Developmental and Expression Evolution in C3 and C4 Atriplex and their Hybrids

Maintaining global food security amidst climate change requires improved photosynthetic efficiency in C3 crops. Above 30°C, photosynthesis and yield are reduced 30-50% by photorespiration. The C4 photosynthetic pathway incorporates biochemical and structural features to minimize photorespiration by concentrating CO2 around Rubisco. Engineering C4 into C3 crops requires an understanding of the molecular and genetic control of C4 photosynthesis. I combined transcriptomic and developmental analyses to investigate the molecular control of leaf development in C3 Atriplex prostrata compared to A. rosea, a C4 species with Kranz anatomy. Plants with Kranz-type C4 photosynthesis have two photosynthetic cell types, mesophyll and bundle sheath. Mesophyll cells contain phosphoenolpyruvate carboxylase, the primary carbon fixation enzyme, whereas bundle sheath contains Rubisco and houses photosynthetic carbon reduction. I used C3 x C4 Atriplex F1 hybrids and allele-specific expression analysis to investigate the role of cis- and trans-regulatory effects on gene expression divergence. The majority (~80%) of expression divergence between C3 and C4 species arose from cis-only regulatory divergence, suggesting that numerous independent expression changes across the genome drove C4 photosynthesis evolution. For genes in the C4 pathway, expression divergence arose from cis-acting changes to individual genes, with little contribution from trans-changes. Genes of interest including regulators of plasmodesmata and chloroplast function did not have distinct patterns of regulatory divergence. Inheritance pattern analysis showed that misexpression was common in F1 hybrids and was supported by phenotypic evidence. I next performed comparative studies of C3 and C4 leaf development. I detected significant species*time interaction effects for a large proportion of genes, implying that evolutionary shifts in expression timing were widespread between these species. Cis-regulation was also implicated in many of these evolutionary shifts in expression timing. C4 Atriplex showed accelerated vascular tissue initiation and formation, supported by earlier expression decline of vascular tissue development and core cell cycle genes. Plasmodesmata-associated genes were differentially expressed between during secondary plasmodesmata formation. Mature leaves of F2 hybrids exhibited variation in biochemistry, leaf tissue patterning, and bundle sheath ultrastructure and would be suitable for future large-scale transcriptomic studies. Combined, these studies facilitated identification of the underlying genetics of C4 evolution and development.Ph.D

Reporter-based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana

The evolution of C(4) photosynthesis proceeded stepwise with each small step increasing the fitness of the plant. An important pre-condition for the introduction of a functional C(4) cycle is the photosynthetic activation of the C(3) bundle sheath by increasing its volume and organelle number. Therefore, to engineer C(4) photosynthesis into existing C(3) crops, information about genes that control the bundle sheath cell size and organelle content is needed. However, very little information is known about the genes that could be manipulated to create a more C(4) -like bundle sheath. To this end, an ethylmethanesulfonate (EMS)-based forward genetic screen was established in the Brassicaceae C(3 ) species Arabidopsis thaliana. To ensure a high-throughput primary screen, the bundle sheath cells of A. thaliana were labeled using a luciferase (LUC68) or by a chloroplast-targeted green fluorescent protein (sGFP) reporter using a bundle sheath specific promoter. The signal strengths of the reporter genes were used as a proxy to search for mutants with altered bundle sheath anatomy. Here, we show that our genetic screen predominantly identified mutants that were primarily affected in the architecture of the vascular bundle, and led to an increase in bundle sheath volume. By using a mapping-by-sequencing approach the genomic segments that contained mutated candidate genes were identified

Mesophyll cells of C4plants have fewer chloroplasts than those of closely related C3plants:C3versus C4mesophyll chloroplasts

The evolution of C4 photosynthesis from C3 ancestors eliminates ribulose bisphosphate carboxylation in the mesophyll (M) cell chloroplast while activating phosphoenolpyruvate (PEP) carboxylation in the cytosol. These changes may lead to fewer chloroplasts and different chloroplast positioning within M cells. To evaluate these possibilities, we compared chloroplast number, size and position in M cells of closely related C3, C3–C4 intermediate and C4 species from 12 lineages of C4 evolution. All C3 species had more chloroplasts per M cell area than their C4 relatives in high-light growth conditions. C3 species also had higher chloroplast coverage of the M cell periphery than C4 species, particularly opposite intercellular air spaces. In M cells from 10 of the 12 C4 lineages, a greater fraction of the chloroplast envelope was pulled away from the plasmalemma in the C4 species than their C3 relatives. C3–C4 intermediate species generally exhibited similar patterns as their C3 relatives. We interpret these results to reflect adaptive shifts that facilitate efficient C4 function by enhancing diffusive access to the site of primary carbon fixation in the cytosol. Fewer chloroplasts in C4 M cells would also reduce shading of the bundle sheath chloroplasts, which also generate energy required by C4 photosynthesis

Initial events during the evolution of C 4 photosynthesis in C 3 species of Flaveria

The evolution of C4 photosynthesis in many taxa involves the establishment of a two-celled photorespiratory CO2 pump, termed C2 photosynthesis. How C3 species evolved C2 metabolism is critical to understanding the initial phases of C4 plant evolution. T

Initial Events during the Evolution of C 4

The evolution of C(4) photosynthesis in many taxa involves the establishment of a two-celled photorespiratory CO(2) pump, termed C(2) photosynthesis. How C(3) species evolved C(2) metabolism is critical to understanding the initial phases of C(4) plant evolution. To evaluate early events in C(4) evolution, we compared leaf anatomy, ultrastructure, and gas-exchange responses of closely related C(3) and C(2) species of Flaveria, a model genus for C(4) evolution. We hypothesized that Flaveria pringlei and Flaveria robusta, two C(3) species that are most closely related to the C(2) Flaveria species, would show rudimentary characteristics of C(2) physiology. Compared with less-related C(3) species, bundle sheath (BS) cells of F. pringlei and F. robusta had more mitochondria and chloroplasts, larger mitochondria, and proportionally more of these organelles located along the inner cell periphery. These patterns were similar, although generally less in magnitude, than those observed in the C(2) species Flaveria angustifolia and Flaveria sonorensis. In F. pringlei and F. robusta, the CO(2) compensation point of photosynthesis was slightly lower than in the less-related C(3) species, indicating an increase in photosynthetic efficiency. This could occur because of enhanced refixation of photorespired CO(2) by the centripetally positioned organelles in the BS cells. If the phylogenetic positions of F. pringlei and F. robusta reflect ancestral states, these results support a hypothesis that increased numbers of centripetally located organelles initiated a metabolic scavenging of photorespired CO(2) within the BS. This could have facilitated the formation of a glycine shuttle between mesophyll and BS cells that characterizes C(2) photosynthesis

Initial Events during the Evolution of C₄ Photosynthesis in C₃ Species of <i>Flaveria</i>

The evolution of C(4) photosynthesis in many taxa involves the establishment of a two-celled photorespiratory CO(2) pump, termed C(2) photosynthesis. How C(3) species evolved C(2) metabolism is critical to understanding the initial phases of C(4) plant evolution. To evaluate early events in C(4) evolution, we compared leaf anatomy, ultrastructure, and gas-exchange responses of closely related C(3) and C(2) species of Flaveria, a model genus for C(4) evolution. We hypothesized that Flaveria pringlei and Flaveria robusta, two C(3) species that are most closely related to the C(2) Flaveria species, would show rudimentary characteristics of C(2) physiology. Compared with less-related C(3) species, bundle sheath (BS) cells of F. pringlei and F. robusta had more mitochondria and chloroplasts, larger mitochondria, and proportionally more of these organelles located along the inner cell periphery. These patterns were similar, although generally less in magnitude, than those observed in the C(2) species Flaveria angustifolia and Flaveria sonorensis. In F. pringlei and F. robusta, the CO(2) compensation point of photosynthesis was slightly lower than in the less-related C(3) species, indicating an increase in photosynthetic efficiency. This could occur because of enhanced refixation of photorespired CO(2) by the centripetally positioned organelles in the BS cells. If the phylogenetic positions of F. pringlei and F. robusta reflect ancestral states, these results support a hypothesis that increased numbers of centripetally located organelles initiated a metabolic scavenging of photorespired CO(2) within the BS. This could have facilitated the formation of a glycine shuttle between mesophyll and BS cells that characterizes C(2) photosynthesis

Data from: C4 anatomy can evolve via a single developmental change

C4 photosynthesis boosts productivity in warm environments. Paradoxically, this complex physiological process evolved independently in numerous plant lineages, despite requiring specialized leaf anatomy. The anatomical modifications underlying C4 evolution have previously been evaluated through interspecific comparisons, which capture numerous changes besides those needed for C4 functionality. Here, we quantify the anatomical changes accompanying the transition between non-C4 and C4 phenotypes by sampling widely across the continuum of leaf anatomical traits in the grass Alloteropsis semialata. Within this species, the only trait that is shared and specific to C4 individuals is an increase in vein density, driven specifically by minor vein development. The minor veins are genetically determined, and their multiple effects facilitate C4 function. For species with the necessary anatomical preconditions, developmental proliferation of veins can therefore be sufficient to produce a functional C4 leaf anatomy, creating an evolutionary entry point to complex C4 syndromes that can become more specialized

Facilitating the adoption of high‐throughput sequencing technologies as a plant pest diagnostic test in laboratories: A step‐by‐step description

International audienceHigh-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of preparing a laboratory for HTS tests within existing quality assurance systems. From nucleic acid extractions to data analysis and interpretation, all of the steps are covered to ensure reliable and reproducible results. These guidelines are relevant for the detection and identification of any plant pest (e.g. arthropods, bacteria, fungi, nematodes, invasive plants or weeds, protozoa, viroids, viruses), and from any type of matrix (e.g. pure microbial culture, plant tissue, soil, water), regardless of the HTS technology (e.g. amplicon sequencing, shotgun sequencing) and of the application (e.g. surveillance programme, phytosanitary certification, quarantine, import control). These guidelines are written in general terms to facilitate the adoption of HTS technologies in plant pest routine diagnostics and enable broader application in all plant health fields, including research. A glossary of relevant terms is provided among the Supplementary Material