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.

Plant protoplasts in the age of synthetic biology.

Protoplasts, which are plant cells with their cell walls removed, have been used for decades in plant research and have been instrumental in genetic transformation and the study of various aspects of plant physiology and genetics. With the advent of synthetic biology, these individualized plant cells are fundamental to accelerate the 'design-build-test-learn' cycle, which is relatively slow in plant research. Despite their potential, challenges remain in expanding the use of protoplasts in synthetic biology. The capacity of individual protoplasts to hybridize to form new varieties, and to regenerate from single cells, creating individuals with new features is underexplored. The main objective of this review is to discuss the use of protoplasts in plant synthetic biology and to highlight the challenges to exploiting protoplast technologies in this new 'age of synthetic biology'.

C4 gene induction during de-etiolation evolved through changes in cis to allow integration with ancestral C3 gene regulatory networks.

C4 photosynthesis has evolved by repurposing enzymes found in C3 plants. Compared with the ancestral C3 state, accumulation of C4 cycle proteins is enhanced. We used de-etiolation of C4 Gynandropsis gynandra and C3 Arabidopsis thaliana to understand this process. C4 gene expression and chloroplast biogenesis in G. gynandra were tightly coordinated. Although C3 and C4 photosynthesis genes showed similar induction patterns, in G. gynandra, C4 genes were more strongly induced than orthologs from A. thaliana. In vivo binding of TGA and homeodomain as well as light-responsive elements such as G- and I-box motifs were associated with the rapid increase in transcripts of C4 genes. Deletion analysis confirmed that regions containing G- and I-boxes were necessary for high expression. The data support a model in which accumulation of transcripts derived from C4 photosynthesis genes in C4 leaves is enhanced because modifications in cis allowed integration into ancestral transcriptional networks.

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.

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

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.

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.

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.

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.

Ancient duons may underpin spatial patterning of gene expression in C4 leaves.

If the highly efficient C4 photosynthesis pathway could be transferred to crops with the C3 pathway there could be yield gains of up to 50%. It has been proposed that the multiple metabolic and developmental modifications associated with C4 photosynthesis are underpinned by relatively few master regulators that have allowed the evolution of C4 photosynthesis more than 60 times in flowering plants. Here we identify a component of one such regulator that consists of a pair of cis-elements located in coding sequence of multiple genes that are preferentially expressed in bundle sheath cells of C4 leaves. These motifs represent duons as they play a dual role in coding for amino acids as well as controlling the spatial patterning of gene expression associated with the C4 leaf. They act to repress transcription of C4 photosynthesis genes in mesophyll cells. These duons are also present in the C3 model Arabidopsis thaliana, and, in fact, are conserved in all land plants and even some algae that use C3 photosynthesis. C4 photosynthesis therefore appears to have coopted an ancient regulatory code to generate the spatial patterning of gene expression that is a hallmark of C4 photosynthesis. This intragenic transcriptional regulatory sequence could be exploited in the engineering of efficient photosynthesis of crops.

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 C3Arabidopsis 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'.

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

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

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.

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.