High temperature is one of the main forms of abiotic stress in nature (Bita and Gerats 2013). Although is often aggravated by additional abiotic stresses such as drought, it is important to study the independent action and biological consequences of high temperature to alleviate the effects of combined abiotic stress in plants (Bita and Gerats 2013; Lamaoui et al. 2018; Imran et al. 2021). In this work, when one-year and three-month-old P. radiata somatic plants were grown at 40°C in the greenhouse, their Ψleaf was significantly lower than the Ψleaf of the plants growing at 23°C. In contrast, we reported in a previous study (Do Nascimento et al. 2020) that the Ψleaf of six-month-old P. radiata somatic plants growing at 23°C was significantly lower (-0.3 MPa) than in those growing at 40°C (-0.26 MPa) in the greenhouse, indicating that the Ψleaf is not constant under the same growing conditions, but it varies with the age of the P. radiata somatic plants as reported in other conifers (Rosner et al. 2019).
Drought triggers an instructional mechanism that guides the epigenetic machinery, causing plants to respond more quickly and effectively to a subsequent drought event (Colaneri and Jones 2013; Akhter et al. 2021). The degree of resistance to drought and, consequently, changes in physiological parameters depend on several factors such as phenological stages and exposure time (Ozturk et al. 2021). Our results in the drought experiment showed that P. radiata somatic plants derived from EMs maturated at high maturation temperature (50°C, 30 min) showed higher Ψleaffinal and a significant lower Efinal when exposed to drought conditions, indicating that these somatic plants showed a much higher degree of regulation of water loss under drought conditions than those obtained from EMs maturated at 23°C. In line with these findings, similar reductions in E and Ψleaf of somatic plants obtained from EMs of P. radiata initiated at high temperatures (40°C, 4 h; 50°C, 30 min; 60°C, 5 min) and maintained under water stress were observed by Castander-Olarieta et al. (2021). In this way, in a short time, the regulation of water loss in isohydric plants, such as P. radiata, allows plants to limit the transpiration losses and, thus, keep Ψleaf within tolerable limits (Martínez-Vilalta and Garcia-Forner 2017), avoiding complete stomatal closure without severe restrictions on carbon assimilation and tissue preservation against dehydration (Meinzer et al. 2014). Furthermore, a reduction in E in Fragaria x ananassa Duch. plants under saline stress was related to a state of pre-adaptation to saline and/or drought stress, preserving tissues from dehydration and a more effective adjustment to the hyperosmotic environment (Orsini et al. 2012).
The abovementioned results confirmed the hypothesis that stresses applied at different stages of SE can induce memory in the ses that is maintained at later stages of plant development (Do Nascimento et al. 2020; Castander-Olarieta et al. 2021; Pérez-Oliver et al. 2021). Similarly, two-year-old P. pinaster Ait. plants derived from EMs induced at high initiation temperature (50°C, 3 h) showed an improvement in defense mechanisms against drought stress with a better osmotic adjustment and higher chlorophyll, soluble sugars and starch contents when exposed to high temperatures (45°C, for three hours per 12 days) in the greenhouse (Pérez-Oliver et al. 2021). Furthermore, in our study, the plants coming from EMs maturated at 50°C compared with those plants coming from EMs maturated at 23°C had a higher gsfinal under irrigation conditions. This high gs has been reported to be beneficial for pine growth (Urban et al. 2017). In P. tabulaeformis Carr. inoculated with ectomycorrhizal fungi, although the increase in gs could increase the loss of water, it also permitted that the seedlings, that grew under drought could be carried out more photosynthesis by means of increasing CO2 absorption (Augé et al. 2015; Gehring et al. 2017; Wang et al. 2021a).
In conifers, low E.L. values associated with high RWC values were related to a greater capacity of plants to survive in drought conditions (Mantova et al. 2021). In this work, under irrigation, the plants obtained from maturation at 50°C showed a significantly higher E.L.initial (%) (10.80 ± 2.02) than those maturated at 23°C (5.77 ± 1.01), but these differences were not observed when plants were submitted to drought. These values of E.L. were considered low when compared with other works with P. radiata and other conifers subjected to biotic and abiotic stress (De Diego et al. 2012). For example, in P. radiata plants subjected to biotic stress conditions (infection by Trichoderma viride and Fusarium circinatum), there was an increase in E.L. (approximately 26%) characterizing damage to cell membranes in the stressed plants when compared to control plants (Amaral et al. 2019). Also, an increase of E.L. (> 40%) in Pseudotsuga menziesii Mirb. Franco plants submitted to drought was reported (Mantova et al. 2021). In addition, the RWC contents were similar in all treatments showing higher than 86%. Likewise, in P. pinaster somatic plants obtained from ses embryos maturated at different temperatures (18, 23, or 28°C) and kept under heat stress (45°C for 3 h/day for 10 days) the RWC was not significantly affected regardless of its origin (Sales et al. 2022). The maintenance of RWC values in Pinus needles is necessary for normal physiological processes, but contrary to our results, in two-year-old P. sylvestris needles obtained from mature trees, the RWC values decreased by about 27% with high-temperature stress (55° C, 10 min) (Gette et al. 2020).
Our results showed that different stress conditions in P. radiata plants did not affect significantly the percentage of 5-mC independently of the maturation temperature or growth condition in the greenhouse. A percentage higher than 37% of 5-mC in all treatments was found. Similar to this, high values of 5-mC were observed in needles of in vitro somatic plants of P. halepensis Mill. (> 40%) initiated at different temperatures (Pereira et al. 2021) and in needles of one-year-old somatic plants of P. radiata (> 37%) obtained from different initiation temperatures (Castander-Olarieta et al. 2020). However, in this last case, the authors reported statistically significant differences between initiation treatments with a significant decrease in the percentage of 5-mC at the highest temperature (60°C, 5 min). Also, contrary to our results, Entrambasaguas et al. (2021) reported that plants of Posidonia oceanica L. and Cymodocea nodosa (Ucria) Aschers. from different ecotypes showed different epigenetic responses with a modification of the global levels of methylation improving their response to an increase in temperature.
In this work, the percentage of 5-hmC and the 5-hmC/5-mC ratio were affected by the greenhouse temperature in somatic plants from maturation at 23°C with a significant decrease when the greenhouse temperature was 40°C. These results are in line with previous findings by Castander-Olarieta et al. (2020) who reported that the percentage of 5-hmC and the 5-hmC/5-mC ratio varied with different temperatures, but in their case, this variation was due to the initiation temperature of the EMs. On contrary to our results, in Brassica napus L. an increase in 5-hmC in response to heat stress was reported (Golubov and Kovalchuk 2017).
We observed that changes in the DNA methylation/hydroxymethylation contents were significantly correlated with changes in the physiological parameters. This fact supports the idea that physiological responses during abiotic stress can be influenced by methylation/hydroxymethylation changes, as previously suggested Castander-Olarieta et al. (2020). In this work, within each treatment, theΨleaffinal significantly correlated with methylated and hydroxymethylated DNA, being water and thermal dependent; since it is an essential component of any biological system (Singh et al. 2020). Furthermore, the presence or absence of irrigation exerted changes in the state of correlation between these variables in agreement with Colaneri and Jones (2013). However, they reported that drought stress, caused for the addition of polyethylene glycol in the culture medium, imposed on Arabidopsis seedlings triggered changes in DNA methylation with hypermethylation of protein-coding genes related to stress responses. Methylation has also been associated with aquaporins, which are proteins that regulate the movement of water across cell membranes, but the methylation does not interfere with the intrinsic permeability of aquaporin to water in Arabidopsis (Santoni et al. 2006).
On the other hand, the correlation between gs and E with 5-mC, 5-hmC, the 5-hmC/5-mC ratio changed in the control somatic plants kept under heat and drought stress. Similar to our results, in Populus nigra L. plants grown in medium culture supplemented with 100 - 1000 µM of 5-Azacytidine, which had a significantly negative correlation with DNA methylation level, showed changes in with gs and E indicating that the phenotypic changes were related to the methylation changes in the regenerated plants (Zhong et al. 2021). Contrariwise, Auler et al. (2021) reported a simultaneous decrease in physiological parameters (gs and photosynthesis), with 5-mC during the application of recurrent water stress in the reproductive stage of Oryza sativa L. plants. In addition, they reported that proteome and the transcripts associated with the guard cells showed a positive correlation between the highest accumulation of proteins and genes with the highest percentages of 5-mC in the vegetative stage and the vegetative and at the reproductive stages. Furthermore, an increase in the DNA methylation associated with phenotypic changes, such as higher antioxidant activity in Hibiscus cannabinus L. seedlings was related to chromium tolerance mechanisms, and these changes then affected the expression of specific genes involved in the chromium stress response (Tang et al. 2021). In this sense, several works reported that DNA methylation modulates the expression of various genes and consequently phenotypic changes in response to stressful environmental conditions (He et al. 2021; Tang et al. 2021; Wang et al. 2021b; Su et al. 2022). Thus, in the future, it would be interesting to combine the results of our experiments with genetic analysis to understand how the methylation of genes responsible for responses to water and heat stress affects the physiological parameters in these conditions.