ABSTRACT: We created a gene expression atlas of major organs and under environmental perturbations (temperature and light) of the lycophyte Selaginella moellendorffii
Project description:We generated a comprehensive RNAseq expression atlas for several stress conditions in order to analyze changes in the gene expression during adaptation to mild stresses. The stresses are divided into two main groups: the “nutrient stresses” and the “environmental stresses”. Nutrient stresses include nutrient depletion (-N, -P, -S, -micronutrients), salt stress (+NaCl), osmotic stress (+mannitol) and control. The environmental stresses consist of high light, prolonged darkness, heat, cold and control.
Project description:We compared the diurnal gene expression across nine clades of Archaeplastida. The organisms were subjected to either 12 hours light / 12 hours darkness (12L/12D) or 16L/8D. The samples were taken every two hours in triplicates.
Project description:Acclimation to singlet oxygen was shown to induce various oxidative stress response genes of which some were also strongly overexpressed in the singlet oxygen resistant mutant sor1. Because sor1 was also more tolerant to other oxidative and electrophilic stress conditions, and because many of the sor1 overexpressed genes are known to be involved in the detoxification of reactive electrophile species, the response of the C. reinhardtii wild-type strain to various oxidative and electrophilic stress conditions was determined. Therefore, cultures were exposed to the reactive oxygen species-producing photosensitizer neutral red, the organic hydroperoxide tert-butylhydroperoxide, the photosynthetic herbicide 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the lipophilic electrophile 2(E)-Hexenal for two hours and the global genetic response was analyzed. Cluster analysis revealed the most similar expression pattern between DBMIB and 2(E)-Hexenal and to a lower degree between NR and tBOOH. Still, there were many common induced genes including several of the oxidative stress response and detoxification genes overexpressed in the sor1 mutant. The 4A+ wild-type strain was grown mixotrophically in a Tris-acetate phosphate to a density of 2x10^6 cells/ml. Then cultures were split into 20 ml subcultures, and exposed to either of the four chemicals DBMIB (2 µM), 2(E)-Hexenal (0.3 mM), neutral red (NR, 3 µM), tert-butylhydroperoxide (tBOOH, 100 µM) or no chemical (control) for 2 hours, in three independent biological replicates. The cells of each replicate were harvested by centrifugation and total RNA was isolated using the RNeasy Mini Kit (Qiagen). DNA microarrays were performed using the ‘One-Color Microarray-Based Gene Expression Analysis’ system and a custom made 4 × 44 K ‘Chlamydomonas Whole Genome DNA Microarrays’ (Agilent Technologies) containing 15143 specific probes designed based on the Chlamydomonas version 4.0 transcript models provided by the DOE Joint Genome Institute (JGI), with an average of three replicates for each probe
Project description:Haematococcus pluvialis is a green microalga of commercial interests due to its ability to produce a high value ketocarotenoid, astaxanthin. As a non-model species that lacks a well annotated genome, omics analyses such as transcriptomics and proteomics analysis have often been used together with physiological and biochemical analysis to explore pathways of interest. However, interpretation of these datasets remains challenging. In this work, TMT-based proteomics and phosphoproteomics analyses were conducted on Haematococcus cells grown under favorable conditions (green stage biomass) and high-light stress conditions (red stage biomass). Phosphoproteins were enriched using titanium dioxide before LC-MS/MS analysis. Our proteomics and phosphoproteomics analyses identified 1394 proteins and 569 phosphosites on 366 phosphoproteins, respectively. Of these, 1315 proteins and 396 phosphosites on 314 phosphoproteins were quantifiable, among which 370 proteins and 121 phosphosites on 94 phosphoproteins were differentially expressed. Using an improved analysis pipeline that combines Blast2GO, KEGG, and DAVID to analyze differentially expressed proteins and phosphoproteins, total identified proteins increased from 255 to 322 and total identified phosphoproteins increased from 59 to 70, which were 26.28% and 18.64%, respectively, higher than with the UniProt analysis alone. Using this pipeline, a previously uncharacterized protein and phosphoprotein were identified as an ATPase subunit B and a phosphofructokinase, respectively, and further confirmed with translated genomic and transcriptomic data. This work provides the first example of phosphoproteomics analysis in H. pluvialis, while the proteomics and phosphoproteomics analysis pipelines described here may be useful to analyze omics data from other non-model algal species.
Project description:Drought is one of the most severe stresses leading to retardation of plant growth and development and loss of crop yield. Here we examined the proteome changes of an important oil seed crop canola Brassica napus under drought stress over a 14 day period. Using iTRAQ LC-MS/MS, we identified 2,244 proteins expressed during drought stress. Among them, 412 proteins showed significant changes in abundance under stress, and 67, 243, 287, and 79 proteins were differentially expressed in 3rd, 7th, 10th, and 14th day of drought stress, respectively. Functional analysis of the 412 proteins indicated that the number of proteins associated with “Metabolism”, “Protein synthesis”, and “Signaling” decreased, while those related to “Photosynthesis” and “Stress and defense” increased in response to drought stress. In particular, the proteome profiles at the 7th and 10th day were similar to each other, although there were much more post-translational modifications (PTM) at the 10th day of drought. Interestingly, 286 of 2,244 proteins exhibited PTMs in response to drought stress, 82 of which were differentially changed in drought-stressed plants, and 60 were observed at the 10th day. Furthermore, comparison of protein expression changes with those of gene transcription showed that there was positive correlation in B. napus, although there were different patterns between transcripts and proteins at each time point. As drought stress prolongs, most of the protein abundance changes may be attributed to gene transcription, and PTMs clearly contribute to the protein diversity and functions.
Project description:Tocopherols (vitamin E) are lipid-soluble antioxidants produced by all plants and algae, and many cyanobacteria, yet their functions in these photosynthetic organisms are still not fully understood. We have previously reported that the vitamin E deficient 2 (vte2) mutant of Arabidopsis thaliana is sensitive to low temperature (LT) due to impaired transfer cell wall (TCW) development and photoassimilate export, associated with massive callose deposition in transfer cells of the phloem. To further understand the roles of tocopherols in LT induced TCW development we compared the global transcript profiles of vte2 and wild type leaves during LT treatment. Tocopherol deficiency had no significant impact on global gene expression in permissive conditions, but significantly affected expression of 77 genes after 48 hours of LT treatment. In vte2 relative to wild type, genes associated with solute transport were repressed, while those involved in various pathogen responses and cell wall modifications, such as GLUCAN SYNTHASE LIKE genes (GSL4 and GSL11), were induced. The vte2 LT experiment comprised 18 chips in a factorial design. vte2 and Col plants were grown under permisive conditions for four weeks, Three independent biological replicates were conducted for Col and vte2 at each of the three time points during LT treatment. 0h represent when they are physiologically and biochemically indistinguishable, and at two time points of LT treatment (48 h, 120 h) selected based on our previous timecourse study of the physiological and biochemical changes of vte2 and Col during LT treatment (Maeda et al., 2006). After 48 h of LT, vascular callose deposition is strongly induced and photoassimilate export capacity is significantly lower in vte2 compared to Col, though the visible whole plant phenotypes and soluble sugar accumulation between the two genotypes do not differ (Maeda et al., 2006). The 120 h LT timepoint represents a relatively late response of vte2 when soluble sugars are significantly higher and callose deposition is even more extensive and wide spread in vte2 (Maeda et al., 2006). Thus the 48 h time point should allow identification of early responses to tocopherol deficiency that are distinct from later, pleiotropic responses resulting from the strongly elevated sugar levels in vte2 after 120 h of LT. The 0 h time point represents the absence of LT treatment (see method) and serves as a critical treatment control. 7°C. Three biological replicated the 9 to 11th oldest rosetta leaves from three independent plants were harvested together into a tube filled with liquid nitrogen one hour into the light cycle after 48 and 120 hours of LT-treatment or without LT-treatment (referred to as 0 hour LT treatment).
Project description:Proteorhodopsin has been an ongoing hot topic for the past decade. However the complete physiological role of this extremely widely distributed protein remains mysterious. In this study we aim to give an insight to the physiology of a proteorhodopsin-containing sea ice bacteria – Psychroflexus torquis using gel-free label-free proteomic approach for the first time. We also addressed the life strategy that used by this organism to successfully inhabit extreme sea ice environment.
Project description:Thiol-based redox regulation is a crucial post-translational mechanism to acclimate plants to changing light availability. Here, we conduct a biotin-switch-based redox proteomics study to systematically investigate dynamics of the thiol-redox network in response to temporal changes in light availability and across genotypes lacking parts of the NTRC/thioredoxin (Trx) systems in the chloroplast. Temporal dynamics revealed light leading to marked decreases in the oxidation states of 75 chloroplast proteins mainly involved in photosynthesis during the first 10 min, followed by their partial re-oxidation after 2-6 hours into the photoperiod. This involved f, m and x-type Trx proteins showing similar light-induced reduction-oxidation dynamics, while NTRC, 2-Cys-Prx and Trx y2 showed an opposing pattern, being more oxidized in the light, compared to the dark. In Arabidopsis trxf1f2, trxm1m2 or ntrc mutants, most protein candidates showed increased oxidation states, compared to the wild type, suggesting their light-dependent dynamics to be related to the NTRC/Trx network. In this context, deficiencies in f- and m-type Trxs were found to have different impacts on the thiol-redox proteome depending on the light environment, being higher in constant and fluctuating light, respectively, while NTRC deficiency having a strong influence in all light conditions. Results indicate the plant redox proteome to be subject to dynamic changes in reductive and oxidative pathways to cooperatively fine-tune photosynthetic and metabolic processes in the light. This involves f-type Trxs and NTRC to play a role in constant light conditions, while both m-type Trxs and NTRC being important to balance changes in protein redox-pattern during dynamic alterations in fluctuating light intensities.
Project description:Light initiates the seedling deetiolation transition by promoting major changes in gene expression mainly regulated by phytochrome (phy) photoreceptors. During the initial dark-to-light transition, phy photoactivation induces rapid changes in gene expression that eventually lead to the photomorphogenic development. Recent reports indicate that this process is achieved by phy-induced degradation of Phy-Interacting bHLH transcription Factors (PIFs) PIF1, PIF3 PIF4 and PIF5, which are partly redundant constitutive repressors of photomorphogenesis that accumulate in darkness. In order to test whether light/phy-regulated gene expression occurs through these PIFs, we have performed whole-genome expression analysis in the pif1pif3pif4pif5 quadruple mutant (pifq). Wild-type and pifq mutant seeds were plated on GM medium without sucrose at room temperature. During this procedure the seeds were routinely exposed to white light (WL) for a total of 1.5 hours after imbibition. Seeds were then stratified for 5 days at 4ºC in darkness, induced to germinate with a 5-min red pulse (Rp) (46 μmol/m2/s) and then incubated in the dark for 3h at 21°C before exposure to a terminal 5-min far red pulse (FRp) (58 μmol/m2/s) to suppress pseudo-dark effects. Seeds were then placed in either dark (D) or constant red light (Rc) (6.7 μmol/ m2/s) at 21°C for 45h (2d-old seedlings). Alternatively, 2d-old dark-grown seedlings were treated with 1h of red light (R1) (7.5 μmol/m2/s). Seed samples were harvested after stratification (5d stratified seeds).
Project description:The trophic condition related proteomes of an oxygenic photosynthetic cyanobacterium Synechocystis sp. PCC 6803 (glucose tolerant strain) were analyzed to get further insights into the metabolic traits governing the early exponential growth under photoautotrophic, mixotrophic and heterotrophic conditions. The cells were grown under constant light autotrophically (AT) in low (air-level) CO2 (ATLC) and in high CO2 (ATHC), mixotrophically (MT) in low (air-level) CO2 in the presence of glucose (MTLC) and heterotrophically in low (air-level) CO2 in the presence of glucose and only 10 min exposure to light every 24 hours (Light-Activated Heterotrophic Growth; LAHGLC). The proteomes of ATHC, MTLC and LAHGLC differed substantially from that of ATLC, particularly by downregulation of the inducible carbon concentrating mechanisms (CCM) and photorespiration related enzymes. On the contrary, under carbon-rich conditions (ATHC, MTLC, LAHGLC) the cells accumulated respiratory NAD(P)H dehydrogenase I complexes in the thylakoid membrane. In glucose supplemented cultures (MTLC, LAHGLC) a distinct NDH-2 protein, NdbA, additionally accumulated in the thylakoid membrane whilst the plasma membrane localized NdbC and ARTO decreased in abundance. The light-harvesting proteins together with the photosystem I and II subunits were uniquely depleted under LAHGLC condition, accumulated under ATHC and in MTLC remained at the level of the reference ATLC condition. The accumulation of P-transporters was unique, revealing only minor differences between ATLC and ATHC but strong up-regulation in MTLC and down-regulation in LAHGLC. This likely implies that MTLC stores energy surplus in the highly energetic bonds of polyphosphate polymer that can be used under unfavorable growth conditions. It is concluded that the rigor of cell growth in MTLC condition results to a great extent from high intracellular inorganic carbon conditions created upon glucose breakdown and release of CO2, beside the direct utilization of glucose-derived carbon skeletons for growth. This combination provides the MTLC cultures with excellent conditions for growth that often exceeds that of the mere photoautotrophic growth at high CO2.