Physiological Changes And Gene Expression Pattern In Response To Low Temperature Stress In Prunus Mume

As a traditional flower,mei(Prunus mume Sieb.et Zucc.)is highly appreciated by the Chinese people.Mei is historically distributed south of the Yellow River,while cultivation further north is limited by low temperature.Based on continuous hardy cultivars breeding and regional trial studies,the understanding of cold tolerance mechanisms in mei is improving.However,the molecular mechanism of mei response to overwintering low temperature stress is unclear.In this study,multi-year multi-area field evaluations of seven mei cultivars and low temperature stress physiological experiments on four mei cultivars were conducted.On these bases,‘Songchun' was selected as the material at three sites and three time points to sequence the transcriptomics,and the physiological changes and gene expression patterns of mei in response to low temperature during the overwintering period were systematically analysed.The main results are as follows:(1)Field evaluations at five test sites showed that ‘Yan Xing' was the hardiest(-31.4 ?)among distant hybridization cultivars,followed by ‘Songchun'(-29.1 ?),‘Fenghou' and ‘Dan Fenghou',‘Meiren' was the least hardy cultivar.Physiological experiments with low temperature stress showed that ‘Songchun' was the highest,followed by,‘Meiren',‘Sanlun Yudie' and ‘Xiaohong Zhusha'.During stress,the relative conductivity and MDA content of ‘Songchun' were lower than those of ‘Xiaohong Zhusha',indicating that the membrane was less damaged in hardy cultivars.Mei can respond to low temperature stress by increasing the cellular soluble sugar content and the activity of protective enzymes(SOD,POD,and CAT).(2)‘Songchun',which was hardy and had different overwintering performance at three test sites,was selected for transcriptome sequencing,and samples were collected at three time points(early stage of leaf falling in fall,final stage of endo-dormancy in winter and early stage of bud swelling in spring)and three test sites(Beijing,Chifeng and Gongzhuling).The total number of DEGs between the three sites on winter sampling days(5813)was more than on fall sampling days(3559)and spring sampling days(3445),suggesting that the largest transcriptional differences among three test sites occurred in winter,consistent with the temperature factor.The transcriptome sequencing data also showed that there were 1559(6.4%),727(3%)and 2410(10%)DEGs at three sites during the cold acclimation,and 3517(14.5%),4177(17.3%),4691(19.1%)DEGs at three sites during the deacclimation.More DEGs responded during the deacclimation at either test site,suggesting that the deacclimation should not be viewed simply as the opposite process of the cold acclimation.(3)After sensed,the low temperature signal was transduced through the Ca2+ signal transduction system and MAPK cascade,in conjunction with hormone signal transduction pathway and phosphatidylinositol signaling system,thus rapidly activating a series of transcription factors(ICE,CBF,WRKY,Hsf,etc.)that regulate downstream the up-or down-regulated expression of genes related to lowtemperature-induced proteins(Pm LEAs,HSP83),starch and sucrose metabolism(BGLU42,GNS1,XYL1,CEL1),photosynthesis(pbs A),antioxidant enzyme metabolism(GLO1,CAT1)and lipid metabolism(ADS3).The activation or repression of these genes regulated cell stability,light adaptation,ROS balance,and membrane lipid composition,promoting the adaptation of ‘Songchun' to low temperatures.These results also indicated that ‘Songchun' had the molecular basis for overwintering in Beijing,Chifeng and Gongzhuling.(4)The JAZ protein is the core node of cross-talk among the jasmonate acid signaling pathway,the gibberellin signaling pathway,and the ICE-CBF signaling pathway.Fifteen members of TIFY family were identified in the mei genome,eight of which belong to the JAZ subfamily.Transcriptome analysis showed that Pm JAZ2 and Pm JAZ4 were co-expressed differentially in three sites during the deacclimation,suggesting that JAZ protein was important for adaptation to overwintering low temperatures in mei.Twelve Pm TIFYs were highly expressed in fall or winter,suggesting that gene function of Pm TIFYs could be related to cold stress response.The expression analysis of the Pm JAZs under controlled cold stress showed that the expression of Pm JAZ2 and Pm JAZ6 were induced by low temperature stress.This is the first transcriptomics study on overwintering mei trees,combined with field evaluations,physiological and molecular biology experiments,to analyse the physiological and transcriptional changes of mei in response to low temperature stress.Ultimately,this study proposed the hypothetical model of differential gene expression events in mei during natural cold acclimation/deacclimation responding to low temperature,which will provide a theoretical basis for the hardy cultivars breeding and “the acclimatization to the north” of mei.