Functional analysis of arabidopsis acyl-coenzyme A-binding protein1 and acyl-coenzyme A-binding protein2 in lipid metabolism

Abstract

In Arabidopsis thaliana, the acyl-CoA-binding protein (ACBP) family consists of six members named AtACBP1 to AtACBP6. AtACBP1 and AtACBP2 are two membrane-anchored proteins which shared 76.9% amino acid similarity. They also contain the conserved acyl-CoA-binding domain which facilitate binding activities to various acyl-CoA esters, and ankyrin repeats that mediate protein-protein interactions. AtACBP1 and AtACBP2 show overlapping functions during embryogenesis but display non-overlapping responses to abiotic stresses. In this study, the roles of AtACBP1 and AtACBP2 in cuticle formation were initially investigated. The β-glucuronidase (GUS) histochemical staining revealed high expression of AtACBP1 and AtACBP2 in stem epidermal cells, which are the factories for wax and cutin synthesis. The ACBP2 RNA interference transgenic lines were generated in the acbp1 mutant background (designated as acbp1/ACBP2-RNAi) to study their potential overlapping function. On stems of the acbp1, acbp2, and acbp1/ACBP2-RNAi lines, wax crystallize patterns were changed and fewer crystals were observed. The leaves and stems of the acbp1, acbp2 and acbp1/ACBP2-RNAi lines were more permeable to toluidine blue than the wild type. Using transmission electron microscopy, an irregular stem cuticle membrane was observed in these lines. Through gas chromatography and GC–mass spectrometry analyses, the acbp1 mutant showed reduction in leaf and stem cuticular wax constituents and stem cutin monomer composition in comparison to the wild type (Col-0), and this defect was complemented by introduction of the AtACBP1 cDNA. The acbp2 mutant also demonstrated reduction in leaf and stem cutin monomer composition and leaf (but not stem) wax. Consistently, the expression of cuticular wax synthetic genes in leaves and cutin synthesis genes in stems of the acbp1, acbp2 and acbp1/ACBP2-RNAi lines decreased and these lines were more susceptible than the wild type to infection of the necrotrophic pathogen Botrytis cinerea. Taken together, these findings suggest that AtACBP1 and AtACBP2 participate in cuticle formation in Arabidopsis. The interactors of AtACBP1 and AtACBP2 were investigated to better understand the function of AtACBP1 and AtACBP2 in lipid metabolism. SMO1-1, SMO1-2 and SMO1-3 were proved to interact with AtACBP1. Transgenic Arabidopsis expressing SMO1-1pro::GUS, SMO1-2pro::GUS and SMO3-1pro::GUS fusion were generated and the GUS histochemical staining showed SMO1s expressed in various tissues including stem, leaf and flower. The expression of SMO1-2 was induced by ABA but not GA3, in the lateral roots, especially in the elongation zone. Transverse sections of anthers indicated expression of three SMO1s in pollen grains and no difference of pollen tube growth was observed in SMO1-2-RNAi Arabidopsis. At3g26110 was shown to interact with AtACBP2 in yeast two-hybrid analysis and bimolecular fluorescence complementation assay. At3g26110::GFP was found to be localized and co-localized with DsRed::AtACBP2 at the PM and ER. Quantitative real-time polymerase chain reactions showed co-expression of At3g26110 and AtACBP2 in flower buds, open flower and siliques. GUS histochemical staining showed At3g26110 specifically expressed in pollen of anther, suggesting its potential role in anther development.