Engineering chloroplast development in rice through cell‐specific control of endogenous genetic circuits

Summary: The engineering of C4 photosynthetic activity into the C3 plant rice has the potential to nearly double rice yields. To engineer a two‐cell photosynthetic system in rice, the rice bundle sheath (BS) must be rewired to enhance photosynthetic capacity. Here, we show that BS chloroplast biogenesis is enhanced when the transcriptional activator, Oryza sativa Cytokinin GATA transcription factor 1 (OsCGA1), is driven by a vascular specific promoter. Ectopic expression of OsCGA1 resulted in increased BS chloroplast planar area and increased expression of photosynthesis‐associated nuclear genes (PhANG), required for the biogenesis of photosynthetically active chloroplasts in BS cells of rice. A further refinement using a DNAse dead Cas9 (dCas9) activation module driven by the same cell‐type specific promoter, directed enhanced chloroplast development of the BS cells when gRNA sequences were delivered by the dCas9 module to the promoter of the endogenous OsCGA1 gene. Single gRNA expression was sufficient to mediate the transactivation of both the endogenous gene and a transgenic GUS reporter fused with OsCGA1 promoter. Our results illustrate the potential for tissue‐specific dCas9‐activation and the co‐regulation of genes needed for multistep engineering of C4 rice

Re-creation of a Key Step in the Evolutionary Switch from C3 to C4 Leaf Anatomy

The C4 photosynthetic pathway accounts for ∼25% of primary productivity on the planet despite being used by only 3% of species. Because C4 plants are higher yielding than C3 plants, efforts are underway to introduce the C4 pathway into the C3 crop rice. This is an ambitious endeavor; however, the C4 pathway evolved from C3 on multiple independent occasions over the last 30 million years, and steps along the trajectory are evident in extant species. One approach toward engineering C4 rice is to recapitulate this trajectory, one of the first steps of which was a change in leaf anatomy. The transition from C3 to so-called “proto-Kranz” anatomy requires an increase in organelle volume in sheath cells surrounding leaf veins. Here we induced chloroplast and mitochondrial development in rice vascular sheath cells through constitutive expression of maize GOLDEN2-LIKE genes. Increased organelle volume was accompanied by the accumulation of photosynthetic enzymes and by increased intercellular connections. This suite of traits reflects that seen in “proto-Kranz” species, and, as such, a key step toward engineering C4 rice has been achieved.Research was funded by a C4 Rice Project grant from The Bill & Melinda Gates Foundation to IRRI (2012–2015; OPPGD1394) and the University of Oxford (2015–2019; OPP1129902)

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter disturbs carbon and nitrogen homeostasis in rice

The chloroplastic 2-oxaloacetate (OAA)/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of OAA. Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing 2-oxoglutarate for the GS/GOGAT (glutamine synthetase/glutamate synthase) reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis: OAA resulting from phosphoenolpyruvate carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-malate dehydrogenase, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. To engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased activity of the transporter in transgenic rice was confirmed by reconstitution of transporter activity into proteoliposomes. Unexpectedly, overexpression of ZmOMT1 in rice negatively affected growth, CO2 assimilation rate, total free amino acid content, tricarboxylic acid cycle metabolites, as well as sucrose and starch contents. Accumulation of high amounts of aspartate and the impaired growth phenotype of OMT1 rice lines could be suppressed by simultaneous overexpression of ZmDiT2. Implications for engineering C4 rice are discussed

From proto-Kranz to C4 Kranz: building the bridge to C4 photosynthesis

In this review, we examine how the specialized “Kranz” anatomy of C4 photosynthesis evolved from C3 ancestors. Kranz anatomy refers to the wreath-like structural traits that compartmentalize the biochemistry of C4 photosynthesis and enables the concentration of CO2 around Rubisco. A simplified version of Kranz anatomy is also present in the species that utilize C2 photosynthesis, where a photorespiratory glycine shuttle concentrates CO2 into an inner bundle-sheath-like compartment surrounding the vascular tissue. C2 Kranz is considered to be an intermediate stage in the evolutionary development of C4 Kranz, based on the intermediate branching position of C2 species in 14 evolutionary lineages of C4 photosynthesis. In the best-supported model of C4 evolution, Kranz anatomy in C2 species evolved from C3 ancestors with enlarged bundle sheath cells and high vein density. Four independent lineages have been identified where C3 sister species of C2 plants exhibit an increase in organelle numbers in the bundle sheath and enlarged bundle sheath cells. Notably, in all of these species, there is a pronounced shift of mitochondria to the inner bundle sheath wall, forming an incipient version of the C2 type of Kranz anatomy. This incipient version of C2 Kranz anatomy is termed proto-Kranz, and is proposed to scavenge photorespiratory CO2. By doing so, it may provide fitness benefits in hot environments, and thus represent a critical first stage of the evolution of both the C2 and C4 forms of Kranz anatomy

C3 plants enhance rates of photosynthesis by reassimilating photorespired and respired CO2

Photosynthetic carbon gain in plants using the C3 photosynthetic pathway is substantially inhibited by photorespiration in warm environments, particularly in atmospheres with low CO2 concentrations. Unlike C4 plants, C3 plants are thought to lack any mechanism to compensate for the loss of photosynthetic productivity caused by photorespiration. Here, for the first time, we demonstrate that the C3 plants rice and wheat employ a specific mechanism to trap and reassimilate photorespired CO2. A continuous layer of chloroplasts covering the portion of the mesophyll cell periphery that is exposed to the intercellular air space creates a diffusion barrier for CO2 exiting the cell. This facilitates the capture and reassimilation of photorespired CO2 in the chloroplast stroma. In both species, 24-38% of photorespired and respired CO2 were reassimilated within the cell, thereby boosting photosynthesis by 8-11% at ambient atmospheric CO2 concentration and 17-33% at a CO2 concentration of 200μmolmol-1. Widespread use of this mechanism in tropical and subtropical C3 plants could explain why the diversity of the world's C3 flora, and dominance of terrestrial net primary productivity, was maintained during the Pleistocene, when atmospheric CO2 concentrations fell below 200μmolmol-1. The significance of this work is that it shows for the first time that plants using the C3 photosynthetic pathway have evolved a mechanism to efficiently trap photorespired CO2 and channel it back into the chloroplast, where it can be reassimilated by Rubisco. Thereby photosynthesis is enhanced by about 10% (current CO2) up to more than 30% (at low CO2 of the late Pleistocene). Widespread use of this mechanism in tropical C3 plants could explain why the diversity of the world's C3 flora, and dominance of terrestrial net primary productivity, was maintained during the Pleistocene, when atmospheric CO2 concentrations fell below 200 ppm

Phylogeny and photosynthetic pathway distribution in Anticharis Endl. (Scrophulariaceae)

C(4) photosynthesis independently evolved >62 times, with the majority of origins within 16 dicot families. One origin occurs in the poorly studied genus Anticharis Endl. (Scrophulariaceae), which consists of ~10 species from arid regions of Africa and southwest Asia. Here, the photosynthetic pathway of 10 Anticharis species and one species from each of the sister genera Aptosimum and Peliostomum was identified using carbon isotope ratios (δ(13)C). The photosynthetic pathway was then mapped onto an internal transcribed spacer (ITS) phylogeny of Anticharis and its sister genera. Leaf anatomy was examined for nine Anticharis species and plants from Aptosimum and Peliostomum. Leaf ultrastructure, gas exchange, and enzyme distributions were assessed in Anticharis glandulosa collected in SE Iran. The results demonstrate that C(3) photosynthesis is the ancestral condition, with C(4) photosynthesis occurring in one clade containing four species. C(4) Anticharis species exhibit the atriplicoid type of C(4) leaf anatomy and the NAD-malic enzyme biochemical subtype. Six Anticharis species had C(3) or C(3)-C(4) δ(13)C values and branched at phylogenetic nodes that were sister to the C(4) clade. The rest of Anticharis species had enlarged bundle sheath cells, close vein spacing, and clusters of chloroplasts along the centripetal (inner) bundle sheath walls. These traits indicate that basal-branching Anticharis species are evolutionary intermediates between the C(3) and C(4) conditions. Anticharis appears to be an important new group in which to study the dynamics of C(4) evolution

Evolutionary Convergence of C4 Photosynthesis:A Case Study in the Nyctaginaceae

C4 photosynthesis evolved over 65 times, with around 24 origins in the eudicot order Caryophyllales. In the Caryophyllales family Nyctaginaceae, the C4 pathway is known in three genera of the tribe Nyctagineae: Allionia, Okenia and Boerhavia. Phylogenetically, Allionia and Boerhavia/Okenia are separated by three genera whose photosynthetic pathway is uncertain. To clarify the distribution of photosynthetic pathways in the Nyctaginaceae, we surveyed carbon isotope ratios of 159 species of the Nyctaginaceae, along with bundle sheath (BS) cell ultrastructure, leaf gas exchange, and C4 pathway biochemistry in five species from the two C4 clades and closely related C3 genera. All species in Allionia, Okenia and Boerhavia are C4, while no C4 species occur in any other genera of the family, including three that branch between Allionia and Boerhavia. This demonstrates that C4 photosynthesis evolved twice in Nyctaginaceae. Boerhavia species use the NADP-malic enzyme (NADP-ME) subtype of C4 photosynthesis, while Allionia species use the NAD-malic enzyme (NAD-ME) subtype. The BS cells of Allionia have many more mitochondria than the BS of Boerhavia. Bundle sheath mitochondria are closely associated with chloroplasts in Allionia which facilitates CO2 refixation following decarboxylation by mitochondrial NAD-ME. The close relationship between Allionia and Boerhavia could provide insights into why NADP-ME versus NAD-ME subtypes evolve, particularly when coupled to analysis of their respective genomes. As such, the group is an excellent system to dissect the organizational hierarchy of convergent versus divergent traits produced by C4 evolution, enabling us to understand when convergence is favored versus when divergent modifications can result in a common phenotype

Ecophysiological shade adaptation in the basal angiosperm, Austrobaileya scandens (Austrobaileyaceae)

Austrobaileya scandens, the sole species of the family Austrobaileyaceae, has been the subject of renewed interest following its placement near the root of the extant angiosperm phylogenetic tree. We present field observations on the growth habit, leaf anatomy, and physiological performance (photosynthesis and stem xylem hydraulics) of Austrobaileya from a premontane rain forest in northern Queensland. Austrobaileya scandens appears to possess functional characters that are commonly associated with flowering plants and ferns adapted to low light, including absence of palisade mesophyll tissue, low leaf photosynthetic rates, and possibly strong reliance on vegetative reproduction for recruitment. Also in line with many but not all shade-tolerant species, the photosynthetic apparatus of A. scandens expressed little physiological ability to upregulate CO2 assimilation rate to increased light availability under greenhouse conditions. Broadly, these features appear to contribute to increasing the collection of light for photosynthesis under light-limiting conditions and the establishment and persistence in forest understory habitats. The ecology and physiology of A. scandens is different from hypotheses that the earliest angiosperms were early-successional xeric shrubs, disturbance-loving herbs characterized by high capacity for photosynthesis and water transport, or aquatic herbs. Our observations of A. scandens, in the context of other early-diverging lineages of flowering plants, indicate that first angio-sperms were woody plants that exploited wet, relatively dark and disturbed (albeit at small scales) habitats