The Mechanism Of Cold Acclimation In Magnolia Wufengensis

Magnolia wufengensis,an arbor species in Magnoliaceae.M.wufengensis has considerable ornamental and economical value owing to its colorful flower(pure,dark,or pale red),varied flower petal numbers(9–25,32,or 46)and various flower shapes(bowl,lotus,cup),especially in Northern China where types of ornamental arbor are quiet limited.However,freezing injury in winter has been a major obstable and threat of M.wufengensis seedling production in northern China.Therefore,developing study of cold tolerance in M.wufengensis is extremely necessary.In this study,we conducted artificial cold acclimation subjected to one-year M.wufengensis seedlings,stem apex were collected and measured cold tolerance-related parameters,and transcriptome analysis were performed based on RNA-seq which uncovered cold-tolerant genes in the regulation of long-term cold acclimation process,key MwWRKYs were selected among numerous DEGs,and role of MwWRKY33 and MwWRKY75 in M.wufengensis were further explored.The main achievements are as following.1.By measuring and analyzing cold tolerance related parameters,as major temperate region perennial plants,cold tolerance of M.wufengensis was developed at non-freezing low temperature(4℃),and under freezing tolerance(0℃)the cold resistance was further strengthened with cold acclimation.2.Basing on Illumina Hi Seq X Ten transcriptome sequencing technology,stem apex of M.wufengensis from different cold acclimation stages were carried out transcriptome sequencing.About 6.43-6.97 G clean data were obtained,number of unigenes were 64,872 with average length1,172 bp.Results indicated a high-quality transcriptome database of Magnolia grandiflora stem apex was obtained,which could provide abundant gene sequence information for subsequent research.3.The common and unique differential genes of M.wufengensis stem apex in response to non-freezing low temperature and freezing low temperature acclimation were analyzed.Photoperiod response,circadian rhythm,photoprotection,plant hormone signaling pathway,cell membrane composition and redox metabolism pathway,stomatal closure were significantly expressed in CA2(4℃)and CA3(0℃).MAPK cascade was more significantly enriched in the non-freezing low temperature(4℃);while ROS was significantly enriched in the freezing low temperature acclimation process,which indicated that ROS accumulation and signal pathway were more important in the freezing temperature pathway(0℃).4.The molecular pathway including signaling,transcriptional factors,hormones and functional key components of M.wufengensis stem apex response to long-term cold acclimation were uncovered.M.wufengensis may initially sense short day and low temperature signaling through cell membrane rigidity and phy B on the membrane,and then conduct the signal from the membrane to the cell nucleus through Ca2 +,plant hormones,ROS and MAPK cascade,then transcription factors,and finally induces cell membrane components,osmotic regulators and anti-freezing proteins and other functional genes regulated the adaptation of M.wufengensis to cold acclimation.5.10 MwWRKYs were cloned and their expression patterns in artificial cold acclimation,natural cold acclimation and artificial cold shock were analyzed.The results showed that 3 of them belonged to group I and 7 belonged to group II.10 MwWRKY was involved in response to different low temperature modes of M.wufengensis in the different ways.6.MwWRKY33 and MwWRKY75 belonging to group I and group II respectively,were further studied.The conserved domain sequences were WRKYGQK.The two genes were expressed in roots,stems,leaves,flowers and buds,indicating that they may participate in various growth and development processes of M.wufengensis.Plantcare online analysis showed the promoter region contained ABA,JA,GA and other hormone cis-acting elements,they were induced by ABA and SA.7.Both MwWRKY33 and MwWRKY75 improved the cold tolerance and salt tolerance of transgenic Arabidopsis.Moreover,it may participate in ABA and SA signaling pathways,and interact with MwVQ4 and MwVQ14 to respond to cold tolerance of M.wufengensis.In conclusion,this study explored the physiological and molecular pathways of M.wufengensis in response to long-term cold acclimation,selected and identified the expression patterns of MwWRKYs during different cooling processes,and verified the functions of MwWRKY33 and MwWRKY75,as well as the possible regulation and interaction modes.All these work provide valuable genetic information for exploring the mechanism of cold acclimation response in woody plants,which has important theoretical and practical significance.