PROTEIN S-ACYL TRANSFERASEs Mediated Root Hair Growth And Pollen Tube Reception In Arabidopsis

Post-translational modifications of proteins are critical for their subcellular targeting,activity,interactome,and stability.Protein S-acylation or palmitoylation is a reversible lipid modification in which long chain fatty acids,usually the 16-carbon palmitate,are covalently attached to a cysteine residue?s?via a thioester bond and by doing so,regulate the activity,stability,its subcellular localization of substrate proteins.Protein palmitoylation is mainly catalyzed by Protein S-Acyl Transferases?PATs?through a conserved catalytic domain DHHC.PATs are encoded by multiple genes in all eukaryotic species.In mammalian and yeast,various proteins involved in cell signaling,such as receptors,SNAREs,GTPases,are subject to protein S-acylation.However,few PAT-substrate pairs have been demonstrated.Plant genomes also encode large numbers of PATs but only three PATs,Arabidopsis PAT10,PAT14,and TIP1/PAT24 were functionally characterized.In this study,we examined the functionality of Arabidopsis PATs that are expressed preferentially in tip-growing cells,i.e.root hairs and pollen tubes.By combining reverse genetic and cellular approaches,we demonstrated that?1?Arabidopsis PAT4 mediates root hair growth through the modification of ROP GTPases.Polar growth of root hairs is critical for plant survival and necessitates fine-tuned ROP signaling.Multiple ROP regulators participate in root hair growth.However,protein S-acyl transferases?PATs?,mediating the S-acylation and membrane partitioning of ROPs,are yet to be found.By using a reverse genetic approach,combining fluorescence probes,pharmacological drugs,site-directed mutagenesis,and genetic analysis with related root-hair mutants,we have identified and characterized an Arabidopsis PAT,which may be responsible for ROP2 S-acylation in root hairs.Arabidopsis PAT,which may be responsible for ROP2S-acylation in root hairs.Specifically,functional loss of PAT4 resulted in reduced root hair elongation,which was rescued by a wild-type but not an enzyme-inactive PAT4.Membrane-associated ROP2 was significantly reduced in pat4,similar to that of S-acylation-deficient ROP2 in wild type.We further showed that PAT4 and SCN1,a ROP regulator,additively mediate ROP2 stability and targeting.Results presented indicate that PAT4-mediated S-acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into the dynamic ROP signaling during plant tip growth.?2?Arabidopsis PAT1,PAT2,PAT3,PAT4,and PAT8 redundantly regulate pollen tube reception.Pollen tube reception is a key step leading to reproductive success in angiosperms.After arriving at the micropyle upon sensing female guidance cues,pollen tubes reach the embryo sac and stop growth presumably by sensing and responding to female cues.One synergid cell degenerates and the pollen tube bursts to release sperm cells for double fertilization.Several cell-surface proteins in synergid cells have been identified to be key components in this process whereas receptors in pollen tubes have recently been identified.All these components are involved in the signaling-interplay between the incoming pollen tube and the embryo sac,mutations of which caused overgrowth of the pollen tube.Key factors mediating pollen tube discharge are still unknown.Arabidopsis PAT1,PAT2,PAT3,PAT4,and PAT8?simplified as PENTAPAT?are expressed preferentially in pollen.Subcellular localization analysis showed that PENTAPAT were targeted to the plasma membrane?PM?of pollen tubes.To overcome functional redundancy,hierarchy mutants,quadruple?1;2;3;4,qua?,and quinary?1;2;3;4;8,penta?mutants were generated by combining T-DNA insertion lines and by genome-editing through CRISPR-CAS9.Functional loss of Arabidopsis PENTAPAT reduced fertility due to failed fertilization.The occurrence of unfertilized ovules were randomly distributed in the siliques of the qua and penta mutants and only when mutants were used as the male parent,suggesting compromised male gametophytic function.Compared with the wild type,both the qua and penta mutants showed no difference in pollen development.By in vitro pollen germination assays,we found that functional loss of PENTAPAT caused a slight reduction of pollen germination and tube growth.The mutant pollen tubes burst more often than those of wild type,suggesting a defect in cell wall composion.However,mutants showed no significant different from wild type during in vivo pollen tube growth.The mutants also showed no defect in pollen tube guidance.To further identify the reason for reduced male gametophytic transmission,we pollinated Pro_DD39:NLS-YFP or Pro_DD45:GUS transgenic plants with either wild-type or penta pollen.Pro_DD39:NLS-YFP or Pro_DD45:GUS labels the synergid cells or the egg cell,respectively.We demonstrated that penta pollen tubes failed to fertilize the egg cells and failed to induce synergid degeneration by GUS staining and by confocal laser scanning microscopy,respectively.By introducing Pro_LAT52:GUS or Pro_LAT52:GFP into the mutant background,we showed that mutant pollen tubes failed to discharge,demonstrating a key role of Arabidopsis PENTAPAT in pollen tube reception.Future reseach should be focused on the identification of PENTAPAT substrates in pollen tubes,whose failed S-acylation resulted in the pollen tube reception defects observed in the penta mutant.