Characterization of the 5'-flanking region of ACBP3 encoding arabidopsis acyl-coenzyme A binding protein 3

Abstract

Arabidopsis thaliana Acyl-CoA-Binding Protein 3, one of six acyl-CoA-binding

proteins, is unique by the C-terminal location of its acyl-CoA-binding (ACB) domain.

It promotes autophagy (ATG)-mediated leaf senescence and confers resistance to

Pseudomonas syringae pv. tomato DC3000. To understand the regulation of ACBP3, a

1.7 kb 5’-flanking region of ACBP3 and its deletion derivatives were characterized

using β-glucuronidase (GUS) reporter gene fusions.

A 374 bp minimal fragment (-151/+223) could drive GUS expression while a

1698 bp fragment (-1475/+223) conferred maximal activity. Further, histochemical

GUS staining analysis on transgenic Arabidopsis harboring the largest (1698 bp)

ACBP3pro::GUS fusion displayed ubiquitous expression in floral organs and vascular

bundles of leaves and stems, consistent with previous results that extracellularly

localized ACBP3 functions in plant defense. A 160 bp region (-434/-274) induced

GUS expression in extended darkness and conferred down-regulation in extended

light. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay

showed that the DNA binding with one finger box (Dof-box, -341/-338) interacted

specifically with leaf nuclear proteins from dark-treated Arabidopsis while GT-1

(-406/-401) binds both dark- and light-treated Arabidopsis, suggesting that Dof and

GT-1 motifs are required to mediate circadian regulation of ACBP3. Moreover, GUS

staining and fluorometric measurements revealed that a 109 bp region (-543/-434)

was responsive to phytohormones and pathogens. Within this 109 bp region, an S-box

of AT-rich sequence (-516/-512) was identified to bind nuclear proteins from

pathogen-infected Arabidopsis leaves, providing the basis for pathogen-inducible

regulation of ACBP3 expression. Hence, three cis-responsive elements (Dof, GT-1

and S-box) in the 5’-flanking region of ACBP3 were demonstrated to participate in

the regulation of ACBP3. The regulation of ACBP3 by circadian control is not

surprising given that defense genes are now known to be circadian-regulated;

infection being anticipated at dawn coinciding with pathogen activity in spore

dispersal during the light period.