RNA-Seq Analysis of Sulfur-Deprived Chlamydomonas Cells Reveals Aspects of Acclimation Critical for Cell Survival

Abstract

The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells. This mutant is null for the regulatory Ser-Thr kinase SNRK2.1, which is required for acclimation of the alga to sulfur deprivation. The transcriptome analyses used microarray hybridization and RNA-seq technology. Quantitative RT-PCR evaluation of the results obtained by these techniques showed that RNA-seq reports a larger dynamic range of expression levels than do microarray hybridizations. Transcripts responsive to sulfur deprivation included those encoding proteins involved in sulfur acquisition and assimilation, synthesis of sulfur-containing metabolites, Cys degradation, and sulfur recycling. Furthermore, we noted potential modifications of cellular structures during sulfur deprivation, including the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that sulfur-deprived cells accumulate proteins with fewer sulfur-containing amino acids. Most of the sulfur deprivation responses are controlled by the SNRK2.1 protein kinase. The snrk2.1 mutant exhibits a set of unique responses during both sulfur-replete and sulfurdepleted conditions that are not observed in wild-type cells; the inability of this mutant to acclimate to S deprivation probably leads to elevated levels of singlet oxygen and severe oxidative stress, which ultimately causes cell death. The transcriptome results for wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as cells become depleted for sulfur and reveal aspects of acclimation that are likely critical for cell survival.

Changes in Transcript Abundance in Chlamydomonas reinhardtii following Nitrogen Deprivation Predict Diversion of Metabolism

“…However, focus on the latter has been largely motivated by the renewed interest in microalgae as biofuel feedstocks (Wijffels and Barbosa, 2010). As sequencing technology has become increasingly fast and affordable, comparison of transcriptomic changes under different experimental conditions by massive parallel sequencing of cDNA libraries is a viable first approach toward identifying genes that define changes in response to N deprivation or other nutrient stresses (González-Ballester et al, 2010). With the goal of gaining a better understanding of the factors underlying or even controlling the process of TAG accumulation following N deprivation, the focus has to be on metabolism and genes that encode enzymes of relevant pathways or regulatory factors.…”

“…At this time, a number of microarrays have been used to interrogate changes in response to environmental factors (Ledford et al, 2004(Ledford et al, , 2007Jamers et al, 2006;Mus et al, 2007;Nguyen et al, 2008;Simon et al, 2008;Yamano et al, 2008;Mustroph et al, 2010). These microarrays could not cover all genes in the genome, but more recently, massively parallel cDNA sequencing approaches were applied to C. reinhardtii, overcoming the shortcomings of microarrays (González-Ballester et al, 2010). Likewise, we have chosen a cDNA sequencing-based approach using 454 and Illumina technologies in parallel that allow the generation of large numbers of ESTs of varying abundance, which can be counted to obtain a measure of gene expression (Weber et al, 2007).…”

Defects in a New Class of Sulfate/Anion Transporter Link Sulfur Acclimation Responses to Intracellular Glutathione Levels and Cell Cycle Control

“…To further test this idea, RNA sequencing (RNA-seq) was used to determine genome-wide transcript abundance in the wild type and smt15-1 under sulfur-replete (+S) conditions or after 6 h in -S. Normally, SULFUR ACCLIMATION (SAC) genes are up-regulated to allow cells to cope with sulfur starvation stress (Zhang et al, 2004;González-Ballester et al, 2010). We found that the induction of SAC genes was either attenuated or decreased in smt15-1 (Table III).…”

RNA sequencing as a tool for understanding biological complexity of abiotic stress in plants

“…The synthetic genes can also be expressed in Phichia Pastoris or Escherichia coli to produce corresponding protein for biochemical studies. This technology could illuminate structural and regulatory gene networks to enlighten how plants reply to fast evolving hints and their environs (González-Ballester et al 2010).…”

“…It allows access of the gene space of unsequenced recalcitrant species. Technological perfections in sequencing platforms; software and smart algorithms will further improve management and mining of large, repetitive plant genomes leading to understanding of plant biochemistry and physiology for application to crop improvement (Lohse et al 2012, Moerkercke et al 2013). …”