Temperature-induced variation in the transcriptome of maritime pine (Pinus pinaster Ait.) embryogenic masses modulates the phenotype of the derived plants

Abstract

A number of studies show that combining somatic embryogenesis with environmental stimuli can induce plant defenses against abiotic stresses, offering a complementary strategy in tree breeding programs. In a previous study, we found that increasing/decreasing the standard temperature of 23 ˚C by 5 ˚C during maritime pine (Pinus pinaster) somatic embryo maturation resulted in epitypes, as the derived plants showed altered phenotypes regarding leaf histology, proline content, photosynthetic rates, and hormone profiles, and that also differentially respond after a short-term heat stress. To elucidate the mechanisms underlying these altered phenotypes, we sequenced the transcriptome of embryonal-suspensor masses (EMs) from the three epitypes, identifying 812 differentially expressed genes (DEGs). Ten genes involved in epigenetic regulation were specifically up-regulated in EMs of the cold epitype. While some of these genes have been linked with somatic embryo maturation, the increased expression of three of these genes, histone deacetylases HDA9, a histone-lysine methyl-transferase (HKMT) and an Argonaute (AGO7), was found to be low temperature-induced epigenetic marks. Among the genes up-regulated in the EMs from the warm epitype, we studied those related to abiotic stress response and observed greater variation in genes involved in abscisic acid (ABA)-mediated response such as those encoding Ras GTPase-activating protein-binding (G3BP) proteins, an AAA-ATPase, and an aspartyl protease (APF2). We also found differential expression in genes encoding for RING-type E3 ubiquitin-transferases, and DNAJ and BAG chaperones. Additionally, the biosynthetic pathways of jasmonic acid, cytokinins and the diterpene pimaradiene were also altered in the warm epitype. However, the increased ABA and cytokinin content observed in the plants derived from this warm epitype cannot be fully explained by the EMs transcriptome profile. Conversely, in the cold epitype, we observed downregulation of genes encoding for an ABA receptor (PYL3), and a xyloglucan endotrans-glucosylase/hydrolase (XTH6). These findings support the hypothesis that the previously reported heat-adapted phenotype of plants derived from the cold epitype (characterized by a faster and higher proline increase, lower increases in ABA levels, no reduction in active cytokinins, and a better net photosynthesis rate recovery) could be attributed to low-temperature-induced epigenetic marks that were absent in the warm epitype.

Keywords: Climate change; Epitypes; Forest-trees; Gene expression; RNA-seq.

Fig. 1

Expression heatmap of maritime pine (Pinus pinaster) based on all the differentially expressed genes in embryonal-suspensor masses (EMs) from 3 genotypes (1007P3a, 1058P4c and 1046P1c) maturated at 18, 23 or 28 ˚C

Fig. 2

Maps of the GO terms enrichment analysis from 812 DEGs detected among transcriptomes of maritime pine embryonal-suspensor masses (EMs) of 3 genotypes maturated at 18, 23 or 28 ˚C. (A) Enriched biological processes; (B) molecular functions of DEGs up-regulated (Log2-FC ≥ 2) in EMs of the 18 ˚C epitypes; and (C) molecular functions of DEGs up-regulated in the 28 ˚C epitypes

Fig. 3

Venn diagram showing the numbers of DEGs detected in pairwise comparisons between the transcriptomes of maritime pine embryonal-suspensor masses (EMs) maturated at 18, 23 or 28 ˚C (A). Maps of GO enrichment analysis of the 2,988 DEGs detected between EMs of the 18 and 28 ˚C epitypes: biological processes (B), and molecular functions (C) of these DEGS

Fig. 4

Expression rates (Log2) estimated for 10 Pinus pinaster genes annotated as regulators of epigenetic modifications, in 3 EMs (1007P3a, 1046P1c and 1058P4c) maturated at 18, 23 or 28 ˚C. * Denotes significantly different expression rates among the three epitypes, while the remaining genes were differentially expressed between 18 (cold) and 28 ˚C (warm) epitypes

Fig. 5

Differential expression rates (Log2-fold-change referred to expression level at control temperature, 23˚C) in maritime pine embryonal-suspensor masses maturated at 18 or 28 ˚C, as determined by qPCR for 10 genes related with epigenetic modulation. From left to right: HI1/5a (Ppinas12549), HI1/5b (Ppinas16086), H2Aa (Ppinas17915), H2Ab (Ppinas18587), HDA2C (Ppinas15640), HDA9 (Ppinas06362), HKMT (Ppinas08395), VIM1 (Ppinas01529), AGO7 (Ppinas00647), and DCL1 (Ppinas06195). Data are mean ± SE of 3 biological replicates analysed in triplicate. * Denotes significant up/down-regulation (Log2-FC ≥ 1/ Log2-FC ≤ -1)

Fig. 6

Differential expression rates (Log2-fold-change referred to expression level at control temperature, 23 ˚C) in maritime pine EMs maturated at 18 or 28 ˚C, as determined by qPCR for 15 genes related with abiotic stress response. Data are mean ± SE of 3 replicates performed for 3 genotypes: 1007P3a (A), 1046P1c (B), and 1058P4c (C). * Denotes significant up/down-regulation (Log2-FC ≥ 1/ Log2-FC ≤ -1)