Characterization of acyl-coenzyme A-binding proteins from rice

Abstract

Acyl-CoA-binding proteins (ACBPs) show conservation in an acyl-CoA-binding domain (ACB domain) which binds acyl-CoA esters. The 10-kDa ACBP represents the highly-conserved prototype that occurs in animals, plants, fungi and protists. Previous phylogenetic analysis of the 10-kDa ACBP from metazoans and nonmetazoans showed lineage-specific duplication and purifying selection during evolution. Besides the 10-kDa ACBPs, large multifunctional proteins containing a predicted ACB domain have been identified in many eukaryotes. However, the evolutionary relationships of these larger ACBPs had not been investigated. The aim of this study is to investigate the phylogeny of plant ACBPs, 10-kDa ACBPs as well as the larger ACBPs, and to characterize the ACBP family from the monocot model plant, rice. Plant protein sequences from 16 plant genomes consisting of an ACB domain were identified using Arabidopsis AtACBP1, AtACBP3, AtACBP4 or AtACBP6 sequences as independent probes. Phylogenetic analyses were conducted by maximum likelihood (ML) analysis and neighbor-joining (NJ) methods. The ACBP family was found to diversify as land plants evolved. Classes I and IV showed lineage-specific gene expansion while Classes II and III were closely-related phylogenetically. Similar to the eudicot, Arabidopsis, six genes (designated OsACBP1 to OsACBP6) encode rice ACBPs, but their distribution into various classes differed from Arabidopsis. Expression profiles of rice ACBPs under normal growth and biotic and abiotic stress conditions, as well as when subject to phytohormone treatments, were examined by quantitative real-time reverse-transcription polymerase chain reactions. Rice ACBP mRNAs showed ubiquitous expression. OsACBP5 is unique because it was highly-expressed throughout the whole reproductive stage. OsACBP1, OsACBP2 and OsACBP3 were abscisic acid (ABA)-and salicylic acid (SA)-inducible. OsACBP4, OsACBP5 were stress (drought and salt) - and ABA/SA-responsive while OsACBP6 was wound-inducible. In vitro acyl-CoA-binding assays and phospholipid binding assays were conducted using bacterial-expressed recombinant (His)6-tagged OsACBPs to determine their potential lipid binding abilities. Other than a common affinity to [14C]linolenoyl-CoA, recombinant rice ACBPs demonstrated specific acyl-CoA ester preferences. All recombinant OsACBPs could bind many species of phosphatidic acid (PA) and phosphatidylcholine (PC) with the exception of recombinant OsACBP4 which was the only OsACBP to bind 16:0 PA. To better understand the potential roles of rice ACBPs in lipid trafficking, the subcellular localization of rice ACBPs was investigated by western blot analysis, transient expression in tobacco leaf cells and stable expression in transgenic Arabidopsis of autofluorescence-tagged OsACBPs, respectively. OsACBP-GFP fusions were observed to be targeted to the cytosol, plasma membrane, endoplasmic reticulum (ER) or peroxisome. Results from phylogeny, gene expression, biochemical analysis and subcellular localization investigation suggest that rice ACBP paralogues, within and across classes, are not redundant proteins. From results on differences in gene expression, acyl-CoA ester/phospholipid binding and subcellular localization between Arabidopsis and rice ACBPs, the function of ACBPs occur to have diverged with the split between eudicots and monocots. Besides performing conserved basal functions, multi-domain rice ACBPs appear to be associated with stress responses which makes them attractive candidates for potential applications in agriculture.