Response of Synechococcus sp. WH7803 LL- and and HL- acclimated cells to oxidative stress (HP and MV)
ABSTRACT: To identify the respective roles of light and ROS in the photoinhibition process and detect a possible light-driven tolerance to oxidative stress, we compared the transcriptomic responses of Synechococcus sp. WH7803 acclimated to low (LL) or high light (HL) to oxidative stress, induced by hydrogen peroxide (H202) or methylviologen (MV). Cultures were acclimated during many generations to continuous low light (LL, 18 ?mol photons m-2 s-1, hereafter LL cells) and high light (HL, 250 ?mol photons m-2 s-1, hereafter HL cells) provided by Sylvania Daylight 58W/154 fluorescent bulbs. For all stress experiments performed in this study, exponentially growing cultures (1 to 3 x 107 cells mL-1), were split into subcultures and submitted to oxidative stress by addition of H2O2 or MV and harvested when PSII quantum yield fell to half of the initial value. For H2O2 experiments, this level of PSII photoinactivation was reached 2 h after submitting LL and HL cultures to 750 µM and 25 µM respectively. Because of the large divergence in dose and kinetics responses to MV between LL- and HL cells, it was not possible to find MV concentrations leading to 50 % decrease of quantum yield at the same time for both light acclimations. Thus, array analyses for MV were performed on HL and LL cultures incubated at the same MV concentration (50 µM) but harvested once PSII quantum yield was halved, i.e. after 1 and 3.5 h of stress respectively. All hybridizations were performed on 4 independent biological replicates and using as reference sample a pool of RNA from all samples investigated in this study. Pairwise comparison were performed to analyze the stress induced by either H2O2 or MV on both LL- and HL cultures (i.e. LL-Ct vs. LL+MV, LL-Ct vs. LL+H2O2, HL-Ct vs. HL+MV, HL-Ct vs. HL+H2O2) as well as to compare the steady state acclimation to different light conditions (i.e. LL-Ct vs. HL-Ct).
Project description:Plant thylakoid membranes contain hundreds of proteins closely interplaying to cope with ever-changing environmental conditions. We investigated how P. sativum (pea) grown at different irradiances optimizes light-use efficiency through the differential accumulation of thylakoid proteins. Thylakoid membranes from plants grown under limiting (LL), normal (NL) and high (HL) light intensity were characterized by combining chlorophyll fluorescence measurements with quantitative proteomic analysis. Protein sequences retrieved from available transcriptomic data considerably improved the protein profiling. We found that increasing growth irradiance affects the electron transport kinetics but not Photosystem (PS) I and II relative abundance. Two acclimation strategies were evident comparing plants acclimated to LL with higher irradiances: 1) in NL, plants turn on photoprotective responses mostly regulating the PSII light-harvesting capacity either accumulating Lhcb4.3 or favouring the xanthophyll cycle; 2) in HL, plants reduce the LHCII pool and enhance the PSII repair cycle. At increasing growth irradiance, plants increase the accumulation of ATP synthase and boost the electron transport to finely tune the ΔpH across the membrane and adjust the thylakoid architecture to optimize protein trafficking. Our results provide a quantitative snapshot on how plants coordinate light-harvesting, electron transport and protein synthesis adjusting the thylakoid membrane proteome in a light-dependent manner
Project description:Corals continuously adjust to short term variation in light availability on shallow reefs. Long-term light alterations can also occur due to natural and anthropogenic stressors, as well as management interventions such as coral transplantation. Although short term photophysiological responses are relatively well-understood in corals, little information is available regarding photoacclimation dynamics over weeks of altered light availability. We coupled photophysiology and metabolomic profiling to explore changes that accompany longer-term photoacclimation in a key Great Barrier Reef coral species (Acropora muricata). High (HL) and low light (LL) acclimated corals were collected from the reef and reciprocally exposed to high and low light ex situ. Rapid light curves using Pulse Amplitude Modulation (PAM) fluorometry revealed photophysiological acclimation of LL to HL and HL to LL shifted corals within 21 days. A subset of colonies sampled at 7 and 21 days for untargeted LC-MS and GC-MS metabolomic profiling revealed metabolic reorganization before acclimation was detected using PAM fluorometry. Metabolomic shifts were more pronounced for LL to HL treated corals than their HL to LL counterparts. Compounds driving metabolomic separation between HL-exposed and LL control colonies included amino acids, organic acids, fatty acids and sterols. Reduced glycerol and campesterol suggest decreased translocation of photosynthetic products from symbiont to host in LL to HL shifted corals, with concurrent increases in fatty acid abundance indicating reliance on stored lipids for energy. We discuss how these data provide novel insight into environmental regulation of metabolism and implications for management strategies that drive rapid changes in light availability.
Project description:The effect of light during the development of freezing tolerance was studied in winter wheat (Triticum aestivum L. var. Mv Emese) and spring wheat variety Nadro. Ten-day-old plants were cold hardened at 5°C for 12 days either under normal (250 mmol m-2 s-1) or low light (20 mmol m-2 s-1) conditions. Samples of Emese (E) and Nadro (N) plants grown at 18°C under normal (NL) and low (LL) light fluences were compared to each other in a simple loop design and E-NL vs. E-LL; N-NL vs. N-LL; E-NL vs. NLL and E-LL vs. N-LL comparisons were made.
Project description:Methionine sulfoxide reductases catalyze the reduction of MetSO back to the correct Met residue. Previously, the gene of Capsicum annuum methionine sulfoxide reductase B2 was isolated and CaMSRB2-overexpressing tomato shows enhanced growth, which may trigger increased resistance to the pathogens. To assess the role of this enzyme in rice, we generated transgenic lines under the control of the rice Rab21 (responsive to ABA protein 21) promoter with/without Bar marker gene. Several physiological tests such as MV and Fv/Fm, indicators of an oxidative stress-inducing agent and a potential maximal PSII quantum yield, respectively strongly suggested CaMSRB2 confers drought tolerance to rice. Using 3′-tiling microarray covering the whole rice genes, we carried out genome-wide expression analyses with CaMsrB2-transformed rice (Oryza sativa L. cv. ILMI). Rice was grown in port for six weeks and treated with drought by water withholding for two days. A total of 15 chips were used for the microarray experiment. RNA was extracted from plants just before and 2 days after the duration of water withdrawal for the control and the comparison, respectively. Experiments were performed with three or two biological replicates.
Project description:To analyze the impact of photosynthetic redox signals, light sources with spectral qualities that preferentially excite either Photosystem I (PSI light) or Photosystem II (PSII light) were used. The light sources have been described in (Fey et al., 2005). Strong reduction and oxidation signals were induced by light shifts from PSI to PSII light (PSI-II) and the reverse light shift (PSII-I), respectivly. The acclimation responses were monitored at 0.5, 2, 8, and 48h after a light shift. Samples taken prior to changing the light quality (0h) served as control. Keywords: photosynthesis, redox regulation, light acclimation, retrograde signalling, long term response Experiments were performed with plant material corresponding to pools of at least 250-500 individuals of Arabidopsis thaliana (Col-0). To abtain healthy and unstressed plants, seedlings were initially grown for 21 days under white light (short day periods, 8-h light/16-h dark) on soil. Plants were then pre-acclimated to PSI-light for 3 days and reference samples were taken. Plants were then shifted to PSII light and tissue was harvested at the described time points. Similarly, samples were harvested before and after the reverse light shift. As additional control, plants acclimated to white light were also analyzed. RNA isolation: Leaf material was harvested and frozen in liquid N2 under the respective light source. Isolation of total RNA was performed as adapted from (Westhoff et al., 1991). Array analyses using the 3292-GST nylon array were performed as described earlier (Richly et al., 2003; Fey et al., 2005). Three independent experiments with different filters and independent cDNA probes were performed (for each timepoint).
Project description:Green plants are more robust to hydrogen peroxide (H2O2) stress and contain high endogeneous H2O2 levels which is generated during photorespiration and photosynthesis. Therefore, exgeneous H2O2 application mostly impose oxidative stress. To reduce endogenous H2O2 background, we adopted a strategy which is to grow Arabidopsis seedlings in the dark to eliminate light-induced H2O2 production, thus to reduce the endogenous H2O2 level. Exogenous H2O2 was then applied to induce transcriptome changes. Global gene expression is studied and compared between samples collected under 7d dark, 7d H2O2 treatment under dark and 7d light conditions. We cultured seedlings in the dark to reduce endogenous H2O2. Three conditions were used for transcriptome profiling: dark grown (dark); dark grown with exogenous H2O2 treatment (H2O2); and light grown (light). Three types of conditions were used for Arabidopsis seedling culture: dark, dark with 5 mM H2O2 treatment and light. Each condition was performed with two biological replicates. The seedlings were harvested at 7 days old.
Project description:Biotic and abiotic stresses limit agricultural yields, and plants are often simultaneously exposed to multiple stresses. Combinations of stresses such as heat and drought or cold and high light intensity, have profound effects on crop performance and yeilds To analyze such responses, we initially compared transcriptome changes in ten Arabidopsis thaliana ecotypes using cold, heat, high light, salt and flagellin treatments as single stress factors or their double combinations. Arabidopsis thaliana plants of ecotypes (Col, Ler, C24, Cvi, Kas1, An1, Sha, Kyo2, Eri and Kond) were subjected to the following stress treatments: Salt, Cold, Heat, High Light (HL), Salt+Heat, Salt+HL, Cold+HL, Heat+HL, as well as FLG (Flagellin, flg22 peptide), Cold+FLG, Heat+FLG
Project description:Photosynthetic diatoms are exposed to rapid and unpredictable changes in irradiance and spectral quality, and must be able to adapt their light harvesting systems to varying light conditions. Molecular mechanisms behind light acclimation in diatoms are largely unknown. We set out to investigate the mechanisms of high light acclimation in Phaeodactylum tricornutum using an integrated approach involving global transcriptional profiling, metabolite profiling and variable fluorescence technique. Algae cultures were acclimated to low light (LL), after which the cultures were transferred to high light (HL). Molecular, metabolic and physiological responses were studied at time points 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h after transfer to HL conditions. The integrated results indicate that the acclimation mechanisms in diatoms can be divided into an initial response phase (0.5 h), an intermediate acclimation phase (3-12 h) and a late acclimation phase (12-48 h). The initial phase is recognized by strong and rapid regulation of genes encoding proteins involved in photosynthesis, pigment metabolism and reactive oxygen species (ROS) scavenging systems. A significant increase in light protecting metabolites occur together with the induction of transcriptional processes involved in protection of cellular structures at this early phase. During the following phases, the metabolite profiling display a pronounced decrease in light harvesting pigments, whereas the variable fluorescence measurements show that the photosynthetic capacity increases strongly during the late acclimation phase. We show that P. tricornutum is capable of swift and efficient execution of photoprotective mechanisms, followed by changes in the composition of the photosynthetic machinery that enable the diatoms to utilize the excess energy available in HL. Central molecular players in light protection and acclimation to high irradiances have been identified. The experiment was designed as a time series, with diatom cultures were harvested at time points 0.5 h, 3 h, 6 h, 12 h, 24 h and 48 h after transfer to high light conditions. The reference samples were kept at low light and harvested in parallel with the treated samples. Three biological replicates were harvested for all samples.
Project description:Arabidopsis thaliana cell suspension cultures (ACSC) were subjected to 30-min, mild chemical treatments with three different singlet oxygen elicitors at low-medium light conditions (150 µE m–2 s–1) with the aim of getting a better understanding of singlet oxygen-mediated defence responses in plants. The three elicitors Indigo Carmine (IC), Methylene Violet (MV) and Rose Bengal (RB) at a concentration of 0.5 µM were chosen because they exhibited different abilities to permeate the plasma membrane and to accumulate in the cell soma or organelles such as chloroplasts. In addition, ACSC were treated with 500 µM H2O2 for comparison. Confocal image analysis of Arabidopsis cells revealed that IC was not retained in cells, whereas MV and RB permeated the plasma membrane and accumulated in the chloroplast envelope and inside chloroplasts, respectively. As a consequence of their different cellular location, the physiological, transcriptional and photosynthetic responses of Arabidopsis cells to singlet oxygen production varied from each other and the activation of programmed cell death (PCD) was observed in ACSC treated with 0.5 µM RB, but not with the other elicitor nor with 500 µM H2O2. The role of chloroplasts in the activation of PCD was further investigated when this physiological response was analyzed in dark-grown cell cultures containing undifferentiated plastids. Interestingly, PCD was only activated in light-grown, but not in dark-grown, Arabidopsis cell cultures, suggesting that singlet oxygen-mediated defence responses were initiated inside chloroplasts. Genome-wide transcriptional profile analyses were performed as well and the results proved that there were only statistically significant changes in the transcript expression of light-grown ACSC treated with 0.5 µM RB and 500 µM H2O2, but not with IC nor with MV. Functional enrichment analyses revealed that GO/Biological process terms associated with defence responses were common in the treatments with 0.5 µM RB and 500 µM H2O2; however, resistance response to pathogen and PCD terms were only significantly over-represented in the RB treatment. Moreover, the analysis of the up-regulated transcripts in ACSC treated with 0.5 µM RB brought out that both specific markers for singlet oxygen from the conditional fluorescence (flu) mutant of Arabidopsis and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present, although there was no evidence for the up-regulation of EDS1 encoding the ENHANCED DISEASE SUSCEPTIBILITY PROTEIN 1. Finally, a co-regulation analysis proved that ACSC treated with 0.5 µM RB exhibited higher correlation with the flu family mutants than with other singlet oxygen producer mutants of Arabidopsis or wild-type plants of Arabidopsis subjected to high light treatments, where singlet oxygen was produced in photosystem II and an acclimatory response was activated instead of PCD. 18 Samples total. Six Samples are controls, 3 Samples each treated with Rose Bengal, Hydrogen Peroxide, Methyl Violet, and Indigo Carmine.
Project description:Series of 6 repetitions of hybridization of treatment (PSII_DCMU) and control (PSII) each. Comparison of plants grown under PSII-specific light and treated with the electron transport inhibitor DCMU versus plants grown under PSII-specific light without DCMU treatment. T. Pfannschmidt, unpublished Keywords: repeat sample