Xiang Jin

Quantitative proteomics of Sesuvium portulacastrum leaves revealed that ion transportation by V-ATPase and sugar accumulation in chloroplast played crucial roles in halophyte salt tolerance.

UNLABELLED Physiological and proteomic responses of Sesuvium portulacastrum leaves under salinity were investigated. Different from glycophytes, this halophyte had optimal growth at 200-300mM NaCl and accumulated more starch grains in chloroplasts under high salinity. Increased contents of soluble sugars, proline, and Na(+) were observed upon salinity. X-ray microanalysis revealed that Na(+) was mainly compartmentalized into cell vacuole. Quantitative proteomics produced 96 salt responsive proteins, and the majority was chloroplast-located proteins. Gene ontology analysis revealed that proteins involved in ion binding, proton transport, photosynthesis and ATP synthesis were overrepresented. The expressions of a Na(+)/H(+) antiporter and several ATP synthase subunits were activated upon high salinity. ATP hydrolysis assay demonstrated that V-ATPase activity at tonoplast was dramatically increased upon NaCl whereas vacuolar H(+)-pyrophosphatase and plasma membrane P-ATPase activities were not increased, which indicated that sodium compartmentalization was mainly performed by enhancing V-ATPase activity rather than P-ATPase and H(+)-pyrophosphatase. Accumulation of soluble sugars as well as sodium compartmentalization maintained the osmotic balance between vacuole and cytoplasm, which finally established ionic homeostasis in saline cells in true halophytes. BIOLOGICAL SIGNIFICANCE Physiological and proteomic analyses of S. portulacastrum leaves under different salinities were investigated. This true halophyte accumulated more soluble sugars, starch, proline and Na(+) under high salinity. Differential proteomics produced 96 salt responsive proteins and the majority was involved in ion binding, proton transport, photosynthesis, and ATP synthesis. A Na(+)/H(+) antiporter and several ATP synthase subunits were induced upon high salinity. ATP hydrolysis assay demonstrated that V-ATPase activity at tonoplast was dramatically increased whereas vacuolar H(+)-pyrophosphatase and plasma membrane ATPase activities were stable upon NaCl. These findings demonstrated that the increased Na(+) was compartmentalized into vacuole by enhancing V-ATPase activity rather than H(+)-ATPase.

Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization.

Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement.

Comprehensive Proteomics Analysis of Laticifer Latex Reveals New Insights into Ethylene Stimulation of Natural Rubber Production

Ethylene is a stimulant to increase natural rubber latex. After ethylene application, both fresh yield and dry matter of latex are substantially improved. Moreover, we found that ethylene improves the generation of small rubber particles. However, most genes involved in rubber biosynthesis are inhibited by exogenous ethylene. Therefore, we conducted a proteomics analysis of ethylene-stimulated rubber latex, and identified 287 abundant proteins as well as 143 ethylene responsive latex proteins (ERLPs) with mass spectrometry from the 2-DE and DIGE gels, respectively. In addition, more than 1,600 proteins, including 404 ERLPs, were identified by iTRAQ. Functional classification of ERLPs revealed that enzymes involved in post-translational modification, carbohydrate metabolism, hydrolase activity, and kinase activity were overrepresented. Some enzymes for rubber particle aggregation were inhibited to prolong latex flow, and thus finally improved latex production. Phosphoproteomics analysis identified 59 differential phosphoproteins; notably, specific isoforms of rubber elongation factor and small rubber particle protein that were phosphorylated mainly at serine residues. This post-translational modification and isoform-specific phosphorylation might be important for ethylene-stimulated latex production. These results not only deepen our understanding of the rubber latex proteome but also provide new insights into the use of ethylene to stimulate rubber latex production.

Quantitative proteomics and transcriptomics reveal key metabolic processes associated with cotton fiber initiation

UNLABELLED An iTRAQ-based proteomics of ovules from the upland cotton species Gossypium hirsutum and its fuzzless-lintless mutant was performed, and finally 2729 proteins that preferentially accumulated at anthesis in wild-type ovules were identified. We confirmed that the gene expression levels of 2005 among these proteins also increased by performing an RNA sequencing transcriptomics. Expression of proteins involved in carboxylic acid metabolism, small-molecule metabolic processes, hormone regulation, and lipid metabolism was significantly enhanced in wild-type ovules. Quantitative real-time PCR verified the increased expression of 26 genes involved in these processes. Cotton 3-hydroxyacyl-CoA dehydratase (GhPAS2) catalyzing the third reaction of very long-chain fatty acid (VLCFA) biosynthesis, accumulated at anthesis in wild-type ovules. Heterogeneous expression of GhPAS2 restored viability to the Saccharomyces cerevisiae haploid psh1-deletion strain deficient in PAS2 activity. Application of VLCFA biosynthesis inhibitor acetochlor (2-chloro-N-[ethoxymethyl]-N-[2-ethyl-6-methyl-phenyl]-acetamide; ACE) and gibberellic acid to the unfertilized cotton ovules significantly suppressed fiber cell protrusion. In this study, the profiling of gene expression at both transcriptome and proteome levels provides new insights into cotton fiber cell initiation. BIOLOGICAL SIGNIFICANCE Cotton fiber initiation determines the ultimate number of fibers per ovule, thereby determining fiber yield. In total, 2729 proteins were preferentially accumulated in wild-type ovules at anthesis. The most up-regulated proteins were assigned to carboxylic acid metabolism, small-molecule metabolic processes, hormone regulation, and lipid metabolism. In consistence with these findings, we characterized GhPAS2 gene coding for the enzyme that catalyzes VLCFA production. VLCFA biosynthesis inhibitor, acetochlor, was shown to significantly suppress fiber initiation. This study provides a genome-scale transcriptomic and proteomic characterization of fiber initial cells, laying a solid basis for further investigation of the molecular processes governing fiber cell development.

The beta subunit of glyceraldehyde 3-phosphate dehydrogenase is an important factor for maintaining photosynthesis and plant development under salt stress—Based on an integrative analysis of the structural, physiological and proteomic changes in chloroplasts in Thellungiella halophila

Thellungiella halophila, a new model halophyte, can survive under highly saline conditions. We performed comparative proteomics of chloroplasts from plants grown under different saline conditions. Seventy-five salt-responsive proteins were positively identified by mass spectrometry, which represented 43 unique ones. These proteins were categorized into 7 main pathways: light reaction, carbon fixation, energy metabolism, antenna proteins, cell structure, and protein degradation and folding. Saline conditions increased the abundance of proteins involved in photosynthesis, energy metabolism and cell structure. The results indicated that Thellungiella could withstand high salinity by maintaining normal or high photosynthetic capacity, reducing ROS production, as well as enhancing energy usage. Meanwhile, the ultrastructural and physiological data also agree with chloroplast proteomics results. Subsequently, the glyceraldehydes 3-phosphate dehydrogenase beta subunit (GAPB) involved in carbon fixation was selected and its role in salt tolerance was clarified by over-expressing it in Arabidopsis. ThGAPB-overexpressing plants had higher total chlorophyll contents, dry weights, water contents and survival rates than that of wild type plants. These results indicated that ThGAPB might improve plant salt tolerance by maintaining higher recycling rates of ADP and NADP(+) to decrease ROS production, helping to maintain photosynthetic efficiency and plant development under saline conditions.

Proteomics of Fusarium oxysporum Race 1 and Race 4 Reveals Enzymes Involved in Carbohydrate Metabolism and Ion Transport That Might Play Important Roles in Banana Fusarium Wilt

Banana Fusarium wilt is a soil–spread fungal disease caused by Fusarium oxysporum. In China, the main virulence fungi in banana are F. oxysporum race 1 (F1, weak virulence) and race 4 (F4, strong virulence). To date, no proteomic analyses have compared the two races, but the difference in virulence between F1 and F4 might result from their differentially expressed proteins. Here we report the first comparative proteomics of F1 and F4 cultured under various conditions, and finally identify 99 protein species, which represent 59 unique proteins. These proteins are mainly involved in carbohydrate metabolism, post-translational modification, energy production, and inorganic ion transport. Bioinformatics analysis indicated that among the 46 proteins identified from F4 were several enzymes that might be important for virulence. Reverse transcription PCR analysis of the genes for 15 of the 56 proteins revealed that their transcriptional patterns were similar to their protein expression patterns. Taken together, these data suggest that proteins involved in carbohydrate metabolism and ion transport may be important in the pathogenesis of banana Fusarium wilt. Some enzymes such as catalase-peroxidase, galactosidase and chitinase might contribute to the strong virulence of F4. Overexpression or knockout of the genes for the F4-specific proteins will help us to further understand the molecular mechanism of Fusarium-induced banana wilt.

Expression Profiling of Mitogen-Activated Protein Kinase Genes Reveals Their Evolutionary and Functional Diversity in Different Rubber Tree (Hevea brasiliensis) Cultivars

Rubber tree (Hevea brasiliensis) is the only commercially cultivated plant for producing natural rubber, one of the most essential industrial raw materials. Knowledge of the evolutionary and functional characteristics of kinases in H. brasiliensis is limited because of the long growth period and lack of well annotated genome information. Here, we reported mitogen-activated protein kinases in H. brasiliensis (HbMPKs) by manually checking and correcting the rubber tree genome. Of the 20 identified HbMPKs, four members were validated by proteomic data. Protein motif and phylogenetic analyses classified these members into four known groups comprising Thr-Glu-Tyr (TEY) and Thr-Asp-Tyr (TDY) domains, respectively. Evolutionary and syntenic analyses suggested four duplication events: HbMPK3/HbMPK6, HbMPK8/HbMPK9/HbMPK15, HbMPK10/HbMPK12 and HbMPK11/HbMPK16/HbMPK19. Expression profiling of the identified HbMPKs in roots, stems, leaves and latex obtained from three cultivars with different latex yield ability revealed tissue- and variety-expression specificity of HbMPK paralogues. Gene expression patterns under osmotic, oxidative, salt and cold stresses, combined with cis-element distribution analyses, indicated different regulation patterns of HbMPK paralogues. Further, Ka/Ks and Tajima analyses suggested an accelerated evolutionary rate in paralogues HbMPK10/12. These results revealed HbMPKs have diverse functions in natural rubber biosynthesis, and highlighted the potential possibility of using MPKs to improve stress tolerance in future rubber tree breeding.

Two-Dimensional Gel Electrophoresis-Based Proteomic Analysis Reveals N-terminal Truncation of the Hsc70 Protein in Cotton Fibers In Vivo

On two-dimensional electrophoresis gels, six protein spots from cotton ovules and fibers were identified as heat shock cognate 70 kD protein (Hsc70). Three spots corresponded to an experimental molecular weight (MW) of 70 kD (spots 1, 2 and 3), and the remaining three spots corresponded to an experimental MW slightly greater than 45 kD (spots 4, 5 and 6). Protein spots 1, 2 and 3 were abundant on gels of 0-day (the day of anthesis) wild-type (WT) ovules, 0-day fuzzless-lintless mutant ovules and 10-day WT ovules but absent from gels of 10-day WT fibers. Three individual transcripts encoding these six protein spots were obtained by using rapid amplification of cDNA ends (RACE). Edman degradation and western blotting confirmed that the three 45 kD Hsc70 protein spots had the same N-terminal, which started from the T271 amino acid in the intact Hsc70 protein. Furthermore, quadrupole time-of-flight mass spectrometry analysis identified a methylation modification on the arginine at position 475 for protein spots 4 and 5. Our data demonstrate that site-specific in vivo N-terminal truncation of the Hsc70 protein was particularly prevalent in cotton fibers, indicating that post-translational regulation might play an important role in cotton fiber development.

Comparative Proteomics Reveals that Phosphorylation of β Carbonic Anhydrase 1 Might be Important for Adaptation to Drought Stress in Brassica napus

Little is known about the mechanism of drought tolerance in rapeseed (Brassica napus L.). In this study, different morphological and physiological responses to drought stress were studied in three rapeseed cultivars. For the cultivar 2AF009 with high drought tolerance, comparative proteomic analyses were conducted to determine the molecular mechanism behind. Approximately 138 differentially abundant proteins (DAPs) and 1232 phosphoproteins containing 4469 phosphopeptides were identified. Furthermore, 337 phosphoproteins containing 547 phosphorylation sites demonstrated significant changes. These drought-responsive DAPs and phosphoproteins were mainly involved in signal transduction, photosynthesis, and glutathione-ascorbate metabolism. Notably, 9 DAPs were also identified as drought-responsive phosphoproteins, especially beta carbonic anhydrase 1 (βCA1), which was represented by eight distinct protein spots with different abundant levels during drought stress. Tyr207 phosphorylated site of βCA1 was down-regulated at the phosphorylation level during drought stress, which was also located in the substrate-binding active region of three-dimensional (3D) structure. Moreover, drought stress inhibited CA activity. We concluded that Tyr207 was the most likely phosphorylation target affecting the enzyme activity, and phosphorylation of βCA1 might be important for the response to drought stress in rapeseed. The study provided a new clue for the drought tolerance mechanism in B.napus.

Subcellular proteome profiles of different latex fractions revealed washed solutions from rubber particles contain crucial enzymes for natural rubber biosynthesis

Rubber particle (RP) is a specific organelle for natural rubber biosynthesis (NRB) and storage in rubber tree Hevea brasiliensis. NRB is processed by RP membrane-localized proteins, which were traditionally purified by repeated washing. However, we noticed many proteins in the discarded washing solutions (WS) from RP. Here, we compared the proteome profiles of WS, C-serum (CS) and RP by 2-DE, and identified 233 abundant proteins from WS by mass spectrometry. Many spots on 2-DE gels were identified as different protein species. We further performed shotgun analysis of CS, WS and RP and identified 1837, 1799 and 1020 unique proteins, respectively. Together with 2-DE, we finally identified 1825 proteins from WS, 246 were WS-specific. These WS-specific proteins were annotated in Gene Ontology, indicating most abundant pathways are organic substance metabolic process, protein degradation, primary metabolic process, and energy metabolism. Protein-protein interaction analysis revealed these WS-specific proteins are mainly involved in ribosomal metabolism, proteasome system, vacuolar protein sorting and endocytosis. Label free and Western blotting revealed many WS-specific proteins and protein complexes are crucial for NRB initiation. These findings not only deepen our understanding of WS proteome, but also provide new evidences on the roles of RP membrane proteins in NRB. SIGNIFICANCE Natural rubber is stored in rubber particle from the rubber tree. Rubber particles were traditionally purified by repeated washing, but many proteins were identified from the washing solutions (WS). We obtained the first visualization proteome profiles with 1825 proteins from WS, including 246 WS-specific ones. These WS proteins contain almost all enzymes for polyisoprene initiation and may play important roles in rubber biosynthesis.