13 research outputs found

    The integration of developmental signals during root procambial patterning in Arabidopsis thaliana

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    The vascular system of plants functions as a transportation route for water, nutrients and signaling molecules while also forming a support structure and generating most of the radial growth by increasing the number of cell files through periclinal cell divisions. These features have transformed life on Earth by enabling plants to colonize land and grow larger. In mature plants, the conductive tissues xylem and phloem are produced from stem cells in the vascular cambium, which develops from the procambium formed during early development. The vascular cylinder of the Arabidopsis root comprises a central xylem axis with a peripheral phloem pole on either side and procambial cells located between the xylem and phloem. Formation of the vascular pattern requires high auxin and cytokinin signaling domains in the xylem and phloem/procambium positions, respectively. However, the gene regulatory network acting downstream of these hormonal cues has remained unknown. I investigated procambium patterning in the Arabidopsis root. Our research group discovered that radial growth is activated in the peripheral phloem domain by six mobile DOF transcription factors that we named PHLOEM EARLY DOF (PEAR) proteins, consisting of PEAR1, PEAR2, and their four homologues. PEAR proteins form an inverse concentration gradient to the HD-ZIP III transcription factors, which inhibit periclinal cell divisions in the central domain partially by inhibiting the movement of PEAR proteins. HD-ZIP III expression is promoted by auxin in the xylem axis and inhibited by endodermis-derived mobile microRNA165/166 in the periphery. The PEAR and HD-ZIP III genes form a feedback loop in which the PEAR proteins promote HD-ZIP III transcription while the HD-ZIP IIIs inhibit PEAR transcription and protein movement. The PEAR-HD-ZIP III regulatory module decodes hormonal and microRNA signals to result in the formation of a highly active peripheral zone and a more quiescent central zone during procambium development. We also determined that a member of the DOF family, DOF2.1, acts downstream of TARGET OF MONOPTEROS 5/LONESOME HIGHWAY-dependent cytokinin biosynthesis to regulate periclinal cell divisions in the outer procambial cells in contact with the xylem axis. Together, PEAR and DOF2.1 proteins control all of the periclinal divisions in the procambium through their activity in partially distinct domains. We also identified SUPPRESSOR OF MAX2 1-LIKE 3 (SMXL3), a member of SMXL subclade 2 which is expressed in the early phloem and procambium cells, as a putative direct target of PEAR2 that is sufficient to promote periclinal divisions. Characterization of SMXL subclade 2 identified SMXL3, 4 and 5 as essential regulators of phloem formation that act very early in development and thus are required for all aspects of phloem development. Phloem specification requires periclinal divisions in the procambium. SMXL3, 4 and 5 act in both the periclinal divisions and phloem specification in a partially redundant manner. Furthermore, analysis of regulators downstream of the PEARs revealed that they not only promote cell proliferation but also specify the identity of the surrounding cells non-cell autonomously, including procambial and phloem pole pericycle identity. Our work highlights the importance of cell-to-cell communication in plant development. The interaction of mobile hormones, transcription factors and microRNAs originating from different tissues is required to coordinate developmental processes in the vascular cylinder. We have assembled the most complete understanding to date of the regulatory network coordinating procambial development and have identified the protophloem sieve elements as the organizers of radial growth during the early stages of vascular development in the Arabidopsis root. These findings can potentially be used to increase yields in forestry and agriculture.Kasvien johtosolukko kuljettaa vettä, ravinteita ja viestimolekyylejä, sekä toimii tukirakenteena, joka tuottaa suurimman osan kasvin paksuuskasvusta solun pitkittäisen akselin suuntaistesti tapahtuvien ns. periklinaalisten solunjakautumisten avulla. Näiden ominaisuuksien ansiosta kasvit pystyivät siirtymään maalle ja kasvamaan kooltaan suuremmiksi, minkä seurauksena elämä maanpäällä muuttui täysin. Täysikasvuisilla kasveilla johtosolukon puu- ja nilasolut kehittyvät kantasoluja sisältävästä jällestä, jota edeltää varhaisen kasvuvaiheen esijälsi. Arabidopsis thalianan eli lituruohon keskuslieriössä puu- eli ksyleemisolut muodostavat akselin keskelle, nilasolut ovat molempien puolien reunoilla ja esijällen solut sijaitsevat näiden välissä. Korkea auksiinipitoisuus ksyleemissä ja korkea sytokiniinipitoisuus nilassa ja esijällessä tarvitaan johtosolukon normaaliin kehitykseen. Näiden hormonaalisten viestien ohjaama geeninsäätelyverkosto on ollut kuitenkin tuntematon. Tutkin esijällen muodostumista lituruohon juuressa. Tutkimusryhmämme havaitsi paksuuskasvun aktivoituvan keskuslieriön reunoilla olevissa varhaisen vaiheen nilan siiviläsoluissa ja tietyissä niitä ympäröivissä soluissa. Määritimme että näiden solujen jakautumisesta on vastuussa kuuden liikkuvan DOF-transkriptiotekijän perhe, jolle me annoimme nimeksi PHLOEM EARLY DOF (PEAR). Nämä koostuvat PEAR1 ja PEAR2 proteiineista ja niiden neljästä homologista. PEAR proteiinit muodostavat vastakkaisen pitoisuusgradientin ennestään tunnettujen HZ-ZIP III transkriptiotekijöiden kanssa. HD-ZIP III proteiinit estävät periklinaalisia solunjakautumisia keskuslieriön keskiosassa osittain estämällä PEAR proteiinien liikkumista. Auksiini edistää HD-ZIP III:n ilmenemistä keskuslieriön keskellä sijaitsevissa ksyleemisoluissa, kun taas keskuslieriön ulkopuolelta liikkuva mikroRNA165/166 heikentää HD-ZIP III:n ilmenemistä sen reunoilla. PEAR ja HD-ZIP III muodostavat takaisinkytkentämekanismin: PEAR lisää HD-ZIP III:n ilmenemistä, kun taas HD-ZIP III estää PEAR geenien ilmenemistä ja PEAR proteiinin liikkumista. PEAR-HDZIP III säätelymoduuli tulkitsee hormonaalisia ja mikroRNA signaaleja minkä seurauksena esijälteen muodostuvat aktiivisesti jakautuva reuna-alue ja hiljainen keskialue. Osoitimme myös, että TARGET OF MONOPTEROS 5/LONESOME HIGHWAY transkriptiotekijäparin aktivoima sytokiniinin biosynteesi aktivoi DOF perheeseen kuuluvan DOF2.1 geenin ilmenemistä. DOF2.1 säätelee periklinaalisia solujakautumisia ksyleemin viereisissä reuna- alueen esijälsisoluissa. Osittain eri alueilla toimivat PEAR ja DOF2.1 proteiinit säätelevät kaikkia esijällen periklinaalisia solunjakautumisia. Tutkimuksessamme havaitsimme, että PEAR2 aktivoi SUPPRESSOR OF MAX2 1-LIKE (SMXL) proteiinien toiseen alaluokkaan kuuluvan SMXL3 geenin ilmenemistä. SMXL3 toimii varhaisen vaiheen nilan ja esijällen soluissa ja on riittävä aktivoimaan periklinaalisia solunjakautumisia. SMXL proteiinien toisen alaluokan jäsenet SMXL3, 4 ja 5 säätelevät lisäksi nilan kehitystä. Ne toimivat jo kehityksen varhaisessa vaiheessa ja täten ovat tarpeellisia kaikkiin nilan kehitysvaiheisiin. Esijällen periklinaaliset solunjakautumiset ovat tärkeä osa nilan kehitystä. Tutkimuksemme osoittaa, että SMXL3, 4 ja 5 toimivat sekä solunjakautumisten että erilaistumisen säätelyssä osittain päällekkäisesti. PEAR proteiinien säätelykohteiden analyysi myös paljasti, että PEAR proteiinit toimivat sekä solunjakautumisten säätelyssä että ympäröivien solujen, kuten esijällen ja nilan viereisten perisyklisolujen, identiteetin määrittämisessä liikkumalla solusta toiseen. Tuloksemme korostavat solujenvälisen viestinnän tärkeyttä: eri solukoista peräisin olevien liikkuvien kasvihormonien, säätelytekijöiden ja mikroRNA-molekyylien vuorovaikutus tarvitaan ohjaamaan kehitystä. Työmme luo tähän mennessä kattavimman ymmärtämyksen säätelyverkostoista, jotka ohjaavat esijällen kehitystä ja osoittaa varhaisen vaiheen nilan siiviläsolujen järjestävän paksuuskasvun aktivoinnin lituruohon juuren johtosolukon varhaisessa kehityksessä. Näitä löydöksiä voidaan mahdollisesti hyödyntää maa- ja metsätaloudessa lisäämään tuotantoa

    Auxin Influx Carriers Control Vascular Patterning and Xylem Differentiation in Arabidopsis thaliana

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    Auxin is an essential hormone for plant growth and development. Auxin influx carriers AUX1/LAX transport auxin into the cell, while auxin efflux carriers PIN pump it out of the cell. It is well established that efflux carriers play an important role in the shoot vascular patterning, yet the contribution of influx carriers to the shoot vasculature remains unknown. Here, we combined theoretical and experimental approaches to decipher the role of auxin influx carriers in the patterning and differentiation of vascular tissues in the Arabidopsis inflorescence stem. Our theoretical analysis predicts that influx carriers facilitate periodic patterning and modulate the periodicity of auxin maxima. In agreement, we observed fewer and more spaced vascular bundles in quadruple mutants plants of the auxin influx carriers aux1lax1lax2lax3. Furthermore, we show AUX1/LAX carriers promote xylem differentiation in both the shoot and the root tissues. Influx carriers increase cytoplasmic auxin signaling, and thereby differentiation. In addition to this cytoplasmic role of auxin, our computational simulations propose a role for extracellular auxin as an inhibitor of xylem differentiation. Altogether, our study shows that auxin influx carriers AUX1/LAX regulate vascular patterning and differentiation in plants.Peer reviewe

    Plant Development and Organogenesis: From Basic Principles to Applied Research

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    The way plants grow and develop organs significantly impacts the overall performance and yield of crop plants. The basic knowledge now available in plant development has the potential to help breeders in generating plants with defined architectural features to improve productivity. Plant translational research effort has steadily increased over the last decade due to the huge increase in the availability of crop genomic resources and Arabidopsis-based sequence annotation systems. However, a consistent gap between fundamental and applied science has yet to be filled. One critical point often brought up is the unreadiness of developmental biologists on one side to foresee agricultural applications for their discoveries, and of the breeders to exploit gene function studies to apply to candidate gene approaches when advantageous on the other. In this book, both developmental biologists and breeders make a special effort to reconcile research on the basic principles of plant development and organogenesis with its applications to crop production and genetic improvement. Fundamental and applied science contributions intertwine and chase each other, giving the reader different but complementary perspectives from only apparently distant corners of the same world

    Branching out in roots: uncovering form, function, and regulation

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    Root branching is critical for plants to secure anchorage and ensure the supply of water, minerals, and nutrients. To date, research on root branching has focused on lateral root development in young seedlings. However, many other programs of postembryonic root organogenesis exist in angiosperms. In cereal crops, the majority of the mature root system is composed of several classes of adventitious roots that include crown roots and brace roots. In this Update, we initially describe the diversity of postembryonic root forms. Next, we review recent advances in our understanding of the genes, signals, and mechanisms regulating lateral root and adventitious root branching in the plant models Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and rice (Oryza sativa). While many common signals, regulatory components, and mechanisms have been identified that control the initiation, morphogenesis, and emergence of new lateral and adventitious root organs, much more remains to be done. We conclude by discussing the challenges and opportunities facing root branching research

    Gene Regulatory Networks in Plant Stem Cells - Investigating cell fate decision-making in the vascular cambium of Arabidopsis

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    Stem cells have fascinated humans for a long time because of their fundamental importance in building and sustaining many forms of life. Stem cells are able to divide and maintain themselves while producing new cell types. Like animal stem cells, plant stem cells are a fascinating research object. They provide the cells for plant growth and allow many plants to continue growing throughout their lives, thereby producing enormous amounts of plant biomass on this planet. In this study, I specifically focussed on the plant stem cells that produce wood and bast. These stem cells form a cylindrical stem cell niche, the vascular cambium, which is found in the root, the stem and the tissue that connects the root and stem, the hypocotyl. In my analyses, I focused on the hypocotyl of the model plant Arabidopsis thaliana, since wood and bast production is very active there and the tissue patterning resembles the patterning of the vascular cambium of trees. A vascular cambium stem cell produces both, wood progenitor cells inward and bast progenitor cells outward. Thus, it needs to “choose” between the following cell fates: becoming a wood progenitor cell, becoming a bast progenitor cell, or remaining a stem cell. The basis on which this first cell fate decision is made has not yet been clarified. To better understand this decision-making process, in a first step I mathematically modelled gene regulatory networks in collaboration with the Mironova laboratory. This has shown that a network of just three genes that regulate each other in a specific way is sufficient to bring about the three cell fate possibilities mentioned above. I then hypothesized that this network could consist of two mutually inhibitory auxin and cytokinin signalling pathway components. Furthermore, these components could be additionally regulated by WUSCHEL-RELATED HOMEOBOX4 (WOX4), an important transcription factor for stem cell regulation in the vascular cambium. Integrated into a 1D model, this network generated bidirectional growth. Using in planta analysis, I was able to show that both auxin and cytokinin signalling levels are low specifically in the vascular cambium. To test my hypothesis, I investigated which genes of the auxin and cytokinin pathways are regulated by WOX4, also in comparison to WUSCHEL, a stem cell regulator of the shoot apical meristem. In the course of my investigations, I identified specific type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs), which negatively regulate cytokinin signalling, as putative WOX4 targets. I found that the expression of the type-A ARR genes ARR5, ARR6, ARR7 and ARR15 is reduced in the hypocotyl vascular cambium. Furthermore, I observed that the expression of ARR6 and ARR15 is downregulated by WOX4. WUSCHEL also exerts this type of regulation on type-A ARRs in the shoot apical meristem and therefore this could represent a general concept for the regulation of plant stem cells. In addition, findings in this study propose that WOX4 itself is regulated by cytokinin via the DNA BINDING WITH ONE FINGER 2.1 (DOF2.1) protein. Importantly, when ARR7 and ARR15 are mutated, the cell fate decisions of vascular cambium stem cells are altered and more wood cells are produced than in wild type plants. In conclusion, this study suggests that a mechanism of cell fate decision making in vascular cambium stem cells is based on the regulation of cytokinin signalling by WOX4 and by type-A ARRs

    Quantitative description & computational modelling of the BRI1 response module controlling root cell elongation growth – from organ-scale to nanodomains

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    Die Familie der Brassinosteroid-Pflanzenhormone (BR) ist an der schnellen Kontrolle des Zellstreckungswachstums beteiligt. Die Aktivierung des plasmamembran-ständigen Brassinosteroidrezeptors BRASSINOSTEROID INSENSITIVE 1 (BRI1) und seines Cofaktors BRI1-ASSOCIATED KINASE (BAK1) führt zur Dissoziation des Inhibitors BAK1-INTERACTING RECEPTOR-LIKE KINASE 3 (BIR3) und schließlich zur Aktivierung von P-Typ ATPasen (AHAs). Die aktivierung der Protonenpumpen (AHAs) führt zu einer Ansäuerung des extrazellulären Raums, einer Hyperpolarisierung der Plasmamembran und einer Lockerung der Zellwand, was schließlich zu Zellstreckungswachstum führt. Dieses Signaltransduktionsmodul ist auf Komponentenebene gut beschrieben. Es ist jedoch noch nicht bekannt, wie diese Akteure räumlich und zeitlich zusammenwirken, um das unterschiedliche Zellstreckungswachstum als Reaktion auf BR in verschiedenen Pflanzengeweben zu vermitteln. Hier zeigen wir durch quantitative in vivo-Ansätze, dass sich die Proteinmenge von AHA2 in der schnell wachsenden Elongationszone von der in der weniger / nicht wachsenden meristematischen und Wurzelhaarzone in der Wurzel von Arabidopsis thaliana unterscheidet. Während das Proteinverhältnis von BIR3 und BRI1 entlang der Wurzelachse gleich bleibt, ist das Verhältnis von AHAs und BRI1 in der meristematischen Zone signifikant niedriger. Unter Einbeziehung der Proteinmengen in ein mathematisches Modell untersuchen wir die Regulation und Dynamik des schnellen BR-Antwortmoduls in silico, einschließlich der Membranhyperpolarisation und des Zellstreckungswachstums. Sowohl das Modell als auch die Experimente unterstreichen die Bedeutung der Protonenpumpen in Kombination mit dem Hormonrezeptor. Unter Berücksichtigung der unterschiedlichen Proteinspiegel kann das Modell das unterschiedliche Wachstumsverhalten entlang der Wurzelachse beschreiben, einschließlich nicht-invasiver Protonenflussmessungen und apoplastischer pH-Messungen. Wir nehmen daher an, dass das Verhältnis von AHAs zu BRI1 der entscheidende Parameter ist, der die differentielle Fähigkeit von Zellen steuert, sich als Reaktion auf BR entlang der Wurzelachse zu verlängern. Es ist auch noch nicht geklärt, ob die Aufteilung der Komplexe in der Plasmamembran (Mikrodomänen) für die physiologische Leistung, nämlich das Zellstreckungswachstums, relevant ist. Unter Verwendung der photoaktivierten Lokalisationsmikroskopie mit Einzelpartikelverfolgung (sptPALM) fanden wir, dass die Mehrheit der BRI1-Rezeptoren in Tabakblättern (Nicotiana benthamiana) ein subdiffusives Verhalten zeigt. Dieses heterologe System ist jedoch vermutlich nicht gut für den Nachweis geringfügiger Änderungen der Rezeptordynamik geeignet, da weder die BR-Behandlung noch das Aufbrechen von Mikrodomänen mit Methyl-Beta-Cyclodextran (MbCD) und die Depolymerisation von Aktinfilamenten die BRI1-Rezeptordynamik signifikant veränderten. Im Gegensatz dazu, schwächt die Störung der Mikrodomänen durch MbCD in Arabidopsis thaliana die Brassinosteroid-Signalübertragung in Bezug auf primäres Wurzelwachstum und Wurzelwellen ab, was darauf hinweist, dass die Integrität der PM-Mikrodomänen für die BRI1- Funktion entscheidend ist. Es ist derzeit nicht klar, ob BRI1, BAK1 und das die Zellwandintegrität erfassende RECEPTOR LIKE PROTEIN 44 (RLP44) gleichzeitig in derselben Mikrodomäne lokalisiert sind und einen Trimerkomplex bilden. Hier liefern wir durch quantitative in vivo FRETFLIM- Messungen mit drei Fluorophoren den Nachweis, dass RLP44, BRI1 und BAK1 in der Plasmamembran von Nicotiana benthamiana-Blattzellen in Abwesenheit von exogenem BR einen Trimerkomplex bilden, wobei der geschätzte Abstand zwischen ihnen unter 15 nm liegt. Der Immunrezeptor FLAGELLIN SENSING 2 (FLS2), der strukturell BRI1 ähnlich ist, ist nicht in einen ähnlichen Komplex mit RLP44 und BAK1 integriert. Unsere Studie belegt, dass BRI1 und FLS2 in der Plasmamembran in unterschiedlichen Nanodomänen lokalisiert sind. Darüber hinaus scheint RLP44 spezifisch für BRI1-haltige Mikrodomänen zu sein, da FRET mit FLS2 nie beobachtet wurde. Da die Fluoreszenzlebensdauer des Donors überwacht wird, umgeht unsere Methode die umfangreichen Berechnungen, die für intensitätsbasierte FRET-Interaktionstests erforderlich sind, und bietet somit eine praktikable Grundlage für die Untersuchung der Subkompartimentierung in der Plasmamembran lebender Pflanzenzellen mit einer Auflösung im Nanomaßstab

    PXY collaboration with other cambial regulators, ER, and MP, is essential for secondary growth

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    Plants grow both in height, and in width. The process of radial expansion, known as secondary growth, generates the majority of the plant biomass through expansion of the vasculature, the plant’s water and nutrient conducting tissues. It is therefore imperative to understand how vascular growth is regulated. Secondary growth is facilitated by a collection of stem cells present in a meristem called the vascular cambium. The cambium gives rise to the water-conducting xylem and nutrient conducting phloem, on opposing sides via periclinal cell divisions. A receptor-like kinase PXY has been found to promote cell division in the cambium, and to control its ability to maintain distinct domains for xylem and phloem. Loss of PXY results in interspersal of these cell types. PXY interacts with other components in regulating secondary growth. It was seen to genetically interact with another receptor-kinase and its family of genes, ER. However, comprehensive exploration of how these two genes and their families interact had not been determined. Similarly, PXY was shown to indirectly suppress the transcription factor MP in stem, but to be promoted by MP in root. Both components were also found to be localized in the same domain on the xylem side of the cambium, where the hormone auxin was shown to accumulate. Disruption of the auxin pattern or removal of PXY or MP results in defects in cambial function, but the basis of these interactions is not fully understood. To address the questions surrounding PXY’s role in secondary growth, a bespoke method for measuring cell sizes and shapes from cross-sections of plants was developed. This method was employed to analyse PXY and ER families single and combinatorial mutants. Finally, a theoretical three-cell mathematical model was proposed examining PXY’s relationship with the transcription factor MP in controlling the accumulation of auxin in the cambium. The results of these studies demonstrated that loss of PXY and ER families results in different consequences in stem and hypocotyl. In hypocotyl and in the absence of the PXY family, ER and its genetic paralogues promote hypocotyl radial growth in part, compensating for loss of PXY by promoting cell size increases, but this was not observed in stem. Moreover, loss of all of the PXY and ER genes results in complete suspension of secondary growth, suggesting that these two genetic pathways are required for the transition between primary and secondary growth. In the investigation of PXY’s relationship with the transcription factor MP in root, it was shown both numerically and analytically that a negative feedback loop between the two provides stability to the system, thus generating a more stable auxin gradient in the cambium. Thus, PXY interacts with both ER and MP to maintain vascular organisation and growth, and these interactions are essential for the induction of secondary expansion, as well as hormone patterning in order to promote cambial activity

    Developmental instability in lateral roots of maize: a multi-scale analysis

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    In the perspective of a second Green Revolution, aiming, unlike the first one, to enhance yields of crops in a low fertility context, the strategies used by plants for an optimal uptake of soil nutrients are at the core of the problem. To solve it and identify ideal breeds among the genetic diversity of crops, plant root systems, their development and their architecture, are called upon to play the leading role. The variability among secondary roots appears as a crucial feature for the optimality of soil exploration and acquisition of mobile and immobile resources, but this phenomenon remains poorly understood. The work presented in this thesis focuses on the lateral roots of maize (Zea mays L.) and attempts to unravel the processes at the origin of intrinsic variations in lateral root development. It relies notably on the phenotyping of individual lateral roots at an unprecedented scale, tracking the daily growth of thousands of them at a high spatial resolution, in order to characterize precisely the spatio-temporal variations existing both between and within root individuals. Individual growth rate profiles were analyzed with a statistical model that identified three main temporal trends in growth rates leading to the definition of three lateral root classes with contrasted growth rates and growth duration. Differences in lateral root diameter at root emergence (originating at the primordium stage) were likely to condition the followed growth trend but did not seem enough to entirely determine lateral root fate. Lastly, these lateral root classes were randomly distributed along the primary root, suggesting that there is no local inhibition or stimulation between neighbouring lateral roots. In order to explain the origin of the observed differences in growth behaviour, we complemented our study with a multi-scale characterization of groups of lateral roots with contrasted growth at a cellular, anatomical and molecular level. A particular focus is set on the analysis of cell length profiles in lateral root apices for which we introduced a segmentation model to identify developmental zones. Using this method, we evidenced strong modulations in the length of the division and elongation zones that could be closely related to variations in lateral root growth. The regulatory role of auxin on the balance between cellular proliferation and elongation processes is demonstrated through the analysis of mutant lines. Ultimately, variations in lateral root growth are traced back to the allocation of carbon assimilates and the transport capacity of the root, suggesting that a feedback control loop mechanism could play a determinant role in the setting out of contrasted lateral root growth trends. (Résumé d'auteur

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