In plants, the transcriptional and post-transcriptional regulation of gene expression mediated by sRNAs  is involved in several biological processes, ranging from organ differentiation to biotic and abiotic stress responses [2–4]. Small RNAs are divided into two main classes based on their biogenesis: the small interfering RNAs (siRNAs) are processed from perfect and long double-stranded RNAs while miRNAs are processed from single-stranded RNA transcripts that fold back onto themselves producing an imperfectly double-stranded stem loop . The endogenous siRNAs are divided into trans-acting-siRNAs (ta-siRNAs) and heterochromatic siRNAs (hc-siRNAs) .
The pathways of gene silencing mediated by sRNAs share, in plants, four consensus biochemical steps : (1) the biosynthesis of a double strand RNA (dsRNA); (2) the cutting of the dsRNA by a Dicer-like protein (DCL) in 18-25 nt-long sRNAs; (3) the O-methylation of the sRNAs by Hua Enhancer (HEN1), to protect them from degradation through the Small RNA Degrading Nuclease (SDN) class of exonucleases ; and (4) the integration of the sRNAs into an Argonaute (AGO) that associates with other proteins to promote gene silencing by partially or fully complementation with target RNA or DNA.
Plants have at least four different DCL proteins and each generates predominantly a particular class of sRNAs: DCL1 cleaves the imperfect double-stranded stem loop generating the miRNAs with around 21-nt ; DCL2 produces viral siRNAs 22-nt long ; DCL3 generates hc-siRNAs with 24-nt ; and DCL4 generates ta-siRNAs 21-nt long .
Plant DCLs contain six domains: one PAZ, two RNaseIII, one DEAD-helicase box (DEXD/H-box), one DUF283, at least one double-stranded RNA-binding (dsRB) domain and one Helicase-C domain . The PAZ domain binds to double-stranded RNAs at the 3' end . The two RNaseIII domains form an intramolecular dimer and the active site of each domain cleaves the dsRNA . The DExD/H-box domain might have an auto-inhibitory function, because removal of this domain increases the cleavage rate of the human dicer . The DUF283 domain displays affinity to bind the double-stranded RNA-binding domains of the A. thaliana dsRNA binding proteins (DRBs)  suggesting a functional role in the selection of the small RNA processing pathway.
The A. thaliana and Oryza sativa genomes have been completely sequenced and annotated [16, 17]. These plant species encode ten and eighteen AGOs, respectively [16, 17]. Both species share common phylogenetic related AGOs that are divided in three clades . In A. thaliana some AGOs are well studied, for example AGO1 binds the miRNAs to mediate the cleavage of targets mRNAs and together with AGO10 both promote the translational repression of the targets but with different selectivity for the miRNAs [19, 20]. AGO4, AGO6 and AGO9 fall in another clade and they are associated with hc-siRNAs to control DNA methylation . AGO7 in the last clade is implicated in the production of the ta-siRNAs .
The AGO proteins generally contain one variable N-terminal region and one conserved C-terminal region constituted by the PAZ, middle (MID) and PIWI domains . The PAZ domain binds to the 3' end of the guide strand of the sRNAs. The PIWI domain is responsible for the Argonaute slicer activity. The cleavage activity is carried out by the active site on the PIWI domain usually presenting an Asp-Asp-His (DDH) motif [19, 24]. The slicer activity of Argonaute requires a perfect complementarity around the cleavage site of the guide-target duplex . The 5' phosphate group of the sRNA guide strand is buried in a deep pocket at interface between the MID domain and PIWI domain .
In A. thaliana, the sRNAs association with the Argonaute proteins is based on the recognition of the 5' end nucleotide. This specificity is mediated by the MID domain . For example AGO1 binds mainly to RNAs with a uridine at their 5' end, whereas AGO2, AGO4, AGO6 and AGO9 recruit RNAs with a 5' end adenosine and the AGO5 predominantly binds to sRNAs with a cytosine [21, 26].
The biogenesis of miRNAs is under feedback regulation such that two key players are themselves regulated by miRNAs. DCL1 mRNA has a complementary sequence for miR162, which leads to the cleavage of DCL1 mRNA . Likewise, AGO1 mRNA contains a complementary sequence for miR168 which leads to AGO1-mediated cleavage of AGO1 mRNA .
Medicago truncatula is a model legume , and its genome is almost completely sequenced (accessed 2 April 2010) . However, almost nothing is known about the identification and function of AGO and DCL genes in legumes species. In M. truncatula several sRNAs were found to be differentially expressed in different organs and abiotic stress conditions [2, 3, 31, 32]. Recently we described the up-regulation of miR398a/b and miR408 under water deficit and the corresponding down regulation of their respective targets, COX5b and plantacyanin . However, no studies have been reported implicating the modulation of small RNA pathways in response to either water deficit or any other abiotic stress in legumes.
In the present study we identify three putative DCL and twelve putative AGO genes in the M. truncatula genome. We also established their phylogenetic relationship with the A. thaliana DCLs (AtDCLs) and AGOs (AtAGOs) and performed their domain characterization. The mRNA levels of these genes were quantified by quantitative real time PCR (qPCR) in vegetative growing plants under water deficit conditions. Our results show that the mRNA levels of the identified AGO and DCL genes are modulated when M. truncatula is subjected to water deprivation.