Molecular Mechanism Of GhNCED And GhABI5 In Regulating Gladiolus Corm Dormancy

Gladiolus is a worldwide important cut flower,and corm dormancy is a main factor that limits its off-season production.In order to investigate the molecular mechanism of Gladiolus corm dormancy and dormancy breaking,Gladiolus cv.'Rose Supreme' was chosen as the material,and the corms were treated with ABA to explore the effect on corm dormancy.Furthermore,transcriptome sequencing of dormant corm at different stage was performed and found the express of GhNCED which is a key enzyme in ABA metabolism,and bZIP family members were changed dramatically.So,we investigated the mechanism of GhNCED and GhABI5 in regulating corm dormancy with VIGS technic,and also investigated their relationship by yeast one hybrid.The main results are listed as follow:1)We preliminarily found that ABA is an essential phytohormone regulating corm dormancy by detecting endogenous ABA during corm dormancy and dormancy breaking,and the effect of exogenous ABA on corm dormancy.2)Different stage of dormant corms were employed for transcriptome sequencing technology.A total number of 276,884,698 clean reads was generated and de novo assembled into 91,293 unique transcripts.About 56.77%of unique transcripts were further extensively annotated by comparing their sequences to protein databases.A total number of 711 significant different expression genes was produced based on Differenet Expression Gene(DEG)analysis,of which 275 genes were constantly up-regulated during corm dormancy breaking,such as CYP707A and PP2C,while 185 genes were constantly down-regulated,like NCED.In addition,the other 251 genes were changed significantly at a certain stage,such as MYB,bHLH,CCCH and bZIP family members.These results suggest that ABA metabolism and ABA signaling are related to corm dormancy and dormancy breaking.3)We detected the gene expression profile of GhNCED by qRT-PCR,the results showed that GhNCED is a constitutively expressing gene and is highly expressed in stolons;its expression is down-regulated along with corm dormancy breaking which is matched the transcriptome data.GhNCED is regulated by ABA,GA and auxin by GhNCED promoter assay;GhNCED exists in the plastid via subcellular localization assay;moreover,silencing GhNCED would promote dormancy breaking.These results suggest that GhNCED has a negatively role on corm dormancy breaking.4)We cloned GhABI5,a bZIP family member,which is a basic leucine zipper motif transcriptional factor(TF).GhABI5 is expressed in vegetative organs(corm,cormel and stolon)as well as in reproductive organs(stamen),and it is up-regulated by ABA or drought.Complementation analysis reveals that GhABI5 rescues the ABA insensitivity of abi5-3 during seed germination and induces the expression of downstream ABA response genes in Arabidopsis thaliana(EMI,EM6 and RD29B).Down-regulation of GABI5 in dormant cormels via Virus Induced Gene Silence(VIGS)promotes sprouting and reduces the expression of downstream genes(GhLEA and GhRD29B).The results of this study reveal that GhABI5 regulates corm dormancy in Gladiolus corm(vegetative organ)in addition to its well-studied function in Arabidopsis seeds(reproductive organ).5)The expression of GhNCED in GhABI5-silenced corm was down-regulated by qRT-PCR.Furthermore,GhABI5 bound the promoter of GhNCED by yeast one hybrid assay.Thus,we hypothesis that GhABI5 forms a feedback to ABA synthesis via directly regulating the expression of GhNCED.Taken together,ABA is an important hormone controlling corm dormancy and dormancy breaking,and its metabolism and signaling pathway are involved in regulating corm dormancy.Both GhNCED and GhABI5 inhibit corm dormancy breaking.In addition,GhABI5 involves in ABA feedback via binding the promoter of GhNCED.