Chilling-stress modifies DNA methylation level in cucumber (Cucumis sativus L.) seedling radicle to regulate elongation rate

Seedlings have been used as an experimental model to understand plant response to chilling-stress. In this study, we investigated if the occurrence or severity of chilling injury correlated with genomic methylation state in cucumber radicles, using Methylation-Sensitive Amplification Polymorphism (MSAP) analysis. Radicles were chilled or treated with a methylation inhibitor, 5-azacytidine (AZA), or a combination thereof. Chilling cucumber radicles at 2.5 degrees C was marked by an immediate cessation in elongation and higher levels of genomic methylation. Rewarming was associated with a partial reversal of the methylation caused by chilling-stress, a decrease in genomic methylation to the same level as that before chilling, and to a resumption in radicle growth, but only to 18.6% of that without chilling. This cessation and recovery of elongation during and after chilling was also observed when radicles were treated with AZA. Under normal and rewarming conditions, 60% and 74% suppression of elongation were detected in AZA-treated radicles, respectively. Growth rate of the radicles was slowest in AZA-treated radicles after exposure to chilling-stress, suggesting a "double" suppressive effect due to the treatment combination. This was paralleled by methylation modifications at novel sites, with no change in the global methylation level, although the latter was expected to decrease as an effect of AZA. This work showed that DNA methylation is associated with, and may partially regulate, radicle elongation rate under chilling stress, through a dynamic alteration of methylation pattern.