Function And Molecular Mechanism Of PbNAC71 Gene Regulating Dwarfing Traits In Pear

Dwarfing and high-density planting is the trend of modern fruit cultivation and an effective way to realize modernization transformation and upgrading of pear industry.Many dwarfing rootstocks and dwarf varieties are currently used for apple production,but there are relatively few resources for the development of dwarf pear cultivars.Therefore,cultivating excellent dwarf rootstock and dwarf varieties of pear is the key to achieve dwarfing and high-density planting.In this study,we found that grafting ’Zaosu’ pear on’Yunnan’ quince(with ’Hardy’ as the interstock)showed an obvious dwarf phenotype.Subsequently,we isaolated a dwarfing candidate gene,PbNAC71,and verified its function in inducing dwarfing of tobacco and ‘OHF-333’pear.Further,the molecular mechanism of PbNAC71 regulating xylem and vessel development was analyzed.It was found that PbNAC71 protein may be modified by ubiquitination,forming a regulatory model of’PbRNF217-PbNAC71-PbWAT1’,which provided important theoretical basis and reference for breeding excellent dwarf varieties and dwarfing rootstock of pear.The main results of this study are as follows:1.PbNAC71 is a negative regulator of growth,and tobacco with overexpression of PbNAC71 and ‘OHF-333’ pear showed obvious dwarfing phenotype.In this study,we found that grafting ’Zaosu’ pear on ’Yunnan’ quince(with ’Hardy’ as the interstock)(Z/Q)showed an obvious dwarf phenotype.Compared with ‘Zaosu’/‘Duli’(Z/D),the plant height of Z/Q was significantly reduced,the short shoot rate of Z/Q was significantly increased,and annual shoot growth of Z/Q was significantly inhibited.The results of hormone analysis showed that the indole-3-acetic acid(IAA)and abscisic acid(ABA)contents decreased and increased significantly in Z/Q annual shoots,respectively.And the trans-zeatin(TZ)content decreased significantly at 20 DAFB.The cross-sectional examination showed the xylem of the Z/Q shoots was significantly smaller than that of the Z/D shoots.According the RNA-sequencing data,the expression levels of four NAC TF-encoding genes were up-regulated.PbNAC71 was significantly up-regulated in all dwarf germplasm,and significantly down-regulated in response to auxin and cytokinin treatments.Therefore,PbNAC71 was selected as a dwarfing candidate gene.Transgenic tobacco with overexpression of PbNAC71 and ‘OHF-333’ pear were obtained.Compared with the wild type,we found that both transgenic tobacco and ‘OHF-333’ pear plant height decreased significantly.These results indicate that PbNAC71 is a negative regulator of pear growth and development,and overexpression of PbNAC71 can lead to dwarf traits in pears.2.PbNAC71 inhibits xylem and vessel development by binding to SNBE elements on the PbWAT1 promoter to reduce its promoter activity.The results of paraffin section showed that the stem xylem and vessel size of transgenic plants decreased significantly.In order to further clarify the mechanism by which PbNAC71 regulates xylem and vessel development,we further analyzed the transcriptome data and found that two secondary wall synthesis related genes PbWAT1 and PbWAT1-related were differentially expressed.The phenotype of the Arabidopsis thaliana mutant showed that only atwat1 showed a dwarfing phenotype.Therefore,PbWAT1 was screened as a candidate downstream target gene for PbNAC71.Through the Arabidopsis mutant replacement assay,we confirmed that PbWAT1 can regulate xylem development and affect plant height.The results of dual luciferase assay showed that PbNAC71 could inhibit the promoter activity of PbWAT1.Through promoter sequence analysis,we found four SNBE elements on the PbWAT1 promoter.By Y1 H,Ch IP-q PCR and EMSA assays,it was confirmed that PbNAC71 can bind SNBE elements on PbWAT1 promoter both in vivo and in vitro.These results suggest that PbNAC71 reduces its promoter activity by binding to SNBE elements on the PbWAT1 promoter.3.PbRNF217 can promote protein ubiquitination of PbNAC71,thereby regulating xylem and vessel development and plant height.Through the yeast two-hybrid screening library,an E3 ubiquitin ligase PbRNF217 was selected for interacting with PbNAC71.Further,the results of yeast two-hybrid,Bi FC and Pull-down assays demonstrated that PbNAC71 can interact with PbRNF217 protein in vivo and in vitro.The results of Western blot showed that the abundance of PbNAC71 protein decreased significantly in the double-transgenic ‘Duli’ roots.Based on the function of E3 ubiquitin ligase,we speculated that PbRNF217 can promote the ubiquitination degradation of PbNAC71 protein.Through in vivo ubiquitination and in vivo protein degradation experiments,we demonstrated that PbRNF217 can promote ubiquitination degradation of PbNAC71 protein dependent on 26 S proteasome pathway.Subsequently,we overexpressed PbRNF217 in transgenic tobacco overexpressing PbNAC71.Compared with single transgenic tobacco,double transgenic tobacco plant height was significantly increased,Nt WAT1 expression was significantly up-regulated,xylem and vessel size were also significantly increased.Moreover,we also found that after proteasome inhibitor MG132 treatment,the growth of double-transgenic tobacco was significantly inhibited,the expression of Nt WAT1 was significantly down-regulated,and the xylem and catheter size were also significantly reduced.These results indicated that the ubiquitination degradation of PbNAC71 protein was the main reason for the significant increase in plant height,xylem size and vessel area of double-transgenic tobacco.