# Recurrent Water Deficit and Epigenetic Memory in _Medicago sativa_ L. Varieties
## 4 Discussion
Plants can respond more efficiently to environmental stress by \"remembering\" a stress episode and react effectively upon subsequent exposures to an environmental challenge There are two types of'memory'--first, it exists a trans-generational transmission of stress memory to the progeny of a plant that experienced a stressor, and second, during the biological cycle of a plant, there is a short-term memory of a stress episode (known as priming). Such a mechanism allows cells to form a molecular epigenetic memory during the first stress, and the occurrence of second stress activates the stress memory and the induction of a new, improved plant response [8]. Epigenetic marks, such as DNA methylation, can be modified and result in an epigenetic response. Thus, environmental stresses, such as drought, salinity, and elevated temperature, can provoke changes in DNA methylation that lead to modified phenotypes and, ultimately, to better plant adaptation [39; 45]. In this work, we attempted to study the priming effect of drought by imposing the two varieties to two consecutive stress episodes allowing a long recovery between the two stress events. A significant decrease in the dry shoot weight was observed in Lamia upon one drought stress episode (CD2). However, there was no further reduction in the second drought stress. In other words, Lamia is maintaining its 'first stress' biomass in the second drought stress and suffers no further decrease. It is plausible, therefore, that a priming effect may be operating in this system, whereby a drought-responsive mechanism is'remembered' and activated in the second stress episode and aids in preventing additional biomass decrease. Moreover, the maintenance of decreased total DNA methylation in D1D2 plants may suggest the operation of a DNA methylation 'priming' mechanism in Lamia. This mechanism provides a better and faster adaptation to recurrent stress not by increasing its biomass but by increasing the flexibility of the plant to respond to the environmental stresses (i.e., maintaining shoot and root water content), as indicated in several other studies [16; 39]. Although Lamia's adaptation may compromise plant productivity in the short term, it represents increased tolerance to recurrent stresses and, therefore, enhances productivity in the long-term [46; 47]. However, whether these drought-induced DNA methylation alterations persist after subsequent recovery, and if they can be transmitted to the next generation needs further investigations. On the other hand, regarding Chiaronia variety, a significant decrease is observed in the shoot biomass between CD2 plants and D1D2 plants, according to the Iepi and hepi values. Interestingly, the total methylation is significantly altered (increased) in D1D2 plants compared to CD2. Consequently, the increase in methylation of D1D2 plants compared to CD2 plants in the Chiaronia variety might suggest a negative effect on the total biomass of D1D2 plants. Consequently, no priming effect is observed in the Chiaronia variety. Several reports highlight that extreme environmental constraints, such as drought are expected to occur more frequently and more intensely in the future [48]. Thus, the identification of genetic material that can be adapted to recurrent stressors is of great importance. Plant adaptation to environmental alterations can be greatly improved through priming, thus reducing economic losses due to stress. However, priming does not always correlate with improved plant performance and, therefore, the memory may only influence the phenotype of specific traits [49]. This study provided baseline knowledge about methylation status, epigenetic memory, shoot and root water content for the two varieties of _M. sativa_, Lamia, and Chiaronia. Future research through Whole Genome Bisulfite Sequencing (WGBS) could reveal the differentially methylated regions and underlying genes that are primarily responsible for drought stress giving an important opportunity for future breeding of Medicago species.