The Role Of Heterotrimeric G Protein In Arabidopsis Thaliana Roots' Avoidance Response To EATP The Role Of Ethylene Responsive Factor ERF_X In EATP-regulatedauxin Abundance And Transport In Arabidopsis Thaliana Seedlings

ATP,which is synthesized in plant cells,can be used as "energy currency" to provide energy for plant growth and development.ATP can also be released to the extracellular matrix.Extracellular ATP(eATP)as an messenger,participate in regulation of plant growth&development or environmental adaptability.So far,the signal transduction of eATP regulated root growth and development is still unclear.Previously,results from our laboratory demonstrated that roots of Arabidopsis thaliana seedlings growing in jointed medium bent upon contact with ATP-containing medium to keep away from eATP,showing a marked avoidance response.To clarify eATP perception and responding,two items were investigated in this work.1.The role of heterotrimeric G protein in Arabidopsis thaliana roots'avoidance response to eATPPrevious results in our lab showed that roots of Arabidopsis thaliana "sense" the existence of eATP and response to eATP by negative tropic growth.We defined it as"e ATP avoidance" growth manner.To verify the signal transduction mechanism of eATP avoidance growth of Arabidopsis thaliana seedling roots,lost-of-function mutants of heterotrimeric G protein ?,?,? subunit were used as materials,the root growth of different seedlings which were growing in ATP-containing jointed mediums was investigated.Results showed that,the growth avoidance response to eATP was weakened in roots of heterotrimeric G protein a subunit null mutants(gp?1-1?gp?1-2).The roots of G?null mutants(agb1-1?agb1-2)or Gy null mutants(agg1-1?agg1-2)responded to ATP similarly to the wild type.These results indicate that heterotrimeric Ga subunit may play an important role in the process of eATP avoidance growth.To further explore the role of Ga in eATP induced physiological responses,DR5-GFP and D?-VENUS fusion genes were transformed into wild-type(WS)and Ga null mutants(gp?1-1,gp?1-2),effect of eATP on concentration and distribution in root cells of auxin was investigated by using confocal laser scanning microscopy.The results showed that the fluorescent intensity of DR5-GFP in the root cells,especially in the quiescent center and surrounding cells increased significantly after 24 hours of ATP treatment.At the same time,the DR5-GFP was asymmetrically localized,the auxin concentration in the inner side cells was higher than that in outer side at the bending area of roots.24 hours after ATP treatment,it was found that the fluorescent intensity of D?-VENUS in root tip cells,especially in the quiescent center and surrounding cells decreased significantly.And the asymmetric distribution of D?-VENUS was detected,the fluorescent intensity in the inner-side of root curve was significantly higher than in the outer-side.These results indicate that the concentration of auxin in the root tip cells of wild-type seedlings was increased,and the accumulation of auxin may inhibit the growth of primary roots.Roots was much more sensitive to auxin,the auxin accumulation may suppress,the cell growth of the inner-side,led root bending which result in avoidance response eventually.In Ga null mutants(gp?1-1,gp?1-2),the fluorescent intensity of DR5-GFP in the root tip cells increased,while the location did not change.In the Ga null mutant seedlings in which D?-VENUS was transformed,the fluorescent intensity of D?-VENUS in root tip cells decreased while their location did not change.The results indicated that eATP induced the increase of auxin concentration in root tip cells of Ga null mutants(gp?1-1,gp?1-2),and the accumulation of auxin may inhibit the root elongation of seedlings as well.However,the asymmetric distribution of auxin,which appeared in wild-type,was not detected in Ga null mutants.So,Ga may regulate the avoidance response of Arabidopsis thaliana seedlings to eATP by participating the re-distribution of auxin.To verify the role of heterotrimeric Ga in ATP-induced asymmetric distribution of auxin,PIN1-GFP,PIN2-GFP,PIN3-GFP,PIN7-GFP and AUX1-YFP fusion genes were transformed into wild-type(WS)and Ga null mutants(gp?1-1,gp?1-2),effect of eATP on content and distribution of anxin transporters were investigated by using confocal laser scanning microscopy.PIN1-GFP was mainly located in the stele,PIN3-GFP and PIN7-GFP was mainly located in stele and columella cells,AUX1-YFP was mainly located in stele,columella cells and epidermal cells.24 h after ATP treatment,the fluorescent intensity of PIN1-GFP,PIN3-GFP,PIN7-GFP and AUX1-YFP decreased markedly while their location did not change.PIN2-GFP was located in epidermis and cortex.After ATP treatment,the fluorescent intensity of PIN2-GFP decreased dramatically,and fluorescent intensity in the inner side of root curve was significantly higher than in the outer side.The decreasing of PIN1,PIN2,PIN3,PIN7 and AUX1 content in root tip cells may result in decreasing of auxin content and subsequent root growth inhibition.The asymmetric distribution of both PIN2 and DR5-GFP in root tip cells suggests that PIN2 may regulated the avoidance response of Arabidopsis thaliana roots to eATP by directing the asymmetric distribution of auxin.After ATP treatment,the fluorescent intensity and distribution of PIN1-GFP,PIN3-GFP,PIN7-GFP and AUX1-YFP were similar between wild type and the two Ga null mutants,indicating that Ga was not involved in eATP regulated expression and localization of PIN1,PIN3,PIN7 and AUX1.The fluorescent intensity of PIN2-GFP decreased after ATP treatment.The distribution of PIN2-GFP did not change significantly in root tip cells of the two Ga null mutants(gp?1-1?gp?1-2),indicating that Ga may be involved in roots' response to eATP through regulating auxin transport which mainly charged by PIN2.To further clarify the role of Ga in ATP-induced root bending,the gene expression pattern in root tip cells was detected by DNA microarray and real-time qPCR analysis.Results of DNA microarray showed that,60 min after ATP treatment,650 genes were up-regulated and 765 genes were down-regulated in WS,622 genes were up-regulated and 702 genes were down-regulated in gpa1-2.Expression of some functional genes,which associated with auxin metabolism or auxin response were significantly up-regulated in WS while not up-regulated in G? null mutants,indicated that Ga may be involved in ATP-regulated expression of functional genes.In order to further explore the role of these functional genes in eATP avoidance response,lost-of-function mutants of WAG1 and WAG2 were used as materials,the root growth rate and orientation of seedlings grown in jointed mediums was investigated.Results showed that,roots of WAG1 null mutants(wag1-1?wag1-2?wag1-3?wag1-5)responded to eATP more sensitively than wild type,and roots of WAG2 mutants(wag2-1?wag2-2)show enhanced avoidance response comparing with wild-type.The result indicated that WAG1 and WAG2 played different role in the eATP avoidance response of Arabidopsis thaliana roots.Based on above results,we suggest the possible signal transduction mechanism of eATP avoidance response of Arabidopsis thaliana roots:"eATP?G??PIN2?auxin asymmetric distribution? root bending".2.The role of ethylene responsive factor ERFX in eATP-regulated root auxin abundance and transport in Arabidopsis thaliana seedlingPrevious studies have found that an ethylene response factor(ERFx)play a key role in eATP regulated growth and development of seedlings.For seedlings growing in light,eATP inhibited root elongation.While,primary root of ERFx null mutants(erfx)did not responsed to ATP.For etiolated seedlings growing in dark,eATP induced"triple response" which appeared in wild-type hypocotyls,was impaired in ERFx null mutant(erfx).To futher explore the mechanism of eATP regulated root and hypocotyl growth,DR5-GFP fusion gene was transformed into wild type and erfx,effect of eATP on content and distribution of auxin was investigated.The results showed that the fluorescent intensity of DR5-GFP in the root cells,especially in the quiescent center and surrounding cells increased significantly 24 hours after ATP treatment.In ERFx null mutants(erfx),the fluorescent intensity of DR5-GFP in the root tip cells did not change,indicating that ERFx may be involved in eATP induced increase of auxin concentration in root tip cells.To verify the role of ERFx in ATP-induced asymmetric auxin distribution,PIN1-GFP,PIN2-GFP,PIN3-GFP,PIN7-GFP and AUX1-YFP fusion genes were transformed into wild-type(Col-0)and ERFx null mutants(erfx),effect of eATP on content and distribution of anxin transporters were investigated.After ATP treatment,the fluorescent intensity and distribution of PIN1-GFP,PIN2-GFP,PIN3-GFP and AUX1-YFP were similar between wild type and the ERFx null mutants(erfx).However,fluorescent intensity of PIN7-GFP in columella cells decreased in wild-type while not in ERFx null mutants(erfx).The results indicated that ERFx regulated PIN7content and distribution may participate in e ATP-regulated root elongation.In etiolated wild-type seedlings,auxin concentration in hypocotyl cells increased significantly after eATP treatment.At the bending curve,DR5-GFP content in the outer side cells was significantly higher than that in the inner side cells.The results indicate that asymmetric auxin distribution may lead bending growth of hypocotyls.The expression and localization of auxin transporters were also affected by eATP in etiolated seedlings.After ATP treatment,in hypocotyl cells of etiolated wild-type seedlings,PIN3-GFP fluorescent intensity in the outer side of the hypocotyl curve was significantly higher than in the inner side.However,the asymmetric distribution of PIN3-GFP did not appear in hypocotyl cells of ERFx null mutants.The expression and distribution of PIN1,PIN2,PIN7 and AUX1 in hypocotyls cells did not significantly change after ATP treatment.These results indicated that ERFx regulated PIN3 content and distribution may participate in eATP-regulated bending growth of hypocotyls.Based on above results,we suggest the possible signal transduction mechanism of root elongation growth of Arabidopsis thaliana seedlings:"eATP?ERFx?PIN7?auxin?root growth".Similarly,the signal transduction mechanism of hypocotyls curvature growth:"eATP?ERF?PIN3?auxin asymmetric distribution?hypocotyls bending growth".