C4 Species Research Articles

Metabolic engineering of the serine/glycine network as a means to improve the nitrogen content of crops.

In plants, L-serine (Ser) biosynthesis occurs through various pathways and is highly dependent on the atmospheric CO2 concentration, especially in C3 species, due to the association of the Glycolate Pathway of Ser Biosynthesis (GPSB) with photorespiration. Characterization of a second plant Ser pathway, the Phosphorylated Pathway of Ser Biosynthesis (PPSB), revealed that it is at the crossroads of carbon, nitrogen, and sulphur metabolism. The PPSB comprises three sequential reactions catalysed by 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoSer aminotransferase (PSAT) and 3-phosphoSer phosphatase (PSP). PPSB was overexpressed in plants exhibiting two different modes of photosynthesis: Arabidopsis (C3 metabolism), and maize (C4 metabolism), under ambient (aCO2) and elevated (eCO2) CO2 growth conditions. Overexpression in Arabidopsis of the PGDH1 gene alone or PGDH1, PSAT1 and PSP1 in combination increased the Ser levels but also the essential amino acids threonine (aCO2), isoleucine, leucine, lysine, phenylalanine, threonine and methionine (eCO2) compared to the wild-type. These increases translated into higher protein levels. Likewise, starch levels were also increased in the PPSB-overexpressing lines. In maize, PPSB-deficient lines were obtained by targeting PSP1 using Cas9 endonuclease. We concluded that the expression of PPSB in maize male gametophyte is required for viable pollen development. Maize lines overexpressing the AtPGDH1 gene only displayed higher protein levels but not starch at both aCO2 and eCO2 conditions, this translated into a significant rise in the nitrogen/carbon ratio. These results suggest that metabolic engineering of PPSB in crops could enhance nitrogen content, particularly under upcoming eCO2 conditions where the activity of GPSB is limited.

Expanding the Triangle of U: Comparative analysis of the Hirschfeldia incana genome provides insights into chromosomal evolution, phylogenomics and high photosynthesis-related traits.

The Brassiceae tribe encompasses many economically important crops and exhibits high intraspecific and interspecific phenotypic variation. After a shared whole-genome triplication (WGT) event (Br-α, ~15.9 million years ago), differential lineage diversification and genomic changes contributed to an array of divergence in morphology, biochemistry, and physiology underlying photosynthesis-related traits. Here, the C3 species Hirschfeldia incana is studied as it displays high photosynthetic rates under high-light conditions. Our aim was to elucidate the evolution that gave rise to the genome of H. incana and its high-photosynthesis traits. We reconstructed a chromosome-level genome assembly for H. incana (Nijmegen, v2.0) using nanopore and chromosome conformation capture (Hi-C) technologies, with 409Mb in size and an N50 of 52Mb (a 10× improvement over the previously published scaffold-level v1.0 assembly). The updated assembly and annotation was subsequently employed to investigate the WGT history of H. incana in a comparative phylogenomic framework from the Brassiceae ancestral genomic blocks and related diploidized crops. Hirschfeldia incana (x=7) shares extensive genome collinearity with Raphanus sativus (x=9). These two species share some commonalities with Brassica rapa and B. oleracea (A genome, x=10 and C genome, x=9, respectively) and other similarities with B. nigra (B genome, x=8). Phylogenetic analysis revealed that H. incana and R. sativus form a monophyletic clade in between the Brassica A/C and B genomes. We postulate that H. incana and R. sativus genomes are results of hybridization or introgression of the Brassica A/C and B genome types. Our results might explain the discrepancy observed in published studies regarding phylogenetic placement of H. incana and R. sativus in relation to the "Triangle of U" species. Expression analysis of WGT retained gene copies revealed sub-genome expression divergence, likely due to neo- or sub-functionalization. Finally, we highlighted genes associated with physio-biochemical-anatomical adaptive changes observed in H. incana which likely facilitate its high-photosynthesis traits under high light. The improved H. incana genome assembly, annotation and results presented in this work will be a valuable resource for future research to unravel the genetic basis of its ability to maintain a high photosynthetic efficiency in high-light conditions and thereby improve photosynthesis for enhanced agricultural production.

C4 grasses employ distinct strategies to acclimate rubisco activase to heat stress.

Rising temperatures due to the current climate crisis will soon have devastating impacts on crop performance and resilience. In particular, CO2 assimilation is dramatically limited at high temperatures. CO2 assimilation is accomplished by rubisco, which is inhibited by the binding of inhibitory sugar phosphates to its active site. Plants therefore utilize the essential chaperone rubisco activase (RCA) to remove these inhibitors and enable continued CO2 fixation. However, RCA does not function at moderately high temperatures (42°C), resulting in impaired rubisco activity and reduced CO2 assimilation. We set out to understand temperature-dependent RCA regulation in four different C4 plants, with a focus on the crop plants maize (two cultivars) and sorghum, as well as the model grass Setaria viridis (setaria) using gas exchange measurements, which confirm that CO2 assimilation is limited by carboxylation in these organisms at high temperatures (42°C). All three species express distinct complements of RCA isoforms and each species alters the isoform and proteoform abundances in response to heat; however, the changes are species-specific. We also examine whether the heat-mediated inactivation of RCA is due to biochemical regulation rather than simple thermal denaturation. We reveal that biochemical regulation affects RCA function differently in different C4 species, and differences are apparent even between different cultivars of the same species. Our results suggest that each grass evolved different strategies to maintain RCA function during stress and we conclude that a successful engineering approach aimed at improving carbon capture in C4 grasses will need to accommodate these individual regulatory mechanisms.

Climate and management changes over 40 years drove more stress‐tolerant and less ruderal weed communities in vineyards

AbstractSpontaneous plant communities have undergone considerable constraints due to human‐mediated changes. Understanding how plant communities are shifting in response to land management and climate changes is necessary to predict future ecosystem functioning and improve the resilience of managed ecosystems, such as agroecosystems. Using Mediterranean weed communities as models of managed plant communities in a climate change hotspot, we quantified the extent to which they have shifted from the 1980s to the 2020s in response to climate and management changes in vineyards. The weed communities of the same 40 vineyards in the Montpellier region were surveyed using the same protocol in spring, summer, and autumn, for two years, with a 40‐year interval (1978–1979 vs. 2020–2021). In four decades, the annual range of temperatures (i.e., the difference between the warmest month's and the coldest month's mean temperatures) increased by 1.2°C and the summer temperatures by 2°C. Weed management diversified over time with the adoption of mowing that replaced the chemical weeding of interrows. Chemical weeding is now mostly limited to the area under the row. Current weed communities were 41% more abundant, 24% more diverse, and with a less even distribution of abundance across species than the 1980s communities at the vineyard level. Modern communities were composed of more annual species (57% of annual species in the 1980s vs. 80% in the 2020s) with lower community‐weighted seed mass and were composed of fewer C4 species. They had higher community‐weighted specific leaf area, higher leaf dry matter content, and lower leaf area than the 1980s weed communities. At the community level, the onset of flowering was earlier and the duration of flowering was longer in the 2020s. Climate change induced more stress‐tolerant communities in the 2020s while the diversification of weed management practices favored less ruderal communities. This study shows that plant communities are shifting in response to climate change and that land management is a strong lever for action to model more diverse and eventually more desirable weed communities in the future.

Transcriptome dynamics in developing leaves from C3 and C4 Flaveria species.

C4 species have evolved more than 60 times independently from C3 ancestors. This multiple and parallel evolution of the complex C4 trait suggests common underlying evolutionary mechanisms, which could be identified by comparative analysis of closely related C3 and C4 species. Efficient C4 function depends on a distinctive leaf anatomy that is characterised by enlarged, chloroplast-rich bundle sheath cells and narrow vein spacing. To elucidate the molecular mechanisms that generate the Kranz anatomy, we analysed a developmental series of leaves from the C4 plant Flaveria bidentis and the closely related C3 species Flaveria robusta by comparing anatomies and transcriptomes. Vascular density measurements of all nine leaf developmental stages identified three leaf anatomical zones whose proportions vary with respect to the developmental stage. We then deconvoluted the transcriptome datasets using non-negative matrix factorisation, which identified four distinct transcriptome patterns in the growing leaves of both species. By integrating the leaf anatomy and transcriptome data, we were able to correlate the different transcriptional profiles with different developmental zones in the leaves. These comparisons revealed an important role for auxin metabolism, in particular auxin homeostasis (conjugation and deconjugation), in establishing the high vein density typical of C4 species.

Metabolomics of related C3 and C4 Flaveria species indicate differences in the operation of photorespiration under fluctuating light.

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.

Bigger genomes provide environment-dependent growth benefits in grasses.

Increasing genome size (GS) has been associated with slower rates of DNA replication and greater cellular nitrogen (N) and phosphorus demands. Despite most plant species having small genomes, the existence of larger GS species suggests that such costs may be negligible or represent benefits under certain conditions. Focussing on the widespread and diverse grass family (Poaceae), we used data on species' climatic niches and growth rates under different environmental conditions to test for growth costs or benefits associated with GS. The influence of photosynthetic pathway, life history and evolutionary history on grass GS was also explored. We found that evolutionary history, photosynthetic pathway and life history all influence the distribution of grass species' GS. Genomes were smaller in annual and C4 species, the latter allowing for small cells necessary for C4 leaf anatomy. We found larger GS were associated with high N availability and, for perennial species, low growth-season temperature. Our findings reveal that GS is a globally important predictor of grass performance dependent on environmental conditions. The benefits for species with larger GS are likely due to associated larger cell sizes, allowing rapid biomass production where soil fertility meets N demands and/or when growth occurs via temperature-independent cell expansion.

Grass leaf structural and stomatal trait responses to climate gradients assessed over the 20th century and across the Great Plains, USA.

Abstract. Using herbarium specimens spanning 133 years and field-collected measurements, we assessed intraspecific trait (leaf structural and stomatal) variability from grass species in the Great Plains of North America. We focused on two widespread, closely related grasses from the tribe Paniceae: Dichanthelium oligosanthes subsp. scribnerianum (C3) and Panicum virgatum (C4). Thirty-one specimens per taxon were sampled from local herbaria from the years 1887 to 2013 to assess trait responses across time to changes in atmospheric [CO2] and growing season precipitation and temperature. In 2021 and 2022, the species were measured from eight grasslands sites to explore how traits vary spatially across natural continental precipitation and temperature gradients. Δ13C increased with atmospheric [CO2] for D. oligosanthes but decreased for P. virgatum, likely linked to increases in precipitation in the study region over the past century. Notably, this is the first record of decreasing Δ13C over time for a C4 species illustrating 13C linkages to climate. As atmospheric [CO2] increased, C:N increased and δ15N decreased for both species and %N decreased for D. oligosanthes. Across a large precipitation gradient, D. oligosanthes leaf traits were more responsive to changes in precipitation than those of P. virgatum. In contrast, only two traits of P. virgatum responded to increases in temperature across a gradient: specific leaf area (increase) and leaf dry matter content (decrease). The only shared significant trend between species was increased C:N with precipitation. Our work demonstrates that these closely related grass species with different photosynthetic pathways exhibited various trait responses across temporal and spatial scales, illustrating the key role of scale of inquiry for forecasting leaf trait responses to future environmental change.

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.

Mesophyll airspace unsaturation drives C4 plant success under vapor pressure deficit stress

A fundamental assumption in plant science posits that leaf air spaces remain vapor saturated, leading to the predominant view that stomata alone control leaf water loss. This concept has been pivotal in photosynthesis and water-use efficiency research. However, recent evidence has refuted this longstanding assumption by providing evidence of unsaturation in the leaf air space of C3 plants under relatively mild vapor pressure deficit (VPD) stress. This phenomenon represents a nonstomatal mechanism restricting water loss from the mesophyll. The potential ubiquity and physiological implications of this phenomenon, its driving mechanisms in different plant species and habitats, and its interaction with other ecological adaptations have. In this context, C4 plants spark particular interest for their importance as crops, bundle sheath cells' unique anatomical characteristics and specialized functions, and notably higher water-use efficiency relative to C3 plants. Here, we confirm reduced relative humidities in the substomatal cavity of the C4 plants maize, sorghum, and proso millet down to 80% under mild VPD stress. We demonstrate the critical role of nonstomatal control in these plants, indicating that the role of the CO2 concentration mechanism in CO2 management at a high VPD may have been overestimated. Our findings offer a mechanistic reconciliation between discrepancies in CO2 and VPD responses reported in C4 species. They also reveal that nonstomatal control is integral to maintaining an advantageous microclimate of relatively higher CO2 concentrations in the mesophyll air space of C4 plants for carbon fixation, proving vital when these plants face VPD stress.

Does the response of Rubisco and photosynthesis to elevated [CO2] change with unfavourable environmental conditions?

Climate change due to anthropogenic CO2 emissions affects plant performance globally. To improve crop resilience, we need to understand the effects of elevated CO2 concentration (e[CO2]) on CO2 assimilation and Rubisco biochemistry. However, the interactive effects of e[CO2] and abiotic stress are especially unclear. This study analyses the CO2 effect on photosynthetic capacity under different water availability and temperature conditions in 42 different crop species, varying in functional group, photosynthetic pathway and phenological stage. We analysed close to 3000 data points extracted from 120 published manuscripts. For C3 species, e[CO2] increases net photosynthesis and intercellular [CO2], while reducing stomatal conductance and transpiration. Vmaxc, Rubisco in vitro extractable maximal activity and content also decrease with e[CO2] in C3 species, while C4 crops are less responsive to e[CO2]. The interaction with drought and/or heat stress does not significantly alter these photosynthetic responses, indicating that the photosynthetic capacity of stressed plants responds to e[CO2]. Moreover, e[CO2] has strong effect on the photosynthetic capacity of grasses mainly in the final stages of development. This study provides insight into the intricate interactions within the plant photosynthetic apparatus under the influence of climate change, enhancing the understanding of mechanisms governing plant responses to environmental parameters.

Native arbuscular mycorrhizal fungi drive ecophysiology through phenotypic integration and functional plasticity under the Sonoran desert conditions.

Knowledge is scarce to what extent environmental drivers and native symbiotic fungi in soil induce abrupt (short-term), systemic (multiple traits), or specific (a subset of traits) shifts in C3 plants' ecophysiological/mycorrhizal responses. We cultivated an emblematic native C3 species (Capsicum annuum var. glabriusculum, "Chiltepín") to look at how the extreme heat of the Sonoran desert, sunlight regimes (low = 2, intermediate = 15, high = 46 mol m2 d-1) and density of native arbuscular mycorrhizal fungi in soil (low AMF = 1% v/v, high AMF = 100% v/v), drive shifts on mycorrhizal responses through multiple functional traits (106 traits). The warming thresholds were relentlessly harsh even under intensive shade (e.g. superheat maximum thresholds reached ranged between 47-63°C), and several pivotal traits were synergistically driven by AMF (e.g. photosynthetic capacity, biomass gain/allometry, and mycorrhizal colonization traits); whereas concurrently, sunlight regimes promoted most (76%) alterations in functional acclimation traits in the short-term and opposite directions (e.g. survival, phenology, photosynthetic, carbon/nitrogen economy). Multidimensional reduction analysis suggests that the AMF promotes a synergistic impact on plants' phenotypic integration and functional plasticity in response to sunlight regimes; however, complex relationships among traits suggest that phenotypic variation determines the robustness degree of ecophysiological/mycorrhizal phenotypes between/within environments. Photosynthetic canopy surface expansion, Rubisco activity, photosynthetic nitrogen allocation, carbon gain, and differential colonization traits could be central to plants' overall ecophysiological/mycorrhizal fitness strengthening. In conclusion, we found evidence that a strong combined effect among environmental factors in which AMF are key effectors could drive important trade-offs on plants' ecophysiological/mycorrhizal fitness in the short term.

Leaf transcriptomes from C3, C3-C4 intermediate, and C4 Neurachne species give insights into C4 photosynthesis evolution.

The C4 photosynthetic pathway is hypothesized to have evolved from the ancestral C3 pathway through progressive changes in leaf anatomy and biochemistry with extant C3-C4 photosynthetic intermediate species representing phenotypes between species demonstrating full C3 and full C4 states. The Australian endemic genus Neurachne is the only known grass group that contains distinct, closely related species that carry out C3, C3-C4 intermediate, or C4 photosynthesis. To explore and understand the molecular mechanisms underlying C4 photosynthesis evolution in this genus, leaf transcriptomes were generated from two C3, three photosynthetic intermediate (proto-Kranz, C2-like, and C2), and two C4 Neurachne species. The data were used to reconstruct phylogenetic relationships in Neurachne, which confirmed two independent C4 origins in the genus. Relative transcript abundances substantiated the photosynthetic phenotypes of individual species and highlighted transcriptional investment differences between species, including between the two C4 species. The data also revealed proteins potentially involved in C4 cycle intermediate transport and identified molecular mechanisms responsible for the evolution of C4-associated proteins in the genus.

The applicability of a SIF-based mechanistic model for estimating GPP at the canopy scale

Mechanistically linking gross primary productivity (GPP) and sun-induced chlorophyll fluorescence (SIF) is an essential step to unleash the full potential of SIF for remote sensing-based predictions of GPP across biomes, climates, and spatiotemporal scales. The latest SIF-based mechanistic light response model that includes the fraction of open photosystem II reaction centers as key parameter (qMLR-SIF model), can accurately reproduce leaf-scale photosynthesis under various conditions. However, it remains unclear to what extent the qMLR-SIF model is suitable for estimating GPP at larger scales such as the canopy scale. Therefore, canopy-scale data of tower-based far-red SIF, GPP and key environmental variables from 10 study sites were collected to analyze the SIF-GPP relationship and to compare the qMLR-SIF model with the widely used Farquhar, von Caemmerer, Berry (FvCB) model and with a light use efficiency (LUE) model for different plant functional types (PFTs), photosynthetic pathways (C3 and C4), and temporal scales (hourly, daily and 4-day). Results showed that the nonlinear SIF-GPP relationship existed in all PFTs and the degree of linearity increased at larger temporal scales. The qMLR-SIF model exhibited wide applicability to quantify canopy GPP for different PFTs (R2 = 0.55–0.80, RMSE = 2.72–11.03 μmol CO2 m-2 s-1), photosynthetic pathways (R2 = 0.70–0.78, RMSE = 5.29–9.05 μmol CO2 m-2 s-1) and temporal scales (R2 = 0.82–0.97, RMSE = 3.42–8.32 μmol CO2 m-2s-1). Compared with the two other models, the qMLR-SIF model performed best overall, which is mainly due to its simpler model structure and the mechanistic link between SIF and photosynthesis. Particularly, the qMLR-SIF model could more accurately estimate GPP in C4 species, with higher R2 (0.78) and lower RMSE (8.46 μmol CO2 m-2s-1). These findings highlight the advantages of the qMLR-SIF model in GPP estimation at the canopy scale, showing its potential in applications at regional and global scales.

Tribulus (Zygophyllaceae) as a case study for the evolution of C2 and C4 photosynthesis.

C2 photosynthesis is a photosynthetic pathway in which photorespiratory CO2 release and refixation are enhanced in leaf bundle sheath (BS) tissues. The evolution of C2 photosynthesis has been hypothesized to be a major step in the origin of C4 photosynthesis, highlighting the importance of studying C2 evolution. In this study, physiological, anatomical, ultrastructural, and immunohistochemical properties of leaf photosynthetic tissues were investigated in six non-C4 Tribulus species and four C4 Tribulus species. At 42°C, T. cristatus exhibited a photosynthetic CO2 compensation point in the absence of respiration (C*) of 21 µmol mol-1, below the C3 mean C* of 73 µmol mol-1. Tribulus astrocarpus had a C* value at 42°C of 55 µmol mol-1, intermediate between the C3 species and the C2 T. cristatus. Glycine decarboxylase (GDC) allocation to BS tissues was associated with lower C*. Tribulus cristatus and T. astrocarpus allocated 86% and 30% of their GDC to the BS tissues, respectively, well above the C3 mean of 11%. Tribulus astrocarpus thus exhibits a weaker C2 (termed sub-C2) phenotype. Increased allocation of mitochondria to the BS and decreased length-to-width ratios of BS cells, were present in non-C4 species, indicating a potential role in C2 and C4 evolution.

Soil basal respiration and nitrogen mineralization from C3 and C4 grass dominated plant communities respond differently to temperature and soil water variation

Key environmental influences on soil basal respiration (Rs) and nitrogen mineralization (ΔIN) are temperature and soil water content (SWC) and both are being altered by climate change. Yet we cannot expect that variation in temperature and SWC will equally affect all ecosystems. We examine the influences of temperature and SWC on Rs and ΔIN in two grassland plant communities dominated by C3 or C4 species. We collected soil samples from these communities and incubated them at temperatures (5 °C, 10 °C, 15 °C, and 25 °C) and four SWC (10%, 20%, 30% and 40% by weight). After four one-month incubation experiments, we found that (1) plant communities, temperature, and SWC significantly influenced Rs and ΔIN; (2) the highest Rs and ΔIN occurred at 25 °C and 30% SWC in the C4 plant community; (3) in the driest soils, N was immobilized in both communities regardless of temperature. We suggest that there is a greater limitation to C and N mineralization in the C3 plant community than in the C4 plant community making the C3 community less sensitive to variation in temperature and SWC.

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.

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.

Botanical composition gradients in silvopastoral systems on temperate native grasslands of Uruguay

AbstractSilvopastoral systems may provide important production and environmental benefits. The loss of cool-season (C3) grasses from temperate grazed native grasslands is associated with selective grazing and excessive solar radiation that limit their survival. Silvopastoral systems integrate trees with grasslands that provide shade to both cattle and herbaceous plants, potentially favoring C3 species. There is limited information about the effect of trees on the species and functional composition of native grasslands in the Campos biome in South America. The objective of this study was to detect gradients in the botanical composition of grasslands as affected by changes in the shade associated with distance to the trees and cardinal orientation in three situations defined by the combination of soil and tree species (Prosopis on Solonetz, Acacia on Brunisols, and Eucalyptus on Brunisols). Soil cover of the herbaceous species under trees was recorded in double transects located in the four cardinal directions. In all situations there were changes in pasture composition in the different shaded regions (total shade, partial shade, or full sun). Under the canopy, there was an increase of cool-season grasses such as Bromus catharticus Vahl, Lolium multiflorum Lam., Stipa hyalina (Nees) Barkworth, and S. setigera J.Presl. At greater distances from trees, cover of warm-season grasses, such as Axonopus affinis Chase and Paspalum notatum Flueggé increased. These gradients suggest that trees in silvopastoral systems can increase the abundance of cool-season species and potentially improve the forage nutritive value of the native pasture.

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.