Malate Transport and Metabolism in Nitrogen-Fixing Legume Nodules

Legumes form a symbiosis with rhizobia, a soil bacterium that allows them to access atmospheric nitrogen and deliver it to the plant for growth. Biological nitrogen fixation occurs in specialized organs, termed nodules, that develop on the legume root system and house nitrogen-fixing rhizobial bacteroids in organelle-like structures termed symbiosomes. The process is highly energetic and there is a large demand for carbon by the bacteroids. This carbon is supplied to the nodule as sucrose, which is broken down in nodule cells to organic acids, principally malate, that can then be assimilated by bacteroids. Sucrose may move through apoplastic and/or symplastic routes to the uninfected cells of the nodule or be directly metabolised at the site of import within the vascular parenchyma cells. Malate must be transported to the infected cells and then across the symbiosome membrane, where it is taken up by bacteroids through a well-characterized dct system. The dicarboxylate transporters on the infected cell and symbiosome membranes have been functionally characterized but remain unidentified. Proteomic and transcriptomic studies have revealed numerous candidates, but more work is required to characterize their function and localise the proteins in planta. GABA, which is present at high concentrations in nodules, may play a regulatory role, but this remains to be explored.

DCT4 - a new member of the dicarboxylate transporter family in C4 grasses

ABSTRACTMalate transport shuttles atmospheric carbon into the Calvin-Benson cycle during NADP-ME C4 photosynthesis. Previous characterizations of several plant dicarboxylate transporters (DCT) showed that they efficiently exchange malate across membranes. Here we identify and characterize a previously unknown member of the DCT family, DCT4, in Sorghum bicolor. We show that SbDCT4 exchanges malate across membranes and its expression pattern is consistent with a role in malate transport during C4 photosynthesis. SbDCT4 is not syntenic to the characterized photosynthetic gene ZmDCT2, and an ortholog is not detectable in the maize reference genome. We found that the expression patterns of DCT family genes in the leaves of Z. mays, and S. bicolor varied by cell type. Our results suggest that sub-functionalization of members of the DCT family for the transport of malate into the bundle sheath (BS) plastids occurred during the process of independent recurrent evolution of C4 photosynthesis in grasses of the PACMAD clade. This study confirms the value of using both syntenic information and gene expression profiles to assign orthology in evolutionarily related genomes.

Involvement of phosphatidylinositol metabolism in aluminum-induced malate secretion in Arabidopsis

To identify the upstream signaling of aluminum-induced malate secretion through aluminum-activated malate transporter 1 (AtALMT1), a pharmacological assay using inhibitors of human signal transduction pathways was performed. Early aluminum-induced transcription of AtALMT1 and other aluminum-responsive genes was significantly suppressed by phosphatidylinositol 4-kinase (PI4K) and phospholipase C (PLC) inhibitors, indicating that the PI4K–PLC metabolic pathway activates early aluminum signaling. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and PI4K reduced aluminum-activated malate transport by AtALMT1, suggesting that both the PI3K and PI4K metabolic pathways regulate this process. These results were validated using T-DNA insertion mutants of PI4K and PI3K-RNAi lines. A human protein kinase inhibitor, putatively inhibiting homologous calcineurin B-like protein-interacting protein kinase and/or Ca-dependent protein kinase in Arabidopsis, suppressed late-phase aluminum-induced expression of AtALMT1, which was concomitant with the induction of an AtALMT1 repressor, WRKY46, and suppression of an AtALMT1 activator, Calmodulin-binding transcription activator 2 (CAMTA2). In addition, a human deubiquitinase inhibitor suppressed aluminum-activated malate transport, suggesting that deubiquitinases can regulate this process. We also found a reduction of aluminum-induced citrate secretion in tobacco by applying inhibitors of PI3K and PI4K. Taken together, our results indicated that phosphatidylinositol metabolism regulates organic acid secretion in plants under aluminum stress.

Split personality of Aluminum Activated Malate Transporter family proteins: facilitation of both GABA and malate transport

ABSTRACTPlant aluminum activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires the presence of a specific amino acid motif in ALMTs that shares similarity with a GABA-binding site in mammalian GABAAreceptors. Here, we explore why TaALMT1-activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA]i) in both wheat root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA]ibecause TaALMT1 facilitates GABA efflux. TaALMT1-expression also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via14CGABA uptake into TaALMT1-expressingXenopus laevisoocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1F213C) prevented both GABA influx and efflux, and uncoupled the relationship between malate efflux and [GABA]i. We conclude that ALMTs are likely to act as both GABA and anion transportersin planta. GABA and malate appear to interact with ALMTs in a complex manner regulating each other’s transport, suggestive of a role for ALMTs in communicating metabolic status.