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.

Ander Castander-Olarieta,Itziar A Montalbán,Isabel Arrillaga,María Dolores Ugarte,Tomás Goicoa,Cátia Pereira,María Jesús Cañal,Paloma Moncaleán,Mónica Meijón,Ester Sales

doi:10.3390/plants9121762

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.

Highlights

Epigenetic mechanisms refer to all the mitotically and/or meiotically heritable changes in patterns of gene expression that occur without alterations in DNA sequence [1]
Epigenetics has been to be essential for long-lifespan organisms with complex lifelife-cycles like forest treedemonstrated species, regulating many developmental and stress-response processes
For example, different developmental transitions like bud set in autumn the annual cycle, for example, different developmental transitions like bud set in autumn and bud burst in spring are governed by changes in DNA methylation patterns [29]

Summary

Introduction

Epigenetic mechanisms refer to all the mitotically and/or meiotically heritable changes in patterns of gene expression that occur without alterations in DNA sequence [1]. The regulation of gene expression is tightly influenced by the accessibility of genes for the transcriptional machinery, and the packaging grade of chromatin is fundamental during these processes [2]. Non-coding RNAs, including small RNAs, are currently gaining special interest as novel epigenetic regulators influencing the distribution of epigenetic marks and the expression of specific genes at both transcriptional ad post-transcriptional levels [4]. DNA methylation is the most studied epigenetic mark due to its occurrence in plants and mammals, its stability, and its role in gene regulation and genome structure maintenance through transposon silencing [5]. Methylation is site specific and usually occurs in the fifth carbon position of cytosines in the following sequences: CG, CHG, and CHH (where H = A, T or C) [6]. DNA methylation has frequently been linked with gene expression repression, its specific location in, for example, CG gene body sequences, has most often been associated with transcriptional activity [7]

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