Investigations on acyl-CoA-binding proteins AtACBP4, AtACBP5 and AtACBP6 in arabidopsis

Abstract

A gene family encoding six members of acyl-CoA-binding proteins (ACBPs) co-exist in Arabidopsis, designated as AtACBP1 to AtACBP6. They have been reported to play pivotal roles in plant lipid metabolism. AtACBP4, AtACBP5 and AtACBP6 represent the cytosolic forms in the AtACBP family. The combinatory and distinct roles of the cytosolic AtACBPs have been studied in pollen and seed development, light-dependent regulation and substrate affinities to acyl-CoA esters, suggesting that they are potential candidates for acyl-CoA binding and trafficking in plant cells. To understand the regulation of AtACBP4 and AtACBP5, the 1.1-kb 5’-flanking region of AtACBP4 and the 1.9-kb 5’-flanking region of AtACBP5 as well as their deletion derivatives were characterized using β-glucuronidase (GUS) reporter gene fusions. Deletion analysis of AtACBP4pro::GUS and AtACBP5pro::GUS constructs indicated the minimal region for AtACBP4 (-145/+103) and AtACBP5 (-181/+81) expression. Electrophoretic mobility shift assay identified a pollen-specific cis-acting element POLLEN1 (AGAAA), which was further confirmed by DNase I footprinting. Transgenic AtACBP4pro::GUS and AtACBP5pro::GUS Arabidopsis showed GUS expression in pollen grains albeit at different developmental stages. Immunoelectron microscopy using AtACBP4- or AtACBP5-specific antibodies indicated that AtACBP4 and AtACBP5 were expressed in the cytoplasm. Scanning and transmission electron microscopy revealed defects in acbp4acbp5 pollen. Wax and cutin analysis revealed that acbp4, acbp5 and acbp4acbp5 buds accumulated cuticular wax and stem cutin monomers in comparison to the wild type. Fatty acid profiling demonstrated a decrease in stearic acid and an increase in linolenic acid in the acbp4 and acbp4acbp5 buds. AtACBP4 expression in the pollen tube was confirmed by GUS-staining of AtACBP4pro::GUS transgenic plants. In vivo pollen tube germination assays revealed that acbp4acbp5acbp6 is defective in pollen tube germination. It was previously shown that AtACBP6pro::GUS Arabidopsis exhibited strong GUS activity in vascular tissues. Herein, immunoelectron microscopy using anti-AtACBP6 antibodies demonstrated AtACBP6 localization in the phloem especially in the companion cells and sieve elements. The presence of gold grains in plasmodesmata indicated its potential role in systemic trafficking. Western blot and RT-PCR analysis detected AtACBP6 (but not AtACBP6 mRNA) in the phloem exudate. Fatty acid profiling revealed an increase in the jasmonate precursor, 12-oxo-cis,cis-10,15-phytodienoic acid, and a reduction in jasmonate or its derivatives in acbp6 phloem exudates in comparison to the wild type. Also, qRT-PCR showed down-regulation of COMATOSE (CTS) in acbp6 rosettes suggesting that AtACBP6 affects CTS function. AtACBP6 likely facilitates the symplastic transport of lipids into the sieve tubes, consistent with its role in pathogen-defense and the wound-inducibility of AtACBP6pro::GUS. PLASMODESMATA-LOCATED PROTEIN8 (PDLP8) was confirmed to interact with AtACBP6 by bimolecular fluorescence complementation assays, subcellular colocalization using autofluorescent tags, isothermal titration calorimetry and pull-down assays. Both AtACBP6 and PDLP8 showed higher expression in the reproductive than vegetative tissues in qRT-PCR, albeit AtACBP6 was generally higher than PDLP8 in microarrays and GUS assays on AtACBP6pro::GUS or PDLP8pro::GUS lines, likely attributed to PDLP8 expression being confined to the plasmodesmata. Western blot analysis using anti-AtACBP6 antibodies showed a reduction in AtACBP6 expression in the pdlp8 mutant, implying that PDLP8 may influence AtACBP6 expression.