Characterisation of lepidopteran geranylgeranyl diphosphate synthase as a putative pesticide target.

Geranylgeranyl pyrophosphate (diphosphate) synthase (GGPPS) plays an important role in various physiological processes in insects, such as isoprenoid biosynthesis and protein prenylation. Here, we functionally characterised the GGPPS from the major agricultural lepidopteran pests Spodoptera frugiperda and Helicoverpa armigera. Partial disruption of GGPPS by CRISPR in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential. Functional expression in vitro of Helicoverpa armigera GGPPS in Escherichia coli revealed a catalytically active enzyme. Next, we developed and optimised an enzyme assay to screen for potential inhibitors, such as the zoledronate and the minodronate, which showed a dose-dependent inhibition. Phylogenetic analysis of GGPPS across insects showed that GGPPS is highly conserved but also revealed several residues likely to be involved in substrate binding, which were substantially different in bee pollinator and human GGPPS. Considering the essentiality of GGPPS and its putative binding residue variability qualifies a GGPPS as a novel pesticide target. The developed assay may contribute to the identification of novel insecticide leads.

The Identification and Evolutionary Trends of the Solute Carrier Superfamily in Arthropods.

The solute carrier (SLC) transporter superfamily comprises an ancient and ubiquitous group of proteins capable of translocating a range of nutrients, endogenous molecules, and xenobiotics. Although the group has been the subject of intense investigation in both bacteria and mammals, its systematic identification in arthropods has not yet been undertaken. Here, we present a genome-wide identification of all 66 human SLC families in 174 arthropod species. A pipeline (SLC_id) was constructed to identify and group SLCs using a combination of hidden Markov model and BLAST searches followed by filtering based on polypeptide length and the number of transmembrane domains. Comparative analysis of the number of transporters in each family across diverse arthropod lineages was accomplished using one-way analysis of variance (ANOVA) and the Computational Analysis of gene Family Evolution (CAFE). These results suggested that many SLC families have undergone expansions or contractions in particular evolutionary lineages. Notably, the sugar transporting SLC2 family was significantly larger in insects compared with arachnids. This difference may have been complemented by a rapid expansion of the SLC60 family in arachnids which also acts on dietary sugars. Furthermore, the SLC33 family underwent a recent and drastic expansion in aphids, although the biological relevance of this expansion was not possible to infer. Information on specific SLC transporter families across arthropod species can be accessed through an R shiny web application at http://chrysalida.imbb.forth.gr : 3838/Arthropod_SLC_Database/. The present study greatly facilitates further investigation of the diverse group of SLC transporters in arthropods.

Arabidopsis A BOUT DE SOUFFLE is a putative mitochondrial transporter involved in photorespiratory metabolism and is required for meristem growth at ambient CO2 levels.

Photorespiratory metabolism is essential in all oxygenic photosynthetic organisms. In plants, it is a highly compartmentalized pathway that involves chloroplasts, peroxisomes, mitochondria and the cytoplasm. The metabolic pathway itself is well characterized, and the enzymes required for its function have been identified. However, very little information is available on the transport proteins that catalyze the high metabolic flux between the involved compartments. Here we show that the A BOUT DE SOUFFLE (BOU) gene, which encodes a mitochondrial carrier, is involved in photorespiration in Arabidopsis. BOU was found to be co-expressed with photorespiratory genes in leaf tissues. The knockout mutant bou-2 showed the hallmarks of a photorespiratory growth phenotype, an elevated CO(2) compensation point, and excessive accumulation of glycine. Furthermore, degradation of the P-protein, a subunit of glycine decarboxylase, was demonstrated for bou-2, and is reflected in strongly reduced glycine decarboxylase activity. The photorespiration defect in bou-2 has dramatic consequences early in the seedling stage, which are highlighted by transcriptome studies. In bou-2 seedlings, as in shm1, another photorespiratory mutant, the shoot apical meristem organization is severely compromised. Cell divisions are arrested, leading to growth arrest at ambient CO(2) . Although the specific substrate for the BOU transporter protein remains elusive, we show that it is essential for the function of the photorespiratory metabolism. We hypothesize that BOU function is linked with glycine decarboxylase activity, and is required for normal apical meristems functioning in seedlings.

Phylogenetic and biochemical evidence supports the recruitment of an ADP-glucose translocator for the export of photosynthate during plastid endosymbiosis.

The acquisition of photosynthesis by eukaryotic cells through enslavement of a cyanobacterium represents one of the most remarkable turning points in the history of life on Earth. In addition to endosymbiotic gene transfer, the acquisition of a protein import apparatus and the coordination of gene expression between host and endosymbiont genomes, the establishment of a metabolic connection was crucial for a functional endosymbiosis. It was previously hypothesized that the first metabolic connection between both partners of endosymbiosis was achieved through insertion of a host-derived metabolite transporter into the cyanobacterial plasma membrane. Reconstruction of starch metabolism in the common ancestor of photosynthetic eukaryotes suggested that adenosine diphosphoglucose (ADP-Glc), a bacterial-specific metabolite, was likely to be the photosynthate, which was exported from the early cyanobiont. However, extant plastid transporters that have evolved from host-derived endomembrane transporters do not transport ADP-Glc but simple phosphorylated sugars in exchange for orthophosphate. We now show that those eukaryotic nucleotide sugar transporters, which define the closest relatives to the common ancestor of extant plastid envelope carbon translocators, possess an innate ability for transporting ADP-Glc. Such an unexpected ability would have been required to establish plastid endosymbiosis.

The carbon and energy sources of the non-photosynthetic plastid in the malaria parasite.

The malaria parasite harbours an indispensable plastid known as the 'apicoplast'. The apicoplast's exact role remains uncertain, but it houses components involved in fatty acid, isoprenoid and haem biosyntheses. These pathways offer opportunities to develop anti-malarials. In the absence of photosynthesis, how apicoplast anabolism is fuelled is unclear. Here we investigated plant-like transporters of the apicoplast and measured their substrate preferences using a novel cell-free assay system to explore the carbon and energy sources of the apicoplast. The transporters exchange triose phosphate and phosphoenolpyruvate for inorganic phosphate, demonstrating that the apicoplast taps into host-derived glucose to fuel its metabolism.

Peroxisomal ATP import is essential for seedling development in Arabidopsis thaliana.

Several recent proteomic studies of plant peroxisomes indicate that the peroxisomal matrix harbors multiple ATP-dependent enzymes and chaperones. However, it is unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether external ATP fuels the energy-dependent reactions within peroxisomes. The existence of transport proteins that supply plant peroxisomes with energy for fatty acid oxidation and other ATP-dependent processes has not previously been demonstrated. Here, we describe two Arabidopsis thaliana genes that encode peroxisomal adenine nucleotide carriers, PNC1 and PNC2. Both proteins, when fused to enhanced yellow fluorescent protein, are targeted to peroxisomes. Complementation of a yeast mutant deficient in peroxisomal ATP import and in vitro transport assays using recombinant transporter proteins revealed that PNC1 and PNC2 catalyze the counterexchange of ATP with ADP or AMP. Transgenic Arabidopsis lines repressing both PNC genes were generated using ethanol-inducible RNA interference. A detailed analysis of these plants showed that an impaired peroxisomal ATP import inhibits fatty acid breakdown during early seedling growth and other beta-oxidation reactions, such as auxin biosynthesis. We show conclusively that PNC1 and PNC2 are essential for supplying peroxisomes with ATP, indicating that no other ATP generating systems exist inside plant peroxisomes.

Functional characterization of the plastidic phosphate translocator gene family from the thermo-acidophilic red alga Galdieria sulphuraria reveals specific adaptations of primary carbon partitioning in green plants and red algae.

In chloroplasts of green plants and algae, CO(2) is assimilated into triose-phosphates (TPs); a large part of these TPs is exported to the cytosol by a TP/phosphate translocator (TPT), whereas some is stored in the plastid as starch. Plastidial phosphate translocators have evolved from transport proteins of the host endomembrane system shortly after the origin of chloroplasts by endosymbiosis. The red microalga Galdieria sulphuraria shares three conserved putative orthologous transport proteins with the distantly related seed plants and green algae. However, red algae, in contrast to green plants, store starch in their cytosol, not inside plastids. Hence, due to the lack of a plastidic starch pool, a larger share of recently assimilated CO(2) needs to be exported to the cytosol. We thus hypothesized that red algal transporters have distinct substrate specificity in comparison to their green orthologs. This hypothesis was tested by expression of the red algal genes in yeast (Saccharomyces cerevisiae) and assessment of their substrate specificities and kinetic constants. Indeed, two of the three red algal phosphate translocator candidate orthologs have clearly distinct substrate specificities when compared to their green homologs. GsTPT (for G. sulphuraria TPT) displays very narrow substrate specificity and high affinity; in contrast to green plant TPTs, 3-phosphoglyceric acid is poorly transported and thus not able to serve as a TP/3-phosphoglyceric acid redox shuttle in vivo. Apparently, the specific features of red algal primary carbon metabolism promoted the evolution of a highly efficient export system with high affinities for its substrates. The low-affinity TPT of plants maintains TP levels sufficient for starch biosynthesis inside of chloroplasts, whereas the red algal TPT is optimized for efficient export of TP from the chloroplast.

Host origin of plastid solute transporters in the first photosynthetic eukaryotes.

BACKGROUND: It is generally accepted that a single primary endosymbiosis in the Plantae (red, green (including land plants), and glaucophyte algae) common ancestor gave rise to the ancestral photosynthetic organelle (plastid). Plastid establishment necessitated many steps, including the transfer and activation of endosymbiont genes that were relocated to the nuclear genome of the 'host' followed by import of the encoded proteins into the organelle. These innovations are, however, highly complex and could not have driven the initial formation of the endosymbiosis. We postulate that the re-targeting of existing host solute transporters to the plastid fore-runner was critical for the early success of the primary endosymbiosis, allowing the host to harvest endosymbiont primary production. RESULTS: We tested this model of transporter evolution by conducting a comprehensive analysis of the plastid permeome in Arabidopsis thaliana. Of 137 well-annotated transporter proteins that were initially considered, 83 that are broadly distributed in Plantae were submitted to phylogenetic analysis. Consistent with our hypothesis, we find that 58% of Arabidopsis transporters, including all carbohydrate transporters, are of host origin, whereas only 12% arose from the cyanobacterial endosymbiont. Four transporter genes are derived from a Chlamydia-like source, suggesting that establishment of the primary plastid likely involved contributions from at least two prokaryotic sources. CONCLUSION: Our results indicate that the existing plastid solute transport system shared by Plantae is derived primarily from host genes. Important contributions also came from the cyanobacterial endosymbiont and Chlamydia-like bacteria likely co-resident in the first algae.

Single, ancient origin of a plastid metabolite translocator family in Plantae from an endomembrane-derived ancestor.

Phylogenetic analyses show the single origin of a plastid metabolite translocator family in the Plantae from a gene encoding an existing endomembrane-derived protein. Red algal secondary endosymbiosis has spread a translocator gene into the ancestor of the "chromalveolate" protists, where it has diversified into a novel clade of proteins.

Shuffling ammonia between mitochondria and plastids during photorespiration.

Surprisingly, glutamine synthetase was recently shown to be dual targeted to chloroplasts and mitochondria in Arabidopsis leaves. It is likely that mitochondrial glutamine synthetase assimilates ammonia, which is generated in large amounts in mitochondria during photorespiration. However, ammonia assimilation is a two-step process and the second step, catalyzed by glutamate synthase, is exclusively located in plastids. Hence, a shuttle for ammonia, possibly in the form of amino acids, is required between mitochondria and plastids. We discuss two alternative shuttles, an ornithine-citrulline shuttle and a glutamine-glutamate shuttle. Both shuttles allow the safe transport of the toxic metabolite ammonium in the form of amino acids. The ornithine-citrulline shuttle also provides a means for the transport of carbon dioxide from mitochondria to plastids, but this shuttle requires more energy than the alternative glutamate-glutamine shuttle.