Genome-Wide Transcription Factor Binding in Leaves from C3 and C4 Grasses.

The majority of plants use C3 photosynthesis, but over 60 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 sequencing 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 C3 Brachypodium 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 influence 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.BBSRC ERC CONACy

An Untranslated cis-Element Regulates the Accumulation of Multiple C4 Enzymes in Gynandropsis gynandra Mesophyll Cells.

C4 photosynthesis is a complex phenotype that allows more efficient carbon capture than the ancestral C3 pathway. In leaves of C4 species, hundreds of transcripts increase in abundance compared with C3 relatives and become restricted to mesophyll (M) or bundle sheath (BS) cells. However, no mechanism has been reported that regulates the compartmentation of multiple enzymes in M or BS cells. We examined mechanisms regulating CARBONIC ANHYDRASE4 (CA4) in C4 Gynandropsis gynandra. Increased abundance is directed by both the promoter region and introns of the G. gynandra gene. A nine-nucleotide motif located in the 5' untranslated region (UTR) is required for preferential accumulation of GUS in M cells. This element is present and functional in three additional 5' UTRs and six 3' UTRs where it determines accumulation of two isoforms of CA and pyruvate,orthophosphate dikinase in M cells. Although the GgCA4 5' UTR is sufficient to direct GUS accumulation in M cells, transcripts encoding GUS are abundant in both M and BS. Mutating the GgCA4 5' UTR abolishes enrichment of protein in M cells without affecting transcript abundance. The work identifies a mechanism that directs cell-preferential accumulation of multiple enzymes required for C4 photosynthesis.We would like to thank the BBSRC for funding.This is the author accepted manuscript. The final version is available from the American Society of Plant Biologists via http://dx.doi.org/10.​1105/​tpc.​15.​0057

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.BBSRC and EPSRC OpenPlant BB/L014130/

Photosynthesis-independent production of reactive oxygen species in the rice bundle sheath during high light is mediated by NADPH oxidase.

When exposed to high light, plants produce reactive oxygen species (ROS). In Arabidopsis thaliana, local stress such as excess heat or light initiates a systemic ROS wave in phloem and xylem cells dependent on NADPH oxidase/respiratory burst oxidase homolog (RBOH) proteins. In the case of excess light, although the initial local accumulation of ROS preferentially takes place in bundle-sheath strands, little is known about how this response takes place. Using rice and the ROS probes diaminobenzidine and 2',7'-dichlorodihydrofluorescein diacetate, we found that, after exposure to high light, ROS were produced more rapidly in bundle-sheath strands than mesophyll cells. This response was not affected either by CO2 supply or photorespiration. Consistent with these findings, deep sequencing of messenger RNA (mRNA) isolated from mesophyll or bundle-sheath strands indicated balanced accumulation of transcripts encoding all major components of the photosynthetic apparatus. However, transcripts encoding several isoforms of the superoxide/H2O2-producing enzyme NADPH oxidase were more abundant in bundle-sheath strands than mesophyll cells. ROS production in bundle-sheath strands was decreased in mutant alleles of the bundle-sheath strand preferential isoform of OsRBOHA and increased when it was overexpressed. Despite the plethora of pathways able to generate ROS in response to excess light, NADPH oxidase-mediated accumulation of ROS in the rice bundle-sheath strand was detected in etiolated leaves lacking chlorophyll. We conclude that photosynthesis is not necessary for the local ROS response to high light but is in part mediated by NADPH oxidase activity

Shared characteristics underpinning C 4 leaf maturation derived from analysis of multiple C 3 and C 4 species of Flaveria

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

The bundle sheath of rice is conditioned to play an active role in water transport as well as sulfur assimilation and jasmonic acid synthesis

Funder: Bill and Melinda Gates Foundation; Id: http://dx.doi.org/10.13039/100000865Summary: Leaves comprise multiple cell types but our knowledge of the patterns of gene expression that underpin their functional specialization is fragmentary. Our understanding and ability to undertake the rational redesign of these cells is therefore limited. We aimed to identify genes associated with the incompletely understood bundle sheath of C3 plants, which represents a key target associated with engineering traits such as C4 photosynthesis into Oryza sativa (rice). To better understand the veins, bundle sheath and mesophyll cells of rice, we used laser capture microdissection followed by deep sequencing. Gene expression of the mesophyll is conditioned to allow coenzyme metabolism and redox homeostasis, as well as photosynthesis. In contrast, the bundle sheath is specialized in water transport, sulphur assimilation and jasmonic acid biosynthesis. Despite the small chloroplast compartment of bundle sheath cells, substantial photosynthesis gene expression was detected. These patterns of gene expression were not associated with the presence or absence of specific transcription factors in each cell type, but were instead associated with gradients in expression across the leaf. Comparative analysis with C3 Arabidopsis identified a small gene set preferentially expressed in the bundle sheath cells of both species. This gene set included genes encoding transcription factors from 14 orthogroups and proteins allowing water transport, sulphate assimilation and jasmonic acid synthesis. The most parsimonious explanation for our findings is that bundle sheath cells from the last common ancestor of rice and Arabidopsis were specialized in this manner, and as the species diverged these patterns of gene expression have been maintained

Compartmentation of photosynthesis gene expression in C4 maize depends on time of day

Compared with the ancestral C3 state, C4 photosynthesis occurs at higher rates with improved water and nitrogen use efficiencies. In both C3 and C4 plants, rates of photosynthesis increase with light intensity and are maximal around midday. We determined that in the absence of light or temperature fluctuations, photosynthesis in maize (Zea mays) peaks in the middle of the subjective photoperiod. To investigate the molecular processes associated with these temporal changes, we performed RNA sequencing of maize mesophyll and bundle sheath strands over a 24-h time course. Preferential expression of C4 cycle genes in these cell types was strongest between 6 and 10 h after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene coexpression analyses suggested members of the DNA binding with one finger (DOF) and MADS (MINICHROMOSOME MAINTENANCE FACTOR 1/AGAMOUS/DEFICIENS/Serum Response Factor)-domain transcription factor families mediate diurnal fluctuations in C4 gene expression, while trans-activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators and peaks in cell-specific C4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.This work was supported by the European Commission project 3to4 (grant agreement no: 289582), by Fundação para a Ciência e Tecnologia (FCT) through research unit GREEN-it “Bioresources for Sustainability” (UID/Multi/04551/2013, UIDB/04551/2020), by POPH-QREN to A.R.B. (SFRH/BD/105739/2014), A.M.G. (SFRH/BD/89743/2012), and N.J.M.S. (IF/01126/2012), by ERACAPS grant C4BREED and BBSRC grants BB/L014130, BBP0031171, and BB/S006370/1, and by European Research Council Grant Revolution RG80867

Synergistic Binding of bHLH Transcription Factors to the Promoter of the Maize NADP-ME Gene Used in C4 Photosynthesis Is Based on an Ancient Code Found in the Ancestral C3 State.

C4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialized form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world's most productive crops. The C4 cycle is accomplished through cell-type-specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we tested whether mechanisms impacting on transcription in C4 plants evolved from ancestral components found in C3 species. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 Panicoid grass species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis

Evolution as a principle for engineering photosynthesis

Trabajo presentado el día 1 de marzo de 2022 en el ciclo de seminarios organizado por el Instituto de Investigación en Inteligencia Artificial, IIA Webinar Series.As the world population continues to expand, it is predicted that crop yields will have to increase by 50% over the next 35 years. Traditional breeding programs cannot keep pace with this current population growth rate. One of the main determinants of crop yield is the capacity of the plant to harvest light and convert it into sugars through photosynthesis. Despite this, photosynthesis improvement is still underexploited for the purpose of increasing yield. Plants have evolved a wide variety of photosynthesis flavours, some of them more efficient than others. This provides an “evolutionary guide” for engineering some of these traits in target crops like rice. In this talk I will describe some of the strategies we use in improving photosynthesis and some of the problems we face where the interaction with researchers in Artificial intelligence could prove beneficial