Legumes develop symbiotic interactions with rhizobial bacteria to form nitrogen-fixing nodules. Bacterial Nod factors (NFs) and plant regulatory pathways modulating NF signalling control rhizobial infections and nodulation efficiency. Here we show that gibberellin (GA) signalling mediated by DELLA proteins inhibits rhizobial infections and controls the NF induction of the infection marker ENOD11 in Medicago truncatula. Ectopic expression of a constitutively active DELLA protein in the epidermis is sufficient to promote ENOD11 expression in the absence of symbiotic signals. We show using heterologous systems that DELLA proteins can interact with the nodulation signalling pathway 2 (NSP2) and nuclear factor-YA1 (NF-YA1) transcription factors that are essential for the activation of NF responses. Furthermore, MtDELLA1 can bind the ERN1 (ERF required for nodulation 1) promoter and positively transactivate its expression. Overall, we propose that GA-dependent action of DELLA proteins may directly regulate the NSP1/NSP2 and NF-YA1 activation of ERN1 transcription to regulate rhizobial infections.

Summary: Organisation and patterning of the vascular network in land plants varies in different taxonomic, developmental and environmental contexts. In leaves, the degree of vascular strand connectivity influences both light and CO₂ harvesting capabilities as well as hydraulic capacity. As such, developmental mechanisms that regulate leaf venation patterning have a direct impact on physiological performance. Development of the leaf venation network requires the specification of procambial cells within the ground meristem of the primordium and subsequent proliferation and differentiation of the procambial lineage to form vascular strands. An understanding of how diverse venation patterns are manifest therefore requires mechanistic insight into how procambium is dynamically specified in a growing leaf. A role for auxin in this process was identified many years ago, but questions remain. In this review we first provide an overview of the diverse venation patterns that exist in land plants, providing an evolutionary perspective. We then focus on the developmental regulation of leaf venation patterns in angiosperms, comparing patterning in eudicots and monocots, and the role of auxin in each case. Although common themes emerge, we conclude that the developmental mechanisms elucidated in eudicots are unlikely to fully explain how parallel venation patterns in monocot leaves are elaborated.

A Medicago truncatula cytokinin-signaling transcription factor is required for early symbiotic nodulation and to regulate key Nod factor signaling and endoredup

Legumes can establish on nitrogen-deprived soils a symbiotic interaction with Rhizobia bacteria, leading to the formation of nitrogen-fixing root nodules. Cytokinin phytohormones are critical for triggering root cortical cell divisions at the onset of nodule initiation. Cytokinin signaling is based on a Two-Component System (TCS) phosphorelay cascade, involving successively Cytokinin-binding Histidine Kinase receptors, phosphorelay proteins shuttling between the cytoplasm and the nucleus, and Type-B Response Regulator (RRB) transcription factors activating the expression of cytokinin primary response genes. Among those, Type-A Response Regulators (RRA) exert a negative feedback on the TCS signaling. To determine whether the legume plant nodulation capacity is linked to specific features of TCS proteins, a genome-wide identification was performed in six legume genomes (Cajanus cajan, pigeonpea; Cicer arietinum, chickpea; Glycine max, soybean; Phaseolus vulgaris, common bean; Lotus japonicus; Medicago truncatula). The diversity of legume TCS proteins was compared to the one found in two non-nodulating species, Arabidopsis thaliana and Vitis vinifera, which are references for functional analyses of TCS components and phylogenetic analyses, respectively. A striking expansion of non-canonical RRBs was identified, notably leading to the emergence of proteins where the conserved phosphor-accepting aspartate residue is replaced by a glutamate or an asparagine. M. truncatula genome-wide expression datasets additionally revealed that only a limited subset of cytokinin-related TCS genes is highly expressed in different organs, namely MtCHK1/MtCRE1, MtHPT1, and MtRRB3, suggesting that this “core” module potentially acts in most plant organs including nodules. Further functional analyses are required to determine the relevance of these numerous non-canonical TCS RRBs in symbiotic nodulation, as well as of canonical MtHPT1 and MtRRB3 core signaling elements.

Plant development is a complex process that relies on molecular and cellular events being co-ordinated in space and time. Microscopy is one of the most powerful tools available to investigate this spatiotemporal complexity. One step towards a better understanding of complexity in plants would be the acquisition of 3D images of entire organs. However, 3D imaging of intact plant samples is not always simple and often requires expensive and/or non-trivial approaches. In particular, the inner tissues of thick samples are challenging to image. Here, we present the Flip-Flap method, a simple imaging protocol to produce 3D images of cleared plant samples at the organ scale. This method allows full 3D reconstruction of plant organs suitable for 3D segmentation and further related analysis and can be easily handled by relatively inexperienced microscopists.

L’interaction symbiotique légumineuses-rhizobium nécessite l'infection des racines de la plante par les bactéries et l’initiation de divisions cellulaires dans le cortex racinaire.Les cytokinines sont des hormones végétales agissant via une signalisation par phosphotransfert qui conduit à l’activation de Régulateurs de Réponse de type B (RRBs), des facteurs de transcription régulant l'expression des gènes de réponse primaire aux cytokinines. Une étude phylogénétique menée sur plusieurs espèces de légumineuses a révélé une expansion génique de la famille des RRBs et l’apparition de formes non-canoniques de ces facteurs de transcription. Chez Medicago truncatula,MtRRB3 est le RRB le plus fortement exprimé dans les racines et les nodosités et est impliqué dans la nodulation. En effet, les plantes dont l’expression de MtRRB3 a été réduite par ARNi ainsi que des mutants rrb3 présentent une diminution significative du nombre de nodosités formées. De plus, l’expression de gènes associés à la nodulation, tels que "Nodulation Signalling Pathway 2" (MtNSP2) et "Cell Cycle Switch 52A"(MtCCS52A), est réduite en réponse aux cytokinines dans ces mutants. Des fusions transcriptionnelles avec le rapporteur GUS montrent que MtRRB3, MtNSP2 et MtCCS52Aprésentent un profil d’expression spatiale largement chevauchant dans les racines et lesnodosités. Des expériences de ChIP-qPCR et de trans-activation en protoplastes indiquent par ailleurs que MtRRB3 peut respectivement interagir avec et activer les promoteurs des gènesMtNSP2 et MtCCS52A. Cette thèse a donc permis d’établir des mécanismes moléculaires impliqués dans les régulations transcriptionnelles médiées par les cytokinines lors de la mise en place des nodosités symbiotiques fixatrices d’azote.