Abstract
The ubiquitin-proteasome proteolysis pathway is responsible for the degradation of abnormal and short-lived proteins to regulate many important biochemical activities in eukaryotes. By employing affymetrix microarray analysis, we have identified a novel ubiquitin ligase E3 gene GhRING2 that is differentially expressed between two Gossypium hirsutum lines-Texas Marker-1 (TM-1) and Chromosome Substitution Line CS-B25. The CS-B25 line has chromosome 25 from G. barbadense substituted into TM-1. The complete nucleotide sequences of GhRING2 along with its 5’-flanking region were obtained by genomic walking. GhRING2 was highly expressed in elongating fiber, and GUS expression directed by the GhRING2 promoter was found in hypocotyls and young stems of transgenic Arabidopsis plants. Using a yeast two-hybrid assay GhRING2 was found to interact with a PROTODERMAL FACTOR1 (GhPDF1) protein. GhPDF1 was expressed preferentially in immature ovules and fiber initials, and the GhPDF1 gene had been suggested to play a role in cell fate determination and fiber development. Pull down and plasmid swap assays further confirmed the interaction between GhRING2 and GhPDF1. The expression and protein interaction data indicate that GhRING2 may be involved in the turnover of GhPDF1 and participation in the transition from initiation to elongation stages during fiber development. Our data strongly suggest that the ubiquitin-proteasome pathway may regulate cotton fiber growth and development. The nucleotide sequence data of GhRING2 in this article have been submitted to the Gen Bank Nucleotide Sequence Data Bases under the accession number BankIt 1,742,008 SeqKM 108,000.
Highlights
Cotton is one of the most important economic crops for textile industries
Affymetrix microarray analysis was conducted by our lab to determine the difference in gene expression between Texas Marker-1 (TM-1) and CS-B25 fibers, and we identified four RING-type ubiquitin ligases (GhRING2-5) that are differentially expressed in 10-DPA CS-B25 fiber [24]
A protein Blast search was performed to find RING proteins homologous to GhRING2 from plants, and eleven closly related ubiquitin E3 ligases with their amino acid sequences, including Theobroma cacao (TcRING), Fragaria vesca (FvRING), Ricinus communis (RcRING), Populus trichocarpa (PtRING), Solanum lycopersicum (LeRING), Vitis vinifera (VvRING), Prunus persica (PpRING), Cicer arietinum (CaRING), Medicago truncatula (MtRING), Arabidopsis lyrata (AlRING) and Arabidopsis thaliana (AtRING) were retrived
Summary
Cotton is one of the most important economic crops for textile industries. Over 90% of the cotton produced worldwide is from Upland cotton (Gossypium hirsutum). We previously cloned two cotton E2 genes (GhUBC1 and GhUBC2) and one E3 gene (GhRING1 encoding a RING-type ubiquitin ligase), and studied their expression in cotton fiber [20] [21]. The E. coli over-expressed GhRING1 was found to have the ubiquitin E3 ligase activity by an in vitro reconstitution assay [21] These data suggested that cotton fiber growth and development are regulated via protein ubiquitination and protein ubiquitination may play a role in fiber differentiation. Affymetrix microarray analysis was conducted by our lab to determine the difference in gene expression between TM-1 and CS-B25 fibers, and we identified four RING-type ubiquitin ligases (GhRING2-5) that are differentially expressed in 10-DPA CS-B25 fiber [24]. The ubiquitin E3 ligase activity of GhRING2 was assayed and its target protein was subsequently identified
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