Dataset Information

Na2CO3-responsive phosphoproteins in Puccinellia tenuiflora leaves

ABSTRACT: Reversible protein phosphorylation also play crucial roles in plant salt resistance. In our syudy, 62 Na2CO3-responsive phosphoproteins were found in leaves by isobaric tags for relative and absolute quantification (iTRAQ) labeling. These Na2CO3-responsive phosphoproteins were sorted into seven functional categories. Among them, most of photosynthetic proteins including 11 light harvesting proteins, five PS II protein, and ten Calvin cycle-related protein (i.e. RuBisCO activase, RuBisCO large subunit, glyceraldehyde-3-phosphate dehydrogenase, and Phosphoglycerate kinase), three ATP synthase subunits, and other carbohydrate and energy metabolism related proteins were identified increased in phosphorylation level. While two starch and sucrose metabolism related protein (e.g. UDP-glucose 6-dehydrogenase), one stress response protein, one membrane and transporting protein, and most of gene expression, protein synthesis and turnover related proteins were identified inhibited phosphorylation under Na2CO3 treatment. In addition, calmodulin, inactive receptor kinase, VWA copine domain containing protein, and tetraspanin family protein participate in signaling pathways were identified enhanced phosphorylation under Na2CO3.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Oryza Sativa (rice)

TISSUE(S): Shoot

SUBMITTER: Shaojun Dai

LAB HEAD: Shaojun Dai

PROVIDER: PXD005471 | Pride | 2021-08-30

REPOSITORIES: Pride

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Publications

Na2CO3-responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics.

Suo Jinwei J Zhang Heng H Zhao Qi Q Zhang Nan N Zhang Yongxue Y Li Ying Y Song Baohua B Yu Juanjuan J Cao Jianguo J Wang Tai T Luo Ji J Guo Lihai L Ma Jun J Zhang Xumin X She Yimin Y Peng Lianwei L Ma Weimin W Guo Siyi S Miao Yuchen Y Chen Sixue S Qin Zhi Z Dai Shaojun S

Genomics, proteomics & bioinformatics 20200601 3

Alkali-salinity exerts severe osmotic, ionic, and high-pH stresses to plants. To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species (ROS) homeostasis, physiological and diverse quantitative proteomics analyses of alkaligrass (Puccinellia tenuiflora) under Na2CO3 stress were conducted. In addition, Western blot, real-time PCR, and transgenic techniques were applied to validate the proteomic results and test t ...[more]

PMID: 32683046

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Project description:The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco containing compartments known as pyrenoids in diverse algal lineages and carboxysomes in prokaryotes. Inside these compartments actively transported bicarbonate is converted into CO2 gas, which saturates the carboxylase with its substrate. Using co-immunoprecipitation experiments in Phaeodactylum tricornutum we have identified the Rubisco linker protein PYCO1. Similar to the green algal Rubisco linker protein EPYC1, PYCO1 is intrinsically disordered, possesses repeats and is positively charged at physiological pH. However, it possesses no sequence similarity to EPYC1, as expected for convergent evolution of a red Rubisco containing pyrenoid. Fluorescent PYCO1 fusion proteins localize as a rod shaped structure in the diatom chloroplast, consistent with the shape of the pyrenoid defined by transmission electron microscopy. To test the hypothesis that PYCO1 is the diatom pyrenoid scaffold we produced pure protein in Escherichia coli. Recombinant PYCO1 protein undergoes homotypic liquid liquid phase separation in a salt dependent manner. Diatom Rubisco specifically partitions into PYCO1 condensates. Heterotypic PYCO1-Rubisco condensates can bind up to three Rubisco hexadecamers per PYCO1 protein. Rubisco carboxylase function is unaffected in the condensates. PYCO1 is highly mobile in homotypic condensates. In contrast PYCO1 condensates saturated with diatom Rubisco have greatly reduced dynamics, with both PYCO1 and Rubisco becoming immobile. Consistently, FRAP experiments indicate that PYCO1 is not mobile in vivo. A combination of Cryo-electron microscopy and site-directed mutagenesis data show that the KWSP motif found in PYCO1 repeats binds to small subunits at the entrance of the Rubisco hexadecamer’s solvent channel. Analysis of mutant PYCO1 proteins show that both the “KWSP” tryptophan and another repeating tyrosine are essential for homotypic phase separation. We speculate that the unusual material properties of the PYCO1-Rubisco condensate are necessary to support the unusual non-spherical shape of the Phaeodactylum pyrenoid. Careful characterization of multiple diverse Rubisco condensates will strengthen translational approaches aiming to introduce pyrenoids and other metabolic condensates into new host organisms.

Project description:Methylmercury (MeHg) is a potent neurotoxin and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. We investigated the gene expression profile in adult female zebrafish whole brain induced by acute (96 hr) MeHg exposure. Fish were exposed by injection to 0 or 0.5 M-BM-5g MeHg/g. Gene expression changes in the brain were examined using a two-color 22,000 feature zebrafish microarray. At a significance level of p<0.01, 79 genes were up-regulated and 76 genes were down-regulated in response to MeHg exposure. Individual genes exhibiting altered expression in response to MeHg exposure implicate effects on glutathione metabolism and GABA-A receptors in the mechanism of MeHg neurotoxicity. Gene ontology (GO) terms significantly enriched among altered genes included protein folding, cell redox homeostasis, and steroid biosynthetic process. The most affected biological functions were related to the nervous system development and function, as well as lipid metabolism and molecular transport. These results support the involvement of oxidative stress and effects on protein structure in the mechanism of action of MeHg in the female brain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxins and investigate responsive genes as potential biomarkers of MeHg exposure. Wild-type strain AB-1 zebrafish (Zebrafish International Resource Center, University of Oregon, Eugene, OR) were cultured at the Columbia Environmental Research Center (CERC), USGS, for MeHg exposures. Adult female zebrafish were injected with 0 M-NM-<g/g or 0.5 M-NM-<g/g MeHg in 2 M-BM-5L Na2CO3 (pH 6.98)/g body weight. After 96 hr, fish were anesthetized using ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma, St. Louis, MO). Whole brains were removed, flash frozen with liquid nitrogen and stored at 80M-BM-0C. For the microarray experiment, two zebrafish brains were pooled per sample. Four pooled samples were taken from fish treated with 0.5 M-NM-<g/g of MeHg, and the other five were taken from control fish treated with sodium carbonate. Array hybridizations were performed using a reference design, where each sample was compared to a reference sample. The reference sample consisted of equal amounts of RNA from control and treated female brains. Five replicates for the control and four replicates for the treated were analyzed. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA).

Dataset Information

Na2CO3-responsive phosphoproteins in Puccinellia tenuiflora leaves

Publications

Na<sub>2</sub>CO<sub>3</sub>-responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics.

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