ABSTRACT: Phosphorus (P) is an essential nutrient that is limiting in many environments. When P is scarce organisms employ strategies for conservation of internal stores, and to efficiently scavenge P from their external surroundings. In this study we investigated the acclimation response of Chlamydomonas reinhardtii to P deficiency, comparing the transcriptional profiles of P starved wild-type cells to the P replete condition. RNA was prepared from P-containing or P-deprived logarithmic growth phase cells and subjected to RNA-Seq analysis. During the 24 hours after the imposition of P starvation we observed that from the 407 significantly changing genes (> 2 fold change, corrected p-value < 0.05) in the wild-type 317 genes were up-regulated, in average 8.36-fold, and 90 genes were down-regulated by 3.43-fold, in average. Many of the upregulated genes encoded enzymes involved in specific responses to P starvation, including PHOX, encoding the major secreted alkaline phosphatase, and multiple putative, high-efficiency phosphate transporter genes. More general responses included the up-regulation of genes involved in photoprotective processes (LHCSR3, LHCSR1, LHCBM9, PTOX1) and genes involved in protein modification and degradation. Down-regulated mRNAs indicated an early stage of the reduction of chloroplast ribosomal proteins, which are considered to be a reservoir for P in the cell. Chlamydomonas reinhardtii strain 21 gr (CC1690, wild-type) grown in TAP medium (Harris 1989) in a rotary incubator (200 rpm) at 25 °C in continuous light (70 µmol m-2 s-1). For 24 hours, either 1.1 mM phosphate or 0 mM were provided with the growth media. P deprivation was achieved by washing cells twice in midlogarithmic growth phase with liquid TAP medium without P (TAP-P) and cells were resuspended at a density of 2.5 mg/ml Chlorophyll in TAP or TAP-P. Cell aliquots were collected for mRNA isolation 24 h after being transferred either to TAP or TAP-P medium.
Project description:C. reinhardtii cells exposed to abiotic stresses (e.g. iron-, nitrogen-, zinc- or phosphorus-deficiency) accumulate TAGs which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipids bodies and accumulation of TAGs. This occurs between 12 and 24 h of iron-starvation. C. reinhardtii cells deprived for iron have more saturated FA, due to the loss of functional FA desaturases, which are diiron enzymes. The abundance of a plastid ACP-acyl desaturase (FAB2) is significantly decreased to the same degree as observed for ferredoxin, which is a substrate of the desaturases. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in saturated FA (C16:4, C18:3 or C18:4). This pattern was observed for MGDG, DGTS or DGDG. When we monitored the absolute levels of glycerolipids, MGDG content dropped significantly after only 2 h of iron-starvation. On the other hand, DGTS and DGDG contents gradually decrease until a minimum is reached after 24-48 h of iron-deprivation. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed significant changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (DGAT or MLDP) are increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that a major remodeling of lipid membranes occurs during iron-starvation in C. reinhardtii. Sampling of Chlamydomonas CC-4532 (2137) cells cultivated photoheterotrophically (TAP) under iron-starvation condition (0 uM Fe-EDTA). Samples were collected from biological duplicates after washing in TAP medium lacking Fe at 0, 0.5, 1, 2, 4, 8, 12, 24 and 48 hours.
Project description:The absence of oxygen (O2) is a stress condition for aerobic organisms and requires extensive acclimation responses. Previously, Chlamydomonas reinhardtii has been used as a reference organism for understanding these acclimation responses. In this work, we use RNA-Seq for a whole genome view of the acclimation of the organism to dark-anoxic conditions. To distinguish the responses dependent on the COPPER RESPONSE REGULATOR 1 (CRR1), which is also involved in hypoxic gene regulation, we compared the transcriptome of crr1 mutants to that of complemented strains. Nearly 10% of the genome (~ 1,400 genes) are affected by hypoxia based on pairwise comparisons of all strains and two time-points. Comparing transcript profiles from early (hypoxic) with those from late (anoxic) time-points indicated that the cells activated oxidative energy generation pathways before employing fermentative enzymes. Probable substrates included not only carbohydrates but also amino acids and fatty acids (FAs). Lipid profiling of the C. reinhardtii cells revealed that they degraded FAs but also accumulated triacylglycerols (TAGs). In contrast to N-deprived cells, the TAGs accumulating in hypoxic cells are enriched in desaturated FAs, which distinguishes the contribution of individual pathways for Chlamydomonas TAG accumulation. In crr1 mutants, about 140 genes were aberrantly regulated , re-affirming the importance of CRR1 for the hypoxic response, but indicating also the contribution of additional O2-sensors and signaling strategies to account for the remaining differentially regulated transcripts. We conclude that nitric oxide (NO) dependent signaling cascades, employing both known and novel components, are operative in C. reinhardtii. The transcriptome of four different Chlamydomonas strains (wild type CC-124, crr1 mutant, crr1:CRR1 rescued strain and crr1dCys rescued strain) are profiled by RNA-Seq in the dark at different times after the transition from light-oxic to dark-anoxic conditions
Project description:Three acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. Each gene also shows increased mRNA abundance in other TAG-accumulating conditions (-S, -P, -Zn, -Fe). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared to the parent strain, D66+, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analysis suggest than an SBP domain transcription factor protein, whose mRNA increases precede that of other genes like DGAT1, is a candidate regulator of the N-deficiency response. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared to the parental strain in N-starvation conditions and is unaffected by other nutrient stresses, suggesting the operation of multiple signaling pathways leading to stress-induced TAG accumulation. Sampling of Chlamydomonas 2137 cultivated in TAP, N-Free TAP or TAP with varying amounts of N.
Project description:Zinc is an essential nutrient because of its role in catalysis and in stabilizing protein structure, but excess zinc can also be deleterious. Four nutritional zinc states have been identified in the alga Chlamydomonas reinhardtii: zinc toxic, zinc replete, zinc deficient and zinc limited. Growth is inhibited in zinc-limited and zinc toxic cells relative to zinc-replete cells, while zinc-deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition responsive changes in gene expression. We identified a subset of genes encoding zinc-handling components, including ZIP family transporters and candidate zinc chaperones. In addition, we noted an impact on two other regulatory pathways, the carbon concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc-deficiency, as a likely consequence of reduced carbonic anhydrase activity, which is validated by mass spectrometry and immunoblot analysis of Cah1, Cah3 and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in air in zinc limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. Surprisingly, we noted also that the Crr1 regulon, which responds to Cu limitation, is also turned on in zinc deficiency, and in fact, Crr1 is required for growth in zinc-limiting conditions. Zinc deficient cells are functionally copper deficient, as evidenced by reduced plastocyanin abundance, even though they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester Cu in a bio-unavailable form, perhaps to prevent mis-metallation of critical zinc sites. Zn-limited wild-type cells were generated by transfer of cells from the first round of growth in medium with no supplemental Zn into TAP medium supplemented or not with 2.5 µM Zn-EDTA The control samples for this study are represented in GSE25622
Project description:The l-galactose (Smirnoff-Wheeler) pathway represents the major route to l-ascorbic acid (vitamin C) biosynthesis in plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-l-galactose phosphorylases converting GDP-l-galactose to l-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of l-ascorbate. Here we report that the l-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the Smirnoff-Wheeler pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-l-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and l-ascorbate levels. We have also shown that enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, Mn superoxide dismutase, dehydroascorbate reductase) are up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a key enzyme in ascorbate biosynthesis in green algae and together with components of the ascorbate recycling system represents the major route in providing protective levels of ascorbate in oxidatively stressed algal cells. Our results suggest that C. reinhardtii cells exposed to oxidative stress conditions produce more ascorbate both by de novo synthesis (Smirnoff-Wheeler pathway) and by recycling via the ascorbate-glutathione cycle. Sampling of Chlamydomonas 2137 exposed to hydrogen peroxide
Project description:We used RNA-Seq to compare the transcriptomes of Fe-replete vs. Fe-deficient vs. Fe-limited Chlamydomonas wild-type cells. Our RNA-Seq data revealed 90 and 49 genes to be specifically expressed under hetero-phototrophic and phototrophic conditions, respectively. Around 30 genes represent putative Fe-deficiency targets, independent of the carbon source used. Many of these Fe-specific responses are conserved between Chlamydomonas and land plants. We identified several transporters (NRAMP4, a CCC1-like proteins and a ferroportin homologue) all of them most likely being involved in intracellular Fe redistribution. RNA-seq of Chlamydomonas Fe-deficient and limited cells indicated that about 40% of differentially expressed genes represent proteins of unknown functions. Whereas Fe-deficiency gave us insides into putative Fe-specific responses, Fe-limitation revealed responses related to increased oxidative stress. Quantitative proteomics on the soluble Chlamydomonas extracts indicated a fair correlation between changes we detected at mRNA levels compared to changes in protein levels in Fe-deficient and Fe-limited Chlamydomonas. We found that Fe-deficient and Fe-limited cells have increased ascorbate levels, a major antioxidant molecule in plants. Ascorbate levels appear to be elevated by de novo synthesis via the L-Galactose pathway and recycling by monodehydroascorbate reductase. Fe-limited cells showed increased transcript and protein levels of enzymatic antioxidant components of the ascorbate-glutathione scavenging system (MSD3, MDAR1 or GSH1). Fe-limited cells showed the increase of several proteases indicative of elevated proteolitic activity under these severe nutrient limitation conditions. Sampling of Chlamydomonas CC-1021 (2137) cells cultivated photoheterotrophically (TAP) or phototrophically (minimal) under Fe-replete (20mM), Fe-deficient (1 mM) and Fe-limited (0.25 mM) conditions.
Project description:When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen-starved in acetate and then "boosted" after two days with additional acetate, the cells become "obese" after eight days, with triacylglyceride-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, sta6 and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the two days prior and two days post boost, and the data were compared with published reports using other strains and growth conditions. During the two hours post boost, ~425 genes are up-regulated ≥2-fold and ~875 genes are down-regulated ≥2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes in the glyoxylate and Calvin Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boost. Four genes--encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)--are selectively up-regulated in sta6. Although the bulk rate of acetate depletion from the medium is not boost-enhanced, three candidate acetate permease-encoding genes in the GPR1_FUN34_YaaH superfamily are boost-up-regulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is down-regulated by boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in sta6. Here we report studies on gene-expression patterns during the path to obesity. The Merchant/Pellegrini and Los Alamos laboratories recently generated and analyzed RNA-Seq transcriptomes of cw15, sta6, and several complemented sta6 strains during two days of N-starvation (0→48 h). In collaboration with these groups, the Goodenough lab generated a second pair of transcriptomes using cw15 and sta6, tracing 0→48 h gene expression patterns under a different set of culture conditions and taking the time course out to 96 h, with an intervening acetate boost. Analysis of these data was deeply informed by cross-comparisons with the Blaby et al. data. Three additional RNA-Seq studies of wild-type strains were also considered.
Project description:Here we show that the phytochrome-less chlorophyte Chlamydomonas reinhardtii retains a functional pathway to synthesize the linear tetrapyrrole (bilin) precursor of the phytochrome chromophore. Reverse genetic, metabolic inactivation and bilin rescue experiments establish that this pathway is needed for heme iron acquisition and for the diurnal transition to phototrophic growth. RNA-Seq measurements reveal a bilin-dependent signaling network that is necessary for the heterotrophic to phototrophic transition. These results imply the presence of a novel bilin sensor pathway that may be widely distributed amongst oxygenic photosynthetic organisms. We isolated RNA from heterotrophic suspension cultures of 4A+ WT and the hmox1 mutant grown in the presence or absence of 0.1 mM BV IXα before and after transfer to low light.
Project description:To understand the molecular basis underlying increased triacylglycerol accumulation in starchless Chlamydomonas mutants, we undertook comparative time course transcriptomics of sta6 (CC-4348), a cw strain purported to represent the parental STA6 strain (CC-4349), and 3 independent STA6 strains (STA6-C2, STA6-C4 and STA6-C6, generated by complementation of sta6, CC-4565, –4566 and –4567) in the context of nitrogen deprivation. Despite the N starvation induced dramatic remodeling of the transcriptome, there are relatively few differences (5 x 102) between sta6 and STA6, the most dramatic of which are in the abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically ICL1, MAS1, TAL1, FBP1 and PCK1, suggestive of increased carbon movement towards hexosephosphate in sta6 by up-regulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicates increased succinate and malate, increased Glc-6-P and decreased fructose-1,6-bisphosphate, which documents the impact of these changes. Comparisons of the independent datasets in multiple strains allowed the delineation of a sequence of events in a global N-starvation response in Chlamydomonas, starting within minutes with up-regulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual up-regulation of genes encoding enzymes of TAG synthesis. Finally, genome re-sequencing analysis indicated that i) the deletion in sta6 extends into the neighboring gene, RBO1, and ii) a commonly-used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype. The transcriptomes of five different Chlamydomonas strains (wild type CC-4349, sta6 mutant (CC-4348),and three indepdent STA6 strains (STA6-C2, STA6-C4 and STA6-C6) are profiled by RNA-Seq in timecourse experiments after the transition to minus nitrogen
Project description:We analysed global gene expression changes in Chlamydomonas reinhardtii in response to 1h UV-B, applied at the same low level that was seen to promote subsequent UV-B stress tolerance, in order to elucidate the transcriptional reprogramming that leads to UV-B acclimation. mRNA profiles generated by deep sequencing from triplicate replicate Chlamydomonas reinhardtii samples sourced from independent cultures either protected from UV-B or exposed to 1h acclimation-level UV-B.