Yassin Refahi

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.

An epidermis-driven mechanism positions and scales stem cell niches in plants

An epidermis control of plant shoot stem cells can explain the scaling and position of the niche expression domains. How molecular patterning scales to organ size is highly debated in developmental biology. We explore this question for the characteristic gene expression domains of the plant stem cell niche residing in the shoot apical meristem. We show that a combination of signals originating from the epidermal cell layer can correctly pattern the key gene expression domains and notably leads to adaptive scaling of these domains to the size of the tissue. Using live imaging, we experimentally confirm this prediction. The identified mechanism is also sufficient to explain de novo stem cell niches in emerging flowers. Our findings suggest that the deformation of the tissue transposes meristem geometry into an instructive scaling and positional input for the apical plant stem cell niche.

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.

Cell size and growth regulation in the Arabidopsis thaliana apical stem cell niche

Significance How does a cell decide when to divide or initiate DNA replication? How does it regulate its own growth? These fundamental questions are not well understood in most organisms; this lack of understanding is particularly true for multicellular eukaryotes. Following classical studies in yeast, we have quantified the key aspects of cell growth and division dynamics in the Arabidopsis apical stem cell niche. Our results disprove various theories for plant stem cell size/cell cycle regulation, such as that cell cycle progression is triggered when a prefixed critical size is attained, and constitute the necessary first step in the development of integrative mechanistic theories for the coordinated regulation of cell cycle progression, cell growth, and cell size in plants. Cell size and growth kinetics are fundamental cellular properties with important physiological implications. Classical studies on yeast, and recently on bacteria, have identified rules for cell size regulation in single cells, but in the more complex environment of multicellular tissues, data have been lacking. In this study, to characterize cell size and growth regulation in a multicellular context, we developed a 4D imaging pipeline and applied it to track and quantify epidermal cells over 3–4 d in Arabidopsis thaliana shoot apical meristems. We found that a cell size checkpoint is not the trigger for G2/M or cytokinesis, refuting the unexamined assumption that meristematic cells trigger cell cycle phases upon reaching a critical size. Our data also rule out models in which cells undergo G2/M at a fixed time after birth, or by adding a critical size increment between G2/M transitions. Rather, cell size regulation was intermediate between the critical size and critical increment paradigms, meaning that cell size fluctuations decay by ∼75% in one generation compared with 100% (critical size) and 50% (critical increment). Notably, this behavior was independent of local cell–cell contact topologies and of position within the tissue. Cells grew exponentially throughout the first >80% of the cell cycle, but following an asymmetrical division, the small daughter grew at a faster exponential rate than the large daughter, an observation that potentially challenges present models of growth regulation. These growth and division behaviors place strong constraints on quantitative mechanistic descriptions of the cell cycle and growth control.

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].

Phyllotactic regularity requires the Paf1 complex in Arabidopsis

In plants, aerial organs are initiated at stereotyped intervals, both spatially (every 137° in a pattern called phyllotaxis) and temporally (at prescribed time intervals called plastochrons). To investigate the molecular basis of such regularity, mutants with altered architecture have been isolated. However, most of them only exhibit plastochron defects and/or produce a new, albeit equally reproducible, phyllotactic pattern. This leaves open the question of a molecular control of phyllotaxis regularity. Here, we show that phyllotaxis regularity depends on the function of VIP proteins, components of the RNA polymerase II-associated factor 1 complex (Paf1c). Divergence angles between successive organs along the stem exhibited increased variance in vip3-1 and vip3-2 compared with the wild type, in two different growth conditions. Similar results were obtained with the weak vip3-6 allele and in vip6, a mutant for another Paf1c subunit. Mathematical analysis confirmed that these defects could not be explained solely by plastochron defects. Instead, increased variance in phyllotaxis in vip3 was observed at the meristem and related to defects in spatial patterns of auxin activity. Thus, the regularity of spatial, auxin-dependent, patterning at the meristem requires Paf1c. Summary: Analysis of divergence angles in VIP mutants reveals that the regularity of organ initiation at the shoot apical meristem requires Paf1c and is related to spatial patterns of auxin activity.

Spatio-temporal registration of 3D microscopy image sequences of arabidopsis floral meristems

The shoot apical meristem (SAM) is at the origin of all the plant above-ground organs (including stems, leaves and flowers) and is a biological object of interest for the understanding of plant morphogenesis. The quantification of tissue growth at a cellular level requires the analysis of 3D microscopic image sequences of developing meristems. To address inter-individual variability, it is also required to compare individuals. This obviously implies the ability to process inter-individual registration, i.e. to compute spatial and temporal correspondences between sequences from different meristems. In the present work, we propose a spatial registration method dedicated to microscopy floral meristem (FM) images, and the identification, for a given still image of a meristem, of its best corresponding time-point in a sequence of an other individual (temporal registration).Recent microscopy techniques allow imaging temporal 3D stacks of developing organs or embryos with a cellular level of resolution and with a sufficient acquisition frequency to accurately track cell lineages. Imaging multiple organs or embryos in different experimental conditions may help decipher the impact of genetic backgrounds and environmental inputs on the developmental program. For this, we need to precisely compare distinct individuals and to compute population statistics. The first step of this procedure is to develop methods to register individuals. From a previous work of cell segmentation from microscopy images, we here demonstrate how to extract the symmetry plane of embryos at early stages, and how to use this information as a geometrical constraint to both register these embryos and obtain a cell-to-cell mapping.