| An important characteristic of temperate deciduous perennial plants is their ability to maintain a dormant state in extreme environments.During the perennial plant life cycle,buds transit through the various stages of dormancy(para-,endo-,and eco-dormancy)(in this paper,the dormancy refers to endodormancy).In this state,the meristem is rendered insensitive to growth-promoting signals for some time before dormancy is released and the plants resume growth.Dormancy regulation in buds is a complex process that is necessary for plant development,flowering and fruiting,adaptability,survival,and geographic distribution.Knowledge about mechanisms regulating dormancy induction,maintenance and release may provide the basis for solving critical problems in agriculture,especially irregular blooming and prolonged flowering periods in deciduous fruit trees cultivated in warm temperate regions.Recent genomics-and transcriptomics-based studies have provided insights into some of the basic aspects of the molecular mechanisms of dormancy regulation.Up to now,it has been accepted that cold signaling and plant hormones(mainly ABA/GA)are crucial in regulating the bud dormancy in fruit trees including pear.However,only the function of Dormancy-associated MADS-box genes(DAMs)involved in bud dormancy has rudimentary illustrated,the molecular mechanism of chilling and GA/ABA in regulating bud dormancy has rarely reported.Furthermore,how does the expression of these genes involved in dormancy turn on and off specifically is still unknown.An increasing volume of evidence indicates that DNA methylation is involved in regulating gene expression and gene silencing in many physiological processes including bud dormancy.In order to better understand the molecular network and mechanism pear dormancy,the study on the transcription regulation and epigenetic regulation was conducted during bud dormancy in 'Suli' pear(Pyrus pyrifolia White Pear Group).The main results were as follows:The role of DAM(Dormancy-associated MADS-box genes)in pear dormancy transition was studied systematically.The 30 MIKCc-type MADS-box genes(MIKC~C)were identified and analyzed to determine their phylogenetic relationships with homologous genes,genome locations,gene structure,tissue-specific transcript profiles,and transcriptional patterns during flower bud dormancy in 'Suli' pear.The roles in regulating bud dormancy varied among the MIKC~C gene family members.Yeast one-hybrid and transient assays showed that PpCBF enhanced PpDAM1 and PpDAM3 transcriptional activity during the induction of dormancy,probably by binding to the C-repeat/DRE binding site,while DAM proteins inhibited the transcriptional activity of PpFT2 during dormancy release.In the small RNA-seq analysis,we identified 185 conserved,24 less-conserved,and 32 pear-specific miRNAs with distinct expression patterns during bud dormancy.Joint analyses of miRNAs and MIKC genes together with degradome data showed that miR6390 targeted PpDAM transcripts and degraded them to release PpFT2.Our data showed that crosstalk among PpCBF,PpDAM,PpFT2 and miR6390 played important roles in regulating endo-dormancy.A model for the molecular mechanism of dormancy transition is proposed:short-term chilling in autumn activates the accumulation of CBF,which directly promotes DAM expression;DAM subsequently inhibits FT2 expression to induce endo-dormancy and miR6390 degrades DAM genes to release endo-dormancy.Based on high-performance liquid chromatography,coupled with tandem mass spectrometry,we developed and established a novel method to quantify four distinct endogenous hormones through ultra-performance liquid chromatography-tandem mass spectrometry(UPLC/ESI-MS/MS),which achieves higher throughput screening and improved resolution than HPLC or HPLC/ESI-MS/MS.The method was simple,rapid,and precise,and was applied for the determination of plant hormones in pear tissue,with recoveries ranging from 70.11%to 89.84%and relative standard deviations ranging from 4.25%to 14.96%.ABA concentrations were measured in pear flower buds during bud dormancy.The endogenous ABA concentration rose sharply with deepened endo-dormancy and then dropped sharply during the endo-dormancy release.To understand further the effect of ABA on dormancy transition,the expression of HD-Zip genes which are important ABA response transcription factors were investigated.A total of 47 HD-Zip genes were identified in the pear genome and further divided into 4 sub-families.The 47 genes were analyzed to determine their phylogenetic relationships with homologous genes,genome locations,gene structure,and transcriptional patterns during flower bud dormancy in'Suli' pear.During bud dormancy transition,PpHB22 and PpDAM1 showed similar transcription patterns.Their transcript levels increased and peaked from 15 November to 15 December during endo-dormancy,and then decreased rapidly.Yeast one-hybrid and transient assays showed that PpHB22 enhanced PpDAMI transcriptional activity during the induction of dormancy,which inferred that PyHB22 might activate PpDAM1 to induce bud dormancy through a CBF-independent pathway in 'Suli' pear.The excised shoots of 'Suli' pear were incubated in 5-azacytidine solution(DNA methylation inhibitor)as a treatment and water without 5-azacytidine as control for different chilling treatments,then DNAs from treated and control buds were sequenced using WGBS and RNA-seq.We found different methylation patterns for three sequence contexts(mCG,mCHG and mCHH)and in different genic regions,potentially with different roles in gene expression.Prcentage of mCG sites were methylated in all three tissues is higher than precentage of mCHG and mCHH sites.The number of mC sites decreased from paradormancy to endodormancy,then increased during the dormancy release.This trend was true for all sequence contexts.Differentially methylated regions(DMRs)also were identified in promoters,function elements and the interior of genes involved in plant hormone signal transduation,starch and sucrose metabolism and other dormancy-associated pathways. |
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