Isolation and functional characterization of the Ca-DREBLP1 gene encoding a dehydration-responsive element binding-factor-like protein 1 in hot pepper (Capsicum annuum L. cv. Pukang).
Through the use of subtractive hybridization analysis, we have identified 14 partial cDNA clones (pCa-DSRs) that are rapidly induced by dehydration in hot pepper (Capsicum annuum L.) roots. The predicted proteins encoded by Ca-DSRs are putatively involved in processes as diverse as primary and secondary metabolism, protein degradation, and stress responses, indicating the complexity of cellular responses to water deficit in hot pepper roots. Particularly, we investigated the detailed structural properties and expression profiles of Ca-DSR2 (Ca-DREBLP1: dehydration-responsive element binding-factor-like protein 1) encoding a protein that contains a single ERF/AP2 DNA-binding domain. Based on the conserved 14th valine and 19th glutamic acid residues in the ERF/AP2 domain, a basic amino acid stretch (PKKPAGRKKFR) near its N-terminal region, and DSAW signature sequence at the end of its ERF/AP2 domain, Ca-DREBLP1 was classified as a member of a DREB1-type subfamily. Gel retardation assays revealed that Ca-DREBLP1 was able to form a specific complex with the DRE/CRT motif, but not with the GCC box. When fused to the GAL4 DNA-binding domain, the Ca-DREBLP1(190-215) mutant could effectively function as a trans-activator in yeast. This suggests that the extreme C-terminal region plays an essential role in transcription activation. In hot pepper plants, Ca-DREBLP1 was rapidly induced by dehydration, high salinity and, to a lesser extent, mechanical wounding, but not by cold stress. Thus, although the structural features of Ca-DREBLP1 resemble those of the DREB1-type proteins of Arabidopsis thaliana and rice plants, its induction patterns are reminiscent of the DREB2-type proteins, indicating that Ca-DREBLP1 is a novel class DREB subfamily in hot pepper.
Hong
Jong-Pil
JP
Department of Biology, College of Science, Yonsei University, 120-749, Seoul, Korea.
Kim
Woo Taek
WT
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002212:Capsicum / Q000235:genetics*; D002467:Cell Nucleus / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification; D018517:Plant Roots / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification; D014867:Water / Q000494:pharmacology
D010940:Plant Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014867:Water; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride