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. ...

SUMMARY KARRIKIN INSENSITIVE2 (KAI2) is an α/β‐hydrolase required for plant responses to karrikins, which are abiotic butenolides that can influence seed germination and seedling growth. Although represented by four angiosperm species, loss‐of‐function kai2 mutants are phenotypically inconsistent and incompletely characterised, resulting in uncertainties about the core functions of KAI2 in plant development. Here we characterised the developmental functions of KAI2 in the grass Brachypodium distachyon using molecular, physiological and biochemical approaches. Bdkai2 mutants exhibit increased internode elongation and reduced leaf chlorophyll levels, but only a modest increase in water loss from detached leaves. Bdkai2 shows increased numbers of lateral roots and reduced root hair growth, and fails to support normal root colonisation by arbuscular‐mycorrhizal (AM) fungi. The karrikins KAR1 and KAR2, and the strigolactone (SL) analogue rac‐GR24, each elicit overlapping but distinct changes to the shoot transcriptome via BdKAI2. Finally, we show that BdKAI2 exhibits a clear ligand preference for desmethyl butenolides and weak responses to methyl‐substituted SL analogues such as GR24. Our findings suggest that KAI2 has multiple roles in shoot development, root system development and transcriptional regulation in grasses. Although KAI2‐dependent AM symbiosis is likely conserved within monocots, the magnitude of the effect of KAI2 on water relations may vary across angiosperms.

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.

The NOOT-BOP-COCH-LIKE (NBCL) genes are orthologs of Arabidopsis thaliana BLADE-ON-PETIOLE1/2. The NBCLs are developmental regulators essential for plant shaping, mainly through the regulation of organ boundaries, the promotion of lateral organ differentiation and the acquisition of organ identity. In addition to their roles in leaf, stipule and flower development, NBCLs are required for maintaining the identity of indeterminate nitrogen-fixing nodules with persistent meristems in legumes. In legumes forming determinate nodules, without persistent meristem, the roles of NBCL genes are not known. We thus investigated the role of Lotus japonicus NOOT-BOP-COCH-LIKE1 (LjNBCL1) in determinate nodule identity and studied its functions in aerial organ development using LORE1 insertional mutants and RNA interference-mediated silencing approaches. In Lotus, LjNBCL1 is involved in leaf patterning and participates in the regulation of axillary outgrowth. Wild-type Lotus leaves are composed of five leaflets and possess a pair of nectaries at the leaf axil. Legumes such as pea and Medicago have a pair of stipules, rather than nectaries, at the base of their leaves. In Ljnbcl1, nectary development is abolished, demonstrating that nectaries and stipules share a common evolutionary origin. In addition, ectopic roots arising from nodule vascular meristems and reorganization of the nodule vascular bundle vessels were observed on Ljnbcl1 nodules. This demonstrates that NBCL functions are conserved in both indeterminate and determinate nodules through the maintenance of nodule vascular bundle identity. In contrast to its role in floral patterning described in other plants, LjNBCL1 appears essential for the development of both secondary inflorescence meristem and floral meristem.

Rhodophiala bifida ( R . bifida ) is a representative of the Amaryllidaceae plant family and is rich in montanine, an alkaloid with high pharmaceutical potential. Despite the interest in these compounds, many steps of the biosynthetic pathway have not been elucidated. In this study, we identified the alkaloids produced in different organs of R . bifida under different growth conditions, set up the conditions for in vitro R . bifida regeneration and initiated the molecular characterization of two R . bifida genes involved in alkaloids biosynthesis: the Norbelladine 4 ′ -O-Methyltransferase ( RbN4OMT ) and the Cytochrome P450 ( RbCYP96T ). We show that montanine is the main alkaloid produced in the different R . bifida organs and developed a direct organogenesis regeneration protocol, using twin-scale explants cultivated on media enriched with naphthalene acetic acid and benzyladenine. Finally, we analyzed the RbN4OMT and RbCYP96T gene expressions in different organs and culture conditions and compared them to alkaloid production. In different organs of R . bifida young, adult and regenerated plants, as well as under various growing conditions, the transcripts accumulation was correlated with the production of alkaloids. This work provides new tools to improve the production of this important pharmaceutical compound and for future biotechnological studies.

SUMMARY In cultivated grasses, tillering, spike architecture and seed shattering represent major agronomical traits. In barley, maize and rice, the NOOT‐BOP‐COCH‐LIKE (NBCL) genes play important roles in development, especially in ligule development, tillering and flower identity. However, compared with dicots, the role of grass NBCL genes is underinvestigated. To better understand the role of grass NBCLs and to overcome any effects of domestication that might conceal their original functions, we studied TILLING nbcl mutants in the non‐domesticated grass Brachypodium distachyon. In B. distachyon, the NBCL genes BdUNICULME4 (CUL4) and BdLAXATUM‐A (LAXA) are orthologous, respectively, to the barley HvUniculme4 and HvLaxatum‐a, to the maize Zmtassels replace upper ears1 and Zmtassels replace upper ears2 and to the rice OsBLADE‐ON‐PETIOLE1 and OsBLADE‐ON‐PETIOLE2/3. In B. distachyon, our reverse genetics study shows that CUL4 is not essential for the establishment of the blade–sheath boundary but is necessary for the development of the ligule and auricles. We report that CUL4 also exerts a positive role in tillering and a negative role in spikelet meristem activity. On the other hand, we demonstrate that LAXA plays a negative role in tillering, positively participates in spikelet development and contributes to the control of floral organ number and identity. In this work, we functionally characterized two new NBCL genes in a context of non‐domesticated grass and highlighted original roles for grass NBCL genes that are related to important agronomical traits.

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In cultivated grasses, tillering, leaf and inflorescence architecture, as well as abscission ability, are major agronomical traits. In barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and brachypodium (Brachypodium distachyon), NOOT-BOP-COCH-LIKE (NBCL) genes are essential regulators of vegetative and reproductive development. Grass species usually possess two-to-four NBCL copies and until now a single study in O. sativa showed that the disruption of all NBCL genes strongly altered O. sativa leaf development. To improve our understanding of the role of NBCL genes in grasses, we extended the study of the two NBCL paralogs BdUNICULME4 (CUL4) and BdLAXATUM-A (LAXA) in the non-domesticated grass B. distachyon. For this, we applied reversed genetics and generated original B. distachyon single and double nbcl mutants by CRISPR-Cas9 approaches and genetic crossing between nbcl TILLING mutants. Through the study of original single laxa CRISPR-Cas9 null alleles, we validated functions previously proposed for LAXA in tillering, leaf patterning, inflorescence and flower development and also unveiled roles for these genes in seed yield. Furthermore, the characterization of cul4laxa double mutants revealed essential functions for nbcl genes in B. distachyon development, especially in the regulation of tillering, stem cell elongation and secondary cell wall composition as well as for the transition towards the reproductive phase. Our results also highlight recurrent antagonist interactions between NBCLs occurring in multiple aspects of B. distachyon development.