title: The transcriptional regulatory mechanism of the peroxisomal ascorbate peroxidase (pAPX) gene cloned from an extreme halophyte, Salicornia brachiata.
abstract: Peroxisomal ascorbate peroxidase detoxifies H₂O₂ leaching out from peroxisomes into the cytoplasm. The present study describes transcript expression and cis-regulation of the SbpAPX gene cloned from an extreme halophyte, Salicornia brachiata, in the steady state and under different stresses. About 2-fold elevated transcript expression was found in salt- and drought-treated shoots at 12 h compared with control, while 1.9-fold increased expression was observed under heat treatment. In roots, the transcript level was down-regulated at 2 h, thereafter increasing with the time of exposure and reaching a maximum at the control level. The SbpAPX promoter has characteristic cis-regulatory ABA-dependent abiotic stress-responsive elements. The full-length promoter (1,024 bp, PP1) and deletion constructs -838 (PP2), -697 (PP3), -433 (PP4) and -185 bp (PP5) were fused with the GUS (β-glucuronidase) gene and transformed into tobacco for functional validation. Expression of GUS increased significantly in transgenic plants under stress. Quantitative expression analysis of GUS in T₁ plants revealed that promoter PP5 is efficient for gene expression. In planta transient expression further suggested that the promoter PP5 contains efficient stress-inducible elements. A steep decline in GUS expression in PP3, and thereafter an elevated expression in PP4 and PP5, suggested the presence of a repressor element between -696 and -433 bp, while an enhancer element was predicted between -838 and -697 bp. Further, transient expression analyses and electrophoretic mobility shift assay revealed that the core sequence of cis-acting motifs ATAA and CCTCAA function as enhancer and repressor binding sites, respectively. Based on the study, a model is proposed for the cis-regulation of the SbpAPX gene. The present study provides a useful insight for understanding gene expression regulation in a halophyte with or without stress. Furthermore, potential stress-responsive promoter-driven expression of introgressed gene(s) can be used for engineering crops with enhanced stress tolerance.
authors.affiliation: Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, G.B. Road, Bhavnagar-364002, Gujarat, India.
authors.forename: Vivekanand
authors.initials: V
authors.lastname: Tiwari
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Chaturvedi
authors.forename: Avinash
authors.initials: A
authors.lastname: Mishra
authors.forename: Bhavanath
authors.initials: B
authors.lastname: Jha
mesh_terms: D060387:Ascorbate Peroxidases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D019663:Chenopodiaceae / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D003198:Computer Simulation; D055864:Droughts; D004742:Enhancer Elements, Genetic / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D017930:Genes, Reporter; D005966:Glucuronidase / Q000378:metabolism; D006358:Hot Temperature; D008954:Models, Biological; D008969:Molecular Sequence Data; D020675:Peroxisomes / Q000201:enzymology*; D018517:Plant Roots / Q000235:genetics; D018520:Plant Shoots / Q000235:genetics; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D055051:Salt-Tolerant Plants / Q000235:genetics*; D013312:Stress, Physiological / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic
chemical_list: D012333:RNA, Messenger; D012097:Repressor Proteins; D060387:Ascorbate Peroxidases; D005966:Glucuronidase
keywords: Abiotic stress; EMSA; Halophyte; Stress-responsive promoter; Transcript regulation; pAPX