Mechanisms Of E3 Ubiquitin Ligase ATL17 Regulating ABA Inhibition Of Primary Root Growth In Arabidopsis Thaliana

Abscisic acid(ABA)plays an important role in promoting the development of plant roots.Nevertheless,the detailed molecular mechanism remains to be elucidated.E3 ubiquitin ligase is involved in the regulation of many plant growth processes by degrading proteins.However,whether ATL17,a E3 ubiquitin ligase RING family member,regulates the primary root development is not clear.In the present study,we identified ATL17 T-DNA insertion homozygous mutants(atl17-1 and atl17-2)of Arabidopsis,and analyze the role of ATL17 in root growth regulated by ABA using these genetic materials.We found that ATL17 localized to the plasma membrane.In addition,GUS staining results revealed that ATL17 was expressed in the root tip and the stele of primary root,and highly expressed in leaves and flowers in Arabidopsis.Phenotypic analysis revealed that no significant differences in primary root elongation existed between wild-type and atl17 mutants in the absence of exogenous ABA.However,the primary roots of the mutants grew significantly longer than those of WT after 0.5 ?M,0.7 ?M or 1 ABA treatment.These results indicate that these mutants are insensitive to ABA-inhibited primary root elongation.Moreover,when overexpressed ATL17 genein the atl17-1 mutants,the primary roots of RE1 and RE2 transgenic plants were more sensitive to ABA,indicating that ATL17 plays an important role in ABA inhibition of primary root development.The lengths of primary roots in different parts were counted after ABA treatment.It was found that the length of the meristem and elongation zone of atl17 mutants was significantly greater than that of WT,while the length of these zones of RE1 and RE2 was smaller than that of WT.These findings imply that ATL17 regulates primary root development by affecting the growth of root meristem and elongation zones.Next,the cell number and cell size in meristem and elongation zones of the mutants and WT main roots were detected.It was proved that the cell number but not the cell size of the mutant was significantly more than WT,suggesting that ATL17 regulates the elongation of the primary root by affecting cell division rather than cell differentiation.Further study shown that exogenous NPA(naphthyl phthalamic acid),the auxin extra transport inhibitor,could attenuate the difference in root elongation between the ATL17 mutants and WT in the presence of ABA,where as NOA(naphtha eneoxyacetic acid),an auxin-internal inhibitor,could not restore the ABA-insensitive phenotype of the atl17 mutants.These results indicate that ATL17 affects the ABA-inhibited primary root development by modulation of the external transport of auxin.With the addition of ethylene synthesis precursor(ACC),the insensitive phenotype of primary root elongation of the atl17 mutants to ABA was restored.This indicates that ATL17 may mediate ABA supressed root growth by influencing ethylene signalingResults from DAB staining experiments indicated there was significant difference in the levels of H2O2 in primary roots between WT and the atl17 mutants after exposure to ABA,These data imply that H2O2 play an important role in ABA suppression of root growth in ArabidopsisThe expression of some genesin roots was detected after treatment with ABA for different time periods.These genes included auxin-related genes AUX1(AUXIN1),PIN1(PIN-FORMED1)and PIN2,ROS-related genes RbohD(Respiratory burst oxidase homologue D)and RbohF,as well as cell cycle associated genes CYCB2;1(B2-type cyclins),CYCD3;1(D3-type cyclins),CYCA2;1(A2-cyclins),CDKA1(Cyclin-dependent kinase Al)and KRP2(Kip related protein 2).The results showed that the expression levels of AUX1,PIN1 and PIN2 were significantly higher in atl17 mutants than in WT,and the expression levels of CYCB2;1 and CDKA1 were remarkably higher than those of WT.However,the expression level of CYCA2;1 was lower than that of WT,those of CYCD3;1,RbohD and RbohF remained unchanged,and the expression of in 7 mutants was lower than that in WT.These results suggested that ATL17 may regulate ABA inhibited primary root development by blocking the expression of AUX1,PIN1,PIN2,CYCB2;1 and CDKA1.Ubiquitination experiments showed that the ATL17 protien has E3 ubiquitin ligase activity.In addition,in vitro experiments revealed that ARF2 could combine with the element of AuxREs(TGTCTC)in the promoter of ATL17.