Systematic characterization of gene function in the photosynthetic alga Chlamydomonas reinhardtii.

Most genes in photosynthetic organisms remain functionally uncharacterized. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. A total of 59% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles place uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism and ciliogenesis. We illustrate the value of this resource by validating phenotypes and gene functions, including three new components of an actin cytoskeleton defense pathway. The data also inform phenotype discovery in land plants; mutants in Arabidopsis thaliana genes exhibit phenotypes similar to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

The mitochondrial alternative oxidase from Chlamydomonas reinhardtii enables survival in high light.

Photosynthetic organisms often experience extreme light conditions that can cause hyper-reduction of the chloroplast electron transport chain, resulting in oxidative damage. Accumulating evidence suggests that mitochondrial respiration and chloroplast photosynthesis are coupled when cells are absorbing high levels of excitation energy. This coupling helps protect the cells from hyper-reduction of photosynthetic electron carriers and diminishes the production of reactive oxygen species (ROS). To examine this cooperative protection, here we characterized Chlamydomonas reinhardtii mutants lacking the mitochondrial alternative terminal respiratory oxidases, CrAOX1 and CrAOX2. Using fluorescent fusion proteins, we experimentally demonstrated that both enzymes localize to mitochondria. We also observed that the mutant strains were more sensitive than WT cells to high light under mixotrophic and photoautotrophic conditions, with the aox1 strain being more sensitive than aox2 Additionally, the lack of CrAOX1 increased ROS accumulation, especially in very high light, and damaged the photosynthetic machinery, ultimately resulting in cell death. These findings indicate that the Chlamydomonas AOX proteins can participate in acclimation of C. reinhardtii cells to excess absorbed light energy. They suggest that when photosynthetic electron carriers are highly reduced, a chloroplast-mitochondria coupling allows safe dissipation of photosynthetically derived electrons via the reduction of O2 through AOX (especially AOX1)-dependent mitochondrial respiration.

Development of a nuclear transformation system for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and genetic complementation of a mutant strain, deficient in arachidonic acid biosynthesis.

Microalgae are considered a promising source for various high value products, such as carotenoids, ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The unicellular green alga Lobosphaera (Parietochloris) incisa is an outstanding candidate for the efficient phototrophic production of arachidonic acid (AA), an essential ω-6 PUFA for infant brain development and a widely used ingredient in the baby formula industry. Although phototrophic production of such algal products has not yet been established, estimated costs are considered to be 2-5 times higher than competing heterotrophic production costs. This alga accumulates unprecedented amounts of AA within triacylglycerols and the molecular pathway of AA biosynthesis in L. incisa has been previously elucidated. Thus, progress in transformation and metabolic engineering of this high value alga could be exploited for increasing the efficient production of AA at competitive prices. We describe here the first successful transformation of L. incisa using the ble gene as a selection marker, under the control of the endogenous RBCS promoter. Furthermore, we have succeeded in the functional complementation of the L. incisa mutant strain P127, containing a mutated, inactive version of the delta-5 (Δ5) fatty acid desaturase gene. A copy of the functional Δ5 desaturase gene, linked to the ble selection marker, was transformed into the P127 mutant. The resulting transformants selected for zeocine resistant, had AA biosynthesis partially restored, indicating the functional complementation of the mutant strain with the wild-type gene. The results of this study present a platform for the successful genetic engineering of L. incisa and its long-chain PUFA metabolism.

The Arabidopsis STRESS RESPONSE SUPPRESSOR DEAD-box RNA helicases are nucleolar- and chromocenter-localized proteins that undergo stress-mediated relocalization and are involved in epigenetic gene silencing.

DEAD-box RNA helicases are involved in many aspects of RNA metabolism and in diverse biological processes in plants. Arabidopsis thaliana mutants of two DEAD-box RNA helicases, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2 were previously shown to exhibit tolerance to abiotic stresses and up-regulated stress-responsive gene expression. Here, we show that Arabidopsis STRS-overexpressing lines displayed a less tolerant phenotype and reduced expression of stress-induced genes confirming the STRSs as attenuators of Arabidopsis stress responses. GFP-STRS fusion proteins exhibited localization to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. The STRS proteins displayed mis-localization in specific gene-silencing mutants and exhibited RNA-dependent ATPase and RNA-unwinding activities. In particular, STRS2 showed mis-localization in three out of four mutants of the RNA-directed DNA methylation (RdDM) pathway while STRS1 was mis-localized in the hd2c mutant that is defective in histone deacetylase activity. Furthermore, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, our findings suggest that the STRS proteins are involved in epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response.

PCR amplification and DNA sequence of mcyA gene: the distribution profile of a toxigenic Microcystis aeruginosa in the Hartbeespoort Dam, South Africa.

Using new polymerase chain reaction (PCR) primers, a once known to be under-transcribed microcystin synthetase A (mcyA) gene from the only known toxigenic cyanobacterium Microcystis aeruginosa dominating the Hartbeespoort Dam was consistently amplified from genomic DNA extracted from a set of algal and cell free water samples collected across this dam. In addition to this, five more mcy genes (mcyBCDEG) were also amplified during this study. The resultant mcyA PCR products (518 bp) were purified and sequenced and gave nucleotide sequence segments of 408 bp sizes. The obtained sequence was aligned to the published mcyA gene sequence available online on the NCBI database and resulted in 100% similarity to a 408 bp mcyA gene sequence segment of M. aeruginosa UWOCC RID-1. Furthermore, it was found that the above sequence segment (408 bp) spans from a common base in M. aeruginosa PCC 7806 and M. aeruginosa PCC 7820 from 141 to 548 bp in the N-methyl transferase (NMT) region signifying their closer relatedness to M. aeruginosa UWOCC strains. This study has for the first time amplified mcyA gene consistently from both intracellular and extracellular DNA extracts obtained from algal and cell free water samples, respectively. Sequence data and the amplified mcy genes showed that M. aeruginosa is widely distributed and dominant in this dam.

Growth, lipid production and metabolic adjustments in the euryhaline eustigmatophyte Nannochloropsis oceanica CCALA 804 in response to osmotic downshift.

We investigated the effects of osmotic downshift induced by the transfer of Nannochloropsis oceanica CCALA 804 from artificial seawater medium (27 g L(-1) NaCl) to the same medium without NaCl or freshwater modified BG-11 medium (mBG-11) as a function of photosynthetically active radiation (170, 350, or 700 µmol photon m(-2) s(-1)). Alterations in growth, total fatty acid (FA) content and FA composition of individual lipid classes, and in relative contents of metabolites relevant to osmotic adjustments were studied. Cells displayed remarkable tolerance to the osmotic downshift apart from some swelling, with no substantial lag or decline in cell division rate. Biomass accumulation and chlorophyll a content were enhanced upon downshifting, especially under the highest irradiance. The highest chlorophyll a and eicosapentaenoic acid (EPA) biomass and culture contents were determined in the cultures grown in mBG-11. Two days after transfer to 0 g L(-1) NaCl, the proportion in total acyl lipids of the major chloroplast galactolipid monogalactosyldiacylglycerol, a major depot of EPA, increased twofold, along with a modest change in the proportion of digalactosyldiacylglycerol (DGDG). EPA percentage decreased in DGDG and increased in the extraplastidial lipid phosphatidylethanolamine. Metabolite profiling by GC-MS analysis revealed a sharp decrease in metabolites potentially involved in osmoregulation, such as mannitol and proline, while proline-cycle intermediates and some free sugars increased. The stress-induced polyamine spermidine decreased ca. one order of magnitude, while its catabolic product-the non-protein amino acid γ-amino butyric acid-increased twofold, as did the stress-related sugars trehalose and talose. Biochemical mechanisms governing osmotic plasticity and implications for optimization of EPA production by N. oceanica CCALA 804 under variable cultivation conditions are discussed.

Inositol polyphosphate phosphatidylinositol 5-phosphatase9 (At5ptase9) controls plant salt tolerance by regulating endocytosis.

Phosphatidylinositol 5-phosphatases (5PTases) that hydrolyze the 5' position of the inositol ring are key components of membrane trafficking system. Recently, we reported that mutation in At5PTase7 gene reduced production of reactive oxygen species (ROS) and decreased expression of stress-responsive genes, resulting in increased salt sensitivity. Here, we describe an even more salt-sensitive 5ptase mutant, At5ptase9, which also hydrolyzes the 5' phosphate groups specifically from membrane-bound phosphatidylinositides. Interestingly, the mutants were more tolerant to osmotic stress. We analyzed the main cellular processes that may be affected by the mutation, such as production of ROS, influx of calcium, and induction of salt-response genes. The At5ptase9 mutants showed reduced ROS production and Ca(2+) influx, as well as decreased fluid-phase endocytosis. Inhibition of endocytosis by phenylarsine oxide or Tyrphostin A23 in wild-type plants blocked these responses. Induction of salt-responsive genes in wild-type plants was also suppressed by the endocytosis inhibitors. Thus, inhibition of endocytosis in wild-type plants mimicked the salt stress responses, observed in the At5ptase9 mutants. In summary, our results show a key non-redundant role of At5PTase7 and 9 isozymes, and underscore the localization of membrane-bound PtdIns in regulating plant salt tolerance by coordinating the endocytosis, ROS production, Ca(2+) influx, and induction of stress-responsive genes.

Inositol polyphosphate 5-phosphatase7 regulates the production of reactive oxygen species and salt tolerance in Arabidopsis.

Plants possess remarkable ability to adapt to adverse environmental conditions. The adaptation process involves the removal of many molecules from organelles, especially membranes, and replacing them with new ones. The process is mediated by an intracellular vesicle-trafficking system regulated by phosphatidylinositol (PtdIns) kinases and phosphatases. Although PtdIns comprise a fraction of membrane lipids, they function as major regulators of stress signaling. We analyzed the role of PtdIns 5-phosphatases (5PTases) in plant salt tolerance. The Arabidopsis (Arabidopsis thaliana) genome contains 15 At5PTases. We analyzed salt sensitivity in nine At5ptase mutants and identified one (At5ptase7) that showed increased sensitivity, which was improved by overexpression. At5ptase7 mutants demonstrated reduced production of reactive oxygen species (ROS). Supplementation of mutants with exogenous PtdIns dephosphorylated at the D5' position restored ROS production, while PtdIns(4,5)P(2), PtdIns(3,5)P(2), or PtdIns(3,4,5)P(3) were ineffective. Compromised salt tolerance was also observed in mutant NADPH Oxidase, in agreement with the low ROS production and salt sensitivity of PtdIns 3-kinase mutants and with the inhibition of NADPH oxidase activity in wild-type plants. Localization of green fluorescent protein-labeled At5PTase7 occurred in the plasma membrane and nucleus, places that coincided with ROS production. Analysis of salt-responsive gene expression showed that mutants failed to induce the RD29A and RD22 genes, which contain several ROS-dependent elements in their promoters. Inhibition of ROS production by diphenylene iodonium suppressed gene induction. In summary, our results show a nonredundant function of At5PTase7 in salt stress response by regulating ROS production and gene expression.

Reduced expression of the v-SNAREs AtVAMP71/AtVAMP7C gene family in Arabidopsis reduces drought tolerance by suppression of abscisic acid-dependent stomatal closure.

Stomatal closure during water stress is a major plant mechanism for reducing the loss of water through leaves. The opening and closure of stomata are mediated by endomembrane trafficking. The role of the vacuolar trafficking pathway, that involves v-SNAREs of the AtVAMP71 family (formerly called AtVAMP7C) in stomatal movements, was analysed. Expression of AtVAMP711-14 genes was manipulated in Arabidopsis plants with sense or antisense constructs by transformation of the AtVAMP711 gene. Antisense plants exhibited decreased stomatal closure during drought or after treatment with abscisic acid (ABA), resulting in the rapid loss of leaf water and tissue collapse. No improvement was seen in plants overexpressing the AtVAMP711 gene, suggesting that wild-type levels of AtVAMP711 expression are sufficient. ABA treatment induced the production of reactive oxygen species (ROS) in guard cells of both wild-type and antisense plants, indicating that correct hormone sensing is maintained. ROS were detected in nuclei, chloroplasts, and vacuoles. ABA treatment caused a significant increase in ROS-containing small vacuoles and also in plastids and nuclei of neighbouring epidermal and mesophyll cells. Taken together, our results show that VAMP71 proteins play an important role in the localization of ROS, and in the regulation of stomatal closure by ABA treatment. The paper also describes a novel aspect of ROS signalling in plants: that of ROS production in small vacuoles that are dispersed in the cytoplasm.

NPR1 protein regulates pathogenic and symbiotic interactions between Rhizobium and legumes and non-legumes.

BACKGROUND: Legumes are unique in their ability to establish symbiotic interaction with rhizobacteria from Rhizobium genus, which provide them with available nitrogen. Nodulation factors (NFs) produced by Rhizobium initiate legume root hair deformation and curling that entrap the bacteria, and allow it to grow inside the plant. In contrast, legumes and non-legumes activate defense responses when inoculated with pathogenic bacteria. One major defense pathway is mediated by salicylic acid (SA). SA is sensed and transduced to downstream defense components by a redox-regulated protein called NPR1. METHODOLOGY/PRINCIPAL FINDINGS: We used Arabidopsis mutants in SA defense pathway to test the role of NPR1 in symbiotic interactions. Inoculation of Sinorhizobium meliloti or purified NF on Medicago truncatula or nim1/npr1 A. thaliana mutants induced root hair deformation and transcription of early and late nodulins. Application of S. meliloti or NF on M. truncatula or A. thaliana roots also induced a strong oxidative burst that lasted much longer than in plants inoculated with pathogenic or mutualistic bacteria. Transient overexpression of NPR1 in M. truncatula suppressed root hair curling, while inhibition of NPR1 expression by RNAi accelerated curling. CONCLUSIONS/SIGNIFICANCE: We show that, while NPR1 has a positive effect on pathogen resistance, it has a negative effect on symbiotic interactions, by inhibiting root hair deformation and nodulin expression. Our results also show that basic plant responses to Rhizobium inoculation are conserved in legumes and non-legumes.