The Plastid-Localized NAD-Dependent Malate Dehydrogenase Is Crucial for Energy Homeostasis in Developing Arabidopsis thaliana Seeds

ABSTRACTIn the absence of photosynthesis, ATP is imported into chloroplasts and non-green plastids by ATP/ADP transporters or formed during glycolysis, the latter requiring continuous regeneration of NAD+, supplied by the plastidial isoform of NAD–MDH. During screening for T-DNA insertion mutants in the plNAD–MDH gene of Arabidopsis, only heterozygous plants could be isolated and homozygous knockout mutants grew only after complementation. These heterozygous plants show higher transcript levels of an alternative NAD+-regenerating enzyme, NADH–GOGAT, and, remarkably, improved growth when ammonium is the sole N-source. In situ hybridization and GUS-histochemical staining revealed that plNAD–MDH was particularly abundant in male and female gametophytes. Knockout plNAD–MDH pollen exhibit impaired tube growth in vitro, which can be overcome by adding the substrates of NADH–GOGAT. In vivo, knockout pollen is able to fertilize the egg cell. Young siliques of selfed heterozygous plants contain both green and white seeds corresponding to wild-type/heterozygous (green) and homozygous knockout mutants (white) in a (1:2):1 ratio. Embryos of the homozygous knockout seeds only reached the globular stage, did not green, and developed to tiny wrinkled seeds. Complementation with the gene under the native promoter rescued this defect, and all seeds developed as wild-type. This suggests that a blocked major physiological process in plNAD–MDH mutants stops both embryo and endosperm development, thus avoiding assimilate investment in compromised offspring.SUMMARYEnergy supply from plastidial glycolysis is dependent upon plastidial NAD–MDH for removal of NADH. A decrease is compensated by NADH–GOGAT. A homozygous plNAD–MDH knockout in gametophytes is overcome by GOGAT substrates, but embryo development stops at the globular stage.