Phytochromes (PHYs) and plant hormones have been emerging as important regulators of fleshy fruit physiology and quality traits; however, the relevance of PHY-hormonal signaling crosstalk in controlling fruit development and metabolism remains elusive. This Thesis assesses the role of PHYs and their interplay with auxins, cytokinins and ethylene during the regulation of tomato (Solanum lycopersicum) fruit development and ripening, with a focus on the control of the plastid biogenesis, sugar metabolism and carotenoid accumulation. In Chapter I, we present evidence that the deficiency in PHY chromophore phytochromobilin (PΦB) biosynthesis, which leads to a global deficiency in functional PHYs, represses fruit chloroplast biogenesis in immature fruits and inhibits fruit sugar accumulation by transcriptionally downregulating sink- and starch biosynthesis-related enzymes. Genetic and physiological evidence suggested the involvement of both auxins and cytokinins as mediators of the negative impact of PΦB deficiency on fruit sink strength and chloroplast formation. During the ripening phase, PΦB deficiency was shown to delay the rise in climacteric ethylene production, affecting the ripening initiation rather than its progression. PHY-hormonal signaling crosstalk was shown to be active not only in the more externally positioned fruit tissues (i.e., pericarp) but also in the most inner fruit regions (i.e., columella). We, therefore, concluded that the global deficiency in functional PHY drastically affects fruit sugar metabolism, chloroplast formation as well as the timing of ripening via an intricate interplay involving phytochromes, auxins, cytokinins and ethylene. In Chapter II, we employed fruit-specific RNAi-mediated silencing of PHY genes to shed light on the specific role played by fruit-localized PHYs and their downstream signaling cascades on tomato fruit physiology and quality traits. Data revealed that fruit-localized SlPHYB2 negatively regulates chlorophyll accumulation in immature fruits whereas SlPHYA positively influences the plastid division machinery. Both SlPHYA and SlPHYB2 were shown to play overlapping, yet distinct, roles in controlling fruit starch metabolism and carotenoid biosynthesis. Our data implicated cytokinin signaling-related proteins as mediators of the SlPHYA-dependent regulation of plastid division machinery, and specific AUXIN RESPONSE FACTORs as intermediates in the PHY-mediated regulation of fruit sugar and carotenoid metabolisms. We concluded that fruit-localized SlPHYA- and SlPHYB2-mediated light perception regulate fruit plastid biogenesis as well as sugar and carotenoid metabolisms via coordinated changes in key components of both auxin and cytokinin signaling cascades. Altogether, this study brings important insights into the combined action of PHYs and hormones in the control of fruit plastid biogenesis and highlights that the interplay between PHY-hormonal signaling cascades influences essential features of tomato fruit quality, such as the sugar and carotenoid accumulation / Fitocromos (PHYs) e fitormônios têm sido caracterizados como importantes reguladores da fisiologia e qualidade de frutos carnosos; todavia, a importância de interações entre a sinalização hormonal e dos PHYs no controle do desenvolvimento e metabolismo de frutos ainda permanece pouco elucidada. Este trabalho de Tese avaliou o papel dos PHYs e das suas interações com as auxinas, as citocininas e o etileno sobre a regulação do desenvolvimento e amadurecimento de frutos de tomateiro (Solanum lycopersicum), particularmente no que tange ao controle da biogênese plastidial e metabolismos de açúcares e de carotenoides. No Capítulo I são apresentadas evidências de que a deficiência na produção de fitocromobilina (PΦB), a qual resulta numa deficiência global in PHYs funcionais, impacta negativamente a biogênese de cloroplastos em frutos imaturos e inibe o acúmulo de açúcares por meio da repressão transcricional de enzimas relacionadas a biossíntese de amido e força de dreno nos frutos. Evidências genéticas e fisiológicas indicaram o envolvimento tanto das auxinas quanto das citocininas como mediadoras do impacto negativo da deficiência de PΦB sobre a força de dreno dos frutos bem como na formação de cloroplastos. Durante a fase de amadurecimento, a deficiência em PΦB atrasou a produção climatérica de etileno, afetando o início do amadurecimento mas não a sua progressão. As interações entre PHYs e hormônios mostraram-se ativas não apenas nos tecidos posicionados mais externamente (i.e., pericarpo) mas também nas regiões mais internas do fruto (i.e., columela). Conclui-se, portanto, que a deficiência global em PHYs funcionais afeta drasticamente o metabolismo de açucares, formação de cloroplastos, bem como o tempo de amadurecimento através de uma interação complexa envolvendo fitocromos, auxinas, citocininas e etileno. No Capítulo II utilizamos o silenciamento fruto-específico de PHYs a fim de desvendar de que forma a fisiologia e parâmetros de qualidade do tomate seriam regulados por PHYs presentes no próprio fruto. Os dados obtidos revelaram que moléculas de SlPHYB2 presentes no próprio fruto regulam negativamente o acúmulo de clorofilas nos frutos imaturos, já as de SlPHYA influenciam positivamente a maquinaria de divisão plastidial, e tanto SlPHYA quanto SlPHYB2 desempenham papel sobrepostos, porém distintos, no controle do metabolismo de amido e acúmulo de carotenoides em frutos de tomateiro. Evidências sugerem que proteínas relacionadas à sinalização de citocininas atuariam como mediadoras do impacto de SlPHYA sobre a maquinaria de divisão plastidial, e que AUXIN RESPONSE FACTORs específicos seriam intermediários no controle dos PHYs sobre os metabolismos de açúcares e carotenoides. Conclui-se, dessa forma, que a percepção de luz mediada por moléculas de SlPHYA e SlPHYB2 presentes no próprio fruto regulam a biogênese plastidial e os metabolismos de açúcares e carotenoides por meio de alterações coordenadas em componentes chaves das cascatas de sinalização de auxinas e citocininas. Quando combinados, os dados obtidos neste estudo apresentam novidades importantes sobre a ação conjunta de PHYs e fitormônios no controle da biogênese plastidial e demonstram que a interação entre esses sinalizadores influencia características essenciais da qualidade de frutos de tomateiro, tais como o acúmulo de açúcares e de carotenoides

Les Bradyrhizobium photosynthétiques sont capables d'induire la formation de nodules fixateurs d'azote chez certaines légumineuses du genre Aeschynomene. La découverte récente que certaines de ces souches ne possèdent pas les gènes canoniques nodABC indique l'existence d'un nouveau processus symbiotique rhizobium-légumineuse indépendant des facteurs Nod. L'objectif de ce travail de thèse a consisté à avancer dans la compréhension des mécanismes mis en jeu lors de cette nouvelle interaction. Dans un premier temps, à travers différentes approches cytologiques, le processus par lequel la bactérie infecte la plante en l'absence de facteurs Nod a été décrit. Dans un deuxième temps, afin de mettre en évidence les bases moléculaires de cette interaction, une banque de 15 000 mutants Tn5 de la souche ORS278 a été criblée sur plante. Ce criblage a permit l'identification de plus d'une centaine de gènes bactériens intervenant durant le processus symbiotique. Les résultats obtenus nous ont conduits à proposer un modèle dans lequel la mise en place de la symbiose Nod-indépendante impliquerait, d'une part, la synthèse bactérienne d'une cytokinine permettant le déclenchement de l'organogenèse nodulaire, et d'autre part, d'autres signaux bactériens intervenant dans l'étape de reconnaissance avec la plante hôte. Enfin, nous avons mis en place une technique de transformation génétique d'Aeschynomene et validé cet outil à travers l'étude de l'expression hétérologue de la noduline précoce MtENOD11. Il peut à présent être envisagé de conduire des études fonctionnelles sur Aeschynomene en vue de caractériser la voie de signalisation Nod-indépendante. / The photosynthetic Bradyrhizobium are able to induce the formation of nitrogen-fixing nodules in some legumes of the Aeschynomene genus. The recent discovery that some of these strains lack the canonical nodABC genes indicates the existence of a new symbiotic rhizobium-legume process that is independent of Nod factors. The aim of this work was to improve our understanding of the mechanisms involved in this new interaction. First, through various cytological approaches, the process by which the bacterium infects the plant in the absence of Nod factors has been described. Second, in order to decipher the molecular basis of this interaction, a library of 15,000 Tn5 mutants of the ORS278 strain was screened on plant. This screening allowed the identification of about one hundred bacterial genes involved in this symbiotic process. These results led us to propose a model in which the establishment of the Nod-independent symbiosis involves, on one han d, the synthesis of a bacterial cytokinin that triggers nodule organogenesis, and on the other hand, others bacterial signals that permit the recognition with the host plant. Finally, we developed a genetic transformation procedure of Aeschynomene and we validated this tool by studying the heterologous expression of the early nodulin MtENOD11. Now, functional studies on Aeschynomene are possible to permit the characterization of the Nod-independent signaling pathway.

Made available in DSpace on 2014-06-11T19:22:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-24Bitstream added on 2014-06-13T18:48:46Z : No. of bitstreams: 1 fioreze_sl_me_botfca.pdf: 515942 bytes, checksum: 36e6e68c9485cd45059afbe0dccf4ee9 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Melhorar o potencial produtivo de plantas cultivadas através de práticas de cultivo é um dos principais desafios da pesquisa frente à crescente demanda de alimentos em nível mundial. Assim, o presente estudo teve por objetivo investigar as alterações morfofisiológicas ocorridas no colmo principal e em perfilhos, bem como caracterizar os processos envolvidos com a plasticidade do desenvolvimento de plantas de trigo em função da densidade de semeadura e da aplicação de regulares vegetais. O experimento foi conduzido no ano agrícola de 2010 na Fazenda Experimental Lageado, Faculdade de Ciências Agronômicas, UNESP, em Botucatu (SP). O delineamento experimental utilizado foi o de blocos casualizados, em esquema de parcelas subdivididas. As parcelas foram formadas por quatro densidades de semeadura (30, 50, 70 e 90 plantas m-1), cultivar IAC 370, enquanto as subparcelas foram formadas pela aplicação de reguladores vegetais [controle, (Ax+GA+CK), Etil-Trinexapac e (Ax+GA+CK) + Etil-Trinexapac]. Foram avaliados a emissão de perfilhos, o acúmulo de matéria seca em perfilhos e no colmo principal, a altura de plantas, as trocas gasosas, características morfofisiológicas da folha bandeira e sua relações com a espiga, além dos componentes da produção para perfilhos e colmo principal e da produtividade final da cultura. Ao final do experimento foram determinadas a participação de perfilhos no desenvolvimento e na produção de grãos, bem como o potencial produtivo de perfilhos em relação ao colmo principal. A aplicação de Etil- Trinexapac resultou em alterações morfofisiológicas em plantas de trigo, como altura de plantas, redução da distância entre fonte e dreno, teor de clorofila e comprimento da folha bandeira, não afetou a produtividade de grãos, tampouco as relações entre colmo principal e perfilhos de trigo. O aumento do número de plantas por unidade de área... / Improve on yield potential of plants by crop management is the main research challenge due to the growing of food demand worldwide. The objective of this work was to study morphological and physiological changes in main stem and tillers, and to characterize the processes involved on developmental plasticity of wheat plants affected by sowing densities under plant growth regulators application. The experiment was carried out at Faculdade de Ciencias Agronômicas – Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu (SP) in 2010 crop season. The experimental design was split-plot with four replications. The plots were consisted by four plant densities (30, 50 70 and 90 plants m-1) and the subplots were consisted by plant growth regulators [control, (Ax+GA+CK), Trinexapac- Ethyl e (Ax+GA+CK) + Trinexapac-Ethyl]. Tiller emission, main stem and tiller dry mass accumulation, plant height, gas exchange, morphological and physiological characteristics of flag leaf, main stem and tiller yield components and yield were determined. The contribution of main stem and tillers on grain yield and yield potential of tillers in relation to main stem were evaluated at the end of the crop cycle. Trinexapac-Ethyl application resulted in decreasing of plant height, source-sink distance, flag leaf length and chlorophyll content, however, the grain yield was not affected. The increase in the number of plants per unit area resulted in linear lower emission of tillers, dry matter accumulation and relationship between main stem and tillers, with direct relation to the yield. The increase in yield of the lower plant populations occurred due to the higher number of grain weight per spike, offsetting the decrease in the number of spikes per square meter. The competition for production factors resulted in decreasing in contribution of tillers on grain yield and yield potential... (Complete abstract click electronic access below)

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-02-20T18:25:57Z No. of bitstreams: 1 2017 - F?bio Ferreira Cruvinel.pdf: 1438823 bytes, checksum: 49768f1c89baea668d06d925b16c5e0c (MD5) / Made available in DSpace on 2018-02-20T18:25:57Z (GMT). No. of bitstreams: 1 2017 - F?bio Ferreira Cruvinel.pdf: 1438823 bytes, checksum: 49768f1c89baea668d06d925b16c5e0c (MD5) Previous issue date: 2017-02-21 / CAPES / The pitaya (Hylocereus undatus (Haw.) Britton & Rose) has an exotic shape, pleasant taste, easy digestibility and antioxidant properties, which have given greater demands in the Brazilian market for this fruit in recent years. The production of seedlings is a determining factor for the successful installation of pitaias orchards and the use of phytoregulators such as BAP (6-benzylaminopurine) and organic substrates, such as millipede-compost, in the production of seedlings of this fruit is still little reported. The objective of this work was to analyze the effect of BAP and millipede-compost on the production of pitaia seedlings. Pitaia cuttings from Fazendinha Agroecol?gica - Km 47, in the town of Serop?dica-RJ, were used. The stakes were standardized with 20 cm in length. The experiment with the phytoregulator was conducted in the greenhouse sector of the UFRRJ and the cuttings planted in substrate composed of sand and bovine manure in a ratio of 1: 1 by volume in a completely randomized design (DIC) in a factorial scheme (2X4) with 4 replicates: (2) By immersion and incision made at the apex of cladodes stakes versus (4) four doses of BAP (0, 50, 100 and 150 mg.L-1) were applied. Non-destructive Growth Analysis was performed and the simple correlations were calculated with the climatic data obtained at the Ecologia Automatic Station in Serop?dica-RJ. For the experiment with the millipede compost, the cutting was conducted in pots with three types of substrates: Gongocomposto, Biomix? and sand + bovine manure (1: 1 by volume) in DIC with five replicates. The results showed a linear tendency in number of shoots and quadratic tendency in other parameters (length, fresh and dry weight) evaluated with use of BAP. There was a correlation between the growth rates of the pitaya with the data of Global Solar Radiation of the 19h (22h UTC) period. The use of the gongocomposto showed larger and more vigorous plants of pitaia. / O fruto da pitaieira (Hylocereus undatus (Haw.) Britton & Rose) apresenta forma ex?tica, sabor agrad?vel, f?cil digestibilidade e propriedades antioxidantes, caracter?sticas essas que proporcionaram maiores demandas no mercado brasileiro por essa fruta nos ?ltimos anos. A produ??o de mudas ? um fator determinante para o sucesso da instala??o de pomares de pitaias e o uso de fitorreguladores como o BAP (6-benzilaminopurina) e substratos org?nicos, como o gongocomposto, na produ??o de mudas desta frut?fera ainda ? pouco relatado. O objetivo deste trabalho foi analisar o efeito do BAP e gongocomposto na produ??o de mudas de pitaia. Utilizaram-se estacas de pitaieira proveniente da Fazendinha Agroecol?gica ? Km 47, no munic?pio de Serop?dica-RJ. As estacas foram padronizadas com 20 cm de comprimento. O experimento com o fitorregulador foi conduzido no setor de olericultura da UFRRJ, em casa de vegeta??o e as estacas plantadas em substrato composto por areia e esterco bovino na propor??o 1:1 em volume em Delineamento Inteiramente Casualizado (DIC), com 4 repeti??es, em esquema fatorial (2X4), sendo (2) por imers?o e incis?o feita no ?pice das estacas dos clad?dios versus (4) quatro doses da citocinina BAP (0, 50, 100 e 150 mg.L-1). Foi realizada a An?lise de Crescimento n?o destrutiva e foram calculadas as correla??es simples com os dados clim?ticos obtidos na Esta??o Autom?tica Ecologia em Serop?dica-RJ. Para o experimento com o gongocomposto a estaquia foi conduzida em vasos com tr?s tipos de substratos: Gongocomposto, Biomix? e areia+esterco bovino (1:1 em volume) em DIC com cinco repeti??es. Os resultados mostraram uma tend?ncia linear positiva no n?mero de brota??es e quadr?tica para o incremento de comprimento, peso fresco e seco das brota??es para o uso da citocinina BAP no incremento de. Houve correla??o entre as taxas de crescimento da pitaia com os dados de Radia??o Solar Global do per?odo das 19h (22h UTC). O uso do gongocomposto proporcionou maiores e mais vigorosas mudas de pitaieira.

Open Access Article

Magistro darbe tiriama in vitro sąlygų įtaka lenkinio maumedžio regeneracijai ir genetinė įvairovė. Darbo objektas – Lenkiniai maumedžiai (Larix polonica Racib.) iš Degsnės sėklinės maumedžių palantacijos ir europinis maumedis (Larix decidua Mill.) iš Girionių parko. Darbo tikslas – Nustatyti sąlygas maumedžio palikuonių regeneracijai gemalų kultūroje ir įvertinti genetinę įvairovę ankstyvose vystymosi stadijose. Darbo metodai – Regeneravusių mikroūglių įvertinimas, DNR nustatymas. Darbo rezultatai: Atlikus maumedžių regeneracijos tyrimus in vitro nustatyta: sterilinimo procedūrą papildžius aseptiku “ACE” pavyko ženkliai padidinti sterilių eksplantų skaičių. Po sterilinimo dauginamąją medžiagą savaitę palaikius tamsoje 3◦C temperatūroje ir tik po to pasodinus į sterilią terpę sulauktas didesnis sudygusių gemalų skaičius. Terpės pH limituojantis eksplantų vystymąsi veiksnys. Citokininas N–izopentenilaminopurinas (2–iP) WPM terpėje tinkamiausias lenkinio maumedžio kultivavimui audinių kultūroje. Oligonukleotidiniai pradmenys A 03, R370 03, R370 04, R370 10 informatyvūs maumedžių polimorfizmo tyrimams, o oligonukleotidinis pradmuo R170 01 neinformatyvus. / Master thesis in vitro conditions on polish larch regeneration and genetic diversity. Object of study – Polish larch (Larix polonica Racib) from Degsnės larch seed plantation and European larch (Larix decidua Mill.) from the park Girionys. Goal of study – Establish conditions for the regeneration of larch seed embryo culture and assessment of genetic diversity in the early stages of development. Methods of study – Regenerate shoot evaluation, DNA identification. Results of study: After larch regeneration studies in vitro set: steriling procedure for adding an antiseptic “Ace” was able to significantly increase the number of aseptic explants. After a week sterile propagating material in the dark brew 3◦C, and only then seated in a sterile environment attracted larger number of germ sprouted. PH factor limit explants development. Cytokinin 2-iP in WPM medium appropriate polish larch cultivation in tissue culture. Oligonucleotidic primers A 03, R370 03 R370 04 R370 10 informative for larch polymorphism analysis, and oligonucleotidic primer R170 01 noninterpretable.

Consistency of yield and quality of seed are traits not yet optimised by the brassica seed industry in New Zealand. As of 2008, seed producers in Canterbury, New Zealand, exported approximately $18m of brassica seed. However, there is a need to increase both seed quantity and/or quality. The plant hormone group, the cytokinins, regulates many stages of plant growth and development, including cell division and enhancement of sink strength, both of which are important processes in seed development and embryonic growth. The two gene families targeted in this project play a key role in maintaining cytokinin homeostasis. Isopentenyl transferase (IPT) catalyzes the rate limiting step in the formation of cytokinins, and cytokinin oxidase/dehydrogenase (CKX) irreversibly inactivates cytokinins. The aim of this project was to identify those cytokinin gene family members expressing specifically during the early phases of pod and seed development. Initially this study used a rapid-cycling Brassica rapa (RCBr) because of its rapid life cycle, then, as the project developed, a commercial crop of forage brassica (B. napus) was studied. Reverse transcriptase PCR (RT-PCR) and BLAST analysis was used to identify putative IPT and CKX genes from RCBr and B. napus. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to measure the expression of individual gene family members during leaf, flower, pod and seed development. BrIPT1, -3, and -5, and BrCKX1, -2, -3, and -5/7 and were shown to express differentially both temporally and spatially within RCBr root, stem, leaf, seed, and pod tissues. BnIPT1, 3 and 7 and BnCKX1, 2, 5 and 7 were also differentially expressed. Particularly strong expression was shown by BrIPT3, BrIPT5 and BrCKX2 in developing seeds. Both Brand BnIPT3 expressed strongly in maturing leaves. In normal plant growth and development, biosynthesis and metabolism of cytokinin is tightly regulated by the plant. Increasing the levels of cytokinins during seed development, either by over expressing IPT3 or IPT5, or decreasing the expression of CKX2, or both, could potentially increase both seed yield and seed vigour.

Improved sequencing technologies have created an explosion of sequence information that is analyzed and proteins are annotated automatically. Annotations are made based on similarity scores to previously annotated sequences, so one misannotation is propagated throughout databases and the number of misannotated proteins grows with the number of sequenced genomes. A systematic approach to correctly identify the function of proteins in the amidohydrolase superfamily is described in this work using Clusters of Orthologous Groups of proteins as defined by NCBI. The focus of this work is COG1816, which contains proteins annotated, often incorrectly, as adenosine deaminase enzymes. Sequence similarity networks were used to evaluate the relationship between proteins. Proteins previously annotated as adenosine deaminases: Pa0148 (Pseudomonas aeruginosa PAO1), AAur_1117 (Arthrobacter aurescens TC1), Sgx9403e and Sgx9403g, were purified and their substrate profiles revealed that adenine and not adenosine was a substrate for these enzymes. All of these proteins will deaminate adenine with values of kcat/Km that exceed 105 M-1s-1. A small group of enzymes similar to Pa0148 was discovered to catalyze the hydrolysis of N-6-substituted adenine derivatives, several of which are cytokinins, a common type of plant hormone. Patl2390, from Pseudoalteromonas atlantica T6c, was shown to hydrolytically deaminate N-6-isopentenyladenine to hypoxanthine and isopentenylamine with a kcat/Km of 1.2 x 107 M^-1 s^-1. This enzyme does not catalyze the deamination of adenine or adenosine. Two small groups of proteins from COG1816 were found to have 6-aminodeoxyfutalosine as their true substrate. This function is shared with 2 small groups of proteins closely related to guanine and cytosine deaminase from COG0402. The deamination of 6-aminofutalosine is part of the alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminofutalosine is deaminated with a catalytic effeciency of 105 M-1s-1 or greater, Km’s of 0.9 to 6.0 µM and kcat’s of 1.2 to 8.6 s-1. Another group of proteins was shown to deaminate cyclic- 3’, 5’ -adenosine monophosphate (cAMP) to produce cyclic-3’, 5’-inosine monophosphate, but will not deaminate adenosine, adenine or adenosine monophosphate. This protein was cloned from a human pathogen, Leptospira interrogans. Deamination may function in regulating the signaling activities of cAMP.

Diversity in plant architecture is largely generated by the post-embryonic regulation of meristem initiation and activity. In a phenomenon known as apical dominance, the active growth of the shoot apical meristem (SAM) exerts significant inhibitory force on the outgrowth of axillary meristems (AMs) into shoot branches. The degree of branching in plants is a determinant of yield in many crop species and is carefully regulated to ensure that plants only branch at specific stages of development or in response to their environment. Apical dominance has been attributed to the action of the hormone auxin, produced in SAM tissues and transported downwards. A second hormone, cytokinin, acts antagonistically to auxin to promote branching. Nonetheless, the exact mechanism by which these hormones operate is still being elucidated and continued research suggested that novel signals are involved. The recent discovery that strigolactones, previously implicated in parasitic weed germination and mycorrhizal associations, are branching inhibitors supports the existence of additional signals controlling branching in plants. In garden pea (Pisum sativum) strigolactones are synthesised by the coordinated action of the carotenoid cleavage dioxygenase (CCD) family enzymes, RMS1 (RAMOSUS1) and RMS5. These are encoded by MAX4 (MORE AXILLARY GROWTH4) and MAX3 in Arabidopsis thaliana respectively. Mutants for MAX genes have increased amounts of auxin travelling in the polar auxin transport stream (PATS) of inflorescence stems but exhibit increased branching that is insensitive to inhibition by this auxin. Two hypotheses for the action of strigolactones have been presented. The first is that strigolactones modulate the levels of auxin transport proteins, preventing axillary buds from establishing an active auxin transport flow into the primary stem, which inhibits growth. The second is that strigolactones act downstream of auxin signalling to inhibit the action of outgrowth-promoters. Consistent with this latter hypothesis, in pea, rice (Oryza sativa) and petunia (Petunia hybrida), the expression of RMS1/MAX4 orthologues is auxin regulated. These genes are also regulated by feedback signalling in strigolactone pathway mutants and this is proposed to involve an additional novel signal. In Arabidopsis, however, research showed that MAX4 is not regulated by feedback or auxin in the shoot and placed doubt on the importance of this regulation for branching control. The strigolactone biosynthetic pathway offers a novel target for the manipulation of plant architecture and yield while controlling the germination of parasitic weed species that are detrimental to agriculture. Therefore, a greater understanding of the pathway and its regulators is beneficial. The majority of the research in this thesis pre-dates the discovery of strigolactones as the RMS/MAX-derived branching inhibitor, yet aimed to clarify the evolutionary conservation and functional importance of the regulation of strigolactone biosynthetic genes by auxin and feedback signalling in Arabidopsis. Quantitative real-time PCR analysis demonstrated that MAX3 and MAX4 are co-ordinately and systemically regulated by auxin and by feedback throughout development. Both auxin and feedback regulation required the AXR1/TIR1 auxin response pathway, which targets Aux/IAA transcriptional repressors for proteasomal degradation. In particular, correct degradation of the Aux/IAA protein IAA12 appears to be necessary for optimal MAX3 and MAX4 expression. Moreover this regulation affects strigolactone-dependent branching inhibition. Therefore it is proposed that auxin inhibits branching, in part, by positively regulating strigolactone synthesis. As feedback requires AXR1, this also suggests that increased auxin level and/or signalling in the PATS in conditions of reduced strigolactone signalling mediates feedback regulation of the strigolactone pathway. Consistent with this, microarray analysis revealed that in addition to the inflorescence, max mutants have increased global auxin-responsive gene expression associated with the PATS in the vegetative stage. The pea RMS1 gene was the first strigolactone pathway gene demonstrated to be auxin-regulated. Sequencing of the RMS1 promoter and comparative bioinformatic analysis with promoters of other strigolactone synthesis genes revealed a number of conserved, putative regulatory cis-elements that could mediate this regulation and cross-talk with additional branching cues. However a 2.5 kb fragment of the RMS1 promoter was not sufficient to drive transcriptional and translational fusions with GFP and the RMS1 coding region in Arabidopsis. The RMS1 coding region driven by the CAMV 35S promoter complemented the max4 mutant but did not affect branching induced by auxin-depleting treatments. Grafting studies with axr1 and iaa12 mutants, and decapitation and auxin-transport inhibition in max4 mutants, demonstrated that auxin signalling has a function in branching control independent from the regulation of strigolactone synthesis genes. Overall, data obtained herein was incorporated into current models for the interaction of the strigolactone pathway with auxin and cytokinin in the control of shoot branching. It is suggested that both strigolactone and auxin have the capacity to regulate the levels or distribution of each other in interlocking feedback loop that intersects with additional developmental, physiological and environmental cues for the precise control of axillary branching in plants.

Diversity in plant architecture is largely generated by the post-embryonic regulation of meristem initiation and activity. In a phenomenon known as apical dominance, the active growth of the shoot apical meristem (SAM) exerts significant inhibitory force on the outgrowth of axillary meristems (AMs) into shoot branches. The degree of branching in plants is a determinant of yield in many crop species and is carefully regulated to ensure that plants only branch at specific stages of development or in response to their environment. Apical dominance has been attributed to the action of the hormone auxin, produced in SAM tissues and transported downwards. A second hormone, cytokinin, acts antagonistically to auxin to promote branching. Nonetheless, the exact mechanism by which these hormones operate is still being elucidated and continued research suggested that novel signals are involved. The recent discovery that strigolactones, previously implicated in parasitic weed germination and mycorrhizal associations, are branching inhibitors supports the existence of additional signals controlling branching in plants. In garden pea (Pisum sativum) strigolactones are synthesised by the coordinated action of the carotenoid cleavage dioxygenase (CCD) family enzymes, RMS1 (RAMOSUS1) and RMS5. These are encoded by MAX4 (MORE AXILLARY GROWTH4) and MAX3 in Arabidopsis thaliana respectively. Mutants for MAX genes have increased amounts of auxin travelling in the polar auxin transport stream (PATS) of inflorescence stems but exhibit increased branching that is insensitive to inhibition by this auxin. Two hypotheses for the action of strigolactones have been presented. The first is that strigolactones modulate the levels of auxin transport proteins, preventing axillary buds from establishing an active auxin transport flow into the primary stem, which inhibits growth. The second is that strigolactones act downstream of auxin signalling to inhibit the action of outgrowth-promoters. Consistent with this latter hypothesis, in pea, rice (Oryza sativa) and petunia (Petunia hybrida), the expression of RMS1/MAX4 orthologues is auxin regulated. These genes are also regulated by feedback signalling in strigolactone pathway mutants and this is proposed to involve an additional novel signal. In Arabidopsis, however, research showed that MAX4 is not regulated by feedback or auxin in the shoot and placed doubt on the importance of this regulation for branching control. The strigolactone biosynthetic pathway offers a novel target for the manipulation of plant architecture and yield while controlling the germination of parasitic weed species that are detrimental to agriculture. Therefore, a greater understanding of the pathway and its regulators is beneficial. The majority of the research in this thesis pre-dates the discovery of strigolactones as the RMS/MAX-derived branching inhibitor, yet aimed to clarify the evolutionary conservation and functional importance of the regulation of strigolactone biosynthetic genes by auxin and feedback signalling in Arabidopsis. Quantitative real-time PCR analysis demonstrated that MAX3 and MAX4 are co-ordinately and systemically regulated by auxin and by feedback throughout development. Both auxin and feedback regulation required the AXR1/TIR1 auxin response pathway, which targets Aux/IAA transcriptional repressors for proteasomal degradation. In particular, correct degradation of the Aux/IAA protein IAA12 appears to be necessary for optimal MAX3 and MAX4 expression. Moreover this regulation affects strigolactone-dependent branching inhibition. Therefore it is proposed that auxin inhibits branching, in part, by positively regulating strigolactone synthesis. As feedback requires AXR1, this also suggests that increased auxin level and/or signalling in the PATS in conditions of reduced strigolactone signalling mediates feedback regulation of the strigolactone pathway. Consistent with this, microarray analysis revealed that in addition to the inflorescence, max mutants have increased global auxin-responsive gene expression associated with the PATS in the vegetative stage. The pea RMS1 gene was the first strigolactone pathway gene demonstrated to be auxin-regulated. Sequencing of the RMS1 promoter and comparative bioinformatic analysis with promoters of other strigolactone synthesis genes revealed a number of conserved, putative regulatory cis-elements that could mediate this regulation and cross-talk with additional branching cues. However a 2.5 kb fragment of the RMS1 promoter was not sufficient to drive transcriptional and translational fusions with GFP and the RMS1 coding region in Arabidopsis. The RMS1 coding region driven by the CAMV 35S promoter complemented the max4 mutant but did not affect branching induced by auxin-depleting treatments. Grafting studies with axr1 and iaa12 mutants, and decapitation and auxin-transport inhibition in max4 mutants, demonstrated that auxin signalling has a function in branching control independent from the regulation of strigolactone synthesis genes. Overall, data obtained herein was incorporated into current models for the interaction of the strigolactone pathway with auxin and cytokinin in the control of shoot branching. It is suggested that both strigolactone and auxin have the capacity to regulate the levels or distribution of each other in interlocking feedback loop that intersects with additional developmental, physiological and environmental cues for the precise control of axillary branching in plants.