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Grain size and weight are crucial yield-related traits in rice (Oryza sativa). Although certain key genes associated with rice grain size and weight have been successfully cloned, the molecular mechanisms underlying grain size and weight regulation remain elusive. Here, we identified a molecular pathway regulating grain size and weight in rice involving the MPS ONE BINDER KINASE ACTIVATOR-LIKE 1A-SERINE/THREONINE-PROTEIN KINASE 38-CYCLIN C (OsMOB1A-OsSTK38-OsCycC) module. OsSTK38 is a nuclear Dbf2-related kinase that positively regulates grain size and weight by coordinating cell proliferation and expansion in the spikelet hull. OsMOB1A interacts with and enhances the autophosphorylation of OsSTK38. Specifically, the critical role of the OsSTK38 S322 site in its kinase activity is highlighted. Furthermore, OsCycC, a component of the Mediator complex, was identified as a substrate of OsSTK38, with enhancement by OsMOB1A. Notably, OsSTK38 phosphorylates the T33 site of OsCycC. The phosphorylation of OsCycC by OsSTK38 influenced its interaction with the transcription factor KNOTTED-LIKE HOMEOBOX OF ARABIDOPSIS THALIANA 7 (OsKNAT7). Genetic analysis confirmed that OsMOB1A, OsSTK38, and OsCycC function in a common pathway to regulate grain size and weight. Taken together, our findings revealed a connection between the Hippo signaling pathway and the cyclin-dependent kinase module in eukaryotes. Moreover, they provide insights into the molecular mechanisms linked to yield-related traits and propose innovative breeding strategies for high-yielding varieties., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

C3 and C4 grasses directly and indirectly provide the vast majority of calories to the human diet, yet our understanding of the molecular mechanisms driving photosynthetic productivity in grasses is largely unexplored. Ground meristem cells divide to form mesophyll or vascular initial cells early in leaf development in C3 and C4 grasses. Here we define a genetic circuit composed of SHORT ROOT (SHR), INDETERMINATE DOMAIN (IDD), and PIN-FORMED (PIN) family members that specifies vascular identify and ground cell proliferation in leaves of both C3 and C4 grasses. Ectopic expression and loss-of-function mutant studies of SHR paralogs in the C3 plant Oryza sativa (rice) and the C4 plant Setaria viridis (green millet) revealed the roles of these genes in both minor vein formation and ground cell differentiation. Genetic and in vitro studies further suggested that SHR regulates this process through its interactions with IDD12 and 13. We also revealed direct interactions of these IDD proteins with a putative regulatory element within the auxin transporter gene PIN5c. Collectively, these findings indicate that a SHR-IDD regulatory circuit mediates auxin transport by negatively regulating PIN expression to modulate minor vein patterning in the grasses., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Identifying rice ( Oryza sativa ) germplasm with improved efficiency of primary metabolism is of utmost importance in order to increase yields. One such approach can be attained through screening genetically diverse populations under altered environmental conditions. Growth or treatment under low carbon dioxide (CO 2 ) concentrations can be used as a means of revealing altered leaf photorespiration, respiration and other metabolic variants., Methods: We developed a pipeline for very high throughput treatment of gamma- and ethyl methanesulfonate- (EMS) induced mutant populations of IR64 rice seedlings at very low CO 2 for 7 days. 1050 seedlings per batch at 5 th leaf stage were exposed to 60 ppm CO 2 for the first day and 30 ppm for the remaining three days. Following this, putative candidates were identified by measuring chlorophyll depletion using SPAD. Screening results showed a distinct difference between the mutants and the WTs., Results and Discussion: The mean chlorophyll loss in WTs ranged from 65% to 11% respectively, whereas in the mutant lines chlorophyll loss ranged from 0 to 100%, suggesting considerable phenotypic variation. Rice mutants with a reduced chlorophyll reduction (<10%) were identified as 'Chlorophyll retention mutants' (CRMs) under low CO 2 stress. In total, 1909 mutant lines (14,000 seedlings) were screened for chlorophyll content under 30 ppm CO 2, with 26 lines selected for detailed screening. These 26 putative candidates were self-seeded to produce an M 5 generation, used to determine the genetic control of the altered response to low CO 2. Gas exchange of light and CO 2 response revealed that there were significant variations among photosynthetic properties in two selected rice mutants. The CO 2 compensation points in the absence of photorespiration and leaf respiration rates were lower than the WTs and anatomical analyses showed that CRM 29 had improved mesophyll cell area. We propose that this approach is useful for generating new material for breeding rice with improved primary metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mubarak, Burgess, Pyke, Quick and Murchie.)

Stomata play a fundamental role in modulating the exchange of gases between plants and the atmosphere. These microscopic structures form in high numbers on the leaf epidermis and are also present on flowers. Although leaf stomata are well studied, little attention has been paid to the development or function of floral stomata. Here, we characterize in detail the spatial distribution and development of the floral stomata of the indica rice variety IR64. We show that stomatal complexes are present at low density on specific areas of the lemma, palea and anthers and are morphologically different compared to stomata found on leaves. We reveal that in the bract-like organs, stomatal development follows the same cell lineage transitions as in rice leaves and demonstrate that the overexpression of the stomatal development regulators OsEPFL9-1 and OsEPF1 leads to dramatic changes in stomatal density in rice floral organs, producing lemma with approximately twice as many stomata (OsEPFL9-1&#95;oe) or lemma where stomata are practically absent (OsEPF1&#95;oe). Transcriptomic analysis of developing florets also indicates that the cellular transitions during the development of floral stomata are regulated by the same genetic network used in rice leaves. Finally, although we were unable to detect an impact on plant reproduction linked to changes in the density of floral stomata, we report alterations in global gene expression in lines overexpressing OsEPF1 and discuss how our results reflect on the possible role(s) of floral stomata., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Improvement of photosynthetic traits in crops to increase yield potential and crop resilience has recently become a major breeding target. Synthetic biology and genetic technologies offer unparalleled opportunities to create new genetics for photosynthetic traits driven by existing fundamental knowledge. However, large 'gene bank' collections of germplasm comprising historical collections of crop species and their relatives offer a wealth of opportunities to find novel allelic variation in the key steps of photosynthesis, to identify new mechanisms and to accelerate genetic progress in crop breeding programmes. Here we explore the available genetic resources in food and fibre crops, strategies to selectively target allelic variation in genes underpinning key photosynthetic processes, and deployment of this variation via gene editing in modern elite material., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. 
For permissions, please email: journals.permissions@oup.com.)

Key Message: Elevated expression of nucleotide-binding and leucine-rich repeat proteins led to closer vein spacing and higher vein density in rice leaves. To feed the growing global population and mitigate the negative effects of climate change, there is a need to improve the photosynthetic capacity and efficiency of major crops such as rice to enhance grain yield potential. Alterations in internal leaf morphology and cellular architecture are needed to underpin some of these improvements. One of the targets is to generate a "Kranz-like" anatomy in leaves that includes decreased interveinal spacing close to that in C 4 plant species. As C 4 photosynthesis has evolved from C 3 photosynthesis independently in multiple lineages, the genes required to facilitate C 4 may already be present in the rice genome. The Taiwan Rice Insertional Mutants (TRIM) population offers the advantage of gain-of-function phenotype trapping, which accelerates the identification of rice gene function. In the present study, we screened the TRIM population to determine the extent to which genetic plasticity can alter vein density (VD) in rice. Close vein spacing mutant 1 (CVS1), identified from a VD screening of approximately 17,000 TRIM lines, conferred heritable high leaf VD. Increased vein number in CVS1 was confirmed to be associated with activated expression of two nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. Overexpression of the two NB-LRR genes individually in rice recapitulates the high VD phenotype, due mainly to reduced interveinal mesophyll cell (M cell) number, length, bulliform cell size and thus interveinal distance. Our studies demonstrate that the trait of high VD in rice can be achieved by elevated expression of NB-LRR proteins limited to no yield penalty., (© 2021. The Author(s).)

We generated antisense constructs targeting two of the five Rubisco small subunit genes (OsRBCS2 and 4) which account for between 30-40 % of the RBCS transcript abundance in leaf blades. The constructs were driven by a maize phosphoenolpyruvate carboxylase (PEPC) promoter known to have enriched expression in mesophyll cells (MCs). In the resulting lines leaf, Rubisco protein content was reduced by between 30-50 % and CO 2 assimilation rate was limited under photorespiratory and non-photorespiratory conditions. A relationship between Rubisco protein content and CO 2 assimilation rate was found. This was associated with a significant reduction in dry biomass accumulation and grain yield of between 37-70%. In addition to serving as a resource for reducing Rubisco accumulation in a cell-preferential manner, these lines allow us to characterize gene function and isoform specific suppression on photosynthesis and growth. Our results suggest that the knockdown of multiple genes is required to completely reduce Rubisco accumulation in MCs., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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 phosphoenolpyruvate carboxylase (PEPC) in the first step of C4 photosynthesis. Here we describe the identification and characterization of low CO2-responsive mutant 1 (lcr1) isolated from an 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-CA 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 was 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 with the wild type. Changes in the abundance of other primary C4 pathway enzymes were observed; accumulation of PEPC protein was significantly increased and accumulation of malate dehydrogenase and malic enzyme 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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

C 4 photosynthesis provides an effective solution for overcoming the catalytic inefficiency of Rubisco. The pathway is characterised by a biochemical CO 2 concentrating mechanism that operates across mesophyll and bundle sheath (BS) cells and relies on a gas tight BS compartment. A screen of a mutant population of Setaria viridis, an NADP-malic enzyme type C 4 monocot, generated using N-nitroso-N-methylurea identified a mutant with an amino acid change in the gene coding region of the ABCG transporter, a step in the suberin synthesis pathway. Here, Nile red staining, TEM, and GC/MS confirmed the alteration in suberin deposition in the BS cell wall of the mutant. We show that this has disrupted the suberin lamellae of BS cell wall and increased BS conductance to CO 2 diffusion more than two-fold in the mutant. Consequently, BS CO 2 partial pressure is reduced and CO 2 assimilation was impaired in the mutant. Our findings provide experimental evidence that a functional suberin lamellae is an essential anatomical feature for efficient C 4 photosynthesis in NADP-ME plants like S. viridis and have implications for engineering strategies to ensure future food security.

Several breeding initiatives have sought to improve flag leaf performance as its health and physiology are closely correlated to rice yield. Previous studies have described natural variation of photosynthesis for flag leaves; however, none has examined their performance under the non-steady-state conditions that prevail in crop fields. Photosynthetic induction is the transient response of photosynthesis to a change from low to high light. Rice flag leaf photosynthesis was measured in both steady- and non-steady-state conditions to characterize natural variation. Between the lowest and highest performing accession, there was a 152% difference for average CO2 assimilation during induction (Ā300), a 77% difference for average intrinsic water use efficiency during induction (iWUEavg), and a 185% difference for the speed of induction (IT50), indicating plentiful variation. No significant correlation was found between steady- and non-steady-state photosynthetic traits. Additionally, measures of neither steady-state nor non-steady-state photosynthesis of flag leaves correlated with the same measures of leaves in the vegetative growth stage, with the exception of iWUEavg. Photosynthetic induction was measured at six [CO2], to determine biochemical and diffusive limitations to photosynthesis in vivo. Photosynthetic induction in rice flag leaves was limited primarily by biochemistry., (© Society for Experimental Biology 2020.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The fast-moving CRISPR technology has allowed plant scientists to manipulate plant genomes in a targeted manner. So far, most of the applications were focused on gene knocking out by creating indels. However, more precise genome editing tools are demanded to assist the introduction of functional single nucleotide polymorphisms (SNPs) in breeding programs. The CRISPR base editing tools were developed to meet this need. In this chapter, we present a cytidine deaminase base editing method for editing the point mutations that control the grain size and seed coat color in rice.

Immunolocalization analysis is a principal tool to study protein expression and subcellular distribution in plant cells or tissues. In this chapter, we present the method of the preparation of lightly fixed fresh rice leaf tissue for immunolocalization analysis and detection of the protein of interest using fluorescent probes by fluorescent microscopy. This method especially does not need the process of embedding plant materials that saves time and prevents alterations of cellular compounds and structure during sample preparation. Using this method, the C 4 rice project compared the expressions of the proteins of interest among C 4 model plants, wild-type rice, and transgenic or mutant plants and successfully selected the transgenic plants with the correct location of each protein to create a C 4 rice prototype.

The morphology of bulliform cells located on the upper epidermis of leaves is one of the most important cell structures affecting leaf shape. Although many mechanisms regulating the development of bulliform cells have been reported, the fine regulatory mechanisms governing this process have rarely been described. To identify novel components regulating rice leaf morphology, a mutant showing a constitutively rolling phenotype from the seedling stage to flowering, known as crm1-D, was selected for further analysis. Anatomical analyses in crm1-D were attributable to the size reduction of bulliform cells. The crm1-D was controlled by a single dominant nuclear gene. Map-based cloning revealed that Roc8, an HD zipper class IV family member, was responsible for the crm1-D phenotype. Notably, the 50-bp sequence in the 3'-untranslated region (3'-UTR) of the Roc8 gene represses Roc8 at the translational level. Moreover, the roc8 knockdown lines notably increased the size of bulliform cells. A series of assays revealed that Roc8 negatively regulates the size of bulliform cells. Unexpectedly, Roc8 was also observed to positively mediate lignin biosynthesis without incurring a production penalty. The above results show that Roc8 may have a practical application in cultivating materials with high photosynthetic efficiency and low lignin content., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Introduction of a C 4 photosynthetic pathway into C 3 rice ( Oryza sativa ) requires installation of a biochemical pump that concentrates CO 2 at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously accumulates four of the core C 4 photosynthetic enzymes from the NADP-malic enzyme subtype, phospho enol pyruvate carboxylase ( Zm PEPC), NADP-malate dehydrogenase ( Zm NADP-MDH), NADP-malic enzyme ( Zm NADP-ME), and pyruvate phosphate dikinase ( Zm PPDK). This led to enhanced enzyme activity and mild phenotypic perturbations but was largely neutral in its effects on photosynthetic rate. Measurements of the flux of 13 CO 2 through photosynthetic metabolism revealed a significant increase in the incorporation of 13 C into malate, consistent with increased fixation of 13 CO 2 via PEP carboxylase in lines expressing the maize PEPC enzyme. However, there was no significant differences in labeling of 3-phosphoglycerate (3PGA) indicating that there was no carbon flux through NADP-ME into the Calvin-Benson cycle. There was also no significant difference in labeling of phospho enol pyruvate (PEP) indicating that there was no carbon flux through PPDK. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein ( Os GDCH) abundance led to a photosynthetic phenotype characteristic of the reduced Os GDCH line and higher labeling of malate, aspartate and citrate than in the quintuple line. There was evidence of 13 C labeling of aspartate indicating 13 CO 2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C 4 cycle, these results demonstrate for the first-time a partial flux through the carboxylation phase of NADP-ME C 4 metabolism in transgenic rice containing two of the key metabolic steps in the C 4 pathway., (Copyright © 2020 Lin, Arrivault, Coe, Karki, Covshoff, Bagunu, Lunn, Stitt, Furbank, Hibberd and Quick.)

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 (∆ 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 ∆ 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 ∆ 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., Conclusion: Current paper generates useful data on stomatal profile of wild rice that is hitherto unknown for the rice science community. It has been proved here that the speciation has resulted in an increased stomatal number accompanied by size reduction during Oryza's evolutionary course; this has resulted in an increased g max but reduced water use efficiency. Although may not be the sole driver of water use efficiency in rice, our data suggests that stomata are a potential target for modifying the currently low water use efficiency in domesticated rice. It is proposed that Oryza barthii can be used in traditional breeding programs in enhancing the stomatal size of elite rice cultivars.

Photosynthetic induction describes the transient increase in leaf CO 2 uptake with an increase in light. During induction, efficiency is lower than at steady state. Under field conditions of fluctuating light, this lower efficiency during induction may cost > 20% of potential crop assimilation. Accelerating induction would boost photosynthetic and resource-use efficiencies. Variation between rice accessions and potential for accelerating induction was analysed by gas exchange. Induction during shade to sun transitions of 14 accessions representing five subpopulations from the 3000 Rice Genome Project Panel (3K RGP) was analysed. Differences of 109% occurred in the CO 2 fixed during the first 300 s of induction, 117% in the half-time to completion of induction, and 65% in intrinsic water-use efficiency during induction, between the highest and lowest performing accessions. Induction in three accessions with contrasting responses (AUS 278, NCS 771 A and IR64-21) was compared for a range of [CO 2 ] to analyse limitations. This showed in vivo capacity for carboxylation at Rubisco (V c,max ), and not stomata, as the primary limitation to induction, with significant differences between accessions. Variation in nonsteady-state efficiency greatly exceeded that at steady state, suggesting a new and more promising opportunity for selection of greater crop photosynthetic efficiency in this key food crop., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

The engineering process of C 4 photosynthesis into C 3 plants requires an increased activity of phosphoenolpyruvate carboxylase (PEPC) in the cytosol of leaf mesophyll cells. The literature varies on the physiological effect of transgenic maize (Zea mays) PEPC (ZmPEPC) leaf expression in Oryza sativa (rice). Therefore, to address this issue, leaf-atmosphere CO 2 and 13 CO 2 exchanges were measured, both in the light (at atmospheric O 2 partial pressure of 1.84 kPa and at different CO 2 levels) and in the dark, in transgenic rice expressing ZmPEPC and wild-type (WT) plants. The in vitro PEPC activity was 25 times higher in the PEPC overexpressing (PEPC-OE) plants (~20% of maize) compared to the negligible activity in WT. In the PEPC-OE plants, the estimated fraction of carboxylation by PEPC (β) was ~6% and leaf net biochemical discrimination against 13 CO 2 [Formula: see text] was ~ 2‰ lower than in WT. However, there were no differences in leaf net CO 2 assimilation rates (A) between genotypes, while the leaf dark respiration rates (R d ) over three hours after light-dark transition were enhanced (~ 30%) and with a higher 13 C composition [Formula: see text] in the PEPC-OE plants compared to WT. These data indicate that ZmPEPC in the PEPC-OE rice plants contributes to leaf carbon metabolism in both the light and in the dark. However, there are some factors, potentially posttranslational regulation and PEP availability, which reduce ZmPEPC activity in vivo.

Rapid metabolite diffusion across the mesophyll (M) and bundle sheath (BS) cell interface in C 4 leaves is a key requirement for C 4 photosynthesis and occurs via plasmodesmata (PD). Here, we investigated how growth irradiance affects PD density between M and BS cells and between M cells in two C 4 species using our PD quantification method, which combines three-dimensional laser confocal fluorescence microscopy and scanning electron microscopy. The response of leaf anatomy and physiology of NADP-ME species, Setaria viridis and Zea mays to growth under different irradiances, low light (100 μmol m -2  s -1 ), and high light (1,000 μmol m -2  s -1 ), was observed both at seedling and established growth stages. We found that the effect of growth irradiance on C 4 leaf PD density depended on plant age and species. The high light treatment resulted in two to four-fold greater PD density per unit leaf area than at low light, due to greater area of PD clusters and greater PD size in high light plants. These results along with our finding that the effect of light on M-BS PD density was not tightly linked to photosynthetic capacity suggest a complex mechanism underlying the dynamic response of C 4 leaf PD formation to growth irradiance., (© 2019 John Wiley & Sons Ltd.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The influence of reduced glycine decarboxylase complex (GDC) activity on leaf atmosphere CO2 and 13CO2 exchange was tested in transgenic Oryza sativa with the GDC H-subunit knocked down in leaf mesophyll cells. Leaf measurements on transgenic gdch knockdown and wild-type plants were carried out in the light under photorespiratory and low photorespiratory conditions (i.e. 18.4 kPa and 1.84 kPa atmospheric O2 partial pressure, respectively), and in the dark. Under approximately current ambient O2 partial pressure (18.4 kPa pO2), the gdch knockdown plants showed an expected photorespiratory-deficient phenotype, with lower leaf net CO2 assimilation rates (A) than the wild-type. Additionally, under these conditions, the gdch knockdown plants had greater leaf net discrimination against 13CO2 (Δo) than the wild-type. This difference in Δo was in part due to lower 13C photorespiratory fractionation (f) ascribed to alternative decarboxylation of photorespiratory intermediates. Furthermore, the leaf dark respiration rate (Rd) was enhanced and the 13CO2 composition of respired CO2 (δ13CRd) showed a tendency to be more depleted in the gdch knockdown plants. These changes in Rd and δ13CRd were due to the amount and carbon isotopic composition of substrates available for dark respiration. These results demonstrate that impairment of the photorespiratory pathway affects leaf 13CO2 exchange, particularly the 13C decarboxylation fractionation associated with photorespiration., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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 grass Alloteropsis 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 the A. semialata nuclear 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 of Alloteropsis. The majority of the 59 LGTs in A. semialata are 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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

With continued economic development in Asia the demand for high yielding varieties with premium grain quality traits is set to increase. This presents a significant challenge to plant breeders because varieties must be tailored to meet regional preferences. It is already apparent that traditional breeding techniques cannot meet this challenge and so emerging genomics technologies will have to be utilized. Genome editing tools afford the ability to efficiently and precisely manipulate the genome. Among these, the bacterial clustered, regularly interspaced, short palindromic repeat (CRISPR) associated protein 9 (Cas9) or CRISPR-Cas9 has emerged as the easiest, most economic, and efficient technology to undertake genome editing in rice. This technique allows precise site-specific gene modification or integration. In this chapter we present a method for utilizing CRISPR-Cas9 for improving grain quality traits in rice; this should enable molecular breeders to quickly and efficiently produce high yielding rice varieties tailored to meet specific cultural and regional requirements for grain quality.

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 2 , 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., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Background: Temperature is one of the most important abiotic factors limiting plant growth and productivity. Many plants exhibit cold acclimation to prepare for the likelihood of freezing as temperatures decrease towards 0 °C. The physiological mechanisms associated with enabling increased tolerance to sub-zero temperatures vary between species and genotypes. Geographically and climatically diverse populations of Arabidopsis lyrata ssp. petraea were examined for their ability to survive, maintain functional photosynthetic parameters and cellular electrolyte leakage integrity after being exposed to sub-zero temperatures. The duration of cold acclimation prior to sub-zero temperatures was also manipulated (2 and 14 days)., Results: We found that there was significant natural variation in tolerances to sub-zero temperatures among populations of A. petraea. The origin of the population affected the acclimation response and survival after exposure to sub-zero temperatures. Cold acclimation of plants prior to sub-zero temperatures affected the maximum quantum efficiency of photosystem II (PSII) (F v /F m ) in that plants that were cold acclimated for longer periods had higher values of F v /F m as a result of sub-zero temperatures. The inner immature leaves were better able to recover F v /F m from sub-zero temperatures than mature outer leaves. The Irish population (Leitrim) acclimated faster, in terms of survival and electrolyte leakage than the Norwegian population (Helin)., Conclusion: The ability to survive, recover photosynthetic processes and cellular electrolyte leakage after exposure to sub-zero temperatures is highly dependent on the duration of cold acclimation.

To assist with efforts to engineer a C4 photosynthetic pathway into rice, forward-genetic approaches are being used to identify the genes modulating key C4 traits. Currently, a major challenge is how to screen for a variety of different traits in a high-throughput manner. Here we describe a method for identifying C4 mutant plants with increased CO2 compensation points. This is used as a signature for decreased photosynthetic efficiency associated with a loss of C4 function. By exposing plants to a CO2 concentration close to the CO2 compensation point of a wild-type plant, individuals can be identified from measurements of chlorophyll a fluorescence. We use this method to screen a mutant population of the C4 monocot Setaria viridis (L.)P.Beauv. generated using N-nitroso-N-methylurea (NMU). Mutants were identified at a frequency of 1 per 157 lines screened. Forty-six candidate lines were identified and one line with a heritable homozygous phenotype selected for further characterisation. The CO2 compensation point of this mutant was increased to a value similar to that of C3 rice. Photosynthesis and growth was significantly reduced under ambient conditions. These data indicate that the screen was capable of identifying mutants with decreased photosynthetic efficiency. Characterisation and next-generation sequencing of all the mutants identified in this screen may lead to the discovery of novel genes underpinning C4 photosynthesis. These can be used to engineer a C4 photosynthetic pathway into rice.

Proliferation of plasmodesmata (PD) connections between bundle sheath (BS) and mesophyll (M) cells has been proposed as a key step in the evolution of two-cell C4 photosynthesis; However, a lack of quantitative data has hampered further exploration and validation of this hypothesis. In this study, we quantified leaf anatomical traits associated with metabolite transport in 18 species of BEP and PACMAD grasses encompassing four origins of C4 photosynthesis and all three C4 subtypes (NADP-ME, NAD-ME, and PCK). We demonstrate that C4 leaves have greater PD density between M and BS cells than C3 leaves. We show that this greater PD density is achieved by increasing either the pit field (cluster of PD) area or the number of PD per pit field area. NAD-ME species had greater pit field area per M-BS interface than NADP-ME or PCK species. In contrast, NADP-ME and PCK species had lower pit field area with increased number of PD per pit field area than NAD-ME species. Overall, PD density per M-BS cell interface was greatest in NAD-ME species while PD density in PCK species exhibited the largest variability. Finally, the only other anatomical characteristic that clearly distinguished C4 from C3 species was their greater Sb value, the BS surface area to subtending leaf area ratio. In contrast, BS cell volume was comparable between the C3 and C4 grass species examined.

The C 4 photosynthetic pathway accounts for ∼25% of primary productivity on the planet despite being used by only 3% of species. Because C 4 plants are higher yielding than C 3 plants, efforts are underway to introduce the C 4 pathway into the C 3 crop rice. This is an ambitious endeavor; however, the C 4 pathway evolved from C 3 on multiple independent occasions over the last 30 million years, and steps along the trajectory are evident in extant species. One approach toward engineering C 4 rice is to recapitulate this trajectory, one of the first steps of which was a change in leaf anatomy. The transition from C 3 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 C 4 rice has been achieved., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Improvements to leaf photosynthetic rates of crops can be achieved by targeted manipulation of individual component processes, such as the activity and properties of RuBisCO or photoprotection. This study shows that simple forward genetic screens of mutant populations can also be used to rapidly generate photosynthesis variants that are useful for breeding. Increasing leaf vein density (concentration of vascular tissue per unit leaf area) has important implications for plant hydraulic properties and assimilate transport. It was an important step to improving photosynthetic rates in the evolution of both C 3 and C 4 species and is a foundation or prerequisite trait for C 4 engineering in crops like rice ( Oryza sativa ). A previous high throughput screen identified five mutant rice lines (cv. IR64) with increased vein densities and associated narrower leaf widths (Feldman et al., 2014). Here, these high vein density rice variants were analyzed for properties related to photosynthesis. Two lines were identified as having significantly reduced mesophyll to bundle sheath cell number ratios. All five lines had 20% higher light saturated photosynthetic capacity per unit leaf area, higher maximum carboxylation rates, dark respiration rates and electron transport capacities. This was associated with no significant differences in leaf thickness, stomatal conductance or CO 2 compensation point between mutants and the wild-type. The enhanced photosynthetic rate in these lines may be a result of increased RuBisCO and electron transport component amount and/or activity and/or enhanced transport of photoassimilates. We conclude that high vein density (associated with altered mesophyll cell length and number) is a trait that may confer increased photosynthetic efficiency without increased transpiration.

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C 3 or C 4 photosynthesis. As part of a multinational effort to introduce C 4 traits into rice to boost crop yield, candidate regulators of C 4 leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C 4 -like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C 4 plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C 3 plants.

Recent efforts to engineer C4 photosynthetic traits into C3 plants such as rice demand an understanding of the genetic elements that enable C4 plants to outperform C3 plants. As a part of the C4 Rice Consortium's efforts to identify genes needed to support C4 photosynthesis, EMS mutagenized sorghum populations were generated and screened to identify genes that cause a loss of C4 function. Stable carbon isotope ratio (δ13C) of leaf dry matter has been used to distinguishspecies with C3 and C4 photosynthetic pathways. Here, we report the identification of a sorghum (Sorghum bicolor) mutant with a low δ13C characteristic. A mutant (named Mut33) with a pale phenotype and stunted growth was identified from an EMS treated sorghum M2 population. The stable carbon isotope analysis of the mutants showed a decrease of 13C uptake capacity. The noise of random mutation was reduced by crossing the mutant and its wildtype (WT). The back-cross (BC1F1) progenies were like the WT parent in terms of 13C values and plant phenotypes. All the BC1F2 plants with low δ13C died before they produced their 6th leaf. Gas exchange measurements of the low δ13C sorghum mutants showed a higher CO2 compensation point (25.24 μmol CO2.mol-1air) and the maximum rate of photosynthesis was less than 5μmol.m-2.s-1. To identify the genetic determinant of this trait, four DNA pools were isolated; two each from normal and low δ13C BC1F2 mutant plants. These were sequenced using an Illumina platform. Comparison of allele frequency of the single nucleotide polymorphisms (SNPs) between the pools with contrasting phenotype showed that a locus in Chromosome 10 between 57,941,104 and 59,985,708 bps had an allele frequency of 1. There were 211 mutations and 37 genes in the locus, out of which mutations in 9 genes showed non-synonymous changes. This finding is expected to contribute to future research on the identification of the causal factor differentiating C4 from C3 species that can be used in the transformation of C3 to C4 plants.

Numerous studies have addressed effects of rising atmospheric CO2 concentration on rice biomass production and yield but effects on crop water use are less well understood. Irrigated rice evapotranspiration (ET) is composed of floodwater evaporation and canopy transpiration. Crop coefficient Kc (ET over potential ET, or ETo) is crop specific according to FAO, but may decrease as CO2 concentration rises. A sunlit growth chamber experiment was conducted in the Philippines, exposing 1.44-m2 canopies of IR72 rice to four constant CO2 levels (195, 390, 780 and 1560 ppmv). Crop geometry and management emulated field conditions. In two wet (WS) and two dry (DS) seasons, final aboveground dry weight (agdw) was measured. At 390 ppmv [CO2] (current ambient level), agdw averaged 1744 g m-2, similar to field although solar radiation was only 61% of ambient. Reduction to 195 ppmv [CO2] reduced agdw to 56±5% (SE), increase to 780 ppmv increased agdw to 128±8%, and 1560 ppmv increased agdw to 142±5%. In 2013WS, crop ET was measured by weighing the water extracted daily from the chambers by the air conditioners controlling air humidity. Chamber ETo was calculated according to FAO and empirically corrected via observed pan evaporation in chamber vs. field. For 390 ppmv [CO2], Kc was about 1 during crop establishment but increased to about 3 at flowering. 195 ppmv CO2 reduced Kc, 780 ppmv increased it, but at 1560 ppmv it declined. Whole-season crop water use was 564 mm (195 ppmv), 719 mm (390 ppmv), 928 mm (780 ppmv) and 803 mm (1560 ppmv). With increasing [CO2], crop water use efficiency (WUE) gradually increased from 1.59 g kg-1 (195 ppmv) to 2.88 g kg-1 (1560 ppmv). Transpiration efficiency (TE) measured on flag leaves responded more strongly to [CO2] than WUE. Responses of some morphological traits are also reported. In conclusion, increased CO2 promotes biomass more than water use of irrigated rice, causing increased WUE, but it does not help saving water. Comparability with field conditions is discussed. The results will be used to train crop models., Competing Interests: The authors have declared that no competing interests exist.

Genetic improvement of photosynthetic performance of cereal crops and increasing the efficiency with which solar radiation is converted into biomass has recently become a major focus for crop physiologists and breeders. The pulse amplitude modulated chlorophyll fluorescence technique (PAM) allows quantitative leaf level monitoring of the utilization of energy for photochemical light conversion and photoprotection in natural environments, potentially over the entire crop lifecycle. Here, the diurnal relationship between electron transport rate (ETR) and irradiance was measured in five cultivars of rice (Oryza sativa) in canopy conditions with PAM fluorescence under natural solar radiation. This relationship differed substantially from that observed for conventional short term light response curves measured under controlled actinic light with the same leaves. This difference was characterized by a reduced curvature factor when curve fitting was used to model this diurnal response. The engagement of photoprotective processes in chloroplast electron transport in leaves under canopy solar radiation was shown to be a major contributor to this difference. Genotypic variation in the irradiance at which energy flux into photoprotective dissipation became greater than ETR was observed. Cultivars capable of higher ETR at midrange light intensities were shown to produce greater leaf area over time, estimated by noninvasive imaging., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a prokaryotic adaptive immune system that has the ability to identify specific locations on the bacteriophage (phage) genome to create breaks in it, and internalize the phage genome fragments in its own genome as CRISPR arrays for memory-dependent resistance. Although CRISPR has been used in the dairy industry for a long time, it recently gained importance in the field of genome editing because of its ability to precisely target locations in a genome. This system has further been modified to locate and target any region of a genome of choice due to modifications in the components of the system. By changing the nucleotide sequence of the 20-nucleotide target sequence in the guide RNA, targeting any location is possible. It has found an application in the modification of plant genomes with its ability to generate mutations and insertions, thus helping to create new varieties of plants. With the ability to introduce specific sequences into the plant genome after cleavage by the CRISPR system and subsequent DNA repair through homology-directed repair (HDR), CRISPR ensures that genome editing can be successfully applied in plants, thus generating stronger and more improved traits. Also, the use of the CRISPR editing system can generate plants that are transgene-free and have mutations that are stably inherited, thus helping to circumvent current GMO regulations.

While genotype-environment interaction is increasingly receiving attention by ecologists and evolutionary biologists, such studies need genetically homogeneous replicates-a challenging hurdle in outcrossing plants. This could be potentially overcome by using tissue culture techniques. However, plants regenerated from tissue culture may show aberrant phenotypes and "somaclonal" variation. Here, we examined somaclonal variation due to tissue culturing using the response to cold treatment of photosynthetic efficiency (chlorophyll fluorescence measurements for F v /F m , F v '/F m ', and Φ PSII , representing maximum efficiency of photosynthesis for dark- and light-adapted leaves, and the actual electron transport operating efficiency, respectively, which are reliable indicators of photoinhibition and damage to the photosynthetic electron transport system). We compared this to variation among half-sibling seedlings from three different families of Arabidopsis lyrata ssp. petraea Somaclonal variation was limited, and we could detect within-family variation in change in chlorophyll fluorescence due to cold shock successfully with the help of tissue-culture derived replicates. Icelandic and Norwegian families exhibited higher chlorophyll fluorescence, suggesting higher performance after cold shock, than a Swedish family. Although the main effect of tissue culture on F v /F m , F v '/F m ', and Φ PSII was small, there were significant interactions between tissue culture and family, suggesting that the effect of tissue culture is genotype-specific. Tissue-cultured plantlets were less affected by cold treatment than seedlings, but to a different extent in each family. These interactive effects, however, were comparable to, or much smaller than the single effect of family. These results suggest that tissue culture is a useful method for obtaining genetically homogenous replicates for studying genotype-environment interaction related to adaptively-relevant phenotypes, such as cold response, in nonmodel outcrossing plants., (Copyright © 2016 Kenta et al.)

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., Competing Interests: The authors have declared that no competing interests exist.

This study set out to identify and characterize transcription factors regulating photosynthesis in rice. Screening populations of rice T-DNA activation lines led to the identification of a T-DNA mutant with an increase in intrinsic water use efficiency (iWUE) under well-watered conditions. Flanking sequence analysis showed that the T-DNA construct was located upstream of LOC&#95;Os07g38240 (OsSAP16) encoding for a stress-associated protein (SAP). A second mutant identified with activation in the same gene exhibited the same phenotype; expression of OsSAP16 was shown to be enhanced in both lines. There were no differences in stomatal development or morphology in either of these mutants, although overexpression of OsSAP16 reduced stomatal conductance. This phenotype limited CO2 uptake and the rate of photosynthesis, which resulted in the accumulation of less biomass in the two mutants. Whole transcriptome analysis showed that overexpression of OsSAP16 led to global changes in gene expression consistent with the function of zinc-finger transcription factors. These results show that the gene is involved in modulating the response of rice to drought stress through regulation of the expression of a set of stress-associated genes.

C4 photosynthesis is characterized by a CO2-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 C3 (rice [Oryza sativa] and wheat [Triticum aestivum]) and C4 (maize [Zea mays] and Setaria viridis) monocot species. Scanning electron microscopy quantification of PD density revealed that C4 species had approximately twice the number of PD per pitfield area compared with their C3 counterparts. 3D immunolocalization of callose at pitfields using confocal microscopy showed that pitfield area per M-BS interface area was 5 times greater in C4 species. Thus, the two C4 species had up to nine times more PD per M-BS interface area (S. viridis, 9.3 PD µm(-2); maize, 7.5 PD µm(-2); rice 1.0 PD µm(-2); wheat, 2.6 PD µm(-2)). Using these anatomical data and measured photosynthetic rates in these C4 species, we have now calculated symplastic C4 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 C4 photosynthesis to C3 crops., (© 2016 American Society of Plant Biologists. All rights reserved.)

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., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Leaves are derived from heterotrophic meristem tissue that, at some point, must make the transition to autotrophy via the initiation of photosynthesis. However, the timing and spatial coordination of the molecular and cellular processes underpinning this switch are poorly characterized. Here, we report on the identification of a specific stage in rice (Oryza sativa) leaf development (P3/P4 transition) when photosynthetic competence is first established. Using a combined physiological and molecular approach, we show that elements of stomatal and vascular differentiation are coordinated with the onset of measurable light absorption for photosynthesis. Moreover, by exploring the response of the system to environmental perturbation, we show that the earliest stages of rice leaf development have significant plasticity with respect to elements of cellular differentiation of relevance for mature leaf photosynthetic performance. Finally, by performing an RNA sequencing analysis targeted at the early stages of rice leaf development, we uncover a palette of genes whose expression likely underpins the acquisition of photosynthetic capability. Our results identify the P3/P4 transition as a highly dynamic stage in rice leaf development when several processes for the initiation of photosynthetic competence are coordinated. As well as identifying gene targets for future manipulation of rice leaf structure/function, our data highlight a developmental window during which such manipulations are likely to be most effective., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The 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 γ-ray-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 next-generation 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., (© 2015 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Phenotypic plasticity in overcoming heat stress-induced damage across hot tropical rice-growing regions is predominantly governed by relative humidity. Expression of transpiration cooling, an effective heat-avoiding mechanism, will diminish with the transition from fully flooded paddies to water-saving technologies, such as direct-seeded and aerobic rice cultivation, thus further aggravating stress damage. This change can potentially introduce greater sensitivity to previously unaffected developmental stages such as floral meristem (panicle) initiation and spikelet differentiation, and further intensify vulnerability at the known sensitive gametogenesis and flowering stages. More than the mean temperature rise, increased variability and a more rapid increase in nighttime temperature compared with the daytime maximum present a greater challenge. This review addresses (1) the importance of vapour pressure deficit under fully flooded paddies and increased vulnerability of rice production to heat stress or intermittent occurrence of combined heat and drought stress under emerging water-saving rice technologies; (2) the major disconnect with high night temperature response between field and controlled environments in terms of spikelet sterility; (3) highlights the most important mechanisms that affect key grain quality parameters, such as chalk formation under heat stress; and finally (4), we model and estimate heat stress-induced spikelet sterility taking South Asia as a case study., (© 2014 John Wiley & Sons Ltd.)

An efficient method for crossing green foxtail (Setaria viridis) is currently lacking. S. viridis is considered to be the new model plant for the study of C4 system in monocots and so an effective crossing protocol is urgently needed. S. viridis is a small grass with C4-NADP (ME) type of photosynthesis and has the advantage of having small genome of about 515 Mb, small plant stature, short life cycle, multiple tillers, and profuse seed set, and hence is an ideal model species for research. The objectives of this project were to develop efficient methods of emasculation and pollination, and to speed up generation advancement. We assessed the response of S. viridis flowers to hot water treatment (48°C) and to different concentrations of gibberellic acid, abscisic acid, maleic hydrazide (MH), and kinetin. We found that 500 μM of MH was effective in the emasculation of S. viridis, whilst still retaining the receptivity of the stigma to pollination. We also report effective ways to accelerate the breeding cycle of S. viridis for research through the germination of mature as well as immature seeds in optimized culture media. We believe these findings will be of great interest to researchers using Setaria.

A high leaf vein density is both an essential feature of C4 photosynthesis and a foundation trait to C4 evolution, ensuring the optimal proportion and proximity of mesophyll and bundle sheath cells for permitting the rapid exchange of photosynthates. Two rice mutant populations, a deletion mutant library with a cv. IR64 background (12,470 lines) and a T-DNA insertion mutant library with a cv. Tainung 67 background (10,830 lines), were screened for increases in vein density. A high throughput method with handheld microscopes was developed and its accuracy was supported by more rigorous microscopy analysis. Eight lines with significantly increased leaf vein densities were identified to be used as genetic stock for the global C4 Rice Consortium. The candidate population was shown to include both shared and independent mutations and so more than one gene controlled the high vein density phenotype. The high vein density trait was found to be linked to a narrow leaf width trait but the linkage was incomplete. The more genetically robust narrow leaf width trait was proposed to be used as a reliable phenotypic marker for finding high vein density variants in rice in future screens.

To boost food production for a rapidly growing global population, crop yields must significantly increase. One of the avenues being recently explored is the improvement of photosynthetic capacity by installing the C4 photosynthetic pathway into C3 crops like rice to drastically increase their yield. Crops with an enhanced photosynthetic mechanism would better utilize the solar radiation that can be translated into yield. This subsequently will help in producing more grain yield, reduce water loss and increase nitrogen use efficiency especially in hot and dry environments. This review provides a summary of the factors that need to be modified in rice so that the C4 pathway can be introduced successfully. It also discusses the differences between the C3 and C4 photosynthetic pathways in terms of anatomy, biochemistry and genetics.