Epigenetic regulation of gene expression is expected to be an important mechanism behind phenotypic plasticity. Whether epigenetic regulation affects species ecophysiological adaptations to changing climate remains largely unexplored. We compared ecophysiological traits between individuals treated with 5-azaC, assumed to lead to DNA demethylation, with control individuals of a clonal grass originating from and grown under different climates, simulating different directions and magnitudes of climate change. We linked the ecophysiological data to proxies of fitness. Main effects of plant origin and cultivating conditions predicted variation in plant traits, but 5-azaC did not. Effects of 5-azaC interacted with conditions of cultivation and plant origin. The direction of the 5-azaC effects suggests that DNA methylation does not reflect species long-term adaptations to climate of origin and species likely epigenetically adjusted to the conditions experienced during experiment set-up. Ecophysiology translated to proxies of fitness, but the intensity and direction of the relationships were context dependent and affected by 5-azaC. The study suggests that effects of DNA methylation depend on conditions of plant origin and current climate. Direction of 5-azaC effects suggests limited role of epigenetic modifications in long-term adaptation of plants. It rather facilitates fast adaptations to temporal fluctuations of the environment. Identification of the mechanisms allowing plants to mitigate negative effects of the change on their performance and survival thus became one of the key recent research topics. Migration, phenotypic plasticity and genetic differentiation have been among the most frequently considered and scrutinized mechanisms3. However, there are other mechanisms such as heritable epigenetic variation that can play a potentially important role in plant responses to novel climates that have been studied only scarcely (but see e.g.7).The ongoing global climate change represents one of the greatest recent challenges for plants8. In contrast to the genetic adaptation, the epigenetic adaptation can arise within a single generation without any underlying genetic change and the epigenetic responses can thus be much faster than the genetic responses9. The epigenetic variation can arise for example by silencing of transposable elements and may thus depend on the genetic set-up of the species11, but can also result from random epimutation13 or be induced by environmental change9. Because environmentally induced DNA methylation variation can be inherited by offspring generations6, the epigenetic changes may contribute to species ability to adapt to the rapidly changing climates15.Epigenetic regulation of gene expression e.g. via DNA methylation, belongs among important mechanisms allowing species to phenotypically respond to current conditions as well as to conditions experienced by the species in previous years and/or even generations19. Many of these studies, however, do not enable separation of the epigenetic and genetic effects. Studies on natural plant populations separating the epigenetic from genetic differentiation are still quite scarce but highly desirable25.One of the common approaches for testing whether the epigenetic inheritance plays a role in the plant adaptation to the climate change is analyzing epigenetic profiles of natural plant populations originating from various environmental conditions exposed to different target conditions. Several studies already provided evidence that the epigenetic variation is correlated with the environmental conditions of plant populations27. Ecophysiological traits are often highly plastic and variable among populations . Importantly, several ecophysiological traits such as stomata density and/or length have also been shown to be under epigenetic control39 and can be transgenerationally inherited41. To what extent is the adaptive ecophysiological differentiation between populations originating from different conditions caused by heritable epigenetic variation, however, remains to be tested. A recently developed method42 allowing to modify epigenetic set-up of established plants and thus directly test the importance of epigenetic memory for species response to the changing climate provides unique opportunity to obtain novel insights into the issue.It has been suggested that particularly adaptive changes in ecophysiological traits related for example to photosynthetic activity and water management should be crucial for maintaining plant fitness under the changing climateFestuca rubra, as a model. In our previous studies using the same species, we have already demonstrated that the ecophysiological traits such as maximum photosystem II efficiency (Fv/Fm), osmotic potential, stomata density and length are highly plastic and/or differ between populations originating from different climatic conditions with stomatal density showing lower levels of plasticity than the other traits43. To what extent are the observed patterns epigenetically coordinated remains to be unraveled. The four above mentioned traits have been selected because they are all known to be closely linked to species performance under the changing environmental conditions . Fv/Fm provides information on overall photosynthetic capacity of the plant48 and is an indicator of damage in PSII reaction center due to stress50. Osmotic potential is key drought resistance trait with high level of plasticity51. Stomatal density and stomatal length regulate gaseous exchange30 and thus are crucial for regulation of photosynthesis46 by driving the trade-off between water loss and carbon acquisition by the plant53.To better understand the role of epigenetic processes in ecophysiological response of plants to the changing climatic conditions, we used a clonal grass, 54. Experimental alteration of DNA methylation status via application of a demethylating agent such as 5-azacytidine (5-azaC) is probably the easiest approach available for plants. 5-azaC inhibits methylation of newly synthetized DNA55. A clonal plant continuously exposed to this agent thus produces ramets with altered (reduced) level of DNA methylation54.The relatively simple yet powerful method for testing the role of DNA methylation in response to the environmental change is comparison of genetically identical but epigenetically distinct individuals subjected to different environments7. Application of the 5-azaC on the plants in our model system led to changes in some growth- and fitness-related traits and the effects interacted with the conditions of plant origin as well as of plant cultivation suggesting that these traits are controlled epigenetically7. Interestingly, plant responses to the conditions of plant origin in terms of growth- and fitness-related traits were stronger in plants treated with 5-azaC than in untreated plants. This could be because the epigenetic memory on the environmental conditions of plant origin was overwritten by the epigenetic adaptations to conditions in the experimental garden, thus equalizing plant performance.Demethylation by 5-azaC has been already applied in our model system leading to changes in plant response to the environment7 to explore variation in the ecophysiological traits. The plants originate from western Norway and were collected in natural grassland ‘climate grid’ with factorially crossed gradients of temperature and precipitation spanning ~ 4 °C and ~ 2100°mm of precipitation . We cultivated control plants and plants treated with 5-azaC in two growth chambers set to simulate two climatic extremes (cold-dry and warm-wet environment) mimicking the original localities. By cultivating the plants originating from the different original climates under the different cultivation climates in the growth chambers, we simulated different directions and magnitudes of climate change. In addition to exploring variation in the ecophysiological traits, we also explored to what extent the ecophysiological traits may explain variation in the previously measured fitness-related traits.In this study, we used the same plants as Münzbergová et al.Specifically, we asked the following questions: (1) What is the effect of experimental application of 5-azaC on the ecophysiological traits?, (2) Do the effects of application of 5-azaC depend on plant origin and cultivating conditions?, (3) Do the effects of application of 5-azaC differ between genotypes and can these differences be explained by their genetic relatedness?, (4) Is there any relationship between the ecophysiological traits and fitness-related traits and does this relationship depend on application of 5-azaC and climate of cultivation?We hypothesized that: (1) the ecophysiological traits will be significantly influenced by the application of 5-azaC. The effect will be the weakest in the least plastic trait, i.e. stomata density. (2) Plant origin as well as cultivation conditions will strongly interact with the application of 5-azaC. The plants treated with 5-azaC will show higher between population differentiation than the untreated plants due to removal of the epigenetic memory on the common garden conditions experienced during the plant precultivation. (3) The effects of application of 5-azaC will differ between the genotypes and these differences could be explained by their genetic relatedness. The strength of this effect will differ between the different cultivating conditions. (4) The ecophysiological traits will be related to the proxies of plant fitness, but the relationship will depend on the conditions of plant cultivation and the application of 5-azaC.Festuca rubra L. is a common perennial grass species of temperate grasslands in Europe. Festuca rubra used in the experiment is a widespread hexaploid type from the F. rubra complex. The species has 42 chromosomes57. The species grows across a wide climatic gradient in grasslands, both as a dominant with only few other species and also as a minor component of species rich stands. It reproduces by seeds as well as vegetatively58.The model plants are not under any legal protection, so no permission was required for their study. The plants have been identified by ZM and VH. The material has been studied already in several previous studies and has been characterized also genetically and in terms of genome size. The material has not been deposited in any herbaria. Collection of plant material was in line with relevant institutional, national, and international guidelines and legislation. Specifically, no permissions were required for any of the actions taken.62). We used plants from the four most extreme localities from this grid. These four localities represent 2 levels of mean annual precipitation  and 2 levels of summer temperature . The exact positions of the localities are as follows, warm-dry: 61.035°N, 9.079°E, warm-wet: 60.690°N, 5.965°E, cold-dry: 61.024°N, 8.124°E and cold-wet: 60.934°N, 6.414°E. The communities are grazed intermediate-rich meadows with comparable slopes, grazing regime and bedrock. For more details about the localities, see58.Plant material originated from a natural climate grid in western Norway  of F. rubra collected at each locality at least 1 m apart from each other. We transported the plants and cultivated them in experimental garden of the Institute of Botany, Czech Academy of Sciences, Průhonice, Czech Republic . Microsatellite analysis of the material confirmed that each experimental plant represents an independent genotype57.The plant material was collected as a part of a previous experiment, data from which were used in several papers7 exploring the effects of 5-azaC treatment on growth- and fitness-related traits (this experiment is independent of the experiment mentioned above). Twelve single ramets (later referred as plants) from each of the 6 genotypes per population were separately planted into 10 × 10 × 10 cm pots in a greenhouse in October 2015. Random half of the plants (six per genotype) was chosen for the 5-azaC application treatment. Each genotype thus had two treatments (5-azaC treated plants and control) with 6 replicates per treatment, i.e. 288 pots in total  with added surfactant  in one-liter solution for 10 months between October 2015 and August 2016. It has been suggested that the effects of 5-azaC spraying may be long-term due to long-term inhibition of 5-DNA-methyltransferase42, no such information has unfortunately been obtained in our study. In addition, application of 5-azaC may theoretically have other effects than only demethylation. Due to all this, we speak about effects of application of 5-azaC rather than effects of demethylation in the text. Previous studies, however, suggest that the main effect of application of 5-azaC is indeed demethylation of the plants42. We thus expect that the 5-azaC application will modify ecophysiology of our model plant and finding such a pattern will indicate that the ecophysiological traits are under epigenetic control . The exact setting of the growth chambers is described in Supplementary Table In September 2016, several ramets (each with three leaves and without visible signs of initiating flower stems) were cut from each of the 288 (6 clones × 6 replicates × 4 populations × 2 5-azaC treatments) plants and were placed into small plastic cups filled with water to set roots. After 2 weeks, two ramets from each initial plant were individually planted with developed roots into 5 × 5 × 8.5 cm pots filled with a mixture of common garden soil and sand in 1:2 ratio. The prepared plants were placed into two growth chambers simulating the two strongest climatic extremes along the climatic grid—warm-wet and cold-dry . In fact, also Hurlbert69 concluded that such experiments can be analyzed with standard statistical approaches as long as the interaction term is used as an estimate of the error term to test the main effect. In our experiment, we were not primarily interested in the effect manipulated at the growth chamber level, i.e. the cultivating conditions. We were primarily interested in the effect of plant origin and 5-azaC application, which are well replicated and also in the interaction between the plant origin, 5-azaC application and cultivating conditions. In such a case, using the standard error terms is well justified. Thus, in line with a range of other studies using similar settings such as unreplicated gardens at different elevations71 or growth chambers76, we suggest that such studies are useful to study various factors possibly interacting with the climate of cultivation as done here.It may be argued that our experiment is pseudoreplicated as it was performed only in 2 growth chambers and the growth chambers may theoretically differ in a range of other variables (e.g. light intensity) leading to possible spurious treatment effectsv/Fm), osmotic potential, stomatal density and stomatal length. Maximum photosystem II (PSII) efficiency  was measured using FluorPen FP-100 MAX/USB  in dark-acclimated (1 h) plants (Maxwell and Johnson 2000). In healthy leaves, Fv/Fm value is usually close to 0.8. Lower values indicate that a proportion of PSII reaction centers is damaged or inactivated leading to photoinhibition78.After 24 weeks, when the plants reached reasonable size to be measured, we measured the ecophysiological photosynthesis-related traits: maximum photosystem II efficiency  with C-52 sample chambers as described previouslyStomatal density and stomatal length were measured using epidermal impressions of the adaxial side of the leaf blade using a clear nail polish. The impressions were mounted on microscope slides with a transparent adhesive tape. Stomatal density was counted as an average of three no overlapping areas (each 500 × 500 µm). Each counting area was in contact with a margin of the leaf to avoid possible variation in the stomata size from the leaf margin to the center. From each of these three areas, three stomata were randomly chosen, and their length was measured, resulting in 9 measured stomata of each sample. Preliminary explorations of the stomatal imprints indicated low variation in the stomata across the leaf margin suggesting that the 9 stomata measured at the leaf margin well represent the stomata of the given leaf (Hadincová pers. obs.). We did not measure the stomata in the leaf centers as the leaves are naturally folded and the stomata are hard to observe in the folded parts with the occurrence of ribs.7 as proxies of fitness. While it is generally assumed that lifetime fitness should be represented e.g. by lifetime seed production80, our model is a long-lived species with extensive clonal reproduction and measuring lifetime fitness is not straightforward. The two traits used here as proxies of fitness are expected to be closely related to seed production and vegetative spread of the individuals81. These data are visualized in Supplementary Fig. To explore the relationship between the physiological traits and plant fitness, we used number of ramets and aboveground biomass, measured in our previous study82. Genotype was nested within population of origin in all the tests (formula: lm (trait ~ (Population/ Genotype) × 5-azaC × Growth chamber)). The data on stomata density and size and maximum photosystem II efficiency (Fv/Fm) fitted the assumption of the model for normality and homoscedasticity of the data. The data on osmotic potential did not fit the assumption and could not be easily transformed to achieve normality. We thus multiplied the values by -1 . After this transformation, these data fitted Gamma distribution and could be analysed with the formula glm (trait ~ (Population/ Genotype) × 5-azaC × Growth chamber, family = “Gamma”).To estimate the effect of application of the 5-azaC, population of plant origin, genotype, growth chamber and all their interactions on all the dependent variables, we used linear model using function lm with type III sum of squares in R version 4.0.383. For this purpose, we calculated pairwise genetic relatedness  between studied genotypes) using data on five microsatellite markers identifying 63 different alleles in the samples57. To calculate differences in the response of the different genotypes to the application of 5-azaC, we calculated average proportional change in each trait between the 5-azaC treated genotypes and the controls. For that we used average trait values for each genotype in each chamber across the 6 replicates. From these values, we generated matrix using Euclidean distance among the plants for each growth chamber and for each trait separately. Then we tested correlation between the matrix of genetic distances and the matrix of distances in response to application of 5-azaC using Mantel test as implemented in R package vegan84, formula: mantel . The tests were done for each growth chamber and each trait separately. To check whether the differences observed could be due to the genetic differentiation in the performance of the control plants, we repeated the test using difference in traits between the control plants. This later test was never significant .Then, we explored dependence of differences in the response to the application of 5-azaC between the genotypes on genetic relatedness of the genotypesBecause the relationships between the genetic similarity and the similarity in response to the application of 5-azaC could be driven by between population differences, we also performed additional tests using partial Mantel tests. Here, we used a matrix of populations of origin (coded 0 for pair of genotypes which do not come from the same population and 1 for pair of genotypes which come from the same population) as a covariate ). If this relationship was significant, we repeated the test using trait values of control plants only (as described above). This control test was never significant .85, in the results separately for each 5-azaC treatment and growth chamber. Because the selection effects may not be linear but rather quadratic86, we added the quadratic version of the traits into our models and tested if the models improved. As the models did not improve in any case (based on AIC), we do not report these results further.Finally, we tested phenotypic selection of the ecophysiological traits. For these analyses, both ecophysiological traits and proxies of fitness (number of ramets and aboveground biomass) were standardized to a mean of zero and a unit variance across the whole dataset . We then tested the effect of the physiological traits alone and in interaction with growth chamber and 5-azaC application on proxies of plant fitness (formula: lm (fitness proxy ~ (ecophys. trait1 + ecophys. trait2 + ⋯) × 5-azaC × Growth chamber). Due to a range of significant interactions, we also calculated these relationships separately for each growth chamber and 5-azaC application treatment and report standardized directional selection gradients (β), calculated as a partial regression coefficient from multiple linear regressions to relative values of proxies of fitness on all standardized ecophysiological traitsThe model plants are not under any legal protection, so no permission was required for their study. The plants have been identified by ZM and VH. The material has been studied already in several previous studies and has been characterized also genetically and in terms of genome size see “”. The maPlants of different genotypes and localities of origin differed in all the measured traits , a significant triple interaction of 5-azaC, plant origin and growth chamber was detected (Table v/Fm (by ± 0.012 on average) than the corresponding control plants. No significant difference was detected in any other combination ) and their response to the application of 5-azaC for Fte Table . No signte Table .Table 2Cv/Fm in the overall tests , their plasticity (the effects of growth chamber) as well as by the genetic differentiation in trait plasticity (the interactions of growth chamber with population and genotype). In contrast, there was no significant main effect of the application of 5-azaC and only three interactions of the application of 5-azaC with the other factors suggesting relatively limited effect of 5-azaC, and thus likely of DNA methylation, on these traits. The interactions of the climate of origin and cultivation with 5-azaC indicate that species response to the changing climatic conditions is modified by 5-azaC.87. If the DNA methylation played a role in species adaptation to the conditions of plant origin, we should observe longer stomata in the control plants than in the plants treated with 5-azaC and not the opposite. Similarly as for the photosynthetic efficiency (discussed below), the 5-azaC application thus changed the plant response in non-intuitive direction.For stomatal length, we found that the 5-azaC treated plants originating from the warm-wet locality had longer stomata than the controls from the same locality. Interestingly, these controls had comparable stomata length as plants originating from very contrasting environment: cold and dry. It is an intriguing finding because longer stomata are usually considered to be selected for in warm and moist environments due to their increased transpirational cooling ability88. In terms of the ecophysiological traits, this would mean that the control plants should show values corresponding to the optimal values in the given conditions, while the 5-azaC treated plants would deviate from this optimal value. This assumption is based on the idea that some of the epigenetic markers related to the long-term adaptation to the conditions of plant origin would be removed and/or changed due to the effect of 5-azaC, which would be observable as a maladaptive response in their home environment. Nonetheless, we observed the opposite response, the value of stomatal length for the given plant origin moved in the direction towards the optimal value in 5-azaC treated plants in comparison to the controls. The response going in an opposite direction than expected is in line with our previous study on growth- and fitness-related traits of the same plants7.The general expectation if DNA methylation plays a positive role in local adaptation and 5-azaC reduced the DNA methylation level would be better performance of the plants that experience their home environment and their DNA is naturally methylated over genetically identical individuals that were treated by the 5-azaC7. This is in line with other studies indicating that epigenetic memory can be gradually lost over several asexual generations90. Epigenetic adjustments of the plants to our common garden and greenhouse could be however maladaptive when plants were moved to the growth chambers simulating their original environments. Therefore, the 5-azaC application could disrupt some of the epigenetic memory that reflected the precultivation period and these plants could perform better than the plants with the memory of precultivation (controls). This assumption is supported by Gonzalez et al.54 who demonstrated that clonal offspring of Trifolium repens grew the same phenotypes as their drought stressed parents unless they were treated via 5-azaC that removed the memory on the drought. After 5-azaC treatment, the plants grew the same phenotype as the offspring of the not stressed parents. An alternative explanation may be that the 5-azaC application changing DNA demethylation can lead to both activation or deactivation of genes underlying the ecophysiological traits93. Therefore, it is possible that demethylation enhances the expression of some genes instead of suppressing it94.One of the possible explanations for the observed patterns could be linked with the design of our study. We cultivated selected plants in a common garden and subsequently in a greenhouse for 2 years before we performed the experiment. This was done in order to let the plants grow to obtain sufficient number of ramets for the experiment. Hence, it is plausible that the plants already lost their epigenetic memory on the field conditions and became epigenetically adjusted to the new experimental conditions39. They demonstrated that stomatal development under different environmental conditions can be induced by changes in DNA methylation status due to the climate change. It was also found that the stomatal traits could be transgenerationally heritable under environmental stress in Arabidopsis thaliana in response to drought41 and their development was shown to be under epigenetic control95. Stomata traits could also be genetically adapted to different climatic conditions of plant origin97. In Kosová et al.43 as well as in this study, we also found genetic differentiation of our populations in the stomatal traits. Our results thus suggest that the expression of the stomatal traits is probably driven by both, genetic as well as epigenetic mechanisms, dependent on the specific climatic conditions of their cultivation.The observed effect of the 5-azaC on stomata length is in line with Tricker et al.For photosynthetic efficiency, the effect of 5-azaC was observed in higher photosynthetic efficiency of the plants treated with 5-azaC originating from the cold-dry environments grown in the same conditions when compared to the control plants. Such response does not reflect species long-term epigenetic adaptations to the climate of origin because the plants with natural methylation patterns (controls) should be better adapted to their original climate than the genetically identical plants treated with 5-azaC if epigenetic variation plays a role in local adaptation. It can be explained in the same way as discussed above for the stomatal length.98 indicating that the photosynthetic traits are closely linked to the variation in DNA methylation in a clonal sea grass. In contrast, others demonstrated that the photosynthetic efficiency was affected by transgenerational memory of drought stress, but this effect was not linked to the changes in the methylome100. The existing knowledge is thus largely ambiguous and more research is needed in this area, especially for extreme climatic conditions, where damage of photosystem II and whole functioning of photosynthesis apparatus is crucial for plant survival102.The effect of 5-azaC on photosynthetic efficiency is in line with Jueterbock et al.105 we did not find any effect of the 5-azaC on osmotic potential. In contrast to this, Hao et al.106 indicated that species have an ability of epigenetic adjustments to osmotic stress. Similarly, Colaneri and Jones107 indicated that methylome is highly responsive to the changes in osmotic potential. None of these studies, however, provided direct functional link between the osmotic potential of a plant and the DNA methylation patterns.Despite the high genetic differentiation and plasticity in the osmotic potential shown in this as well as in the previous studies108 but the epigenomes can also be more closely aligned to the local environmental variation than to the genotypic variation109. In our study, response to the 5-azaC application was genotype specific, but genetically more related plants showed more similar response to the 5-azaC only for Fv/Fm. According to these results , we suggest that the response to 5-azaC, and thus DNA methylation patterns, were determined both by the specific genetic structure as well as the local environments112.Epigenetic variation depends on the specific genetic profiles of the different plants114. This expectation was supported by the significant main effects of the traits in half of our tests. In line with Carlson et al.31, we found that the direction and intensity of the relationship depend on the specific conditions with cultivating conditions having much stronger effect than the 5-azaC application. The highest number of relationships was detected in the warm-wet conditions and the control plants and the lowest in cold-dry conditions. While it could be generally expected that the warm-wet conditions are less stressful for plants than the cold-dry conditions, it may be the opposite in our system of alpine species growing along a moisture gradient from very wet to wet, rather than wet to dry. A higher number of significant correlations in the warm-wet conditions may thus be in line with an expectation that the variation in ecophysiological traits plays a role for our fitness proxies (plant biomass and number of ramets) only if the plants are under some level of stress117. These significant correlations may allow evolutionary adaptations of the plants to changing climatic conditions63.We expected that the observed changes in the ecophysiological traits translate into changes in plant fitness indicating potential selection on these traitsv/Fm value was the only parameter showing significant negative relationship with both number of ramets and biomass in the overall test. However, an opposite relationship would be expected—the higher values of Fv/Fm, indicating lower damage of the photosystem, the higher plant fitness121. However, Madriaza et al.122 found such a positive relationship only in plants damaged by herbivores, while no such relationship has been found in control plants. The negative relationship shown here seems to be in contrast to all previous findings and may indicate a trade-off in the plants. They either invest into growth at the expense of lower quality of the photosynthetic apparatus or vice versa.The F123. The opposite relationship in cold-dry chamber in 5-azaC treated plants may be caused by differential variation in the trait values between the control and 5-azaC treated plants. Alternatively, this may mean that the relationship is affected by some other traits affected by 5-azaC application  due to trait interactions124. This result may also indicate that control plants with naturally methylated DNA perform better when stomata are bigger and fewer possibly suggesting that DNA methylation may have an adaptive value. We, however, also found even a stronger negative relationship between stomatal length and number of ramets in 5-azaC treated plants from the cold-dry chamber. While this supports the general assumption that stomata size and density are important determinants of fitness126, its specific effects are clearly dependent on actual conditions43 and likely affected by DNA methylation.In stomatal traits, the only significant main effect was positive effect of stomatal length on aboveground biomass. In other cases, the effects of stomatal traits interacted with cultivating conditions. Plant fitness proxies were higher in case of longer stomata in control warm-wet chamber, while fitness proxies decreased with stomatal length in cold-dry chamber in plants receiving 5-azaC treatment. Few larger stomata are expected to be less costly than many smaller stomata and can indicate that water supply is optimal in these conditions127 at least under some conditions. Low osmotic potential indicates accumulation of osmotic solutes in the plants e.g. as a protection against drought or frost51. The positive relationship thus indicates that the less stressed plants, not needing to invest to lowered osmotic potential, could invest into growth.Increasing values of fitness proxies with increased osmotic potential indicates close link between the traits129. We have also provided an evidence in our previous studies that the foliar application of 5-azaC reduces overall DNA methylation level by 4–30% in treated Trifolium repens, Taraxacum brevicorniculatum and Fragaria vesca plants130. In a grass, Oryza sativa, foliar application of 75 µM 5-azaC solution reduced their overall DNA methylation level by more than 50%131. This indicates that the 5-azaC methylation works even in monocots, and may be even stronger than in dicots, and is thus likely to work in our monocot species as well. In rare circumstances, 5-azaC can induce hypermethylation of particular loci132, hence likely silence some genes, but the overall effect of 5-azaC is reduction of DNA methylation level, which is in most cases followed by upregulation of previously epigenetically silenced genes. Therefore, we suggest that the majority of the 5-azaC effects in our study can be ascribed to its demethylating effect than to some other, less likely functions of the 5-azaC on DNA methylation status of plants and on other plant functions.In the study, we assumed that the application of 5-azaC leads primarily to DNA demethylation but did not collect our own data to support the assumption. We suggest that the assumption is reasonable as it is generally accepted that the 5-azaC reduces DNA global methylation through the effect on DNA methyltransferases, an enzyme that bonds methyl group on DNA135. Multiple rounds of the 5-azaC application over the period of 10 months as done here may have even increased the noise in the system. This could lead to low statistical power of our study as we only worked with 6 genotypes per climate of origin, i.e. 24 genotypes in total . On the other hand, 5-azaC application has been applied to each genotype in 6 replicates, reducing the chance that the effects detected per genotype are just consequences of the random demethylation. Overall, the fact that we did detected effects of 5-azaC application after accounting for our hierarchical model structure indicates that the 5-azaC application had some predictable effects in our system.While we detected significant effects of the 5-azaC application on ecophysiology of the plants (in interaction with other factors), the effects were relatively weak and highly variable. This is likely due to the fact that the 5-azaC is randomly incorporated into the DNA instead of cytosine during its replication resulting in reduced activity of DNA methyltransferase, which is followed by hypomethylation at random sequences and thus random demethylation patterns137 and from seeds to flowering several months later138 suggesting long lasting effect of the 5-azaC on the DNA methylation. On the other hand, Kumpatla and Hall139 reported only temporary effect of the 5-azaC on the DNA methylation. Although we have no empirical data to support our assumption, we consider it to be very unlikely that the DNA methylation status of the 5-azaC treated plants would completely restore to its original status, notably the DNA methylation patterns induced by the original environment. Hence, considering also the slow growth of our plant species and the detected 5-azaC effects, we assume that the 5-azaC was still effective when we assessed the ecophysiological traits.Another important question is whether the effect of 5-azaC applied 6 months before measurements could still be effectively altering ecophysiological traits in our study. There is no general consensus on how persistent the effect of 5-azaC on DNA methylation is over time. 5-azaC induced demethylation of DNA was shown to be passed to progeny65 is extensively discussed in the methods part of the paper.The possible suspicion that our experiment is pseudoreplicated as defined by HurlbertF. rubra were not strongly affected by the 5-azaC application. In few cases, we, however, found significant effects of the 5-azaC application on the ecophysiological traits under specific conditions of cultivation of plants of a specific origin. Under the assumption that the 5-azaC reduced the level of DNA methylation, the effects detected indicate that the DNA methylation does not mediate long-term adaptations of the species to their environment of origin. These findings do not support increasing body of evidence that heritable epigenetic differentiation can be an important driver of plant adaptation and/or population differentiation reflecting long-term natural conditions143. In contrast, our results suggest that the heritable epigenetic differentiation, modified by the 5-azaC application, may be an important driver of species response to conditions of a previous season66, allowing for fast responses to the rapidly changing conditions. This is in line with McGuigan et al.136 suggesting that epigenetic mechanisms may be enhancing fitness extremely rapidly. This fast ability to epigenetically adjust to the environment may be adaptive in case the conditions repeat in time, but will likely be maladaptive in case of increased variation in climate.Ecophysiological traits of 145. It is thus possible, that despite our conclusion of the lack of an effect of DNA methylation in the long-term adaptation of plants, the increased level of mutations in the methylated sites may be important for these adaptations.Epigenetic regulation of ecophysiological processes thus seems to play a limited role in the long-term adaptations of plants. However, as the 5-azaC is blocking the function of 5-methyl cytosine and methyl cytosine is an endogenous mutagen in DNA, the sites of DNA methylation are mutational hotspotsThe observed variation in the ecophysiological traits translated into differences in our fitness proxies (plant biomass and number of ramets) but the effects were context dependent. In addition, most of these effects point to an opposite direction than expected indicating that the ecophysiological traits-fitness proxies’ relationships are very complex. Further studies need to attempt obtaining more mechanistic insights into the epigenetic regulation of the different ecophysiological traits as well as into the trait-fitness proxies’ relationships. These studies always need to be performed under different conditions to account for the strong context-dependency.Supplementary Information.


SR Protein Kinases (SRPKs), discovered approximately 30 years ago, are widely known as splice factor kinases due to their decisive involvement in the regulation of various steps of mRNA splicing. However, they were also shown to regulate diverse cellular activities by phosphorylation of serine residues residing in serine-arginine/arginine-serine dipeptide motifs. Over the last decade, SRPK1 has been reported as both tumor suppressor and promoter, depending on the cellular context and has been implicated in both chemotherapy sensitivity and resistance. Moreover, SRPK2 has been reported to exhibit contradictory functions in different cell contexts promoting either apoptosis or tumor growth. The aim of the current review is to broaden and deepen our understanding of the SRPK function focusing on the subcellular localization of the kinases. There is ample evidence that the balance between cytoplasmic and nuclear SRPK levels is tightly regulated and determines cell response to external signals. Specific cell states coupled to kinase levels, spatial specific interactions with substrates but also changes in the extent of phosphorylation that allow SRPKs to exhibit a rheostat-like control on their substrates, could decide the proliferative or antiproliferative role of SRPKs. There are more than 170 review articles in PubMed on signaling proteins that can function both as oncogenes or tumor suppressors in human cancer. The pro- and anti-tumorigenic activities depend on the cellular context and are usually ascribed on the different expression levels of these proteins and/or their involvement in different signaling pathways. Among these molecules are members of the CMGC family of protein kinases that share the common property to phosphorylate SR proteins and modulate their activity, thus regulating alternative splicing. DYRK2 has been reported to trigger antitumor and pro-apoptotic signals, while in parallel other studies identified DYRK2 as a highly overexpressed kinase in various cancer types and DYRK2 inhibitors were shown to exhibit antiproliferative properties . This puSRPKs have also been related both to chemotherapy sensitivity and resistance . Moreover, SRPK1, the most-studied member of SRPKs, has been reported to function as both an oncogene and tumor suppressor depending on its expression level . Wang etThe total SRPKs’ protein abundance in a cell has been considered as the determinant factor in their function. Yet, SRPKs are mobile proteins that can rapidly transit between the cytoplasm and nucleus. Their high response sensitivity to external signals, such as for example stress signals, is best evidenced by the fact that inefficient fixation with low concentrations of formaldehyde during the immunofluorescence process for just 5 min was suffient to stimulate nuclear translocation of SRPK1 in HeLa cells . Hence, Here, we review the signaling pathways and the respective post-translational modifications that mediate the cytoplasmic-to-nuclear relocalization of SRPKs and discuss how these molecular mechanisms result in a well-defined change in nuclear kinase levels and activity which in turn may elicit specific functional outcomes. We also re-examine carefully the literature to shed some light onto the question why SRPKs have been reported both to promote and suppress cell proliferation and also to provide an explanation on the enigmatic and rather contradictory role of the kinases in mediating either sensitivity or resistance of cancer cells to chemotherapeutic drugs.Our current knowledge regarding the subcellular localization of SRPKs is largely based on experiments in highly proliferating cells such as cancer and yeast cells. In cancer cells SRPK1 localizes predominantly in the cytoplasm with a more or less faint staining in the nucleus, while the nuclear staining profile of SRPK2 is more apparent .The predominant cytoplasmic localization of SRPK1 and SRPK2 strongly supports the notion that these kinases mainly function outside the nucleus . Accordirtin-SR2 . Besidesrtin-SR2 , includirtin-SR2 . The nucrtin-SR2 . FurtherThe “steady-state” cytoplasmic localization of SRPKs is altered by external signals that dissociate the kinases from the chaperone complexes, resulting in their translocation to the nucleus. Kinase activity is a prerequisite for the nuclear translocation of SRPKs. Blocking SRPK activity either by inactivating mutations or by the selective SRPK1/2 inhibitor, SRPIN340, efficiently impeded the entry of the kinases into the nucleus .Schizosaccharomyces pombe dsk1 were observed in SDS-PAGE. The fast migrating form was enriched in interphase cells, while the slowly migrating form was prominent in mitotic cells. The disappearance of the slowly migrating band following phosphatase treatment strongly suggested that mitotic dsk1 was phosphorylated.Earlier reports indicated that cell cycle signals triggered the translocation of SRPKs to the nucleus at the late G2 phase in yeast and HeLa cells  and suggActivation of the PI3K-Akt signaling pathway by EGF was then shown to be involved in SRPK1/2 nuclear translocation. Specifically, On the other hand, nuclear translocation of SRPKs is closely related to the DNA damage response. In most of the above-mentioned phosphorylation events, phosphorylation was both necessary and sufficient to induce the relocation of the kinases from the cytoplasm to the nucleus, since the phosphorylation-defective mutants were restricted in the cytoplasm, whereas the phosphorylation-mimicking mutants localized in the nucleus even in the absence of any treatment. Yet, in 5-FU treated HeLa cells, while the phosphorylation-defective double mutant (SRPK1326/408A) was almost completely restricted to the cytoplasm, the phosphorylation-mimicking double mutant (SRPK1326/408D) partially localized in the nucleus in the absence of 5-FU treatment, suggesting that phosphorylation of these residues was necessary but not sufficient for nuclear translocation of SRPK1 and additional modification(s) was (were) required . In thisFinally, SRPK2 was also found to be O-GlcNAcylated at Ser490, Thr492, and Thr498 in non-treated MCF-7 and HEK293T cells . O-GlcNAin vitro assays revealed that activated Akt1 induced phosphorylation of SRPK1 at a very low stoichiometry -treated HeLa cells and conferred partial resistance to the drug . SimilarThere has been considerable debate within the literature as to whether SRPKs are related to chemotherapy sensitivity or resistance. The conflicting results were derived mainly from studies on SRPK1 in cisplatin-treated cells . In all −/− cells). TSC2 is a negative regulator of mTORC1 and therefore in these cells the mTORC1/S6K1/CK1 pathway is constitutively activated, SRPK2 is highly phosphorylated and as expected was found almost exclusively to the nucleus  gene  nuclear SRPKs are critical in sustaining the non-proliferative state and/or mediating drug toxicity. This suggests that SRPKs have a complicated role in cancer that seems to depend on the cell state, the subcellular localization and the threshold levels of the kinases. Deciphering this coordinated mode of action not only can provide valuable insights into the roles of SRPKs but can have a significant impact on medical research.Based on our recent data  we formu


Oxygen is the most common drug used in the neonatal intensive care. It has a narrow therapeutic range in preterm infants. Too high (hyperoxemia) or low oxygen (hypoxemia) is associated with adverse neonatal outcomes. It is not only prudent to maintain oxygen saturations in the target range, but also to avoid extremes of oxygen saturations. In routine practice when done manually by the staff, it is challenging to maintain oxygen saturations within the target range. Automatic control of oxygen delivery is now feasible and has shown to improve the time spent with in the target range of oxygen saturations. In addition, it also helps to avoid extremes of oxygen saturation. However, there are no studies that evaluated the clinical outcomes with automatic control of oxygen delivery. In this narrative review article, we aim to present the current evidence on automatic oxygen control and the future directions. Oxygen is a drug with a narrow therapeutic range in vulnerable preterm neonates. Avoiding both hypoxemia and hyperoxemia is important especially in neonates as both are associated with short-term and long-term adverse outcomes , 2. HypoStudies using A-FiO2 have consistently shown to improve the proportion of time spent in the TR of SpO2, reduce hypoxemia and hyperoxemia in preterm infants on non-invasive or invasive respiratory support. Whilst the A-FiO2 systems have been commercially available, it has not yet established itself in the routine care in the neonatal ICU . This isIn this review article we will make a case for importance of SpO2 targeting in preterm infants, clinical implications of intermittent hypoxemia/hyperoxemia, current evidence for the use of A-FiO2, the types of algorithms available in clinical practice, challenges in implementation of technology and the future directions.In the last decade, five large randomized controlled trials –18 were post-hoc analysis of Canadian oxygen trial (COT study), intermittent prolonged hypoxemia (SpO2 < 80%) for at least 1 min was associated with increase in composite outcome of death after 36 weeks or major neuro-disability  at 18 months corrected age , integrates over time and velocity and determines the oxygen output. The adaptive model algorithms consider the infant's physiology that may have the effect on oxygen dissociation curve. A non-linear model is created based on FiO2-oxygen saturation relationship. The controller adjusts its model of this relationship to achieve target saturations .Ⓡ infant ventilator , p = 0.008] , p = 0.009].The details of the currently available studies are shown in = 0.008] . A-FiO2 p < 0.005) and time spent below the TR  were higher as compared to CLiO2. The difference in the hypoxemia and hyperoxemia episodes may be related to the different design of the algorithm.Various algorithms are available with A-FiO2. Only two studies compared different A-FiO2 algorithms. Schwarz et al. compared fast and slow CLAC algorithms  and SalvAlthough A-FiO2 has consistently shown to be superior to the M-FiO2 in maintaining the SpO2 in the TR, we do not know if this physiological benefit is associated with improved clinical benefits. It can be hypothesized that better control in maintaining SpO2 in TR, reduction in hypoxemia and hyperoxemia may concomitantly result in improved short- and long-term clinical outcomes. There are currently no studies available that has looked at use of A-FiO2 to improve clinical outcomes. For a clinical outcome study with A-FiO2, it is imperative that parallel arm RCT design is chosen. The study should also capture the entire period on respiratory support and supplemental oxygen. A large RCT with aim to recruit 2,340 preterm infants (<28 weeks) is currently underway (NCT03168516) . In thisAn improvement in saturation targeting with A-FiO2 was not associated with improved tissue oxygenation in studies by Dani et al. and Waitz et al. , 49.Alarms are necessary evils in any intensive care units . Alarm oN = 293) and post  implementation of A-FiO2 in preterm infants , showed that around 19 neonatal units (9.9%) units used A-FiO2 . Sixty-eThe main challenges to implementation of A-FiO2 in NICU are lack of devices delivering A-FiO2, unfamiliarity with the devices and the lack of clinical outcome studies. Most of the new neonatal ventilators have A-FiO2 options on them. However, without appropriate expertise and training, the introduction and implementation of any change can be a failure. There are few reports that A-FiO2 can result in inadvertent higher FiO2 when the probe was displaced and mask desaturations.One of the concerns with regards to use of A-FiO2 is that it may mask clinical deteriorations. A-FiO2 is better than M-FiO2 at reducing hypoxemic episodes by automatically increasing the FiO2. However, the hypoxemic events may occur in relation to clinical deterioration like sepsis and just by increasing the FiO2 during these episodes, such events may be masked. This is generally not an issue especially if the staffing level is such that there is continuous close observation of these infants. This can also be overcome by appropriate staff training and using appropriate FiO2 alarms. In our unit we have addressed this by staff education and training. The CLiO2 system provides base FiO2 which is a trend, and a trend upwards may be indicative of deteriorating clinical condition. There is continuous scrutiny and medical staff are alerted when the there is an upwards trend of more than 5%.Another potential limitation with A-FiO2 is its inability to differentiate hypoxemic events secondary to apneic episodes. A-FiO2 would provide sufficient oxygen to keep the SpO2 in TR, whereas with severe apneic episodes the infant may need other intervention like stimulation and positive pressure support. This issue can be overcome again by close observation of the infant and appropriate vital parameter alarm limits. Again, in these scenarios the role of staff education and training cannot be over emphasized.It is often perceived at the bedside that FiO2 tends to be higher with A-FiO2 than M-FiO2. Some cross over studies with A-FiO2, did not show any statistical difference in the median FiO2 , 36, 42,Whilst on M-FiO2, the staff proactively intervene for hypoxia than hyperoxia episodes . Also, iIn most of the A-FiO2 devices, the SpO2 can be monitored on the device in which the algorithm is incorporated. Some of the A-FiO2 devices albeit having the monitoring functionality does not have SpO2 alarms incorporated. This necessitates having additional SpO2 monitoring system with alarms to alert the staff of the deviation from TR. Despite using the same SpO2 technology, on occasions there seems to be discrepancy in SpO2 between the two monitoring devices. In our practice, we instruct our nursing staff to reposition/replace SpO2 probe which seems to resolve this discrepancy on most occasions. Resolution of discrepancy on most occasions reassures us that this discrepancy is not to the extent of clinical significance (hypoxia/hyperoxia), still it could result in staff and parental anxiety. However, this can be overcome by incorporating SpO2 monitoring with alarm limits on the same device.Cost of the equipment is reported as another major limitation. Although, most of the newer neonatal ventilators are equipped with A-FiO2, the older versions may not have this facility. The discussion around cost-effectiveness should consider the clinical benefits with this technology. However, we are clearly lacking clinical studies looking at the short- and long-term outcomes of A-FiO2. When staff work load is considered, A-FiO2 has shown to be associated with significant reduction in the number of manual adjustments required thus allowing staff to focus on other aspects of clinical care .Not all neonates of the exact same maturity are alike. The recent AAP guidelines recommends TR between 90 and 95%. However, it also underlines that there is no ideal TR and that it is patient specific and vary with gestation, chronological age and the underlying condition . StudiesAt birth, preterm infants slowly transition from fetal to neonatal life and often need interventions to support with this transition. Oxygen supplementation is often needed for these infants to maintain recommended SpO2 levels in the first 10 min of life. Hence use of pulse oximetry is recommended by the resuscitation council to monitor and titrate oxygen supplementation . With paEven with advances in neonatal resuscitation it remains a challenge to meet the SpO2 targets during the first 10 min of life. In a study with preterm infants ≤30 weeks the median percentages of time spent above and below the target were 44 and 51%, respectively . A-FiO2 Need for RCTs that are adequately powered for short term and long-term outcomes. These studies should also report the cost effectiveness of the intervention, considering all the health outcomes and staff workload. The future studies should consider recruitment as soon as possible after birth to limit extremes of oxygenation during early period of the life.Studies are needed with characterization of all the existing algorithms with both invasive and non-invasive respiratory support.Innovations are needed to provide commercial algorithms that could support moving SpO2 targets (like during first 10 min of birth).Role of automated oxygen during elective neonatal intubation and reduction in hypoxemia during these procedures.Use of automated oxygen in preterm infants receiving nasal cannula low flow oxygen.Establish a role of A-FiO2 in low resource-staff limited settings.There is overwhelming evidence that A-FiO2 achieves higher proportion of time in SpO2 TR, reduces duration and episodes of hypoxemia and hyperoxemia. Although the impact on clinical outcomes associated with A-FiO2 is yet to be proven, from the available studies we can presume that there is no harm. Merely adopting the recommendations of targeting SpO2 (90–95%) will not suffice. It is essential that this is achieved. If not, this will be justice half done and infact we may not see the actual clinical benefits of SpO2 targeting. A-FiO2 is a promising technology that helps to achieve this target. However, the clinical benefits of it are still unknown.VN, PL, and ML conceptualized the review, drafted the initial manuscript, reviewed, and revised the manuscript. TB reviewed and revised the manuscript. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.