Induction of Radiata Pine Somatic Embryogenesis at High Temperatures Provokes a Long-Term Decrease in DNA Methylation/Hydroxymethylation and Differential Expression of Stress-Related Genes

Abstract

Based on the hypothesis that embryo development is a crucial stage for the formation of stable epigenetic marks that could modulate the behaviour of the resulting plants, in this study, radiata pine somatic embryogenesis was induced at high temperatures (23 °C, eight weeks, control; 40 °C, 4 h; 60 °C, 5 min) and the global methylation and hydroxymethylation levels of emerging embryonal masses and somatic plants were analysed using LC-ESI-MS/ MS-MRM. In this context, the expression pattern of six genes previously described as stress-mediators was studied throughout the embryogenic process until plant level to assess whether the observed epigenetic changes could have provoked a sustained alteration of the transcriptome. Results indicated that the highest temperatures led to hypomethylation of both embryonal masses and somatic plants. Moreover, we detected for the first time in a pine species the presence of 5-hydroxymethylcytosine, and revealed its tissue specificity and potential involvement in heat-stress responses. Additionally, a heat shock protein-coding gene showed a down-regulation tendency along the process, with a special emphasis given to embryonal masses at first subculture and ex vitro somatic plants. Likewise, the transcripts of several proteins related with translation, oxidative stress response, and drought resilience were differentially expressed.

Keywords: 5-hydroxymethylcytosine; 5-methylcytosine; Pinus radiata; epigenetics; heat; heat shock protein; memory; priming; somatic embryo; somatic plant.

Figure 1

Fold-relative gene expression of six stress-related genes (APFI, SOD, HSP20, 50SR, DI19 and ADH) along the different stages of Pinus radiata somatic embryogenesis (T1, T2, T3, T4 and T5) between samples originating from different temperature conditions during induction (23 °C, 8 weeks; 40 °C, 4 h; 60 °C, 5 min). Data are presented as mean values ± SE. * Denotes significant differences at p < 0.05 among high temperature treatments with respect to the control temperature of 23 °C.