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.

Proteome-Wide Analysis of Heat-Stress in Pinus radiata Somatic Embryos Reveals a Combined Response of Sugar Metabolism and Translational Regulation Mechanisms

“…It controls methylation through regulation of the intracellular concentration of adenosylhomocysteine, a competitive inhibitor of S -adenosyl- L -methionine-dependent methyl transferase reactions. These data suggest that heat-stress might have derived in hypomethylation of Se’s, as observed in embryonal masses and somatic plants originating from the same temperature treatments in previous studies ( Castander-Olarieta et al, 2020c ). Similar results were obtained by Lamelas et al (2020) , indicating that DNA methylation, together with post-transcriptional regulation mechanisms may be involved in the maintenance of acquired thermotolerance and in the establishment of an epigenetic memory.…”

“…Those treatments can alter the morphology of embryonal masses (EMs) and Se’s in Pinus radiata and Pinus halepensis , as well as their hormonal and metabolic profiles ( Moncaleán et al, 2018 ; Castander-Olarieta et al, 2019 , 2020a , b ; Pereira et al, 2020 ). Besides, the initial culture conditions can have long-lasting effects, modulating the development and drought stress resilience of somatic plants years later ( Castander-Olarieta et al, 2020b ), presumably by changes in the expression of stress-related genes and epigenetic marks ( Castander-Olarieta et al, 2020c ). However, very few proteomic studies have been carried out in pine species addressing heat-stress, and, as far as we know, this is the first report carried out along SE applying such high temperatures.…”

Priming Maritime Pine Megagametophytes during Somatic Embryogenesis Improved Plant Adaptation to Heat Stress

“…A priming treatment at 28 • C during SE of radiata pine also increased differentiation of somatic embryos, which showed altered hormonal profiles [31,32], and finally resulted in plants with different water use efficiency levels when assayed at the greenhouse [33]. Priming using higher temperatures (up to 60 • C) improved somatic embryos production and altered amino acid [34] and CK profiles [35] of embryogenic masses (EMs), while regenerated plants showed differential patterns of gene expression and hormone content [36,37]. Similar experiments were carried out with Aleppo pine [38,39].…”

The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets

“…In this sense, the optimization of initiation and proliferation stages of SE in P. radiata (Montalbán et al, 2010(Montalbán et al, , 2012 and P. halepensis (Montalbán et al, 2013) has been our objective in recent years. Recently, some studies focused on promoting and improving the embryogenic process in terms of quality and quantity of somatic embryos (ses) obtained by reduction of water availability in P. radiata (Garcia-Mendiguren et al, 2016;Castander-Olarieta et al, 2020) and P. halepensis (Pereira et al, 2016(Pereira et al, , 2017do Nascimento et al, 2020) have been carried out. However, the rate of germination (%) and the subsequent conversion into plantlets is sometimes low (do Nascimento et al, 2020), and additional research is needed to increase somatic plantlet regeneration (Montalbán and Moncaleán, 2019).…”