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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 Zea mays, and S. bicolor varied by cell type. Our results suggest that subfunctionalization, of members of the DCT family, for the transport of malate into the bundle sheath plastids, occurred during the process of independent recurrent evolution of C4 photosynthesis in grasses of the PACMAD clade. We also show that this subfunctionalization 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.

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. This study confirms the value of using both syntenic information and gene expression profiles to assign orthology in evolutionarily related genomes.

C4 photosynthesis in grasses requires the coordinated movement of metabolites through two specialized leaf cell types, mesophyll (M) and bundle sheath (BS), to concentrate CO2 around Rubisco. Despite the importance of transporters in this process, few have been identified or rigorously characterized. In maize (Zea mays), DCT2 has been proposed to function as a plastid-localized malate transporter and is preferentially expressed in BS cells. Here, we characterized the role of DCT2 in maize leaves using Activator-tagged mutant alleles. Our results indicate that DCT2 enables the transport of malate into the BS chloroplast. Isotopic labeling experiments show that the loss of DCT2 results in markedly different metabolic network operation and dramatically reduced biomass production. In the absence of a functioning malate shuttle, dct2 lines survive through the enhanced use of the phosphoenolpyruvate carboxykinase carbon shuttle pathway that in wild-type maize accounts for ∼ 25% of the photosynthetic activity. The results emphasize the importance of malate transport during C4 photosynthesis, define the role of a primary malate transporter in BS cells, and support a model for carbon exchange between BS and M cells in maize.

C₄ and C₃ photosynthesis differ in the efficiency with which they consume water and nitrogen. Engineering traits of the more efficient C₄ photosynthesis into C₃ crops could substantially increase crop yields in hot, arid conditions. To identify differences between C₄ and C₃ photosynthetic mechanisms, we profiled metabolites and gene expression in the developing leaves of Zea mays (maize), a C₄ plant, and Oryza sativa (rice), a C₃ plant, using a statistical method named the unified developmental model (UDM). Candidate cis-regulatory elements and transcription factors that might regulate photosynthesis were identified, together with differences between C₄ and C₃ nitrogen and carbon metabolism. The UDM algorithms could be applied to analyze and compare development in other species. These data sets together with community viewers to access and mine them provide a resource for photosynthetic research that will inform efforts to engineer improvements in carbon fixation in economically valuable grass crops.

The Runx3 transcription factor is essential for development and diversification of the dorsal root ganglia (DRGs) TrkC sensory neurons. In Runx3-deficient mice, developing TrkC neurons fail to extend central and peripheral afferents, leading to cell death and disruption of the stretch reflex circuit, resulting in severe limb ataxia. Despite its central role, the mechanisms underlying the spatiotemporal expression specificities of Runx3 in TrkC neurons were largely unknown. Here we first defined the genomic transcription unit encompassing regulatory elements (REs) that mediate the tissue-specific expression of Runx3. Using transgenic mice expressing BAC reporters spanning the Runx3 locus, we discovered three REs—dubbed R1, R2, and R3—that cross-talk with promoter-2 (P2) to drive TrkC neuron-specific Runx3 transcription. Deletion of single or multiple elements either in the BAC transgenics or by CRISPR/Cas9-mediated endogenous ablation established the REs’ ability to promote and/or repress Runx3 expression in developing sensory neurons. Our analysis reveals that an intricate combinatorial interplay among the three REs governs Runx3 expression in distinct subtypes of TrkC neurons while concomitantly extinguishing its expression in non-TrkC neurons. These findings provide insights into the mechanism regulating cell type-specific expression and subtype diversification of TrkC neurons in developing DRGs.

C4 photosynthesis is a complex metabolic pathway responsible for carbon fixation in major feed, food and bioenergy crops. Although many enzymes driving this pathway have been identified, regulatory mechanisms underlying this system remain elusive. C4 photosynthesis contributes to photosynthetic efficiency in major bioenergy crops such as sugarcane, Miscanthus, switchgrass, maize and sorghum, and international efforts are underway to engineer C4 photosynthesis into C3 crops. A fundamental understanding of the C4 network is thus needed. New experimental and informatics methods can facilitate the accumulation and analysis of high-throughput data to define components of the C4 system. The use of new model plants, closely related to C4 crops, will also contribute to our understanding of the mechanisms that regulate this complex and important pathway.

Durum and bread wheat need transgenic traits such as herbicide and disease resistance due to recent evolution of herbicide resistant grass weeds and an intractable new strain of stem rust. Transgenic wheat varieties have not been commercialized partly due to potential transgene movement to wild/weedy relatives, which occurs naturally to closely related Aegilops and other spp. Recombination does not occur in the F 1 hybrid between wheat and its relatives due to the presence of the Ph 1 gene on wheat chromosome arm 5BL, which acts as a chaperone, preventing promiscuous homoeologous pairing to similar, but not homologous chromosomes of the wild/weedy species. Thus recombination must occur during backcrossing after the wheat Ph 1 gene has been eliminated. Based on these findings, we speculate that Ph 1 could be used to prevent gene introgression into weedy relatives. We propose two methods to prevent such transgene establishment: (1) link the transgene in proximity to the wheat Ph 1 gene and (2) insert the transgene in tandem with the lethal barnase on any chromosome arm other than 5BL, and insert barstar , which suppresses barnase on chromosome arm 5BL in proximity to Ph 1. The presence of Ph 1 in backcross plants containing 5BL will prevent the homoeologous establishment of barnase coupled to the desired transgene in the wild population. 5BL itself will be eliminated during repeated backcrossing to the wild parent, and progeny bearing the desired transgene in tandem with barnase but without the Ph 1- barstar complex will die.

Leaves of C4 crops usually have higher radiation, water and nitrogen use efficiencies compared to the C3 species. Engineering C4 traits into C3 crops has been proposed as one of the most promising ways to repeal the biomass yield ceiling. To better understand the function of C4 photosynthesis, and to identify candidate genes that are associated with the C4 pathways, a comparative transcription network analysis was conducted on leaf developmental gradients of three C4 species including maize, green foxtail and sorghum and one C3 species, rice. By combining the methods of gene co-expression and differentially co-expression networks, we identified a total of 128 C4 specific genes. Besides the classic C4 shuttle genes, a new set of genes associated with light reaction, starch and sucrose metabolism, metabolites transportation, as well as transcription regulation, were identified as involved in C4 photosynthesis. These findings will provide important insights into the differential gene regulation between C3 and C4 species, and a good genetic resource for establishing C4 pathways in C3 crops.

Background Comparisons between C3 and C4 grasses often utilize C3 species from the subfamilies Ehrhartoideae or Pooideae and C4 species from the subfamily Panicoideae, two clades that diverged over 50 million years ago. The divergence of the C3 panicoid grass Dichanthelium oligosanthes from the independent C4 lineages represented by Setaria viridis and Sorghum bicolor occurred approximately 15 million years ago, which is significantly more recent than members of the Bambusoideae, Ehrhartoideae, and Pooideae subfamilies. D. oligosanthes is ideally placed within the panicoid clade for comparative studies of C3 and C4 grasses. Results We report the assembly of the nuclear and chloroplast genomes of D. oligosanthes, from high-throughput short read sequencing data and a comparative transcriptomics analysis of the developing leaf of D. oligosanthes, S. viridis, and S. bicolor. Physiological and anatomical characterizations verified that D. oligosanthes utilizes the C3 pathway for carbon fixation and lacks Kranz anatomy. Expression profiles of transcription factors along developing leaves of D. oligosanthes and S. viridis were compared with previously published data from S. bicolor, Zea mays, and Oryza sativa to identify a small suite of transcription factors that likely acquired functions specifically related to C4 photosynthesis. Conclusions The phylogenetic location of D. oligosanthes makes it an ideal C3 plant for comparative analysis of C4 evolution in the panicoid grasses. This genome will not only provide a better C3 species for comparisons with C4 panicoid grasses, but also highlights the power of using high-throughput sequencing to address questions in evolutionary biology. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1080-3) contains supplementary material, which is available to authorized users.

C₄ and C₃ photosynthesis differ in the efficiency with which they consume water and nitrogen. Engineering traits of the more efficient C₄ photosynthesis into C₃ crops could substantially increase crop yields in hot, arid conditions. To identify differences between C₄ and C₃ photosynthetic mechanisms, we profiled metabolites and gene expression in the developing leaves of Zea mays (maize), a C₄ plant, and Oryza sativa (rice), a C₃ plant, using a statistical method named the unified developmental model (UDM). Candidate cis-regulatory elements and transcription factors that might regulate photosynthesis were identified, together with differences between C₄ and C₃ nitrogen and carbon metabolism. The UDM algorithms could be applied to analyze and compare development in other species. These data sets together with community viewers to access and mine them provide a resource for photosynthetic research that will inform efforts to engineer improvements in carbon fixation in economically valuable grass crops.

Comparisons between C3 and C4 grasses often utilize C3 species from the subfamilies Ehrhartoideae or Pooideae and C4 species from the subfamily Panicoideae, two clades that diverged over 50 million years ago. The divergence of the C3 panicoid grass Dichanthelium oligosanthes from the independent C4 lineages represented by Setaria viridis and Sorghum bicolor occurred approximately 15 million years ago, which is significantly more recent than members of the Bambusoideae, Ehrhartoideae, and Pooideae subfamilies. D. oligosanthes is ideally placed within the panicoid clade for comparative studies of C3 and C4 grasses. We report the assembly of the nuclear and chloroplast genomes of D. oligosanthes, from high-throughput short read sequencing data and a comparative transcriptomics analysis of the developing leaf of D. oligosanthes, S. viridis, and S. bicolor. Physiological and anatomical characterizations verified that D. oligosanthes utilizes the C3 pathway for carbon fixation and lacks Kranz anatomy. Expression profiles of transcription factors along developing leaves of D. oligosanthes and S. viridis were compared with previously published data from S. bicolor, Zea mays, and Oryza sativa to identify a small suite of transcription factors that likely acquired functions specifically related to C4 photosynthesis. The phylogenetic location of D. oligosanthes makes it an ideal C3 plant for comparative analysis of C4 evolution in the panicoid grasses. This genome will not only provide a better C3 species for comparisons with C4 panicoid grasses, but also highlights the power of using high-throughput sequencing to address questions in evolutionary biology.