Seeds germinating underground display a specific developmental programme, termed skotomorphogenesis, to ensure survival of the emerging seedlings until they reach the light. They rapidly elongate the hypocotyl and maintain the cotyledons closed, forming a hook with the hypocotyl in order to protect apical meristematic cells from mechanical damage. Such crucial events for the fate of the seedling are tightly regulated and although some transcriptional regulators and phytohormones are known to be implicated in this regulation, we are still far from a complete understanding of these biological processes. Our work provides information on the diverse roles in skotomorphogenesis of the core components of microRNA biogenesis in Arabidopsis, HYL1, DCL1, and SE. We show that hypocotyl elongation is promoted by all these components, probably through the action of specific miRNAs. Hook development also depends on these proteins however, remarkably, HYL1 exerts its role in an opposite way to DCL1 and SE. Interestingly, we found that a specific HYL1 domain involved in protein-protein interaction is required for this function. Genetic evidences also point to the phosphorylation status of HYL1 as important for this function. We propose that HYL1 help maintain the hook closed during early skotomorphogenesis in a microprocessor-independent manner by repressing the activity of HY5, the transcriptional master regulator that triggers light responses. This work uncovers a previously unnoticed link between components of the miRNA biogenesis machinery, the skotomorphogenic growth, and hook development in Arabidopsis.Plasmids containing pHYL1::HYL1-CFP and pSE::YFP-SE (Fang and Spector, 2007) used for transformation of hyl1-2 or se-1 plants, respectively, were a kind gift from Dr Fang and Dr Spector. Agrobacterium tumefaciens strain GV3101 containing each construct with the respective translational fusion under the

DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. Substrate pri-miRNA recognition by DCL1 requires two double stranded RNA binding domains located at the C-terminus of the protein. We have previously shown that the first of these domains, DCL1-A, is intrinsically disordered and folds upon binding pri-miRNA. Integrating NMR and SAXS data, we study here the conformational landscape of free DCL1-A through an ensemble description. Our results reveal that secondary structure elements, corresponding to the folded form of the protein, are transiently populated in the unbound state. The conformation of one of the dsRNA binding regions in the free protein shows that, at a local level, RNA recognition proceeds through a conformational selection mechanism. We further explored the stability of the preformed structural elements via temperature and urea destabilization. The C-terminal helix is halfway on the folding pathway in free DCL1-A, constituting a potential nucleation site for the final folding of the protein. In contrast, the N-terminal helix adopts stable non-native structures that could hinder the correct folding of the protein in the absence of RNA. This description of the unfolded form allows us to understand details of the mechanism of binding-induced folding of the protein.

23MicroRNAs are small RNA molecules with big impact in many eukaryotic biological 24 processes. In plants, their role as regulators of important developmental programs such as 25 leaf size and shape, flower organs or phase transitions, among others, have been evidenced 26 by mutants in specific miRNAs and by mutants in components of their biogenesis. However, 27 we are still far from understanding the scope of this regulatory system so other crucial 28 developmental phases might be influenced by the microRNA pathway. 29 Skotomorphogenesis is an essential developmental program that takes place after seeds 30 germinate underground in order to display a proper response when seedlings reach the light. 31 In this work, we found that the core components of microRNA pathway, DCL1, HYL1 and 32 SERRATE, promote hypocotyl elongation during skotomorphogenesis. Hook unfolding, 33 another characteristic phenotype displayed by dark-grown seedlings is also regulated by 34 these proteins but, surprisingly, they act in different ways. Thus, HYL1 represses hook 35 unfolding while DCL1 and SE promote it since the hooks of mutants on each component are 36 more or less open than those of wild-type during skotomorphogenesis, respectively. Genetic 37 and physiological analyses on HYL1 mutants provide evidence that repression of hook 38 unfolding is carried out through the HYL1 protein-protein interaction domain. Furthermore, 39 the data indicates that phosphorylated HYL1 is necessary for this function. Molecular and 40 genetic analyses also suggest that HYL1 regulates the activity of the master 3 41 photomorphogenic regulator HY5 in darkness to ensure a proper early skotomorphogenic 42 growth. In summary, while our data show a role for miRNAs in darkness, it also suggests a 43 microprocessor-independent role of HYL1 as a repressor of hook unfolding assigning a 44 biological function to phosphorylated HYL1. This work uncovers a previously unnoticed link 45 between components of the miRNA biogenesis machinery, the skotomorphogenic growth 46 and hook development in Arabidopsis. 48Author summary 49 Seeds germinating underground display a specific developmental program, termed 50 skotomorphogenesis, to ensure survival of the emerging seedlings until they reach the light. 51They rapidly elongate the hypocotyl and maintain the cotyledons closed, forming a hook with 52 the hypocotyl in order to protect apical meristematic cells from mechanical damage. Such 53 crucial events for the fate of the seedling are tightly regulated and although some 54 transcriptional regulators and phytohormones are known to be implicated in this regulation, 55 we are still far from a complete understanding of these biological processes. Our work 56 provides new information on the diverse roles in skotomorphogenesis of the core components 57 of microRNA biogenesis in Arabidopsis, HYL1, SE, and DCL1. We show that hypocotyl 58 elongation is promoted by all these components, probably through the action of specific 59 miRNAs. Hook development is also...