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Much of what we know about the role of auxin in plant development derives from exogenous manipulations of auxin distribution and signaling, using inhibitors, auxins and auxin analogs. In this context, synthetic auxin analogs, such as 1-Naphtalene Acetic Acid (1-NAA), are often favored over the endogenous auxin indole-3-acetic acid (IAA), in part due to their higher stability. While such auxin analogs have proven to be instrumental to reveal the various faces of auxin, they displays in some cases distinct bioactivities compared to IAA. Here, we focused on the effect of auxin analogs on the accumulation of PIN proteins in Brefeldin A (BFA)-sensitive endosomal aggregations (BFA bodies), and the correlation with the ability to elicit Ca(2+) responses. For a set of commonly used auxin analogs, we evaluated if auxin-analog induced Ca(2+) signaling inhibits PIN accumulation in. We found that not all auxin analogs elicited a Ca(2+) response, and that their differential ability to elicit Ca(2+) responses correlated partially with their ability to inhibit BFA-body formation. However, in tir1/afb and cngc14, 1-NAA-induced Ca(2+) signaling was strongly impaired, while 1-NAA’s still could inhibit PIN accumulation in BFA bodies. This demonstrates that TIR1/AFB-CNGC14-dependent Ca(2+) signaling does not inhibit BFA body formation.

Much of what we know about the role of auxin in plant development derives from exogenous manipulations of auxin distribution and signaling, using inhibitors, auxins, and auxin analogs. In this context, synthetic auxin analogs, such as 1-naphthalene acetic acid (1-NAA), are often favored over the endogenous auxin, indole-3-acetic acid (IAA), in part due to their higher stability. While such auxin analogs have proven instrumental in revealing the various faces of auxin, they display in some cases bioactivities distinct from IAA. Here, we focused on the effect of auxin analogs on the accumulation of PIN proteins in brefeldin A-sensitive endosomal aggregations (BFA bodies), and correlation with the ability to elicit Ca2+ responses. For a set of commonly used auxin analogs, we evaluated if auxin analog-induced Ca2+ signaling inhibits PIN accumulation. Not all auxin analogs elicited a Ca2+ response, and their differential ability to elicit Ca2+ responses correlated partially with their ability to inhibit BFA-body formation. However, in tir1/afb and cngc14, 1-NAA-induced Ca2+ signaling was strongly impaired, yet 1-NAA still could inhibit PIN accumulation in BFA bodies. This demonstrates that TIR1/AFB-CNGC14-dependent Ca2+ signaling does not inhibit BFA body formation in Arabidopsis roots. Synthetic auxin analog-induced Ca(2+)signaling is independent of inhibition of PIN accumulation in brefeldin A-sensitive endosomal aggregations.

Modular, repetitive structures are a key component of complex multicellular body plans across the tree of life. Typically, these structures are prepatterned by temporal oscillations in gene expression or signaling. Although a clock-and-wavefront mechanism was identified and plant leaf phyllotaxis arises from a Turing-type patterning for vertebrate somitogenesis and arthropod segmentation, the mechanism underlying lateral root patterning has remained elusive. To resolve this enigma, we combined computational modeling with in planta experiments. Intriguingly, auxin oscillations automatically emerge in our model from the interplay between a reflux-loop-generated auxin loading zone and stem-cell-driven growth dynamics generating periodic cell-size variations. In contrast to the clock-and-wavefront mechanism and Turing patterning, the uncovered mechanism predicts both frequency and spacing of lateral-root-forming sites to positively correlate with root meristem growth. We validate this prediction experimentally. Combined, our model and experimental results support that a reflux-and-growth patterning mechanism underlies lateral root priming.

Sterols are very well known for their important roles in membranes and signaling in eukaryotes. Plants stand out among eukaryotes by the large variety of sterols that they can produce, and employing them across a wide spectrum of physiological processes. Therefore, it is critical to understand the wiring of the biosynthetic pathways by which plants generate these distinct sterols, to allow manipulating them and dissect their precise physiological roles. Many enzymatic steps show a deep evolutionary conservation, while others are executed by completely different enzymes. Here, we review the complexity and variation of the biosynthetic routes of the most abundant phytosterols in the green lineage and how different enzymes in these pathways are conserved and diverged from humans,yeast and even bacteria. Based on their evolutionary conservation we discuss the use of human and yeast sterol biosynthesis inhibitors in plants, as an argument for the development of plant-tailored inhibitors of sterol biosynthesis.

During periderm differentiation, phellem cells undergo secondary cell wall thickening by suberin deposition, forming a compact barrier shielding plant organs from the environment. Development and suberization of periderm cells are tightly controlled processes, however their regulation is still poorly understood. To identify novel regulators of these processes, we have conducted histological and transcriptomic analyses to follow periderm development in two model systems: Arabidopsis and cork oak (Q. suber). After a detailed spatio-temporal study of periderm formation in Arabidopsis roots, a transcriptomic analysis using TRAP-Seq targeting periderm cells at the onset of suberization was performed with the aim to identify tissue-specific regulators. We found 2433, out of 4631 differentially expressed genes overrepresented in suberizing cells. Through a functional enrichment analysis, several terms were found to be represented, as suberin biosynthesis, response to karrikin, cell wall metabolic process and response to abscisic acid. Possible candidate genes involved in control of phellem suberization will be identified and selected for further functional validation. Furthermore, in cork oak, histochemical analyses on 1-week old tap roots, showed a positive effect of drought and heat stress on periderm development. This system is now being used as a model to identify regulators of cork formation in this species. A comparative transcriptomic analysis based on different growth conditions and root differentiation stages was performed, revealing contrasting transcriptome changes between different root zones. Genes potentially involved in the crosstalk between cork development and response to environmental changes are being identified by integrating the results obtained from both plant systems. This knowledge might further contribute to design new strategies for the improvement of cork production under abiotic stress conditions.

Many signal perception mechanisms are connected to Ca(2+)-based second messenger signaling to modulate specific cellular responses. The well-characterized plant hormone auxin elicits a very rapid Ca(2+) signal. However, the cellular targets of auxin-induced Ca(2+) are largely unknown. Here, we screened a biologically annotated chemical library for inhibitors of auxin-induced Ca(2+) entry in plant cell suspensions to better understand the molecular mechanism of auxin-induced Ca(2+) and to explore the physiological relevance of Ca(2+) in auxin signal transduction. Using this approach, we defined a set of diverse, small molecules that interfere with auxin-induced Ca(2+) entry. Based on annotated biological activities of the hit molecules, we found that auxin-induced Ca(2+) signaling is, among others, highly sensitive to disruption of membrane proton gradients and the mammalian Ca(2+) channel inhibitor bepridil. Whereas protonophores nonselectively inhibited auxin-induced and osmotic stress–induced Ca(2+) signals, bepridil specifically inhibited auxin-induced Ca(2+). We found evidence that bepridil severely alters vacuolar morphology and antagonized auxin-induced vacuolar remodeling. Further exploration of this plant-tailored collection of inhibitors will lead to a better understanding of auxin-induced Ca(2+) entry and its relevance for auxin responses.

Cyclic Nucleotide Gated Ion Channels (CNGCs) are non-selective cation channels that are involved in regulating responses to both biotic and abiotic stresses in plants. CNGC2 has been implicated in plant immunity and Ca 2+ signaling through the study of the autoimmune phenotypes exhibited by the null mutant, defense, no death1 (dnd1) . However, dnd1 also shows additional phenotypes that are unique among autoimmune mutants. This suggests that CNGC2 plays multiple biological roles beyond pathogen defense. In this study, we cloned the gene that encodes the first suppressor of dnd1 (cngc2), REPRESSOR OF DEFENSE, NO DEATH1 (RDD1) , which encodes an auxin biosynthesis gene, YUCCA6 (YUC6) . We found that dnd1 (cngc2) is defective in auxin-mediated root growth inhibition and gravitropic responses in roots. Consistently with these auxin resistance phenotypes, we found dnd1 shows a dampened response to exogenous auxin compared to wildtype plants using the auxin inducible DR5::GUS and DII:VENUS reporter systems. Finally, auxin-induced Ca 2+ influx was examined using the Förster resonance energy transfer (FRET)-based, genetically encoded Ca 2+ indicator yellow cameleon (YC)-Nano65. We captured severe defects in auxin-induced Ca 2+ increase in dnd1 . These defects were rescued by the rdd1 mutation. Our findings highlight the unexpected involvement of CNGC2 in auxin-induced Ca 2+ signaling and calls into question the current interpretation of dnd1 phenotypes in defense signaling.

© 2018 Elsevier Ltd Efficient soil exploration by roots represents an important target for crop improvement and food security [1, 2]. Lateral root (LR) formation is a key trait for optimizing soil foraging for crucial resources such as water and nutrients. Here, we report an adaptive response termed xerobranching, exhibited by cereal roots, that represses branching when root tips are not in contact with wet soil. Non-invasive X-ray microCT imaging revealed that cereal roots rapidly repress LR formation as they enter an air space within a soil profile and are no longer in contact with water. Transcript profiling of cereal root tips revealed that transient water deficit triggers the abscisic acid (ABA) response pathway. In agreement with this, exogenous ABA treatment can mimic repression of LR formation under transient water deficit. Genetic analysis in Arabidopsis revealed that ABA repression of LR formation requires the PYR/PYL/RCAR-dependent signaling pathway. Our findings suggest that ABA acts as the key signal regulating xerobranching. We conclude that this new ABA-dependent adaptive mechanism allows roots to rapidly respond to changes in water availability in their local micro-environment and to use internal resources efficiently. Orman-Ligeza et al. report that lateral root formation is repressed in soil air spaces. This novel adaptive response appears to be regulated by the abiotic stress signal ABA. They propose that this mechanism enables plants to fine-tune root branching to local variation in soil structure and water availability.

Lateral root (LR) emergence represents a highly coordinated process in which the plant hormone auxin plays a central role. Reactive oxygen species (ROS) have been proposed to function as important signals during auxin-regulated LR formation, however their mode of action is poorly understood. Here, we report that Arabidopsis roots exposed to ROS show increased LR numbers due to the activation of LR pre-branch sites and LR primordia (LRP). Strikingly, ROS treatment can also restore LR formation in pCASP1:shy2-2 and aux1 lax3 mutant lines in which auxin-mediated cell wall accommodation and remodeling in cells overlying the sites of LR formation is disrupted. Specifically, ROS are deposited in the apoplast of these cells during LR emergence, following a spatio-temporal pattern that overlaps the combined expression domains of extracellular ROS donors of the RESPIRATORY BURST OXIDASE HOMOLOGS (RBOH). We also show that disrupting (or enhancing) expression of RBOH in LRP and/or overlying root tissues decelerates (or accelerates) the development and emergence of LRs. We conclude that RBOH-mediated ROS production facilitates LR outgrowth by promoting cell wall remodeling of overlying parental tissues.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Upward leaf movement (hyponastic growth) is frequently observed in response to changing environmental conditions and can be induced by the phytohormone ethylene. Hyponasty results from differential growth (i.e. enhanced cell elongation at the proximal abaxial side of the petiole relative to the adaxial side). Here, we characterize Enhanced Hyponasty-D, an activation-tagged Arabidopsis (Arabidopsis thaliana) line with exaggerated hyponasty. This phenotype is associated with overexpression of the mitotic cyclin CYCLINA2;1 (CYCA2;1), which hints at a role for cell divisions in regulating hyponasty. Indeed, mathematical analysis suggested that the observed changes in abaxial cell elongation rates during ethylene treatment should result in a larger hyponastic amplitude than observed, unless a decrease in cell proliferation rate at the proximal abaxial side of the petiole relative to the adaxial side was implemented. Our model predicts that when this differential proliferation mechanism is disrupted by either ectopic overexpression or mutation of CYCA2;1, the hyponastic growth response becomes exaggerated. This is in accordance with experimental observations on CYCA2;1 overexpression lines and cyca2;1 knockouts. We therefore propose a bipartite mechanism controlling leaf movement: ethylene induces longitudinal cell expansion in the abaxial petiole epidermis to induce hyponasty and simultaneously affects its amplitude by controlling cell proliferation through CYCA2;1. Further corroborating the model, we found that ethylene treatment results in transcriptional down-regulation of A2-type CYCLINs and propose that this, and possibly other regulatory mechanisms affecting CYCA2;1, may contribute to this attenuation of hyponastic growth.

Upward leaf movement (hyponastic growth) is frequently observed in response to changing environmental conditions and can be induced by the phytohormone ethylene. Hyponasty results from differential growth (i.e. enhanced cell elongation at the proximal abaxial side of the petiole relative to the adaxial side). Here, we characterize Enhanced Hyponasty-d, an activation-tagged Arabidopsis (Arabidopsis thaliana) line with exaggerated hyponasty. This phenotype is associated with overexpression of the mitotic cyclin CYCLINA2;1 (CYCA2;1), which hints at a role for cell divisions in regulating hyponasty. Indeed, mathematical analysis suggested that the observed changes in abaxial cell elongation rates during ethylene treatment should result in a larger hyponastic amplitude than observed, unless a decrease in cell proliferation rate at the proximal abaxial side of the petiole relative to the adaxial side was implemented. Our model predicts that when this differential proliferation mechanism is disrupted by either ectopic overexpression or mutation of CYCA2;1, the hyponastic growth response becomes exaggerated. This is in accordance with experimental observations on CYCA2;1 overexpression lines and cyca2;1 knockouts. We therefore propose a bipartite mechanism controlling leaf movement: ethylene induces longitudinal cell expansion in the abaxial petiole epidermis to induce hyponasty and simultaneously affects its amplitude by controlling cell proliferation through CYCA2;1. Further corroborating the model, we found that ethylene treatment results in transcriptional down-regulation of A2-type CYCLINs and propose that this, and possibly other regulatory mechanisms affecting CYCA2;1, may contribute to this attenuation of hyponastic growth.

Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain-containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism.

Overall root architecture is the combined result of primary and lateral root growth and is influenced by both intrinsic genetic programs and external signals. One of the main questions for root biologists is how plants control the number of lateral root primordia and their emergence through the main root. We recently identified S-phase kinase-associated protein2 (SKP2B) as a new early marker for lateral root development. Here, we took advantage of its specific expression pattern in Arabidopsis (Arabidopsis thaliana) in a cell-sorting and transcriptomic approach to generate a lateral root-specific cell sorting SKP2B data set that represents the endogenous genetic developmental program. We first validated this data set by showing that many of the identified genes have a function during root growth or lateral root development. Importantly, genes encoding peroxidases were highly represented in our data set. Thus, we next focused on this class of enzymes and showed, using genetic and chemical inhibitor studies, that peroxidase activity and reactive oxygen species signaling are specifically required during lateral root emergence but, intriguingly, not for primordium specification itself.

In multicellular organisms, morphogenesis relies on a strict coordination in time and space of cell proliferation and differentiation. In contrast to animals, plant development displays continuous organ formation and adaptive growth responses during their lifespan relying on a tight coordination of cell proliferation. How developmental signals interact with the plant cell-cycle machinery is largely unknown. Here, we characterize plant A2-type cyclins, a small gene family of mitotic cyclins, and show how they contribute to the fine-tuning of local proliferation during plant development. Moreover, the timely repression of CYCA2;3 expression in newly formed guard cells is shown to require the stomatal transcription factors FOUR LIPS/MYB124 and MYB88, providing a direct link between developmental programming and cell-cycle exit in plants. Thus, transcriptional downregulation of CYCA2s represents a critical mechanism to coordinate proliferation during plant development.

Lateral root initiation and development has been increasingly studied over the last two decades. This postembryonic organogenic process guarantees the spatial development and plasticity of the root system in response to environmental cues and is crucial for the plant's growth and development. Several independent large-scale transcriptome studies in different species resulted in a wealth of data that can be instructive to understand this process at the molecular level. Here, we present an easy and flexible spreadsheet tool, called Visual Lateral Root Transcriptome Compendium, that combines publicly available data sets involved in Arabidopsis (Arabidopsis thaliana) lateral root development and links them with additional information on tissue-specific expression and cell cycle involvement, thus allowing the extraction of novel information from existing data sets in a visual and user-friendly manner. We believe that this tool will be valuable not only for root biologists but also for a broader range of scientists as it enables a fast indication of the potential involvement of a given gene during de novo organogenesis.

Upward leaf movement (hyponastic growth) is frequently observed in response to changing environmental conditions and can be induced by the phytohormone ethylene. Hyponasty results from differential growth (i.e. enhanced cell elongation at the proximal abaxial side of the petiole relative to the adaxial side). Here, we characterize Enhanced Hyponasty-d, an activation-tagged Arabidopsis (Arabidopsis thaliana) line with exaggerated hyponasty. This phenotype is associated with overexpression of the mitotic cyclin CYCLINA2;1 (CYCA2;1), which hints at a role for cell divisions in regulating hyponasty. Indeed, mathematical analysis suggested that the observed changes in abaxial cell elongation rates during ethylene treatment should result in a larger hyponastic amplitude than observed, unless a decrease in cell proliferation rate at the proximal abaxial side of the petiole relative to the adaxial side was implemented. Our model predicts that when this differential proliferation mechanism is disrupted by either ectopic overexpression or mutation of CYCA2;1, the hyponastic growth response becomes exaggerated. This is in accordance with experimental observations on CYCA2;1 overexpression lines and cyca2;1 knockouts. We therefore propose a bipartite mechanism controlling leaf movement: ethylene induces longitudinal cell expansion in the abaxial petiole epidermis to induce hyponasty and simultaneously affects its amplitude by controlling cell proliferation through CYCA2;1. Further corroborating the model, we found that ethylene treatment results in transcriptional down-regulation of A2-type CYCLINs and propose that this, and possibly other regulatory mechanisms affecting CYCA2;1, may contribute to this attenuation of hyponastic growth.

CSIC - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), In Arabidopsis, the root clock regulates the spacing of lateral organs along the primary root through oscillating gene expression. The core molecular mechanism that drives the root clock periodicity and how it is modified by exogenous cues such as auxin and gravity remain unknown. We identified the key elements of the oscillator (AUXIN RESPONSE FACTOR 7, its auxin-sensitive inhibitor IAA18/POTENT, and auxin) that form a negative regulatory loop circuit in the oscillation zone. Through multilevel computer modeling fitted to experimental data, we explain how gene expression oscillations coordinate with cell division and growth to create the periodic pattern of organ spacing. Furthermore, gravistimulation experiments based on the model predictions show that external auxin stimuli can lead to entrainment of the root clock. Our work demonstrates the mechanism underlying a robust biological clock and how it can respond to external stimuli., This work was funded by the Ministerio de Economía y Competitividad of Spain (MINECO) and/or the ERDF (BFU2016-80315-P to M.A.M.-R., BIO2017-82209-R to J.C.d.P., and TIN2016-81079-R to A.R.-P.), the Comunidad de Madrid and/or ERDF and ESF (2017-T1/BIO-5654 to K.W. and S2017/BMD-3691 to A.R.-P.), the Howard Hughes Medical Institute and the NIH (R35-GM131725 to P.N.B.), the Fonds Wetenschappelijk Onderzoek (FWO Flanders) (G022516N, G020918N, and G024118N to T.B.), and the “Severo Ochoa Program for Centres of Excellence in R&D” from the Agencia Estatal de Investigacion of Spain [SEV-2016-0672 (2017–2021)] to K.W., P.P.-G., and M.A.M.-R. through CBGP. M.M. was supported by a postdoctoral contract associated to SEV-2016-0672, E.B.-A. by Ayudante de Investigacion contract PEJ-2017-AI/BIO-7360 from the Comunidad de Madrid, A.S.-C. and L.S.-R. by FPI contracts from MINECO (BES-2014-068852 and BES-2017-080155, respectively), J.C. by a Juan de la Cierva contract from MINECO (FJCI-2016-28607), P.P.-G. by a Juan de la Cierva contract from MINECO (FJCI-2015-24905) and Programa Atraccion Talento from Comunidad Madrid (2017-T2/BIO-3453), A.S. by a Torres Quevedo contract from MINECO (PTQ-15-07915), and K.W. by program PGC2018-093387-A-I00 from the Ministerio de Ciencia e Innovacion (MICIU)

Growth of plants depends on cell division and elongation of the divided cells. After cell division, plant cells are fixed immediately within their tissue context. Thus, a carefully positioning and orientation of the division plane is crucial for plants to properly pattern and grow. Anticlinal cell divisions result in more cells within a cell file and lead to longitudinal growth; while periclinal and radial divisions (PRD) result in the addition of cell files during radial growth. So far, how a cell controls the position and orientation of its division plane remains poorly understood. In this thesis we focused on a complex of two proteins called TMO5 and LHW that are capable of shifting the division plane orientation towards PRD. Our main aim was to identify which genes are activated by the TMO5/LHW complex and characterize these to understand how plant cells control the positioning and orientation of their cell divisions to allow normal growth. After introducing the main concepts and background information in Chapter 2, we continue to show in Chapter 3 how we generated and characterized an inducible line of TMO5 and LHW (dGR). We compared this line to the existing TMO5-LHW misexpression and overexpression lines and concluded we created an excellent tool to study cell division orientation. Because TMO5 and LHW are transcription factors, we next studied the transcriptional changes upon induction. While it is commonly assumed that the plant hormone cytokinin is required to control these divisions, we could prove that indeed cytokinin response is triggered upon induction of TMO5/LHW. In Chapter 4 we used the transcriptional data to select putative targets, which were verified using a misexpression screen. We identified DOF2.1 as a factor capable of inducing PRD and continued to show how DOF2.1 functions downstream of TMO5-LHW and controls division plane orientation during vascular development. Analysis of loss-of-function mutants of DOF2.1 and the two closest family members revealed these Dof-type TF family members act in a redundant manner to control PRD. Because our misexpression screen only yielded one factor controlling PRD, we reanalyzed our transcriptional data in Chapter 5 using an inferred network analysis algorithm. We generated a gene regulatory network which indicated that a novel family of bHLHs acts as key transcriptional hubs downstream of TMO5-LHW. We could confirm that some of these factors are indeed TMO5-LHW regulated and are part of the downstream cytokinin response. Overexpression of these bHLH did not result in any obvious phenotype; however we showed that they interact with members of a different family of bHLH TFs. This suggests that they might require this interaction in order to carry out their function. Loss-of-function of single genes did not reveal a clear phenotype, indicating that there is a high amount of redundancy within the novel bHLH subfamily. Analysis of higher order mutants will be required to identify the function of these bHLH genes. In conclusion, the identification of DOF2.1 and its involvement in controlling PRD brings us one step closer to identify the key players that reorient and shift the division plane. However, the actual role of the novel bHLH subfamily acting downstream of TMO5-LHW is yet to be unraveled.

Plant roots are required to anchor the plant in the soil, acquire water and nutrients and respond to biotic and abiotic stimuli. Root branching contributes largely to the morphological plasticy of the root system. Auxin is an important plant hormone involved in lateral root (LR) development and root gravitropism. In this thesis, an in silico translational approach was employed to identify selected auxin-related genes in barely. We focused our work on auxin transporters that are involved in plant response to environmental stimuli. AUX1 and LAX3 were identified in barley and subsequently characterised. These experiments revealed the maintenance of AUX/LAX developmental function between highly divergent plant species. Reactive oxygen species (ROS) were recently proposed to contribute to auxin-mediated LR formation, however the nature of their involvement remains elusive. We show that H2O2 accumulates in middle lamellae of cells overlying LR primordia and progressively creates a fine layer around the emerging LR primordia. Our results lead us to propose that ROS contribute to LR emergence by influencing cell wall plasticity. We finally show that the Respiratory burst oxidase homologs (Rboh) gene family are likely contributors to a fine-tuned extracellular ROS balance during LR emergence. Abscisic acid (ABA) is another plant hormone known to modulate LR development and to regulate responses to environmental stimuli. We demonstrated that ABA response pathways act downstream of water deficit in the acropetal root zone and that ABA is likely to mediate LR repression. Those results lead to a model of root branching adaptation to local soil porosity. (AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2015

The plasticity of branching patterns of plants plays a crucial role in the capture of resources from unpredictable, heterogeneous and dynamic soil environments. This plasticity relies on the plant ability to regulate the site and time of lateral root initiation. It may involve various steps of their developmental program. In this thesis, we first described the scheme of lateral root formation in barley and compared it with the reference model available for Arabidopsis thaliana. We could reveal several important differences which make the cereal case unique: the contribution of the endodermis, the division of cortex cells and the circumferential position of lateral root initiation sites. We then developed a novel method to analyse early steps of the regulation of lateral root formation. This method is based on the time-lapse analysis of growth and branching events following the application of a transient treatment that blocks the formation of lateral roots. We first showed, in Hordeum vulgare and Zea mays seedlings grown in aeroponics that the distal segment of the seminal root formed during a transient water shortage is devoid of lateral roots. Using a detailed microscopic analysis, we showed that this treatment blocks lateral root formation within a small window of developmental stages. This was confirmed by the time-lapse analysis which pointed to a region of the seminal root that matches the occurrence of these stages. We showed further that abscissic acid produces the same response as the water shortage and that auxin is able to prevent this response. While those data are insufficient to set the basis of a mechanistic model of transduction of the drought signal into an inhibition of lateral root formation, several observation lead us to propose that ABA could act upstream of the establishment of auxin maxima that precede the formation of lateral roots. ABA could be involved in the modulation of auxin transport as suggested by other studies, but further experiments are needed to test this hypothesis. Chez l’orge (H. vulgare), l'application d'un déficit hydrique transitoire induit une inhibition caractéristique du développement des racines latérales qui se traduit par la formation d’une zone dépourvue de ramifications. Ce travail avait pour objectif de mieux comprendre les mécanismes de cette inhibition. Le développement des racines latérales de l'orge et du maïs présente des différences majeures par rapport au modèle de référence établi pour A. thaliana. L’initiation des racines latérales chez les deux céréales a lieu face aux pôles de protophloème et l'endoderme participe activement à la formation du primordium et est à la base de la coiffe, de l'épiderme, du cortex et du péricycle de la racine latérale. Nous avons également montré que les cellules du cortex de la racine séminale situées face à un primordium de racine latérale subissent des divisions transversales qui préparent vraisemblablement la réorganisation du cortex nécessaire au passage de la racine latérale. L’analyse microscopique détaillée de la zone non ramifiée nous a permis de montrer que le déficit hyfrique bloque les étapes précoces de l'initiation. Cette observation a été confirmée par le positionnement de la zone inhibée le long de l’axe séminal, grâce à une méthode inédite basée sur l'imagerie time-lapse et la modélisation inverse. Nous avons finalement montré que l'acide abscissique provoque le même type de réponse et que l'auxine peut en atténuer les effets. Sur cette base, nous avons émis l'hypothèse que l'acide abscissique est un premier messager de cette réponse et qu'il induit une redistribution des flux d'auxines dont une des conséquences est la réduction de la ramification racinaire. Nos résultats suggèrent également que l'acide abscissique et l'auxine agissent d'une manière dose-dépendante, ce qui sous-tend la possibilité de manipuler de manière graduelle la densité de ramification. (AGRO 3) -- UCL, 2011

Sugars are the fuel of life to plants and other organisms. They are the result of the fixed energy from the sun that feeds the plant cells to sustain growth and development. Sucrose is the main sugar transported in the plant and, as such reaches the cells where it is cleaved in glucose and fructose. Many vital processes are governed by sugars, but complex networks had to evolve allowing the plant to trigger the right program depending on its energy status. Surprisingly, one sugar phosphate is emerging to connect carbon status with the growth program. That sugar is trehalose-6-phosphate, the intermediate on the trehalose metabolism pathway. This molecule is a signal of sucrose status in the cell and it is also an inhibitor of the main regulator of starvation responses in plants. The mechanism mediating trehalose-6-phosphate balancing of energy levels is waiting to be unraveled. The aim of this work was to scout into the possible role of the trehalose biosynthesis pathway in root development and stress tolerance. The focus was on the 10 active phosphatase members that convert the intermediate trehalose-6-phosphate into trehalose. By a forward genetics approach, we could identify two potential candidates important for lateral root development. Interestingly, they appear to function in an antagonistic manner in this organogenesis process and are regulated by auxin. Additionally, two other enzymes in the trehalose pathway were found to regulate abscisic acid responses. With these results, we open a big window of research searching on the molecular and hormonal components interacting with the trehalose pathway.

To feed an ever-increasing world population while the availability of fertile soils is shrinking, there is a rising need for an increase in plant biomass production. One of the strategies to enhance plant yield is the optimization of the root system. A deep and highly branched root system for instance is more efficient in scavenging the minerals scattered throughout the soil, and its increased foraging capacity can help to overcome periods of water scarcity. With Arabidopsis thaliana as a model, scientists have made important progress in disentangling the mechanisms controlling root branching. However, when it comes to economically important crops such as maize, our understanding of processes as lateral root formation is still fragmentary. The aim of this project was to characterize the early events occurring during lateral root initiation in maize at a histological and genetic level. Further, we wanted to identify genes which could enhance maize root branching, and thereby would increase the resistance of plants to water deprivation. Therefore a transcriptomic approach was used in combination with the synchronized induction of lateral roots. We selected genes with a potentially conserved role in lateral root initiation in maize and Arabidopsis. The effect of the transgene expression on root growth was characterised in Arabidopsis seedlings, while analysis of transgenic rice plants was performed to study the consequences of the transgene on biomass and seed yield under water deprivation.