# Recurrent Water Deficit and Epigenetic Memory in _Medicago sativa_ L. Varieties
## 1 Introduction  
Climate changes affect crop production as well as the availability of natural resources, such as land and water. More than 60% of the annual yield fluctuations of important crops have been attributed toclimatic variability [1]. Thus, the release of new, improved varieties combining high productivity and increased adaptability to the ongoing environmental changes has become an imperative [2]. Recent findings highlight a new source of heritable variation in plants, derived from epigenetic regulations, that play an essential role in directing the expression of agronomically important traits and adaptation of plants to a changing environment due to global climate change [3]. In this respect, since the epigenetic regulatory mechanisms are greatly affected by the environment, they could be a potential source of variation for adaptation [4]. Furthermore, as these mechanisms influence flowering regulation and reproductive development, they constitute an important factor in the plant breeding process [5]. Plant tolerance to external conditions depends on a complex network of molecular mechanisms, including massive changes in gene expression programs, regulated by epigenetic factors that modify chromatin structure. Chromatin remodeling to open (permissive) or closed (repressive) forms and subsequent gene activation or gene silencing, respectively, is mediated by DNA methylation, chromatin histone modifications, and the action of small RNAs [6; 7; 8]. Among the epigenetic regulatory mechanisms, DNA methylation is one of the most well-studied, especially in relation to plant adaptation to a broad range of abiotic stresses [9; 10]. DNA methylation, the process whereby a methyl group is added to a cytosine or adenine nucleotide of DNA, plays a key role in the epigenetic regulation of eukaryotic gene expression [11; 12; 13]. Growing evidence indicates that the ability of plants to respond to environmentally adverse conditions such as drought stress is strongly dependent on changes in the DNA methylation status, which can be tissue- and cell type-specific, and can be propagated mitotically within a single generation. Importantly, these changes also can be heritable and stably transmitted to the offspring of the next generation [14]. A genome-wide study by [15] showed that drought-tolerant and drought-susceptible _Oryza sativa_ varieties displayed differential site-specific DNA methylation upon drought imposed at the tillering stage, and nearly 70% of these DNA methylation changes were restored upon recovery under non-drought conditions [15]. In _Arabidopsis thaliana_[16] found that plants, which had been previously exposed multiple times to drought stress conditions, had the ability to respond to new stress by more rapid adaptive changes to gene expression patterns compared to plants which had not been exposed previously to drought conditions. Evidence already exists for _Arabidopsis thaliana_ showing transgenerational effects associated with low humidity stress. These are transmitted to at least one generation by changes in the DNA methylation status at various sites of the _SPEECHLESS_ (_SPCH_) and _FAMA_ genes encoding basic-helix-loop-helix (bHLH) transcription factors [17; 18]. Reports are emerging regarding other crops such as _Zea mays_. [PERSON] et al. [19] studied the response of maize plants to mild water stress, by modulating stress-induced chromatin mark changes that control memory genes in order to activate or suppress them in upcoming stress events. _Medicago sativa_ L. (_M. sativa_) or alfalfa is a perennial legume species that is extensively cultivated for grazing, hay, and silage production worldwide [20], and a key component of many crop rotation systems [21]. It is characterized by high forage production and high nutritional value for animal feed, with higher and lower content of protein and fibers, respectively, compared to other forage crops [22]. Generally, it is considered a drought-tolerant forage legume due to its deep rooting system [20] with wide within-species variation [23].