Plastidial Phosphoglucomutase Research Articles

Plant growth: An active or passive role for starch reserves?

Starch metabolism is linked to plant growth, yet blocking its biosynthesis has species-specific consequences. In a new study, plastidial phosphoglucomutase is knocked out in aspen trees using CRISPR-Cas9, limiting starch production and altering photosynthesis, but growth, bud break and wood production proceed unaffected.

Aspen growth is not limited by starch reserves

All photosynthetic organisms balance CO2 assimilation with growth and carbon storage. Stored carbon is used for growth at night and when demand exceeds assimilation. Gaining a mechanistic understanding of carbon partitioning between storage and growth in trees is important for biological studies and for estimating the potential of terrestrial photosynthesis to sequester anthropogenic CO2 emissions.1,2 Starch represents the main carbon storage in plants.3,4 To examine the carbon storage mechanism and role of starch during tree growth, we generated and characterized low-starch hybrid aspen (Populus tremula×tremuloides) trees using CRISPR-Cas9-mediated gene editing of two PHOSPHOGLUCOMUTASE (PGM) genes coding for plastidial PGM isoforms essential for starch biosynthesis. We demonstrate that starch deficiency does not reduce tree growth even in short days, showing that starch is not a critical carbon reserve during diel growth of aspen. The low-starch trees assimilated up to ∼30% less CO2 compared to the wild type under a range of irradiance levels, but this did not reduce growth or wood density. This implies that aspen growth is not limited by carbon assimilation under benign growth conditions. Moreover, the timing of bud set and bud flush in the low-starch trees was not altered, implying that starch reserves are not critical for the seasonal growth-dormancy cycle. The findings are consistent with a passive starch storage mechanism that contrasts with the annual Arabidopsis and indicate that the capacity of the aspen to absorb CO2 is limited by the rate of sink tissue growth.

Influence of starch deficiency on photosynthetic and post-photosynthetic carbon isotope fractionations.

Carbon isotope (13C) fractionations occurring during and after photosynthetic CO2 fixation shape the carbon isotope composition (δ13C) of plant material and respired CO2. However, responses of 13C fractionations to diel variation in starch metabolism in the leaf are not fully understood. Here we measured δ13C of organic matter (δ13COM), concentrations and δ13C of potential respiratory substrates, δ13C of dark-respired CO2 (δ13CR), and gas exchange in leaves of starch-deficient plastidial phosphoglucomutase (pgm) mutants and wild-type plants of four species (Arabidopsis thaliana, Mesembryanthemum crystallinum, Nicotiana sylvestris, and Pisum sativum). The strongest δ13C response to the pgm-induced starch deficiency was observed in N. sylvestris, with more negative δ13COM, δ13CR, and δ13C values for assimilates (i.e. sugars and starch) and organic acids (i.e. malate and citrate) in pgm mutants than in wild-type plants during a diel cycle. The genotype differences in δ13C values could be largely explained by differences in leaf gas exchange. In contrast, the PGM-knockout effect on post-photosynthetic 13C fractionations via the plastidic fructose-1,6-bisphosphate aldolase reaction or during respiration was small. Taken together, our results show that the δ13C variations in starch-deficient mutants are primarily explained by photosynthetic 13C fractionations and that the combination of knockout mutants and isotope analyses allows additional insights into plant metabolism.

Diurnal periodicity of assimilate transport shapes resource allocation and whole-plant carbon balance.

Whole-plant carbon balance comprises diurnal fluctuations of photosynthetic carbon gain and respiratory losses, as well as partitioning of assimilates between phototrophic and heterotrophic organs. Because it is difficult to access, the root system is frequently neglected in growth models, or its metabolism is rated based on generalizations from other organs. Here, whole-plant cuvettes were used for investigating total-plant carbon exchange with the environment over full diurnal cycles. Dynamics of primary metabolism and diurnally resolved phloem exudation profiles, as proxy of assimilate transport, were combined to obtain a full picture of resource allocation. This uncovered a strong impact of periodicity of inter-organ transport on the efficiency of carbon gain. While a sinusoidal fluctuation of the transport rate, with minor diel deflections, minimized respiratory losses in Arabidopsis wild-type plants, triangular or rectangular patterns of transport, found in mutants defective in either starch or sucrose metabolism, increased root respiration at the end or beginning of the day, respectively. Power spectral density and cross-correlation analysis revealed that only the rate of starch synthesis was strictly correlated to the rate of net photosynthesis in wild-type, while in a sucrose-phosphate synthase mutant (spsa1), this applied also to carboxylate synthesis, serving as an alternative carbon pool. In the starchless mutant of plastidial phospho-gluco mutase (pgm), none of these rates, but concentrations of sucrose and glucose in the root, followed the pattern of photosynthesis, indicating direct transduction of shoot sugar levels to the root. The results demonstrate that starch metabolism alone is insufficient to buffer diurnal fluctuations of carbon exchange.

Subcellular reprogramming of metabolism during cold acclimation in Arabidopsis thaliana.

Metabolite changes in plant leaves during exposure to low temperatures involve re-allocation of a large number of metabolites between sub-cellular compartments. Therefore, metabolite determination at the whole cell level may be insufficient for interpretation of the functional significance of cellular compounds. To investigate the cold-induced metabolite dynamics at the level of individual sub-cellular compartments, an integrative platform was developed that combines quantitative metabolite profiling by gas chromatography coupled to mass spectrometry (GC-MS) with the non-aqueous fractionation technique allowing separation of cytosol, vacuole and the plastidial compartment. Two mutants of Arabidopsis thaliana representing antipodes in the diversion of carbohydrate metabolism between sucrose and starch were compared to Col-0 wildtype before and after cold acclimation to investigate interactions of cold acclimation with subcellular re-programming of metabolism. A multivariate analysis of the data set revealed dominant effects of compartmentation on metabolite concentrations that were modulated by environmental condition and genetic determinants. While for both, the starchless mutant of plastidial phospho-gluco mutase (pgm) and a mutant defective in sucrose-phosphate synthase A1, metabolic constraints, especially at low temperature, could be uncovered based on subcellularly resolved metabolite profiles, only pgm had lowered freezing tolerance. Metabolic profiles of pgm point to redox imbalance as a possible reason for reduced cold acclimation capacity.

Exaggerated root respiration accounts for growth retardation in a starchless mutant of Arabidopsis thaliana

The knock-out mutation of plastidial phosphoglucomutase (pgm) causes a starchless phenotype in Arabidopsis thaliana, and results in a severe growth reduction of plants cultivated under diurnal conditions. It has been speculated that high soluble sugar levels accumulating during the light phase in leaf mesophyll might cause a reduction of photosynthetic activity or that shortage of reduced carbon during the night is the reason for the slow biomass gain of pgm. Separate simultaneous measurements of leaf net photosynthesis and root respiration demonstrate that photosynthetic activity per unit fresh weight is not reduced in pgm, whereas root respiration is strongly elevated. Comparison with a mutant defective in the dominating vacuolar invertase (AtβFruct4) revealed that high sucrose concentration in the cytosol, but not in the vacuole, of leaf cells is responsible for elevated assimilate transport to the root. Increased sugar supply to the root, as observed in pgm mutants, forces substantial respiratory losses. Because root respiration accounts for 80% of total plant respiration under long-day conditions, this gives rise to retarded biomass formation. In contrast, reduced vacuolar invertase activity leads to reduced net photosynthesis in the shoot and lowered root respiration, and affords an increased root/shoot ratio. The results demonstrate that roots have very limited capacity for carbon storage but exert rigid control of supply for their maintenance metabolism.

Loss of starch granule initiation has a deleterious effect on the growth of arabidopsis plants due to an accumulation of ADP-glucose.

STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.

Carbon transitions from either Calvin cycle or transitory starch to heteroglycans as revealed by 14C‐labeling experiments using protoplasts from Arabidopsis

Plants metabolize transitory starch by precisely coordinated plastidial and cytosolic processes. The latter appear to include the action of water-soluble heteroglycans (SHGin ) whose monosaccharide pattern is similar to that of apoplastic glycans (SHGex ) but, unlike SHGex , SHGin strongly interacts with glucosyl transferases. In this study, we analyzed starch metabolism using mesophyll protoplasts from wild-type plants and two knock-out mutants [deficient in the cytosolic transglucosidase, disproportionating isoenzyme 2 (DPE2) or the plastidial phosphoglucomutase (PGM1)] from Arabidopsis thaliana. Protoplasts prelabeled by photosynthetic (14) CO2 fixation were transferred to an unlabeled medium and were darkened or illuminated. Carbon transitions from the Calvin cycle or from starch to both SHGin and SHGex were analyzed. In illuminated protoplasts, starch turn-over was undetectable but darkened protoplasts continuously degraded starch. During illumination, neither the total (14) C content nor the labeling patterns of the sugar residues of SHGin were significantly altered but both the total amount and the labeling of the constituents of SHGex increased with time. In darkened protoplasts, the (14) C-content of most of the sugar residues of SHGin transiently and strongly increased and then declined. This effect was not observed in any SHGex constituent. In darkened DPE2-deficient protoplasts, none of the SHGin constituents exhibited an essential transient increase in labeling. In contrast, some residues of SHGin from the PGM1 mutant exhibited a transient increase in label but this effect significantly differed from that of the wild type. Two conclusions are reached: first, SHGin and SHGex exert different metabolic functions and second, SHGin is directly involved in starch degradation.

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.

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 13CO2 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 vivo13C-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.

Defects in leaf carbohydrate metabolism compromise acclimation to high light and lead to a high chlorophyll fluorescence phenotype in Arabidopsis thaliana

BackgroundWe have studied the impact of carbohydrate-starvation on the acclimation response to high light using Arabidopsis thaliana double mutants strongly impaired in the day- and night path of photoassimilate export from the chloroplast. A complete knock-out mutant of the triose phosphate/phosphate translocator (TPT; tpt-2 mutant) was crossed to mutants defective in (i) starch biosynthesis (adg1-1, pgm1 and pgi1-1; knock-outs of ADP-glucose pyrophosphorylase, plastidial phosphoglucomutase and phosphoglucose isomerase) or (ii) starch mobilization (sex1-3, knock-out of glucan water dikinase) as well as in (iii) maltose export from the chloroplast (mex1-2).ResultsAll double mutants were viable and indistinguishable from the wild type when grown under low light conditions, but - except for sex1-3/tpt-2 - developed a high chlorophyll fluorescence (HCF) phenotype and growth retardation when grown in high light. Immunoblots of thylakoid proteins, Blue-Native gel electrophoresis and chlorophyll fluorescence emission analyses at 77 Kelvin with the adg1-1/tpt-2 double mutant revealed that HCF was linked to a specific decrease in plastome-encoded core proteins of both photosystems (with the exception of the PSII component cytochrome b559), whereas nuclear-encoded antennae (LHCs) accumulated normally, but were predominantly not attached to their photosystems. Uncoupled antennae are the major cause for HCF of dark-adapted plants. Feeding of sucrose or glucose to high light-grown adg1-1/tpt-2 plants rescued the HCF- and growth phenotypes. Elevated sugar levels induce the expression of the glucose-6-phosphate/phosphate translocator2 (GPT2), which in principle could compensate for the deficiency in the TPT. A triple mutant with an additional defect in GPT2 (adg1-1/tpt-2/gpt2-1) exhibited an identical rescue of the HCF- and growth phenotype in response to sugar feeding as the adg1-1/tpt-2 double mutant, indicating that this rescue is independent from the sugar-triggered induction of GPT2.ConclusionsWe propose that cytosolic carbohydrate availability modulates acclimation to high light in A. thaliana. It is conceivable that the strong relationship between the chloroplast and nucleus with respect to a co-ordinated expression of photosynthesis genes is modified in carbohydrate-starved plants. Hence carbohydrates may be considered as a novel component involved in chloroplast-to-nucleus retrograde signaling, an aspect that will be addressed in future studies.

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.

A Suite of Lotus japonicus Starch Mutants Reveals Both Conserved and Novel Features of Starch Metabolism

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.

Glucose 1‐phosphate is efficiently taken up by potato (Solanum tuberosum) tuber parenchyma cells and converted to reserve starch granules

Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [(14)C]glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.

The Debate on the Pathway of Starch Synthesis: A Closer Look at Low-Starch Mutants Lacking Plastidial Phosphoglucomutase Supports the Chloroplast-Localized Pathway

ADPglucose (ADPGlc) is the substrate for starch synthesis in the plastids of higher plants. The glucosyl moiety is used by starch synthases to elongate the glucans that comprise starch. Recently, there has been renewed debate about the ADPGlc synthesis, with the widely accepted or classical pathway

Sucrose Synthase Controls Both Intracellular ADP Glucose Levels and Transitory Starch Biosynthesis in Source Leaves

The prevailing model on transitory starch biosynthesis in source leaves assumes that the plastidial ADPglucose (ADPG) pyrophosphorylase (AGP) is the sole enzyme catalyzing the synthesis of the starch precursor molecule, ADPG. However, recent investigations have shown that ADPG linked to starch biosynthesis accumulates outside the chloroplast, presumably in the cytosol. This finding is consistent with the occurrence of an 'alternative' gluconeogenic pathway wherein sucrose synthase (SuSy) is involved in the production of ADPG in the cytosol, whereas both plastidial phosphoglucomutase (pPGM) and AGP play a prime role in the scavenging of starch breakdown products. To test this hypothesis, we have compared the ADPG content in both Arabidopsis and potato wild-type (WT) leaves with those of the starch-deficient mutants with reduced pPGM and AGP. These analyses provided evidence against the 'classical' model of starch biosynthesis, since ADPG levels in all the starch-deficient lines were normal compared with WT plants. Whether or not SuSy is involved in the synthesis of ADPG accumulating in leaves was tested by characterizing both SuSy-overexpressing and SuSy-antisensed transgenic leaves. Importantly, SuSy-overexpressing leaves exhibited a large increase of both ADPG and starch levels compared with WT leaves, whereas SuSy-antisensed leaves accumulated low amounts of both ADPG and starch. These findings show that (i) ADPG produced by SuSy is linked to starch biosynthesis; (ii) SuSy exerts a strong control on the starch biosynthetic process; and (iii) SuSy, but not AGP, controls the production of ADPG accumulating in source leaves.

Metabolic control analysis and regulation of the conversion of sucrose to starch in growing potato tubers

ABSTRACTStarch is of great importance both as a carbon storage reserve in plants and as a biotechnologically important product. The potato tuber is an attractive model system for the study of starch metabolism, because it is a relatively homogenous tissue in which conversion of sucrose to starch represents the dominant metabolic flux. All the major genes of the potato tuber sucrose to starch pathway have been cloned in recent years, allowing the generation of a suite of antisense transgenic lines to be produced in which the activity of each individual enzyme in the pathway is progressively decreased. Investigations of these plants have provided a complete picture of the distribution of control in this important pathway. Sucrose synthase, UGPase, hexokinase, cytosolic phosphoglucomutase, plastidial phosphoglucomutase, the amyloplastidial adenylate translocator, AGPase, starch synthase and starch branching enzyme have flux control coefficients (FCCs) of 0.10, approximating 0.00, approximating 0.00, 0.15, 0.23, 0.98, 0.35, 0.12 and approximating 0.00 for starch accumulation. These results show that the majority of the control on starch accumulation in potato tubers resides in the transfer of adenylate between the cytosol and the amyloplast, with a minor contribution being made by the first two steps of the plastidial starch synthesis pathway (the reactions catalysed by plastidial phosphoglucomutase and AGPase). This contrasts with leaves, in which the majority of the control has been found to reside in the reactions catalysed by plastidial phosphoglucomutase and AGPase. In leaves, ATP for starch synthesis is generated within the plastid via photophosphorylation. Several studies have attempted to increase the rate of starch synthesis by overexpressing pathway enzymes in tubers. The results of these studies and the role of other ATP producers in the starch synthetic process are reviewed. In the same time period methods of non‐aqueous fractionation have been adapted to potato tuber tissue in order to ascertain subcellular metabolite levels. Results obtained from these studies allow the calculation of mass action ratios of the constitutive enzymes of the sucrose to starch transition. When taken together with the known regulatory properties of these enzymes the combination of broad control analysis studies and assessment of the mass action ratios of the respective enzymes allows a comprehensive description of this important metabolic network. Some illustrative examples of how this network responds to environmental change are presented. Finally implications of this whole pathway evaluation for more general studies of plant metabolic pathways and networks are discussed.

Photosynthetic metabolism is severely impaired on the parallel reduction of plastidial and cytosolic isoforms of phosphoglucomutase

The aim of this work was to examine the role of phosphoglucomutase (PGM) activities (EC 5.4.2.2) in photosynthetic carbon partitioning. We have previously described the generation of transgenic potato ( Solanum tuberosum cv. Desiree) lines expressing either the StcPGM or the StpPGM genes, encoding cytosolic and plastidial isoforms of PGM, in the antisense orientation under the control of the 35S promoter. These studies suggested that the cytosolic isoform of this enzyme might be redundant in the leaf and that another activity may be able to fulfil this role. Here we investigate the effect on photosynthetic metabolism of repressing both isoforms of PGM. This manipulation resulted in a marked reduction in biomass in addition to a restricted photosynthesis, an alteration in carbon partitioning and mild changes in the levels of metabolic intermediates. The results are consistent with the hypothesis that the plastidial PGM can substitute for its cytosolic counterpart during photosynthetic sucrose synthesis.

Carbon assimilation and metabolism in potato leaves deficient in plastidial phosphoglucomutase.

We have previously described the generation of transgenic potato ( Solanum tuberosum L. cv. Desiree) lines expressing the S. tuberosum plastidial phosphoglucomutase ( StpPGM) gene in the antisense orientation under the control of the 35S promoter and characterised heterotrophic metabolism in these lines [E. Tauberger et al. (2000) Plant J 23:43-53]. The aim of the current work was to examine the role of plastidial phosphoglucomutase (pPGM, EC 5.4.2.2) in photosynthetic carbon partitioning. Here we characterise the metabolism of leaves of the same lines and show that reducing the activity of this enzyme has profound effects on carbon partitioning, characterised by a strong (up to 50%) reduction in the rate of starch accumulation accompanied by a minor reduction in the rate of sucrose accumulation. Gas-exchange and (14)CO(2)-feeding experiments revealed that the transgenic lines exhibited a decreased rate of photosynthesis and a corresponding reduced assimilation of radiolabel into starch, even in lines exhibiting only a minor decrease in pPGM activity. In illuminated leaves, decreasing the amount of pPGM resulted in decreased amounts of triose-phosphates, hexose-phosphates and inorganic phosphate without changes in the level of 3-phosphoglycerate. Most importantly, the deduced ratio of phosphoesters to inorganic phosphate increased, indicating the likelihood that photosynthesis was phosphate-limited in these lines. Determination of a more complete metabolic profile of leaf material from these lines revealed a large number of changes in the levels of amino and organic acids, consistent with an inhibition of triose-phosphate export from the chloroplast, but little change in the energy status of the transformants. We discuss the implications of these changes with respect to both consequences of inhibiting starch synthesis and of inhibiting photosynthesis, and conclude that a high activity of pPGM is required both to prevent phosphate limitation of photosynthesis and for co-ordination of plastidially and cytosolically compartmented photosynthetic metabolism.

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.