Characterization of the 5'-flanking regions of arabidopsis acyl-coenzyme a-binding protein1 and acyl-coenzyme a-binding protein2

Abstract

In the model plant Arabidopsis thaliana, a gene family of six members encodes acyl-Coenzyme A (CoA)-binding proteins (ACBPs). They are designated as ACBP1 to ACBP6 and they bind to acyl-CoA esters with different affinities. Two membrane-localized members, ACBP1 and ACBP2, share over 70% amino acid identity. Previous studies have shown they are involved in a variety of developmental processes and stress responses in Arabidopsis, including embryogenesis, heavy metal and cold stress responses. Also, ACBP1 and ACBP2 participate in ABA signaling. ACBP1 was demonstrated to function in seed germination and seedling establishment by interacting with PLDα1, an important regulator in lipid-based abscisic acid (ABA) signaling. ACBP2-overexpressing Arabidopsis were drought tolerant through the up-regulation of two NAD(P)H oxidases, AtrbohD and AtrbohF.

In this study, the 5’-flanking regions of ACBP1 and ACBP2 were analyzed. Computer prediction identified various cis-elements, including those associated with tissue-specific expression, besides gene enhancers and hormone- or stress-responsive motifs. The β-glucuronidase (GUS) fusion deletion derivatives from the distal end of the ACBP1 and ACBP2 5’-flanking regions were generated and the wild-type Arabidopsis was transformed with these constructs. GUS histochemical staining was then carried out to study the transcriptional regulation of these two promoters. Different DNA fragments were identified in spatial and temporal expression of ACBP1 and ACBP2.

The promoter signatures of ACBP1 and ACBP2 were further characterized in response to internal and external stumili. The 5’-flanking region of ACBP1 was induced by Pb(II). GUS staining and activity assays identified the -1479/-160 and -378/-158 regions as important for Pb(II) induction in shoots and roots, respectively. Three Pas elements were identified to have different binding activities in seedlings during Pb(II) treatment. In addition, the 5’-flanking regions of ACBP1 and ACBP2 were induced by ABA in seedlings. The -460/-237 region of the ACBP1 promoter was responsible for both basal expression and ABA induction, whereas the -861/-341 region of the ACBP2 promoter functions in both basal expression and ABA induction. Furthermore, the promoter strengths of ACBP1 and ACBP2 were in the same order of magnitude as the 35S promoter in seeds. ABA and gibberellins were found to antagonistivally regulate the expression of ACBP1 and ACBP2 during seed germination.

The function of ACBP1 and ACBP2 in salt and osmotic stress was also analyzed. The acbp1 and acbp2 single mutants were insensitive to NaCl and mannitol treatments, whereas the ACBP1-OXs and ACBP2-OXs were sensitive to these treatments. These phenotypic differences have been demonstrated to be ABA-dependent. Also, ACBP1 interacted with ABA-RESPONSIVE BINDING PROTEIN1 (AREB1), a major transcription factor, in ABA signaling. The function of ACBPs in flooding stress was investigated. The acbp2 mutant and acbp1/ACBP2 RNAi transgenic Arabidopsis were more submergence tolerant than wild-type Arabidopsis. The induction of ACBP1 and ACBP2 under submergence treatment was observed by GUS staining and real-time quantitative PCR. Furthermore, the differential expression patterns of other ACBPs (ACBP3 to ACBP6) demonstrate the potential roles of ACBPs in flooding stress.