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Summary Global demand for food may rise by 60% mid‐century. A central challenge is to meet this need using less land in a changing climate. Nearly all crop carbon is assimilated through Rubisco, which is catalytically slow, reactive with oxygen, and a major component of leaf nitrogen. Developing more efficient forms of Rubisco, or engineering CO2 concentrating mechanisms into C3 crops to competitively repress oxygenation, are major endeavors, which could hugely increase photosynthetic productivity (≥ 60%). New technologies are bringing this closer, but improvements remain in the discovery phase and have not been reduced to practice. A simpler shorter‐term strategy that could fill this time gap, but with smaller productivity increases (c. 10%) is to increase leaf Rubisco content. This has been demonstrated in initial field trials, improving the productivity of C3 and C4 crops. Combining three‐dimensional leaf canopies with metabolic models infers that a 20% increase in Rubisco increases canopy photosynthesis by 14% in sugarcane (C4) and 9% in soybean (C3). This is consistent with observed productivity increases in rice, maize, sorghum and sugarcane. Upregulation of Rubisco is calculated not to require more nitrogen per unit yield and although achieved transgenically to date, might be achieved using gene editing to produce transgene‐free gain of function mutations or using breeding. [ABSTRACT FROM AUTHOR]

The content and localization of Rubisco large subunit (RbcL), as well as the activities of NADP-MDH and NADP-ME enzymes involved in the C4 carbon-concentrating mechanism (CCM) were studied under elevated (800 ppm, eCO2) CO2 concentration at optimal (25°C) and elevated (32°C, eT) temperatures in the leaves of C4-NADP species Kochia prostrata. In control plants Rubisco was revealed not only in the bundle sheath cells (BS), typical of C4 plants but also in the mesophyll cells (M), indicating a C4-like type of photosynthesis. In addition, the presence of starch grains in the BS and the adjacent layer of mesophyll cells (M(I)) suggests the Calvin cycle activity. eT and/or eCO2 markedly reduced the number of starch grains in these cells, probably as a result of decreased activity of C4 CCM (NADP-MDH and NADP-ME activities). NADP-MDH activity was sensitive to temperature, while that of NADP-ME decreased under eT or eCO2 but most conspicuously when they acted together. Under eCO2, regardless of temperature, the appearance of starch grains was observed in the cells of the second mesophyll layer (M(II)), which also indicated Calvin cycle activity. Thus, K. prostrata exhibits the signs of C4-like photosynthesis and, possibly, an active C3 cycle in the M(II) cells under eCO2 alone or combined with eT. [ABSTRACT FROM AUTHOR]

C4 plants are expected to have faster stomatal movements than C3 species because they tend to have smaller guard cells. However, little is known about how the evolution of C4 photosynthesis influences stomatal dynamics in relation to guard cell size and environmental factors. We studied photosynthetically diverse populations of the grass Alloteropsis semialata, showing that the origin of C4 photosynthesis in this species was associated with a shortening of stomatal guard and subsidiary cells. However, for a given cell size, C4 and C3–C4 intermediate individuals had similar or slower light‐induced stomatal opening speeds than C3 individuals. Conversely, when exposed to decreasing light, stomata in C4 plants closed as fast as those in non‐C4 plants. Polyploid formation in some C4 plants led to larger stomatal cells and was associated with slower stomatal opening. Conversely, diversification of C4 diploid plants into wetter environments was associated with an acceleration of stomatal opening. Overall, there was significant relationship between light‐saturated photosynthesis and stomatal opening speed in the C4 plants, implying that photosynthetic energy production was limiting for stomatal opening. Stomatal dynamics in this wild grass therefore arise from the evolving interplay between photosynthetic physiology and the size and biochemical function of stomatal complexes. Summary statement: Stomatal dynamics in a wild grass arise from the evolving interplay between photosynthetic physiology and the size and biochemical function of stomatal complexes. [ABSTRACT FROM AUTHOR]

C4 crops have more efficient photosynthetic pathways that enable their higher photosynthetic capacities as well as nitrogen and water use efficiencies than C3 crops. Previous research has demonstrated that the genomes of C3 species include and express every gene needed for the C4 photosynthesis pathway. However, very little is known about the dynamics and evolutionary history of such genetic evolution in C4 plants. In this study, the genes encoding five key photosynthetic pathway enzymes in the genomes of C3 (rice), C4 (maize, sorghum, and foxtail millet), and CAM (pineapple) crops were identified and compared systematically. The numbers of genes in these photosynthetic enzymes were highest in the C4 crops like sorghum and foxtail millet, while only eight genes were identified in the CAM plant. However, 16 genes were identified in the C3 crop rice. Furthermore, we performed physical, chemical, gene structure and, cis-element analyses to obtain complete insights into these key genes. Tissue-specific expressions showed that most of the photosynthetic genes are expressed in the leaf tissues. Comparisons of the expression characteristics confirmed that the expression patterns of non-photosynthetic gene copies were relatively conserved among the species, while the C4 gene copies in the C4 species acquired new tissue expression patterns during evolution. Additionally, multiple sequence features that could affect C4 gene expressions and subcellular localization were found in the coding and promoter regions. Our research also highlights the variations in how different genes have evolved within the C4 photosynthetic pathway, and we confirmed that specific high expressions in the leaves and right distribution within the cells were crucial for the development of the C4 photosynthetic abilities. The findings of this study are expected to aid in understanding the evolutionary process of the C4 photosynthetic pathway in grasses as well as offer insights for modifying the C4 photosynthetic pathways in wheat, rice, and other significant C3 cereal crops. [ABSTRACT FROM AUTHOR]

Eleocharis vivipara, an amphibious sedge in the Cyperaceae family, has several remarkable properties, most notably its alternate use of C3 photosynthesis underwater and C4 photosynthesis on land. However, the absence of genomic data has hindered its utility for evolutionary and genetic research. Here, we present a high‐quality genome for E. vivipara, representing the first chromosome‐level genome for the Eleocharis genus, with an approximate size of 965.22 Mb mainly distributed across 10 chromosomes. Its Hi–C pattern, chromosome clustering results, and one‐to‐one genome synteny across two subgroups indicates a tetraploid structure with chromosome count 2n = 4x = 20. Phylogenetic analysis suggests that E. vivipara diverged from Cyperus esculentus approximately 32.96 million years ago (Mya), and underwent a whole‐genome duplication (WGD) about 3.5 Mya. Numerous fusion and fission events were identified between the chromosomes of E. vivipara and its close relatives. We demonstrate that E. vivipara has holocentromeres, a chromosomal feature which can maintain the stability of such chromosomal rearrangements. Experimental transplantation and cross‐section studies showed its terrestrial culms developed C4 Kranz anatomy with increased number of chloroplasts in the bundle sheath (BS) cells. Gene expression and weighted gene co‐expression network analysis (WGCNA) showed overall elevated expression of core genes associated with the C4 pathway, and significant enrichment of genes related to modified culm anatomy and photosynthesis efficiency. We found evidence of mixed nicotinamide adenine dinucleotide ‐ malic enzyme and phosphoenolpyruvate carboxykinase type C4 photosynthesis in E. vivipara, and hypothesize that the evolution of C4 photosynthesis predates the WGD event. The mixed type is dominated by subgenome A and supplemented by subgenome B. Collectively, our findings not only shed light on the evolution of E. vivipara and karyotype within the Cyperaceae family, but also provide valuable insights into the transition between C3 and C4 photosynthesis, offering promising avenues for crop improvement and breeding. [ABSTRACT FROM AUTHOR]

Summary: Societal Impact Statement: Climate change continues to intensify the challenges of food production as agricultural systems face more variable and extreme weather. Coupled with increasing human population, growers must balance increasing crop yields with nutrient content to prevent global malnutrition. Photosynthetic diversity may permit some crops to tolerate climate change and elevated CO2 whilst maintaining both crop yield quantity and quality. This review examines how photosynthetic diversity interacts with crop production and nutritional stability under elevated CO2 and climate change, and highlights opportunities for photosynthetic diversity to inspire agricultural solutions. Summary: Innovative agricultural solutions are desperately needed to achieve food security for a growing human population amidst the imminent pressures of climate change that threaten more variable and extreme weather, placing additional pressures on already precarious agricultural systems. Not only are crop yields at risk under climate change but rising global atmospheric CO2 concentrations are concurrently driving a carbon dilution effect that threatens to reduce the nutritional quality of our crops to further global malnutrition. Plants using different photosynthetic metabolisms, however, experience these negative impacts to yield and nutrition to different degrees. Thus, photosynthetic diversity may offer solutions to combat malnutrition under climate change and elevated CO2 concentrations, whether that be through targeting existing resilient species for agricultural programmes or applying agricultural biotechnology to engineer photosynthetic diversity into existing crops. Here, we discuss how each major photosynthetic type is predicted to fare under elevated CO2 concentrations and climate change and explore agricultural opportunities to maintain both yield and nutrient stability. [ABSTRACT FROM AUTHOR]

While considerable knowledge exists about the enzymes pivotal for C4 photosynthesis, much less is known about the cis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C4 enzymes for five different grass species. This study spans four C4 species, covering three distinct photosynthetic subtypes: Zea mays and Sorghum bicolor (NADP-dependent malic enzyme), Panicum miliaceum (NAD-dependent malic enzyme), Urochloa fusca (phosphoenolpyruvate carboxykinase), along with the C3 outgroup Oryza sativa. We studied the cis-regulatory landscape of enzymes essential across all C4 species and those unique to C4 subtypes, measuring cell-type-specific biases for C4 enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C4 evolution. Besides promoter proximal ACRs, we found that, on average, C4 genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C4 evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution of cis-regulation at these C4 loci. This study illuminates the dynamic and complex nature of cis-regulatory elements evolution in C4 photosynthesis, particularly highlighting the intricate cis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C3 crop performance under changing climatic conditions. [ABSTRACT FROM AUTHOR]

C3 photosynthesis can be complemented with a C4 carbon concentrating mechanism (CCM) to minimize photorespiratory losses. C4 photosynthesis is often more efficient than C3 under steady‐state conditions. However, the C4 CCM depends on inter‐cellular metabolite concentration gradients, which must increase following increases in light intensity and could decrease rates of C4 photosynthesis under fluctuating light. Additionally, incomplete flux through photorespiration could prove beneficial to C4 assimilation during light induction of the CCM. Here, we compare metabolic profiles in the closely related C3Flaveria robusta and C4Flaveria bidentis during a light transient from low to high light to determine if these non‐steady state accumulation patterns provide insight to the induction of the metabolite gradients needed to drive C4 intermediate transport and if there is incomplete cycling of photorespiratory intermediates. In these C3 and C4 species, metabolite steady‐state pool sizes suggest that C4 transport acids maintain concentration gradients across the bundle sheath and mesophyll cell types under these light fluctuations. However, there was incomplete flux through photorespiration in the C4F. bidentis, which could reduce photorespiratory CO2 loss via glycine decarboxylation and help maintain higher rates of assimilation during following induction periods. [ABSTRACT FROM AUTHOR]

Summary Societal Impact Statement Climate change continues to intensify the challenges of food production as agricultural systems face more variable and extreme weather. Coupled with increasing human population, growers must balance increasing crop yields with nutrient content to prevent global malnutrition. Photosynthetic diversity may permit some crops to tolerate climate change and elevated CO2 whilst maintaining both crop yield quantity and quality. This review examines how photosynthetic diversity interacts with crop production and nutritional stability under elevated CO2 and climate change, and highlights opportunities for photosynthetic diversity to inspire agricultural solutions. Summary Innovative agricultural solutions are desperately needed to achieve food security for a growing human population amidst the imminent pressures of climate change that threaten more variable and extreme weather, placing additional pressures on already precarious agricultural systems. Not only are crop yields at risk under climate change but rising global atmospheric CO2 concentrations are concurrently driving a carbon dilution effect that threatens to reduce the nutritional quality of our crops to further global malnutrition. Plants using different photosynthetic metabolisms, however, experience these negative impacts to yield and nutrition to different degrees. Thus, photosynthetic diversity may offer solutions to combat malnutrition under climate change and elevated CO2 concentrations, whether that be through targeting existing resilient species for agricultural programmes or applying agricultural biotechnology to engineer photosynthetic diversity into existing crops. Here, we discuss how each major photosynthetic type is predicted to fare under elevated CO2 concentrations and climate change and explore agricultural opportunities to maintain both yield and nutrient stability.

Summary: The superior productivity of C4 plants is achieved via a metabolic C4 cycle which acts as a CO2 pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. The importance of chloroplast NADH dehydrogenase‐like complex (NDH) operating cyclic electron flow (CEF) around Photosystem I (PSI) for C4 photosynthesis has been shown in reverse genetics studies but the contribution of CEF and NDH to cell‐level electron fluxes remained unknown.We have created gene‐edited Setaria viridis with null ndhO alleles lacking functional NDH and developed methods for quantification of electron flow through NDH in BS and mesophyll cells.We show that CEF accounts for 84% of electrons reducing PSI in BS cells and most of those electrons are delivered through NDH while the contribution of the complex to electron transport in mesophyll cells is minimal. A decreased leaf CO2 assimilation rate and growth of plants lacking NDH cannot be rescued by supplying additional CO2.Our results indicate that NDH‐mediated CEF is the primary electron transport route in BS chloroplasts highlighting the essential role of NDH in generating ATP required for CO2 fixation by the C3 cycle in BS cells. [ABSTRACT FROM AUTHOR]

Summary: C4 photosynthesis is a complex trait requiring multiple developmental and metabolic alterations. Despite this complexity, it has independently evolved over 60 times. However, our understanding of the transition to C4 is complicated by the fact that variation in photosynthetic type is usually segregated between species that diverged a long time ago.Here, we perform a genome‐wide association study (GWAS) using the grass Alloteropsis semialata, the only known species to have C3, intermediate, and C4 accessions that recently diverged. We aimed to identify genomic regions associated with the strength of the C4 cycle (measured using δ13C), and the development of C4 leaf anatomy.Genomic regions correlated with δ13C include regulators of C4 decarboxylation enzymes (RIPK), nonphotochemical quenching (SOQ1), and the development of Kranz anatomy (SCARECROW‐LIKE). Regions associated with the development of C4 leaf anatomy in the intermediate individuals contain additional leaf anatomy regulators, including those responsible for vein patterning (GSL8) and meristem determinacy (GIF1).The parallel recruitment of paralogous leaf anatomy regulators between A. semialata and other C4 lineages implies the co‐option of these genes is context‐dependent, which likely has implications for the engineering of the C4 trait into C3 species. [ABSTRACT FROM AUTHOR]

C4 NAD‐malic enzyme (NAD‐ME) species occurs in drier regions and exhibit different drought responses compared to C4 NADP‐malic enzyme (NADP‐ME) species. However, a physiological mechanism explaining the geographical discrepancies remains uncertain. This study examined gas exchange patterns that might explain different distributions observed between two subtypes of C4 photosynthesis. We measured the response of leaf gas exchange to vapour pressure deficit (VPD) and CO2 in plants from six distinct C4 clades having closely related NAD‐ME and NADP‐ME species using a Li‐Cor 6400 gas exchange system. We found that NAD‐ME species exhibited greater relative reductions in stomatal conductance with increases in VPD than NADP‐ME species but observed no consistent subtype differences in C4 cycle activity as indicated by the initial slope of the A response to intercellular CO2 concentration. Based on these results, we hypothesise the greater response of gs to increasing VPD may enable NAD‐ME plants to outperform NADP‐ME plants in hot, dry environments where VPD is normally high. Summary statement: NAD‐malic enzyme (NAD‐ME) C4 species have more sensitive stomatal closures in response to increasing vapour pressure deficit than NADP‐malic enzyme C4 species which may explain why NAD‐ME grass species are found in drier areas than NADP‐ME species. This provides insight into the role of plant eco‐physiology in shaping biogeography. [ABSTRACT FROM AUTHOR]

The dramatic decrease in atmospheric CO2 concentration during Oligocene was proposed as directly linked to C4 evolution. However, it remains unclear how the decreased CO2 concentration directly facilitate C4 evolution, besides its role as a selection pressure. We conducted a systematic transcriptomics and metabolomics analysis under short-term low CO2 condition and found that Arabidopsis grown under this condition showed 1) increased expression of most genes encoding C4-related enzymes and transporters; 2) increased expression of genes involved in photorespiration and pathways related to carbon skeleton generation for ammonium refixation; 3) increased expression of genes directly involved in ammonium refixation. Furthermore, we found that in vitro treatment of leaves with NH4 + induced a similar pattern of changes in C4 related genes and genes involved in ammonium refixation. These data support the view that Arabidopsis grown under short-term low CO2 conditions rewired its metabolism to supply carbon skeleton for ammonium recycling, during which process the expression of C4 genes were up-regulated as a result of a hitchhiking process. This study provides new insights into the adaptation of the C3 model plant Arabidopsis under low CO2 conditions and suggests that low CO2 can facilitate the evolution of C4 photosynthesis beyond the commonly assumed role of being a selection pressure. [ABSTRACT FROM AUTHOR]

The C4 Poaceae are a diverse group in terms of both evolutionary lineage and biochemistry. There is a distinct pattern in the distribution of C4 grass groups with aridity; however, the mechanistic basis for this distribution is not well understood. Additionally, few studies have investigated the functional strategies of co‐occurring C4 grass species for dealing with aridity in their natural environments. We explored the coordination of leaf‐level gas exchange, water use, and morphology among five co‐occurring semiarid C4 grasses belonging to divergent clades, biochemical subtypes, and size classes at three sites along a natural aridity gradient. More specifically, we measured predawn and midday water potential, stomatal conductance, water use efficiency, and photosynthesis. Leaf tissue was also collected for the analysis of stable isotopes of carbon and oxygen as well as for measurement of specific leaf area (SLA) and leaf width. Species differences in responsiveness of stomata to changes in vapor pressure deficit (VPD) were also assessed. It was expected that NAD‐me species would maintain higher rates of photosynthesis, higher water use efficiency, and have more responsive stomata than other co‐occurring species based on observed biogeographic patterns and past greenhouse studies. We found that Aristidoideae and Chloridoideae NAD‐me‐type grasses had greater stomatal sensitivity to VPD, consistent with a more isohydric strategy. However, midgrasses had both greater apparent water access and water use efficiency, regardless of subtype or lineage. PCK‐type species had less responsive stomata and maintained lower levels of photosynthesis with increasing aridity. There were strong interspecific differences in δ13C, leaf width, and SLA; however, these were not significantly correlated with water use efficiency. C4 grasses in our study did not fit discretely into functional groups as defined by lineage, biochemistry, or size class. Interspecific differences, evolutionary legacy, and biochemical pathway are likely to interact to determine water use and photosynthetic strategies of these plants. Control of water loss via highly responsive stomata may form the basis for dominance of certain C4 grass groups in arid environments. These findings build on our understanding of contrasting strategies of C4 grasses for dealing with aridity in their natural environments. [ABSTRACT FROM AUTHOR]

Summary: Increasing atmospheric CO2 is changing the dynamics of tropical savanna vegetation. C3 trees and grasses are known to experience CO2 fertilization, whereas responses to CO2 by C4 grasses are more ambiguous.Here, we sample stable carbon isotope trends in herbarium collections of South African C4 and C3 grasses to reconstruct 13C discrimination.We found that C3 grasses showed no trends in 13C discrimination over the past century but that C4 grasses increased their 13C discrimination through time, especially since 1950. These changes were most strongly linked to changes in atmospheric CO2 rather than to trends in rainfall climatology or temperature.Combined with previously published evidence that grass biomass has increased in C4‐dominated savannas, these trends suggest that increasing water‐use efficiency due to CO2 fertilization may be changing C4 plant–water relations. CO2 fertilization of C4 grasses may thus be a neglected pathway for anthropogenic global change in tropical savanna ecosystems. [ABSTRACT FROM AUTHOR]

The Chenopodiaceae family is one of the largest and most ancient families in arid regions of the globe. The representatives of this family, being dominants and edificators of many desert plant communities, play a significant role in forming the vegetation cover, particularly the species of the Salsoloideae subfamily. A characteristic feature of the anatomical structure of Chenopodiaceae species is the presence of Kranz cells. The discovery of a unique pathway of primary carboxylation, known as the C4 dicarboxylic acid cycle, has led to a rapid development of biochemical and anatomical research. It is known that the Chenopodiaceae family has the largest number of species with the C4 photosynthesis type and the greatest diversity in C4 leaf anatomy. This study aimed to identify the anatomical structural features of the dominant species of the Chenopodiaceae family growing in arid regions. The study objects are species of the Chenopodiaceae family growing in arid territories of the Kyzylorda region in Kazakhstan. Cross-sections of samples were made using a "Semi-automatic Rotary Microtome M530" (MEDITE M530). The thickness of the cross-sections was 40 μm. The cross-sections were examined using a Levenhuk Zoom&Joy microscope (China), and images were taken with a Levenhuk D740T 5.1 camera using the LevenhukLite software. The study of the anatomical structure of assimilative organs revealed the following types: Cоrispermоid-type, Ventrо-dоrsal type, Kоchiоid-type, Atriplicоid-type, Salsina-type, Shоberia-type, Salsоlоid-type, and Climacоptera-type. The anatomical structure of Atriplex dimorphostegia was clarified. Haloxylon aphyllum was transferred from the Kranz-ventrodorsal type to the Salsoloid type. For the first time, a detailed anatomical structure of the leaves of Salsola sogdiana and Caroxylon nitrarium species was described. [ABSTRACT FROM AUTHOR]

C4 crops have more efficient photosynthetic pathways that enable their higher photosynthetic capacities as well as nitrogen and water use efficiencies than C3 crops. Previous research has demonstrated that the genomes of C3 species include and express every gene needed for the C4 photosynthesis pathway. However, very little is known about the dynamics and evolutionary history of such genetic evolution in C4 plants. In this study, the genes encoding five key photosynthetic pathway enzymes in the genomes of C3 (rice), C4 (maize, sorghum, and foxtail millet), and CAM (pineapple) crops were identified and compared systematically. The numbers of genes in these photosynthetic enzymes were highest in the C4 crops like sorghum and foxtail millet, while only eight genes were identified in the CAM plant. However, 16 genes were identified in the C3 crop rice. Furthermore, we performed physical, chemical, gene structure and, cis-element analyses to obtain complete insights into these key genes. Tissue-specific expressions showed that most of the photosynthetic genes are expressed in the leaf tissues. Comparisons of the expression characteristics confirmed that the expression patterns of non-photosynthetic gene copies were relatively conserved among the species, while the C4 gene copies in the C4 species acquired new tissue expression patterns during evolution. Additionally, multiple sequence features that could affect C4 gene expressions and subcellular localization were found in the coding and promoter regions. Our research also highlights the variations in how different genes have evolved within the C4 photosynthetic pathway, and we confirmed that specific high expressions in the leaves and right distribution within the cells were crucial for the development of the C4 photosynthetic abilities. The findings of this study are expected to aid in understanding the evolutionary process of the C4 photosynthetic pathway in grasses as well as offer insights for modifying the C4 photosynthetic pathways in wheat, rice, and other significant C3 cereal crops.

Abstract C3 photosynthesis can be complemented with a C4 carbon concentrating mechanism (CCM) to minimize photorespiratory losses. C4 photosynthesis is often more efficient than C3 under steady‐state conditions. However, the C4 CCM depends on inter‐cellular metabolite concentration gradients, which must increase following increases in light intensity and could decrease rates of C4 photosynthesis under fluctuating light. Additionally, incomplete flux through photorespiration could prove beneficial to C4 assimilation during light induction of the CCM. Here, we compare metabolic profiles in the closely related C3 Flaveria robusta and C4 Flaveria bidentis during a light transient from low to high light to determine if these non‐steady state accumulation patterns provide insight to the induction of the metabolite gradients needed to drive C4 intermediate transport and if there is incomplete cycling of photorespiratory intermediates. In these C3 and C4 species, metabolite steady‐state pool sizes suggest that C4 transport acids maintain concentration gradients across the bundle sheath and mesophyll cell types under these light fluctuations. However, there was incomplete flux through photorespiration in the C4 F. bidentis, which could reduce photorespiratory CO2 loss via glycine decarboxylation and help maintain higher rates of assimilation during following induction periods.

Leaf day respiration rate (RL) plays a crucial role in the global carbon cycle. However, RL of C4 species has not been sufficiently studied and its response to environmental factors is largely unknown. This work studied the response of RL of three C4 species, Setaria viridis, Sorghum sudanense, and Zea mays, to alterations in the vapour pressure deficit (VPD) and irradiance of the growth environment. RL was estimated using the Kok method (RL Kok) and an improved method that combined gas exchange and chlorophyll fluorescence measurements (RL Yin). On average, shade treatment led to a 24% reduction in RL Yin and a 20% reduction in respiration in the dark (RDk), while a consistent VPD effect on RL was not observed. RL and RDk were positively correlated with nitrogen content per leaf area and net CO2 assimilation rate but were not correlated with the capacity of carboxylation enzymes. We found a non-significant light inhibition of respiration (1 ​± ​2%), contradicting the assumption that respiration is inhibited by light and affected by light intensity. Our findings indicate that assuming RL to be equal to RDk at the same temperature is a straightforward but reliable approach to model respiration of the examined C4 species.

Photosynthesis is the predominant metabolic process for energy obtention in plants. Here we describe a case study where a set of anatomical, biochemical, and molecular characters are used to reconstruct the evolution of the C4 photosynthetic pathway, within the evolutionary framework provided by the genus Flaveria. Our main educational goal was to engage biology undergraduate students to solve a photosynthesis-related phylogenetic problem by stimulating them to assemble informative characters of diverse nature. This case study was successfully implemented as a seminar in a massive university introductory course, during the COVID19 lockdown. Using an online learning environment, most small groups achieved the Students Learning Outcomes, which were assessed through a questionnaire and an open-ended question. Individual performance was also evaluated through a specific exercise in the course's midterm test. Finally, students were surveyed for their perception about the seminar through a poll. We concluded that this seminar could be easily adopted to promote the learning of complex and interdisciplinary content like the C4 photosynthetic pathway in undergraduate botanical education, even in a large online introductory course. [ABSTRACT FROM AUTHOR]

Main Conclusion: This study reveals miRNA indirect regulation of C4 genes in sugarcane through transcription factors, highlighting potential key regulators like SsHAM3a. C4 photosynthesis is crucial for the high productivity and biomass of sugarcane, however, the miRNA regulation of C4 genes in sugarcane remains elusive. We have identified 384 miRNAs along the leaf gradients, including 293 known miRNAs and 91 novel miRNAs. Among these, 86 unique miRNAs exhibited differential expression patterns, and we identified 3511 potential expressed targets of these differentially expressed miRNAs (DEmiRNAs). Analyses using Pearson correlation coefficient (PCC) and Gene Ontology (GO) enrichment revealed that targets of miRNAs with positive correlations are integral to chlorophyll-related photosynthetic processes. In contrast, negatively correlated pairs are primarily associated with metabolic functions. It is worth noting that no C4 genes were predicted as targets of DEmiRNAs. Our application of weighted gene co-expression network analysis (WGCNA) led to a gene regulatory network (GRN) suggesting miRNAs might indirectly regulate C4 genes via transcription factors (TFs). The GRAS TF SsHAM3a emerged as a potential regulator of C4 genes, targeted by miR171y and miR171am, and exhibiting a negative correlation with miRNA expression along the leaf gradient. This study sheds light on the complex involvement of miRNAs in regulating C4 genes, offering a foundation for future research into enhancing sugarcane's photosynthetic efficiency. [ABSTRACT FROM AUTHOR]

Summary: C4 photosynthesis is a key innovation in land plant evolution, but its immediate effects on population demography are unclear. We explore the early impact of the C4 trait on the trajectories of C4 and non‐C4 populations of the grass Alloteropsis semialata.We combine niche models projected into paleoclimate layers for the last 5 million years with demographic models based on genomic data.The initial split between C4 and non‐C4 populations was followed by a larger expansion of the ancestral C4 population, and further diversification led to the unparalleled expansion of descendant C4 populations. Overall, C4 populations spread over three continents and achieved the highest population growth, in agreement with a broader climatic niche that rendered a large potential range over time. The C4 populations that remained in the region of origin, however, experienced lower population growth, rather consistent with local geographic constraints. Moreover, the posterior transfer of some C4‐related characters to non‐C4 counterparts might have facilitated the recent expansion of non‐C4 populations in the region of origin.Altogether, our findings support that C4 photosynthesis provided an immediate demographic advantage to A. semialata populations, but its effect might be masked by geographic contingencies. [ABSTRACT FROM AUTHOR]

Identification of signaling pathways that control C4 photosynthesis development is essential for introducing the C4 pathway into C3 crops. Species with dual photosynthesis in their life cycle are interesting models to study such regulatory mechanisms. The species used here Halimocnemis mollissima Bunge, belonging to the Caroxyleae tribe, displays C3 photosynthesis in its cotyledons and a NAD-ME subtype of C4 photosynthesis in the First leaves (FLs) onwards. We explored the long-distance signaling pathways that are probably implicated in the shoot–root coordination associated with the manifestation of the C4 traits, including efficient resource usage by comparing the mRNA content of hypocotyls before and after the C4 first leave's formation. Histological examination showed the presence of C3 anatomy in cotyledons and C4 anatomy in the FLs. Our transcriptome analyses verified the performance of the NAD-ME subtype of C4 in FLs and revealed differential transcript abundance of several potential mobile regulators and their associated receptors or transporters in two developmentally different hypocotyls of H. mollissima Bunge. These differentially expressed genes (DEGs) belong to diverse functional groups, including various transcription factor (TF) families, phytohormones metabolism, and signaling peptides, part of which could be related to hypocotyl development. Our findings support the higher nitrogen and water use efficiency associated with C4 photosynthetic and provide insights into the coordinated above- and under-ground tissue communication during the developmental transition of C3–C4 photosynthesis in this species. [ABSTRACT FROM AUTHOR]

Abstract The C4 Poaceae are a diverse group in terms of both evolutionary lineage and biochemistry. There is a distinct pattern in the distribution of C4 grass groups with aridity; however, the mechanistic basis for this distribution is not well understood. Additionally, few studies have investigated the functional strategies of co‐occurring C4 grass species for dealing with aridity in their natural environments. We explored the coordination of leaf‐level gas exchange, water use, and morphology among five co‐occurring semiarid C4 grasses belonging to divergent clades, biochemical subtypes, and size classes at three sites along a natural aridity gradient. More specifically, we measured predawn and midday water potential, stomatal conductance, water use efficiency, and photosynthesis. Leaf tissue was also collected for the analysis of stable isotopes of carbon and oxygen as well as for measurement of specific leaf area (SLA) and leaf width. Species differences in responsiveness of stomata to changes in vapor pressure deficit (VPD) were also assessed. It was expected that NAD‐me species would maintain higher rates of photosynthesis, higher water use efficiency, and have more responsive stomata than other co‐occurring species based on observed biogeographic patterns and past greenhouse studies. We found that Aristidoideae and Chloridoideae NAD‐me‐type grasses had greater stomatal sensitivity to VPD, consistent with a more isohydric strategy. However, midgrasses had both greater apparent water access and water use efficiency, regardless of subtype or lineage. PCK‐type species had less responsive stomata and maintained lower levels of photosynthesis with increasing aridity. There were strong interspecific differences in δ13C, leaf width, and SLA; however, these were not significantly correlated with water use efficiency. C4 grasses in our study did not fit discretely into functional groups as defined by lineage, biochemistry, or size class. Interspecific differences, evolutionary legacy, and biochemical pathway are likely to interact to determine water use and photosynthetic strategies of these plants. Control of water loss via highly responsive stomata may form the basis for dominance of certain C4 grass groups in arid environments. These findings build on our understanding of contrasting strategies of C4 grasses for dealing with aridity in their natural environments.

The dramatic decrease in atmospheric CO2 concentration during Oligocene was proposed as directly linked to C4 evolution. However, it remains unclear how the decreased CO2 concentration directly facilitate C4 evolution, besides its role as a selection pressure. We conducted a systematic transcriptomics and metabolomics analysis under short-term low CO2 condition and found that Arabidopsis grown under this condition showed 1) increased expression of most genes encoding C4-related enzymes and transporters; 2) increased expression of genes involved in photorespiration and pathways related to carbon skeleton generation for ammonium refixation; 3) increased expression of genes directly involved in ammonium refixation. Furthermore, we found that in vitro treatment of leaves with NH4+ induced a similar pattern of changes in C4 related genes and genes involved in ammonium refixation. These data support the view that Arabidopsis grown under short-term low CO2 conditions rewired its metabolism to supply carbon skeleton for ammonium recycling, during which process the expression of C4 genes were up-regulated as a result of a hitchhiking process. This study provides new insights into the adaptation of the C3 model plant Arabidopsis under low CO2 conditions and suggests that low CO2 can facilitate the evolution of C4 photosynthesis beyond the commonly assumed role of being a selection pressure.

In this study, Zea mays L. was grown in indoor controlled environment chambers with a uniform daytime temperature of 30°C, and night-time temperatures of 30, 25, 20, or 15°C. Responses of net photosynthesis (PN) of mature leaves at high PPFD to intercellular CO2 concentrations (Ci) were measured at 20, 25, and 30°C using a new method that generates a complete PN vs. Ci curve in less than four minutes. Results indicated that photosynthesis measured at both Ci values of 25 and 150 μmol mol-1 increased strongly with measurement temperature in plants grown with night temperatures of 25 and 30°C, but there was a much smaller change in photosynthesis with temperature in plants grown with night temperatures of 15 or 20°C. These results indicate that the acclimation of photosynthesis to temperature in this C4 species is substantially affected by night temperature.

Summary: Photosynthetic efficiency is reduced by the dual role of Rubisco, which acts either as a carboxylase or as an oxygenase, the latter leading to photorespiration. C4 photosynthesis evolved as a carbon‐concentrating mechanism to reduce photorespiration. To engineer C4 into a C3 plant, it is essential to understand how C4 genes, such as phosphoenolpyruvate carboxylase (PEPC1), are regulated to be expressed at high levels and in a cell‐specific manner.Yeast one‐hybrid screening was used to show that OsPRI1, a rice bHLH transcription factor involved in iron homeostasis, binds to the Setaria viridis PEPC1 promoter. This promoter drives mesophyll‐specific gene expression in rice. The role of OsPRI1 in planta was characterized using a rice line harbouring SvPEPC1pro::GUS.We show that OsPRI1 activates the S. viridis PEPC1 promoter by binding to an N‐box in the proximal promoter, and that GUS activity is highly reduced in SvPEPC1pro::GUS lines when OsPRI1 is mutated. Cross‐species comparisons showed that the SvPRI1 homolog binds to the SvPEPC1 promoter but the maize ZmPRI1 does not bind to the ZmPEPC1 promoter.Our results suggest that elements of the iron homeostasis pathway were co‐opted to regulate PEPC1 gene expression during the evolution of some but not all C4 species. [ABSTRACT FROM AUTHOR]

Summary: Radiation use efficiency (RUE) is a key crop adaptation trait that quantifies the potential amount of aboveground biomass produced by the crop per unit of solar energy intercepted. But it is unclear why elite maize and grain sorghum hybrids differ in their RUE at the crop level. Here, we used a non‐traditional top‐down approach via canopy photosynthesis modelling to identify leaf‐level photosynthetic traits that are key to differences in crop‐level RUE.A novel photosynthetic response measurement was developed and coupled with use of a Bayesian model fitting procedure, incorporating a C4 leaf photosynthesis model, to infer cohesive sets of photosynthetic parameters by simultaneously fitting responses to CO2, light, and temperature.Statistically significant differences between leaf photosynthetic parameters of elite maize and grain sorghum hybrids were found across a range of leaf temperatures, in particular for effects on the quantum yield of photosynthesis, but also for the maximum enzymatic activity of Rubisco and PEPc.Simulation of diurnal canopy photosynthesis predicted that the leaf‐level photosynthetic low‐light response and its temperature dependency are key drivers of the performance of crop‐level RUE, generating testable hypotheses for further physiological analysis and bioengineering applications. [ABSTRACT FROM AUTHOR]

Plant glycerate kinase (GK) was previously considered an exclusively chloroplastic enzyme of the glycolate pathway (photorespiration), and its sole predicted role was to return most of the glycolate-derived carbon (as glycerate) to the Calvin cycle. However, recent discovery of cytosolic GK revealed metabolic links for glycerate to other processes. Although GK was initially proposed as being solely regulated by substrate availability, subsequent discoveries of its redox regulation and the light involvement in the production of chloroplastic and cytosolic GK isoforms have indicated a more refined regulation of the pathways of glycerate conversion. Here, we re-evaluate the importance of GK and emphasize its multifaceted role in plants. Thus, GK can be a major player in several branches of primary metabolism, including the glycolate pathway, gluconeogenesis, glycolysis, and C4 metabolism. In addition, recently, the chloroplastic (but not cytosolic) GK isoform was implicated as part of a light-dependent plant immune response to pathogen attack. The origins of glycerate are also discussed here; it is produced in several cell compartments and undergoes huge fluctuations depending on light/dark conditions. The recent discovery of the vacuolar glycerate transporter adds yet another layer to our understanding of glycerate transport/metabolism and that of other two- and three-carbon metabolites. [ABSTRACT FROM AUTHOR]

Summary: In leaves of C4 plants, the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C4 acids in mesophyll (M) cells and subsequent decarboxylation in the bundle sheath (BS). In C4 grasses, proliferation of plasmodesmata between these cell types is thought to increase cell‐to‐cell connectivity to allow efficient metabolite movement. However, it is not known whether C4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C4 photosynthesis is not clear. How M and BS cells in C4 leaves become highly connected is also not known.We investigated these questions using 3D‐ and 2D‐electron microscopy on the C4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C3 relatives.The M–BS interface of C4G. gynandra showed higher plasmodesmal frequency compared with closely related C3 species. Formation of these plasmodesmata was induced by light. Pharmacological agents that perturbed photosynthesis reduced the number of plasmodesmata, but this inhibitory effect could be reversed by the provision of exogenous sucrose.We conclude that enhanced formation of plasmodesmata between M and BS cells is wired to the induction of photosynthesis in C4G. gynandra. [ABSTRACT FROM AUTHOR]

Summary: C4 plants typically operate a CO2 concentration mechanism from mesophyll (M) cells into bundle sheath (BS) cells. NADH dehydrogenase‐like (NDH) complex is enriched in the BS cells of many NADP‐malic enzyme (ME) type C4 plants and is more abundant in C4 than in C3 plants, but to what extent it is involved in the CO2 concentration mechanism remains to be experimentally investigated.We created maize and rice mutants deficient in NDH function and then used a combination of transcriptomic, proteomic, and metabolomic approaches for comparative analysis.Considerable decreases in growth, photosynthetic activities, and levels of key photosynthetic proteins were observed in maize but not rice mutants. However, transcript abundance for many cyclic electron transport (CET) and Calvin–Benson cycle components, as well as BS‐specific C4 enzymes, was increased in maize mutants. Metabolite analysis of the maize ndh mutants revealed an increased NADPH : NADP ratio, as well as malate, ribulose 1,5‐bisphosphate (RuBP), fructose 1,6‐bisphosphate (FBP), and photorespiration intermediates.We suggest that by optimizing NADPH and malate levels and adjusting NADP‐ME activity, NDH functions to balance metabolic and redox states in the BS cells of maize (in addition to ATP supply), coordinating photosynthetic transcript abundance and protein content, thus directly regulating the carbon flow in the two‐celled C4 system of maize. [ABSTRACT FROM AUTHOR]

In this study, Zea mays L. was grown in indoor controlled environment chambers with a uniform daytime temperature of 30°C, and night-time temperatures of 30, 25, 20, or 15°C. Responses of net photosynthesis (PN) of mature leaves at high PPFD to intercellular CO2 concentrations (Ci) were measured at 20, 25, and 30°C using a new method that generates a complete PN vs. Ci curve in less than four minutes. Results indicated that photosynthesis measured at both Ci values of 25 and 150 µmol mol-1 increased strongly with measurement temperature in plants grown with night temperatures of 25 and 30°C, but there was a much smaller change in photosynthesis with temperature in plants grown with night temperatures of 15 or 20°C. These results indicate that the acclimation of photosynthesis to temperature in this C4 species is substantially affected by night temperature. [ABSTRACT FROM AUTHOR]

Abstract Background Pennisetum giganteum (AABB, 2n = 4x = 28) is a C4 plant in the genus Pennisetum with origin in Africa but currently also grown in Asia and America. It is a crucial forage and potential energy grass with significant advantages in yield, stress resistance, and environmental adaptation. However, the mechanisms underlying these advantageous traits remain largely unexplored. Here, we present a high-quality genome assembly of the allotetraploid P. giganteum aiming at providing insights into biomass accumulation. Results Our assembly has a genome size 2.03 Gb and contig N50 of 88.47 Mb that was further divided into A and B subgenomes. Genome evolution analysis revealed the evolutionary relationships across the Panicoideae subfamily lineages and identified numerous genome rearrangements that had occurred in P. giganteum. Comparative genomic analysis showed functional differentiation between the subgenomes. Transcriptome analysis found no subgenome dominance at the overall gene expression level; however, differentially expressed homoeologous genes and homoeolog-specific expressed genes between the two subgenomes were identified, suggesting that complementary effects between the A and B subgenomes contributed to biomass accumulation of P. giganteum. Besides, C4 photosynthesis-related genes were significantly expanded in P. giganteum and their sequences and expression patterns were highly conserved between the two subgenomes, implying that both subgenomes contributed greatly and almost equally to the highly efficient C4 photosynthesis in P. giganteum. We also identified key candidate genes in the C4 photosynthesis pathway that showed sustained high expression across all developmental stages of P. giganteum. Conclusions Our study provides important genomic resources for elucidating the genetic basis of advantageous traits in polyploid species, and facilitates further functional genomics research and genetic improvement of P. giganteum.

Abstract Photosynthetic phosphoenolpyruvate carboxylase (PEPC) catalyses the irreversible carboxylation of phosphoenolpyruvate (PEP), producing oxaloacetate (OAA). This enzyme catalyses the first step of carbon fixation in C4 photosynthesis, contributing to the high photosynthetic efficiency of C4 plants. PEPC is also involved in replenishing tricarboxylic acid cycle intermediates, such as OAA, being involved in the C/N balance. In plants, PEPCs are classified in two types: bacterial type (BTPC) and plant-type (PTPC), which includes photosynthetic and non-photosynthetic PEPCs. During C4 evolution, photosynthetic PEPCs evolved independently. C4 PEPCs evolved to be highly expressed and active in a spatial-specific manner. Their gene expression pattern is also regulated by developmental cues, light, circadian clock as well as adverse environmental conditions. However, the gene regulatory networks controlling C4 PEPC gene expression, namely its cell-specificity, are largely unknown. Therefore, after an introduction to the evolution of PEPCs, this review aims to discuss the current knowledge regarding the transcriptional regulation of C4 PEPCs, focusing on cell-specific and developmental expression dynamics, light and circadian regulation, as well as response to abiotic stress. In conclusion, this review aims to highlight the evolution, transcriptional regulation by different signals and importance of PEPC in C4 photosynthesis and its potential as tool for crop improvement.

We present anatomical imaging techniques and measurements that are useful additions to the existing morphological toolbox for the identification of seven coastal grasses and one rush. Our method is simple and inexpensive and requires the removal of only one leaf, which prevents removal of plants from their habitats. This method can also be used in seasons when reproductive structures (important for current morphological ID efforts) are not present. We find significant quantitative differences in leaf width, adaxial to abaxial ratios (ADAB ratio, which we introduce as a new parameter), vascular bundle cross-sectional areas, and interveinal distances, as well as providing images for qualitative analysis of these plants' anatomies; including differences in bundle sheath structures, fiber distributions, epidermal and cuticular properties, and chloroplast positioning. [ABSTRACT FROM AUTHOR]

C4 photosynthesis has evolved independently multiple times in grass lineages with nine anatomical and three biochemical subtypes. Chloridoideae represents one of the separate events and contains species of two biochemical subtypes, NAD-ME and PEP-CK. Assessment of C4 photosynthesis diversification is limited by species sampling. In this study, the biochemical subtypes together with anatomical leaf traits were analyzed in 19 species to reveal the evolutionary scenario for diversification of C4 photosynthesis in tribe Zoysieae (Chloridoideae). The effect of habitat on anatomical and biochemical diversification was also evaluated. The results for the 19 species studied indicate that 11 species have only NAD-ME as a decarboxylating enzyme, while eight species belong to the PEP-CK subtype. Leaf anatomy corresponds to the biochemical subtype. Analysis of Zoysieae phylogeny indicates multiple switches between PEP-CK and NAD-ME photosynthetic subtypes, with PEP-CK most likely as the ancestral subtype, and with multiple independent PEP-CK decarboxylase losses and its secondary acquisition. A strong correlation was detected between C4 biochemical subtypes studied and habitat annual precipitation wherein NAD-ME species are confined to drier habitats, while PEP-CK species prefer humid areas. Structural adaptations to arid climate include increases in leaf thickness and interveinal distance. Our analysis suggests that multiple loss of PEP-CK decarboxylase could have been driven by climate aridization followed by continued adaptive changes in leaf anatomy. [ABSTRACT FROM AUTHOR]

C₄ is one of three known photosynthetic processes of carbon fixation in flowering plants. It evolved independently more than 61 times in multiple angiosperm lineages and consists of a series of anatomical and biochemical modifications to the ancestral C3 pathway increasing plant productivity under warm and light‐rich conditions. The C4 lineages of eudicots belong to seven orders and 15 families, are phylogenetically less constrained than those of monocots and entail an enormous structural and ecological diversity. Eudicot C4 lineages likely evolved the C4 syndrome along different evolutionary paths. Therefore, a better understanding of this diversity is key to understanding the evolution of this complex trait as a whole. By compiling 1207 recognised C4 eudicots species described in the literature and presenting trait data among these species, we identify global centres of species richness and of high phylogenetic diversity. Furthermore, we discuss climatic preferences in the context of plant functional traits. We identify two hotspots of C4 eudicot diversity: arid regions of Mexico/Southern United States and Australia, which show a similarly high number of different C4 eudicot genera but differ in the number of C4 lineages that evolved in situ. Further eudicot C4 hotspots with many different families and genera are in South Africa, West Africa, Patagonia, Central Asia and the Mediterranean. In general, C4 eudicots are diverse in deserts and xeric shrublands, tropical and subtropical grasslands, savannas and shrublands. We found C4 eudicots to occur in areas with less annual precipitation than C4 grasses which can be explained by frequently associated adaptations to drought stress such as among others succulence and salt tolerance. The data indicate that C4 eudicot lineages utilising the NAD‐ME decarboxylating enzyme grow in drier areas than those using the NADP‐ME decarboxylating enzyme indicating biochemical restrictions of the later system in higher temperatures. We conclude that in most eudicot lineages, C4 evolved in ancestrally already drought‐adapted clades and enabled these to further spread in these habitats and colonise even drier areas. [ABSTRACT FROM AUTHOR]

In nature, plants are exposed to a dynamic light environment. Fluctuations in light decreased the photosynthetic light utilization efficiency (PLUE) of leaves, and much more severely in C4 species than in C3 species. However, little is known about the plasticity of PLUE under dynamic light in C4 species. Present study focused on the influence of planting density to the photosynthesis under dynamic light in maize (Zea mays L.), a most important C4 crop. In addition, the molecular mechanism behind photosynthetic adaptation to planting density were also explored by quantitative proteomics analysis. Results revealed that as planting density increases, maize leaves receive less light that fluctuates more. The maize planted at high density (HD) improved the PLUE under dynamic light, especially in the middle and later growth stages. Quantitative proteomics analysis showed that the transfer of nitrogen from Rubisco to RuBP regeneration and C4 pathway related enzymes contributes to the photosynthetic adaptation to lower and more fluctuating light environment in HD maize. This study provides potential ways to further improve the light energy utilization efficiency of maize in HD. Summary statement: 1.As planting density increases, maize leaves receive less light that fluctuates more.2.The maize grown at high density improved the light utilization efficiency under dynamic light, especially in the middle and later growth stages.3.The transfer of nitrogen from Rubisco to RuBP regeneration and C4 pathway related enzymes contributes to the photosynthetic adaptation to lower and more fluctuating light environment in maize grown at high density. [ABSTRACT FROM AUTHOR]

Traditional "Green Revolution" cereal breeding strategies to improve yield are now reaching a plateau in our principal global food crop rice. Photosynthesis has now become a major target of international consortia to increase yield potential. Synthetic biology is being used across multiple large projects to improve photosynthetic efficiency. This review follows the genesis and progress of one of the first of these consortia projects, now in its 13th year; the Bill and Melinda Gates funded C4 Rice Project. This project seeks to install the biochemical and anatomical attributes necessary to support C4 photosynthesis in the C3 crop rice. Here we address the advances made thus far in installing the biochemical pathway and some of the key targets yet to be reached. [ABSTRACT FROM AUTHOR]

Previous studies have found that maximum quantum yield of CO2 assimilation (Φ CO2,max,app) declines in lower canopies of maize and miscanthus, a maladaptive response to self-shading. These observations were limited to single genotypes, leaving it unclear whether the maladaptive shade response is a general property of this C4 grass tribe, the Andropogoneae. We explored the generality of this maladaptation by testing the hypothesis that erect leaf forms (erectophiles), which allow more light into the lower canopy, suffer less of a decline in photosynthetic efficiency than drooping leaf (planophile) forms. On average, Φ CO2,max,app declined 27% in lower canopy leaves across 35 accessions, but the decline was over twice as great in planophiles than in erectophiles. The loss of photosynthetic efficiency involved a decoupling between electron transport and assimilation. This was not associated with increased bundle sheath leakage, based on 13C measurements. In both planophiles and erectophiles, shaded leaves had greater leaf absorptivity and lower activities of key C4 enzymes than sun leaves. The erectophile form is considered more productive because it allows a more effective distribution of light through the canopy to support photosynthesis. We show that in sorghum, it provides a second benefit, maintenance of higher Φ CO2,max,app to support efficient use of that light resource.

Abstract C₄ is one of three known photosynthetic processes of carbon fixation in flowering plants. It evolved independently more than 61 times in multiple angiosperm lineages and consists of a series of anatomical and biochemical modifications to the ancestral C3 pathway increasing plant productivity under warm and light‐rich conditions. The C4 lineages of eudicots belong to seven orders and 15 families, are phylogenetically less constrained than those of monocots and entail an enormous structural and ecological diversity. Eudicot C4 lineages likely evolved the C4 syndrome along different evolutionary paths. Therefore, a better understanding of this diversity is key to understanding the evolution of this complex trait as a whole. By compiling 1207 recognised C4 eudicots species described in the literature and presenting trait data among these species, we identify global centres of species richness and of high phylogenetic diversity. Furthermore, we discuss climatic preferences in the context of plant functional traits. We identify two hotspots of C4 eudicot diversity: arid regions of Mexico/Southern United States and Australia, which show a similarly high number of different C4 eudicot genera but differ in the number of C4 lineages that evolved in situ. Further eudicot C4 hotspots with many different families and genera are in South Africa, West Africa, Patagonia, Central Asia and the Mediterranean. In general, C4 eudicots are diverse in deserts and xeric shrublands, tropical and subtropical grasslands, savannas and shrublands. We found C4 eudicots to occur in areas with less annual precipitation than C4 grasses which can be explained by frequently associated adaptations to drought stress such as among others succulence and salt tolerance. The data indicate that C4 eudicot lineages utilising the NAD‐ME decarboxylating enzyme grow in drier areas than those using the NADP‐ME decarboxylating enzyme indicating biochemical restrictions of the later system in higher temperatures. We conclude that in most eudicot lineages, C4 evolved in ancestrally already drought‐adapted clades and enabled these to further spread in these habitats and colonise even drier areas.

In flowering plants, C4 photosynthesis is superior to C3 type in carbon fixation efficiency and adaptation to extreme environmental conditions, but the mechanisms behind the assembly of C4 machinery remain elusive. This study attempts to dissect the evolutionary divergence from C3 to C4 photosynthesis in five photosynthetic model plants from the grass family, using a combined comparative transcriptomics and deep learning technology. By examining and comparing gene expression levels in bundle sheath and mesophyll cells of five model plants, we identified 16 differentially expressed signature genes showing cell-specific expression patterns in C3 and C4 plants. Among them, two showed distinctively opposite cell-specific expression patterns in C3 vs. C4 plants (named as FOGs). The in silico physicochemical analysis of the two FOGs illustrated that C3 homologous proteins of LHCA6 had low and stable pI values of ~6, while the pI values of LHCA6 homologs increased drastically in C4 plants Setaria viridis (7), Zea mays (8), and Sorghum bicolor (over 9), suggesting this protein may have different functions in C3 and C4 plants. Interestingly, based on pairwise protein sequence/structure similarities between each homologous FOG protein, one FOG PGRL1A showed local inconsistency between sequence similarity and structure similarity. To find more examples of the evolutionary characteristics of FOG proteins, we investigated the protein sequence/structure similarities of other FOGs (transcription factors) and found that FOG proteins have diversified incompatibility between sequence and structure similarities during grass family evolution. This raised an interesting question as to whether the sequence similarity is related to structure similarity during C4 photosynthesis evolution. [ABSTRACT FROM AUTHOR]

SUMMARY: Most studies assume midday gas exchange measurements capture the leaf's daytime performance. However, stomatal conductance (gs) and photosynthesis (An) fluctuate diurnally due to endogenous and environmental rhythms, which can affect intrinsic water use efficiency (iWUE). Six Sorghum lines with contrasting stomatal anatomical traits were grown in environmentally controlled conditions, and leaf gas exchange was measured three times a day. Stomatal anatomy and kinetic responses to light transients were also measured. The highest An and gs and the lowest iWUE were observed at midday for most lines. Diurnally averaged iWUE correlated positively with morning and midday iWUE and negatively with the time taken for stomata to close after transition to low light intensity (kclose). There was significant variation among sorghum lines for kclose, and smaller kclose correlated with lower gs and higher stomatal density (SD) across the lines. In turn, gs was negatively correlated with SD and regulated by the operational stomatal aperture regardless of stomatal size. Altogether, our data suggest a common physiology to improve iWUE in sorghum related to the control of water loss without impacting photosynthesis relying on higher SD, lower stomatal aperture and faster stomatal closing in response to low light intensity. Significance Statement: Sorghum lines that maintain high diurnal water use efficiency had higher stomatal density and exhibit faster stomatal closure rather than faster stomatal opening, which aligns with a strategy favouring water conservation over maximising photosynthesis. [ABSTRACT FROM AUTHOR]