Protein S-acyl Transferase10Is Cirtical For Development And Salt Tolerance In Arabidopsis

In order to obtain the full function, proteins have to go through series posttranslationalmodifications, which could dynamicly regulate the activity and function of proteins. ProteinS-acylation (PAT), also known as palmitoylation, is an important reversible posttranslatioanalmodification. Palmitoylation is catalyzed by palmitate transferase, adding a16carbonsaturated lipid group, often palmitate, to the sulfhydryl group of a cystin residue.Palmitoylation regulates enzyme activity, protein stability, subcellular localization, andintracellular sorting. Recent studies showed that many plant proteins are palmitoylated.However, due to the redundancy of this family, little is known about the function of PATs.Here, we report that the tonoplast-localized PAT10is critical for development and salttolerance in Arabidopsis thaliana. The function and substrate analysis of PAT10couldprovide new evidence for stress-induced signaling transduction pathway and inspire us forlearning functions of other PATs. The main results and conclusions presented in this thesis areas follows:(1) PAT10regulates Arabidopsis developmentpat10mutant displayed pleiotropic developmental defects compared with the wild typeunder soil growth condition. Rosette leaves were smaller than wild type during vegetativegrowth, both leaf cell size and cell growth were reduced, and leaves showed early senescenseas the tip turned yellow before flowering. Druing floral transition, pat10showed nocorrelation between flowering time and rosette leaf number such that they floweredtemporally later but had fewer rosette leaves than the wild type, likely a consequence of slowdevelopment. The flower organs of pat10were smaller, and the siliques can not elongatednormally, almost completely sterile.Although we culd obtain homozygous mutants from self-pollinated heterozygousmutants, but the ratio was much lower than expected. Though reciprocal crosses, weconfirmed that the tansmision ratio of male gametophyte was compromised in heterozygousmutants. Alexander staining and DAPI staining showed that both pollen activity and pollen development were not impaired, but scaning electron micrpscopy (SEM) showed that thepollen coat from homozygous pat10was defected, and this result was confirmed bytransmission electron microscopy (TEM). Also, the number of lipid bodies in pat10mutantwas reduced.The transmission ratio of female gametophyte was not affected in heterzogous pat10mutants, but pistil from pat10homozygous mutant can not support the growth and guaidenceof wild type pollen tubes, which indicated sporophytic female defect.Based on the pleiotropic developmental defects, we supposed that PAT10expressedconstitutively during Arabidopsis development. Both ProPAT10:GUS and RNA in situhybridization confirmed that PAT10expressed in multiple organs and tissues, whichcorrelates the pleiotropic defects of mutant.(2) The subcellular localization of PAT10In order to be sure that all the developmental defects were caused by PAT10loss-of-function, we performed complementation analysis. Genomic fragment of PAT10andGFP fusion construct was introduced into heterozygous mutant, and homozygous pat10withexogenous PAT10-GFP was found in the next generation. Such plants displayed the same aswild type plants, so we concluded that the developmental defects of pat10were induced byPAT10loss-of-function. Using this complementation plants, we tested the subcellularlocalization of PAT10by introducing marker protein through crossing. Co-labeling analysisshowed that PAT10was localized at tonoplast.PAT10is a member of PAT family in Arabidopsis, but we can not conclude that thefunction of PAT10was determined by its enzyme activity. Animal studies suggested that theconserved cystin residue within the enzyme domain is important for the activity of PAT. Sowe generated a construct with C192S mutation, the subccellular localization of PAT10was notcompromised by this site mutation, but this construct can not rescue the phenotype of pat10mutant which indicated PAT10functions through its palmitate transferase activity.(3) The substrates of PAT10As PAT10is tonoplast-localization, we proposed that maybe PAT10was involed invacuole function. Under salt stress condition, we found pat10mutant was more sensitive toabiotic stress than wild type, not only indicated by the growth condition, but also revealed by the upper-regulation of stress-inducible genes. Recent studies showed that CBL2/3/6weretonoplast-localization and involved in stress response, also these protein were anchored totonoplast through palmitoylation which were confirmed by the treatment of palmitoylationinhibitor (2-bromopalmitate,2-BP). So we believed that CBL2/3/6were substrates of PAT10.We expressed CBL2/3/6in both wild type and pat10mutant protoplast to test ourhypothesis. CBL2/3/6showed tonoplast-localization in wild type, but in protoplast frompat10mutant, CBL2/3/6turned cytosolic. We used2-BP to treat wild type protoplast, CBLslost their tonoplast-localization and turned out to be cytosolic. In order to exclude thepossibility that the localization change was due to the reduce of palmitoylation level within acell, we tested some other proteins which had been demonstrated as the substrates ofpalmitoylation. Both endosome marker ARA6and plasma membrane marker CBL9displayedthe same localization in wild type and pat10mutant, indicating the localization change ofCBL2/3/6was specific in pat10mutant. Also the localizations of Golgi marker ERD2andTGN marker ARA7were not compromised in mutant, which means the endomembranesystem in mutant was not defected.