Udo Gowik

The Sorghum bicolor genome and the diversification of grasses

Sorghum, an African grass related to sugar cane and maize, is grown for food, feed, fibre and fuel. We present an initial analysis of the ∼730-megabase Sorghum bicolor (L.) Moench genome, placing ∼98% of genes in their chromosomal context using whole-genome shotgun sequence validated by genetic, physical and syntenic information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally recalcitrant heterochromatin explains the ∼75% larger genome size of sorghum compared with rice. Although gene and repetitive DNA distributions have been preserved since palaeopolyploidization ∼70 million years ago, most duplicated gene sets lost one member before the sorghum–rice divergence. Concerted evolution makes one duplicated chromosomal segment appear to be only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and microRNA duplications may contribute to sorghum’s drought tolerance.

cis-Regulatory Elements for Mesophyll-Specific Gene Expression in the C4 Plant Flaveria trinervia, the Promoter of the C4 Phosphoenolpyruvate Carboxylase Gene

C4 photosynthesis depends on the strict compartmentalization of CO2 assimilatory enzymes. cis-regulatory mechanisms are described that ensure mesophyll-specific expression of the gene encoding the C4 isoform of phosphoenolpyruvate carboxylase (ppcA1) of the C4 dicot Flaveria trinervia. To elucidate and understand the anatomy of the C4 ppcA1 promoter, detailed promoter/reporter gene studies were performed in the closely related C4 species F. bidentis, revealing that the C4 promoter contains two regions, a proximal segment up to −570 and a distal part from −1566 to −2141, which are necessary but also sufficient for high mesophyll-specific expression of the β-glucuronidase reporter gene. The distal region behaves as an enhancer-like expression module that can direct mesophyll-specific expression when inserted into the ppcA1 promoter of the C3 plant F. pringlei. Mesophyll expression determinants were restricted to a 41-bp segment, referred to as mesophyll expression module 1 (Mem1). Evolutionary and functional studies identified the tetranucleotide sequence CACT as a key component of Mem1.

An mRNA blueprint for C4 photosynthesis derived from comparative transcriptomics of closely related C3 and C4 species

C4 photosynthesis involves alterations to the biochemistry, cell biology, and development of leaves. Together, these modifications increase the efficiency of photosynthesis, and despite the apparent complexity of the pathway, it has evolved at least 45 times independently within the angiosperms. To provide insight into the extent to which gene expression is altered between C3 and C4 leaves, and to identify candidates associated with the C4 pathway, we used massively parallel mRNA sequencing of closely related C3 (Cleome spinosa) and C4 (Cleome gynandra) species. Gene annotation was facilitated by the phylogenetic proximity of Cleome and Arabidopsis (Arabidopsis thaliana). Up to 603 transcripts differ in abundance between these C3 and C4 leaves. These include 17 transcription factors, putative transport proteins, as well as genes that in Arabidopsis are implicated in chloroplast movement and expansion, plasmodesmatal connectivity, and cell wall modification. These are all characteristics known to alter in a C4 leaf but that previously had remained undefined at the molecular level. We also document large shifts in overall transcription profiles for selected functional classes. Our approach defines the extent to which transcript abundance in these C3 and C4 leaves differs, provides a blueprint for the NAD-malic enzyme C4 pathway operating in a dicotyledon, and furthermore identifies potential regulators. We anticipate that comparative transcriptomics of closely related species will provide deep insight into the evolution of other complex traits.

A plastidial sodium-dependent pyruvate transporter

Pyruvate serves as a metabolic precursor for many plastid-localized biosynthetic pathways, such as those for fatty acids, terpenoids and branched-chain amino acids. In spite of the importance of pyruvate uptake into plastids (organelles within cells of plants and algae), the molecular mechanisms of this uptake have not yet been explored. This is mainly because pyruvate is a relatively small compound that is able to passively permeate lipid bilayers, which precludes accurate measurement of pyruvate transport activity in reconstituted liposomes. Using differential transcriptome analyses of C3 and C4 plants of the genera Flaveria and Cleome, here we have identified a novel gene that is abundant in C4 species, named BASS2 (BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2). The BASS2 protein is localized at the chloroplast envelope membrane, and is highly abundant in C4 plants that have the sodium-dependent pyruvate transporter. Recombinant BASS2 shows sodium-dependent pyruvate uptake activity. Sodium influx is balanced by a sodium:proton antiporter (NHD1), which was mimicked in recombinant Escherichia coli cells expressing both BASS2 and NHD1. Arabidopsis thaliana bass2 mutants lack pyruvate uptake into chloroplasts, which affects plastid-localized isopentenyl diphosphate synthesis, as evidenced by increased sensitivity of such mutants to mevastatin, an inhibitor of cytosolic isopentenyl diphosphate biosynthesis. We thus provide molecular evidence for a sodium-coupled metabolite transporter in plastid envelopes. Orthologues of BASS2 can be detected in all the genomes of land plants that have been characterized so far, thus indicating the widespread importance of sodium-coupled pyruvate import into plastids.

What can next generation sequencing do for you? Next generation sequencing as a valuable tool in plant research.

Next generation sequencing (NGS) technologies have opened fascinating opportunities for the analysis of plants with and without a sequenced genome on a genomic scale. During the last few years, NGS methods have become widely available and cost effective. They can be applied to a wide variety of biological questions, from the sequencing of complete eukaryotic genomes and transcriptomes, to the genome-scale analysis of DNA-protein interactions. In this review, we focus on the use of NGS for plant transcriptomics, including gene discovery, transcript quantification and marker discovery for non-model plants, as well as transcript annotation and quantification, small RNA discovery and antisense transcription analysis for model plants. We discuss the experimental design for analysis of plants with and without a sequenced genome, including considerations on sampling, RNA preparation, sequencing platforms and bioinformatics tools for data analysis. NGS technologies offer exciting new opportunities for the plant sciences, especially for work on plants without a sequenced genome, since large sequence resources can be generated at moderate cost.

The Path from C3 to C4 Photosynthesis

The C4 photosynthetic carbon cycle is an elaborated addition to the C3 photosynthetic pathway. It evolved as an adaptation to high light intensities, high temperatures, and dryness. Therefore, C4 plants dominate grassland floras and biomass production in the warmer climates of the tropical and

Evolution of C4 Photosynthesis in the Genus Flaveria: How Many and Which Genes Does It Take to Make C4?

We carried out a comparative transcriptome analysis in leaves of five closely related C3, C4, and C3-C4 intermediate Flaveria species to gain insight into the extent to which gene expression patterns were altered in the evolutionary progression from C3 to C4. The comparative analysis indicates that C4 evolution affected far more biological functions than only photosynthesis. Selective pressure exerted by a massive decline in atmospheric CO2 levels 55 to 40 million years ago promoted the evolution of a novel, highly efficient mode of photosynthetic carbon assimilation known as C4 photosynthesis. C4 species have concurrently evolved multiple times in a broad range of plant families, and this multiple and parallel evolution of the complex C4 trait indicates a common underlying evolutionary mechanism that might be elucidated by comparative analyses of related C3 and C4 species. Here, we use mRNA-Seq analysis of five species within the genus Flaveria, ranging from C3 to C3-C4 intermediate to C4 species, to quantify the differences in the transcriptomes of closely related plant species with varying degrees of C4-associated characteristics. Single gene analysis defines the C4 cycle enzymes and transporters more precisely and provides new candidates for yet unknown functions as well as identifies C4 associated pathways. Molecular evidence for a photorespiratory CO2 pump prior to the establishment of the C4 cycle-based CO2 pump is provided. Cluster analysis defines the upper limit of C4-related gene expression changes in mature leaves of Flaveria as 3582 alterations.

Comparative genomic analysis of C4 photosynthetic pathway evolution in grasses

BackgroundSorghum is the first C4 plant and the second grass with a full genome sequence available. This makes it possible to perform a whole-genome-level exploration of C4 pathway evolution by comparing key photosynthetic enzyme genes in sorghum, maize (C4) and rice (C3), and to investigate a long-standing hypothesis that a reservoir of duplicated genes is a prerequisite for the evolution of C4 photosynthesis from a C3 progenitor.ResultsWe show that both whole-genome and individual gene duplication have contributed to the evolution of C4 photosynthesis. The C4 gene isoforms show differential duplicability, with some C4 genes being recruited from whole genome duplication duplicates by multiple modes of functional innovation. The sorghum and maize carbonic anhydrase genes display a novel mode of new gene formation, with recursive tandem duplication and gene fusion accompanied by adaptive evolution to produce C4 genes with one to three functional units. Other C4 enzymes in sorghum and maize also show evidence of adaptive evolution, though differing in level and mode. Intriguingly, a phosphoenolpyruvate carboxylase gene in the C3 plant rice has also been evolving rapidly and shows evidence of adaptive evolution, although lacking key mutations that are characteristic of C4 metabolism. We also found evidence that both gene redundancy and alternative splicing may have sheltered the evolution of new function.ConclusionsGene duplication followed by functional innovation is common to evolution of most but not all C4 genes. The apparently long time-lag between the availability of duplicates for recruitment into C4 and the appearance of C4 grasses, together with the heterogeneity of origins of C4 genes, suggests that there may have been a long transition process before the establishment of C4 photosynthesis.

Predicting C4 Photosynthesis Evolution: Modular, Individually Adaptive Steps on a Mount Fuji Fitness Landscape

An ultimate goal of evolutionary biology is the prediction and experimental verification of adaptive trajectories on macroevolutionary timescales. This aim has rarely been achieved for complex biological systems, as models usually lack clear correlates of organismal fitness. Here, we simulate the fitness landscape connecting two carbon fixation systems: C3 photosynthesis, used by most plant species, and the C4 system, which is more efficient at ambient CO2 levels and elevated temperatures and which repeatedly evolved from C3. Despite extensive sign epistasis, C4 photosynthesis is evolutionarily accessible through individually adaptive steps from any intermediate state. Simulations show that biochemical subtraits evolve in modules; the order and constitution of modules confirm and extend previous hypotheses based on species comparisons. Plant-species-designated C3-C4 intermediates lie on predicted evolutionary trajectories, indicating that they indeed represent transitory states. Contrary to expectations, we find no slowdown of adaptation and no diminishing fitness gains along evolutionary trajectories.

The role of photorespiration during the evolution of C4 photosynthesis in the genus Flaveria

C4 photosynthesis represents a most remarkable case of convergent evolution of a complex trait, which includes the reprogramming of the expression patterns of thousands of genes. Anatomical, physiological, and phylogenetic and analyses as well as computational modeling indicate that the establishment of a photorespiratory carbon pump (termed C2 photosynthesis) is a prerequisite for the evolution of C4. However, a mechanistic model explaining the tight connection between the evolution of C4 and C2 photosynthesis is currently lacking. Here we address this question through comparative transcriptomic and biochemical analyses of closely related C3, C3–C4, and C4 species, combined with Flux Balance Analysis constrained through a mechanistic model of carbon fixation. We show that C2 photosynthesis creates a misbalance in nitrogen metabolism between bundle sheath and mesophyll cells. Rebalancing nitrogen metabolism requires anaplerotic reactions that resemble at least parts of a basic C4 cycle. Our findings thus show how C2 photosynthesis represents a pre-adaptation for the C4 system, where the evolution of the C2 system establishes important C4 components as a side effect. DOI: http://dx.doi.org/10.7554/eLife.02478.001