Much of humanity relies on rice (Oryza sativa) as a food source, but cultivation is water intensive and the crop is vulnerable to drought and high temperatures. Under climate change, periods of reduced water availability and high temperature are expected to become more frequent, leading to detrimental effects on rice yields. We engineered the high-yielding rice cultivar 'IR64' to produce fewer stomata by manipulating the level of a developmental signal. We overexpressed the rice epidermal patterning factor OsEPF1, creating plants with substantially reduced stomatal density and correspondingly low stomatal conductance. Low stomatal density rice lines were more able to conserve water, using c. 60% of the normal amount between weeks 4 and 5 post germination. When grown at elevated atmospheric CO , rice plants with low stomatal density were able to maintain their stomatal conductance and survive drought and high temperature (40°C) for longer than control plants. Low stomatal density rice gave equivalent or even improved yields, despite a reduced rate of photosynthesis in some conditions. Rice plants with fewer stomata are drought tolerant and more conservative in their water use, and they should perform better in the future when climate change is expected to threaten food security.

A fundamental tenet of multicellular eukaryotic evolution is that vertical inheritance is paramount, with natural selection acting on genetic variants transferred from parents to offspring. This lineal process means that an organism’s adaptive potential can be restricted by its evolutionary history, the amount of standing genetic variation, and its mutation rate. Lateral gene transfer (LGT) theoretically provides a mechanism to bypass many of these limitations, but the evolutionary importance and frequency of this process in multicellular eukaryotes, such as plants, remains debated. We address this issue by assembling a chromosome-level genome for the grassAlloteropsis semialata, a species surmised to exhibit two LGTs, and screen it for other grass-to-grass LGTs using genomic data from 146 other grass species. Through stringent phylogenomic analyses, we discovered 57 additional LGTs in theA. semialatanuclear genome, involving at least nine different donor species. The LGTs are clustered in 23 laterally acquired genomic fragments that are up to 170 kb long and have accumulated during the diversification ofAlloteropsis.The majority of the 59 LGTs inA. semialataare expressed, and we show that they have added functions to the recipient genome. Functional LGTs were further detected in the genomes of five other grass species, demonstrating that this process is likely widespread in this globally important group of plants. LGT therefore appears to represent a potent evolutionary force capable of spreading functional genes among distantly related grass species.

CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants. The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest. The present study utilized CRISPR/Cas9 and CRISPR-Cpf1 technology to knock out an early developmental gene EPFL9 (Epidermal Patterning Factor like-9, a positive regulator of stomatal development in Arabidopsis) orthologue in rice. Germ-line mutants that were generated showed edits that were carried forward into the T2 generation when Cas9-free homozygous mutants were obtained. The homozygous mutant plants showed more than an eightfold reduction in stomatal density on the abaxial leaf surface of the edited rice plants. Potential off-target analysis showed no significant off-target effects. This study also utilized the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to target the same OsEPFL9 gene to test the activity of this class-2 CRISPR system in rice and found that Cpf1 is also capable of genome editing and edits get transmitted through generations with similar phenotypic changes seen with CRISPR-Cas9. This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.

C4 photosynthesis is a complex trait that boosts productivity in tropical conditions. Compared with C3 species, the C4 state seems to require numerous novelties, but species comparisons can be confounded by long divergence times. Here, we exploit the photosynthetic diversity that exists within a single species, the grass Alloteropsis semialata, to detect changes in gene expression associated with different photosynthetic phenotypes. Phylogenetically informed comparative transcriptomics show that intermediates with a weak C4 cycle are separated from the C3 phenotype by increases in the expression of 58 genes (0.22% of genes expressed in the leaves), including those encoding just three core C4 enzymes: aspartate aminotransferase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxylase. The subsequent transition to full C4 physiology was accompanied by increases in another 15 genes (0.06%), including only the core C4 enzyme pyruvate orthophosphate dikinase. These changes probably created a rudimentary C4 physiology, and isolated populations subsequently improved this emerging C4 physiology, resulting in a patchwork of expression for some C4 accessory genes. Our work shows how C4 assembly in A. semialata happened in incremental steps, each requiring few alterations over the previous step. These create short bridges across adaptive landscapes that probably facilitated the recurrent origins of C4 photosynthesis through a gradual process of evolution.

Rice contains genetically and ecologically diverse wild and cultivated species that show a wide variation in plant and leaf architecture. A systematic characterization of leaf anatomy is essential in understanding the dynamics behind such diversity. Therefore, leaf anatomies of 24 Oryza species spanning 11 genetically diverse rice genomes were studied in both lateral and longitudinal directions and possible evolutionary trends were examined. A significant inter-species variation in mesophyll cells, bundle sheath cells, and vein structure was observed, suggesting precise genetic control over these major rice leaf anatomical traits. Cellular dimensions, measured along three growth axes, were further combined proportionately to construct three-dimensional (3D) leaf anatomy models to compare the relative size and orientation of the major cell types present in a fully expanded leaf. A reconstruction of the ancestral leaf state revealed that the following are the major characteristics of recently evolved rice species: fewer veins, larger and laterally elongated mesophyll cells, with an increase in total mesophyll area and in bundle sheath cell number. A huge diversity in leaf anatomy within wild and domesticated rice species has been portrayed in this study, on an evolutionary context, predicting a two-pronged evolutionary pathway leading to the ‘sativa leaf type’ that we see today in domesticated species.

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.

Background: Stomata in rice control a number of physiological processes by regulating gas and water exchange between the atmosphere and plant tissues. The impact of the structural diversity of these micropores on its conductance level is an important area to explore before introducing stomatal traits into any breeding program in order to increase photosynthesis and crop yield. Therefore, an intensive measurement of structural components of stomatal complex (SC) of twenty three Oryza species spanning the primary, secondary and tertiary gene pools of rice has been conducted. Results: Extensive diversity was found in stomatal number and size in different Oryza species and Oryza complexes. Interestingly, the dynamics of stomatal traits in Oryza family varies differently within different Oryza genetic complexes. Example, the Sativa complex exhibits the greatest diversity in stomatal number, while the Officinalis complex is more diverse for its stomatal size. Combining the structural information with the Oryza phylogeny revealed that speciation has tended towards increasing stomatal density rather than stomatal size in rice family. Thus, the most recent species (i.e. the domesticated rice) eventually has developed smaller yet numerous stomata. Along with this, speciation has also resulted in a steady increase in stomatal conductance (anatomical, g max) in different Oryza species. These two results unambiguously prove that increasing stomatal number (which results in stomatal size reduction) has increased the stomatal conductance in rice. Correlations of structural traits with the anatomical conductance, leaf carbon isotope discrimination (Δ 13 C) and major leaf morphological and anatomical traits provide strong supports to untangle the ever mysterious dependencies of these traits in rice. The result displayed an expected negative correlation in the number and size of stomata; and positive correlations among the stomatal length, width and area with guard cell length, width on both abaxial and adaxial leaf surfaces. In addition, g max is found to be positively correlated with stomatal number and guard cell length. The Δ 13 C values of rice species showed a positive correlation with stomatal number, which suggest an increased water loss with increased stomatal number. Interestingly, in contrast, the Δ 13 C consistently shows a negative relationship with stomatal and guard cell size, which suggests that the water loss is less when the stomata are larger. Therefore, we hypothesize that increasing stomatal size, instead of numbers, is a better approach for breeding programs in order to minimize the water loss through stomata in rice.

In C4 species β-carbonic anhydrase (CA), localized to the cytosol of the mesophyll cells, accelerates the interconversion of CO2 to HCO3  -, the substrate used by PEP carboxylase in the first step of C4 photosynthesis. Here we describe the identification and characterization of a low CO2  responsive mutant 1 (lcr1) isolated from a N-Nitroso-N-methylurea (NMU) treated Setaria viridis mutant population. Forward genetic investigation revealed that the mutated gene Sevir.5G247800 of lcr1 possessed a single nucleotide transition from Cytosine to Thymine in a β-carbonic anhydrase gene causing an amino acid change from Leucine to Phenylalanine. This resulted in severe reduction in growth and photosynthesis in the mutant. Both the CO2 compensation point and carbon isotope discrimination values of the mutant were significantly increased. Growth of the mutants were stunted when grown under ambient pCO2 but recovered at elevated pCO2. Further bioinformatics analyses revealed that the mutation has led to functional changes in one of the conserved residues of the protein, situated near the catalytic site. CA transcript accumulation in the mutant was 80% lower, CA protein accumulation 30% lower and CA activity ~98% lower compared to WT. Changes in the abundance of other primary C4 pathway enzymes were observed; accumulation of PEP carboxylase (PEPC) protein was significantly increased and accumulation of Malate Dehydrogenase (MDH) and Malic Enzyme (ME) decreased. The reduction of CA protein activity and abundance in lcr1 restricts the supply of bicarbonate to PEPC limiting C4 photosynthesis and growth. This study establishes Sevir.5G247800 as the major CA allele in Setaria for C4 photosynthesis and provides important insights into the function of CA in C4 photosynthesis that would be required to generate a rice plant with a functional C4 biochemical pathway.