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)

Abstract

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.

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog

“…Using differential display PCR and subtractive hybridization analysis, we previously identified a broad spectrum of partial cDNA clones from hot pepper seedlings, which were rapidly induced by dehydration (Choi et al, 2002;Park et al, 2003;Hong and Kim, 2005). One of the isolated clones, pCaDRS3 (GenBank accession no.…”

“…One of the isolated clones, pCaDRS3 (GenBank accession no. CK327619), renamed CaPUB, encoded a partial polypeptide homologous to a U-box-containing protein (Hong and Kim, 2005) and was used to isolate a full-length pCaPUB cDNA clone. The partial cDNA fragment, pCaDRS3, was radioactively labeled and used as a probe to screen the l-uni-Zap II cDNA library constructed from water-stressed leaves of hot pepper plants.…”

“…We are interested in elucidating the adaptive response of hot pepper plants against abiotic stresses, such as water deficit. Recently, we have isolated and characterized a broad spectrum of cDNAs from hot pepper plants whose expression is enhanced rapidly in response to drought (Choi et al, 2002;Park et al, 2003;Hong and Kim, 2005). Among the identified cDNAs, pCaDRS3 (GenBank accession no.…”

“…Among the identified cDNAs, pCaDRS3 (GenBank accession no. CK327619) encodes a partial protein that is homologous to a U-box-containing enzyme with putative Ub ligase activity (Hong and Kim, 2005). In this study, we have isolated a full-length CaDRS3 for C. annuum putative U-box protein 1 (CaPUB1) and analyzed the detailed expression pattern of its mRNA in response to various abiotic stresses, hormones, and wounding.…”

Cloning and characterization of cold, salt and drought inducible C-repeat binding factor gene from a highly cold adapted ecotype of Lepidium latifolium L.

“…DREB2-like genes, belong to the A-2 subgroup, are mainly involved in regulation of genes responsive to osmotic stress (Sakuma et al 2002;Nakashima and Yamaguchi-Shinozaki 2006). Arabidopsis homologs of DREB1/CBF genes have been isolated and characterised in evolutionarily diverse plants with different levels of adaptation to cold stress such as, common wheat (Triticum aestivum L.), rice (Oryza sativa L.), rye (Secale cereale L.), maize (Zea mays L.), canola (Brassica napus), etc., and overexpression of DREB1/CBF genes in transgenic plants increases tolerance to drought, high salt, and freezing stresses (Dubouzet et al 2003;Shen et al 2003;Qin et al 2004;Zhang et al 2004a;Hong and Kim 2005;Benedict et al 2006;Zhao et al 2006;Chinnusamy et al 2010). So, CBF genes may be useful for improving the stress tolerance of crops (Yamaguchi- .…”