DCT4—A New Member of the Dicarboxylate Transporter Family in C4 Grasses

Abstract

Malate 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. We also show that this sub-functionalization is lineage independent. Our results challenge the dogma that key C4 genes must be orthologues of one another among C4 species, and shed new light on the evolution of C4 photosynthesis.

Investigating thecis-Regulatory Basis of C3and C4Photosynthesis in Grasses at Single-Cell Resolution

“…S. bicolor has a BS-specific copy of SbDIT4, which shares a clade with ZmDIT1 . These results are consistent with earlier studies that found similar patterns and gene expression profiles of these copies of the DITs in Z. mays and S. bicolor (33,36,55). Although previous studies have documented changes in cell-type-specific gene expression for the BS-specific copies of the DITs, the mechanisms underlying these changes remain unclear.…”

“…However, upon further inspection of the phylogenies of the DITs in S. bicolor, we noticed a pattern where the most BS-biased copy, SbDIT4 (Sobic.004G035500), was phylogenetically more closely related to the ZmDIT1. Something which has been previously reported (33,36). These results indicate that over evolutionary time, even members of the same C 4 photosynthetic subtype, which likely share a C4 ancestor, can use different paralogous loci to achieve cell-type-specific expression.…”

The metabolite transporters of C4 photosynthesis.

“…Although DCT2 is likely involved in malate uptake in maize, different C4 species have subfunctionalised different DCT subfamily members. For example, other NADP-ME subtype C4 species Sorghum bicolor (sorghum), Setaria italica, and Setaria viridis show strong bundle sheath-specific expression of DCT4, a duplicate of DCT1 that is not found in maize [44][45][46] (Figure 4C). In contrast to NADP-ME, the NAD-ME species Panicum virgatum (Figure 4C) and Cleome gynandra, and the PCK species Urochloa fusca, have low expression of DCT genes [46][47][48] .…”

“…For example, other NADP-ME subtype C4 species Sorghum bicolor (sorghum), Setaria italica, and Setaria viridis show strong bundle sheath-specific expression of DCT4, a duplicate of DCT1 that is not found in maize [44][45][46] (Figure 4C). In contrast to NADP-ME, the NAD-ME species Panicum virgatum (Figure 4C) and Cleome gynandra, and the PCK species Urochloa fusca, have low expression of DCT genes [46][47][48] . The absence of this transporter from species that do not take up malate into bundle sheath chloroplasts further supports the proposed role of DCT2/4 in malate uptake into the bundle sheath chloroplasts of NADP-ME C4 species.…”

Transport Proteins Enabling Plant Photorespiratory Metabolism

“…The malate transport could be coordinated with aspartate transport, but, if malate would be exchanged with aspartate, a second transporter for aspartate uptake into the plastid would be necessary [ 99 ]. In S. bicolor , another member of the gene family, DCT4, seems to be the predominant transporter in the bundle sheath [ 100 ]. For the export of pyruvate out of the bundle sheath plastid, no transporter could be identified so far.…”