Analysis Of Physiological And Molecular Mechanisms Of Cold-resistance In Casuarina Equisetifolia

Casuarina species are valued as pioneer trees for coastal shelter forest construction,which play an important role in the restoration of island ecosystems,the defensive of natural disasters and the improvement of coastal sandy area soil.However,low temperature is one of the main limiting factors for its cultivation expansion,fast growth,and high yield.Therefore,it is important to study the mechanisms of cold-resistance in Casuarina trees,and accelerate the molecular selection and breeding of cold-resistance Casuarina cultivars.In this study,the research on the two group of Casuarina equisetifolia seedlings that one was treated by-5°C for 5 h and the other untreated were carried out by the method of RNA-Seq.The differentially expressed genes and cold resistance related genes were analyzed.The differential expression of the cold resistance related genes in cold-tolerant and cold-intolerant was investigated.Meanwhile,the physiological indexes relating to cold resistence and the influnce of mycorrhizal fugi on cold resistant physiological indexes of C.equisetifolia under low temperature wrer analyzed.This study provided a useful basis for enriching the physiological and molecular mechanism of Casuarina trees incoping with cold stress,and for molecular selection and breeding of cold-tolerant Casuarina clones.The main results are as the followings:（1）The relative conductivity in sixclones of C.equisetifolia were measured under low temperature stresses at-2--11℃.The low half-lethal temperature(LT₅₀)were calculated by fitting the temperature with the relative conductivity based on the logistic equation.The results showed that ZS7 and HN1 were with cold-tolerant and cold-intolerant,respectively.We employed RNA-Seqtechnology to study the genome-wide expression profiles during cold stress in Casuarina equisetifolia.Cold-treated（ZS7）seedlings（at-5°C for 5 h）were sequenced by using the Illuminas equencing platform.A total of 118,270 unigenes were obtained by denovo assembly with the sequence information of 139.4 Mbp,and 47.8%of all unigenes were annotated by COG、KEGG、GO and nr databases.10,291 EST-SSR loci were identified from assembled 118,270 unigenes.The distribution frequency of SSRs was 8.7%.The most common repeat motifs were dinucleotide with AG/CT（54.24%）and trinucleotide with AAG/CTT（13.02%）.The 15 highly polymorphic Casuarina SSR markers were proved to be transferable across Casuarina and Allocasuarina species.The comparison between CD and CK revealed that 4639 genes were differently expressed.GO enrichment and KEGG enrichment analysis showed that these DEGs were mainly relating to biosynthesis of secondary metabolites,plant hormone signal transduction,oxidation-reduction process,regulation of transcription,carbohydrate metabolism etc.Multiple differtial expressin genes were identified,such as CAT2、MSD1、FBA1 etc.Conclusively,the action of the TFs,trigger a cascade of down stream gene activation.These gene products participate in physiological processes of changing the composition of membrane,antioxidant enzyme activities,content of osmoticsubstances to enhance the cold tolerance of C.equisetifolia.（2）The expression patterns of 6 cold resistance related genes（RAP2.7、ABR1、AtHSFA6B、AtbZIP44、GRXC6 and HSP18.2）in cold-tolerant and cold-sensitive Casuarina germplasm were analyzed.Quantitative Real-Time PCR（qPCR）was employed to investigate the differential expression of 6 cold resistance related genes in cold-tolerant（ZS7）and cold-sensitive（HN1）C.equisetifolia clones under continuous low temperature stresses at-2℃,-5℃,and-8℃,as well as precise expression patterns under low temperature stresses at-5℃for 1 h,2 h,5 h,8 h,16 h,24 h,48 h and 72 h.The specific primers for qPCR were designed based on the EST sequences of 6 cold resistance related genes identified from former transcriptome analysis.Under normal temperature condition,the relative expression of all six genes in two C.equisetifolia clones had no significant differences.However,under low temperature stress at-2℃for 2 h,expression of these genes in cold-sensitive clone were strongly inhibited,while in cold-tolerant clone were activated.Under low temperature stress at-5℃for 2 h,expression of these genes were further inhibited in cold-sensitive clone,also in cold-tolerant clone.Under low temperature stress at-8℃for 2 h,expression of all six genes in two C.equisetifolia clones were inhibited continuously.The precise expression patterns analysis revealed that expression of all six genes were significantly up-regulated in cold-tolerant C.equisetifolia clone under low temperature stress at-5℃for 8 h,and the expression level reached the maximum at 8 h,24 h and 48 h,instead it were significantly down-regulated in cold-sensitive C.equisetifolia clone at 1¹6 h,and the expression level reached the minimum at 1⁵ h.On the expression level of cold resistance related genes,different clones showed significantly different responding mechanisms to low temperature stress.Low temperature induces the expression of genes belonging to transcription factors and ROS family,and genes related with the response to ROS in cold-tolerant C.equisetifolia to resist or adapt to cold stress,instead it was inhibited in the cold-intolerant C.equisetifolia,thus significantly decreased its adaptation to cold stress.（3）The physiological response in cold-tolerant and cold-sensitive Casuarina germplasm under successive low temperature stresses,as well as precise expression patterns under low temperature stresses were analyzed.Under successive low temperature stresses,the rising amplitude of H₂O₂ and MDA content were significantly lower than those of HN1.The decrease amplitude of total chlorophyll,soluble protein,proline contents,the activities of SOD,POD,CAT,APX and GR,GSH,GSSG,GSH+GSSG content in ZS7 were also significantly lower than those of HN1.The GSH/GSSG ratio in ZS7 increased gradually,while HN1 were firstly decreased and then increased.Under low temperature stresses at-5℃,the rising amplitude and decrease amplitude in physiological indexes of the two clones were different.Meanwhile,the time to peak of physiological indexes were different.The activities of SOD,POD,CAT,APX and GR,content of total chlorophyll,soluble protein,proline contents and GSH in ZS7higher than these in HN1 generally.Different clones showed significantly different responding mechanisms to low temperature stress.Compared with the cold-intolerant clone,the cold-tolerant clone resisted low temperature by decrease the accumulation of H₂O₂ and MDA,promoting antioxidant enzyme activities and antioxidant contents,and there by reduce the damage of the active oxygen to the leaf and enhance the cold resistance.（4）Two ectomycorrhiza Pisolithus tinctorius and Suillus bovinus were used to inoculated the seedlings of.The effects of mycorrhiza on the cold tolerance of the seedlings under 0℃and-5℃were studied.The results shows that,C.equisetifolia were inoculated with the strain PtLS7522 of Pisolithus tinctorius.Under low temperature stress SOD,POD,CAT activity of inoculated seedlings were significantly higher than that of contrast.MDA content and cell membrane permeability were significantly lower than that of contrast.With mycorrhizal fungi could sinificantly improve the protection of enzyme activity,reduce cell membrane permeability and MDA concent of C.equisetifolia to improve their resistance to cold stress.