced levels of cytosolic phosphorylase and the observed changes with respect to sprouting and flowering are discussed.     

The sources of carbon and reducing power for fatty acid synthesis in the heterotrophic plastids of developing sunflower (Helianthus annuus L.) embryos

The provision of carbon substrates and reducing power for fatty acid synthesis in the heterotrophic plastids of developing embryos of sunflower (Helianthus annuus L.) has been investigated. Profiles of oil and storage protein accumulation were determined and embryos at 17 and 24 days after anthesis (DAA) were selected to represent early and late periods of oil accumulation. Plastids isolated from either 17 or 24 DAA embryos did not incorporate label from [1-(14)C]glucose 6-phosphate (Glc6P) into fatty acids. Malate, when supplied alone, supported the highest rates of fatty acid synthesis by the isolated plastids at both stages. Pyruvate supported rates of fatty acid synthesis at 17 DAA that were comparable to those supported by malate, but only when incubations also included Glc6P. The stimulatory effect of Glc6P on pyruvate utilization at 17 DAA was related to the rapid utilization of Glc6P through the oxidative pentose phosphate pathway (OPPP) at this stage. Addition of pyruvate to incubations containing [1-(14)C]Glc6P increased OPPP activity (measured as (14)CO(2) release), while the addition of malate suppressed it. Observations of the interactions between the rate of metabolite utilization for fatty acid synthesis and the rate of the OPPP are consistent with regulation of the OPPP by redox control of the plastidial glucose 6-phosphate dehydrogenase activity through the demand for NADPH. During pyruvate utilization for fatty acid synthesis, flux through the OPPP increases as NADPH is consumed, whereas during malate utilization, in which NADPH is produced by NADP-malic enzyme, flux through the OPPP is decreased.     

Competitive inhibition of spinach leaf phosphoglucose isomerase isoenzymes by erythrose 4-phosphate

In plant cells, the reversible isomerization between fructose 6-phosphate (Fru6P) and glucose 6-phosphate (Glc6P) is catalyzed by a cytosolic and a chloroplastic isoenzyme of phosphoglucose isomerase (PGI, EC 5.3.1.9). The extractable activities of both PGI isoenzymes are in large excess compared with the flux required for product synthesis, but the measured Glu6P/Fru6P ratio in illuminated chloroplasts and in whole leaves is always displaced from equilibrium. Cytosolic (PGI 2) and stromal (PGI 1) isoenzymes were purified from spinach leaves and used to investigate the possibility of metabolic regulation at this step. Several metabolites were found to inhibit PGI, but within the physiological concentration range, only erythrose 4-phosphate (Ery4P) inhibited significantly. The inhibition was competitive, with K_i values below 10 μM for PGI 2 and 1. The physiological significance of the inhibition of PGI by Ery4P was assessed in isolated intact spinach chloroplasts. We conclude that, in vivo, this inhibition is probably responsible for the observed displacement from equilibrium in the chloroplasts, but limits the carbon flow towards starch synthesis only when Fru6P is low. In contrast, the inhibition by Ery4P is unlikely to play any role in the cytosolic carbon metabolism because both Fru6P concentration and PGI activity, are much higher than in the chloroplast stroma.     

The role of amylomaltase in maltose metabolism in the cytosol of photosynthetic cells

Transitory starch is stored during the day inside chloroplasts and then broken down at night for export. Recent data indicate that maltose is the major form of carbon exported from the chloroplast at night but its fate in the cytosol is unknown. An amylomaltase gene (malQ) cloned from Escherichia coli is necessary for maltose metabolism in E. coli. We investigated whether there is an amylomaltase in the cytosol of plant leaves and the role of this enzyme in plants. Two mutants of Arabidopsis thaliana (L) Heynh. were identified in which the gene encoding a putative amylomaltase enzyme [disproportionating enzyme 2, DPE2 (DPE1 refers to the plastid version of this enzyme)] was disrupted by a T-DNA insertion. Both dpe2-1 and dpe2-2 plants exhibited a dwarf phenotype and accumulated a large amount of maltose. In addition, dpe2 mutants accumulated starch and a water-soluble, ethanol/KCl-insoluble maltodextrin in their chloroplasts. At night, the amount of sucrose in dpe2 plants was lower than that in wild-type plants. These results show that Arabidopsis has an amylomaltase that is involved in the conversion of maltose to sucrose in the cytosol. We hypothesize that knocking out amylomaltase blocks the conversion from maltose to sucrose, and that the higher amount of maltose feeds back to limit starch degradation reactions in chloroplasts. As a result, dpe2 plants have higher maltose, higher starch, and higher maltodextrin but lower nighttime sucrose than wild-type plants. Finally, we propose that maltose metabolism in the cytosol of Arabidopsis leaves is similar to that in the cytoplasm of E. coli.Abbreviations F6P fructose 6-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - GTase glucanotransferase     

Decreased sucrose-6-phosphate phosphatase level in transgenic tobacco inhibits photosynthesis,alters carbohydrate partitioning,and reduces growth

The aim of this work was to examine the role of sucrose-6-phosphate phosphatase (SPP; EC 3.1.3.24) in photosynthetic carbon partitioning. SPP catalyzes the final step in the pathway of sucrose synthesis; however, until now the importance of this enzyme in plants has not been studied by reversed-genetics approaches. With the intention of conducting such a study, transgenic tobacco plants with reduced SPP levels were produced using an RNA interference (RNAi) strategy. Transformants with less than 10% of wild-type SPP activity displayed a range of phenotypes, including those that showed inhibition of photosynthesis, chlorosis, and reduced growth rates. These plants had strongly reduced levels of sucrose and hexoses but contained 3–5 times more starch than the control specimens. The leaves were unable to export transient starch during extended periods of darkness and as consequence showed a starch- and maltose-excess phenotype. This indicates that no alternative mechanism for carbon export was activated. Inhibition of SPP resulted in an approximately 1,000-fold higher accumulation of sucrose-6-phosphate (Suc6P) compared to wild-type leaves, whereas the content of hexose-phosphates was reduced. Although the massive accumulation of Suc6P in the cytosol of transgenic leaves was assumed to impair phosphate-recycling into the chloroplast, no obvious signs of phosphate-limitation of photosynthesis became apparent. 3-Phosphoglycerate (3-PGA) levels dropped slightly and the ATP/ADP ratio was not reduced in the transgenic lines under investigation. It is proposed that in SPP-deficient plants, long-term compensatory responses give rise to the observed acceleration of starch synthesis, increase in total cellular Pi content, decrease in protein content, and related reduction in photosynthetic activity.     

Starch synthesis and carbohydrate oxidation in amyloplasts from developing wheat endosperm

The rates of incorporation of various metabolites into starch by isolated amyloplasts from developing endosperm of spring wheat (Triticum aestivum L. cv. Axona) were examined. Of the metabolites tested that were likely to be present in the cytosol at concentrations sufficient to sustain starch synthesis, only glucose 1-phosphate (Glc1P) supported physiologically relevant rates of starch synthesis. Incorporation of Glc1P into starch was both dependent on the presence of ATP and intact organelles. The rate of incorporation of hexose into starch became saturated at a Glc1P concentration of less than 1 mol·m^-3 in the presence of 1 mol·m^-3 ATP. Starch synthesis from 5 mol · m^-3 ADP-glucose supplied to the organelles occurred at rates 15-fold higher than from similar concentrations of Glc1P, but it is argued that this is probably of little physiological relevance. The net incorporation of hexose units into starch from GlclP was inhibited 50% by 100 mmol.m^-3 carboxyatractyloside. Carbohydrate oxidation in the amyloplast was stimulated by the addition of 2-oxoglutarate and glutamine, and in such circumstances incorporation of¹⁴C-labelled metabolites into starch was reduced. Glucose 6-phosphate proved to be a better substrate for oxidative pathways than Glc1P. Our results suggest that Glc1P is the primary substrate for starch synthesis in developing wheat endosperm, and that ATP required for starch synthesis is imported via an adenylate translocator.     

Loss of the major isoform of phosphoglucomutase results in altered calcium homeostasis in Saccharomyces cerevisiae

Phosphoglucomutase (PGM) is a key enzyme in glucose metabolism, where it catalyzes the interconversion of glucose 1-phosphate (Glc-1-P) and glucose 6-phosphate (Glc-6-P). In this study, we make the novel observation that PGM is also involved in the regulation of cellular Ca(2+) homeostasis in Saccharomyces cerevisiae. When a strain lacking the major isoform of PGM (pgm2Delta) was grown on media containing galactose as sole carbon source, its rate of Ca(2+) uptake was 5-fold higher than an isogenic wild-type strain. This increased rate of Ca(2+) uptake resulted in a 9-fold increase in the steady-state total cellular Ca(2+) level. The fraction of cellular Ca(2+) located in the exchangeable pool in the pgm2Delta strain was found to be as large as the exchangeable fraction observed in wild-type cells, suggesting that the depletion of Golgi Ca(2+) stores is not responsible for the increased rate of Ca(2+) uptake. We also found that growth of the pgm2Delta strain on galactose media is inhibited by 10 microM cyclosporin A, suggesting that activation of the calmodulin/calcineurin signaling pathway is required to activate the Ca(2+) transporters that sequester the increased cytosolic Ca(2+) load caused by this high rate of Ca(2+) uptake. We propose that these Ca(2+)-related alterations are attributable to a reduced metabolic flux between Glc-1-P and Glc-6-P due to a limitation of PGM enzymatic activity in the pgm2Delta strain. Consistent with this hypothesis, we found that this "metabolic bottleneck" resulted in an 8-fold increase in the Glc-1-P level compared with the wild-type strain, while the Glc-6-P and ATP levels were normal. These results suggest that Glc-1-P (or a related metabolite) may participate in the control of Ca(2+) uptake from the environment.     

Loss of cytosolic phosphoglucomutase compromises gametophyte development in Arabidopsis

Cytosolic phosphoglucomutase (cPGM) interconverts glucose-6-phosphate and glucose-1-phosphate and is a key enzyme of central metabolism. In this study, we show that Arabidopsis (Arabidopsis thaliana) has two cPGM genes (PGM2 and PGM3) encoding proteins with high sequence similarity and redundant functions. Whereas pgm2 and pgm3 single mutants were undistinguishable from the wild type, loss of both PGM2 and PGM3 severely impaired male and female gametophyte function. Double mutant pollen completed development but failed to germinate. Double mutant ovules also developed normally, but approximately half remained unfertilized 2 d after pollination. We attribute these phenotypes to an inability to effectively distribute carbohydrate from imported or stored substrates (e.g. sucrose) into the major biosynthetic (e.g. cell wall biosynthesis) and respiratory pathways (e.g. glycolysis and the oxidative pentose phosphate pathway). Disturbing these pathways is expected to have dramatic consequences for germinating pollen grains, which have high metabolic and biosynthetic activities. We propose that residual cPGM mRNA or protein derived from the diploid mother plant is sufficient to enable double mutant female gametophytes to attain maturity and for some to be fertilized. Mature plants possessing a single cPGM allele had a major reduction in cPGM activity. However, photosynthetic metabolism and growth were normal, suggesting that under standard laboratory conditions cPGM activity provided from one wild-type allele is sufficient to mediate the photosynthetic and respiratory fluxes in leaves.