Project description:Intertidal zone organisms can experience transient freezing temperatures during winter low tides, but their extreme cold tolerance mechanisms are not known. Petrolisthes cinctipes is a temperate mid-high intertidal zone crab species that can experience wintertime habitat temperatures below the freezing point of seawater. We examined how cold tolerance changed during the initial phase of thermal acclimation to cold and warm temperatures, as well as the persistence of cold tolerance during long-term thermal acclimation. Thermal acclimation for as little as 6 hours at 8˚C enhanced crab tolerance during a 1h exposure to -2°C relative to crabs acclimated to 18˚C. Potential mechanisms for this enhanced tolerance were elucidated using cDNA microarrays to probe for differences in gene expression in cardiac tissue of warm and cold acclimated crabs during the first day of thermal acclimation. No changes in gene expression were detected until 12h of thermal acclimation. Genes strongly upregulated in warm acclimated crabs represented immune response and extracellular / intercellular processes, suggesting that warm acclimated crabs had a generalized stress response and may have been remodelling tissues or altering intercellular processes. Genes strongly upregulated in cold acclimated crabs included many that are involved in glucose production suggesting that cold acclimation involves increasing intracellular glucose as a cryoprotectant. Structural cytoskeletal proteins were also strongly represented among the genes upregulated in only cold acclimated crabs. There were no consistent changes in composition or the level of unsaturation of membrane phospholipid fatty acids with cold acclimation, which suggests that neither short- nor long-term changes in cold tolerance are mediated by changes in membrane fatty acid composition. Overall, our study demonstrates that initial changes in cold tolerance are likely not regulated by transcriptomic responses, but that gene expression-related changes in homeostasis begin within 12 hours – the length of a tidal cycle. all array data and raw images archived at the Porcelain Crab Array Database (http://array.sfsu.edu)

Project description:Intertidal zone organisms can experience transient freezing temperatures during winter low tides, but their extreme cold tolerance mechanisms are not known. Petrolisthes cinctipes is a temperate mid-high intertidal zone crab species that can experience wintertime habitat temperatures below the freezing point of seawater. We examined how cold tolerance changed during the initial phase of thermal acclimation to cold and warm temperatures, as well as the persistence of cold tolerance during long-term thermal acclimation. Thermal acclimation for as little as 6 hours at 8˚C enhanced crab tolerance during a 1h exposure to -2°C relative to crabs acclimated to 18˚C. Potential mechanisms for this enhanced tolerance were elucidated using cDNA microarrays to probe for differences in gene expression in cardiac tissue of warm and cold acclimated crabs during the first day of thermal acclimation. No changes in gene expression were detected until 12h of thermal acclimation. Genes strongly upregulated in warm acclimated crabs represented immune response and extracellular / intercellular processes, suggesting that warm acclimated crabs had a generalized stress response and may have been remodelling tissues or altering intercellular processes. Genes strongly upregulated in cold acclimated crabs included many that are involved in glucose production suggesting that cold acclimation involves increasing intracellular glucose as a cryoprotectant. Structural cytoskeletal proteins were also strongly represented among the genes upregulated in only cold acclimated crabs. There were no consistent changes in composition or the level of unsaturation of membrane phospholipid fatty acids with cold acclimation, which suggests that neither short- nor long-term changes in cold tolerance are mediated by changes in membrane fatty acid composition. Overall, our study demonstrates that initial changes in cold tolerance are likely not regulated by transcriptomic responses, but that gene expression-related changes in homeostasis begin within 12 hours – the length of a tidal cycle. all array data and raw images archived at the Porcelain Crab Array Database (http://array.sfsu.edu) n=264 specimens were divided into warm (18°C, n=96), cold (8°C, n=96), and control (13°C, n=72) acclimation groups. Crabs were sampled from the 13°C group at 0 h (the start of the experiment) and 24 h, the termination of the experiment. Crabs were sampled from the warm and cold acclimation groups at 6, 12, 18, and 24 hours following the start of thermal acclimation. At each time point, heart tissue from n=16 crabs from each group was dissected, flash frozen and stored at −80°C. A pooled total aRNA sample was prepared for each group by mixing equal quantities of total RNA from n=5 individuals in each group in order to have the same amount of biological diversity within each pooled RNA sample. For microarray hybridizations we used n=25 slides in an incomplete loop design where each sample was hybridized n=5 times, 2-3 times labelled with each Cy dye

Project description:The regulation of the photosynthetic apparatus in higher plants is highly connected to the organization of the thylakoid membrane into appressed and non-appressed regions. The two photosystems (I and II) are the spatially strongest segregated protein complexes by this thylakoid heterogenous organization, with PSII predominantly present in the appressed membranes (grana), while PSI is confined to the stroma-exposed non-appressed thylakoid membranes (lamellae). Stacking of thylakoid membranes in grana is highly dynamic and influenced by the levels of PSII and LHCII protein phosphorylation which, in turn, are dependent on changes in light intensity and quality and thus, ultimately, on the redox state of the electron transfer chain. Dynamics of this lateral heterogeneity controls the spillover of excitation energy from PSII to PSI and optimizes the photochemistry in constantly fluctuating light conditions. Recently, a family of membrane-integral thylakoid proteins named CURVATURE THYLAKOID 1 (CURT1) have been proposed as new key components in shaping both the width and the number of layers per granum in response to changes in light intensity. CURT1B, in particular, has been known for a long time as a relatively abundant phosphorylated and acetylated protein, but so far the role and dynamics of these post-translational modifications (PTMs) have not been revealed. To this end, we have quantified by means of targeted proteomics the amount of CURT1 proteins and the level of CURT1B PTMs after short-term fluctuating light treatments in wild type Arabidopsis thaliana and the knock-out mutants of the kinases STN7 and STN8 and of the phosphatase TAP38. The CURT1B protein was first localized to a specific curvature domain largely depleted of the chlorophyll-protein complexes. Moreover, we have found that CURT1B is either phosphorylated or acetylated in the N-terminus, but never both. While the level of acetylation is never affected by the light treatments, the phosphorylation increases in light conditions that lead to sudden increase in PSII core protein phosphorylation, and these dynamics are largely abolished in stn8 plants but not in stn7 or tap38. Intriguingly, the phosphorylation dynamics as well as the level of the other CURT1 proteins, are instead highly affected in psb33 plants, which lack LHCII phosphorylation dynamics, and in the psal plants, which show constitutively phosphorylated LHCII. These results provide important information for assessing the relationships between PSII-LHCII phosphorylation, CURT1B phosphorylation and the dynamics of the appressed thylakoids, which in turn allow optimal photosynthesis and provide photoprotection under fluctuations in light intensity.

Dataset Information

Proteomic analysis of the thylakoid membrane in Arabidopsis in the lab versus the field

Publications

Comparative proteomics of thylakoids from <i>Arabidopsis</i> grown in laboratory and field conditions.

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