Ivan Reyna-Llorens

A Specific Transcriptome Signature for Guard Cells from the C4 Plant Gynandropsis gynandra

Transcript profiling from closely related C3 and C4 species identifies a core guard cell signature as well as two patterns of gene expression associated with C4 photosynthesis across the C4 leaf. C4 photosynthesis represents an excellent example of convergent evolution that results in the optimization of both carbon and water usage by plants. In C4 plants, a carbon-concentrating mechanism divided between bundle sheath and mesophyll cells increases photosynthetic efficiency. Compared with C3 leaves, the carbon-concentrating mechanism of C4 plants allows photosynthetic operation at lower stomatal conductance, and as a consequence, transpiration is reduced. Here, we characterize transcriptomes from guard cells in C3 Tareneya hassleriana and C4 Gynandropsis gynandra belonging to the Cleomaceae. While approximately 60% of Gene Ontology terms previously associated with guard cells from the C3 model Arabidopsis (Arabidopsis thaliana) are conserved, there is much less overlap between patterns of individual gene expression. Most ion and CO2 signaling modules appear unchanged at the transcript level in guard cells from C3 and C4 species, but major variations in transcripts associated with carbon-related pathways known to influence stomatal behavior were detected. Genes associated with C4 photosynthesis were more highly expressed in guard cells of C4 compared with C3 leaves. Furthermore, we detected two major patterns of cell-specific C4 gene expression within the C4 leaf. In the first, genes previously associated with preferential expression in the bundle sheath showed continually decreasing expression from bundle sheath to mesophyll to guard cells. In the second, expression was maximal in the mesophyll compared with both guard cells and bundle sheath. These data imply that at least two gene regulatory networks act to coordinate gene expression across the bundle sheath, mesophyll, and guard cells in the C4 leaf.

Shared characteristics underpinning C4 leaf maturation derived from analysis of multiple C3 and C4 species of Flaveria

Abstract Most terrestrial plants use C3 photosynthesis to fix carbon. In multiple plant lineages a modified system known as C4 photosynthesis has evolved. To better understand the molecular patterns associated with induction of C4 photosynthesis, the genus Flaveria that contains C3 and C4 species was used. A base to tip maturation gradient of leaf anatomy was defined, and RNA sequencing was undertaken along this gradient for two C3 and two C4Flaveria species. Key C4 traits including vein density, mesophyll and bundle sheath cross‐sectional area, chloroplast ultrastructure, and abundance of transcripts encoding proteins of C4 photosynthesis were quantified. Candidate genes underlying each of these C4 characteristics were identified. Principal components analysis indicated that leaf maturation and the photosynthetic pathway were responsible for the greatest amount of variation in transcript abundance. Photosynthesis genes were over‐represented for a prolonged period in the C4 species. Through comparison with publicly available data sets, we identify a small number of transcriptional regulators that have been up‐regulated in diverse C4 species. The analysis identifies similar patterns of expression in independent C4 lineages and so indicates that the complex C4 pathway is associated with parallel as well as convergent evolution.

Recruitment of pre-existing networks during the evolution of C4 photosynthesis

During C4 photosynthesis, CO2 is concentrated around the enzyme RuBisCO. The net effect is to reduce photorespiration while increasing water and nitrogen use efficiencies. Species that use C4 photosynthesis have evolved independently from their C3 ancestors on more than 60 occasions. Along with mimicry and the camera-like eye, the C4 pathway therefore represents a remarkable example of the repeated evolution of a highly complex trait. In this review, we provide evidence that the polyphyletic evolution of C4 photosynthesis is built upon pre-existing metabolic and genetic networks. For example, cells around veins of C3 species show similarities to those of the C4 bundle sheath in terms of C4 acid decarboxylase activity and also the photosynthetic electron transport chain. Enzymes of C4 photosynthesis function together in gluconeogenesis during early seedling growth of C3 Arabidopsis thaliana. Furthermore, multiple C4 genes appear to be under control of both light and chloroplast signals in the ancestral C3 state. We, therefore, hypothesize that relatively minor rewiring of pre-existing genetic and metabolic networks has facilitated the recurrent evolution of this trait. Understanding how these changes are likely to have occurred could inform attempts to install C4 traits into C3 crops. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’.

Ancient coding sequences underpin the spatial patterning of gene expression in C4 leaves

Photosynthesis is compromised in most plants because an enzymatic side-reaction fixes O2 instead of CO2. The energetic cost of oxygenation led to the evolution of C4 photosynthesis. In almost all C4 leaves compartmentation of photosynthesis between cells reduces oxygenation and so increases photosynthetic efficiency. Here we report that spatial expression of most C4 genes is controlled by intragenic cis-elements rather than promoter sequence. Two DNA motifs that co-operatively specify the patterning of genes required for C4 photosynthesis are identified. They are conserved in plants and algae that use the ancestral C3 pathway. As these motifs are located in exons they represent duons determining both gene expression and amino acid sequence. Our findings provide functional evidence for the importance of transcription factors recognising coding sequence as previously defined by genome-wide binding studies. Furthermore, they indicate that C4 evolution is based on ancient DNA motifs found in exonic sequence.

Droplet-based microfluidic analysis and screening of single plant cells

Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.

A transcription factor binding atlas for photosynthesis in cereals identifies a key role for coding sequence in the regulation of gene expression

The gene regulatory architecture associated with photosynthesis is poorly understood. Most plants use the ancestral C3 pathway, but our most productive cereal crops use C4 photosynthesis. In these C4 cereals, large-scale alterations to gene expression allow photosynthesis to be partitioned between cell types of the leaf. Here we provide a genome-wide transcription factor binding atlas for grasses that operate either C3 or C4 photosynthesis. Most of the >950,000 sites bound by transcription factors are preferentially located in genic sequence rather than promoter regions, and specific families of transcription factors preferentially bind coding sequence. Cell specific patterning of gene expression in C4 leaves is associated with combinatorial modifications to transcription factor binding despite broadly similar patterns of DNA accessibility between cell types. A small number of DNA motifs bound by transcription factors are conserved across 60 million years of grass evolution, and C4 evolution has repeatedly co-opted at least one of these hyper-conserved cis-elements. The grass cistrome is highly divergent from that of the model plant Arabidopsis thaliana.Abstract The majority of plants use C3 photosynthesis, but over sixty independent lineages of angiosperms have evolved the C4 pathway. In most C4 species, photosynthesis gene expression is compartmented between mesophyll and bundle sheath cells. We performed DNaseI-SEQ to identify genome-wide profiles of transcription factor binding in leaves of the C4 grasses Zea mays, Sorghum bicolor and Setaria italica as well as C3Brachypodium distachyon. In C4 species, while bundle sheath strands and whole leaves shared similarity in the broad regions of DNA accessible to transcription factors, the short sequences bound varied. Transcription factor binding was prevalent in gene bodies as well as promoters, and many of these sites could represent duons that impact gene regulation in addition to amino acid sequence. Although globally there was little correlation between any individual DNaseI footprint and cell-specific gene expression, within individual species transcription factor binding to the same motifs in multiple genes provided evidence for shared mechanisms governing C4 photosynthesis gene expression. Furthermore, interspecific comparisons identified a small number of highly conserved transcription factor binding sites associated with leaves from species that diverged around 60 million years ago. These data therefore provide insight into the architecture associated with C4 photosynthesis gene expression in particular and characteristics of transcription factor binding in cereal crops in general. One sentence summary Genome-wide patterns of transcription factor binding in vivo defined by DNaseI for leaves of C3 and C4 grasses