Studies On The Molecular Mechanism Of ABA And Related Phytohormones-regulated Pear Bud Dormancy

Pear is the third largest fruit in China after Malus and Citrus,in both cultivation area and yield.In winter,pear has a key feature of transition from growth to bud dormancy i.e.’a pause in visible growth’.It is a unique physiological phenomenon of perennial woody plants including deciduous fruit trees,which can help plants to survive from low temperature damage and safely pass through winter.Dormancy are classified into para-dormancy,endo-dormancy and eco-dormancy according to the different causing factors.The endo-dormant bud cannot break even under suitable environmental conditions,and it can only restore its growth ability after satisfying its chilling requirement.After the endo-dormancy release,the buds still remain in a state of growth inhibition due to the low temperature.When the temperature gradually rises,the external environmental conditions become suitable to resume growth.If the winter low temperature accumulation can not fulfill the minimum chilling requirement of fruit tree,the tree will not be able to pass endo-dormancy normally and the next year will appear irregular flowering,flower fertility declines etc.and ultimately affect the fruit production.So,the regulation mechanisms of bud dormancy are of great significance to the development of fruit tree industry.In this study,the expression of phytohormone related genes during dormancy was analyzed by transcriptome analysis,we found that the expression of ABA signal transduction genes significantly changed,then we explored the role of ABA in the’Suli’dormancy process by measuring the ABA content during flower bud dormancy and applying exogenous ABA on cutting shoots during different dormancy stages.Further clarified the regulation of DAM by CBF genes,which function downstream of ABA.In addition,it was found that BR might play an important role in the upstream of ABA.Molecular mechanisms of ABA and related phytohormones on regulating pear bud dormancy were partially elucidated.The main results are as follows:1)Pear buds in different dormancy stages were selected for second-generation sequencing and mixed samples were used for third-generation sequencing.The third-generation results were corrected by the second-generation sequencing results then compared with pear genome.We obtained 34,996 transcripts,of which 21,799 were alternatively spliced isoforms of known transcripts,and each of the identified genes have 1.89 splicing isforms,which were significantly higher than the genome result.After the second-generation sequencing results were compared,16.448 differentially expressed genes were screened,and 20 phytohormone signal transduction-related genes were screened by mfuzz cluster analysis,11 of them were ABA signal pathway related genes.Combining with these results we hypothesized that ABA may regulate the dormancy process of pear buds through signal transduction pathway genes.Then we checked the ABA content during dormancy,the results showed that ABA increased during pear flower bud endodormancy establishment and decreased towards endodormancy release.During the para-to endo-dormancy transition,PpNCED-2 and PpNCED-3 had high expression levels,while PpCYP707As expression levels were low.However,during endodormancy,the expression of PpCYP707A-3 sharply increased with increasing cold accumulation.At the same time,the ABA content of pear buds declined,and the percentage of bud breaks rapidly increased.On the other hand,the expression levels of PpPYLs,PpPP2Cs,PpSnRK2s,and PpABI4/ABI5s were also changed during the pear flower bud dormancy cycle.Furthermore,exogenous ABA application to para-dormant buds significantly reduced the bud breaks and accelerated the transition to endodormancy.During the whole treatment time,the expression level of PpPP2C-12 decreased to a greater extent in ABA-treated buds than in control.However,the expression levels of PpSnRK2-1,PpSnRK2-4,and PpABI5-1 were higher in ABA-treated buds.Our results indicated that PpCYP707A-3 and PpNCEDs play pivotal roles on the regulation of endodormancy release.while ABA signal transduction pathway also appears to be involved in the process.2)Since CBF functions downstream of ABA.and CBF can transcriptionally regulate the expression of DAM genes.To study whether ABA regulates dormancy through CBF,we identified four CBF and three DAM genes from the pear cultivar’Wonhwang’,and checked their expressions during natural dormancy and artificial chilling treatments.Under natural conditions,PpDAMl expression decreased from the start of chilling accumulation,while the other two DAM and three CBF genes peaked during endodormancy release.Under chilling treatment,the expressions of PpDAM1,PpDAM2 and PpCBF1 genes were similar to those under natural conditions.Different biochemical methods revealed that PpCBF2/4 can bind to the promoter of PpDAMl and activate its expression and that PpCBF1/4 can activate PpDAM3.Interestingly,we found that PpCBF2 can activate PpCBF3/4 transcription by directly binding to their promoters.Combining with other results of our research group,we found that ABA can indirectly activate the expression of DAM gene through CBF.At the same time,we identified that the major CBF and DAM genes regulating internal dormancy are PpCBFl and PpDAM2.3)The BR content of ’Suli’ flower bud during dormancy were analyzed by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS-MS).We only identified TY for the BR content in pear bud is low,and the content of TY during dormancy did not change significantly.Then we studied the expressions of BR-related genes during dormancy by using real-time quantitative PCR,and found some differentially expressed genes,which may play a role in dormancy regulation.By applying exogenous brassinosteroid on the shoots at different dormancy stages,we found that BR could promote the release of dormancy only after a certain chilling accumulation.Then the expression pattern of BR/GA/ABA-related genes in BR-treated shoots were studied.The results showed that BR can inhibit the expressions of ABA-synthesis genes NCEDs and promote the expression of ABA-degrading genes CYP707As.In addition,GA synthesis gene GA20ox is up-regulated in BR-treated shoots.The GA degradation gene GA2ox was down-regulated,indicating that BR may affect the dormancy process by balancing the content between ABA and GA.