A proteomic approach to apoplastic proteins involved in cell wall regeneration in protoplasts of Arabidopsis suspension-cultured cells

To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.     

An integrative analysis of four CESA isoforms specific for fiber cellulose production between Gossypium hirsutum and Gossypium barbadense

Cotton fiber is an excellent model system of cellulose biosynthesis; however, it has not been widely studied due to the lack of information about the cellulose synthase (CESA) family of genes in cotton. In this study, we initially identified six full-length CESA genes designated as GhCESA5–GhCESA10. Phylogenetic analysis and gene co-expression profiling revealed that CESA1, CESA2, CESA7, and CESA8 were the major isoforms for secondary cell wall biosynthesis, whereas CESA3, CESA5, CESA6, CESA9, and CESA10 should involve in primary cell wall formation for cotton fiber initiation and elongation. Using integrative analysis of gene expression patterns, CESA protein levels, and cellulose biosynthesis in vivo, we detected that CESA8 could play an enhancing role for rapid and massive cellulose accumulation in Gossypium hirsutum and Gossypium barbadense. We found that CESA2 displayed a major expression in non-fiber tissues and that CESA1, a housekeeping gene like, was predominantly expressed in all tissues. Further, a dynamic alteration was observed in cell wall composition and a significant discrepancy was observed between the cotton species during fiber elongation, suggesting that pectin accumulation and xyloglucan reduction might contribute to cell wall transition. In addition, we discussed that callose synthesis might be regulated in vivo for massive cellulose production during active secondary cell wall biosynthesis in cotton fibers.     

RNA-Seq Reveals Dynamic Changes of Gene Expression in Key Stages of Intestine Regeneration in the Sea Cucumber Apostichopus japonicas

Background

Sea cucumbers (Holothuroidea; Echinodermata) have the capacity to regenerate lost tissues and organs. Although the histological and cytological aspects of intestine regeneration have been extensively studied, little is known of the genetic mechanisms involved. There has, however, been a renewed effort to develop a database of Expressed Sequence Tags (ESTs) in Apostichopus japonicus, an economically-important species that occurs in China. This is important for studies on genetic breeding, molecular markers and special physiological phenomena. We have also constructed a library of ESTs obtained from the regenerative body wall and intestine of A. japonicus. The database has increased to ∼30000 ESTs.

Results

We used RNA-Seq to determine gene expression profiles associated with intestinal regeneration in A. japonicus at 3, 7, 14 and 21 days post evisceration (dpe). This was compared to profiles obtained from a normally-functioning intestine. Approximately 5 million (M) reads were sequenced in every library. Over 2400 up-regulated genes (>10%) and over 1000 down-regulated genes (∼5%) were observed at 3 and 7dpe (log₂Ratio≥1, FDR≤0.001). Specific “Go terms” revealed that the DEGs (Differentially Expressed Genes) performed an important function at every regeneration stage. Besides some expected pathways (for example, Ribosome and Spliceosome pathway term), the “Notch signaling pathway,” the “ECM-receptor interaction” and the “Cytokine-cytokine receptor interaction” were significantly enriched. We also investigated the expression profiles of developmental genes, ECM-associated genes and Cytoskeletal genes. Twenty of the most important differentially expressed genes (DEGs) were verified by Real-time PCR, which resulted in a trend concordance of almost 100% between the two techniques.

Conclusion

Our studies demonstrated dynamic changes in global gene expression during intestine regeneration and presented a series of candidate genes and enriched pathways that contribute to intestine regeneration in sea cucumbers. This provides a foundation for future studies on the genetics/molecular mechanisms associated with intestine regeneration.     

The role of phytohormones in cotton fiber development

Since 1974, when Beasley and Ting discovered that fertilized ovules of cotton can be cultured in media supplemented with GA along with auxin, the effect of all types of phytohormones on fiber development has been widely studied. Many phytohormones, including GA, IAA, brassinosteroid (Br), ABA, ethylene (Et), and cytokinins (Ck), all have been demonstrated to play important roles during cotton fiber development. In recent years, the rapid development of genomic analysis and the accumulation of high-quality cotton ESTs allowed us to probe phytohormonal gene expression during fiber development. Many phytohormonal genes, including GA-, IAA-, ABA-, Br-, Et-, and Ck-related genes, participating in phytohormone biosynthesis pathways and signal transduction pathway accumulated in the process of cotton fiber development.