UV-B-induced gene expression changes in Chlamydomonas reinhardtii
ABSTRACT: 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.
Project description:Like many microalgae unicellular green alga, Chlamydomonas reinhardtii also accumulates energy rich storage lipid and starch under nutrient-limited conditions, such as phosphorus (P) and nitrogen (N) limitation. To dissect the transcriptional regulation of lipid and starch biosynthesis in response to nutrient limitation, we have characterized the biological role of a candidate regulator: the MYB family transcription factor, PSR1 (phosphorus starvation response 1). Genetically modified (psr1 compromised) and wild-type lines were grown under P limiting and sufficient conditions and assayed at two time points by SOLiD RNA-seq.
Project description:RNA populations in Chlamydomonas reinhardtii Keywords: Highly parallel pyrosequencing Small RNAs were prepared from Chlamydomonas reinhardtii total extracts,ligated to a 3' adaptor and a 5' acceptor sequentially, and then RT-PCR amplified. PCR products were reamplified using a pair of 454 cloning primers and provided to 454 Life Sciences (Branford, CT) for sequencing. For technical details, see Tao Zhao, Guanglin Li, Shijun Mi, Shan Li, Gregory J. Hannon, Xiu-Jie Wang, and Yijun Qi. 2007. A Complex System of Small RNAs in the Unicellular Green Alga Chlamydomonas reinhardtii. Genes & Development
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: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: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: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:This SuperSeries is composed of the following subset Series: GSE20859: Change in gene expression in Chlamydomonas reinhardtii upon heat shock GSE20860: Change in gene expression in Chlamydomonas reinhardtii upon feeding with hemin and Mg-protoporphyrin Refer to individual Series
Project description: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:Zn-limited cells accumulate Cu in intracellular structures in a reversable manner. We used RNAseq analysis to monitor gene expression in Zn-limited cells, and during a time course (0 - 24 h) after Zn resupply. Comparison of Chlamydomonas reinhardtii gene expression when Zn deficient cells are resupplied with 2.5 µM Zn. Time points 0 (before resupply), 1.5, 3, 4.5, 12, and 24 hours after resupply. Four biological replicates per condition.