Chilling- and Freezing- Induced Alterations in Cytosine Methylation and Its Association with the Cold Tolerance of an Alpine Subnival Plant, <i>Chorispora bungeana</i>

<div><p>Chilling (0–18°C) and freezing (<0°C) are two distinct types of cold stresses. Epigenetic regulation can play an important role in plant adaptation to abiotic stresses. However, it is not yet clear whether and how epigenetic modification (i.e., DNA methylation) mediates the adaptation to cold stresses in nature (e.g., in alpine regions). Especially, whether the adaptation to chilling and freezing is involved in differential epigenetic regulations in plants is largely unknown. <i>Chorispora bungeana</i> is an alpine subnival plant that is distributed in the freeze-thaw tundra in Asia, where chilling and freezing frequently fluctuate daily (24 h). To disentangle how <i>C</i>. <i>bungeana</i> copes with these intricate cold stresses through epigenetic modifications, plants of <i>C</i>. <i>bungeana</i> were treated at 4°C (chilling) and -4°C (freezing) over five periods of time (0–24 h). Methylation-sensitive amplified fragment-length polymorphism markers were used to investigate the variation in DNA methylation of <i>C</i>. <i>bungeana</i> in response to chilling and freezing. It was found that the alterations in DNA methylation of <i>C</i>. <i>bungeana</i> largely occurred over the period of chilling and freezing. Moreover, chilling and freezing appeared to gradually induce distinct DNA methylation variations, as the treatment went on (e.g., after 12 h). Forty-three cold-induced polymorphic fragments were randomly selected and further analyzed, and three of the cloned fragments were homologous to genes encoding alcohol dehydrogenase, UDP-glucosyltransferase and polygalacturonase-inhibiting protein. These candidate genes verified the existence of different expressive patterns between chilling and freezing. Our results showed that <i>C</i>. <i>bungeana</i> responded to cold stresses rapidly through the alterations of DNA methylation, and that chilling and freezing induced different DNA methylation changes. Therefore, we conclude that epigenetic modifications can potentially serve as a rapid and flexible mechanism for <i>C</i>. <i>bungeana</i> to adapt to the intricate cold stresses in the alpine areas.</p></div

5-Methylcytosine-binding level assays and the expression analysis of three target genes.

<p>(A) The levels of <i>CbADH1</i>, <i>CbUGT</i> and <i>CbPGIP</i> bound to 5-methylcytosine in chilling (4°C) and freezing (<i>-</i>4°C) treatments. (B) The expression level of these three genes at 23<i>°C</i>, chilling (<i>4°C</i>) and freezing (<i>-</i>4°C).</p

The plant material collection site and a photograph of <i>C</i>. <i>bungeana</i>.

<p>(A) The black-filled triangle displays the geographic position of the collection site on the map, approximately 43°05’N, 86°49’E, altitude 3,800–3,900 m, near the No. 1 glacier in the source area of the Urumqi River in the Tianshan Mountains, Xinjiang, China. (B) Photograph of <i>C</i>. <i>bungeana</i>, flowering in snow and ice. (C) A researcher was working in the alpine talus of the collection site.</p

Principal component analysis (PCA) of the different cold treatments.

<p>Principal component analysis (PCA) of the comparisons of DNA methylation changes among the 23°C, chilling (4°C) and freezing (-4°C) treatments at each treatment time. (A) 0.5 h, (B) 3 h, (C) 12 h, (D) 24 h. The trends in DNA methylation changes at 23°C, 4°C and -4°C are represented by red circles, cyan circles and purple circles, respectively. Numbers 1–5 represent five individual plants at 23°C, 6–10 represent five individual plants at 4°C, and 11–15 represent five individual plants at -4°C. The percentage of the variance that is explained by each principal component is given in the iconograph near the axes. The first two components together explain greater than 50% of the variations in all of the PCA modes.</p

Non-metric multidimensional scaling (NMDS) analysis for variation in methylation epigenotypes.

<p>Non-metric multidimensional scaling representing the variation in methylation epigenotypes between samples. The dynamic pattern of epigenetic divergence among the 0, 0.5, 3, 12 and 24 h in 23°C, 4°C and -4°C treatments based on presence (1) ⁄ absence (0) scores of 16 polymorphic methylation-sensitive amplified fragment-length polymorphism (MS-AFLP) markers. The first two components of the NMDS analysis are extracted and plotted against each other; the small symbols are individual plants (n = 5), while the large symbols with the cross indicate the mean ± SD of the treatment group. (A) At 4°C for 0–24 h. (B) At -4°C for 0–24 h. <i>R</i>² values represent the proportion of the variance that is explained by the first two components. Axis 1 explains the majority of the total variation in both (A) and (B).</p

Isolation and directional sequencing of MS-AFLP fragments.

<p>Methylation occurs if bands only appear in 0h rather than any other time of the cold treatment (0.5, 3, 12, 24h). However, demethylation occurs if the opposite is true.</p><p>The homologs of <i>Arabidopsis</i> genes were presented for functional description.</p

Results of the generalized linear model (GLM) testing of the effects of cold treatments on DNA methylation variations of <i>C</i>. <i>bungeana</i> genome in response to chilling (4°C) and freezing (-4°C) for five periods of time (0–24 h).

<p>In the model, dummy indicator variables are manually constructed for eight cold treatments (at 4°C and -4°C for 0.5, 3, 12 and 24 h). The difference between each of the eight cold treatments and the 23°C (for 0.5, 3, 12 and 24 h) is assessed by comparing the full model with the model from which the respective term is removed using the Generalized likelihood ratio test (GLRT). The effects of markers (different primer pairs) and treatment times are also examined.</p><p>¹, Significant effects (<i>p</i><0.05) are printed in bold, while the number in italic font indicates marginal significance (0.05<<i>p</i><0.1).</p><p>Results of the generalized linear model (GLM) testing of the effects of cold treatments on DNA methylation variations of <i>C</i>. <i>bungeana</i> genome in response to chilling (4°C) and freezing (-4°C) for five periods of time (0–24 h).</p