Effects Of Different Treatments On Flowering Of Pineapple And Ethylene Induced Spinal Study On Flowering Mechanism Of Dill

Natural flowering is a common problem in pineapple [Ananas comosus(L.)Merr.]cultivation,which seriously restricts the development of pineapple industry.To adjust the harvest time of pineapple fruit,exogenous ethylene(ethephon)has been widely used to induce pineapple flowering.However,economic losses often occur due to inappropriate flower induction,which results in a lower flowering rate or no flowering,and the molecular mechanisms of flowering induction in pineapple remain unclear.In addition,the mechanism of exogenous ethylene induced pineapple flower formation is unique,which is different from model plants(Arabidopsis thaliana and rice)and other woody fruit trees(e.g.citrus and apple).Therefore,understanding the mechanism of ethylene-induced pineapple flowering can not only provide theoretical guidance for artificial flower induction of pineapple,but also enrich the theory of plant flowering regulated by ethylene,which has important theoretical and practical significance.In this study,we applied different treatments on pineapple plants to observe their effects on natural flowering.On the other hand,to understand the global changes in gene expression during ethylene-induced pineapple flowering,we collected the shoot apexes or inflorescences at different time points(0 d,8 h,1 d,4 d,7 d,14 d,21 d and28 d after ethephon treatment),for histological observation,endogenous hormone determination,and combined single-molecule real-time(SMRT)sequencing and Illumina RNA sequencing.Furthermore,we overexpressed AcETR2 a,AcETR2 b 、AcCTR1 in Arabidopsis and pineapple.The results are as follows:1.Uniconazole(200 mg/L)and paclobutrazol(50 mg/L,200 mg/L)treatments significantly inhibit the natural flowering of pineapple;furthermore,100 mg/L,200mg/L uniconazole and 200 mg/L,400 mg /L paclobutrazol treatment also significantly delayed ethylene-induced pineapple flowering.2.Histological observations showed that there were no new structures at 4 days after ethephon treatment(DAET),there were four bract primordia(BP)at 7 DAET,then more bracts and the flower primordia(FP)were observed at 14 DAET.The contents of indol-3-acetic acid(IAA),methyl indole-3-acetate(ME-IAA),and indole-3-carboxaldehyde(ICA)were lower at 1 DAET compared to at 0 DAET,the the content of ME-IAA kept decreasing until it dropped to the lowest level at 14 DAET.In contrast,the contents of IAA and ICA changed little from 1 to 4 DAET,but both obviously increased at 7 DAET and then sharply decreased to the lowest level at14 DAET.However,it should be noted that no 3-Indolebutyric acid(IBA)was detected from 0 to 14 DAET.The change profiles of abscisic acid(ABA),jasmonic acid(JA),and dihydrojasmonic acid(H2JA)were similar;they all significantly decreased from 0 to 1 DAET,were similar at 4 and 7 DAET compared to at 1 DAET,and then were significantly(P< 0.05)higher at 14 DAET compared to at 7 DAET;however,the content at 14 DAET was similar to that at 0 DAET.Both jasmonoyl-L-isoleucine(JA-ILE)and salicylic acid(SA)contents were significantly(P<0.05)higher at 1 DAET compared with at 0 DAET,but then sharply decreased at 4DAET and changed little until 14 DAET,compared with at 4 DAET.The content of trans-zeatin(t Z)was also higher at 1 DAET than at 0 DAET,but the difference was not significant.The highest t Z content was observed at 7 DAET,which was significantly(P<0.05)higher than at 0 DAET.3.In this study,we obtained 29,745 polished high-quality isoforms,of which 523 had not yet been annotated within the A.comosus genome.Furthermore,2,049 alternative splicing(AS)events,78 fusion genes,139 long-chain non-coding RNAs(lnc RNAs),and 11,184 alternative polyadenylation(APA)events were identified by SMRT sequencing.Comparative analysis of these transcriptome databases revealed3,690 differentially expressed genes(DEGs)between 0 d and the other time points,which may be play important roles during ethylene-induced pineapple flowering.4.Overexpression of AcETR2 a significantly delayed the flowering time of Arabidopsis thaliana,but the overexpression of AcETR2 b had no effect on this flowering.However,the function of the two ethylene receptors should be furthure studied in pineapple.The AcCTR1-1 and AcCTR1-2 in pineapple were cloned,and it was found that the expression of AcCTR1-1 and AcCTR1-2 reached the lowest 8h after ethylene treatment;the promoters of AcCTR1-1 and AcCTR1-2 were amplified and analyzed Analysis of cis-acting elements,constructed p GBKT7-AcCTR1-1 and p GBKT7-AcCTR1-2 vectors,and screened with different concentrations of 3-AT.In summary,as inhibitors of the effect of gibberellin,uniconazole and paclobutrazol inhibit pineapple natural flowering and ethylene-induced pineapple flowering,which indicates that gibberellin may play an important role in the flowering of both pineapples;ethephon treatment causes significant changes in the content of a variety of endogenous hormones indicate that ethylene may not only play a role in the process of pineapple flowering induced by ethylene.The data obtained through the third-generation transcriptome sequencing enriches the pineapple genome data.The differentially expressed genes obtained through the second-generation transcriptome comparison sequencing may play an important role in ethylene-induced flowering,and AcETR2 a may play an important role in this process.The functions of AcCTR1-1 and AcCTR1-2 need to be verified in Arabidopsis pineapple in future studies.