Investigating the cis-regulatory basis of C3 and C4 photosynthesis in grasses at single-cell resolution.

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.

Regulatory Network of the Late-Recruited Primary Decarboxylase C4NADP-ME in Sugarcane.

In agronomically important C4 grasses, efficient CO2 delivery to Rubisco is facilitated by NADP-malic enzyme (C4NADP-ME), which decarboxylates malate in bundle sheath cells. However, understanding the molecular regulation of the C4NADP-ME gene in sugarcane (Saccharum spp.) is hindered by its complex genetic background. Enzymatic activity assays demonstrated that decarboxylation in sugarcane Saccharum spontaneum predominantly relies on the NADP-ME pathway, similar to sorghum (Sorghum bicolor) and maize (Zea mays). Comparative genomics analysis revealed the recruitment of eight core C4 shuttle genes, including C4NADP-ME (SsC4NADP-ME2), in the C4 pathway of sugarcane. Contrasting to sorghum and maize, the expression of SsC4NADP-ME2 in sugarcane is regulated by different transcription factors (TFs). We propose a gene regulatory network for SsC4NADP-ME2, involving candidate TFs identified through gene co-expression analysis and yeast one-hybrid experiment. Among these, ABA INSENSITIVE5 (ABI5) was validated as the predominant regulator of SsC4NADP-ME2 expression, binding to a G-box within its promoter region. Interestingly, the core element ACGT within the regulatory G-box was conserved in sugarcane, sorghum, maize, and rice (Oryza sativa), suggesting an ancient regulatory code utilized in C4 photosynthesis. This study offers insights into SsC4NADP-ME2 regulation, crucial for optimizing sugarcane as a bioenergy crop.

Midday water use efficiency in sorghum is linked to faster stomatal closure rate, lower stomatal aperture and higher stomatal density.

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.

Transcriptome and small RNA analysis unveils novel insights into the C4 gene regulation in sugarcane.

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.

Identifying genomic regions associated with C4 photosynthetic activity and leaf anatomy in Alloteropsis semialata.

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.

Carbon isotope trends across a century of herbarium specimens suggest CO2 fertilization of C4 grasses.

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.

C4 photosynthesis provided an immediate demographic advantage to populations of the grass Alloteropsis semialata.

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.

The bHLH transcription factor OsPRI1 activates the Setaria viridis PEPC1 promoter in rice.

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.

Plasmodesmal connectivity in C4 Gynandropsis gynandra is induced by light and dependent on photosynthesis.

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 C4 G. 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 C4 G. gynandra.

Chloroplast NADH dehydrogenase-like complex-mediated cyclic electron flow is the main electron transport route in C4 bundle sheath cells.

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.

Contrasting leaf-scale photosynthetic low-light response and its temperature dependency are key to differences in crop-scale radiation use efficiency.

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.

Regulatory NADH dehydrogenase-like complex optimizes C4 photosynthetic carbon flow and cellular redox in maize.

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.

Stomatal dynamics in Alloteropsis semialata arise from the evolving interplay between photosynthetic physiology, stomatal size and biochemistry.

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.

Stomatal response to VPD in C4 plants with different biochemical sub-pathways.

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.

Reducing stomatal density by expression of a synthetic Epidermal Patterning Factor increases leaf intrinsic water use efficiency and reduces plant water use in a C4 crop.

Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience and expanding cultivation on marginal lands. Engineering lower stomatal density to reduce stomatal conductance (gs) has improved WUE in multiple C3 crop species. However, reducing gs in C3 species often reduces photosynthetic carbon gain. A different response is expected in C4 plants because they possess specialized anatomy and biochemistry which concentrates CO2 at the site of fixation. This modifies the photosynthesis (AN) relationship with intracellular CO2 concentration (ci) so that photosynthesis is CO2-saturated and reductions in gs are unlikely to limit AN. To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C4 crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum, led to reduced stomatal densities, reduced gs, reduced plant water use and avoidance of stress during a period of water deprivation. In addition, moderate reduction in stomatal density did not increase stomatal limitation to AN. However, these positive outcomes were associated with negative pleiotropic effects on reproductive development and photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene to specific tissues could provide a pathway to improved agronomic outcomes.

Differences in stomatal sensitivity to CO2 and light influence variation in water use efficiency and leaf carbon isotope composition in two genotypes of the C4 plant Zea mays.

The ratio of net CO2 uptake (Anet) and stomatal conductance (gs) is an intrinsic measurement of leaf water use efficiency (WUEi) however its measurement can be challenging for large phenotypic screens. Measurements of leaf carbon isotope composition (δ13Cleaf) may be a scalable tool to approximate WUEi for screening because it in part reflects the competing influences of Anet and gs on the CO2 partial pressure (pCO2) inside the leaf over time. However, in C4 photosynthesis the CO2 concentrating mechanism complicates the relationship between δ13Cleaf and WUEi. Despite this complicated relationship, several studies have shown genetic variation in δ13Cleaf across C4 plants. Yet there has not been a clear demonstration of whether Anet or gs are the causal mechanisms controlling WUEi and δ13Cleaf. Our approach was to characterize leaf photosynthetic traits of two Zea mays recombinant inbred lines (Z007E0067 and Z007E0150) which consistently differ for δ13Cleaf even though they have minimal confounding genetic differences. We demonstrate that these two genotypes contrasted in WUEi driven by differences in the speed of stomatal responses to changes in pCO2 and light that lead to unproductive leaf water loss. These findings provide support that differences in δ13Cleaf in closely related genotypes do reflect greater WUEi and further suggests that differences in stomatal kinetic response to changing environmental conditions is a key target to improve WUEi.

New insights into plasmodesmata: complex 'protoplasmic connecting threads'.

Intercellular communication in plants, as in other multicellular organisms, allows cells in tissues to coordinate their responses for development and in response to environmental stimuli. Much of this communication is facilitated by plasmodesmata (PD), consisting of membranes and cytoplasm, that connect adjacent cells to each other. PD have long been viewed as passive conduits for the movement of a variety of metabolites and molecular cargoes, but this perception has been changing over the last two decades or so. Research from the last few years has revealed the importance of PD as signaling hubs and as crucial players in hormone signaling. The adoption of advanced biochemical approaches, molecular tools, and high-resolution imaging modalities has led to several recent breakthroughs in our understanding of the roles of PD, revealing the structural and regulatory complexity of these 'protoplasmic connecting threads'. We highlight several of these findings that we think well illustrate the current understanding of PD as functioning at the nexus of plant physiology, development, and acclimation to the environment.

Sugar sensing in C4 source leaves: a gap that needs to be filled.

Plant growth depends on sugar production and export by photosynthesizing source leaves and sugar allocation and import by sink tissues (grains, roots, stems, and young leaves). Photosynthesis and sink demand are tightly coordinated through metabolic (substrate, allosteric) feedback and signalling (sugar, hormones) mechanisms. Sugar signalling integrates sugar production with plant development and environmental cues. In C3 plants (e.g. wheat and rice), it is well documented that sugar accumulation in source leaves, due to source-sink imbalance, negatively feeds back on photosynthesis and plant productivity. However, we have a limited understanding about the molecular mechanisms underlying those feedback regulations, especially in C4 plants (e.g. maize, sorghum, and sugarcane). Recent work with the C4 model plant Setaria viridis suggested that C4 leaves have different sugar sensing thresholds and behaviours relative to C3 counterparts. Addressing this research priority is critical because improving crop yield requires a better understanding of how plants coordinate source activity with sink demand. Here we review the literature, present a model of action for sugar sensing in C4 source leaves, and suggest ways forward.

Differential subgenome expression underlies biomass accumulation in allotetraploid Pennisetum giganteum.

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.

Multiple Roles of Glycerate Kinase-From Photorespiration to Gluconeogenesis, C4 Metabolism, and Plant Immunity.

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.