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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.
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.
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 x phs1a and mex1 x 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 x 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.