The work is focused on the lateral roots, which are an indispensable part of the root system and play a key role in communication with rhizosphere and the efficient exploitation of natural resources. Tetratricopeptide-repeat Thioredoxin-like 3 gene (TTL3) was identified by forward screening based on its gene expression pattern in the search for genes associated with the development of lateral roots in Arabidopsis thaliana. TTL3 also known as VIT (VH1 interacting TPR containing protein), belongs to a family of four paralogues in Arabidopsis showing a very similar arrangement of domains. It seems that this type of proteins is widespread in plants and provides protein-protein signaling as an important component of phytohormone response pathways. The presented diploma thesis is focused on characterization and comparison of expression patterns of the four members of TTL family, through creation of pTTL::uidA (GUS) transcriptional fusions. The promoter area was estimated to be contained in about 2000bp upwards the TTLs coding sequences and transcriptional fusions with uidA gene were individually constructed and transformed into Arabidopsis via Agrobacterium tumefaciens. The expression patterns of analyzed genes were microscopically confirmed and allowed comparison among members of TTL gene family with regard...

The length of the primary root and the density of lateral roots determine the architecture of the root. In this thesis the effect of NAA, ABA and the NO donor SNP alone as well as the combination of ABA or NAA with SNP on lateral root development was investigated. The interaction between CPTIO, a NO scavenger, and NAA or SNP is also reported. Following preliminary experiments in which it was observed that the aerial part of the seedling influenced LR growth and that there was a possible inhibitory effect of light on cultured root tips, experiments were conducted with excised roots tips in the dark. NAA was shown to have the potential to initiate LRs across a wide concentration gradient with the total number of LRs and initiated lateral root primordia (LRP) remaining constant across the range of concentrations tested. Over the last decade, nitric oxide (NO), a bioactive molecule, has been reported to be involved in the regulation of many biological pathways. The presence of NO in the system provided via sodium nitroprusside (SNP), promoted LRP initiation based on the NAA concentration gradient; but without changing the total LR initiation, that is LRs plus primordia density remained constant along the concentration gradient of NAA. The absence of LR and LRP in the treatments of CPTIO (a NO scavenger) with SNP or NAA suggests that NO regulates LRP initiation triggered by NAA, which is in agreement with the recent paper published after the commencement of this study (Correa-Aragunde et al., 2006). In agreement with previous studies, ABA inhibited lateral root development by reducing LR density and the number of LRs. The experiments with fluridone, an ABA biosynthesis inhibitor, may indicate that endogenous ABA was at sufficient concentrations in the excised root tips to inhibit primordia initiation. In this study, evidence is presented for the first time to show that SNP can relieve the inhibitory effect of ABA on LR density and number of LRs suggesting the NO, released from SNP, acts downstream of ABA. Overall these data confirm a critical role for NO in LR initiation.

The development of plants challenged by environmental stress alters plant architecture through several pathways, including those involving plant hormone responses and reactive oxygen species (ROS) production. Auxin, a phytohormone associated with every aspect of development, and abscisic acid (ABA), a phytohormone involved in abiotic stress responses, both interact with ROS. These ROS are used as secondary messengers to activate transcription of abiotic stress genes, and also in developmental responses such as cell elongation. To understand the mechanisms involved in the abiotic stress response and how the response intersects with auxin, ABA, and ROS, I examined COPPER INDUCED IN LEAVES 1 (<i>CIL1</i>) from <i>Brassica carinata</i> and its Arabidopsis orthologue, AUXIN INDUCED IN ROOTS 12 (AIR12). Expression of both genes increases in response to auxin and recent work has placed both <i>CIL1</i> and AIR12 within a family of plant-specific cytochrome b561 proteins thought to be involved with transmission of ROS signals. This suggests a link between auxin and ROS production resulting from abiotic stress. Antisense <i>CIL1 B. carinata</i> plants produced fewer lateral roots and were resistant to salinity stress during vegetative growth. Mutant air12 plants showed a 50% reduction in lateral root number, lateral root length, and H2O2 root distribution. Growth in the presence of H2O2 was able to restore lateral root length to control levels. In silica analysis of the <i>CIL1</i> and AIR12 amino acid sequences detected an attachment site for glucosylphosphatidylinositol, predicting that the protein is targeted to the extracellular leaflet of the plasma membrane where it could be cleaved and released into the apoplast. Subcellular localization using p35S::GFP-CIL1 and p35S::GFP-AIR12 translational fusions confirmed that CIL1 and AIR12 localize to the plasma membrane and are released into the apoplast. Organ localization of AIR12 using the pAIR12::GFP-AIR12 construct in stably transformed Arabidopsis showed fusion protein accumulation in the apex of the primary root and in the vascular tissue. Fusion protein also localized to cells flanking emerging lateral roots. Investigation of pAIR12::GUS Arabidopsis showed GUS accumulation in the apex of elongating lateral roots. I demonstrate that AIR12 is an extracellular protein and that air12 seedlings are susceptible to salt stress, but not osmostic stress and display increased and decreased sensitivity to ABA during germination and primary root elongation, respectively, suggesting that AIR12 acts downstream of abiotic stress recognition.

The development of plants challenged by environmental stress alters plant architecture through several pathways, including those involving plant hormone responses and reactive oxygen species (ROS) production. Auxin, a phytohormone associated with every aspect of development, and abscisic acid (ABA), a phytohormone involved in abiotic stress responses, both interact with ROS. These ROS are used as secondary messengers to activate transcription of abiotic stress genes, and also in developmental responses such as cell elongation. To understand the mechanisms involved in the abiotic stress response and how the response intersects with auxin, ABA, and ROS, I examined COPPER INDUCED IN LEAVES 1 (<i>CIL1</i>) from <i>Brassica carinata</i> and its Arabidopsis orthologue, AUXIN INDUCED IN ROOTS 12 (AIR12). Expression of both genes increases in response to auxin and recent work has placed both <i>CIL1</i> and AIR12 within a family of plant-specific cytochrome b561 proteins thought to be involved with transmission of ROS signals. This suggests a link between auxin and ROS production resulting from abiotic stress. Antisense <i>CIL1 B. carinata</i> plants produced fewer lateral roots and were resistant to salinity stress during vegetative growth. Mutant air12 plants showed a 50% reduction in lateral root number, lateral root length, and H2O2 root distribution. Growth in the presence of H2O2 was able to restore lateral root length to control levels. In silica analysis of the <i>CIL1</i> and AIR12 amino acid sequences detected an attachment site for glucosylphosphatidylinositol, predicting that the protein is targeted to the extracellular leaflet of the plasma membrane where it could be cleaved and released into the apoplast. Subcellular localization using p35S::GFP-CIL1 and p35S::GFP-AIR12 translational fusions confirmed that CIL1 and AIR12 localize to the plasma membrane and are released into the apoplast. Organ localization of AIR12 using the pAIR12::GFP-AIR12 construct in stably transformed Arabidopsis showed fusion protein accumulation in the apex of the primary root and in the vascular tissue. Fusion protein also localized to cells flanking emerging lateral roots. Investigation of pAIR12::GUS Arabidopsis showed GUS accumulation in the apex of elongating lateral roots. I demonstrate that AIR12 is an extracellular protein and that air12 seedlings are susceptible to salt stress, but not osmostic stress and display increased and decreased sensitivity to ABA during germination and primary root elongation, respectively, suggesting that AIR12 acts downstream of abiotic stress recognition.

Xylem is an inherent feature of all vascular plants and functions in water transport and mechanical support. In order to efficiently transport water, xylem cells are reinforced by secondary walls before they undergo programmed cell death and their cell contents are removed by autolysis to create a hollow tube. During their differentiation, xylem cells express various hydrolytic enzymes, such as proteases, nucleases and lipases, but only in a few examples has their role in xylem cell death been characterized. This thesis focuses on the regulatory aspects of xylem cell death and the autolytic cell clearance in vessel elements and fibers of hybrid aspen (Populus tremula L. x tremuloides Michx.) and in vessel elements of Arabidopsis thaliana. Using comparative transcriptomic analysis, candidate genes for fiber-specific cell death processes were identified. Further, a hypothesis is presented on the regulation of thermospermine levels in the vasculature by a negative feedback-loop involving auxin and the class III Homeodomain-Leucine Zipper (HD-ZIP III) transcription factor HOMEOBOX8 (PtHB8). The role of the Arabidopsis METACASPASE9 (AtMC9) in xylem cell death was characterized using molecular tools, such as reporter lines and fluorescent fusion proteins, and electron microscopy (TEM). This showed that cell death initiation is not controlled by AtMC9. Instead, evidence is presented for the involvement of AtMC9 in the post mortem autolysis of vessel elements that follows tonoplast rupture and leads to the formation of the hollow conduit. Cell death-associated genes were further observed to be expressed during the emergence of lateral roots in Arabidopsis thaliana. This led to the discovery that cells overlying a lateral root primordium undergo cell death, which was demonstrated by detection of DNA degradation and TEM analysis. It is concluded that cell death facilitates emergence of lateral roots through the overlying tissues in a concerted manner with cell wall remodelling. Together, these findings show that although individual hydrolytic enzymes may be dispensable for plant growth and development, their common regulators are the tool for understanding their function and importance. / Xylem är en karakteristisk vävnad i alla kärlväxter som leder vatten och mineraler samt har mekanisk stödfunktion. För att effektivt kunna transportera vatten förstärks xylemceller med sekundära cellväggar innan de dör genom programmerad celldöd. Deras cellinnehåll bryts ner genom autolys för att skapa ett ihåligt rör. Xylemceller uttrycker under sin differentiering olika hydrolytiska enzymer, såsom proteaser, lipaser och nukleaser, men bara för ett fåtal av dessa har funktionen under xylemcelldöd kartlagts. Denna avhandling fokuserar på reglering av xylemcelldöden och den autolytiska nedbrytningen av cellen, i såväl kärlelement och fibrer av hybridasp (Populus tremula L. x tremuloides Michx.) som i kärlelement av backtrav (Arabidopsis thaliana). Med hjälp av jämförande transkriptomanalys identifierades kandidatgener för fiber-specifika celldödsprocesser i hybridasp. Vidare utvecklades en hypotes om reglering av termosperminnivåer i vaskulaturen genom en negativ feedback-loop, som omfattar auxin reglering och klass III homeodomän-leucinzipper (HD-ZIP III) transkriptionsfaktorn HOMEOBOX8 (PtHB8). Funktionen av Arabidopsis METACASPASE9 (AtMC9) under xylemcelldöd karakteriserades med molekylära verktyg, såsom reporterlinjer och fluorescerande fusionsproteiner och elektronmikroskopi (TEM). Dessa analyser visade att celldödens initiering inte styrs av AtMC9. Istället presenteras bevis för en roll av AtMC9 i autolysen av kärlelement som sker post mortem efter att vakuolen har gått sönder och som slutför bildandet av det tomma kärlet. Genuttryck som associeras med celldöd observerades också under utvecklingen av laterala rötter i Arabidopsis thaliana. Detta ledde till upptäckten att celler som ligger ovanför ett lateralrotprimordium dör en programmerad celldöd och visar tecken på DNA-nedbrytning och autolys i TEM-analyser. Slutsatsen av denna studie är att celldöd i samspel med cellväggsmodifiering underlättar utväxten av laterala rötter genom de överliggande cellagren. Sammantaget tyder dessa upptäckter på att även om enstaka hydrolyserande enzymer inte är nödvändiga för växternas tillväxt och utveckling, så kan deras gemensamma reglering nyttjas för att förstå deras funktion och betydelse.

The length of the primary root and the density of lateral roots determine the architecture of the root. In this thesis the effect of NAA, ABA and the NO donor SNP alone as well as the combination of ABA or NAA with SNP on lateral root development was investigated. The interaction between CPTIO, a NO scavenger, and NAA or SNP is also reported. Following preliminary experiments in which it was observed that the aerial part of the seedling influenced LR growth and that there was a possible inhibitory effect of light on cultured root tips, experiments were conducted with excised roots tips in the dark. NAA was shown to have the potential to initiate LRs across a wide concentration gradient with the total number of LRs and initiated lateral root primordia (LRP) remaining constant across the range of concentrations tested. Over the last decade, nitric oxide (NO), a bioactive molecule, has been reported to be involved in the regulation of many biological pathways. The presence of NO in the system provided via sodium nitroprusside (SNP), promoted LRP initiation based on the NAA concentration gradient; but without changing the total LR initiation, that is LRs plus primordia density remained constant along the concentration gradient of NAA. The absence of LR and LRP in the treatments of CPTIO (a NO scavenger) with SNP or NAA suggests that NO regulates LRP initiation triggered by NAA, which is in agreement with the recent paper published after the commencement of this study (Correa-Aragunde et al., 2006). In agreement with previous studies, ABA inhibited lateral root development by reducing LR density and the number of LRs. The experiments with fluridone, an ABA biosynthesis inhibitor, may indicate that endogenous ABA was at sufficient concentrations in the excised root tips to inhibit primordia initiation. In this study, evidence is presented for the first time to show that SNP can relieve the inhibitory effect of ABA on LR density and number of LRs suggesting the NO, released from SNP, acts downstream of ABA. Overall these data confirm a critical role for NO in LR initiation.

Root branching allows plants to explore rhizosphere, to gain efficiently water, mineral nutrients or enter in various biotic interactions. Initiation of lateral root formation is localized to pericycle cells, flanking the xylem poles of diarch vascular bundle in Arabidopsis thaliana. Right in these pericycle cells, there is the expression pattern of the gene trap line MGT180. In this theseis , I have provided the evidence that the expression pattern of MGT180 is related with AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED18 (AHL18; At3g60870). AHL18 belongs to a gene family of 29 transcriptional factors of Arabidopsis. AHL18 has not been functionally characterized yet, the analysis of singlemutant ahl18 and some others revealed no significant phenotype. However, one of doublemutants, E15, showed a significant phenotype. This phenotype was evident mainly in the aboveground part of plants, and was not corresponding to any phenotype of AHL mutation described so far. There is a known redundancy among some AHL genes, confirmed by crossing of ahl18 and ahl28 leading to E15 plant. Translation phusion AHL18-mRUBY and AHL22-mRUBY under native promotors should reveal, where these two related proteins act, and if they fiction in autonomous manner or not. Key words Arabidopsis thaliana, lateral root, AHL, pericycle

Photosynthetic plants have a remarkable ability to modify their metabolism and development according to ever changing environmental conditions. The root system displays continuous growth of the primary root and formation of lateral roots enabling efficient water and nutrient uptake and anchorage of the plant in soil. With regard to lateral roots, development is post-embryonic, originating from the pericycle of the primary root. Coordinated activity of several molecular signalling pathways controlled by the hormone auxin is important throughout all stages of lateral root development.At first, two adjacent Xylem Pole Pericycle (XPP) cells are activated and the nuclei of these cells migrate towards a common cell wall.This is followed by XPP cells acquiring volume thus swelling up.The XPP cells then undergo anticlinal cell division, followed by a series of periclinal and anticlinal divisions,leading to lateral root primordia.These break through the radial cell layers and emerge out the primary root. Although root system plasticity is well-described in response to environmental cues such as ion nutrition in the soil, little is known on how root development is shaped according to the endogenous energy status of the plant.In this study, we were able to connect limited perturbations in photosynthetic energy supply to lateral root development.We established two experimental systems – treatment with low light and unexpected darkness which led to short-term energy imbalance in the plant.These short perturbations administered, showed an increase in the emerged lateral root density and decrease in root hexose availability and activation of the low energy marker gene ASN1 (ASPARAGINE SYNTHETASE 1).Although not demonstrated, presumably, these disturbances in the plant energy homeo-stasis activates SnRK1 (SNF1 RELATED KINASE 1),an evolutionary conserved kinase mediat-ing metabolic and transcriptional responses towards low energy conditions. In A. thaliana, two catalytic α-subunits of this kinase (SnRK1.α1 and SnRK1.α2) are functionally active and form ternary complexes with the regulatory β- and γ- subunits. Whereas unexpected darkness results in an increase in emerged lateral root density, the snrk1.α1 loss-of-function mutant displayed decrease in emerged lateral root density. As this effect is not that pronounced in the snrk1.α2 loss-of-function mutant, the α1 catalytic subunit is important for the observed lateral root phenotype under short-term energy perturbations. Moreover, root expression patterns of SnRK1.α1:GFP supports a role of this catalytic subunit in lateral root development. Furthermore, the lateral root response during short-term perturbations requires the SnRK1 downstream transcriptional regulator bZIP63 (BASIC LEU-CINE ZIPPER 63), as demonstrated here by a loss-of-function approach. Phenotypic studies showed that in comparison to wild-type, bzip63 mutants displayed decreased lateral root density upon low-light and unexpected darkness conditions. Previous work has demonstrat-ed that SnRK1 directly phosphorylates bZIP63 at three serine residues. Alanine-exchange mutants of the SnRK1 dependent bZIP63 phosphorylation sites behave similarly to bzip63 loss-of-function mutants and do not display increased lateral root density upon short-term unexpected darkness. This data strongly supports an impact of SnRK1-bZIP63 signalling in mediating the observed lateral root density phenotype. Plants expressing a bZIP63:YFP fu-sion protein showed specific localization patterns in primary root and in all developmental stages of the lateral root. bzip63 loss-of-function mutant lines displayed reduced early stage lateral root initiation events under unexpected darkness as demonstrated by Differen-tial Interference Contrast microscopy (DIC) and the use of a GATA23 reporter line. This data supports a role of bZIP63 in early lateral root initiation. Next, by employing Chromatin Immunoprecitation (ChIP) sequencing, we were able to iden-tify global binding targets of bZIP63, including the auxin-regulated transcription factor (TF) ARF19 (AUXIN RESPONSE FACTOR 19), a well-described central regulator of lateral root development. Additional ChIP experiments confirmed direct binding of bZIP63 to an ARF19 promoter region harboring a G-Box cis-element, a well-established bZIP63 binding site. We also observed that short-term energy perturbation upon unexpected darkness induced tran-scription of ARF19, which was impaired in the bzip63 loss-of-function mutant. These results propose that bZIP63 mediates lateral root development under short-term energy perturba-tion via ARF19. In conclusion, this study provides a novel mechanistic link between energy homeostasis and plant development. By employing reverse genetics, confocal imaging and high-throughput sequencing strategies, we were able to propose a SnRK1-bZIP63-ARF19 signalling module in integrating energy signalling into lateral root developmental programs. / Photosynthestisch aktive Pflanzen haben die bemerkenswerte Fähigkeit, ihren Stoffwechsel und ihre Entwicklung an sich ständig ändernde Umweltbedingungen anzupassen. Das pflanz- liche Wurzelsystem weist ein kontinuierliches Primärwurzelwachstum und eine Ausbildung von Seitenwurzeln auf, wodurch eine effiziente Wasser- und Nährstoffaufnahme sowie die Verankerung der Pflanze im Boden ermöglicht werden. Die Entwicklung der Seitenwurzeln verläuft post-embryonal, ausgehend vom Perizykel der Primärwurzel. Die koordinierte Aktivi- tät mehrerer molekularer Signalwege, die durch das Hormon Auxin gesteuert werden, ist in allen Stadien der Seitenwurzelentwicklung wichtig. Bei diesem Prozess werden zunächst zwei benachbarte Xylem-Pol-Perizykel-Zellen (XPP) aktiviert, deren Zellkerne zu einer gemeinsa- men Zellwand migrieren. Daraufhin schwillt das Volumen der XPP-Zellen an, bevor sich diese zunächst antiklinal teilen. Durch sukzessive periklinale und antiklinale Teilungen entstehen so Seitenwurzel-Primordien. Diese durchbrechen die radialen Zellschichten und treten aus der Primärwurzel aus. Während die Plastizität des Wurzelsystems als Reaktion auf Umwelteinflüsse, wie z.B. die Ver- sorgung mit Ionen aus dem Boden, bereits umfassend erforscht wurde, so ist die Abhängigkeit der Wurzelentwicklung vom endogenen Energiezustand der Pflanze weitgehend unbekannt. In dieser Arbeit konnten wir geringfügige Störungen der photosynthetischen Energieversor- gung mit der Seitenwurzelentwicklung in Verbindung bringen. Pflanzen wurden Schwachlicht oder unerwarteter Dunkelheit ausgesetzt und damit ein kurzzeitiges Energieungleichgewicht erzeugt. Hierdurch zeigte sich eine Zunahme der Seitenwurzeldichte bei gleichzeitiger Ab- nahme der Verfügbarkeit von Hexosen in der Wurzel und Aktivierung des Energieverarmungs- Markergens ASN1 (ASPARAGIN-SYNTHETASE 1). Obwohl dieser Mechanismus noch nicht ge- klärt ist, aktiviert die Störung der pflanzlichen Energie-Homöostase vermutlich SnRK1 (SNF1 RELATED KINASE 1), eine evolutionär konservierte Kinase, die metabolische und transkriptio- nelle Reaktionen auf niederenergetische Bedingungen vermittelt. In Arabidopsis sind zwei ka- talytische α-Untereinheiten dieser Kinase (SnRK1.α1 und SnRK1.α2) funktionell aktiv und bil- den ternäre Komplexe mit den regulatorischen β- und γ-Untereinheiten. Während eine uner- wartete Dunkelheit zu einer Zunahme der Dichte der auswachsenden Seitenwurzeln führt, zeigte die Snrk1.α1 Funktionsverlustmutante den gegenteiligen Effekt. Da dieser Effekt in der Funktionsverlustmutante von snrk1.α2 weniger stark ausgeprägt ist, scheint die katalytische Untereinheit α1 für den beobachteten Seitenwurzel-Phänotyp unter kurzfristigen Energiestö- rungen eine wichtige Rolle zu spielen. Das Expressionsmuster von SnRK1.α1:GFP in der Wur- zel unterstützt die mögliche Rolle dieser katalytischen Untereinheit bei der Seitenwurzelent- wicklung weiter. Darüber hinaus erfordert die Seitenwurzelbildung während kurzfristiger Störung des pflanzli- chen Energiehaushalts den SnRK1-nachgeschalteten Transkriptionsregulator bZIP63 (BASIC LEUCINE ZIPPER 63). Phänotypische Studien zeigten, dass bzip63-Funktionsverlust-Mutanten im Vergleich zum Wildtyp nach der Kultivierung unter Schwachlicht oder nach unerwarteter Dunkelheit eine geringere Seitenwurzeldichte aufwiesen. Frühere Arbeiten haben gezeigt, dass SnRK1 bZIP63 direkt an drei Serinresten phosphoryliert. Alanin-Austauschmutanten der SnRK1-abhängigen bZIP63-Phosphorylierungsstellen verhielten sich ähnlich wie bzip63-Funk- tionsverlustmutanten und zeigten bei kurzzeitiger unerwarteter Dunkelheit keine erhöhte Seitenwurzeldichte. Diese Daten weisen deutlich auf einen Einfluss des SnRK1-bZIP63-Signal- wegs auf den beobachteten Seitenwurzeldichte Phänotyp hin. Pflanzen, die ein bZIP63:YFP- Fusionsprotein exprimieren, zeigten ein spezifisches bZIP63 Lokalisierungsmuster in der Pri- märwurzel, sowie in allen Entwicklungsstadien der Seitenwurzel. bzip63-Funktionsverlustmu- tantenlinien zeigten reduzierte Seitenwurzel- Initiationsereignisse bei unerwarteter Dunkel- heit, wie durch Differentialinterferenzkontrast-Mikroskopie (DIC) und der Verwendung einer GATA23-Reporterlinie nachgewiesen wurde. Diese Ergebnisse deuten auf eine Rolle von bZIP63 bei der frühen Seitenwurzel-Initiierung hin. Durch die Anwendung der Chromatin-Immunopräzipitation (ChIP)-Sequenzierungsmethode konnten wir daraufhin globale Bindungsziele von bZIP63 identifizieren, einschließlich des au- xinregulierten Transkriptionsfaktors ARF19 (AUXIN RESPONSE FACTOR 19), einem gut be- schriebenen zentralen Regulator der Seitenwurzelentwicklung. Zusätzliche ChIP-Experimente bestätigten die direkte Bindung von bZIP63 an eine ARF19-Promotorregion, die ein G-Box cis- Element, eine bekannte bZIP63-Bindungsstelle, beherbergt. Wir beobachteten auch, dass kurzfristige Energiestörungen bei unerwarteter Dunkelheit die Transkription von ARF19 indu- zierte, die in der bzip63-Funktionsverlustmutante beeinträchtigt war. Diese Ergebnisse legen nahe, dass bZIP63 die Seitenwurzelentwicklung unter kurzfristiger Energiestörung über ARF19 vermittelt. Zusammenfassend lässt sich sagen, dass diese Studie eine neuartige mechanistische Verbin- dung zwischen Energiehomöostase und Pflanzenentwicklung herstellt. Durch den Einsatz von reverser Genetik, konfokaler Mikroskopie und Hochdurchsatz-Sequenzierungsstrategien konnten wir einen SnRK1-bZIP63-ARF19-Signalweg zur Integration von Energiesignalen in Sei- tenwurzelentwicklungsprogramme aufdecken.

In Medicago truncatula, the MtNPF1.7 transporter has been shown to be essential for root morphology and nodulation development. The allelic MtNPF1.7 mutants, Mtnip-1 (A497V), Mtnip-3 (E171K), and Mtlatd (W341STOP), show altered lateral root growth and compromised legume-rhizobium symbiosis. To assess the role of a series of distinct amino acids in the transporter's function, in silico structural predictions were combined with in planta complementation of the severely defective Mtnip-1 mutant plants. The findings support hypotheses about the functional importance of the ExxE(R/K) motif including an essential role for the first glutamic acid of the motif in proton(s) and possibly substrate transport. The results also question the existence of a putative TMH4-TMH10 salt bridge, which may not form in MtNPF1.7. Results reveal that a motif conserved among MFS proteins, Motif A, is essential for function. Hypothetically, the Motif A participates in intradomain packing of transmembrane helices and stabilizing one conformation during transport. The mutated valine (A497V) in Mtnip-1 may interfere with the lateral helix. Mutating a residue (L253) on the lateral helix with reduced side chain restored Mtnip-1 function. The predicted residue (Q351) for substrate binding is not essential for protein function. To probe the possibility that MtNPF1.7 transports auxin, two heterologous assay systems were attempted. The first was a Xenopus laevis oocyte assay. However, MtNPF1.7 expressed in oocytes failed to show substrate transport, which may due to low expression levels of proteins on the membrane or may be caused by other factors. Second, yeast (Saccharomyces cerevisiae) strains expressing MtNPF1.7 were constructed. They showed an increased flux of radiolabeled IAA and differential susceptibility to 5-fluoroindole-3-acetic acid (F-IAA), a toxic IAA-like compound. These results suggested that MtNPF1.7 may function as an auxin transporter in yeast. Unexpectedly, the Mtnip-1 (A497V) and Mtnip-3 (E171K) proteins when expressed in yeast also showed influx of F-IAA transport in yeast, suggesting the auxin transport remains functional in the mutants, potentially contradicting other results suggesting Mtnip-1 is non-functional. To address the possible role of MtNPF1.7 in auxin responses, the auxin reporter DR5-GUS was used and mutant plants were subjected to gravitropism tests. The auxin reporter DR5::GUS displayed different expression in Mtnip-1 mutant in comparison with DR5::GUS expression in wild-type. In addition, the spatial expression of pMtNPF1.7-GUS partly co-localized with the expression of DR5:GUS in roots and nodules. The Mtnip-1 plants showed altered gravitropic responses to gravistimulus compared to wild-type and Mtnip-3 plants. The results suggest that MtNPF1.7, recognized as a nitrate transporter, may be able to transport auxin and may participate in auxin distribution during M. truncatula root and nodule development. These research findings helps elucidate the complex function of plant NPF transporters.

Dans l’optique d’une seconde Révolution Verte, visant, à la différence de la première, à accroître les rendements des cultures dans un contexte de faible fertilité, les stratégies mises en place par les plantes pour une assimilation optimale des nutriments du sol se trouvent au cœur du problème. Afin de le résoudre et d’identifier les variétés idéales parmi la diversité génétique des plantes cultivées, les systèmes racinaires, leur développement et leur architecture, sont appelés à jouer le premier rôle. La variabilité au sein des racines latérales semble s’avérer une caractéristique cruciale pour l’optimisation de l’exploration du sol et de l’acquisition de ses ressources mobiles et immobiles, mais ce phénomène est encore mal appréhendé.Le travail présenté dans cette thèse se concentre sur les racines latérales du maïs (Zea mays L.) dans un effort pour révéler les processus à l’origine des variations intrinsèques dans le développement racinaire. Il s’appuie en particulier sur le phénotypage des racines latérales à une échelle sans précédent, suivant la croissance journalière de milliers d’entre elles à haute résolution spatiale, pour caractériser précisément les variations spatio-temporelles entre et au sein des individus racinaires. Les profils individuels de vitesse de croissance ont été analysés à l’aide d’un modèle statistique qui a identifié trois principales tendances temporelles dans les vitesses de croissance menant à la définition de trois classes de racines latérales avec une vitesse et durée de croissance distinctes. Des différences de diamètre à l’émergence de ces racines (dont l’origine remonte au stade du primordium) conditionnent probablement la tendance ultérieur de croissance mais ne suffisent pas à déterminer le destin de la racine. Finalement, ces classes racinaires sont distribuées aléatoirement le long de la racine primaire, ce qui suggère qu’aucune stimulation ou inhibition locale n’existe entre racines voisines.Pour expliquer l’origine des variations observées dans la croissance, ce travail a été complété par une caractérisation multi-échelle de groupes de racines latérales présentant une croissance distincte, à un niveau cellulaire, anatomique et moléculaire. Un effort particulier a été dirigé à l’analyse des profils de longueur de cellules dans des apex racinaires pour lequel nous avons introduit un modèle de segmentation pour identifier des zones développementales. Grâce à cette méthode, une forte modulation dans la longueur des zones de division et d’élongation a été mise en évidence, en lien avec les variations de la croissance des racines latérales. Le rôle régulateur de l’auxine sur l'équilibre entre les processus de prolifération et d’élongation cellulaire a été montré avec l’utilisation de lignées mutantes. En fin de compte, les variations de la croissance entre racines latérales sont remontées jusqu’à l’allocation d’assimilats carbonés et la capacité de transport de la racine, ce qui suggère l’existence d’un mécanisme de rétroaction qui pourrait jouer un rôle déterminant dans la mise en place de tendances contrastées dans la croissance des racines latérales. / 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.