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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.

During Arabidopsis embryogenesis, cell-cell signaling plays an essential role in establishing an organized body plan centered around two major axes of development: apical-basal and radial. Two topics of great interest are how the layered structure is initiated and maintained during and after embryogenesis and how communication between layers is achieved to allow for coordinated development. Recent research involving Receptor-Like Kinases (RLKs) in plants suggests that their roles in integrating various signals are important in many aspects of development, including embryonic and post-embryonic patterning. The research presented here describes the roles of two pairs of RLKs with independent roles in two different signaling environments. The first RLK pair, GSO1 and GSO2, function in root development at the transition to photoautotrophic nutrition to integrate sugar signals and regulate root growth. GSO1 and GSO2 regulate root epidermal cell identity by controlling the pattern of cell division of stem cells. The second pair of RLKs, RPK1 and TOAD2, function to control root development by regulation of meristem proliferation and a coordinated response to signaling molecules of the CLE family. The response of wild-type roots to treatment with CLE peptides (A-type) is meristem growth arrest, resulting in short roots. toad2 mutants are insensitive to the effect of CLE peptides in reducing meristem size and TOAD2 also regulates RPK1 upon CLE stimulation. Although responding to different signals, the two pairs of RLK share a common output of regulating cell proliferation in and around the root meristem, especially in the epidermis of the root.

Chez les plantes les protéines de la famille GRAS régissent un grand nombre de processus allant du développement racinaire à la transduction de signaux hormonaux. Deux protéines de cette famille de facteurs de transcription, SCR et SHR jouent un rôle essentiel dans le développement racinaire d’Arabidopsis thaliana en régulant la formation des tissus internes. Chez le riz, il existe deux co-orthologues putatifs pour ces deux gènes, OsSCR1, OsSCR2, OsSHR1 et OsSHR2. Nous avons caractérisé la fonction de trois membres de cette famille au cours du développement racinaire et aérien du riz. OsSHR1 et OsSHR2 sont impliqués dans la variation du nombre de couches de cortex et interfèrent aussi dans la formation des cellules bulliformes et du sclerenchyme dans les feuilles. OsSCR2 a un rôle majeur dans le contrôle du nombre de ramifications aériennes. Nos résultats ont aussi permis de démontrer qu’OsSCR2 et OsSHR2 ont des fonctions spécifiques au riz. / Summary to be coming

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.

O ambiente epifítico é considerado um dos habitats mais desafiadores para as plantas, pois a disponibilidade hídrica e nutricional pode ser bastante escassa. Além disso, as plantas que colonizam este ambiente estão mais expostas aos fatores ambientais, dentre eles a luminosidade se mostra bastante importante, uma vez que esta atua tanto como fonte de energia na fotossíntese quanto como sinal ambiental em repostas fotomorgênicas. Sabe-se, por exemplo, que a luz exerce forte influência sobre a morfogênese radicular de plantas em geral, porém impacta ainda mais o desenvolvimento de orquídeas epífitas, dada a frequente exposição de suas raízes aéreas à incidência luminosa. Tendo em vista que a auxina ácido indolil-3-acético (AIA), o ácido abscísico (ABA) e o etileno são moduladores cruciais no controle da arquitetura radicular na maioria das plantas, sendo também mediadores chave em várias respostas fotomorfogênicas, este estudo propôs-se a investigar o possível envolvimento destes hormônios durante diferentes respostas morfo-fisiológicas desencadeadas pela exposição à luz do sistema radicular de plantas de C. fimbriatum. A ausência de incidência luminosa sobre às raízes resultou em maiores taxas de crescimento e volume radicular, porém, com menor acúmulo de biomassa em relação às raízes expostas à luz. O incremento na biomassa em raízes expostas à luz esteve correlacionado ao espessamento da parede celular na região cortical, o qual ocorreu em resposta especificamente à luz azul. Em termos gerais, a exposição das raízes à luz induziu o aumento nos níveis de AIA e ABA, enquanto que os teores de ACC foram superiores em raízes protegidas da incidência luminosa. Estes resultados sugerem que a luz pode modular o desenvolvimento radicular de C. fimbriatum através de um fino controle hormonal que depende de ajustes coordenados dos níveis de AIA ABA e ACC. Também foi investigado o potencial envolvimento das auxinas e do ABA durante a remobilização de carboidratos entre pseudobulbos e folhas de plantas que tiveram seus sistemas radiculares expostos à (ou protegidos da) luz. Os resultados revelaram que a manutenção das raízes sob condições de escuro levou ao aumento dos teores de AIA e de todas as fontes de carbono estudadas (especialmente de glicose e frutose) nos pseudobulbos, enquanto que as raízes cobertas apresentaram apenas um leve aumento no conteúdo de AIA. O tratamento concomitante das raízes com a condição de escuro e a aplicação de um inibidor do transporte polar de auxina causou uma diminuição abrupta nos teores de AIA em todos os órgãos analisados e a elevação do conteúdo de ABA no sistema radicular. De maneira interessante, essa última condição experimental induziu um conspícuo acúmulo de carboidratos nos pseudobulbos, principalmente de sacarose. Assim, os dados deste trabalho reforçam a importante participação do AIA e ABA como possíveis mediadores da sinalização desencadeada pela luz incidente no sistema radicular de C. fimbriatum, cujas respostas induzidas regulam não somente a morfogênese de tecidos radiculares, mas também influenciam na regulação da partição de carbono no sistema caulinar por meio de um provável mecanismo de sinalização à longa distância / The epiphytic environment is considered one of the most challenging for plants, due to frequent scarcity of water and nutrients. Furthermore, the plants that colonized this biotope are usually more exposed to the environmental cues. Light is considered one of the most important signals controlling plant development because it can act as both an energy source for photosynthesis and an environmental signal for photomorphogenic responses. Besides, light can influence the root morphology of most plants, with even stronger impacts expected in aerial roots of epiphytic orchids due their frequent exposition to direct light. Since indole-3-acetic acid (IAA), abscisic acid (ABA) and ethylene are crucial hormonal signals modulating the root architecture in most plant species, and key mediators during numerous photomorphogenic responses, this study investigated the potential involvement of these hormones in different morpho-physiological responses regulated by either the darkness treatment or the light exposure of Catasetum fimbriatum root system. The absence of light incidence on the roots resulted in higher root volume and growth rate, but lower dry mass accumulation than the light-exposed ones. The higher accumulation of biomass in the light-exposed roots was closed correlated with a more intense cell wall thickening in the root cortex, which appeared to be specifically induced by the blue light. In general, root exposure to light induced increasing levels of ABA and AIA, while the ACC content was higher in roots protected from light. This suggests that light might modulate C. fimbriatum root development through a fine-tuned hormonal mediation, which depends on coordinated adjustments of IAA, ABA and ACC levels. This study also investigated the potential involvement of auxin and ABA during the (re)mobilization of carbohydrates in pseudobulbs and leaves of plants that had their root systems either exposed to (or protected from) light. The results revealed that covering the roots increased in pseudobulbs the levels of AIA and all carbon sources studied (specially glucose and fructose), while the covered roots showed slightly higher levels of AIA. The concomitant treatment with NPA and root covering caused a sharp decrease of AIA levels in all organs and an ABA increase in the root system. Interestingly, this last condition induced a conspicuous carbohydrate accumulation in pseudobulbs, with sucrose as the predominant form. The data obtained in this study reinforce the remarkable participation of IAA and ABA as possible mediators of the signaling cascades triggered by the light incidence on C. fimbriatum root system, which was capable of inducing photomorphogenic responses not only in root tissues, but was also able to influence the carbon portioning in the shoot system by a potential long-distance signaling mechanism

To maintain optimal cytosolic Ca2+ concentrations, cells employ three distinct strategies: 1) tightly regulated influx of Ca2+; 2) efficient efflux of Ca2+ from the cell; 3) sequestration of Ca2+ in organelles. Ca2+efflux and influx are mediated by diverse transporter systems, such as pumps, channels and antiporters. Vacuolar localized Ca2+/H+ exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca2+ homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. I compared and contrasted the properties of Nterminal CAX1 variants in plants and yeast expression systems to determine if autoinhibition of CAX1 is occurring in planta. Although several plant transporters appear to contain N-terminal autoinhibitory domains, my work is the first to document clearly N-terminal-dependent regulation of a Ca2+ transporter in transgenic plants. In my second study, I characterized another Arabidopsis CAX, CAX4. CAX4 is expressed in the root apex and lateral root primordia and expression increased when Ni2+ or Mn2+ levels were elevated or Ca2+ was depleted. Transgenic plants expressing increased levels of CAX4 displayed symptoms consistent with increased sequestration of Ca2+ and Cd2+ into the vacuole. When CAX4 was highly expressed in an Arabidopsis cax1 mutant line with weak vacuolar Ca2+/H+ antiport activity, a 29% increase in Ca2+/H+ antiport was measured. A cax4 loss-of-function mutant and CAX4 RNA interference lines displayed altered root growth in response to Cd2+, Mn2+, and auxin. The DR5::GUS auxin reporter detected reduced auxin responses in the cax4 lines. My results indicate that CAX4 is a cation/H+ antiporter that plays an important function in root growth under heavy metal stress conditions. My third study is to characterize a zebrafish CAX. In this study, I have initiated characterization of a zebrafish CAX by initially expressing the open reading frame in yeast. Zebrafish Cax1 was localized on the yeast endomembranes and displayed Ca2+/H+ activity. In zebrafish embryos, Cax1 was specifically expressed in neural crest cells and morpholino knockdowns of cax1 caused defects in neural crest development as measured by alterations in pigmentation, defects in jaw development and reduction in expression of the neural crest marker, pax7. Collectively, our findings provide a previously unexpected role of CAX transporters in animal growth and development.

A basic understanding of the molecular events occurring during zygotic embryogenesis is required to fully understand how and why only a very small percentage of somatic embryos develop past the late embryogeny phase of embryogenesis. In this work, we have identified genes that have been demonstrated to be required for late embryonic development in the model plant system Arabidopsis thaliana. These genes were subsequently isolated and cloned from Loblolly pine embryos. These isolated clones were sequenced and analyzed to reveal significant homology to the known Arabidopsis ABA responsive genes ABI3, ABI4, and ABI5. Expression analyses of all three genes were completed, and compared to reported data of ABA accumulation, as well as, expression of other ABA responsive genes during the same stages of embryogenesis. Six putative root development genes were isolated and cloned from Loblolly pine embryos. These isolated clones were sequenced and analyzed to reveal significant homology to the known Arabidopsis root development genes WOODENLEG, SHORT ROOT, SCARECROW, HOBBIT, BODENLOS, and MONOPTEROS. Full-length cDNAs were isolated and cloned for WOODENLEG, SHORT ROOT, SCARECROW and BODENLOS. Expression analyses of all six genes were completed throughout mid to late embryogenesis in Loblolly pine.

The har1-1 mutant of Lotus japonicus line Gifu is characterised by increased nodulation and significantly inhibited root growth in the presence of its microsymbiont Mesorhizoboium loti (for example strain NZP2235). A sexual cross between the mutant and another L. japonicus genotype Funakura (with wild-type root and nodule morphology) demonstrated Mendelian recessive segregation of both phenotypes (for root and nodule) in 242 F2 individuals. No separation of phenotypes was observed, suggesting a single mutation with pleiotropic effects. Reciprocal grafting showed that the har1-1 controlled phenotype is governed by the shoot. Using a skeletal genetic map of arbitrary molecular markers produced from a Gifu x Funakura cross, the har1-1 locus was positioned between two markers at about 7 and 13 cM distance. Single nucleotide polymorphisms (SNPs) and transgene sequences were detected by allele-specific PCR in DNA isolated from small (1 mg mass) individual seeds and half-cotyledon of the model legume Lotus japonicus, allowing fast determination of a seedling’s genomic status. This permitted a shortening of the breeding cycle for multi-trait seed lines. Fast neutron mutagenesis of Lotus japonicus wild-type genotype Gifu resulted in the first time isolation of a stable mutant (FNN5-2) unable to form nitrogen-fixing nodules in symbiosis with Mesorhizobium loti, though being infected by mycorrhizal fungi. The mutation behaves as a loss-of-function recessive, and has no other apparent phenotypic effects. Molecular characterization indicates a partial loss of the LjNFR1 LysM type receptor kinase gene. Additionally part of the LjNIN gene (encoding a putative transcription factor needed for nodulation) is also missing. Transcript levels for both genes are severely reduced. As LjNIN and LjNFR1 are in the same chromosomal region we tested whether this terminal portion is lacking. PCR analysis confirms that genes within the relevant interval (such as LjPAL1 (encoding phenylalanine ammonia lyase) and LjEIL2 (encoding an ethylene insensitive-like response regulator)) are present, suggesting that the mutational event induced by the fast neutrons was either a double hit coincidently involving two nodulation-related genes, a major genome rearrangement, or a major segmental inversion. To develop an integrated nodule developmental model based on gene interactions in autoregulation, nodulation and plant hormone response deficient lines, HE double mutants have been built using the har1-1 mutant (hypernodulation and aberrant root) and the ethylene insensitive transgenic line Etr1-1. The homozygous loss-of-function mutant har1-1 has increased nodulation and decreased root growth. Ethylene insensitivity mediated by the transgene 35S::AtETR1-1 restores the normal root growth. The HE double mutants were confirmed by triple response test and allele- or gene-specific PCR. The current results in this study indicate that a) HE double mutants shown the same nodulation pattern as har1-1 and normal root formation as Etr1-1, suggesting that nodule and root control diverge at some stage with root control being ethylene-mediated and the Har1 gene, the orthologue of GmNARK is involved in nodulation. b) Grafting demonstrated that the shoot is the source of ethylene suppression of the har1-1 induced inhibition of root growth. c) The mutated Etr1-1 gene was able to replace AVG in BAP root inhibition; d) IPT-dependent cytokinin overproduction led to aberrant root architecture in har1-1; e) Crosstalk between ethylene and cytokinin in HE double mutant by qRT-PCR.