Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti Chilling-stress modifes DNA methylation level in cucumber (Cucumis sativus L.) seedling radicle to regulate elongation rate Bixuan Chen a,b,1 , Mikal E. Saltveit a , Diane M. Beckles a, ⁎ a Department of Plant Sciences, University of California, Davis, CA, 95616, USA b Graduate Group Horticulture & Agronomy, University of California, Davis, CA, 95616, USA ARTICLE INFO Keywords: 5-Azacytidine DNA methylation Chilling-stress Methylation-sensitive amplifcation polymorphism ABSTRACT 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 Amplifcation Polymorphism (MSAP) analysis. Radicles were chilled or treated with a methylation inhibitor, 5-azacytidine (AZA), or a combination thereof. Chilling cu- cumber radicles at 2.5 °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 efect due to the treatment combination. This was paralleled by methylation modifcations at novel sites, with no change in the global methylation level, although the latter was expected to decrease as an efect 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. 1. Introduction Plants that originated from tropical and subtropical regions exhibit abnormal physiology when exposed to temperatures ranging from 0 to ˜12.5 °C; a physiological disorder called chilling injury (CI) (Lyons, 1973). Symptoms of CI include damage to meristematic tissue, ab- normal fruit ripening, impaired root growth, increased membrane permeability, and the development of necrotic lesions (Harrington and Kihara, 1960; Jennings and Saltveit, 1994; Saltveit and Morris, 1990). The regulation of CI at the molecular level has long been of interest, but is not yet well understood (Albornoz et al., 2019). Modifcation of DNA methylation is a ubiquitous epigenetic me- chanism detected as part of the response to chilling-stress in various tissues in diverse species (Fan et al., 2013; Liu et al., 2017; Pan et al., 2011; Steward et al., 2000). As a chilling-sensitive species, cucumber (Cucumis sativus L.) was identifed as having a large proportion of dif- ferentially methylated regions (DMRs) in the gene-rich euchromatin regions in shoot apices that developed at low temperatures (Lai et al., 2017). This is likely regulated via transposable element (TE)-related small RNA-directed DNA methylation (RdDM) (Lai et al., 2017). Al- though DNA methylation patterns are tissue-specifc, it seemed likely that a similar mechanism of regulation would be found in chilled cu- cumber seedling radicles. Chilled cucumber radicles have been used to understand some as- pects of postharvest chilling injury (PCI) (Cao et al., 2018; Minchin and Simon, 1973). It is a simple and quickly responding system, but more importantly, it responds to chilling-stress similarly to other sensitive species that are harvested as vegetables and fruit (Saltveit, 2001; Tijskens et al., 1994). Low temperatures interrupt cell division (Lloyd and Seagull, 1985), which is particularly active at the meristem, therefore, a reduced rate of radicle growth in seedlings is a straight- forward indicator of chilling injury. Further, the cucumber genome is relatively small (˜367 Mb), compared to tomato (˜925 Mb), the model organism in many PCI studies, thus reducing the complexity of genomic data analysis (Arumuganathan and Earle, 1991; Sato et al., 2012). Cucumber is also a valuable commodity. Annual global production in https://doi.org/10.1016/j.scienta.2019.03.023 Received 26 September 2018; Received in revised form 10 March 2019; Accepted 11 March 2019 ⁎ Corresponding author at: Department of Plant Sciences MS3, One Shields Avenue, University of California, Davis, CA, 95616, USA. E-mail address: dmbeckles@ucdavis.edu (D.M. Beckles). 1 Current address: United Genetics Seeds Co., Hollister, CA 95023, USA. Scientia Horticulturae 252 (2019) 14–19 0304-4238/ © 2019 Elsevier B.V. All rights reserved. T