| Camellia japonica var.decumbens is a naturally occurring highly cold resistant variety of Camellia japonica which is suitable for snowy and cold regions of honshu coast,Japan.It is mainly distributed in northern iwate,akita and shiga prefectures.The C.japonica var.decumbens is a small evergreen broad-leaved tree with graceful branches.The leaves are dark green and shiny,the flowers are colorful,often meet the snow in full bloom Camellia can be isolated,planted,potted for viewing,and can also be used in gardens,special gardens and landscaping.In addition,the flowers and seeds of Camellia have higher medicinal value,and the seeds can extracted essential oils.Therefore,Camellia not only has high ornamental value,but also has economic application value.However,Camellia is difficult to distribut in the northern region due to temperature restrictions.So it is mostly distributed in the Yangtze River basin.C.japonica var.decumbens is suitable materials for studying the low-temperature adaptation of Camellia.It is distributed in the high latitudes(39°N)of Japan and could bloom beautiful flowers in winter.In this study,the C.japonica var.decumbens were experimental materials of introduced in Japan to study the response mechanism in low temperature stress used habitat climate survey,physiological and biochemical analysis,and transcriptome sequencing analysis in phenotype,internal structure and molecular level The results are summarized as follows(1)In order to investigate the feasibility of C.japonica var decumbens could be introduction to Zhengzhou According to the principle of ’climate similarity ",in view of climate factors such as temperature,precipitation and relative humidity were investigated and analyzed infour urban on C.japonica var decumbens provenances and introduced.The result was as follow:temperature and relative humidity were no significant difference,but precipitation exists significant difference in Zhengzhou and C.japonica var decumbens provenance.Therefore,C japonica var introduced to Zhengzhou,that must be through the necessary control of water management,which C.japonica var decumbens could be gradually adapt to the local climate(2)The leaves of C.japonica var.decumbens from three different provenance of Tsu(C.japonica var decumbens 1),Tokyo(C.japonica var.decumbens 5)and Morioka(C.japonica var.decumbens 9)were used as the test materials to analyze and sequence the cold resistance.Which methods we used to determine leaves physiological and biochemical index of wintering period,determine leaves anatomical structure index by paraffin section technology,and determine leaves semi-lethal temperature(LT50)by Logistic equation on natural low temperature conditions in Zhengzhou.The results show that the physiological and biochemical index and leaves anatomical structure index were exist obvious difference in three provenance of C.japonica var.decumbens,and the semi-lethal temperature of C.japonica var.decumbens 1,5 and 9 were 10.61℃,9.8℃ and 11.93℃ in turn Through principal component analysis and variance analysis of physiological and biochemical index,it was show that the content of chlorophyll and soluble protein,and the activity of POD were closely related to cold resistance in C.japonica var.decumbens.The accumulation of high level of chlorophyll and soluble protein played a roles in the cold resistance in C.japonica var.decumbens 9.Through variance analysis of leaves anatomical structure index,it was show that the thickness of palisade tissue and upper epideimis,and cell tense ratio of C.japonica var.decumbens 9 were exist obvious different than C.japonica var.decumbens 1 and 5.The two layers of palisade tissue played another roles in the cold resistance in C.japonica var.decumbens 9.Comprehensive analysis of the test results,the sequence of cold resistance of three provenance of C.japonica var.decumbens was:C.japonica var.decumbens 9>C.japonica var.decumbens 1>C.japonica var.decumbens 5(3)To analyze the cold tolerance differences in Camellia species of Camellia C.japonica `Naidong’ and C.japonica var.decumbens,under natural low temperatures in Zhengzhou(during Oct.2013 to Jan.2014)and the artificially controlled low temperature stress(4℃,0℃,-4℃,-8℃,-12℃)in climate chamber,separately.Physiological and biochemical indexes such as chlorophyll,soluble protein,proline,POD,SOD,CAT and MDA were measured in C.japonica ’Naidong’ and C.japonica var.decumbens.Low semi-lethal temperature(LT50)were calculated using logistic equation and the cold resistance were comprehensively evaluated using subordinate function method in C.japonica ’Naidong’ and C.japonica var.decumbens.Natural low temperature and the artificially controlled low temperature stress test of two Camellia the results were comprehensive analysed found that,the change rule of these substances was similar.With the increase of low temperature stress,the content of chlorophyll in C.japonica ’Naidong’ was always higher than C.japonica var.decumbens,and the natural stress of C.japonica ’Naidong’ was more volatile.The Soluble protein,POD activity and CAT activity decreased first,then increased after reaching a certain threshold.But the thresholds of physiological indexes have differences between two species of Camellia.The soluble protein and POD activity of C.japonica ’Naidong’ was began to rise by-8℃ treatment,but the soluble protein and POD activity of C.japonica var.decumbens was began to rise by 0℃.The CAT activity both of them were began to rise processing by 4℃.After processing of natural low temperature treatment,the LT50 of C.japonica ’Naidong’ was-14.08℃,the LT50 of C.japonica var.decumbens;was-11.93℃.Combined with the comprehensive evaluation used subordinate function method,the cold resistance of C.japonica ’Naidong’ is better than C.japonica var.decumbens(4)We analyzed the transcriptomic changes caused by cold stress in a cold-tolerant accession.Samples were collected at the end of each temperature treatment(T1,T3,T5,T7 and T9 represent the temperatures 25℃,0℃,-4℃,-8℃ and-12℃,respectively).Sample T1 at 25℃ was used as control.Based on transcriptome analysis,2828,2384,3099 and 3075 differentially expressed genes(DEGs)were up-regulated,and 3184,2592,2373 and 2615 DEGs were down-regulated by analyzing T3/T1,T5/T1,T7/T1 and T9/T1,respectively.A gene ontology(GO)analysis revealed an enrichment of GO terms such as response to stimulus,metabolic process,catalytic activity or binding.Out of the larger number of DEGs,67 functional and regulatory DEGs stood out,since they were functionally characterized in other models.These genes are cold-responsive transcription factors(26)or involved in cold sensor or signal transduction(17)and in the stabilization of the plasma membrane and osmosensing response(24).These results suggest rapid and multiple molecular mechanisms of perception,transduction and responses to cold stress in cold acclimation of Camellia japonica var.decumbens.They could also serve as a valuable resource for relevant research on cold-tolerance and help to explore cold-related genes to foster the understanding of low-temperature tolerance and plant-environment interactions. |
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