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Pyruvate transporter is BASS2 Many of the plastid-localized metabolic pathways of plants, including the C 4 photosynthetic pathway that operates in many crop plants, depend critically on the import of pyruvate. The pyruvate transporter has proved elusive, but has now been identified as the bile acid:sodium symporter family protein 2 (BASS2). The BASS2 protein is found in the chloroplast envelope membrane, and is highly abundant in C 4 plants. Orthologues of BASS2 are present in all the genomes of land plants characterized so far, thus indicating the widespread importance of sodium-coupled pyruvate import in plastids. Pyruvate serves as a metabolic precursor for many plastid-localized biosynthetic pathways, such as those for fatty acids 1 , terpenoids 2 and branched-chain amino acids 3 . 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 4 , which precludes accurate measurement of pyruvate transport activity in reconstituted liposomes. Using differential transcriptome analyses of C 3 and C 4 plants of the genera Flaveria and Cleome , here we have identified a novel gene that is abundant in C 4 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 C 4 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.

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.

Background The 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. Results We 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. Conclusions We 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.

Massively parallel sequencing of DNA by pyrosequencing technology offers much higher throughput and lower cost than conventional Sanger sequencing. Although extensively used already for sequencing of genomes, relatively few applications of massively parallel pyrosequencing to transcriptome analysis have been reported. To test the ability of this technology to provide unbiased representation of transcripts, we analyzed mRNA from Arabidopsis (Arabidopsis thaliana) seedlings. Two sequencing runs yielded 541,852 expressed sequence tags (ESTs) after quality control. Mapping of the ESTs to the Arabidopsis genome and to The Arabidopsis Information Resource 7.0 cDNA models indicated: (1) massively parallel pyrosequencing detected transcription of 17,449 gene loci providing very deep coverage of the transcriptome. Performing a second sequencing run only increased the number of genes identified by 10%, but increased the overall sequence coverage by 50%. (2) Mapping of the ESTs to their predicted full-length transcripts indicated that all regions of the transcript were well represented regardless of transcript length or expression level. Furthermore, short, medium, and long transcripts were equally represented. (3) Over 16,000 of the ESTs that mapped to the genome were not represented in the existing dbEST database. In some cases, the ESTs provide the first experimental evidence for transcripts derived from predicted genes, and, for at least 60 locations in the genome, pyrosequencing identified likely protein-coding sequences that are not now annotated as genes. Together, the results indicate massively parallel pyrosequencing provides novel information helpful to improve the annotation of the Arabidopsis genome. Furthermore, the unbiased representation of transcripts will be particularly useful for gene discovery and gene expression analysis of nonmodel plants with less complete genomic information.

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 ¹⁸O ₂-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.

The primary endosymbiotic origin of the plastid in eukaryotes more than 1 billion years ago led to the evolution of algae and plants. We analyzed draft genome and transcriptome data from the basally diverging alga Cyanophora paradoxa and provide evidence for a single origin of the primary plastid in the eukaryote supergroup Plantae. C paradoxa retains ancestral features of starch biosynthesis, fermentation, and plastid protein translocation common to plants and algae but lacks typical eukaryotic light-harvesting complex proteins. Traces of an ancient link to parasites such as Chlamydiae were found in the genomes of C paradoxa and other Plantae. Apparently, Chlamydia-like bacteria donated genes that allow export of photosynthate from the plastid and its polymerization into storage polysaccharide in the cytosol.

Compatible solutes are small molecules that are involved in acclimation to various abiotic stresses, especially high salinity. Among the red algae, the main photosynthetic products floridoside and isofloridoside (galactosylglycerols) are known also to contribute to the osmotic acclimation of cells. However, the genes encoding (iso)floridoside biosynthetic enzymes are still unknown. To identify candidate genes, we examined the genome of the floridoside‐ and isofloridoside‐accumulating extremophilic red alga Galdieria sulphuraria belonging to the Cyanidiales. We hypothesized that two candidate genes, Gasu_10960 and Gasu_26940, code for enzymes involved in floridoside and isofloridoside biosynthesis. These proteins comprise a sugar phosphate synthase and a sugar phosphate phosphatase domain. To verify their biochemical activity, both genes were in vitro translated into the entire proteins. The protein translation mixture containing Gasu_10960 synthesized small amounts of isofloridoside, whereas the Gasu_26940 translation mix also produced small amounts of floridoside. Moreover, the expression of Gasu_10960 in a salt‐sensitive mutant of the cyanobacterium Synechocystis sp. PCC 6803 resulted in increased salt tolerance as a consequence of the presence of isofloridoside in the complemented cells. Thus, our experiments suggest that the Gasu_26940 and Gasu_10960 genes of G. sulphuraria encode the enzymatically active floridoside and isofloridoside phosphate synthase/phosphatase fusion proteins, respectively, crucial for salt acclimation.

The establishment of kleptoplasty (retention of \"stolen plastids\") in the digestive tissue of the sacoglossan Elysia chlorotica Gould was investigated using transmission electron microscopy. Cellular processes occurring during the initial exposure to plastids were observed in laboratory raised animals ranging from 1-14 days post metamorphosis (dpm). These observations revealed an abundance of lipid droplets (LDs) correlating to plastid abundance. Starvation of animals resulted in LD and plastid decay in animals <5 dpm that had not yet achieved permanent kleptoplasty. Animals allowed to feed on algal prey (Vaucheria litorea C. Agardh) for 7 d or greater retained stable plastids resistant to cellular breakdown. Lipid analysis of algal and animal samples supports that these accumulating LDs may be of plastid origin, as the often algal-derived 20∶5 eicosapentaenoic acid was found in high abundance in the animal tissue. Subsequent culturing of animals in dark conditions revealed a reduced ability to establish permanent kleptoplasty in the absence of photosynthetic processes, coupled with increased mortality. Together, these data support an important role of photosynthetic lipid production in establishing and stabilizing this unique animal kleptoplasty.

Photorespiration is frequently considered a wasteful and inefficient process. However, mutant analysis demonstrated that photorespiration is essential for recycling of 2-phosphoglycolate in C₃ and C₄ land plants, in algae, and even in cyanobacteria operating carboxysome-based carbon (C) concentrating mechanisms. Photorespiration links photosynthetic C assimilation with other metabolic processes, such as nitrogen and sulfur assimilation, as well as C₁ metabolism, and it may contribute to balancing the redox poise between chloroplasts, peroxisomes, mitochondria and cytoplasm. The high degree of metabolic interdependencies and the pleiotropic phenotypes of photorespiratory mutants impedes the distinction between core and accessory functions. Newly developed synthetic bypasses of photorespiration, beyond holding potential for significant yield increases in C₃ crops, will enable us to differentiate between essential and accessory functions of photorespiration.

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. Environmental pressures sometimes cause different organisms to independently evolve the same traits. A dramatic example of this phenomenon, which is called convergent evolution, can be seen in the modes used by plants to convert carbon dioxide from the air into starch during photosynthesis. Early plants existed in an environment with high levels of carbon dioxide in the air. Over time, carbon dioxide levels decreased, so plants evolved more efficient types of photosynthesis to cope. A very efficient type of photosynthesis, called C4 photosynthesis essentially represents a carbon dioxide concentration mechanism. It has evolved at least 62 times independently in 19 different families of flowering plants. Scientists have shown that a less advanced, low-efficiency version of photosynthetic carbon dioxide concentration, called C2 photosynthesis, is a stepping-stone to C4 photosynthesis. It is also known that the evolution of C4 photosynthesis required changes to the expression patterns of thousands of genes, but the exact mechanism that leads from C2 photosynthesis to C4 photosynthesis is not clear. To explore this in greater detail, Mallmann, Heckmann et al. studied plants from the genus Flaveria, which belongs to the same family as sunflowers and asters. Under identical greenhouse conditions, plants that use three different photosynthetic pathways—C3 photosynthesis, C4 photosynthesis, or an intermediate between the two—were grown and their gene expression patterns were compared. Computer simulations were used to model the metabolism of plants that relied on C2 photosynthesis. Based on the modeling, it appears that C2 photosynthesis shifts the balance of nitrogen metabolism between two types of cell that are critical to photosynthesis. To rebalance the nitrogen, several genes are expressed to trigger an ammonia recycling mechanism. The same genes are turned on during C4 photosynthesis, and this recycling mechanism include parts of the C4 process. The findings of Mallmann, Heckmann et al. suggest that the initial steps in C4 photosynthesis evolved to prevent nitrogen imbalance. Over time, this mechanism was co-opted to become part of a more efficient form of photosynthesis, which may explain why so many different plants evolved from C2 to C4 photosynthesis.