Developmental Patterning by Mechanical Signals in Arabidopsis

A central question in developmental biology is whether and how mechanical forces serve as cues for cellular behavior and thereby regulate morphogenesis. We found that morphogenesis at the Arabidopsis shoot apex depends on the microtubule cytoskeleton, which in turn is regulated by mechanical stress. A combination of experiments and modeling shows that a feedback loop encompassing tissue morphology, stress patterns, and microtubule-mediated cellular properties is sufficient to account for the coordinated patterns of microtubule arrays observed in epidermal cells, as well as for patterns of apical morphogenesis

Alignment between PIN1 Polarity and Microtubule Orientation in the Shoot Apical Meristem Reveals a Tight Coupling between Morphogenesis and Auxin Transport

Morphogenesis during multicellular development is regulated by intercellular signaling molecules as well as by the mechanical properties of individual cells. In particular, normal patterns of organogenesis in plants require coordination between growth direction and growth magnitude. How this is achieved remains unclear. Here we show that in Arabidopsis thaliana, auxin patterning and cellular growth are linked through a correlated pattern of auxin efflux carrier localization and cortical microtubule orientation. Our experiments reveal that both PIN1 localization and microtubule array orientation are likely to respond to a shared upstream regulator that appears to be biomechanical in nature. Lastly, through mathematical modeling we show that such a biophysical coupling could mediate the feedback loop between auxin and its transport that underlies plant phyllotaxis

Caractérisation biochimique de protéines de la famille Pentatricopeptide repeat chez Arabidopsis thaliana et le colza

Les protéines PPR (PentatricoPeptide Repeat), particulièrement nombreuses chez les plantes supérieures, se révèlent être des acteurs majeurs de l expression des gènes des chloroplastes et des mitochondries. Leur fonctionnement à l échelle moléculaire est encore inconnu ; il est cependant proposé qu elles puissent servir d adaptateurs moléculaires pour recruter des activités enzymatiques spécifiques sur des cibles ARN précises. Afin de tester la validité de cette hypothèse, l objet de ce travail de thèse a consisté à rechercher les partenaires protéiques et les cibles ARN de certaines protéines PPR d Arabidopsis à l aide d approches biochimiques. La fonction de la protéine restauratrice PPRB, codée au locus Rfo, a également été étudiée chez le colza, afin de mieux comprendre son incidence sur l expression du gène mitochondrial orf138 inducteur de la stérilité mâle cytoplasmique Ogura et contribuer à l élucidation du mécanisme de restauration de la fertilité. Nous avons tout d abord produit des transformants stables d Arabidopsis permettant de détecter 9 protéines PPR différentes grâce à leur fusion à de courts épitopes peptidiques. Ce matériel a permis de révéler la présence de quatre d entre elles dans des complexes multiprotéiques in vivo. Le faible rendement du protocole de double purification choisi pour l isolement ultérieur des complexes protéiques a limité l identification de partenaires protéiques. Les cibles ARN, ont été recherchées à l aide de la méthode du RIP-Chip. Cette analyse révèle pour la protéine PPR336, une capacité à lier de nombreux ARN mitochondriaux, avançant ainsi une nouvelle fonction pour une protéine PPR. Enfin, nous avons pu montrer que parmi les différentes protéines PPR fortement similaires également codées au locus Rfo, et accumulées dans les anthères de jeunes boutons floraux, seule la protéine PPRB induit la disparition de l ORF138 de façon quasi-totale et homogène. Nous avons révélé la corrélation de l activité de restauration de PPRB avec sa capacité à lier le transcrit orf138, dont l accumulation et la maturation ne sont pas affectées, en ciblant très probablement sa région 5 UTR. Nous proposons donc que cette liaison soit à l origine d un blocage de l initiation de la traduction du transcrit orf138 empêchant ainsi la synthèse de la protéine responsable de la SMC-Ogura.PentatricoPeptide Repeats proteins, whose family has literally exploded in higher plants, are key factors of mitochondria and chloroplast gene expression. Their molecular functions remain to be elucidated. The PPR proteins are proposed to constitute molecular adaptors that could recruit catalytic proteins to a specific site on RNA targets. In order to test the validity of this assumption, the aim of this work was to try to determine their protein partners and their RNA targets with biochemical approaches. The role of the restorer protein, PPRB, encoded at the Rfo locus, was also studied in rapeseed in order to gain insight into its role during the expression of the orf138 mitochondrial gene which induces Ogura male sterility. First, Arabidopsis stable transformants expressing nine PPR proteins fused to small tags were produced. Analysis of stromal and mitochondrial extracts by size exclusion chromatography allowed us to observe that 4 of the detected PPR proteins were involved in large multi-protein complexes. We used a tandem affinity purification strategy in order to identify their putative partners but its poor yield, limited the identification of partners. We used a RIP-Chip strategy for the characterization of PPR proteins RNA targets. This strategy revealed that PPR336 was able to bind a lot of RNA targets, thus suggesting a new function for PPR proteins. Finally we showed that the PPRB protein accumulates in young buds anthers, like other PPR proteins encoded at the Rfo locus. However, it was the only PPR protein to induce the disappearance of ORF138 protein in these tissues. We revealed the correlation of restoration activity with an ability to bind the orf138 transcript, whose accumulation and maturation are unchanged in restored plants, probably by binding the 5 UTR region of the mRNA. We proposed that the binding could impair the translation initiation and inhibit ORF138 protein synthesis.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Graph metric learning quantifies morphological differences between two genotypes of shoot apical meristem cells in Arabidopsis.

We present a method for learning spectrally descriptive edge weights for graphs. We generalize a previously known distance measure on graphs (graph diffusion distance [GDD]), thereby allowing it to be tuned to minimize an arbitrary loss function. Because all steps involved in calculating this modified GDD are differentiable, we demonstrate that it is possible for a small neural network model to learn edge weights which minimize loss. We apply this method to discriminate between graphs constructed from shoot apical meristem images of two genotypes of Arabidopsis thaliana specimens: wild-type and trm678 triple mutants with cell division phenotype. Training edge weights and kernel parameters with contrastive loss produce a learned distance metric with large margins between these graph categories. We demonstrate this by showing improved performance of a simple k -nearest-neighbour classifier on the learned distance matrix. We also demonstrate a further application of this method to biological image analysis. Once trained, we use our model to compute the distance between the biological graphs and a set of graphs output by a cell division simulator. Comparing simulated cell division graphs to biological ones allows us to identify simulation parameter regimes which characterize mutant versus wild-type Arabidopsis cells. We find that trm678 mutant cells are characterized by increased randomness of division planes and decreased ability to avoid previous vertices between cell walls

A correlative microscopy approach relates microtubule behaviour, local organ geometry, and cell growth at the Arabidopsis shoot apical meristem

Cortical microtubules (CMTs) are often aligned in a particular direction in individual cells or even in groups of cells and play a central role in the definition of growth anisotropy. How the CMTs themselves are aligned is not well known, but two hypotheses have been proposed. According to the first hypothesis, CMTs align perpendicular to the maximal growth direction, and, according to the second, CMTs align parallel to the maximal stress direction. Since both hypotheses were formulated on the basis of mainly qualitative assessments, the link between CMT organization, organ geometry, and cell growth is revisited using a quantitative approach. For this purpose, CMT orientation, local curvature, and growth parameters for each cell were measured in the growing shoot apical meristem (SAM) of Arabidopsis thaliana. Using this approach, it has been shown that stable CMTs tend to be perpendicular to the direction of maximal growth in cells at the SAM periphery, but parallel in the cells at the boundary domain. When examining the local curvature of the SAM surface, no strict correlation between curvature and CMT arrangement was found, which implies that SAM geometry, and presumed geometry-derived stress distribution, is not sufficient to prescribe the CMT orientation. However, a better match between stress and CMTs was found when mechanical stress derived from differential growth was also considered

A transient radial cortical microtubule array primes cell division in Arabidopsis

International audienceBecause all cells experience mechanical stress, they have to develop resistance mechanisms to survive. In plants, this notably involves guidance of cellulose deposition by cortical microtubules in the direction of maximal tensile stress. Although the formation of new walls during cell division tends to follow maximal tensile stress direction too, analyses of individual cells over time reveal a much more variable behavior. The origin of such variability, as well as the exact role of interphasic microtubule behavior before cell division have remained mysterious so far. To approach this question, we took advantage of the Arabidopsis stem, where the tensile stress pattern is both highly anisotropic and stable. Although cortical microtubules generally align with maximal tensile stress, we detected a specific time window, ca. 3 hours before cell division, where cells form a radial pattern of cortical microtubules. This pattern was observed in different growth conditions, and was not related to cell geometry or polar auxin transport. Interestingly, this cortical radial pattern correlated with the well-documented increase of cytoplasmic microtubule accumulation before cell division. This radial organization was prolonged in cells of the trm678 mutant, where cortical microtubules are partially disorganized.Whereas division plane orientation in trm678 is noisier, we found that cell division symmetry was in contrast more precise. We propose that an increased cytoplasmic microtubule accumulation in late G2 disrupts cortical microtubules alignment with tissue stress, allowing the cell to transiently explore its own geometry in order to select a future division plane with correct orientation and symmetry. SIGNIFICANCE STATEMENTIn all kingdoms, cells divide according to their own geometry as well as external cues. We discovered a transient stage in plant cells, where part of the division machinery becomes blind to mechanical forces originating from the tissue. Using quantitative imaging and mutant analysis, we propose that this new pre-mitotic stage allows cells to take their own geometry into account and increase the precision of the following division.</div

Mechanical Stress Acts via Katanin to Amplify Differences in Growth Rate between Adjacent Cells in Arabidopsis

The presence of diffuse morphogen gradients in tissues supports a view in which growth is locally homogenous. Here we challenge this view: we used a high-resolution quantitative approach to reveal significant growth variability among neighboring cells in the shoot apical meristem, the plant stem cell niche. This variability was strongly decreased in a mutant impaired in the microtubule-severing protein katanin. Major shape defects in the mutant could be related to a local decrease in growth heterogeneity. We show that katanin is required for the cell's competence to respond to the mechanical forces generated by growth. This provides the basis for a model in which microtubule dynamics allow the cell to respond efficiently to mechanical forces. This in turn can amplify local growth-rate gradients, yielding more heterogeneous growth and supporting morphogenesis

FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images

Cell biology heavily relies on the behavior of fibrillar structures, such as the cytoskeleton, yet the analysis of their behavior in tissues often remains qualitative. Image analysis tools have been developed to quantify this behavior, but they often involve an image pre-processing stage that may bias the output and/or they require specific software. Here we describe FibrilTool, an ImageJ plug-in based on the concept of nematic tensor, which can provide a quantitative description of the anisotropy of fiber arrays and their average orientation in cells, directly from raw images obtained by any form of microscopy. FibrilTool has been validated on microtubules, actin and cellulose microfibrils, but it may also help analyze other fibrillar structures, such as collagen, or the texture of various materials. The tool is ImageJ-based, and it is therefore freely accessible to the scientific community and does not require specific computational setup. The tool provides the average orientation and anisotropy of fiber arrays in a given region of interest (ROI) in a few seconds