Molucar Mechanism Of Freezing-Tolerance Differentiation Of Alien Invasive Crofton Weed (Ageratina Adenophora) Through Evolution Of ICE-CBFs Transcription Pathway During Its Invasion Northward

Crofton weed (Ageratina adenophora (Spreng.) R.M.King & H.Rob., Syn: Eupatorium adenophorum Spreng.) was early introduced to Southeast Asia. It firstly spread into the tropical and subtropical areas of southwest China around 1940s and has become one of the worst alien invasive weeds in China. The previous studies in Weed Research Laboratory, Nanjing Agricultural University showed that during its invasion to China this weed had involved into different freezing-tolerant populations through freezing-tolerant adaptability differentiation, which may be associated with the adaptive differentiation of the ICE1-CBFs transcription pathway. Hence, the cold responsive transcription factor CBF1 (C-repeat binding factor), its upstream regulator ICE] (inducer of CBF expression) and downstream COR gene were cloned, and further the expression patterns of CBF1, ICE1 genes were compared among different populations under cold treatment. Cold-tolerant HGG had higher expression level of these two genes than cold-susceptible BSG. However, it’s still unclear about what causes the difference in the gene expression levels.ICE] (inducer of CBF expression) gene, a positive upstream regulator for an expression of CBFs, regulates an expression of a series of downstream cold induced genes by associating with CRT/DRE DNA regulation element, and it can improve the ability of plants resistant to freezing stress. However, how the ICEI transcription is regulated in freezing-tolerance adaption remains unknown. A survey on the distribution of Crofton weed in China was conducted, and a serious of molecular biology experiments were performed:to clone and analyze the expression patterns ofAaCBF2, AaCBF3 and AaCOR, amplify the promoter sequences of AaICE1 and AaCBFS and compare the methylation sites of AaICE1 and AaCBF3 genes and promoter sequences. The main purposes aim to clarify the reason leading to the different expression patterns of AaI, AaCBFs and AaCOR-, and then explain the molecular mechanism of freezing-tolerance differentiation of Crofton weed during its invasion to China.1-Geographical distribution of freezing-tolerance evolution populations of Crofton weed.Eighteen more Crofton weed populations were collected and their cold-tolerance was assessed. Adding to the 16 populations which had been assessed before, the freezing-tolerant ability of a total of the 34 geographical populations were compared based on the injury index, using Imaging-PAM and Handy-PEA. The correlation analysis between Crofton weed freezing-tolerant abilities and latitude, extreme minimum temperature and coldest month average temperature were also carried out. Then the distribution pattern of Crofton weed in China was described. Results showed that all Crofton weed populations had differentiated into different freezing-tolerant abilities. BSG (Baise, Guangxi) and six other populations were cold-sensitive; DLY (Dali,Yunnan), JHY (Jinghong, Yunnan) and 17 other populations were moderate freezing-tolerant, while HGG (Huangguoshu, Guizhou) and seven other populations were freezing-tolerant. The freezing-tolerant abilities of different Crofton weed populations had. significantly positive correlation with latitude (P<0.001), and were significantly negatively correlated with extreme minimum temperature (P<0.01) and the coldest month average temoerature (P<0.01). The freezing-tolerant populations mainly distributed in the northern parts of the sampling sites in China, cold-sensitive and moderate freezing-tolerant populations mainly distributed in the southern and middle part of China. This indicates that the freezing-tolerant abilities of Crofton weed populations differentiated during its invasion to China and exhibited the trend of invasion to the northern China with the time extension.2. Differentiation of ICE-CBFs transcription pathway among different freezing-tolerant populations.Cloning of AaCBF"3 and AaCBF2 genes. The AaCBF3 and AaCBF2 genes were cloned from HGG using the methord of RACE. The full-length cDNA of AaCBF3 and AaCBF2 are 908bp and 923bp long respectively, with a 720bp and 654bp ORF (open reading frame) encoding 240 and 218 amino acids. Both the two genes have no introns. The AaCBF3 and AaCBF2 genes belong to the typical AP2 super-family, which plays a key role in DNA binding and the activation of COR gene. Comparing the AaCBF3 and AaCBF2 gene sequences isolated from freezing-tolerant population (HGG), cold-susceptible population (BSG) and two intermediate freezing-tolerant populations of Yunnan Jinghong (JHY) and Dali (DLY), the sequences are completely the same.Expression patterns of AaCBF2, AaCBF3 and AaCOR:the relative expression level of AaCBF3 and AaCBF2 genes of different geographical populations were performed by Quantitative RT-PCR. Results showed that there was no expression of either AaCBF3 or AaCBF2 gene. Expression of AaCBF3 gene reached the peak at 4 hours under treatment of 4℃. The expression was the highest in freezing-tolerant population HGG, while BS expression level was the lowest. AaCOR, a downstream gene of AaCBF3, expressed slower than AaCBF3, all reached the peak after 12h treatment, and HGG population had higher expression level of AaCOR than other populations while BSG the lowest. AaCBF2 gene expressed rapidly under cold treatment, gene expression reached the maximum value after lhour under cold treatment of 4℃in cold-sensitive population BSG, the performance was the same in freezing-tolerant population HGG but the expression level was significantly lower. Expression pattern of moderate-resistant populations DLY and JHY was the same, gene expression amount reached the maximum at 0.5hour under treatment of 4℃, slightly decreased at lhour and maintained at high level during 2-8hour treatment, but the expression level was lower than the BSG population. These results all had the same expression patterns with these in other plants reported, indicating that AaCBF3, AaCBF2 and AaCOR genes were closely related to the freezing-tolerant abilities of Crofton weed.3-Molecular mechanism of the differention of ICE-CBFs transcription pathway.Comparison of the 5’flanking sequences of AalCEl and AaCBF3 genes:the 5’ flanking sequences of AalCEl and AaCBF3 genes of different Crofton weed populations were cloned using the method of Tail-PCR, and sequences of 1043bp and 977bp were obtained respectively. The 5’flanking sequence of AaCBF3 gene containes one TATA box (-99～-94 bp), one G-box (-116～-110 bp), three MYC recognition sites (-271--266bp,-217～-212bp and-171～-166bp) and one MYB recognition sites (-214～-209bp), AaICE1 DNA gene 5’flanking sequence contains one TATA box (-108～-103bp). Different populations of Crofton weed shared the same AaICE1 and AaCBF3 gene 5’ flanking sequences.Comparison of the methylated sites of AaCBF3 and AaICE1 genes and the promoters:methylated sites of AaICE1 and AaCBF3 gene of various populations were detected after the genomic DNA treated by bisulfite. It was found that the methylation level of AaICE1 gene was various in different geographical populations, and the difference of methylated sites located in the frontier 623bp region. The methylation sites of AaICE1 in freezing-tolerant populations, moderate freezing-tolerant populations and freezing-susceptible populations were 50,61-63 and 66, respectively. Correlation analysis showed that AaICE1 gene methylation level, especially the degree of methylation in the front 623 bp, was closely related to the freezing tolerance level of Crofton weed populations (P<0.001). Hence, the stronger was the cold resistance of the population, the lower AaICE1 gene methylation level, indicating that the demethylation of AaICE1 contributed to the differential of ICE-CBFs transcription pathway.So we can come to the conclusion that AaICE1 gene, an upstream regulater of cold-responsive genes, can regulate its expression level by controlling its methylation level, leading to the adaptive cold-tolerance differentiation of Crofton weed during its invasion to China.