Subcellular Localization And Functional Mechanism Analyses Of Class ? ACBP

Acyl-coenzyme A-binding proteins(ACBPs)possess a highly conserved acyl-CoA binding domain(ACB),which can bind acyl-CoA with carbon chain lengths ranging from C12 to C26.They are widely distributed in all eukaryotes and some prokaryotes.ACBP affects plant growth and development by regulating lipid metabolism,including early embryogenesis and leaf senescence,as well as responding to various biological and abiotic stresses,such as heavy metal stress,oxidative stress,low temperature stress and pathogen infection.In this study,we constructed phylogenetic trees of 112 plant class ? ACBP,and investigated the subcellular localization of class ? ACBP from the major branches of the phylogenetic tree by transient expression in tobacco leaves,onion epidermal cells and stable expression in the transgenic Arabidopsis.The yeast two-hybrid assays and luciferase complementation imaging experiments demonstrated the interaction between rice OsACBP6 and peroxisome ABC transporter.The effect of the deletion of OsACBP6 on primary root development was explored by paraffin section technology.The main research contents and results are as follows:(1)Using rice LOC_Os03g61930(OsACBP6)and Arabidopsis AT3G05420(AtACBP4)amino acid sequences as probes,112 homologous sequences were obtained from the PLAZA website.The maximum likelihood method was used for phylogenetic analysis,and the Micromonas Commoda class IV ACBP(MCO016765)as an outgroup.Phylogenetic analysis based on multiple alignments of full-length amino acid sequences showed that with the evolution of terrestrial plants,the class ? ACBP in higher plants diverged with the separation of eudicots and monocots.(2)The Populus trichocarpa 35S::Potri.005G026900::GFP and 35S::Potri.013G018800::GFP?Cucumis sativus L.35S::Cucsa.091180::GFP?Glycine max 35S::Glyma.03G236500::GFP?35S::Glyma.19G234400::GFP and 35S::Glyma.20G235500::GFP?Zea may s 35S::Zm00001d046434::GFP and 35S::Zm00001d034758::GFP?Sorghum bicolor 35S::Sobic.001G023100::GFP and Triticum aestivum 35S::TraesCS5D02G515900::GFP recom-binant plasmid were constructed and used in three experimental systems for subcellular localization.Transient expression in tobacco leaf epidermal cells showed that,Potri.005 G026900::GFP?Potri.013G018800::GFP?Cucsa.091180::GFP?Glyma.03G236500::GFP?Glyma.19G234400::GFP?Glyma.20G235500::GFP?Zm00001d034758::GFP?Sobic.001G023100::GFP were localized in the cytoplasm.While Zm00001d046434::GFP and TraesCS 5D02G515900::GFP exhibit randomly distributed dotted fluorescent signals.The above genes and the peroxisome matrix marker fluorescent protein DsRed-SKL were transiently expressed in onion epidermal cells,which proved Potri.013G018800::GFP,Sobic.001G023100::GFP,Zm00001d046434::GFP,Zm00001d034758::GFP,TraesCS5D02G515900::GFP were localized in the peroxisome matrix,and other gene localizations was the same as in tobacco transient expression.Transgenic Arabidopsis thaliana expressing the above-mentioned peroxisome-localized gene was obtained by flora dip method.Subcellular localization of these GFP fusions in the protoplast were consistent with the transient expression results of onion epidermal cells.The above results suggest that monocots have at least one class ? ACBP located in peroxisomes,and that class ? ACBP of eudicots tend to retain cytoplasmic localization.(3)Yeast two-hybrid assays showed that OsACBP6 interacted with peroxisome ABC transporter LOC_Os01g73530 and LOC_Os05g01700 through the ACB domain.Luciferase complementation imaging experiment further confirmed that OsACBP6 only interacted with LOC_Os01g73530.The above results indicate that the protein-protein interaction between OsACBP6 and LOC_Os01g73530 occurs through the ACB functional domain,which provides more clues for further exploring the functional mechanism of OsACBP6.(4)By using paraffin sections,the primary root of OsACBP6 loss-of-function mutant osacbp6 displayed irregular cell morphology in the meristem and elongation zone.Furthermore,there was no significant difference in the cell length amongst the osacbp6,wild-type rice(Oiyza sativa var.japojnica CV.Dongjin)and OsACBP6 complemented lines,indicating that the slow growth of osacbp6 primary root was not resulted from the compromised cell elongation.