Growth polarity in leaves – a final discussion Insights into the growth processes of leaf lamina have come from studies on several species including Arabidopsis, Antirrhinum, tobacco and maize. A feature common to the growth of leaf in these distantly related species is the existence of a pronounced growth gradient in the proximo-distal axis -growth at the tip (distal part) is arrested at an early stage while the basal region (proximal part) continues to grow for the longest duration. This is because the cell division is arrested first at the tip at an early stage of development and the arrest progressively spreads towards the base. Along with the strong proximo-distal growth gradient, a milder growth gradient also exists in the medio-lateral axis, such that the cell division arrest travels slightly faster on the leaf margins imparting an overall convex shape to the arrest front. The temporal and spatial progression of the arrest front has not only been implicated in shaping up of a leaf but is also of paramount importance in the maintenance of a flat surface during leaf growth. Although the patterning mechanisms described above seem to operate during leaf growth in many6 species, the molecular mechanisms governing these processes is still in its infancy. Moreover, patterning of leaf growth has been studied only in a handful of model species and, therefore, the information from the vast body of natural variation remains neglected. Proximo-distal growth patterning by CINCINNATA Mutant leaves with altered rates or shapes of the arrest front progression deviate significantly from the normal shape and overall flat structure. Mutation in the CIN gene in Antirrhinum and its orthologues in Arabidopsis cause buckling of the leaf due to excess cell proliferation, which in turn is caused by a delayed progression of the arrest front. CIN-like genes code for TCP transcription factors and are expressed in a broad zone of a growing leaf somewhat distal to the proliferation zone. Even though several direct and indirect targets of CIN-like genes have been identified in various plant species, their role in regulating leaf maturity and surface curvature has remained unclear. The comparison of global transcription profile of wild type and cincinnata mutant of Antirrhinum showed that the expression of genes involved in either signaling or biosynthesis of the major growth hormones were altered in the mutant. By combining DNA-protein interaction, expression analysis, chromatin immuno-precipitation and RNA in situ hybridization, we show that CIN maintains surface flatness by regulating the signaling or level of major plant hormones in developing leaves. CIN promotes cytokinin signaling by directly binding to and thereby promoting the expression of a cytokinin receptor, AmHK4, in a spatio¬temporal manner. Furthermore, it also seems to affect GA level indirectly in young leaves by regulating the spatio-temporal as well as levels of GA-biosynthetic and GA-degrading enzymes. Thus, CIN seems to accelerate maturity in leaf cells along the tip-to-base direction through its effect on the cytokinin and GA signaling pathways. In addition, CIN suppresses auxin signaling more at the margin than in the centre by establishing a margin-to-medial expression gradient of a homologue of the auxin suppressor IAA3, thereby suppressing excess cell proliferation on the margin. Our results uncover an underlying mechanism in a developing leaf that controls curvature of the leaf surface by promotion of timely exit from cell proliferation in the proximo-distal as well as the medio-lateral axes via multiple hormone pathways. Divergent growth polarities in the proximo-distal axis of leaves The morphogenetic gradient in the proximo-distal axis of a leaf is brought about by the dynamic expression of several heterochronic regulators which can include TCP and GRF classes of transcription factors. Interestingly, many of these transcription factors are also regulated post-transcriptionally by micro RNAs. In case of the studied model species, these factors seem to be associated with basipetal growth. The early arrest in cell proliferation at the tip and continued cell division at the base has served as a paradigm in studying leaf growth and has been used to conceptualize the growth of leaves with different shapes. However, the possibility of the existence of different patterning mechanisms during leaf growth in the highly diverse plant kingdom remains unexplored. Our survey of leaf growth patterns in 75 dicot species reveals the existence of four distinct proximo-distal polarities in growth patterns. Using the law of simple allometry, we also show that the differentially growing regions of leaves bear a constant relationship between them during growth. A combination of cell-size studies, histochemical staining and expression analysis reveals a strong correlation among growth pattern, cell size and the cell proliferation status. The cell size studies also indicate that there is a wide variation in the final cell sizes of leaves and the relative contribution of cell division and cell expansion to the final leaf size can be highly variable. Furthermore, we find that the varying growth patterns are linked to changes in the expression pattern of miR396, which controls the expression pattern of cell division regulatory transcription factors, the GRFs. Mis-expressing miR396 at the base of the young Arabidopsis leaf caused an early exit from cell division while reducing the expression of the miR396 at the tip allowed cell division to continue for a longer duration near the tip. Our results demonstrate that leaves with similar shapes can be differently patterned and that this divergent patterning is linked to the expression differences in the regulatory micro RNA, miR396 In conclusion, this study shows that regulators like CIN maintain surface flatness of the Antirrhinum leaf during growth by promoting timely exit from cell division along the proximo-distal and the medio-lateral axes; and it does so by regulating multiple hormone pathways. Although the basic mechanism of patterned cell division and differentiation seems to be conserved among species, the polarities of growth can vary. The variability in the growth polarities could be brought about by changes in the trans-regulation or cis-regulatory changes in the patterning genes.

L’objectif de ce projet doctoral a été de coupler la chromatographie en phase supercritique (SFC) avec un spectromètre de masse haute résolution pour l’analyse de substances naturelles apolaires. La SFC est une technique dite « verte » contrairement à la chromatographie liquide en phase normale (NPLC), très consommatrice de solvants organiques toxiques pour l’environnement, puisque la phase mobile est principalement constituée de CO2. Le CO2 ayant une faible viscosité, cela implique une diffusivité, des débits élevés et des temps d’analyse courts. Notre attention a été focalisée sur des molécules apolaires : les lipides. Le but était de mettre au point des dosages dans des matrices alimentaires et biologiques et de débuter une approche lipidomique d’étude de la maladie de Parkinson. La première partie a été dédiée au développement du système SFC avec une détection UV, prêté par le constructeur Agilent Technologies. La première étude s’est portée sur 6 composés de la famille des vitamines A. Une phase d’optimisation a été réalisée afin d’obtenir une séparation satisfaisante des composés, en testant différents paramètres chromatographiques comme le type de phase stationnaire ou encore la composition de la phase mobile, afin d’obtenir une résolution optimale. Ensuite, des études de linéarité et de répétabilité ont été réalisées et des limites de détection et de quantification ont été déterminées afin d’obtenir une méthode fiable et robuste. Une deuxième partie a concerné la mise en place du couplage entre la SFC et un spectromètre de masse de type quadripôle-temps de vol (Q-TOF), afin d’améliorer la spécificité et la sensibilité des analyses. Différentes sources d’ionisation ont été utilisées : ESI, APCI et APPI. Chacune des sources présente des modes d’ionisation différents, qui permettent de pouvoir balayer une large gamme de polarité des analytes. Nous avons choisi 8 dérivés de la vitamine E, composés apolaires pour lesquels la SFC paraît être la technique d’analyse idéale. La séparation de ces composés a été optimisée de façon à obtenir une bonne résolution chromatographique et un temps d’analyse minimal. L’ionisation des composés est réalisée avec les 3 sources disponibles en faisant varier les paramètres de sources ou encore le solvant « make-up », de façon à obtenir une sensibilité optimale. La source APPI a été finalement choisie après une étude sur les performances de la méthode. Cette source présente une bonne répétabilité, linéarité et des limites de détection de l’ordre de celles retrouvées dans la littérature par HPLC-MS. Nous avons ensuite réalisé la quantification des ces composés dans 2 types de matrices alimentaire et biologique : l’huile de soja et le plasma de rat. Une troisième partie a été débutée sur le profilage de lipides à polarités variées par SFC-MS. Cette technique se révèle idéale de par la faible polarité de ces composés et leur absence d’absorbance dans le domaine UV. En effet, l’intégrité des lipides peut être altérée suite aux dommages causés par les radicaux libres, qui sont potentiellement impliqués dans de nombreuses maladies neurodégénératives. Il parait primordial de développer des outils analytiques présentant une haute sensibilité et résolution et la possibilité d’accéder aux informations structurales. La source d’ionisation ESI nous a permis de détecter 12 lipides sur les 20 sous-classes analysées en mode positif et 8 lipides en mode négatif. Une application a été réalisée sur un échantillon de plasma humain. Il serait intéressant à l’avenir d’effectuer cette étude en utilisant la source APPI, source propice à l’analyse structurale de lipides et présentant une bonne sensibilité et répétabilité. Ce couplage SFC-MS, présentant une bonne sensibilité et répétabilité, sera par la suite étendu à l’analyse de lipides dans diverses matrices biologiques et pourra à l’avenir être appliqué à l’étude de nouveaux biomarqueurs et au screening rapide d’un grand nombre d’échantillons / The aim of this PhD project was to couple supercritical fluid chromatography (SFC) with a high resolution mass spectrometer for apolar natural compounds analysis. Because mobile phase is principally constituted of CO2, SFC is called “green technic” contrary to normal phase liquid chromatography (NPLC), which uses lot of organic solvents toxic for environment. The CO2 presents a low viscosity, in this way high diffusivity and flow rate, and lower analysis times are obtained. Our work was focused on apolar molecules: the lipids. The aim was to quantify molecules in alimentary and biological matrices and to a lipidomic approach to study Parkinson disease. The first part was to develop the system SFC with a UV detection on a system on loan by Agilent Technologies. This first study was carried out on 6 vitamin A compounds. An optimization of chromatographic parameters has been realized in order to obtain a good separation of the compounds. Then, linearity, repeatability, detection and quantification limits have been determined in order to have a reliable and robust method. A second part concerned the coupling of SFC and a quadrupole time-of-flight mass spectrometer (Q-TOF), in order to improve specificity and sensitivity of analysis. Different ionization sources have been tested: ESI, APCI and APPI. Each ion source presents different ionization mode, which permits to analyze a wide range of polarities of compounds. We have chosen 8 vitamin E derivatives, which are apolar compounds for which SFC seems to be well suited. Separation compounds have been optimized in order to have a good chromatographic resolution and a short analysis time. This compounds ionization is realized with the 3 sources, varying ionization parameters and make-up solvent, to have an optimal sensitivity. The APPI source has been chosen after a performance evaluation method. This source presents a good repeatability, linearity and detection limit in the same order of magnitude than those found in the literature by HPLC-MS. Then we have quantified these compounds in alimentary and biological matrices: a soya oil and plasma rat. A third study has been started on lipid profiling with various polarities by SFC-MS. This technic is well suited because of the low polarity of this molecules and their lack of absorbance in the UV range. The integrity of lipids can be altered with damages caused by free radicals, and are potentially involved in neurodegenerative diseases. It is essential to develop analytical systems with a high sensitivity and resolution and the possibility to access to structural information. The ESI source permits to detect 12 lipids on the 20 sub-classes analyzed in positive ion mode and 8 lipids in negative mode. An application has been realized on human plasma. In the future, it will be interesting to analyze these lipids with the APPI source, which is good choice for structural analysis of lipids, with good sensitivity and repeatability. Studies with this SFC-MS system, presenting good sensitivity and repeatability, will be extended to lipid analysis in biological matrices and could be applied to new biomarkers study and for fast screening of a large number of samples