Unknown,Transcriptomics,Genomics,Proteomics

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Zinc deficiency impacts CO2 assimilation and disrupts copper homeostasis in Chlamydomonas reinhardtii

ABSTRACT: 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 ÂµM Zn-EDTA The control samples for this study are represented in GSE25622

ORGANISM(S): Chlamydomonas reinhardtii

SUBMITTER: Sabeeha Merchant

PROVIDER: E-GEOD-41096 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Zinc deficiency impacts CO2 assimilation and disrupts copper homeostasis in Chlamydomonas reinhardtii.

Malasarn Davin D Kropat Janette J Hsieh Scott I SI Finazzi Giovanni G Casero David D Loo Joseph A JA Pellegrini Matteo M Wollman Francis-André FA Merchant Sabeeha S SS

The Journal of biological chemistry 20130225 15

Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient, and limited. Growth is inhibited in zinc-limited and zinc-toxic cells relative to zinc-replete cells, whereas zinc deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zi ...[more]

PMID: 23439652

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Project description:Interest in exploiting algae as a biofuel source and the role of nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can be easily manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements on the ionomes of cells would result in optimized cell growth. We characterized the ionomes of multiple wild-type Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel our measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between laboratories using the same supplement. Visual observation did not reveal defects in cell motility or mating efficiency. Ni2+-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased amount of Ni2+ supplementation due to the introduction of an EDTA buffer system in the revised medium. Equilibrium modeling of the revised supplement predicts less metal precipitation in the revised medium. Other advantages include more facile preparation of trace element stock solutions that can readily be adapted for deficiency studies, a reduction in total chemical use, a more consistent batch-to-batch formulation, and long-term stability (up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient-deficiencies. In N and S deficiency, cells accumulate TAG as well in the new medium as previously demonstrated. Fe and Zn deficiency also induced TAG accumulation as suggested by Nile Red and Bodipy staining. This ionomic approach can be used to efficiently optimize culturing conditions for other algal species to improve growth and assay cell physiology. Sampling of Chlamydomonas CC-1021 (2137) cultivated in TAP medium supplemented with a revised trace element recipe based on ionomic data.

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

Dataset Information

Zinc deficiency impacts CO2 assimilation and disrupts copper homeostasis in Chlamydomonas reinhardtii

Publications

Zinc deficiency impacts CO2 assimilation and disrupts copper homeostasis in Chlamydomonas reinhardtii.

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