The plastid-localized NAD-dependent malate dehydrogenase is crucial for energy homeostasis in developing Arabidopsis thaliana seeds.

In 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.

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase.

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C(3) plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck-Halliwell-Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants.

Induction of the AOX1D isoform of alternative oxidase in A. thaliana T-DNA insertion lines lacking isoform AOX1A is insufficient to optimize photosynthesis when treated with antimycin A.

Plant respiration is characterized by two pathways for electron transfer to O(2), namely the cytochrome pathway (CP) that is linked to ATP production, and the alternative pathway (AP), where electrons from ubiquinol are directly transferred to O(2) via an alternative oxidase (AOX) without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T-DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOX1A. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOX1A expression in the wild-type, led to an increased expression of AOX1D in leaves of the aox1a-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosynthesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild-type, which was only marginally affected by the inhibitor. It thus appears that AOX1D was unable to fully compensate for the loss of AOX1A when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex (GDC), suggests that the aox1a mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOX1A to optimize photosynthesis when treated with antimycin A. We suggest that the aox1a mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.

Transcriptional regulation of NADP-dependent malate dehydrogenase: comparative genetics and identification of DNA-binding proteins.

The transcriptional regulation of NADP-malate dehydrogenase (NADP-MDH) was analyzed in Arabidopsis ecotypes and other Brassicaceae. The amount of transcript increased twofold after transfer into low temperature (12 degrees C) or high light (750 microE) in all species. Analysis of the genomic DNA reveals that the NADP-MDH gene (At5g58330 in A. thaliana) in Brassicaceae is located between two other genes (At5g58320 and At5g58340 in Arabidopsis), both encoded on the opposite DNA strand. No promoter elements were identified in 5' direction of the NADP-MDH gene, and the expression of NADP-MDH was not affected in knock-out plants carrying a DNA insert in the 5' region. A yeast-one hybrid approach yielded only three DNA-binding proteins for the 500-bp fragment located upstream of the ATG sequence, but 34 proteins for its coding region. However, in Chlamydomonas and in some Poaceae, which do not possess any genes within the 1200 bp upstream region, typical promoter elements were identified. Alignments of genomic DNA reveal that, in contrast to Poaceae, the introns are highly conserved within Brassicaceae. We conclude that in Brassicaceae the majority of regulatory elements are located within the coding region. The NADP-MDH gene of both families evolved from a common precursor, similar to the gene in Chlamydomonas. Changes in the selection pressure allowed the insertion of At5g58340 into the promoter region of a common ancestor. When the demand for transcriptional regulation increased, At5g58340 disappeared in Poaceae, and a promoter developed in the 5' region. In contrast, Brassicaceae maintained At5g58340 and shifted all regulatory elements into the coding region of NADP-MDH.

Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution.

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.