The Arabidopsis mutant dct is deficient in the plastidic glutamate/malate translocator DiT2

Abstract

SummaryThe Arabidopsis mutant dicarboxylate transport (dct ) is one of the classic mutants in the photorespiratory pathway. It requires high CO 2 levels for survival. Physiologic and biochemical characterization of dct indicated that dct is de®cient in the transport of dicarboxylates across the chloroplast envelope membrane. Hence, re-assimilation of ammonia generated by the photorespiratory cycle is blocked. However, the defective gene in dct has not been identi®ed at the molecular level. Here, we report on the molecular characterization of the defective gene in dct, on the complementation of the mutant phenotype with a wild-type cDNA, and on the functional characterization of the gene product, DiT2, in a recombinant reconstituted system. Furthermore, we provide the kinetic constants of recombinant DiT1 and DiT2, and we discuss these data with respect to their functions in ammonia assimilation. Moreover, an analysis of the transcript levels of DiT1 and DiT2 in different C 3 -and C 4 -type plant species is presented, and we demonstrate that the substrate speci®city of DiT2 from the C 4 -plant Flaveria bidentis is similar to its counterpart from C 3 plants.

Understanding metabolite transport and metabolism in C4 plants through RNA-seq

“…Abundant cytosolic CA activity can be achieved by upregulating the cytosolic CA [5 ] or by retargeting the already abundant plastidic CA [31,32]. Its mesophyll specificity is regulated post-transcriptionally [36]. The importance of the enzyme apparently varies among species.…”

“…The transport protein catalyzing the import of malate and/or the export of pyruvate is unknown at the molecular level ( Table 1). The dicarboxylate transporter DiT2 (alternate name DCT) was considered a candidate transporter for malate in maize [35], however, the Flaveria trinervia DiT2 is unable to catalyze the reaction based on in vitro analysis of the transport substrates [36]. DiT2 is moderately upregulated in conjunction with NADP-ME [6][7][8] and DiT2 is strongly expressed in maize bundle sheath cells (Table 1) and S. bicolor bundle sheath cells [25].…”

“…DiT2 is moderately upregulated in conjunction with NADP-ME [6][7][8] and DiT2 is strongly expressed in maize bundle sheath cells (Table 1) and S. bicolor bundle sheath cells [25]. Mutant analysis of maize showed symptoms consistent with a role in malate import in the C 4 cycle [37 ] raising the question whether the original assay was unsuitable for detecting the exchange [36] or whether Flaveria and maize may use different transporters (Figure 1d). The molecular identity of the pyruvate exporter is currently unknown but it cannot be excluded that pyruvate may rely on diffusion to pass the chloroplast membrane in its electroneutral form, pyruvic acid [38,39], especially given the high concentrations of pyruvate generated by NADP-ME.…”

“…OAA is reduced in the chloroplasts after counter-exchange with malate by dicarboxylate translocator 1 (DiT1) across the plastid envelope [45]. Alternatively, the OAA is converted to aspartate (Asp) in chloroplasts by plastidic aspartate transaminase (AspAT) [12,46] after import by DiT2 [36] in exchange for aspartate. Both enzymes and transporters are moderately upregulated [7]; in maize, AspAT, MDH, and DiT1 are mesophyll specific while DiT2 is bundle sheath specific ( Table 1).…”

High-throughput colorimetric method for the parallel assay of glyoxylic acid and ammonium in a single extract

“…Mutations in enzymes of this pathway usually are lethal, underlining the importance of this salvage pathway [2][3][4]. Although all enzymes and some of the metabolite transporters involved in this highly compartmentalized pathway have been identified [5][6][7][8][9], information about the majority of the transport proteins and the processes regulating the pathway is still missing [1,4], possibly because mutations in the corresponding genes cause subtle metabolic phenotypes. Thus, a high-throughput method to quantify metabolites known to accumulate in photorespiratory mutants, glyoxylate and ammonium, is desirable.…”