Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Systems and trans-system level analysis identifies conserved Fe-deficiency responses in the plant lineage

ABSTRACT: 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.

ORGANISM(S): Chlamydomonas reinhardtii

SUBMITTER: Sabeeha Merchant

PROVIDER: E-GEOD-35305 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas.

Boyle Nanette R NR Page Mark Dudley MD Liu Bensheng B Blaby Ian K IK Casero David D Kropat Janette J Cokus Shawn J SJ Hong-Hermesdorf Anne A Shaw Johnathan J Karpowicz Steven J SJ Gallaher Sean D SD Johnson Shannon S Benning Christoph C Pellegrini Matteo M Grossman Arthur A Merchant Sabeeha S SS

The Journal of biological chemistry 20120308 19

Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumul ...[more]

PMID: 22403401

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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 M-bM-^IM-%2-fold and ~875 genes are down-regulated M-bM-^IM-%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 (0M-bM-^FM-^R48 h). In collaboration with these groups, the Goodenough lab generated a second pair of transcriptomes using cw15 and sta6, tracing 0M-bM-^FM-^R48 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: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

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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, M-bM-^@M-^S4566 and M-bM-^@M-^S4567) 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

Dataset Information

Systems and trans-system level analysis identifies conserved Fe-deficiency responses in the plant lineage

Publications

Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas.

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