An optimized protocol for isolation of high-quality RNA through laser capture microdissection of leaf material.

Laser Capture Microdissection is a powerful tool that allows thin slices of specific cell types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded RNA. Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here, we describe an optimized procedure to limit RNA degradation that is based on the use of low-melting-point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach, high-quality RNA suitable for down-stream applications such as quantitative reverse transcriptase-polymerase chain reactions or RNA-sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue.The work was supported by a C4 Rice project grant from The Bill and Melinda Gates Foundation to the University of Oxford (2015-­2019)

Rice bundle sheath cell shape is regulated by the timing of light exposure during leaf development

Plant leaves contain multiple cell types which achieve distinct characteristics whilst still coordinating development within the leaf. The bundle sheath possesses larger individual cells and lower chloroplast content than the adjacent mesophyll, but how this morphology is achieved remains unknown. To identify regulatory mechanisms determining bundle sheath cell morphology we tested the effects of perturbing environmental (light) and endogenous signals (hormones) during leaf development of Oryza sativa (rice). Total chloroplast area in bundle sheath cells was found to increase with cell size as in the mesophyll but did not maintain a ‘set‐point’ relationship, with the longest bundle sheath cells demonstrating the lowest chloroplast content. Application of exogenous cytokinin and gibberellin significantly altered the relationship between cell size and chloroplast biosynthesis in the bundle sheath, increasing chloroplast content of the longest cells. Delayed exposure to light reduced the mean length of bundle sheath cells but increased corresponding leaf length, whereas premature light reduced final leaf length but did not affect bundle sheath cells. This suggests that the plant hormones cytokinin and gibberellin are regulators of the bundle sheath cell‐chloroplast relationship and that final bundle sheath length may potentially be affected by light‐mediated control of exit from the cell cycle

Insights into C4 metabolism from comparative deep sequencing.

C4 photosynthesis suppresses the oxygenation activity of Ribulose Bisphosphate Carboxylase Oxygenase and so limits photorespiration. Although highly complex, it is estimated to have evolved in 66 plant lineages, with the vast majority lacking sequenced genomes. Transcriptomics has recently initiated assessments of the degree to which transcript abundance differs between C3 and C4 leaves, identified novel components of C4 metabolism, and also led to mathematical models explaining the repeated evolution of this complex phenotype. Evidence is accumulating that this complex and convergent phenotype is partly underpinned by parallel evolution of structural genes, but also regulatory elements in both cis and trans. Furthermore, it appears that initial events associated with acquisition of C4 traits likely represent evolutionary exaptations related to non-photosynthetic processes.We thank the BBSRC for grant BB/1002243/1 and the EU 3to4 program for financial support.This is the accepted manuscript. The final version is available at http://www.sciencedirect.com/science/article/pii/S1369526615000680

Regulatory gateways for cell-specific gene expression in C4 leaves with Kranz anatomy.

C4 photosynthesis is a carbon-concentrating mechanism that increases delivery of carbon dioxide to RuBisCO and as a consequence reduces photorespiration. The C4 pathway is therefore beneficial in environments that promote high photorespiration. This pathway has evolved many times, and involves restricting gene expression to either mesophyll or bundle sheath cells. Here we review the regulatory mechanisms that control cell-preferential expression of genes in the C4 cycle. From this analysis, it is clear that the C4 pathway has a complex regulatory framework, with control operating at epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels. Some genes of the C4 pathway are regulated at multiple levels, and we propose that this ensures robust expression in each cell type. Accumulating evidence suggests that multiple genes of the C4 pathway may share the same regulatory mechanism. The control systems for C4 photosynthesis gene expression appear to operate in C3 plants, and so it appears that pre-existing mechanisms form the basis of C4 photosynthesis gene expression

Fluorescent reporters for functional analysis in rice leaves.

Fluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed the analysis of processes ranging from gene expression and protein localization to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualize fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins (mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato) that are robustly expressed and detectable in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolor imaging of different subcellular compartments. Overall, we conclude that mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato are suitable for high-resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice

TransRate: reference-free quality assessment of de novo transcriptome assemblies.

TransRate is a tool for reference-free quality assessment of de novo transcriptome assemblies. Using only the sequenced reads and the assembly as input, we show that multiple common artifacts of de novo transcriptome assembly can be readily detected. These include chimeras, structural errors, incomplete assembly, and base errors. TransRate evaluates these errors to produce a diagnostic quality score for each contig, and these contig scores are integrated to evaluate whole assemblies. Thus, TransRate can be used for de novo assembly filtering and optimization as well as comparison of assemblies generated using different methods from the same input reads. Applying the method to a data set of 155 published de novo transcriptome assemblies, we deconstruct the contribution that assembly method, read length, read quantity, and read quality make to the accuracy of de novo transcriptome assemblies and reveal that variance in the quality of the input data explains 43% of the variance in the quality of published de novo transcriptome assemblies. Because TransRate is reference-free, it is suitable for assessment of assemblies of all types of RNA, including assemblies of long noncoding RNA, rRNA, mRNA, and mixed RNA samples

Light-regulated and cell-specific methylation of the maize PEPC promoter

The molecular mechanisms governing PEPC expression in maize remain to be fully defined. Differential methylation of a region in the PEPC promoter has been shown to correlate with transcript accumulation, however, to date, investigations into the role of DNA methylation in maize PEPC expression have relied on the use of methylation-sensitive restriction enzymes. Bisulphite sequencing was used here to provide a single-base resolution methylation map of the maize PEPC promoter. It is shown that four cytosine residues in the PEPC promoter are heavily methylated in maize root tissue. In leaves, de-methylation of these cytosines is dependent on illumination and is coincident with elevated PEPC expression. Furthermore, light-regulated de-methylation of these cytosines occurs only in mesophyll cells. No methylation was discovered in the 0.6 kb promoter required for mesophyll-specific expression indicating that cytosine methylation is not required to direct the cell-specificity of PEPC expression. This raises interesting questions regarding the function of the cell-specific cytosine de-methylation observed in the upstream region of the PEPC promoter

Focus Issue Editorial: Synthetic Biology.

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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

Computational modelling predicts substantial carbon assimilation gains for C3 plants with a single-celled C4 biochemical pump.

Achieving global food security for the estimated 9 billion people by 2050 is a major scientific challenge. Crop productivity is fundamentally restricted by the rate of fixation of atmospheric carbon. The dedicated enzyme, RubisCO, has a low turnover and poor specificity for CO2. This limitation of C3 photosynthesis (the basic carbon-assimilation pathway present in all plants) is alleviated in some lineages by use of carbon-concentrating-mechanisms, such as the C4 cycle-a biochemical pump that concentrates CO2 near RubisCO increasing assimilation efficacy. Most crops use only C3 photosynthesis, so one promising research strategy to boost their productivity focuses on introducing a C4 cycle. The simplest proposal is to use the cycle to concentrate CO2 inside individual chloroplasts. The photosynthetic efficiency would then depend on the leakage of CO2 out of a chloroplast. We examine this proposal with a 3D spatial model of carbon and oxygen diffusion and C4 photosynthetic biochemistry inside a typical C3-plant mesophyll cell geometry. We find that the cost-efficiency of C4 photosynthesis depends on the gas permeability of the chloroplast envelope, the C4 pathway having higher quantum efficiency than C3 for permeabilities below 300 μm/s. However, at higher permeabilities the C4 pathway still provides a substantial boost to carbon assimilation with only a moderate decrease in efficiency. The gains would be capped by the ability of chloroplasts to harvest light, but even under realistic light regimes a 100% boost to carbon assimilation is possible. This could be achieved in conjunction with lower investment in chloroplasts if their cell surface coverage is also reduced. Incorporation of this C4 cycle into C3 crops could thus promote higher growth rates and better drought resistance in dry, high-sunlight climates