On the road to C₄ rice: advances and perspectives

Abstract

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.

Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.)

“…All hits of the same gene satisfying the criteria were plotted based onlog (E-value); only hits of top -log (E-value) class were considered if clear differentiation among them was visualized, otherwise all hits were used. biosynthetic pathway of C4 photosynthesis (adapted from [40]…”

A partial C4photosynthetic biochemical pathway in rice

“…The very low incorporation of 13 C into citrate and isocitrate in wild-type rice plants is In addition to the metabolic enzymes of the C 4 pathway, there is a need to identify and 489 overproduce the metabolite transporters required to support C 4 photosynthesis (Weber 490 and von Caemmerer 2010; Ermakova et al 2019). While many of the necessary 491 transporters may be present at low levels in C 3 chloroplasts (Weber and von 492…”

“…properties for diffusion of CO 2 (von Caemmerer and Furbank 2003) and increasing 509 plasmodesmatal frequency at the BSC / MC interface to support metabolite diffusion 510 may be necessary (Ermakova et al 2019). The genetic regulators of many of these 511 changes are not known, and so future goals include identification and incorporation of 512 necessary genes for anatomical modifications into a version of the current biochemical 513 prototype, with the ultimate goal of engineering an efficient C 4 pathway in rice.…”

“…A major recent research objective has been the engineering of a C 4 photosynthetic 56 pathway into rice (https://C4rice.com; Kajala et al, 2011;von Caemmerer et al 2012; 57 Ermakova et al 2019), potentially leading to an increase in radiation use efficiency and 58 yield of up to 50% (Hibberd et al 2008). The C 4 pathway represents a complex 59 combination of both biochemical and anatomical adaptations that suppresses 60 photorespiration by effectively saturating ribulose bisphosphate carboxylase/oxygenase 61 (Rubisco) with CO 2 .…”

Functional reconstitution of a bacterial CO2concentrating mechanism inE. coli

“…As CCMs accelerate CO2 fixation, there is great interest in transplanting them into crops 73 (Ermakova et al, 2020;McGrath and Long, 2014 components -energy-coupled carbon uptake and carboxysome structures that encapsulate rubisco with a carbonic 91 anhydrase (CA) enzyme (Mangan et al, 2016;McGrath and Long, 2014 Δrpi), however, makes Ru5P a metabolic "dead-end" (Figure 2A). Expression of 141 phosphoribulokinase (prk) and rubisco creates a "detour" pathway converting Ru5P and CO2 into 142 two units of the central metabolite 3-phosphoglycerate (3PG), enabling Ru5P metabolism and 143 growth ( Figure 2A).…”