Lateral roots are a key component of the plant root system architecture that help anchorage in soil and acquisition of water and nutrients. In the dicot Arabidopsis, lateral roots initiate post-embryonically from a specialised set of cells at the xylem pole of the pericycle cell layer termed ‘founder cells (FC)’, overlaid by three distinct tissue layers – endodermis, cortex, epidermis. The FCs undergo a coordinated series of asymmetric cell divisions (ACD) to form a primordium that grows and emerges through these overlying layers as a mature lateral root. Different auxin signaling modules, as well as tight regulation of the cell geometry are important during early organogenesis. In this study, we were interested to identify molecular components that influence cell wall remodeling properties and cell geometry in the FC and regulates asymmetric cell division during early lateral root initiation. Transcriptomic analysis of FCs (De Smet et al., 2008) identified a candidate gene EXPANSINA1 (EXPA1) of expansin superfamily, known for their unique ability to alter linkage between the cell wall polymers and cause wall loosening. In vivo expression studies showed that EXPA1 is expressed in FCs prior asymmetric cell division. The mutant expa1-1 exhibits perturbed ACD with a delay in kinetics of primordia from Stage I to II, loss in radial expansion of FCs in response to auxin which is important for organised formative divisions. To understand if these defects are due to altered properties of the cell wall, and role of auxin in this process, we developed an optimised technique to study the chemical properties of the FC cell wall junctions based on confocal Raman spectroscopy. This showed altered interactions in expa1-1 between the major cell wall polymers xyloglucans and pectins locally in the pericycle cell wall upon auxin treatment, that could influence cell geometry during early lateral root development. Additionally, sugar monomer analysis of digested whole roots showed interesting alterations in representative global wall sugar levels in the root which although diluted due to lack of tissue specificity warrants further study. In conclusion, the combination of molecular and biochemical analyses reveals that auxin dependent regulation of EXPA1 plays an important role in lateral root FC and is required for organised asymmetric cell division.

The enzymatic breakdown of indoleacetic acid in the presence of aqueous pea root extracts containing an "indoleacetic acid-oxidase" system was studied. A method based on the "Salkowski" colour reaction was used for the estimation of residual indoleacetic acid. A comparison was made between the "indoleacetic acid-oxidase" activity of extracts made from the roots of plants grown in water and the activity of extracts made from the roots of plants grown in a solution of indoleacetic acid. It was found that the activity of the extracts from the "indoleacetic acid-grown" roots was higher than that of the extracts from the "water-grown" roots. Thus the "maximum rate attained in the reaction" was greater, and the "length of the lag period" shorter, when extracts of the "indoleacetic acid-grown" roots were used. Further, the relationship between the length of the lag period and the maximum rate attained appeared to be slightly different for reaction mixtures containing the two types of extract. The inhibitory activity of solutions prepared from the "water-grown" root extracts and the "indoleacetic acid-grown" root extracts by i. boiling, ii. seitz filtration, or iii. ultrafiltration, wasdetermined. The inhibitor solutions prepared from the "indoleaceticacid-grown" root extracts were always found to be less active thanthe corresponding inhibitor solutions prepared from the "water-grown" root extracts. It was thought that this difference in inhibitorlevel may have accounted for the different level of "enzyme activity"demonstrated for the extracts of the "water-grown" and "indoleacetic acid-grown" roots. The significance of the enhanced "indoleacetic acid-oxidase" activity of "indoleacetic acid-grown" root extracts was discussedin relation to the hypothesis of adaptive enzyme formation. It was concluded that the enzyme was not formed adaptively in the pea root tissues in response to the applied indoleacetic acid.

Circadian clocks are endogenous oscillators that control many physiological processes and confer functional and adaptive advantages in various organisms. These molecular oscillators comprise several interlocked feedback loops at the gene expression level. In plants, the circadian clock was recently shown to be organ specific. The root clock seemed to involve only a morning loop whereas the shoot clock also includes an evening loop in a more complex structure. My work aimed at refining the differences and similarities between the shoot and root clocks, using a combination of experimental and theoretical approaches. I developed an imaging method to obtain more data from the shoot and root clocks over time in various conditions. Some previous results were confirmed: the free running periods (FRPs) are longer in roots compared to shoots under constant light (LL). In addition, the amplitude of clock gene expression rhythms is lower in roots compared to shoots. However, the expression of several evening genes is circadian in roots, contrary to previous conclusions. This was confirmed with qPCR, and was observed in both light- and dark-grown roots. Yet light affects clock gene expression in roots, so an automatic covering system was designed to keep the roots in darkness and obtain data in more physiological conditions. Clock genes behaved differently in shoots and light-grown roots that were in the same environmental conditions, and may be differentially affected by blue and red light. However shoot and root clocks were more similar under constant darkness (DD). My imaging and RT-qPCR data, together with new microarray results and preliminary studies on clock mutants suggest that shoot and root circadian systems may have a similar structure but different input pathways. Entrainment is a fundamental property of circadian systems, which can be reset by cues such as light/dark (LD) cycles. I demonstrated that light can directly entrain the root clock in decapitated plants. The root clock could be entrained by a broad range of T cycles using low light intensity. In addition, rhythms were preferably entrained by low light than by any putative signal from shoots in experiments using conflicting LD cycles of different strengths. My results indicate that direct entrainment by LD cycles could be the main mechanism that synchronise the shoot and root clocks at constant temperature. This is physiologically relevant because dark-grown roots can perceive light channelled by the exposed tissues, in a fibre optic way. I also showed for the first time that clock and output genes could be rapidly entrained by temperature cycles in roots. Several mathematical models of the shoot circadian clock were used to try and fit the root clock data by optimising some parameters. The best set of parameters gave a good qualitative fit to root data under LD, LL and DD. It reproduced the long FRP observed in roots under LL and captured the entrainment under LD with lower amplitude in roots. The parameters that were changed for these simulations were all related to light input, which supports the idea of similar clock structures in shoots and roots but with different input pathways. Together my results confirmed that the plant circadian clock is organ specific and suggest that it is organ autonomous.

X-ray micro computed tomography (µCT) is increasingly applied in plant biology as an imaging system that is valuable for the study of root development in soil, since it allows the three-dimensional and non-destructive visualisation of plant root systems. Variations in the X-ray attenuation values of root material and the overlap in measured intensity values between roots and soil caused by water and organic matter represent major challenges to the extraction of root system architecture. We propose a novel technique to recover root system information from X-ray CT data, using a strategy based on a visual tracking framework embedding a modiffed level set method that is evolved using the Jensen-Shannon divergence. The model-guided search arising from the visual tracking approach makes the method less sensitive to the natural ambiguity of X-ray attenuation values in the image data and thus allows a better extraction of the root system. The method is extended by mechanisms that account for plagiatropic response in roots as well as collision between root objects originating from different plants that are grown and interact within the same soil environment. Experimental results on monocot and dicot plants, grown in different soil textural types, show the ability of successfully extracting root system information. Various global root system traits are measured from the extracted data and compared to results obtained with alternative methods.

The root system is the plant’s principal organ for water and mineral nutrient supply. Root growth follows an endogenous, developmental programme. Yet, this programme can be modulated by external cues which makes root system architecture (RSA), the spatial configuration of all root parts, a highly plastic trait. Presence or absence of nutrients such as nitrate (N), phosphate (P), potassium (K) and sulphate (S) serve as environmental signals to which a plant responds with targeted proliferation or restriction of main or lateral root growth. In turn, RSA serves as a quantitative reporter system of nutrient starvation responses and can therefore be used to study nutrient sensing and signalling mechanisms. In this study, I have analysed root architectural responses of various Arabidopsis thaliana genotypes (wildtype, mutants and natural accessions) to single and multiple nutrient deficiency treatments. A comprehensive analysis of combinatorial N, P, K an S supply allowed me to dissect the effect of individual nutrients on individual root parameters. It also highlighted the existence of interactive effects arising from simultaneous environmental stimuli. Quantification of appropriate RSA parameters allowed for targeted testing of known regulatory genes in specific nutritional settings. This revealed, for example, a novel role for CIPK23, AKT1 and NRT1.1 in integrating K and N effects on higher order lateral root branching and main root angle. A significant contribution to phenotypic variation also arose from P*K interactions. I could show that the iron (Fe) concentration in the external medium is an important driving force of RSA responses to low-P and low-K. In fact, P and K deprivation caused Fe accumulation in distinct parts of the root system, as demonstrated by Fe staining and synchrotron X-Ray fluorescence. Again, selected K, P and Fe transport and signalling mutants were tested for aberrant low-K and/or low-P phenotypes. Most notably, the two paralogous ER-localised multicopper oxidases LPR1 and LPR2 emerged as important signalling components of P and K deprivation, potentially integrating Fe homeostasis with meristematic activity under these conditions. In addition to the targeted characterisation of specific genotype-environment interactions, I investigated novel RSA responses to low-K via a non-targeted approach based on natural variation. A morphological gradient spanned the entire genotype set, linking two extreme strategies of low-K responses. Strategy I accessions responded to low-K with a moderate reduction of main root growth but a severe restriction of lateral root elongation. In contrast, strategy II genotypes ceded main root growth in favour of lateral root proliferation. The genetic basis of these low-K responses was then subsequently mapped onto the A. thaliana genome via quantitative trait loci (QTL) analysis using recombinant inbred lines derived from parental accessions that either adopt strategy I (Col-0) or II (Ct-1). In sum, this study addresses the question how plants incorporate environmental signals to modulate developmental programmes that underly RSA formation. I present evidence for novel phenotypic responses to nutrient deprivation and for novel genetic regulators involved in nutrient signalling and crosstalk.

The overall aim of this research is to develop a new root-image based approach to modelling water uptake by plants. The approach developed employs a digitized image of the root zone to determine an ‘effective root density ratio’ that is subsequently used to yield a spatially variable sink term. The moisture flow model chosen is based on Richards’ Equation added a sink term to facilitate inclusion of a water uptake model (i.e. 1D, 2D and 2D axisymmetric format). A numerical solution was achieved via the finite element method for spatial discretisation along with a finite difference time-marching scheme. The numerical evaluation of the root density ratio was coded in Matlab. The resulting values were then used to define the spatial variation of the sink term within the finite element code. Initial applications of the new model operating in a one-dimensional mode provided some confidence with respect to the implementation of the new image-based root density approach to simulate moisture migration patterns beneath a uniform cover of vegetation. A new two-dimensional axi-symmetric form of the model was then developed and applied to simulate moisture migration near established trees. The model was validated by direct comparison to the field measurements recorded. The study provided an assessment of the significance of water content (and therefore suction) changes on the stability of unsaturated soil slopes. Two typical root architectures were considered to investigate the influence of root architecture on slope stability. In particular, effects of root architecture were emphasized. In conclusion, a new root-image based approach to modelling water uptake by plants has been developed. In general, it is hoped that the current research has provided a useful further contribution on modelling water-uptake process and on the overall assessment of slope stability.

The C₄ photosynthetic cycle acts to concentrate CO₂ around the enzyme Rubisco. By doing so, C₄ photosynthesis leads to increased radiation, water and nitrogen use efficiencies. As such, C₄ photosynthesis is the most productive form of photosynthesis known. Because it enables such high levels of productivity there are large international efforts to introduce C₄ photosynthesis into non-C₄ crop species such as rice. Kranz anatomy is a characteristic leaf cellular arrangement of concentric rings of bundle sheath and mesophyll cells around closely spaced veins and is crucial to C₄ photosynthesis in almost all known examples. Despite the fact that Kranz has evolved on over 60 times independently little is known about the genetic regulation of Kranz development, as attempts to elucidate Kranz regulators using conventional mutagenesis screens have provided few insights. However, the advent of next generation DNA sequencing technologies has enabled the interrogation of genetic networks at a previously unprecedented scale. The work in this thesis describes a genome-wide transcriptomic analysis of leaf development in maize, a C₄ species, that develops both Kranz-type and non-Kranz-type leaves. Detailed bioinformatics analyses identified candidate regulators of both Kranz development and additional aspects of maize leaf development. Three of the identified Kranz candidates were functionally characterised in both C₄ and non-C₄ species. Furthermore, expression and phylogenetic analyses of GOLDEN2-LIKE (GLK) genes, a small transcription factor family previously implicated in C₄ development in maize, were extended to determine the generality of GLK function in C₄ evolution.

L'objectif de cette thèse était d'élucider le rôle des transporteurs de saccharose dans les racines d’A. thaliana pour mieux comprendre la répartition du carbone dans la plante entière.Nous nous sommes focalisés sur l’étude des deux principaux transporteurs de saccharose exprimés dans les racines : AtSUC1 et AtSUC2. Une étude du mutant KO suc1 et une étude des plantes greffées présentant la mutation KO du gène AtSUC2 uniquement dans les racines ont été réalisées sur des plantes au stade adulte (30 à 32 jours après semis) cultivées en rhizobox. De plus, les localisations de l’expression des gènes et des protéines de ces transporteurs ont été réalisées dans les racines de plantes adultes pour la première fois. La localisation de l’expression d’AtSCU1 dans les pointes racinaires, nous a permis de conclure sur un rôle potentiel d’AtSUC1 dans le déchargement du saccharose du phloème dans les zones de croissance des racines et/ou un rôle potentiel de senseur du signal glucidique ou du déficit hydrique, probablement en lien avec l’ABA. Les résultats montrent qu’AtSUC2 aurait un rôle dans le développement racinaire, certainement via le déchargement du saccharose du phloème dans les zones d’élongation des racines et dans le rechargement du phloème le long des racines (notamment dans les départs de racines latérales). De plus, AtSUC2 pourrait avoir un rôle dans l’augmentation locale d’hexoses liée à l’émergence des racines latérales. Enfin, nos résultats indiquent une contradiction entre la localisation de l’expression du gène AtSUC2 retrouvée dans le parenchyme cortical et la localisation de la protéine qui n’est pas retrouvée dans ces cellules. Néanmoins les deux approches s’accordent sur la localisation dans les cellules compagnes. / The aim of this thesis was to elucidate the role of sucrose transporters in the roots of Arabidopsis thaliana to have a better comprehension of the carbon partitioning in the whole plant.We focused on the study of the two main sucrose transporters expressed in the roots: AtSUC1 and AtSUC2. A study of the KO mutant suc1 and a study on grafted plants with the KO mutation of the AtSUC2 gene only in the roots (micro-graft) were carried out on adult plants (30 to 32 days after sowing) grown in rhizobox. In addition, the localization of the expression of the genes and proteins of transporters were carried out in the roots of adult plants for the first time. The localization of AtSCU1 expression in the root tips allowed us to conclude on a potential role of AtSUC1 in the sucrose phloem unloading in root growth areas and/or a potential role of sensor of carbohydrate signal or of water deficit, probably related to ABA. The results also show that AtSUC2 has a role in root development and certainly via sucrose phloem unloading in root elongation’s areas and in the phloem reloading along the roots (especially in sites of emergence of lateral roots). In addition, AtSUC2 may have a role in the local increase of hexoses associated with the emergence of lateral roots. Finally, our results indicate a contradiction between the localization of the AtSUC2 gene expression found in the cortical parenchyma and the localization of the protein that is not found in these cells. Nevertheless the two approaches agree on the localization in the companion cells.

Chez les plantes supérieures, la distribution des sucres entre organes sources et puits requiert l'activité de transporteurs membranaires de sucre. Les flux de sucre variant au cours du développement de la plante et en réponse à des stress, il est logique de penser que les transporteurs de sucres sont impliqués dans ces changements. Le but du travail était de suivre la répartition des sucres et l'expression des transporteurs correspondants dans les racines de la plante modèle Arabidopsis thaliana, en réponse à un stress osmotique. Afin de pouvoir récolter des racines, un système de culture en hydroponie a été mis en place. Après avoir vérifié l'homogénéité des plantes cultivées dans ce système, une étude de l'expression des gènes de transporteurs de sucre a été effectuée au cours du développement des plantes et de d'une journée (24h), en utilisant la technique de macroarray. Cette étude a révélé l'expression dans les racines de 3 transporteurs de saccharose (AtSUC1, AtSUC2 et AtSUC5), 2 transporteurs de polyols (AtPLT5 et AtPLT6) et 2 transporteurs d'hexoses (AtSTP7 et AtSTP13). Le suivi de la teneur en sucres solubles et en amidon ainsi que du transport à longue distance de [U-14C]-saccharose a permis d'émettre des hypothèses quant au rôle des transporteurs de sucres exprimés dans la racine. Afin de mimer un stress hydrique en hydroponie, un protocole d'application progressif d'un agent osmotique (polyéthylène glycol 6000) dans le milieu de culture a été élaboré. Ce système a permis de mettre en évidence que 5 des gènes de transporteurs de sucre identifiés dans la racine ont une expression qui varie dans ces conditions. Trois d'entre eux sont fortement réprimés : AtSUC1 / In plants, sugar allocation between source and sink organs is based on the activity of membrane transporters for sugars. As sugar fluxes are changing during development and in response to stress, sugar transporters are supposed to be involved in those changes. The aim of this work was to study sugar allocation and the corresponding transporters gene expression in the roots of the model plant Arabidopsis thaliana during an osmotic stress. In order to have access to the roots, an hydroponic culture system was developped. The homogeneity of the plants obtained with this system was checked and the expression of sugar tranporter genes was followed during development and during a 24h period was studied by a macro-array technique. The expression in the roots of the following genes was found: 3 sucrose transporters (AtSUC1, AtSUC2 and AtSUC5), 2 polyol transporters (AtPLT5 and AtPLT6) and 2 hexose transporters (AtSTP7 and AtSTP13). The sugar and starch content and the long distance of 14C-sucrose were measured and used to build some hypothesis on the role of sugar transporters in the roots. To mimick a water stress, an osmotic stress due to the progressive addition of Polyethylene-glycol was applied. This system demonstrated that 5 of the identified transporter genes display a change in expression: 3 are repressed (AtSUC1, AtSUC5 and AtPLT6) and 2 are over expressed (AtSUC2 and AtSTP13). Moreover, soluble sugar and starch accumulate in the leaves and 14C-sucrose transport to the roots is decreased in plants subjected to an osmotic stress. The respective role of transporters is discussed. The gene expression data were also confirmed with plants grown in rhizoboxes.

Chez les plantes, le RET (Root Extracellular Trap) est une structure cellulo-moléculaire jouant un rôle central dans la défense racinaire face aux stress abiotiques et biotiques. De nombreuses similitudes de composition ont été observées entre le RET et le NET (Neutrophil Extracellular Trap) du système immunitaire des mammifères, connu pour capturer et tuer certains microorganismes bactériens et fongiques. Le RET est composé de cellules frontières et de leurs sécrétions (composés de haut et de bas poids moléculaire) comprenant des polysaccharides de la paroi cellulaire, des protéoglycannes et des métabolites secondaires. Il contient également des protéines antimicrobiennes et de l'ADN extracellulaire, tout comme le NET. Dans le cadre de mon projet de thèse, nous avons caractérisé la composition moléculaire et la structuration de cette entité de défense chez deux légumineuses, le soja (Glycine max (Merr) L.) et le pois (Pisum sativum L.), par des approches d’imagerie cellulaire photonique et électronique. Nous avons également étudié l’impact du RET du soja sur des pathogènes telluriques, à savoir Phytophthora parasitica et Aphanomyces euteiches. Nous avons ainsi pu mettre en évidence la présence de différents morphotypes de cellules frontières et de mucilage au sein du RET de soja et de pois. Pour la première fois, nous avons montré la présence d’hétéromannanes, de xyloglucane et de cellulose dans le RET, formant une ossature stabilisant le mucilage et reliant les cellules frontières entre elles. Ces polysaccharides structuraux semblent être essentiels à l’intégrité structurale et fonctionnelle du RET. Enfin, nos résultats ont montré que le RET de soja était impliqué dans la défense précoce de la racine contre P. parasitica. Cette étude apporte de nouvelles connaissances relatives à la composition moléculaire et la structure du RET, nous amenant ainsi à comparer le RET à d’autres modèles que le NET des mammifères, tels que les biofilms bactériens et les mucilages de graines. En effet, de nombreuses similitudes existent entre ces différents complexes en termes de composition et de fonctionnement, qui méritent d’être explorer plus en détail dans l’avenir. / In higher plants, the RET (Root Extracellular Trap) is a complex made up of border cells and secretions, released by root tips and believed to play a central role in biotic and abiotic stress tolerance. This structure is quite similar to the Neutrophil Extracellular Trap (NET) known as one of the first lines of defense in mammals, able to trap and kill microbial pathogens. RET secretions consist of high and low-molecular weight compounds including cell wall polysaccharides, proteoglycans and secondary metabolites. They also contain a variety of anti-microbial proteins and extracellular DNA much like the NET. During my thesis work, we investigated the release and morphology of root border cells in soybean (Glycine max (Merr) L.) using light and scanning electron microscopy. The molecular composition of the mucilage was also investigated using immunocytochemistry, anti-cell wall glycan antibodies and confocal microscopy. Immunocytochemistry was also applied to pea (Pisum sativum L.) border cells and secretions to examine the occurrence of specific polysaccharides. We also studied the impact of soybean RET on the soilborne pathogens, Phytophthora parasitica and Aphanomyces euteiches. Our findings showed that root tips of soybean released three border cell morphotypes all of which secreted substantial amounts of mucilage. Immunocytochemical data showed that mucilage was enriched in pectin and the two hemicellulosic polysaccharides xyloglucan and heteromannan. Mucilage also contained cellulose, histone and extracellular DNA. Interestingly, the structural polysaccharides formed a fibrous network surrounding the cells and holding them together, supporting their role in maintaining mucilage architecture and integrity. In addition, we found that xyloglucan and cellulose were also secreted into the mucilage of pea, connecting border cells together. Finally, our findings revealed that RET prevented P. parasitica zoospores from colonizing soybean root tip, by stopping their penetration and inducing their death. Overall the study revealed novel insights into the composition, structure and function of plant RETs. Currently, the RET is much less studied than its mammal counterpart, the NET, but structural and functional similarities exist between these two traps. Interestingly, similarities do also exist between the RET and other important biological complexes, including bacterial biofilms and seed mucilage, that deserve to be further investigated and compared in the context of immunity.