# Recurrent Water Deficit and Epigenetic Memory in _Medicago sativa_ L. Varieties
## 4 Discussion  
In the present study, we investigated the effect of repeated drought stress on two _M. sativa_ varieties, at the phenotypic and epigenetic level, in order to determine which is the most drought-tolerant variety and unravel any association of drought tolerance, priming events, and DNA methylation mechanisms. Plant growth and development is greatly affected by drought stress. Thus, one of the major breeding goals is the improvement of drought stress tolerance. The immediate response to drought stress is relatively well studied, but in nature, drought stress often occurs on a regular basis, and response to this type of stress is much more complicated and less understood. Recent studies have indicated that drought stress priming enhances plant adaptation to subsequent stress by activating different strategies, such as maintaining the photosynthetic capacity [33] accumulating more antioxidants [34] and altering gene expression [35]. The fact that drought priming can improve plant performance to subsequent stress by maintaining its biomass is documented in several studies [36; 37]. Drought was imposed by two rounds of reduced water availability (phase I and phase II) with a long recovery phase in between. According to our findings, the Lamia variety seemed to respond better to drought conditions as judged by the comparative evaluation of dry shoot and root weight between the two varieties. Specifically, the Lamia variety displayed significantly higher values of dry shoot and root weight than Chaironia after the two consecutive drought episodes (Figures 2 and 3). Since increased weight is directly linked to higher economic profit for Medicago breeders, the importance of this observation is evident. Moreover, in Lamia, the shoot water content remains constant among the three treatments, whereas Chaironia displayed increased water content in the D1D2 plants that could be attributed to low growth rates and to the overall stunted growth resulting from stress. Collectively, these results suggest that Lamia is probably less affected by drought treatment compared to Chiaironia in terms of compromised yield and quality upon drought, which would make it a preferred variety for breeding purposes. The results obtained by the MSAP method showed that in the Lamia variety, a significant decrease was observed in DNA methylation upon the second stress incident (D1D2) as compared to the original control (CC) according to Iepi and hepi values. The decrease in DNA methylation might be associated with an attempt to activate specific drought-responsive pathways, as described in previous reports [14; 19; 38; 39; 40]. Certainly, further research into identifying the gene sequences underlying these differential DNA methylations and their expression patterns is needed in order to provide evidence for this hypothesis. A decrease was also observed in the D1D2 as compared to CD2, but it was not significant. Overall it may be suggested that the Lamia variety has a trend for DNA methylation decrease when it undergoes repeated drought episodes. Chiaironia, on the other hand, undergoes a decline of DNA methylation compared to controls in the first stress episode and then an increase in the second stress, which might reflect an attempt of global transcriptional reprogramming (simultaneous activation and de-activation of genes) rather than maintenance of reduced methylation status observed in the Lamia (Table 4). In several studies, the decrease of methylation/hypomethylation/demethylation has been proven to be associated with adapted responses to various stresses [41; 42; 43]. Similar findings regarding the decrease of methylation percentages, as a probable indication of how drought-resistant plants can copewith stress, are presented for _Lolium perenne_ by [12] and for rice genotypes by [44]. Furthermore, in Oryza sativa, it was found that hypomethyllation is more frequent in drought-tolerant genotypes [42].