Fabrice Besnard

Cytokinin signalling inhibitory fields provide robustness to phyllotaxis

How biological systems generate reproducible patterns with high precision is a central question in science. The shoot apical meristem (SAM), a specialized tissue producing plant aerial organs, is a developmental system of choice to address this question. Organs are periodically initiated at the SAM at specific spatial positions and this spatiotemporal pattern defines phyllotaxis. Accumulation of the plant hormone auxin triggers organ initiation, whereas auxin depletion around organs generates inhibitory fields that are thought to be sufficient to maintain these patterns and their dynamics. Here we show that another type of hormone-based inhibitory fields, generated directly downstream of auxin by intercellular movement of the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), is involved in regulating phyllotactic patterns. We demonstrate that AHP6-based fields establish patterns of cytokinin signalling in the meristem that contribute to the robustness of phyllotaxis by imposing a temporal sequence on organ initiation. Our findings indicate that not one but two distinct hormone-based fields may be required for achieving temporal precision during formation of reiterative structures at the SAM, thus indicating an original mechanism for providing robustness to a dynamic developmental system.

Auxin at the Shoot Apical Meristem

Plants continuously generate new tissues and organs through the activity of populations of undifferentiated stem cells, called meristems. Here, we discuss the so-called shoot apical meristem (SAM), which generates all the aerial parts of the plant. It has been known for many years that auxin plays a central role in the functioning of this meristem. Auxin is not homogeneously distributed at the SAM and it is thought that this distribution is interpreted in terms of differential gene expression and patterned growth. In this context, auxin transporters of the PIN and AUX families, creating auxin maxima and minima, are crucial regulators. However, auxin transport is not the only factor involved. Auxin biosynthesis genes also show specific, patterned activities, and local auxin synthesis appears to be essential for meristem function as well. In addition, auxin perception and signal transduction defining the competence of cells to react to auxin, add further complexity to the issue. To unravel this intricate signaling network at the SAM, systems biology approaches, involving not only molecular genetics but also live imaging and computational modeling, have become increasingly important.

Noise and Robustness in Phyllotaxis

A striking feature of vascular plants is the regular arrangement of lateral organs on the stem, known as phyllotaxis. The most common phyllotactic patterns can be described using spirals, numbers from the Fibonacci sequence and the golden angle. This rich mathematical structure, along with the experimental reproduction of phyllotactic spirals in physical systems, has led to a view of phyllotaxis focusing on regularity. However all organisms are affected by natural stochastic variability, raising questions about the effect of this variability on phyllotaxis and the achievement of such regular patterns. Here we address these questions theoretically using a dynamical system of interacting sources of inhibitory field. Previous work has shown that phyllotaxis can emerge deterministically from the self-organization of such sources and that inhibition is primarily mediated by the depletion of the plant hormone auxin through polarized transport. We incorporated stochasticity in the model and found three main classes of defects in spiral phyllotaxis – the reversal of the handedness of spirals, the concomitant initiation of organs and the occurrence of distichous angles – and we investigated whether a secondary inhibitory field filters out defects. Our results are consistent with available experimental data and yield a prediction of the main source of stochasticity during organogenesis. Our model can be related to cellular parameters and thus provides a framework for the analysis of phyllotactic mutants at both cellular and tissular levels. We propose that secondary fields associated with organogenesis, such as other biochemical signals or mechanical forces, are important for the robustness of phyllotaxis. More generally, our work sheds light on how a target pattern can be achieved within a noisy background.

Meristem size contributes to the robustness of phyllotaxis in Arabidopsis

Highlight Phyllotaxis describes the regular position of leaves and flowers along plant stems. It is demonstrated that errors in this pattern can be related to meristem size and day length.

The AHP6 cytokinin signaling inhibitor mediates an auxin-cytokinin crosstalk that regulates the timing of organ initiation at the shoot apical meristem

Phyllotaxis, the spatio-temporal pattern of organogenesis at the shoot apical meristem, emerges in large part from inhibitory fields consisting in auxin-depleted areas centered on organs. We recently demonstrated the existence of an additional hormone-based inhibitory field generated by Arabidopsis Histidine Phosphotransfer Protein 6 (AHP6), an inhibitor of cytokinin signaling. We have shown that the spatio-temporal distribution of AHP6 in the meristem is essential for optimizing the rhythmicity of organ initiation. Here, we further analyzed AHP6 expression using fluorescent whole mount mRNA in situ hybridization and demonstrate a precise control of AHP6 level and expression domain over time. While we previously showed a regulation of AHP6 directly downstream of auxin, we show here that AHP6 transcription is unlikely influenced by cytokinin distribution in the meristem. Finally, we provide evidence that cytokinins and auxin might act synergistically during organ initiation, providing a plausible explanation for how AHP6 regulates phyllotaxis.

Pattern identification and characterization reveal permutations of organs as a key genetically controlled property of post-meristematic phyllotaxis

In vascular plants, the arrangement of organs around the stem generates geometric patterns called phyllotaxis. In the model plant, Arabidopsis thaliana, as in the majority of species, single organs are initiated successively at a divergence angle from the previous organ close to the canonical angle of 137.5°, producing a Fibonacci spiral. Given that little is known about the robustness of these geometric arrangements, we undertook to characterize phyllotaxis by measuring divergence angles between organs along the stems of wild-type and specific mutant plants with obvious defects in phyllotaxis. Sequences of measured divergence angles exhibit segments of non-canonical angles in both genotypes, albeit to a far greater extent in the mutant. We thus designed a pipeline of methods for analyzing these perturbations. The latent structure models used in this pipeline combine a non-observable model representing perturbation patterns (either a variable-order Markov chain or a combinatorial model) with von Mises distributions representing divergence angle uncertainty. We show that the segments of non-canonical angles in both wild-type and mutant plants can be explained by permutations in the order of insertion along the stem of two or three consecutive organs. The number of successive organs between two permutations reveals specific patterns that depend on the nature of the preceding permutation (2- or 3-permutation). We also highlight significant individual deviations from 137.5° in the level of baseline segments and a marked relationship between permutation of organs and defects in the elongation of the internodes between these organs. These results demonstrate that permutations are an intrinsic property of spiral phyllotaxis and that their occurrence is genetically regulated.

A combinatorial model of phyllotaxis perturbations in Arabidopsis thaliana

Phyllotaxis is the geometric arrangement of organs in plants, and is known to be highly regular. However, experimental data (from Arabidopsis thaliana) show that this regularity is in fact subject to specific patterns of permutations. In this paper we introduce a model for these patterns, as well as algorithms designed to identify these patterns in noisy experimental data. These algorithms thus incorporate a denoising step which is based on Gaussian-like distributions for circular data for which a common dispersion parameter has been previously estimated. The application of the proposed algorithms allows us to confirm the plausibility of the proposed model, and to characterize the patterns observed in a specific mutant. The algorithms are available in the OpenAlea software platform for plant modelling [10].

Toward universal forward genetics: Using a draft genome sequence of the nematode oscheius tipulae to identify mutations affecting vulva development

Understanding evolution requires the comparison of more than a few model species, and exploration of the genotype/phenotype relationship is limited... Mapping-by-sequencing has become a standard method to map and identify phenotype-causing mutations in model species. Here, we show that a fragmented draft assembly is sufficient to perform mapping-by-sequencing in nonmodel species. We generated a draft assembly and annotation of the genome of the free-living nematode Oscheius tipulae, a distant relative of the model Caenorhabditis elegans. We used this draft to identify the likely causative mutations at the O. tipulae cov-3 locus, which affect vulval development. The cov-3 locus encodes the O. tipulae ortholog of C. elegans mig-13, and we further show that Cel-mig-13 mutants also have an unsuspected vulval-development phenotype. In a virtuous circle, we were able to use the linkage information collected during mutant mapping to improve the genome assembly. These results showcase the promise of genome-enabled forward genetics in nonmodel species.

Necessity and contingency in developmental genetic screens: LIN-3, Wnt and semaphorin pathways in vulval induction of the nematode Oscheius tipulae

Abstract Genetic screens in the nematode Caenorhabditis elegans identified the EGF/Ras and Notch pathways as central for vulval precursor cell fate patterning. Schematically, the anchor cell secretes EGF, inducing the P6.p cell to a 1° vulval fate; P6.p in turn induces its neighbors to a 2° fate through Delta-Notch signaling and represses Ras signaling. In the nematode Oscheius tipulae, the anchor cell successively induces 2° then 1° vulval fates. Here we report on the molecular identification of mutations affecting vulval induction in O. tipulae. A single Induction Vulvaless mutation was found, which we identify as a cis-regulatory deletion in a tissue-specific enhancer of the O. tipulae lin-3 homolog, confirmed by CRISPR/Cas9 mutation. In contrast to this predictable Vulvaless mutation, mutations resulting in an excess of 2° fates unexpectedly correspond to the plexin/semaphorin pathway, which was not implicated in vulval fate induction in C. elegans. Hyperinduction of P4.p and P8.p in these mutants likely results from mispositioning of these cells due to a lack of contact inhibition. The third signaling pathway found by forward genetics in O. tipulae is the Wnt pathway: decrease in Wnt pathway activity results in loss of vulval precursor competence and induction, and 1° fate miscentering on P5.p. Our results suggest that the EGF and Wnt pathways have qualitatively similar activities in vulval induction in C. elegans and O. tipulae, albeit with quantitative differences in the effects of mutation. This study highlights both necessity and contingency in forward genetic screens. 100-word summary Genetic screens in the nematode Caenorhabditis elegans identified EGF and Notch pathways as key for vulval precursor cell fate patterning. Here we report on the molecular identification of mutations affecting vulval induction in another nematode, Oscheius tipulae. The single mutation with reduced induction is identified as a cis-regulatory deletion in the O. tipulae lin-3 homolog, confirmed by CRISPR/Cas9 mutation. In contrast to this predictable Vulvaless mutation, mutations resulting in an excess of 2° vulval fates unexpectedly correspond to the plexin/semaphorin pathway, not implicated in vulval induction in C. elegans. This study highlights both necessity and contingency in forward genetic screens.