The Changes In Eco-physiological And Epigenetic Features Of Leymus Chinensis(Trin).Tzvelev.in Responses To Warming And Nitrogen Addition

Human activity has a profound effect on the global environment and caused frequent occurrence of climatic fluctuations. To survive, plants need to adapt to the changing environmental conditions through altering their morphological and physiological traits. One known mechanism for phenotypic innovation to be achieved is environment-induced rapid yet inheritable epigenetic changes. Therefore, the use of molecular techniques to address the epigenetic mechanisms underpinning stress adaptation in plants is an important and challenging topic in biological research. In this study, we investigated the impact of warming, nitrogen (N) addition, and warming+nitrogen (N) addition stresses on the cytosine methylation status of Leymus chinensis Tzvel. at the population level by using the amplified fragment length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP) and retrotransposon based sequence-specific amplification polymorphism (SSAP) techniques. Our results showed that, although the percentages of cytosine methylation changes in SSAP are significantly higher than those in MSAP, all the treatment groups showed similar alteration patterns of hypermethylation and hypomethylation. It meant that the abiotic stresses have induced the alterations in cytosine methylation patterns, and the levels of cytosine methylation changes around the transposable element are higher than the other genomic regions. In addition, the identification and analysis of differentially methylated loci (DML) indicated that the abiotic stresses have also caused targeted methylation changes at specific loci and these DML might have contributed to the capability of plants in adaptation to the abiotic stresses. The main results in our study are as follows.1AFLP marker results showed that the three groups of treatment which are warming, N addition and warming+N addition in Leymus chinensis does not exist obvious genetic diversity compared with no treatment groups. That enables us to presume that all of these materials used in this study lacked appreciable genetic heterogeneity and the levels and patterns of methylation alterations to be detected should represent pure epigenetic variations.2MSAP and SSAP molecular markers enabled us to assess the stress-associated epigenetic changes in the four types of cytosine methylation alterations (CG hypo, CHG hypo, CG hyper and CHG hyper). The results indicated that, although the three treatment groups had similar frequencies of methylation variation patterns, the types of CG hyper and CHG hyper are higher than the CG hypo and CHG hypo in most of these comparisons. This indicated that the treatment groups have hypermethylated genomes than the untreated control. In addition, the percentages of all four patterns (CG hypo, CHG hypo, CG hyper and CHG hyper) in SSAP analysis were obviously higher than those in MSAP. This result indicated that the regions around the transposable elements had higher frequencies of cytosine methylation changes than other regions of the genome. Furthermore, comparing the percentages of the differentially methylated loci (DML) revealed that the warming+N addition group had the highest proportion of DML and the warming group the least. Similarly, we also found that the percentages of DML in SSAP apparently exceeded those in MSAP, suggesting higher rates of cytosine methylation alterations adjacent to the transposable elements.3AMOVA was performed in this study based on both the MSAP and SSAP datasets. As the MSAP marker revealed,19.1%of the total variance presented among the four groups, it implies that the four groups had obvious differences in cytosine methylation patterns (FST=0.19). In contrast, only a small amount of variation (6.76%) occurred among the four groups in SSAP analysis, suggesting that the regions around the transposable elements of the four groups have diverged only slightly in cytosine methylation variation patterns (FST=0.07).4Seven fragments which recovered, cloned and sequenced from thirty-six methylated DNA fragments of MSAP showed significant homology to known sequences or genes. Specifically, fragments F1, F21and F22were homologous to genomic sequences of Oryza sativa. F5, F16and F20showed similarity to Hordeum vulgare subsp. vulgare and F19shared homology with sequences of Triticum aestivum. Additionally, fragments F1, F19and F20were mapped to the upstream and downstream of beta-expansin1a precursor, glycosyltransferase and ethylene responsive transcription factor, respectively. The other fragments located on the gene body regions of tubby-like F-box protein, UNR-interacting protein, gag-pol polyprotein and DUF295family protein, respectively.5The content of chlorophyll a under N addition treatment increased significantly, but chlorophyll b reduced. Total chlorophyll content is still significantly increased, that showed an obvious increasing trend of chlorophyll content in Naddition treatment Leymus chinensis. Proline and soluble suger content in Leymus chinensis were obviously increased under N addition treatment. The N content of Leymus chinensis was significantly increased under N addition treatment, but P content decreased slightly, showing that the N addition in the soil directly affects the N content of Leymus chinensis. Growth indices of Leymus chinensis under N addition treatment all showed increased except the biomass per unit area.According to the above findings, we believe that abiotic stresses related to global warming and nitrogen deposition readily evoke alterations of cytosine methylation, and which may provide a molecular basis for rapid adaptation by the affected plant populations to the changed environments.