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In illuminated chloroplasts, one mechanism involved in reduction-oxidation (redox) homeostasis is the malate-oxaloacetate (OAA) shuttle. Excess electrons from photosynthetic electron transport in the form of nicotinamide adenine dinucleotide phosphate, reduced are used by NADP-dependent malate dehydrogenase (MDH) to reduce OAA to malate, thus regenerating the electron acceptor NADP. NADP-MDH is a strictly redox-regulated, light-activated enzyme that is inactive in the dark. In the dark or in nonphotosynthetic tissues, the malate-OAA shuttle was proposed to be mediated by the constitutively active plastidial NAD-specific MDH isoform (pdNAD-MDH), but evidence is scarce. Here, we reveal the critical role of pdNAD-MDH in Arabidopsis (Arabidopsis thaliana) plants. A pdnad-mdh null mutation is embryo lethal. Plants with reduced pdNAD-MDH levels by means of artificial microRNA (miR-mdh-1) are viable, but dark metabolism is altered as reflected by increased nighttime malate, starch, and glutathione levels and a reduced respiration rate. In addition, miR-mdh-1 plants exhibit strong pleiotropic effects, including dwarfism, reductions in chlorophyll levels, photosynthetic rate, and daytime carbohydrate levels, and disordered chloroplast ultrastructure, particularly in developing leaves, compared with the wild type. pdNAD-MDH deficiency in miR-mdh-1 can be functionally complemented by expression of a microRNA-insensitive pdNAD-MDH but not NADP-MDH, confirming distinct roles for NAD- and NADP-linked redox homeostasis. [ABSTRACT FROM AUTHOR]

Cytosolic phosphoglucomutase (cPGM) interconverts glucose-6-phosphate and glucose-l-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 effeciively 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. [ABSTRACT FROM AUTHOR]

The aim of this work was to evaluate the function of isoamylase in starch granule biosynthesis in Arabidopsis leaves. A reverse-genetic approach was used to knockoutAtISA1, one of three genes in Arabidopsis encoding isoamylase-type debranching enzymes. The mutant (Atisa1-1) lacks functionalAtISA1transcript and the major isoamylase activity (detected by native gels) in crude extracts of leaves. The same activity is abolished by mutation at theDBE1locus, which encodes a second isoamylase-type protein, AtISA2. This is consistent with the idea that ISA1 and ISA2 proteins are subunits of the same enzymein vivo.Atisa1-1,Atisa2-1 (dbe1), and theAtisa1-1/Atisa2-1 double mutant all have identical phenotypes. Starch content is reduced compared with the wild type but substantial quantities of the soluble glucan phytoglycogen are produced. The amylopectin of the remaining starch and the phytoglycogen in the mutants are structurally related to each other and differ from wild-type amylopectin. Electron micrographs reveal that the phytoglycogen-accumulating phenotype is highly tissue-specific. Phytoglycogen accumulates primarily in the plastids of the palisade and spongy mesophyll cells. Remarkably, other cell types appear to accumulate only starch, which is normal in appearance but is altered in structure. As phytoglycogen accumulates during the day, its rate of accumulation decreases, its structure changes and intermediates of glucan breakdown accumulate, suggesting that degradation occurs simultaneously with synthesis. We conclude that the AtISA1/AtISA2 isoamylase influences glucan branching pattern, but that this may not be the primary determinant of partitioning between crystalline starch and soluble phytoglycogen. [ABSTRACT FROM AUTHOR]

The aim of this work was to understand the initial steps of starch breakdown inside chloroplasts. In the non-living endosperm of germinating cereal grains, starch breakdown is initiated by α-amylase secreted from surrounding cells. However, loss of α-amylase from Arabidopsis does not prevent chloroplastic starch breakdown (Yu, T.-S., Zeeman, S. C., Thorneycroft, D., Fulton, D. C., Dunstan, H., Lue, W.-L., Hegemann, B., Tung, S.-Y., Umemoto, T., Chapple, A., Tsai, D.-L., Wang, S.-M, Smith, A. M., Chen, J., and Smith, S. M. (2005)1 Biol. Chem. 280,9773-9779), implying that other enzymes must attack the starch granule. Here, we present evidence that the debranching enzyme isoamylase 3 (ISA3) acts at the surface of the starch granule. Atisa3 mutants have more leaf starch and a slower rate of starch breakdown than wild-type plants. The amylopectin of Atisa3 contains many very short branches and ISA3-GFP localizes to granule-like structures inside chloroplasts. We suggest that ISA3 removes short branches from the granule surface. To understand how some starch is still degraded in Atisa3 mutants we eliminated a second debranching enzyme, limit dextrinase (pullulanase-type). Atlda mutants are indistinguishable from the wild type. However, the Atisa3/Atlda double mutant has a more severe starch-excess phenotype and a slower rate of starch breakdown than Atisa3 single mutants. The double mutant accumulates soluble branched oligo- saccharides (limit dextrins) that are undetectable in the wild-type and the single mutants. Together these results suggest that glucan debranching occurs primarily at the granule surface via 15A3, but in its absence soluble branched glucans are debranched in the stroma via limit dextrinase. Consistent with this model, chloroplastic a-amylase AtAMY3, which could release soluble branched glucans, is induced in Atisa3 and in the Atisa3/Atlda double mutant. [ABSTRACT FROM AUTHOR]