Identification Of Interactions Between Aux/IAA And ARF Family Proteins In Chinese Hickory (Carya Cathayensis Sarg.) And Their Potential Roles In Regulating Grafting Survival

Chinese hickory(Carya cathayensis Sarg.)is a high-quality nut and woody oil tree species native to China.Growing problems such as big tree size,poor stress resistances,low level of improvement has restricted the development of Chinese hickory industry.Grafting can be regraded as an effective way to solve the problems emerged in Chinese hickory industry.Revealing the mechanism of Chinese hickory during grafting will provide both theoretical and practical references for industrial transformation and upgrading of Chinese hickory industry.The key roles of auxin in regulating grafing success of Chinese hickory has been confirmed in previous studies.In auxin signaling pathway,individual Aux/IAA(auxin/indole-3-acetic acid)and ARF(auxin response factor)proteins interacts with each other to regulate the expression of aunxin responsive genes.However,the interactions between the Aux/IAA and ARF family proteins in Chinese hickory and whether they participate in regulating grafting survival is less studied.In this study,CcARF family genes were cloned and structurally characterized,the complex interactions between CcARFs and CcIAAs family proteins were examined by Yeast Two-hybrid System(Y2H)and Bimolecular Fluorescent Complimentary(Bi FC)techniques,differentially expressed CcARFs and CcIAAs genes during grafting were examined by transcriptome sequencing and quantitative real-time PCR(q RT-PCR),and the interactions between grafing-related CcARFs and CcIAAs were discussed.The main findings are as follows:(1)The coding sequences of 25 CcARFs were identified,which encoded proteins with the number of amino acids of 394 to 1123 and the number of exons of 1 to 15.CcARFs belonged to hydrophilic and unstable proteins,and were closely related with the homologous proteins in Carya illinoinensis.Various phytohormone-related cis-acting elements were detected on the promotors of CcARFs.(2)Subcellular localization analysis showed that most CcARFs and CcIAAs proteins were localized in the nucleus.However,CcARF19 a and CcARF19 b were localized in the cytoplasm,while CcARF7 a,CcIAA16 and CcIAA28 were localized in both nucleus and membrane.(3)Self-activation was detected in 7 of the 22 CcIAAs-BD bait proteins.Interactions between the 15 CcIAAs without self-activation and 25 CcARFs by Y2 H showed that significant interactions were observed among 113 CcARFs-CcIAAs combinations and weak interactions were observed among 14 CcARFs-CcIAAs combinations.Among the 25 CcARFs,CcARF2 d,CcARF4 a,CcARF9 b and CcARF16 c interacted with more than 10 CcIAAs.Among the 15 CcIAAs,CcIAAx,CcIAA28,CcIAA29 a,CcIAA29 b,CcIAA16 and CcIAA27 b interacted with more than 10 CcARFs.The interactions of partial CcARFs-CcIAAs combinations were further verified by Bi FC technique.(4)The expression levels and trends of CcARFs and CcIAAs varied during grafting.5 CcARFs and5 CcIAAs were identified as differentially expressed genes during grafting,among which CcIAAs were down-regulated while CcIAAs were up-regulated.Among differentially expressed genes,interactions between the five combinations of CcARF2d-CcIAA27a1,CcARF4a-CcIAA27a1,CcARF4a-CcIAA4,CcARF6a-CcIAA27a1 and CcARF16d-CcIAA4 were detected.CcIAA27a1 and CcARFs were mainly expressed in stems,highly expressed duing the whole grafting,and interacted with each other at protein level,implying their potential roles in regulating grafting survival of Chinese hickory.