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 O2, namely the cytochrome pathway （CP） that is linked to ATP production, and the alternative pathway （AP）, where electrons from ubiquinol are directly transferred to O2 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, AOXIA. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOXIA expression in the wild-type, led to an increased expression of AOXID in leaves of the aoxla-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosyn- thesis, 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 AOX1 D was unable to fully compensate for the loss of AOXIA 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 aoxla 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 AOXIA to optimize pho- tosynthesis when treated with antimycin A. We suggest that the aoxla mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.

Contribution of the Alternative Respiratory Pathway to PSII Photoprotection in C3 and C4 Plants

The mechanism by which the mitochondrial alternative oxidase （AOX） pathway contributes to photosystem II （PSII） photoprotection is in dispute. It was generally thought that the AOX pathway protects photosystems by dissipating excess reducing equivalents exported from chloroplasts through the malate/oxaloacetate （Mal/OAA） shuttle and thus preventing the over-reduction of chloroplasts. In this study, using the aoxla Arabidopsis mutant and nine other C3 and C4 plant species, we revealed an additional action model of the AOX pathway in PSII photoprotection. Although the AOX pathway contributes to PSII photoprotection in C3 leaves treated with high light, this contribution was observed to disappear when photorespiration was suppressed. Disruption or inhibition of the AOX pathway significantly decreased the photorespiration in C3 leaves. Moreover, the AOX pathway did not respond to high light and contributed little to PSII photoprotection in C4 leaves possessing a highly active Mal/OAA shuttle but with little photorespiration. These results demonstrate that the AOX pathway contributes to PSII photoprotection in C3 plants by maintaining photo- respiration to detoxify glycolate and via the indirect export of excess reducing equivalents from chloro-plasts by the MaI/OAA shuttle. This new action model explains why the AOX pathway does not contribute to PSII photoprotection in C4 plants.

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 stain- ing 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.