A graph-based approach for simultaneous semantic and instance segmentation of plant 3D point clouds.

Accurate simultaneous semantic and instance segmentation of a plant 3D point cloud is critical for automatic plant phenotyping. Classically, each organ of the plant is detected based on the local geometry of the point cloud, but the consistency of the global structure of the plant is rarely assessed. We propose a two-level, graph-based approach for the automatic, fast and accurate segmentation of a plant into each of its organs with structural guarantees. We compute local geometric and spectral features on a neighbourhood graph of the points to distinguish between linear organs (main stem, branches, petioles) and two-dimensional ones (leaf blades) and even 3-dimensional ones (apices). Then a quotient graph connecting each detected macroscopic organ to its neighbors is used both to refine the labelling of the organs and to check the overall consistency of the segmentation. A refinement loop allows to correct segmentation defects. The method is assessed on both synthetic and real 3D point-cloud data sets of Chenopodium album (wild spinach) and Solanum lycopersicum (tomato plant).

What shoots can teach about theories of plant form.

Plants generate a large variety of shoot forms with regular geometries. These forms emerge primarily from the activity of a stem cell niche at the shoot tip. Recent efforts have established a theoretical framework of form emergence at the shoot tip, which has empowered the use of modelling in conjunction with biological approaches to begin to disentangle the biochemical and physical mechanisms controlling form development at the shoot tip. Here, we discuss how these advances get us closer to identifying the construction principles of plant shoot tips. Considering the current limits of our knowledge, we propose a roadmap for developing a general theory of form development at the shoot tip.

A broad mutational target explains a fast rate of phenotypic evolution.

The rapid evolution of a trait in a clade of organisms can be explained by the sustained action of natural selection or by a high mutational variance, that is the propensity to change under spontaneous mutation. The causes for a high mutational variance are still elusive. In some cases, fast evolution depends on the high mutation rate of one or few loci with short tandem repeats. Here, we report on the fastest evolving cell fate among vulva precursor cells in Caenorhabditis nematodes, that of P3.p. We identify and validate causal mutations underlying P3.p's high mutational variance. We find that these positions do not present any characteristics of a high mutation rate, are scattered across the genome and the corresponding genes belong to distinct biological pathways. Our data indicate that a broad mutational target size is the cause of the high mutational variance and of the corresponding fast phenotypic evolutionary rate.

Heritable characteristics or traits of a group of organisms, for example the large brain size of primates or the hooves of a horse, are determined by genes, the environment, and by the interactions between them. Traits can change over time and generations when enough mutations in these genes have spread in a species to result in visible differences. However, some traits, such as the large brain of primates, evolve faster than others, but why this is the case has been unclear. It could be that a few specific genes important for that trait in question mutate at a high rate, or, that many genes affect the trait, creating a lot of variation for natural selection to choose from. Here, Besnard, Picao-Osorio et al. studied the roundworm Caenorhabditis elegans to better understand the causes underlying the different rates of trait evolution. These worms have a short life cycle and evolve quickly over many generations, making them an ideal candidate for studying mutation rates in different traits. Previous studies have shown that one of C. elegans' six cells of the reproductive system evolves faster than the others. To investigate this further, Besnard, Picao-Osorio et al. analysed the genetic mutations driving change in this cell in 250 worm generations. The results showed that five mutations in five different genes ­ all responsible for different processes in the cells ­ were behind the supercharged evolution of this particular cell. This suggests that fast evolution results from natural selection acting upon a collection of genes, rather than one gene, and that many genes and pathways shape this trait. In conclusion, these results demonstrate that how traits are coded at the molecular level, in one gene or many, can influence the rate at which they evolve.

Necessity and Contingency in Developmental Genetic Screens: EGF, Wnt, and Semaphorin Pathways in Vulval Induction of the Nematode Oscheius tipulae.

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 primary (1°) vulval fate; P6.p in turn induces its neighbors to a secondary (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 clustered regularly interspaced short palindromic repeats/Cas9 mutation. In contrast to this predictable Vulvaless mutation, mutations resulting in an excess of 2° fates unexpectedly correspond to the plexin/semaphorin pathway. 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; a 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. Thus, the derived induction process in C. elegans with an early induction of the 1° fate appeared during evolution, after the recruitment of the EGF pathway for vulval induction.

Toward Universal Forward Genetics: Using a Draft Genome Sequence of the Nematode Oscheius tipulae To Identify Mutations Affecting Vulva Development.

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.

Meristem size contributes to the robustness of phyllotaxis in Arabidopsis.

Using the plant model Arabidopsis, the relationship between day length, the size of the shoot apical meristem, and the robustness of phyllotactic patterns were analysed. First, it was found that reducing day length leads to an increased meristem size and an increased number of alterations in the final positions of organs along the stem. Most of the phyllotactic defects could be related to an altered tempo of organ emergence, while not affecting the spatial positions of organ initiations at the meristem. A correlation was also found between meristem size and the robustness of phyllotaxis in two accessions (Col-0 and WS-4) and a mutant (clasp-1), independent of growth conditions. A reduced meristem size in clasp-1 was even associated with an increased robustness of the phyllotactic pattern, beyond what is observed in the wild type. Interestingly it was also possible to modulate the robustness of phyllotaxis in these different genotypes by changing day length. To conclude, it is shown first that robustness of the phyllotactic pattern is not maximal in the wild type, suggesting that, beyond its apparent stereotypical order, the robustness of phyllotaxis is regulated. Secondly, a role for day length in the robustness of the phyllotaxis was also identified, thus providing a new example of a link between patterning and environment in plants. Thirdly, the experimental results validate previous model predictions suggesting a contribution of meristem size in the robustness of phyllotaxis via the coupling between the temporal sequence and spatial pattern of organ initiations.

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.

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.

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.

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.

Organogenesis from stem cells in planta: multiple feedback loops integrating molecular and mechanical signals.

In multicellular organisms, the coordination of cell behaviors largely relies on biochemical and biophysical signals. Understanding how such signals control development is often challenging, because their distribution relies on the activity of individual cells and, in a feedback loop, on tissue behavior and geometry. This review focuses on one of the best-studied structures in biology, the shoot apical meristem (SAM). This tissue is responsible for the production of all the aerial parts of a plant. In the SAM, a population of stem cells continuously produces new cells that are incorporated in lateral organs, such as leaves, branches, and flowers. Organogenesis from stem cells involves a tight regulation of cell identity and patterning as well as large-scale morphogenetic events. The gene regulatory network controlling these processes is highly coordinated in space by various signals, such as plant hormones, peptides, intracellular mobile factors, and mechanical stresses. Many crosstalks and feedback loops interconnecting these pathways have emerged in the past 10 years. The plant hormone auxin and mechanical forces have received more attention recently and their role is more particularly detailed here. An integrated view of these signaling networks is also presented in order to help understanding how robust shape and patterning can emerge from these networks.

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.