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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]

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]