Andrea Bräutigam

Gene Transfer from Bacteria and Archaea Facilitated Evolution of an Extremophilic Eukaryote

Hot, Toxic Eukaryote Unusually, the single-celled eukaryote red alga, Galdieria sulphuraria, can thrive in hot, acidic springs. This organism is endowed with extraordinary metabolic talents and can consume a variety of strange carbohydrates, as well as turn on photosynthesis when the food runs out. Schönknecht et al. (p. 1207; see the Perspective by Rocha) discerned from phylogenetic analysis of its genome that during its evolution, G. sulphuraria appears to have commandeered at least 75 bacterial and archaeal genes by horizontal gene transfer and then applied gene expansion to boost its metabolic repertoire. A mosaic of genes acquired from various phyla enable a red alga to grow abundantly in hot, acidic, and toxic niches. [Also see Perspective by Rocha] Some microbial eukaryotes, such as the extremophilic red alga Galdieria sulphuraria, live in hot, toxic metal-rich, acidic environments. To elucidate the underlying molecular mechanisms of adaptation, we sequenced the 13.7-megabase genome of G. sulphuraria. This alga shows an enormous metabolic flexibility, growing either photoautotrophically or heterotrophically on more than 50 carbon sources. Environmental adaptation seems to have been facilitated by horizontal gene transfer from various bacteria and archaea, often followed by gene family expansion. At least 5% of protein-coding genes of G. sulphuraria were probably acquired horizontally. These proteins are involved in ecologically important processes ranging from heavy-metal detoxification to glycerol uptake and metabolism. Thus, our findings show that a pan-domain gene pool has facilitated environmental adaptation in this unicellular eukaryote.

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.

Comprehensive transcriptome analysis of the highly complex Pisum sativum genome using next generation sequencing

BackgroundThe garden pea, Pisum sativum, is among the best-investigated legume plants and of significant agro-commercial relevance. Pisum sativum has a large and complex genome and accordingly few comprehensive genomic resources exist.ResultsWe analyzed the pea transcriptome at the highest possible amount of accuracy by current technology. We used next generation sequencing with the Roche/454 platform and evaluated and compared a variety of approaches, including diverse tissue libraries, normalization, alternative sequencing technologies, saturation estimation and diverse assembly strategies. We generated libraries from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings, comprising a total of 450 megabases. Libraries were assembled into 324,428 unigenes in a first pass assembly.A second pass assembly reduced the amount to 81,449 unigenes but caused a significant number of chimeras. Analyses of the assemblies identified the assembly step as a major possibility for improvement. By recording frequencies of Arabidopsis orthologs hit by randomly drawn reads and fitting parameters of the saturation curve we concluded that sequencing was exhaustive. For leaf libraries we found normalization allows partial recovery of expression strength aside the desired effect of increased coverage. Based on theoretical and biological considerations we concluded that the sequence reads in the database tagged the vast majority of transcripts in the aerial tissues. A pathway representation analysis showed the merits of sampling multiple aerial tissues to increase the number of tagged genes. All results have been made available as a fully annotated database in fasta format.ConclusionsWe conclude that the approach taken resulted in a high quality - dataset which serves well as a first comprehensive reference set for the model legume pea. We suggest future deep sequencing transcriptome projects of species lacking a genomics backbone will need to concentrate mainly on resolving the issues of redundancy and paralogy during transcriptome assembly.

Systems Analysis of a Maize Leaf Developmental Gradient Redefines the Current C4 Model and Provides Candidates for Regulation

This work reports on a comprehensive systems biology analysis of maize leaves, concluding that C4 photosynthesis is established from sink tissue without an intermediate phase of C3 or C2 photosynthesis. We systematically analyzed a developmental gradient of the third maize (Zea mays) leaf from the point of emergence into the light to the tip in 10 continuous leaf slices to study organ development and physiological and biochemical functions. Transcriptome analysis, oxygen sensitivity of photosynthesis, and photosynthetic rate measurements showed that the maize leaf undergoes a sink-to-source transition without an intermediate phase of C3 photosynthesis or operation of a photorespiratory carbon pump. Metabolome and transcriptome analysis, chlorophyll and protein measurements, as well as dry weight determination, showed continuous gradients for all analyzed items. The absence of binary on–off switches and regulons pointed to a morphogradient along the leaf as the determining factor of developmental stage. Analysis of transcription factors for differential expression along the leaf gradient defined a list of putative regulators orchestrating the sink-to-source transition and establishment of C4 photosynthesis. Finally, transcriptome and metabolome analysis, as well as enzyme activity measurements, and absolute quantification of selected metabolites revised the current model of maize C4 photosynthesis. All data sets are included within the publication to serve as a resource for maize leaf systems biology.

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 Proteomics of Chloroplast Envelopes from C3 and C4 Plants Reveals Specific Adaptations of the Plastid Envelope to C4 Photosynthesis and Candidate Proteins Required for Maintaining C4 Metabolite Fluxes

C4 plants have up to 10-fold higher apparent CO2 assimilation rates than the most productive C3 plants. This requires higher fluxes of metabolic intermediates across the chloroplast envelope membranes of C4 plants in comparison with those of C3 plants. In particular, the fluxes of metabolites involved in the biochemical inorganic carbon pump of C4 plants, such as malate, pyruvate, oxaloacetate, and phosphoenolpyruvate, must be considerably higher in C4 plants because they exceed the apparent rate of photosynthetic CO2 assimilation, whereas they represent relatively minor fluxes in C3 plants. While the enzymatic steps involved in the C4 biochemical inorganic carbon pump have been studied in much detail, little is known about the metabolite transporters in the envelope membranes of C4 chloroplasts. In this study, we used comparative proteomics of chloroplast envelope membranes from the C3 plant pea (Pisum sativum) and mesophyll cell chloroplast envelopes from the C4 plant maize (Zea mays) to analyze the adaptation of the mesophyll cell chloroplast envelope proteome to the requirements of C4 photosynthesis. We show that C3- and C4-type chloroplasts have qualitatively similar but quantitatively very different chloroplast envelope membrane proteomes. In particular, translocators involved in the transport of triosephosphate and phosphoenolpyruvate as well as two outer envelope porins are much more abundant in C4 plants. Several putative transport proteins have been identified that are highly abundant in C4 plants but relatively minor in C3 envelopes. These represent prime candidates for the transport of C4 photosynthetic intermediates, such as pyruvate, oxaloacetate, and malate.

EST-analysis of the thermo-acidophilic red microalga Galdieria sulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts

When we think of extremophiles, organisms adapted to extreme environments, prokaryotes come to mind first. However, the unicellular red micro-alga Galdieria sulphuraria (Cyanidiales) is a eukaryote that can represent up to 90% of the biomass in extreme habitats such as hot sulfur springs with pH values of 0–4 and temperatures of up to 56 °C. This red alga thrives autotrophically as well as heterotrophically on more than 50 different carbon sources, including a number of rare sugars and sugar alcohols. This biochemical versatility suggests a large repertoire of metabolic enzymes, rivaled by few organisms and a potentially rich source of thermo-stable enzymes for biotechnology. The temperatures under which this organism carries out photosynthesis are at the high end of the range for this process, making G. sulphuraria a valuable model for physical studies on the photosynthetic apparatus. In addition, the gene sequences of this living fossil reveal much about the evolution of modern eukaryotes. Finally, the alga tolerates high concentrations of toxic metal ions such as cadmium, mercury, aluminum, and nickel, suggesting potential application in bioremediation. To begin to explore the unique biology of G. sulphuraria, 5270 expressed sequence tags from two different cDNA libraries have been sequenced and annotated. Particular emphasis has been placed on the reconstruction of metabolic pathways present in this organism. For example, we provide evidence for (i) a complete pathway for lipid A biosynthesis; (ii) export of triose-phosphates from rhodoplasts; (iii) and absence of eukaryotic hexokinases. Sequence data and additional information are available at http://genomics.msu.edu/galdieria.

PLGG1, a plastidic glycolate glycerate transporter, is required for photorespiration and defines a unique class of metabolite transporters

Photorespiratory carbon flux reaches up to a third of photosynthetic flux, thus contributes massively to the global carbon cycle. The pathway recycles glycolate-2-phosphate, the most abundant byproduct of RubisCO reactions. This oxygenation reaction of RubisCO and subsequent photorespiration significantly limit the biomass gains of many crop plants. Although photorespiration is a compartmentalized process with enzymatic reactions in the chloroplast, the peroxisomes, the mitochondria, and the cytosol, no transporter required for the core photorespiratory cycle has been identified at the molecular level to date. Using transcript coexpression analyses, we identified Plastidal glycolate glycerate translocator 1 (PLGG1) as a candidate core photorespiratory transporter. Related genes are encoded in the genomes of archaea, bacteria, fungi, and all Archaeplastida and have previously been associated with a function in programmed cell-death. A mutant deficient in PLGG1 shows WT-like growth only in an elevated carbon dioxide atmosphere. The mutant accumulates glycolate and glycerate, leading to the hypothesis that PLGG1 is a glycolate/glycerate transporter. This hypothesis was tested and supported by in vivo and in vitro transport assays and 18O2-metabolic flux profiling. Our results indicate that PLGG1 is the chloroplastidic glycolate/glycerate transporter, which is required for the function of the photorespiratory cycle. Identification of the PLGG1 transport function will facilitate unraveling the role of similar proteins in bacteria, archaea, and fungi in the future.