Summary The international C4 rice consortium aims to introduce into rice a high capacity photosynthetic mechanism, the C4 pathway, to increase yield. The C4 pathway is characterised by a complex combination of biochemical and anatomical specialisation that ensures high CO2 partial pressure at RuBisCO sites in bundle sheath (BS) cells. Here we report an update of the progress of the C4 rice project. Since its inception in 2008 there has been an exponential growth in synthetic biology and molecular tools. Golden Gate cloning and synthetic promoter systems have facilitated gene building block approaches allowing multiple enzymes and metabolite transporters to be assembled and expressed from single gene constructs. Photosynthetic functionalisation of the BS in rice remains an important step and there has been some success overexpressing transcription factors in the cytokinin signalling network which influence chloroplast volume. The C4 rice project has rejuvenated the research interest in C4 photosynthesis. Comparative anatomical studies now point to critical features essential for the design. So far little attention has been paid to the energetics. C4 photosynthesis has a greater ATP requirement, which is met by increased cyclic electron transport in BS cells. We hypothesise that changes in energy statues may drive this increased capacity for cyclic electron flow without the need for further modification. Although increasing vein density will ultimately be necessary for high efficiency C4 rice, our modelling shows that small amounts of C4 photosynthesis introduced around existing veins could already provide benefits of increased photosynthesis on the road to C4 rice.

Every day almost one billion people suffer from chronic hunger, and the situation is expected to deteriorate with a projected population growth to 9 billion worldwide by 2050. In order to provide adequate nutrition into the future, rice yields in Asia need to increase by 60%, a change that may be achieved by introduction of the C(4) photosynthetic cycle into rice. The international C(4) Rice Consortium was founded in order to test the feasibility of installing the C(4) engine into rice. This review provides an update on two of the many approaches employed by the C(4) Rice Consortium: namely, metabolic C(4) engineering and identification of determinants of leaf anatomy by mutant screens. The aim of the metabolic C(4) engineering approach is to generate a two-celled C(4) shuttle in rice by expressing the classical enzymes of the NADP-ME C(4) cycle in a cell-appropriate manner. The aim is also to restrict RuBisCO and glycine decarboxylase expression to the bundle sheath (BS) cells of rice in a C(4)-like fashion by specifically down-regulating their expression in rice mesophyll (M) cells. In addition to the changes in biochemistry, two-celled C(4) species show a convergence in leaf anatomy that include increased vein density and reduced numbers of M cells between veins. By screening rice activation-tagged lines and loss-of-function sorghum mutants we endeavour to identify genes controlling these key traits.

C 4 photosynthesis is characterized by a CO 2 -concentrating mechanism between mesophyll (M) and bundle sheath (BS) cells of leaves. This generates high metabolic fluxes between these cells, through interconnecting plasmodesmata (PD). Quantification of these symplastic fluxes for modeling studies requires accurate quantification of PD, which has proven difficult using transmission electron microscopy. Our new quantitative technique combines scanning electron microscopy and 3D immunolocalization in intact leaf tissues to compare PD density on cell interfaces in leaves of C 3 (rice [Oryza sativa] and wheat [Triticum aestivum]) and C 4 (maize [Zea mays] and Setaria viridis) monocot species. Scanning electron microscopy quantification of PD density revealed that C 4 species had approximately twice the number of PD per pitfield area compared with their C 3 counterparts. 3D immunolocalization of callose at pitfields using confocal microscopy showed that pitfield area per M-BS interface area was 5 times greater in C 4 species. Thus, the two C 4 species had up to nine times more PD per M-BS interface area (S. viridis, 9.3 PD mm 22 ; maize, 7.5 PD mm 22 ; rice 1.0 PD mm 22 ; wheat, 2.6 PD mm 22 ). Using these anatomical data and measured photosynthetic rates in these C 4 species, we have now calculated symplastic C 4 acid flux per PD across the M-BS interface. These quantitative data are essential for modeling studies and gene discovery strategies needed to introduce aspects of C 4 photosynthesis to C 3 crops.

Summary Introduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two‐cell metabolic prototype for an NADP‐malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP‐malate dehydrogenase, pyruvate orthophosphate dikinase and NADP‐malic enzyme from Zea mays, driven by cell‐preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13CO2 labelling demonstrated a 10‐fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP‐malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.

High plasmodesmata density in C4 grasses is a result of larger pit fields and/or more abundant plasmodesmata per pit field area.

The glycine decarboxylase complex (GDC) plays a critical role in the photorespiratory C2 cycle of C3 species by recovering carbon following the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase/oxygenase. Loss of GDC from mesophyll cells (MCs) is considered a key early step in the evolution of C4 photosynthesis. To assess the impact of preferentially reducing GDC in rice MCs, we decreased the abundance of OsGDCH (Os10g37180) using an artificial microRNA (amiRNA) driven by a promoter that preferentially drives expression in MCs. GDC H- and P-proteins were undetectable in leaves of gdch lines. Plants exhibited a photorespiratory-deficient phenotype with stunted growth, accelerated leaf senescence, reduced chlorophyll, soluble protein and sugars, and increased glycine accumulation in leaves. Gas exchange measurements indicated an impaired ability to regenerate ribulose 1,5-bisphosphate in photorespiratory conditions. In addition, MCs of gdch lines exhibited a significant reduction in chloroplast area and coverage of the cell wall when grown in air, traits that occur during the later stages of C4 evolution. The presence of these two traits important for C4 photosynthesis and the non-lethal, down-regulation of the photorespiratory C2 cycle positively contribute to efforts to produce a C4 rice prototype.

One-sentence summary: 15 RNAi reduction of PEP carboxylase activity in Setaria viridis results in low CO 2 assimilation 16 rates and increased plasmodesmata density at the mesophyll-bundle sheath interface.

These authors contributed equally to this work. SUMMARYThe specification of vascular patterning in plants has interested plant biologists for many years. In the last decade a new context has emerged for this interest. Specifically, recent proposals to engineer C 4 traits into C 3 plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C 4 plants is determined. High vein density with Kranz anatomy, whereby photosynthetic cells are arranged in encircling layers around vascular bundles, is one of the major traits that differentiate C 4 species from C 3 species. To identify genetic factors that specify C 4 leaf anatomy, we generated ethyl methanesulfonate-and cray-mutagenized populations of the C 4 species sorghum (Sorghum bicolor), and screened for lines with reduced vein density. Two mutations were identified that conferred low vein density. Both mutations segregated in backcrossed F 2 populations as homozygous recessive alleles. Bulk segregant analysis using nextgeneration sequencing revealed that, in both cases, the mutant phenotype was associated with mutations in the CYP90D2 gene, which encodes an enzyme in the brassinosteroid biosynthesis pathway. Lack of complementation in allelism tests confirmed this result. These data indicate that the brassinosteroid pathway promotes high vein density in the sorghum leaf, and suggest that differences between C 4 and C 3 leaf anatomy may arise in part through differential activity of this pathway in the two leaf types.