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

Gene Expression Profiling of Shoot-Derived Calli from Adult Radiata Pine and Zygotic Embryo-Derived Embryonal Masses

<div><p>Background</p><p>Although somatic embryogenesis has an unprecedented potential for large-scale clonal propagation of conifers, the ability to efficiently induce the embryonal cultures required for somatic embryo production has long been a challenge. Furthermore, because early stage zygotic embryos remain the only responsive explants for pines, it is not possible to clone individual trees from vegetative explants at a commercial scale. This is of particular interest for adult trees because many elite characteristics only become apparent following sexual maturation.</p><p>Findings</p><p>Shoot explants collected from adult radiata pine trees were cultured on four induction media differing in plant growth regulator composition, either directly after collection or from <i>in vitro</i>-generated axillary shoots. Six callus lines were selected for microscopic examination, which failed to reveal any embryonal masses (EM). qPCR expression profiling of five of these lines indicated that explant type influenced the absolute level of gene expression, but not the type of genes that were expressed. The analysis, which also included three EM lines induced from immature zygotic embryos, encompassed five categories of genes reflective of metabolic, mitotic and meristematic activity, along with putative markers of embryogenicity. Culture medium was found to have no significant impact on gene expression, although differences specific to the explant’s origin were apparent. Expression of transcriptional factors associated with vegetative meristems further suggested that all of the callus lines possessed a substantive vegetative character. Most notable, however, was that they all also expressed a putative embryogenic marker (LEC1).</p><p>Conclusions</p><p>While limited in scope, these results illustrate the utility of expression profiling for characterizing tissues in culture. For example, although the biological significance of LEC1 expression is unclear, it does present the possibility that these callus lines possess some level of embryogenic character. Additionally, expression of vegetative meristem markers is consistent with their vegetative origin, as are differences in expression patterns as compared with EM.</p></div

Induction medium composition.

<p>Induction medium composition.</p

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

WOX4 gene 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

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

YLS8 reference gene expression within three genotypes of EM induced from immature zygotic embryos of radiata pine.

<p>Average of three biological replicates with the standard deviation presented as bars, expressed as the number of YLS8 transcripts per 10 ng of total RNA. Lettering designates no significant differences based on one-way ANOVA analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128679#pone.0128679.s001" target="_blank">S1 File</a>).</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

List of qPCR primers.

<p>*Average LRE-derived amplification efficiency (n = 32)</p><p>List of qPCR primers.</p