PpIDD4,PpIDD12 And PpIDD13 Are Involved In DELLA-dependent Feedback Regulation Of Gibberellin Biosynthesis And PpIDD11 Regulates Pistil Stigma Elongation In Peach

Peach（Prunus persica）is an important economic fruit tree in Rosaceae.However,the annual growth of peach branches and the difficulty of tree management are the problems that restrict the rapid development of peach industry.The pruning of redundant branches increased the labor cost of orchard,wasted tree nutrition and polluted the environment.On the other hand,peach blossoms have important ornamental value,and the study of peach floral organ development is of great significance to improve its ornamental value.Previous studies have shown that gibberellin（GA）is an important factor for the rapid growth of peach branches,and also affects the flower bud differentiation and flowering process of peach,but its biosynthesis regulation mechanism in peach is not clear.INDETERMINATE DOMAIN（IDD）is a plant-specific transcription factor involved in the regulation of floral morphogenesis and gibberellin feedback regulation.In this study,we identified IDD gene family members in peach,and proved that three IDD members（PpIDD4,-12 and-13）can interact with PpDELLA1 and participate in DELLA-dependent GA biosynthesis feedback regulation.At the same time,this paper also identified an IDD gene（IDD11）regulating pistil stigma development.The main research results are as follows:1.PpGA20ox1 and PpGA3ox1 were proved to be key members involved in the feedback regulation of GA biosynthesis in peach by using the dwarf mutant ‘FHSXT’ as the experimental material,which contains large accumulation of PpDELLA1.Previous work showed that a large amount of PpDELLA1 protein and active GAs were accumulated in ‘FHSXT’,suggesting that the feedback regulation of DELLA-dependent GA biosynthesis in ‘FHSXT’ was significantly activated.In order to clarify whether PpGA20ox1 and PpGA3ox1 are key members involved in the feedback regulation of GA biosynthesis in peach,we first developed a method to significantly improve leaf transformation efficiency by weak light treatment,and PpGA20ox1 pro-GUS was transferred into the leaves of self-bred progeny of ‘FHSXT’,with non-dwarf ‘QMH’ as the control.The results showed that PpGA20ox1 promoter was significantly activated in the leaves of ‘FHSXT’.At the same time,this study showed that the transcription levels of GA biosynthetic genes PpGA20ox1 and PpGA3ox1 were down-regulated under GA₃ treatment,while the effect was opposite under PBZ（GA biosynthesis inhibitor）treatment.2.Three IDD members（PpIDD4,-12 and-13）in peach interact with PpDELLA1 protein.In this study,14 IDD family members were identified in peach genome.The phylogenetic tree showed that 14 IDD members belonged to six subgroups of seven different subgroups,including Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ and Ⅶ.Among them,three members of PpIDDs（PpIDD4,-12 and-13）in peach belonged to the same group as At IDD2（GAF1）involved in the feedback regulation of GA biosynthesis in Arabidopsis,and PpIDD11 belonged to the same group as ZmID1 and OsID that regulate flower development.Y2 H and Bi FC assays verified that PpIDD4,-12 and-13 could interact with PpDELLA1.This suggests that there may be multiple IDD-DELLA complexes involved in regulating GA biosynthesis in peach.3.Three IDD members（PpIDD4,-12 and-13）and PpDELLA1 in peach activate the expression of PpGA20ox1 and PpGA3ox1 in the form of complexes.Eight and nine IDD candidate cis-acting elements were identified by analyzing the promoter regions of PpGA20ox1 and PpGA3ox1,respectively.The results showed that PpIDD4 and-13 mainly bind to site 3 of PpGA20ox1 promoter and site 1 of PpGA3ox1 promoter,while PpIDD12 bind to site 5 of PpGA20ox1 promoter and site 5 of PpGA3ox1 promoter.The core sequence of the cis-acting elements recognized by the three PpIDD proteins was TTGTC.Meanwhile,dual luciferase assay showed that the expression of PpGA20ox1pro-LUC or PpGA3ox1pro-LUC could not be activated by PpIDD or PpDELLA1 alone,while the expression of PpGA20ox1pro-LUC or PpGA3ox1pro-LUC could be activated by PpDELLA1 when co-transformed with PpIDD4,-12 or-13.4.PpIDD4,-12 and-13 can promote the growth of Arabidopsis.Tissue-specific analysis showed that PpDELLA1,PpIDD4,-12 and-13 were highly expressed in shoot and stem.Transgenic Arabidopsis lines overexpressing PpIDD4,-12 and-13 were constructed.Overexpression of PpIDD4,-12 and-13 promoted plant growth and hypocotyl elongation.Through the hypocotyl test of GA₃ / PBZ treatment,it was found that GA₃ treatment promoted the hypocotyl elongation of PpIDD4,-12 and-13 overexpressed Arabidopsis lines,and PBZ treatment inhibited the hypocotyl elongation.Among them,the hypocotyl of PpIDD12 overexpressed Arabidopsis lines responded most significantly to GA.5.PpIDD11 regulates pistil stigma elongation.The flower development related gene PpIDD11 was cloned from ‘QMH’ peach.The subcellular localization of PpIDD11 in tobacco showed that PpIDD11 protein was located in the nucleus,and yeast transformation assay showed that it has transcriptional self-activation activity.RT-q PCR analysis showed that PpIDD11 was mainly expressed in floral organ,especially in pistil.The transgenic lines overexpressing PpIDD11 showed higher stigma,shorter pods and lower seed setting rate than the wild type.At the same time,PpIDD11 overexpressed Arabidopsis lines showed curly rosette leaves and dwarf plants.The transcriptome sequencing analysis showed that there were 3378 up-regulated genes and 3156 downregulated genes in PpIDD11 overexpressed Arabidopsis lines,including LOX4,LOX3,GLC,E6L1 and other floral organ development regulatory genes.In summary,14 IDD gene family members were identified from peach,which verified that PpIDD4,-12 and-13 regulated GA biosynthesis key genes PpGA20ox1 and PpGA3ox1 by forming complexes with PpDELLA1,and realized feedback of GA biosynthesis in peach.At the same time,PpIDD11 can promote pistil elongation and affect flower development related gene expression in Arabidopsis.In this study,the regulatory mechanism of IDD family members involved in GA biosynthesis and pistil stigma elongation in peach was analyzed,which provided a theoretical basis for further study on branch elongation and floral organ development in peach.