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.

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.

Correction: Deep Evolutionary Comparison of Gene Expression Identifies Parallel Recruitment of Trans-Factors in Two Independent Origins of C4 Photosynthesis.

[This corrects the article DOI: 10.1371/journal.pgen.1004365.].

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.

C4 Photosynthesis in the Rice Paddy: Insights from the Noxious Weed Echinochloa glabrescens.

The C4 pathway is a highly complex trait that increases photosynthetic efficiency in more than 60 plant lineages. Although the majority of C4 plants occupy disturbed, arid, and nutrient-poor habitats, some grow in high-nutrient, waterlogged conditions. One such example is Echinochloa glabrescens, which is an aggressive weed of rice paddies. We generated comprehensive transcriptome datasets for C4 E. glabrescens and C3 rice to identify genes associated with adaption to waterlogged, nutrient-replete conditions, but also used the data to better understand how C4 photosynthesis operates in these conditions. Leaves of E. glabrescens exhibited classical Kranz anatomy with lightly lobed mesophyll cells having low chloroplast coverage. As with rice and other hygrophytic C3 species, leaves of E. glabrescens accumulated a chloroplastic phosphoenolpyruvate carboxylase protein, albeit at reduced amounts relative to rice. The arid-grown species Setaria italica (C4) and Brachypodium distachyon (C3) were also found to accumulate chloroplastic phosphoenolpyruvate carboxylase. We identified a molecular signature associated with C4 photosynthesis in nutrient-replete, waterlogged conditions that is highly similar to those previously reported from C4 plants that grow in more arid conditions. We also identified a cohort of genes that have been subjected to a selective sweep associated with growth in paddy conditions. Overall, this approach highlights the value of using wild species such as weeds to identify adaptions to specific conditions associated with high-yielding crops in agriculture.

Telomere stability and development of ctc1 mutants are rescued by inhibition of EJ recombination pathways in a telomerase-dependent manner.

The telomeres of linear eukaryotic chromosomes are protected by caps consisting of evolutionarily conserved nucleoprotein complexes. Telomere dysfunction leads to recombination of chromosome ends and this can result in fusions which initiate chromosomal breakage-fusion-bridge cycles, causing genomic instability and potentially cell death or cancer. We hypothesize that in the absence of the recombination pathways implicated in these fusions, deprotected chromosome ends will instead be eroded by nucleases, also leading to the loss of genes and cell death. In this work, we set out to specifically test this hypothesis in the plant, Arabidopsis. Telomere protection in Arabidopsis implicates KU and CST and their absence leads to chromosome fusions, severe genomic instability and dramatic developmental defects. We have analysed the involvement of end-joining recombination pathways in telomere fusions and the consequences of this on genomic instability and growth. Strikingly, the absence of the multiple end-joining pathways eliminates chromosome fusion and restores normal growth and development to cst ku80 mutant plants. It is thus the chromosomal fusions, per se, which are the underlying cause of the severe developmental defects. This rescue is mediated by telomerase-dependent telomere extension, revealing a competition between telomerase and end-joining recombination proteins for access to deprotected telomeres.

Deep evolutionary comparison of gene expression identifies parallel recruitment of trans-factors in two independent origins of C4 photosynthesis.

With at least 60 independent origins spanning monocotyledons and dicotyledons, the C4 photosynthetic pathway represents one of the most remarkable examples of convergent evolution. The recurrent evolution of this highly complex trait involving alterations to leaf anatomy, cell biology and biochemistry allows an increase in productivity by ∼ 50% in tropical and subtropical areas. The extent to which separate lineages of C4 plants use the same genetic networks to maintain C4 photosynthesis is unknown. We developed a new informatics framework to enable deep evolutionary comparison of gene expression in species lacking reference genomes. We exploited this to compare gene expression in species representing two independent C4 lineages (Cleome gynandra and Zea mays) whose last common ancestor diverged ∼ 140 million years ago. We define a cohort of 3,335 genes that represent conserved components of leaf and photosynthetic development in these species. Furthermore, we show that genes encoding proteins of the C4 cycle are recruited into networks defined by photosynthesis-related genes. Despite the wide evolutionary separation and independent origins of the C4 phenotype, we report that these species use homologous transcription factors to both induce C4 photosynthesis and to maintain the cell specific gene expression required for the pathway to operate. We define a core molecular signature associated with leaf and photosynthetic maturation that is likely shared by angiosperm species derived from the last common ancestor of the monocotyledons and dicotyledons. We show that deep evolutionary comparisons of gene expression can reveal novel insight into the molecular convergence of highly complex phenotypes and that parallel evolution of trans-factors underpins the repeated appearance of C4 photosynthesis. Thus, exploitation of extant natural variation associated with complex traits can be used to identify regulators. Moreover, the transcription factors that are shared by independent C4 lineages are key targets for engineering the C4 pathway into C3 crops such as rice.

Transcript residency on ribosomes reveals a key role for the Arabidopsis thaliana bundle sheath in sulfur and glucosinolate metabolism.

Leaves of angiosperms are made up of multiple distinct cell types. While the function of mesophyll cells, guard cells, phloem companion cells and sieve elements are clearly described, this is not the case for the bundle sheath (BS). To provide insight into the role of the BS in the C3 species Arabidopsis thaliana, we labelled ribosomes in this cell type with a FLAG tag. We then used immunocapture to isolate these ribosomes, followed by sequencing of resident mRNAs. This showed that 5% of genes showed specific splice forms in the BS, and that 15% of genes were preferentially expressed in these cells. The BS translatome strongly implies that the BS plays specific roles in sulfur transport and metabolism, glucosinolate biosynthesis and trehalose metabolism. Much of the C4 cycle is differentially expressed between the C3 BS and the rest of the leaf. Furthermore, the global patterns of transcript residency on BS ribosomes overlap to a greater extent with cells of the root pericycle than any other cell type. This analysis provides the first insight into the molecular function of this cell type in C3 species, and also identifies characteristics of BS cells that are probably ancestral to both C3 and C4 plants.

Evolutionary convergence of cell-specific gene expression in independent lineages of C4 grasses.

Leaves of almost all C4 lineages separate the reactions of photosynthesis into the mesophyll (M) and bundle sheath (BS). The extent to which messenger RNA profiles of M and BS cells from independent C4 lineages resemble each other is not known. To address this, we conducted deep sequencing of RNA isolated from the M and BS of Setaria viridis and compared these data with publicly available information from maize (Zea mays). This revealed a high correlation (r=0.89) between the relative abundance of transcripts encoding proteins of the core C4 pathway in M and BS cells in these species, indicating significant convergence in transcript accumulation in these evolutionarily independent C4 lineages. We also found that the vast majority of genes encoding proteins of the C4 cycle in S. viridis are syntenic to homologs used by maize. In both lineages, 122 and 212 homologous transcription factors were preferentially expressed in the M and BS, respectively. Sixteen shared regulators of chloroplast biogenesis were identified, 14 of which were syntenic homologs in maize and S. viridis. In sorghum (Sorghum bicolor), a third C4 grass, we found that 82% of these trans-factors were also differentially expressed in either M or BS cells. Taken together, these data provide, to our knowledge, the first quantification of convergence in transcript abundance in the M and BS cells from independent lineages of C4 grasses. Furthermore, the repeated recruitment of syntenic homologs from large gene families strongly implies that parallel evolution of both structural genes and trans-factors underpins the polyphyletic evolution of this highly complex trait in the monocotyledons.

An open science peer review oath.

One of the foundations of the scientific method is to be able to reproduce experiments and corroborate the results of research that has been done before. However, with the increasing complexities of new technologies and techniques, coupled with the specialisation of experiments, reproducing research findings has become a growing challenge. Clearly, scientific methods must be conveyed succinctly, and with clarity and rigour, in order for research to be reproducible. Here, we propose steps to help increase the transparency of the scientific method and the reproducibility of research results: specifically, we introduce a peer-review oath and accompanying manifesto. These have been designed to offer guidelines to enable reviewers (with the minimum friction or bias) to follow and apply open science principles, and support the ideas of transparency, reproducibility and ultimately greater societal impact. Introducing the oath and manifesto at the stage of peer review will help to check that the research being published includes everything that other researchers would need to successfully repeat the work. Peer review is the lynchpin of the publishing system: encouraging the community to consciously (and conscientiously) uphold these principles should help to improve published papers, increase confidence in the reproducibility of the work and, ultimately, provide strategic benefits to authors and their institutions.