Embryonal Masses Induced at High Temperatures in Aleppo Pine: Cytokinin Profile and Cytological Characterization

Abstract

Aleppo pine (Pinus halepensis Mill.), a native species of the Mediterranean region, has been suggested as a species that when introduced in degraded areas could facilitate the long-term colonization and expansion of late-successional species. Due to climate changes, plants need to withstand extreme environmental conditions through adaptation and changings in developmental pathways. Among other paths, plants undergo changes in developmental pathways controlled by phytohormones. At the same time, somatic embryogenesis has been widely used as a model to understand the mechanisms involved in plant response to different stresses. In this study, in order to induce a strong effect of temperature stress on plants regenerated from somatic embryos, higher temperatures (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) than the control (23 °C) were applied during the induction stage of somatic embryogenesis in Pinus halepensis. A morphological characterization of the embryogenic cultures showed small differences in the number of starch grains, lipid bodies, and phenolic compounds between treatments. Results showed that high temperatures (60 °C) led to higher rates at the maturation stage of somatic embryogenesis when compared to the control (23 °C), strengthening the productivity through the increase in the number of somatic embryos obtained. Finally, analysis of endogenous concentration of cytokinins showed that different conditions applied during the initiation phase of somatic embryogenesis led to different hormonal profiles; isoprenoid cytokinins showed a clear defined pattern with the higher total hormone concentration being found in embryonal masses induced at 50 °C for 30 min, while different aromatic cytokinins presented different individual responses to the treatments applied. These differences corroborate the idea that cytokinins could be potential regulators of stress–response processes during initial steps of somatic embryogenesis.

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

“…Although not significant, the number of Se’s increased under high temperatures, a long-observed trend in our laboratory for both P. radiata and P. halepensis EMs initiated under high temperatures ( García-Mendiguren et al, 2016b ; Castander-Olarieta et al, 2019 ; Pereira et al, 2020 ). The same pattern was detected in similar temperature-based experiments during initiation of Pinus pinaster SE ( Arrillaga et al, 2019 ), confirming the role of temperature as an activator of embryo development.…”

“…For example, the function of some ribosomal proteins is linked with cytokinin molecular circuits ( Černý et al, 2013 ). Interestingly, the application of the same high temperature conditions at initiation of P. radiata and P. halepensis SE provoked changes in the cytokinin profile of EMs ( Castander-Olarieta et al, 2020a , b ; Pereira et al, 2020 ). It would be of special interest for future studies to confirm if those changes are maintained in Se’s and study their relationship with the translasome machinery.…”

“…In previous studies we have reported that high temperatures can determine the success of the different stages of SE, provoking negative effects during initiation but increasing the production of somatic embryos during maturation ( García-Mendiguren et al, 2016b ; Castander-Olarieta et al, 2019 ). 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 ).…”

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

“…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].…”

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

“…In previous studies we have observed that the application of high temperatures during initiation of Pinus radiata and Pinus halepensis SE modulates not only the success of the different stages of the process, but also the morphology and biochemical status (hormones, metabolites) of embryonal masses (EMs) and somatic embryos (Se’s) [ 24 , 25 , 26 , 27 ]. Moreover, those changes can have long-lasting effects determining the ex-vitro behaviour of somatic plants years later at both control and stress conditions [ 25 ].…”