Cloning And Functional Analysis Of Genes Involved In Fatty Acid Metabolism In Arabidopsis Thaliana

Fatty acids are important constituents of cell membrane, participate in cellular signaling,posttranslational modification and essential constituents of triacylglycerol in seed oil.Investigation of genes in Arabidopsis fatty acid metabolism would make foundation forrevealing mechanisms of plant lipid biosynthesis, transport and regulation, and providefurther information for genetic modification of seed oil composition and content in oil crops.The first part focuses on the fatty acyl thioesterase A (FATA) family consisting of twomembers in Arabidopsis. We initially studied the expression of FATA family members inArabidopsis organs. The results showed that FATA1was expressed at low levels in all organsexamined and FATA2was detected in all organs examined, especially with high accumulationin siliques. The transient expression of35S:FATA2-eGFP fusion in Arabidopsis green leafprotoplasts showed that FATA2localized in chloroplasts. A T-DNA insertion mutant line ofFATA2(named fata2) was obtained and used for phenotypic observation. Semi-quantitativeRT-PCR assay showed that the expression level of FATA2decreased significantly in fata2compared with that in wild type. Furthermore, the fata2mutants produced longer siliqueswith more seeds, whereas the sizes of the seeds were slightly smaller than those of the wildtype. Compositional analysis of seed oil revealed that except subtly decreased C24:0andunchanged C22:0, all rest fatty acids were increased by from10to60%in fata2dry seedscompared with those in wild-type. Together, our results indicated that FATA2played importantroles in lipid metabolism in seeds and in silique development in Arabidopsis thaliana.In the second part, we studied the Arabidopsis AtELO family consisting of four membersinvolved in very long chain fatty acid synthesis. Here we initially found AtELO1, AtELO3andAtELO4displayed characteristic expression pattern by real time PCR in Arabidopsis, butAtELO2was nearly expressed in any organs. Then the transient expression of AtELO-eGFPfusions in Arabidopsis green leaf protoplasts showed that AtELO1, AtELO3and AtELO4localized in ER where VLCFA synthesis took place. Finally we found that all fatty acidcompositions were decreased10%to30%in seeds of atelo1T-DNA insertional mutants andwere increased dramatically in leaves of atelo3T-DNA insertional mutants. In seeds ofPro35S:AtELO1plants, except C20:0and C20:3, all remaining components were significantly increased. Taken together, our study revealed biological functions of AtELOmembers and may lay the foundation for further genetic manipulation to generate oil cropswith high oil content.The final part focuses on Arabidopsis palmitoyl acyl transferase (PAT) family. PATs is a afamily of DHHC (Asp-His-His-Cys) palmitoyltransferases, which is required inpalmitoylation through adding palmitate to cysteine in target protein by a thioester bond.Arabidopsis genome consists of20putative PATs. However, little is known about theircharacteristics such as genetic relationship and expression profile. In this study, we initiallypresent the phylogeny and gene structures of of PAT family members. They containedconserved DHHC domain and30%to60%of their cDNA sequences were identical.Theexpression pattern analysis of each individual PAT member showed that nineteen PATmembers differently expressed in examined organs, such as seedlings, leaves, roots, stems,flowers, young and old siliques. However, At2g40990was hardly detected in any examinedorgans. Among these nineteen expressed putative PATs, some members were expressed atvery high levels in certain tissues and some exhibited more even distribution in differenttissues. This is the first report on the expression patterns of all these putative PAT genes,which will provide important fundamental data for further identification of their biologicalfunctions.