Preliminary Study On Subcellular Localization And Function Of SAV4

The plant hormone auxin plays a major regulatory role in embryogenesis,apical dominance,lateral root formation,hypocotyl elongation,tropic response to light and gravity,vascular tissue differentiation and lateral branch differentiation of buds.A mutant sav4(Shade Avoidance 4)with defects in shade-induced hypocotyl elongation and basal auxin transport was previously identified in our laboratory.By observing the localization of SAV4-YFP in the transgenic plant SAV4::SAV4-YFP/sav4,it was found that SAV4 was localized in the nucleus and the cell membrane on the side of the bud.According to protein structure prediction,SAV4 contains ARM(Armadillo repeat domain)and TPR(Tetratricopeptide repeat like)domains,which can be involved in the interactions between proteins.In this paper,we investigate the subcellular localization and function of SAV4,SAV4 and WAG1 negatively regulate the root waving and the mechanism of SAV4 affecting hypocotyl negative gravitation was studied from three aspects.First:We found that the ARM domain determines the nuclear localization of SAV4,and the ARM and TPR domains jointly determine the polar membrane localization of SAV4.We found that none of the transgenic plants without polar membrane localization or nuclear localization could recover the phenotype of SAV4 under shading,indicating that nuclear and polar membrane localization of SAV4 was very important for the function of SAV4 under shading.The previous results showed that the SAV4 protein was modified by palmitylation,and we constructed the transgenic plant SAV4::SAV4-M5C-YFP/sav4 with the palmitylation site of mutant SAV4.It was found that SAV4-M5C-YFP had polar membrane localization and nuclear localization,and could also restore the phenotype under the shade of sav4.These results indicated that the palmitylation site mutation did not affect the subcellular localization and shading function of SAV4.However,we found that SAV4-M5C-YFP degrades faster in vacuole after dark treatment,indicating that palmitylation modification may stabilize the positioning of SAV4 at the top of the plasma membrane.Second:The interaction between SAV4 and WAG1 was screened by yeast hybrid in the early experiment.In this paper,yeast double-hybrid experiment was used to further explore the interaction domain between SAV4 and WAG1,and it was found that WAG1 only interacted with full-length SAV4.We also proved that SAV4 can directly interact with WAG1 through Pull down and tobacco BIFC experiments.We found that S AV4 was also involved in the negative regulation of root waving.We found that the root waving of sav4 was more obvious than that of the wild type,which was similar to the phenotype of wag1,indicating that SAV4 and WAG1 may jointly influence the root waving.Further studies showed that wag1 mutation could lead to decreased localization of the polar membrane of SAV4,suggesting that WAG1 may affect the localization of the top of the plasma membrane of SAV4.Through in vitro Pull down experiment,we found that WAG1 enhanced the interaction between SAV4 and PIN2HL(the hydrophilic ring of PIN2).Moreover,yeast double-hybrid experiments proved that WAG1 may not enhance its interaction with SAV4 through phosphorylation of PIN2JL.In addition,this paper verified the interaction between WAG1 and PIN2 by using Pull down experiment,and found that SAV4 did not affect the interaction between the two.Combined with the above results,we speculate that WAG1 may affect auxin distribution and root development through influencing the localization of SAV4 and the interaction between SAV4 and PIN2.Thirdly:previous yeast double hybridization experiments showed that SAV4 interacted with PGP19C(cytoplasmic free fragment of PGP19C).In this study,we demonstrated that SAV4 can interact with PGP19C through tobacco BIFC experiment.The hypocotyl of sav4 would show a bent phenotype due to the asymmetrical distribution of auxins in the hypocotyl under dark conditions,and the negative gravity response of the hypocotyl would be enhanced after gravity stimulation.We found that pgp19 had a similar phenotype to sav4 mutant.We hypothesized that SAV4 regulates auxin lateral transport in hypocotyls by affecting the function of PGP19.In order to test whether there is genetic interaction between SAV4 and PGP 19,we further tested the phenotype of sav4pgp19 after dark treatment,and found that the hypocotyl curve of the double mutant was stronger than that of sav4 and pgp19,and the negative gravitation of the hypocotyl of sav4pgp19 was more obvious than that of the of the single mutant after gravity stimulation.These results suggest that SAV4 may affect auxin distribution in hypocotyls through other proteins besides PGP19.In this paper,we find that the enhancement of negative gravitaty of sav4 is related to the polarization of PIN3 during the gravitaty response.In sav4 mutants,the recovery of PIN3 polarization induced by gravity stimulation is slowed down,leading to a hyperflexural phenotype after gravity stimulation.