;We characterized three Arabidopsis genes, AtpOMT1, AtpDCT1 and AtpDCT2, localized on chromosome 5 and homologous to spinach chloroplastic 2-oxoglutarate/malate transporter (OMT) gene. The yeast-expressed recombinant AtpOMT1 protein transported malate and 2-oxoglutarate but not glutamate. By contrast, the recombinant AtpDCT1 protein transported 2-oxoglutarate and glutamate at similar affinities in exchange for malate. These findings suggested that AtpOMT1 is OMT and AtpDCT1 is a general dicarboxylate transporter (DCT). The recombinant proteins could also transport oxaloacetate at the same binding sites for dicarboxylates. In particular, the AtpOMT1 had a K m value for oxaloacetate one order of magnitude lower than those for malate and 2-oxoglutarate. Although the transcripts for the three genes were accumulated in all tissues examined, the expression of the genes in leaf tissues was light inducible. The expression of the three genes was also induced by nitrate supplement but the induction was most prominent and transient in AtpOMT1 similar to nitrate reductase gene. These findings lead to a proposition that AtpOMT1 functions as an oxaloacetate transporter in the malate-oxaloacetate shuttle across chloroplast membranes. We identified T-DNA insertional mutants of AtpOMT1 and AtpDCT1. Although the AtpOMT1 mutants could grow normally in normal air, the AtpDCT1 mutants were non-viable under the same conditions. The AtpDCT1 mutants were able to grow under the high CO 2 condition to suppress photorespiration. These findings suggested that at least AtpDCT1 is a necessary component for photorespiratory nitrogen recycling.

SUMMARYTransport of dicarboxylates across the chloroplast envelope plays an important role in transferring carbon skeletons to the nitrogen assimilation pathway and exporting reducing equivalent to the cytosol to prevent photo-inhibition (the malate valve). It was previously shown that the Arabidopsis plastidic 2-oxoglutarate/ malate transporter (AtpOMT1) and the general dicarboxylate transporter (AtpDCT1) play crucial roles at the interface between carbon and nitrogen metabolism. However, based on the in vitro transport properties of the recombinant transporters, it was hypothesized that AtpOMT1 might play a dual role, also functioning as an oxaloacetate/malate transporter, which is a crucial but currently unidentified component of the chloroplast malate valve. Here, we test this hypothesis using Arabidopsis T-DNA insertional mutants of AtpOMT1. Transport studies revealed a dramatically reduced rate of oxaloacetate uptake into chloroplasts isolated from the knockout plant. CO 2 -dependent O 2 evolution assays showed that cytosolic oxaloacetate is efficiently transported into chloroplasts mainly by AtpOMT1, and supported the absence of additional oxaloacetate transporters. These findings strongly indicate that the high-affinity oxaloacetate transporter in Arabidopsis chloroplasts is AtpOMT1. Further, the knockout plants showed enhanced photo-inhibition under high light due to greater accumulation of reducing equivalents in the stroma, indicating malfunction of the malate valve in the knockout plants. The knockout mutant showed a phenotype consistent with reductions in 2-oxoglutarate transport, glutamine synthetase/glutamate synthase activity, subsequent amino acid biosynthesis and photorespiration. Our results demonstrate that AtpOMT1 acts bi-functionally as an oxaloacetate/malate transporter in the malate valve and as a 2-oxoglutarate/malate transporter mediating carbon/nitrogen metabolism.

In C(4) plants, mesophyll (M) chloroplasts are randomly distributed along the cell walls, while bundle sheath (BS) chloroplasts are typically located in either a centripetal or centrifugal position. We investigated whether these intracellular positions are affected by environmental stresses. When mature leaves of finger millet (Eleusine coracana) were exposed to extremely high intensity light, most M chloroplasts aggregatively re-distributed to the BS side, whereas the intracellular arrangement of BS chloroplasts was unaffected. Compared with the homologous light-avoidance movement of M chloroplasts in C(3) plants, it requires extremely high light (3,000-4,000 micromol m(-2) s(-1)) and responds more slowly (distinctive movement observed in 1 h). The high light-induced movement of M chloroplasts was also observed in maize (Zea mays), another C(4) species, but with a distinct pattern of redistribution along the sides of anticlinal walls, analogous to C(3) plants. The aggregative movement of M chloroplasts occurred at normal light intensities (250-500 micromol m(-2) s(-1)) in response to environmental stresses, such as drought, salinity and hyperosmosis. Moreover, the re-arrangement of M chloroplasts was observed in field-grown C(4) plants when exposed to mid-day sunlight, but also under midsummer drought conditions. The migration of M chloroplasts was controlled by actin filaments and also induced in a light-dependent fashion upon incubation with ABA, which may be the physiological signal transducer. Together these results suggest that M and BS cells of C(4) plants have different mechanisms controlling intracellular chloroplast positioning, and that the aggregative movement of C(4) M chloroplasts is thought to be a protective response under environmental stress conditions.

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