Analysis Of The Function And Mechanism Of AcSPL1 In The Process Of Ethylene-induced Pineapple Flower Formation

In order to prolong the time of pineapple and achieve the annual supply in the market,the use of ethylene and its derivatives to "urge the blossom" of pineapple to adjust the time to market has become a widely used cultivation technology in current production.However,the molecular mechanism of ethylene signal inducing flowering in pineapple remains an unsolved mystery.It has become one of the most important problems to explore the mechanism of ethylene induced flowering in pineapple production.In model plants,SPL plays an important role in the flowering pathway of age-dependent,but it is not clear whether SPL is involved in ethylene induced flowering of pineapple.In this study,homogenous 15-month-old plants were used in flower forcing and the terminal buds of the pineapple were collected after treatment,and then the flowering ability was evaluated.We analyzed the expression pattern of AcSPL genes in response to ethephon treatment.The biological functions of AcSPL1 were verified through homologous and heterologous transformations,and the interaction proteins related to AcSPL1 were explored through yeast two-hybrid and Bi FC,and illustrated the AcSPL1–dependent regulatory pathway in pineapple flowering.And the major conclusions are listed as following.1.The key AcSPL gene that flowering-induced in response to ethylene was identified.Genome-Wide analysis of AcSPL transcription factor family revealed that a total of16 AcSPLs distributed in 12 chromosomes were identified from the reference genome of pineapple using bioinformatics.The AcSPLs were widely expressed in flower buds,flower organs,pulp,roots and leaves,suggesting that AcSPLs gene family may be involved in the life activities of various tissues of pineapple.The expression differences of AcSPLs at different stages of ethylene-induced flowering were analyzed by qRT-PCR.A total of 5 AcSPLs were were confirmed to respond to ethylene induction,and the expression level of AcSPL1 was significantly induced by exogenous ethylene in the early flowering stage of pineapple.The results showed that the AcSPL gene family was highly conserved during the evolutionary process,and the expression of AcSPL1 was significantly induced by exogenous ethephon.2.The biological function of AcSPL1 gene was determined by stable genetic transformation of homologous(pineapple callus)and heterologous(Arabidopsis thaliana)The full-length sequence of the AcSPL1 gene was cloned,and the open reading frame was 1086 bp which encoded 362 amino acids.In situ hybridization and qRT-PCR showed that ethylene application could significantly increase the expression of AcSPL1 gene during flower transformation.Subcellular localization analysis showed that AcSPL1 gene was localized in the nucleus,which was consistent with the localization characteristics of transcription factors.Ecotopic over-expression of AcSPL1 gene in Arabidopsis could shorten the flowering time of transgenic Arabidopsis thaliana and cause changes in plant short stature and narrow leaf shape.Pineapple genetic transformation experiment showed that the homologous over-expression of AcSPL1 gene in pineapple could accelerate the flowering process.These results demonstrated that AcSPL1 was involved in the flowering process of pineapple,and could significantly advance the flowering time of pineapple and Arabidopsis thaliana.3.AcSPL1 interacting proteins in pineapple were isolated and the interactions between AcSPL1 and AcERF72 were verified in vitro and in vivo.In this study,pineapple cDNA expression library was constructed,and the recombination rate and conversion rate of the library reached 100%.Using AcSPL1 as bait protein,25 candidate interacting proteins with different biological functions were obtained by screening yeast cDNA expression library.The results of yeast two-hybrid,Bi FC showed that AcSPL1 could interact with AcERF72 in vitro and in vivo.The interactions between AcSPL1 and AcERF72 indicated that AcSPL1 could respond to ethylene signal and affect the flowering of pineapple.AcSPL1 was used as a starting point to preliminarily analyze its biological function through molecular genetics.The results showed that AcSPL1 was involved in plant development and flowering process,effectively promoted plant flowering and changed leaf type.The interaction between AcSPL1 and AcERF72 protein revealed the molecular mechanism of AcSPL1 in response to ethylene induction and involved in the regulation of flowering in pineapple.This study may serve as a comprehensive resource for elucidating the mechanism of SPL gene family in ethylene-induced flowering of pineapple.