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Primary carbohydrate metabolism in plants includes several sugar and sugar-derivative transport processes. Over recent years, evidences have shown that in starch-related transport processes, in addition to glucose 6-phosphate, maltose, glucose and triose-phosphates, glucose 1-phosphate also plays a role and thereby increases the possible fluxes of sugar metabolites in planta. In this study, we report the characterization of two highly similar transporters, At1g34020 and At4g09810, in Arabidopsis thaliana, which allow the import of glucose 1-phosphate through the plasma membrane. Both transporters were expressed in yeast and were biochemically analyzed to reveal an antiport of glucose 1-phosphate/phosphate. Furthermore, we showed that the apoplast of Arabidopsis leaves contained glucose 1-phosphate and that the corresponding mutant of these transporters had higher glucose 1-phosphate amounts in the apoplast and alterations in starch and starch-related metabolism., (© The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Starch phosphorylation by starch-related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50-kDa starch-binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α-glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

The process of starch granule formation in leaves of Arabidopsis ( Arabidopsis thaliana ) is obscure. Besides STARCH SYNTHASE4 (SS4), the PLASTIDIAL PHOSPHORYLASE (PHS1) also seems to be involved, since dpe2-1/phs1a double mutants lacking both PHS1 and the cytosolic DISPROPORTIONATING ENZYME2 (DPE2) displayed only one starch granule per chloroplast under normal growth conditions. For further studies, a dpe2-1/phs1a/ss4 triple mutant and various combinations of double mutants were generated and metabolically analyzed with a focus on starch metabolism. The dpe2-1/phs1a/ss4 mutant revealed a massive starch excess phenotype. Furthermore, these plants grown under 12 h of light/12 h of dark harbored a single large and spherical starch granule per plastid. The number of starch granules was constant when the light/dark regime was altered, but this was not observed in the parental lines. With regard to growth, photosynthetic parameters, and metabolic analyses, the triple mutant additionally displayed alterations in comparison with ss4 and dpe2-1/phs1a The results clearly illustrate that PHS1 and SS4 are differently involved in starch granule formation and do not act in series. However, SS4 appears to exert a stronger influence. In connection with the characterized double mutants, we discuss the generation of starch granules and the observed formation of spherical starch granules., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

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.

Carbohydrate metabolism in plants is tightly linked to photosynthesis and is essential for energy and carbon skeleton supply of the entire organism. Thus, the hexose phosphate pools of the cytosol and the chloroplast represent important metabolic resources that are maintained through action of phosphoglucose isomerase (PGI) and phosphoglucose mutase interconverting glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. Here, we investigated the impact of disrupted cytosolic PGI (cPGI) function on plant viability and metabolism. Overexpressing an artificial microRNA targeted against cPGI (amiR-cpgi) resulted in adult plants with vegetative tissue essentially free of cPGI activity. These plants displayed diminished growth compared with the wild type and accumulated excess starch in chloroplasts but maintained low sucrose content in leaves at the end of the night. Moreover, amiR-cpgi plants exhibited increased nonphotochemical chlorophyll a quenching during photosynthesis. In contrast to amiR-cpgi plants, viable transfer DNA insertion mutants disrupted in cPGI function could only be identified as heterozygous individuals. However, homozygous transfer DNA insertion mutants could be isolated among plants ectopically expressing cPGI. Intriguingly, these plants were only fertile when expression was driven by the ubiquitin10 promoter but sterile when the seed-specific unknown seed protein promoter or the Cauliflower mosaic virus 35S promoter were employed. These data show that metabolism is apparently able to compensate for missing cPGI activity in adult amiR-cpgi plants and indicate an essential function for cPGI in plant reproduction. Moreover, our data suggest a feedback regulation in amiR-cpgi plants that fine-tunes cytosolic sucrose metabolism with plastidic starch turnover., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

In leaves of two starch-related single-knockout lines lacking either the cytosolic transglucosidase (also designated as disproportionating enzyme 2, DPE2) or the maltose transporter (MEX1), the activity of the plastidial phosphorylase isozyme (PHS1) is increased. In both mutants, metabolism of starch-derived maltose is impaired but inhibition is effective at different subcellular sites. Two constitutive double knockout mutants were generated (designated as dpe2-1×phs1a and mex1×phs1b) both lacking functional PHS1. They reveal that in normally grown plants, the plastidial phosphorylase isozyme participates in transitory starch degradation and that the central carbon metabolism is closely integrated into the entire cell biology. All plants were grown either under continuous illumination or in a light-dark regime. Both double mutants were compromised in growth and, compared with the single knockout plants, possess less average leaf starch when grown in a light-dark regime. Starch and chlorophyll contents decline with leaf age. As revealed by transmission electron microscopy, mesophyll cells degrade chloroplasts, but degradation is not observed in plants grown under continuous illumination. The two double mutants possess similar but not identical phenotypes. When grown in a light-dark regime, mesophyll chloroplasts of dpe2-1×phs1a contain a single starch granule but under continuous illumination more granules per chloroplast are formed. The other double mutant synthesizes more granules under either growth condition. In continuous light, growth of both double mutants is similar to that of the parental single knockout lines. Metabolite profiles and oligoglucan patterns differ largely in the two double mutants.

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., (© 2013 Scandinavian Plant Physiology Society.)

Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases, DPE2 and PHS2 (or, in all other species, Pho2). In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids. In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Both photoautotrophic and heterotrophic tissues from plants are capable of synthesizing and degrading starch. To analyse starch metabolism in the two types of tissue from the same plant, several starch-related mutants from Arabidopsis thaliana were grown hydroponically together with the respective wild type control. Starch contents, patterns of starch-related enzymes, and the monomer patterns of the cytosolic starch-related heteroglycans were determined. Based on the phenotypical data obtained, three comparisons were made: First, data from leaves and roots of the mutants were compared with the respective wild type controls. Secondly, data from leaves and roots from the same plant were compared. Third, we included data obtained from soil-grown plants and compared them with those from hydroponically grown plants. Thus, phenotypical features reflecting altered gene expression can be distinguished from those that are due to the specific growth conditions. Implications on the carbon fluxes in photoautotrophic and heterotrophic cells are discussed.

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.