Characterization of a novel RING-type ubiquitin E3 ligase GhRING2 differentially expressed in cotton fiber

Authors: SOMA, SHIVA - Mississippi State University; HSU, CHUAN-YU - Mississippi State University; Saha, Sukumar; Jenkins, Johnie; MA, DIN-POW - Mississippi State University

Submitted to: American Journal of Plant Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/17/2014
Publication Date: 10/15/2014
Publication URL: https://handle.nal.usda.gov/10113/60159
Citation: Soma, S., Hsu, C., Saha, S., Jenkins, J.N., Ma, D. 2014. Characterization of a novel RING-type ubiquitin E3 ligase GhRING2 differentially expressed in cotton fiber. American Journal of Plant Sciences. 5:3364-3379.

Interpretive Summary: We discovered in our previous study that CS-B25, a chromosome substitution lines for the chromosome 25 from Gossypium barbadense 3-79, has improved fiber traits (increased fiber strength, length and reduced micronaire), indicating that the chromosome 25 harbored some genes affecting economically important fiber traits. Since CS-B25 has better fiber qualities and it is nearly isogenic to the parent G. hirsutum TM-1 for 25 pairs of chromosomes except the substituted pair of chromosome 25 from G. barbadense 3-79, a comparative analysis of gene expression in fiber between TM-1 and CS-B25 would help to identify genes associated with good fiber traits on the chromosome 25. Affymetrix microarray analysis was thus conducted to ascertain the difference in gene expression between TM-1 and CS-B25 fibers. We discovered four RING-type ubiquitin ligases (GhRING2-5) that are differentially expressed in 10-DPA CS-B25 fiber. In this research the full length cDNA and genomic sequences of the GhRING2 gene were cloned and characterized. The expression of GhRING2 in fiber cells of TM-1 and CS-B25 at different developmental stages was determined. The ubiquitin E3 ligase activity of GhRING2 was also assayed and its target protein was subsequently identified. Discovery of genes at the molecular level associated with fiber development will expedite the genetic improvement of Upland cotton.

Technical 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.