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

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

Embryogenic marker expression within primordial- and axillary-shoot derived tissues of radiata pine.

<p>Expressed as the number of mRNA transcripts per 10 ng of total RNA, with standard deviations presented as bars. Lettering designates significant differences based on one-way ANOVA analysis with post-hoc Tukey HSD (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s003" target="_blank">S3 File</a>). Note that the level of LEC1 expression within the three axillary shoot-derived genotypes is comparable to that seen in EM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.g005" target="_blank">Fig 5</a>), albeit of greater variability.</p

Cell division gene expression within three genotypes of EM induced from immature zygotic embryos of radiata pine.

<p>Average of three biological replicates expressed as the number of transcripts per 10 ng of total RNA, with standard deviations presented as bars. Lettering designates significant differences in histone 4 expression based on one-way ANOVA analysis with post-hoc Tukey HSD (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s001" target="_blank">S1 File</a>).</p

Morphology of tissues induced from primordial and axillary-shoot explants of radiata pine.

<p>A) PS-B explant cultured on EDM Pic for 6 weeks (5x). B) PS-A apical shoot explant cultured on EDM-NAA for 6 weeks (4x). C) Axl-C axillary shoot explant cultured on EDM-Pic for 9 weeks (4x). D) PS-A callus composed of loosely associated round cells growing on EDM-4 (100x). E) EM induced from immature zygotic embryos (5x). F) Axl-D cell aggregate stained with KI (400x). G) EM-A early stage somatic embryo stained with KI (400x). H) Bright-field and I) fluorescence microphotographs of an Axl-D cell aggregate stained with fluorescin diacetate (200x).</p

Histone 4 and PCNA expression within primordial and axillary shoot-derived tissues of radiata pine.

<p>Expressed as the number of YLS8 transcripts per 10 ng of total RNA, with standard deviations presented as bars. Lettering designates significant differences based on one-way ANOVA analysis with post-hoc Tukey HSD (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s003" target="_blank">S3 File</a>).</p

Meristematic marker expression within cultures derived from primordial and axillary shoot explants of radiata pine.

<p>Expressed as the number of mRNA transcripts per 10 ng of total RNA, with standard deviations presented as bars. Lettering designates significant differences based on one-way ANOVA analysis with post-hoc Tukey HSD (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s003" target="_blank">S3 File</a>).</p

Expression of embryogenic markers within three genotypes of EM induced from immature zygotic embryos of radiata pine.

<p>Average of three biological replicates expressed as the number of transcripts per 10 ng of total RNA, with standard deviations presented as bars. Lettering designates no significant differences based on one-way ANOVA analysis with post-hoc Tukey HSD (p<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s001" target="_blank">S1 File</a>).</p