MicroRNAs (miRNAs) are small regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins. miRNAs are transcribed as much larger primary transcripts (pri-miRNAs), the function of which is not fully understood. Here we show that plant pri-miRNAs contain short open reading frame sequences that encode regulatory peptides. The pri-miR171b of Medicago truncatula and the pri-miR165a of Arabidopsis thaliana produce peptides, which we term miPEP171b and miPEP165a, respectively, that enhance the accumulation of their corresponding mature miRNAs, resulting in downregulation of target genes involved in root development. The mechanism of miRNA-encoded peptide (miPEP) action involves increasing transcription of the pri-miRNA. Five other pri-miRNAs of A. thaliana and M. truncatula encode active miPEPs, suggesting that miPEPs are widespread throughout the plant kingdom. Synthetic miPEP171b and miPEP165a peptides applied to plants specifically trigger the accumulation of miR171b and miR165a, leading to reduction of lateral root development and stimulation of main root growth, respectively, suggesting that miPEPs might have agronomical applications.

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.

Most land plant species live in symbiosis with arbuscular mycorrhizal fungi. These fungi differentiate essential functional structures called arbuscules in root cortical cells from which mineral nutrients are released to the plant. We investigated the role of microRNA393 (miR393), an miRNA that targets several auxin receptors, in arbuscular mycorrhizal root colonization. Expression of the precursors of the miR393 was down-regulated during mycorrhization in three different plant species: Solanum lycopersicum, Medicago truncatula, and Oryza sativa. Treatment of S. lycopersicum, M. truncatula, and O. sativa roots with concentrations of synthetic auxin analogs that did not affect root development stimulated mycorrhization, particularly arbuscule formation. DR5-GUS, a reporter for auxin response, was preferentially expressed in root cells containing arbuscules. Finally, overexpression of miR393 in root tissues resulted in down-regulation of auxin receptor genes (transport inhibitor response1 and auxin-related F box) and underdeveloped arbuscules in all three plant species. These results support the conclusion that miR393 is a negative regulator of arbuscule formation by hampering auxin perception in arbuscule-containing cells.

Arbuscular mycorrhizal (AM) symbiosis associates most plants with fungi of the phylum Glomeromycota. The fungus penetrates into roots and forms within cortical cell branched structures called arbuscules for nutrient exchange. We discovered that miR171b has a mismatched cleavage site and is unable to downregulate the miR171 family target gene, LOM1 (LOST MERISTEMS 1). This mismatched cleavage site is conserved among plants that establish AM symbiosis, but not in non-mycotrophic plants. Unlike other members of the miR171 family, miR171b stimulates AM symbiosis and is expressed specifically in root cells that contain arbuscules. MiR171b protects LOM1 from negative regulation by other miR171 family members. These findings uncover a unique mechanism of positive post-transcriptional regulation of gene expression by miRNAs and demonstrate its relevance for the establishment of AM symbiosis.

One-sentence summary: 40The loss-of-function of MtNOOT1 and MtNOOT2 leads to the complete loss of nodule identity, prevents 41 the symbiotic process, and results in the absence of nitrogen fixation in Medicago truncatula. 56VZ, SC, GEDO, and PR analyzed the data. KM, KS and PR wrote the article. 58This work was supported by the CNRS, by the grants ANR SVSE 6.2010.1 (LEGUMICS) and ANR-14- 62Agriculture (Dufrenoy Grant, 2011). This work has benefited from the facilities and expertise of the IMAGIF 63Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr) which is supported by the Conseil Général de 64 l'Essonne. 66The author responsible for distribution of materials integral to the findings presented in this article in 67 accordance with the policy described in the instructions for authors (www.plantphysiol.org) is: Pascal 68 Ratet (pascal.ratet@u-psud.fr). 70 71 Acknowledgments 72The Institute of Plant Sciences Paris-Saclay (IPS2, France) benefits from the support of the LabEx Saclay 97fixing root-like structures that were no longer able to host symbiotic rhizobia. This study provides original 98 insights into the molecular basis underlying nodule identity in legumes forming indeterminate nodules. 100 INTRODUCTION 102The symbiotic interaction between legumes and rhizobia results in the formation of root nodules 103 dedicated to host nitrogen-fixing rhizobia. This unique ability to form root nodules is restricted to the 104 Rosids I clade. The predisposition of plants to enter symbiosis with nitrogen-fixing rhizobia seems to have 105 evolved once, between 70 and 100 million years ago and to have derived from an ancestral and 106 widespread symbiosis, the arbuscular mycorrhizal symbiosis (AMS, Soltis et al., 1995; Smith and Read, 107 2008;Bonfante and Genre, 2010;Humphreys et al., 2010;Werner et al., 2014). 109Genetic approaches using nodule-deficient (nod -) and non-functional nodule (fix -) mutant plants 110 allowed the identification of many genes essential for the early steps of root nodule symbiosis. 111Recognition between symbiotic partners, rhizobial infection and nodule organogenesis are initiated by the 112 host plant perception of rhizobial lipo-chitooligosacharidic compounds Jones et al., 113 2007;Kouchi et al., 2010; Horvath et al., 2011;Ovchinnikova et al., 2011; 114 . These compounds are called Nod factors 115and they are structurally similar to the mycorrhization factors (Myc factors) required for AMS initiation 116 (Maillet et al., 2011). 118In the Papilionaceae family, determinate nodules formed in the Phaseoleae, Loteae and 119Dalbergieae tribes have no persistent apical nodule meristem (NM). However, indeterminate nodules 120 formed in the Trifolieae and Fabeae tribes have a persistent apical NM. Indeterminate nodules are highly-121 structured and present different zones; the NM, the infection zone, the nitrogen fixation zone and the older 122 senescent zone (from top to bottom; . The ability of indeterminate nodules to grow 123 continuously results from the presence of the NM. ...

Contents 22I.22II.24III.25IV.27V.29VI.1031References32 Summary Plants have evolved a remarkable faculty of adaptation to deal with various and changing environmental conditions. In this context, the roots have taken over nutritional aspects and the root system architecture can be modulated in response to nutrient availability or biotic interactions with soil microorganisms. This adaptability requires a fine tuning of gene expression. Indeed, root specification and development are highly complex processes requiring gene regulatory networks involved in hormonal regulations and cell identity. Among the different molecular partners governing root development, microRNAs (miRNAs) are key players for the fast regulation of gene expression. miRNAs are small RNAs involved in most developmental processes and are required for the normal growth of organisms, by the negative regulation of key genes, such as transcription factors and hormone receptors. Here, we review the known roles of miRNAs in root specification and development, from the embryonic roots to the establishment of root symbioses, highlighting the major roles of miRNAs in these processes.

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SummaryPlants are able to lose organs selectively through a process called abscission. This process relies on the differentiation of specialized territories at the junction between organs and the plant body that are called abscission zones (AZ). Several genes control the formation or functioning of these AZ.We have characterized BLADE-ON-PETIOLE (BOP) orthologues from several legume plants and studied their roles in the abscission process using a mutant approach.Here, we show that the Medicago truncatula NODULE ROOT (NOOT), the Pisum sativum COCHLEATA (COCH) and their orthologue in Lotus japonicus are strictly necessary for the abscission of not only petals, but also leaflets, leaves and fruits. We also showed that the expression pattern of the M. truncatula pNOOT::GUS fusion is associated with functional and vestigial AZs when expressed in Arabidopsis. In addition, we show that the stip mutant from Lupinus angustifolius, defective in stipule formation and leaf abscission, is mutated in a BOP orthologue.In conclusion, this study shows that this clade of proteins plays an important conserved role in promoting abscission of all aerial organs studied so far.