The Study On Physiological And Molecular Mechanism At Flowering Stage Of Camellia Oleifera Response To Low Temperature Stress

Tea-oil tree(Camellia oleifera Abel.)is an important woody edible oil tree species in South China,and tea oil is the best edible vegetable oil.The development of C.oleifera industry is of great significance to ensure the safety of grain and oil and industrial poverty alleviation in mountainous areas.C.oleifera is a kind of self-incompatibility plant.It blooms in autumn and winter,and often encounters continuous cold weather in the flowering period.As it is vulnerable to low temperature stress,resulting in low fruit setting rate and low yield,which seriously affects the healthy development of C.oleifera industry in China.Therefore,it has become an important subject of C.oleifera scientific research to study the mechanism of cold resistance and to seek effective methods to prevent and control cold injury.At present,studies on the low temperature stress of C.oleifera at home and abroad mainly focus on the effects of low temperature on flowering and pollination,the physiological response to low temperature stress of C.oleifera seedlings,and evaluation of cold resistance among different cultivars.However,due to the lack of in-depth study on the physiological mechanism of cold resistance of C.oleifera at flowering stage and systematic theoretical guidance,the prevention technology of cold injury of C.oleifera at flowering stage is still pending.This study using4-year-old potted plants of C.oleifera including late-flowering cultivar'Huashuo'and early-flowering cultivar'Huaxin'which were widely popularized in Hunan Province as objectives,and studied the responses of different organs(leaves and flowers)of these two cultivars to low temperature stress through morphological and anatomical observation,physiological and biochemical determination,metabolic transcriptome analysis,etc.The purpose of this study is to provide a theoretical basis for the promotion of C.oleifera cultivars and screening of key candidate genes for cold resistance in order to assist resistance breeding,and to provide a reference for the high yield cultivation techniques of C.oleifera.The main contents and results of this study are as follows:1.Phenotypic physiological observation of two C.oleifera cultivars under different temperatures.The comparison of flowering,pollen viability,fruit setting rate,fruit growth and leaf anatomical structure of two C.oleifera cultivars under simulated low temperature(6?),simulated normal temperature(25?)and field conditions were studied by using 4-year-old potted plants of'Huashuo'and'Huaxin'as material.The results showed that the flowering time of two cultivars was delayed,and the flowering period was prolonged under simulated low temperature.Besides,the pollen viability was reduced at 6?,and some flower buds were in'dormancy'state.At6?,the leaf thickness was increased while the chlorophyll content was decreased.Besides,the stomata closed and the chloroplast ultrastructure of'Huaxin'suffered a certain degree of damage.To some extent,the changes of leaf anatomical structure reflected that the cold resistance of'Huashuo'was stronger than that of'Huaxin'.Compared with treated at 6?and field conditions,the fruit setting rates of two cultivars were significantly increased.In addition,the young fruits of two cultivars had no dormancy period and began to grow rapidly at 25?.Therefore,temperature is the main environmental factor that causes the dormancy of C.oleifera young fruit.In addition,a large number of honey-like mucilaginous substances appeared in the flower base of two C.oleifera cultivars under long-term low temperature stress,which was a unique physiological response of C.oleifera to cold stress.2.The 4-year-old potted plants of two cultivars('Huashuo'and'Huaxin')were used to study the influence of low temperature of 6?(25?as control)on the photosynthesis,chlorophyll fluorescence parameters,Ru BP carboxylase activity,antioxidant enzyme activity,hormone content of the leaves of C.oleifera.The results showed that the net photosynthetic rate(P_n),stomatal conductance(G_s),transpiration rate(T_r),Ru BP carboxylase activity,electron transfer rate(ETR),maximum photochemical efficiency(F_v/F_m),actual photochemical quantum efficiency(?_PSII)and photochemical quenching coefficient(q P)of the two cultivars decreased under low temperature stress,while the activities of initial fluorescence(F_o),superoxide dismutase(SOD)activity,sucrose synthetase activity and IAA content were increased.The catalase(CAT)activity,polyphenol oxidase(PPO)activity,the salicylic acid(SA)and brassinolide(BR)content decreased first and then increased,while the peroxidase(POD)and ethylene content(ETH)changed in the opposite direction.In conclusion,the decrease of photosynthetic efficiency of two C.oleifera cultivars under low temperature stress was mainly caused by stomatal factors,and the activity of antioxidant enzymes in C.oleifera leaves changed with stress time.The cold resistance of two cultivars under long time low temperature stress had certain enhancement,and the leaves of'Huaxin'were more sensitive to low temperature than'Huashuo'.3.In order to study the molecular mechanism of cold acclimation of C.oleifera,Illumina NGS technology was used to perform transcriptomic analyses of C.oleifera'Huaxin'leaves under long-term cold stress to identify genes related to cold tolerance and explore key candidate genes for chilling-tolerance.Nine c DNA libraries were sequenced,and 58.31 Gb high-quality reads were obtained with an average of 5.92 Gb reads for each sample.A total of 191,150 transcripts were obtained after assembly.Among them,100,703 unigenes were generated,and 44,610 unigenes were annotated.In total,1564 differentially expressed genes(DEGs)were identified related to cold tolerance.The current study performed Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses combined with determination of physiological and revealed a group of cold-responsive genes related to hormone regulation,photosynthesis,membrane systems,and osmoregulation,and these genes encoded many key proteins in physiological metabolism,such as protein kinase,transcription factors(TFs),fatty acid desaturase(FAD),lipid-transfer proteins(LTPs),sugar metabolizing related enzymes and flavonoid biosynthetic enzymes,etc.In addition,the expression levels of 12 key candidate DEGs were verified using quantitative real-time polymerase chain reaction(q RT-PCR),and the results were consistent with the RNA-seq.In summary,exploring key candidate genes provided scientific basis for molecular breeding of cold resistance of C.oleifera.4.Studies on the physiological and molecular mechanism of C.oleifera flowers in response to low temperature.In order to explore the molecular mechanism of the honey-like mucilaginous substances at the flower base of C.oleifera under long-term low temperature stress,flower buds of two C.oleifera cultivars('Huashuo'and'Huaxin')were used as the test material,using UPLC-MS/MS and Illumina NGS technology performed the target metabonomics and transcriptomics for C.oleifera flower buds treated under low temperature stress for different times(1,7 and 25 days).Exploring the regulatory mechanism of C.oleifera flower buds in response to low temperature stress through comparing the changes of sugar and hormone content combining with gene expression between two C.oleifera cultivars and in different stages of low temperature treatment,the purpose is to provide scientific basis for the prevention of cold injury in the flowering period.The main results are as follows:1)D-fructose,D-galactose,glucose and sucrose were main detected saccharides in the flower bud under cold stress,and they increased with stress time.In addition,the accumulation speed of saccharides accelerated in the late stage of cold stress.The four kinds of saccharides in the flower buds of'Huaxin'were higher than that of'Huashuo',indicating that the flower bud of Huaxin had strong tolerance to low temperature.Auxin synthesis was induced in the early stage of low temperature stress,and long-time cold stress induced the accumulation of ABA and SA,and there were differences of hormone responses to cold stress between two cultivars.2)Twenty-four c DNA libraries were sequenced,and 197.74 Gb high-quality reads were obtained with an average of 8.24 Gb reads for each sample.A total of319,549 transcripts were obtained after assembly.Among them,234,040 unigenes were generated,and the length of N50 is 778bp.3)The changes at transcription level in the flower buds of C.oleifera were related to different stages of low temperature stress.The key DEGs associated with cold stress responses in the early stage mainly included early auxin response genes,antioxidant enzyme genes and flavonoid metabolism related genes.The key DEGs associated with cold stress responses in the late stage mainly encoded protein kinases,carbohydrate metabolism related enzymes.Therefore,in the early stage of cold stress,the buds responded to cold stress mainly by improving the activity of antioxidant enzymes and accumulate secondary metabolite such as flavonoids,while in the late stage of cold stress,they mainly response to cold stress by accumulating saccharides.4)The differences at transcription level in the flower buds between two C.oleifera cultivars were studied at 25 DAY,and the results showed that'Huaxin'was more active than'Huashuo'at transcription level.The expression of large numbers of DEGs was up-regulated,and these DEGs were mainly associated with signal transduction and saccharides metabolism.The expression of above genes were closely related to the dynamic changes of saccharides content,indicating that the response differences to cold stress between two cultivars may be caused by different accumulation of saccharides.The results above were consistent with the phenotype observation that'Huaxin'bloomed firstly under low temperature and some honey-like mucilaginous substances appeared at the base of flowers in the late flowering period.5)Transcriptome analysis identified the key candidate genes for cold resistance of C.oleifera buds,including genes of phenylalanine ammonia lyase(PAL),glutathione S-transferase(GST)and peroxidase(POD);the main genes related to flavonoid metabolism were ANS,F3'H and CHS,which were significantly different in the early stage of cold acclimation;the main genes related to sugar metabolism were?-glucosidase genes(Bglu),?-galactosidase genes(GLBs),?-amylase gene(BAMYs),hexokinase genes(HXKs)and sucrose transporter genes(SUTs),which were significantly different in the late stage of cold acclimation.q RT-PCR was used to detect the relative expression of 12 key candidate DEGs,and the results were basically consistent with the RNA-seq.