Les tissus vasculaires sont d’une grande importance pour la physiologie et le développement de la plante. Le xylème et le phloème composent ce tissu. En assurant le transport des sèves brutes et élaborées, ils permettent ainsi de redistribuer minéraux et métabolites entre les différents organes de la plante. La régulation de la mise en place de ces deux tissus est hautement contrôlée tant d’un point de vue génétique que spatio-temporel. Parmi les gènes impliqués dans la mise en place des tissus vasculaires, le gène OCTOPUS (OPS) intervient très précocement au cours du développement dans la mise en place du patron vasculaire ainsi que différenciation du phloème(Bauby et al., 2007; Truernit et al., 2012). OPS appartient à une famille de gènes spécifiques des plantes supérieures. Cependant la fonction moléculaire de la protéine OPS reste inconnue. Une combinaison d’approches physiologique, biochimique, moléculaire, cytologique et génétique a permis de montrer que le gène OPS est un nouveau régulateur positif de la voie signalisation des brassinostéroïdes (BR). Les BR sont des hormones végétales impliquées dans de nombreux processus développementaux incluant l’élongation cellulaire ou encore l’organisation des tissus vasculaires. Bien que la découverte des BR date seulement de 1970 (Mitchell et al., 1970; Mitchell and Gregory,1972; Grove et al., 1979), l’intérêt commun que les scientifiques lui ont porté fait que sa voie de signalisation est aujourd’hui très détaillée. Parmi les composants de la voie des BR, la protéine kinase BIN2 en est le régulateur clef (Li et al., 2001) réprimant la voie par son action sur les facteurs de transcription BES1 et BZR1(Yin et al., 2002; Wang et al., 2002). OPS interagit physiquement avec la protéine BIN2 au niveau de la membrane plasmique, pouvant créer une inhibition par délocalisation de la protéine BIN2 de son site d’activité. A ce titre, les défauts de phloème observés chez le mutant opssont restaurés lorsque l’activité de BIN2 est abolie ou lorsque la voie est induite en dessous de BIN2. Ainsi nous montrons que la voie des BR est directement impliquée dans la différenciation du phloème. Plus généralement, une étude de la redondance fonctionnelle d’autres membres de la famille OPS semble indiquer qu’ils pourraient exercer une fonction similaire de régulateurs positifs de la voie des BR au sein d’autres tissus de la plante / Vascular tissues play an important role in plant physiology and development. Vascular tissues consist on xylem and phloem that ensure sap transport and permit to redistribute mineral and metabolites between different organs. Regulation of vascular tissues establishment is highly controlled in space and time. Among genes involved in vascular tissues formation, OCTOPUS (OPS) gene operates early during development to control vascular patterning and to induce phloem differentiation(Bauby et al., 2007; Truernit et al., 2012). OPS belongs to a multigenic family conserved in high plant. However, molecular function of OPS protein remains unclear. A combination of physiological, biochemical, molecular, cytological and genetic approaches allowed to show that OPS gene is a new positive regulator of brassinosteroid (BR) signaling pathway. BR are phytohormones involved in many developmental processes such as cell elongation or vascular tissues organization. Although BR were discovered in 1970 (Mitchell et al., 1970; Mitchell and Gregory, 1972; Grove et al., 1979), interest of researchers for this hormone permitted its detailed description. Among the component of BR signaling, the BIN2 kinase is the key regulator (Li et al., 2001) which represses the pathway through its action on BES1 and BZR1 transcription factors(Yin et al., 2002; Wang et al., 2002). OPS interacts physically with BIN2 at the plasma membrane which could create an inhibition by delocalization of the BIN2 protein from its activity place. As such, opsphloem defects are restored when the activity of BIN2 is inhibited or when the pathway is induced downstream of BIN2. Thus, we show that BR pathway is directly involved in phloem differentiation. More generally, a study of the functional redundancy of other OPS family members suggests that they could have a similar function as positive regulators of BR pathway in other plant tissues.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第25513号 / 生博第529号 / 新制||生||70(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 中野 雄司, 教授 河内 孝之, 教授 荒木 崇 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Made available in DSpace on 2017-07-25T19:31:00Z (GMT). No. of bitstreams: 1 Marina Oliveira.pdf: 1283767 bytes, checksum: 902cc2416d1d157460b534a7f6b62ed8 (MD5) Previous issue date: 2016-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The expansion of cultivated blueberry fields in Brazil is directly linked to the provision of quality seedlings to the market, which may result in the increase of production. The plant micropropagation technique enables the production and provides high quality plants, physiologically viable and healthy and free from viruses in a short space of time. Woody plants presente difficulty in the in vitro rooting, in this context the development of a more efficient system of rooting results in plants with higher physiological quality and reduction of losses during the acclimatization phase. In the first experimente the treatments consisted in 8 doses of BIOBRAS 16 (0; 0,1; 0,2; 0,3; 0,4; 0,5; 0,6; 0,7 mg L-1) added to the culture medium at the time of preparation. The following characteristics were evaluated: number of corns, callus diameter, number of roots, the total root length, total length, number of shoots, length of shoots, number of leaves and fresh mass. The lowest concentration of BIOBRAS 16 used (0,1 mg L-1) presented a higher percentage of rooting, root length and number of leaves, but also stimulated the development of calluses. The dose of 0,4 mg L-1 stimulated the formation of shoots and length thereof. There was no significant difference for other parameters evaluated. In the second experiment the treatments consisted in diferent doses of BIOBRAS 16 (0,1; 0,3; 0,5 mg L-1) associated to different doses of indole acetic acid (1; 3; 5 L-1 μM) added to the culture medium during preparation. The parameters evaluated were the same as the experiment 1. The use of BIOBRAS 16 associated with indolacetic acid showed a positive effect for the development of in vitro characteristics of blueberry. For rooting, the concentration of 0,3 mg L-1 BIOBRAS 16 + 5 μM AIA showed a higher percentage. Concentrations of 0,5 mg L-1 BIOBRAS 16 + 5 μM of AIA induced the formation and callus diameter, undesirable feature when the goal is rooting. For the parameters number of shoots and number of leaves, the best result was the concentration of 0,1 mg L-1 BIOBRAS 16 + 5 μM of AIA. / A expansão da área cultivada de mirtileiro no Brasil está diretamente vinculada a oferta de mudas de qualidade ao mercado, que poderá resultar em aumento da produção. A técnica da micropropagação de plantas viabiliza a produção e fornece plantas de elevada qualidade, fisiologicamente viáveis, sadias e isentas de vírus emcurto espaço de tempo. Plantas lenhosas apresentam dificuldade de enraizamento in vitro, nesse contexto o desenvolvimento de um sistema de enraizamento mais eficiente resulta em mudas com maior qualidade fisiológica e diminuição de perdas durante a fase de aclimatização. No primeiro experimento os tratamentos consistiram em 8 doses de BIOBRAS 16 (0; 0,1; 0,2; 0,3; 0,4; 0,5; 0,6; 0,7 mg L-1) adicionados no meio de cultura no momento do preparo. As seguintes características foram avaliadas: número de calos, diâmetro do calo, número de raízes, comprimento total de raízes, comprimento total, número de brotações, comprimento de brotações, número de folhas e massa fresca. A menor concentração de BIOBRAS 16 utilizada (0,1 mg L-1) apresentou maior percentual de enraizamento (%), comprimento de raízes (cm) e número de folhas, porém também estimulou o desenvolvimento de calos. A dose de 0,4 mg L-1 estimulou a formação de brotações e o comprimento das mesmas. Não houve diferença significativa para os demais parâmetros avaliados. No segundo experimento os tratamentos consistiram em diferentes doses de BIOBRAS 16® (0,1; 0,3; 0,5 mg L-1) associadas a diferentes doses de ácido incolacético (1; 3; 5 μM L-1) adicionados ao meio de cultura no momento do preparo. As características avaliadas foram número de calos,diâmetro do calo, número de raízes, comprimento total de raízes, comprimento total, número de brotações, comprimento de brotações, número de folhas e massa fresca. A utilização de BIOBRAS 16 associado a ácido indolacético mostrou efeito positivo para características de desenvolvimento in vitro de mirtileiro. Para enraizamento, as concentrações de 0,3 mg L-1 de BIOBRAS 16 + 5 μM de AIA apresentaram maior percentual. As concentrações de 0,5 mg L-1 de BIOBRAS 16® + 5 μM de AIA induziram a formação e diâmetro de calos, característica indesejável quando o objetivo é o enraizamento. Para as características número de brotações e número de folhas, o melhor resultado foi da concentração de 0,1 mg L-1 de BIOBRAS 16 + 5 μM de AIA.

Apomixis, asexual or clonal seed production in plants, can decrease the cost of producing hybrid seed and enable currently open pollinated crops to be converted to more vigorous and higher yielding hybrids that can reproduce themselves through their own seed. Sexual reproduction may be triggered by a programmed stress signaling event that occurs in both the meiocyte, just prior to meiosis, and later in the egg just prior to embryo sac maturation. The prevention of stress signaling and the activation of a pro-growth signal prior to meiosis triggered apomeiosis, the first half of apomixis. The same approach was used prior to embryo sac maturation to trigger parthenogenesis, the second half of apomixis. This discovery suggests that apomixis exists as a program that can be activated by the appropriate metabolic signal at the appropriate developmental stages. Therefore, apomixis may be alternative mode of reproduction rather a ‘broken’ form of sexual reproduction.

Receptor kinases (RKs) are transmembrane proteins that have been shown to regulate an array of important processes in A. thaliana, including polar cell growth, plant reproduction, and many other plant growth processes. In this thesis, I examine RECEPTOR KINASE 7 (RK7) and FERONIA (FER), two closely related transmembrane RKs, and their effects on plant reproduction. The RK7 gene when knocked out (rk7) in conjunction with FER resulted in delayed plant growth, decreased seed yield, and a lower percentage of the seeds germinating as compared to the single FER knockout. Transgenic plants with GUS reporter driven by RK7 promoter and RK7 promoter expressed GFP-tagged RK7 (RK7-GFP) were generated to study, respectively, the expression property of the RK7 gene and characterize the location of the RK7 protein. RK7 expression increased in the papillary cells as a direct result of pollination. Transgenic plants with RK7-GFP showed that RK7 protein localizes to the plasma membrane of stigma cells and pollination induces prominent internalization of this protein. RK7 is also expressed during seedling growth. rk7 mutant seedlings had a much weaker physiological response to brassinosteroids than wild type plants, implicating an involvement of RK7 in brassinosteroid signaling. Taken together this data point to the importance of RK7 in plant growth and reproduction through its ability to receive and transduce signals.

Plants are fundamental to life on earth, crucially providing the basis of our food supply. As world population continues to grow, so too does the pressure on our agricultural systems, with one of the biggest challenges being the control of plant pathogens to ensure a healthy crop. The interaction between plant and pathogen is complex, with subtleties at the molecular level dictating the boundary between health and disease. This is exemplified by pathogen effectors; secreted proteins which enter the plant cell and interact with host targets to facilitate infection. AVR2 is one such effector, secreted by the oomycete pathogen Phytophthora infestans; the pathogen responsible for potato late blight. AVR2 interacts with a family of kelch-repeat containing phosphatases in potato, the BSLs, implicated in brassinosteroid pathway signalling – a major hormone signalling pathway in plants associated with growth and development. This work investigates the role of AVR2 and its host targets in pathogen virulence, with focus on the link between the brassinosteroid pathway and immunity in S. tuberosum. StBSL1 is shown to be a ‘susceptibility factor’ in P. infestans infection – a host protein with a positive effect on pathogen virulence. AVR2 stabilises BSL1 in planta, and both AVR2 and BSL1 are shown to suppress primary defence responses in the plant. Transcriptional analysis of brassinosteroid-treated S. tuberosum is used to identify a set of marker genes for active BR signalling. Strikingly, AVR2 is shown to upregulatethis pathway, and specifically upregulates the transcription factor StHBI1-like, identified as a suppressor of immunity. These findings reveal a novel mechanism in oomycete effector biology; the exploitation of crosstalk between the brassinosteroid pathway and immune signalling in plants.

Douglas-fir (Pseudotsuga menziesii) is an economically important softwood that is clonally propagated for reforestation purposes by somatic embryogenesis. The molecular basis of embryogenesis in conifers is largely unknown and this prevents progress in somatic embryogenesis protocols. In angiosperms, the LEAFY COTYLEDON1 (LEC1) gene, encoding the HAP3 subunit of the eukaryotic CCAAT box-binding factor, is important in embryo formation, and necessary for somatic embryogenesis. A candidate gene strategy was employed to isolate the Douglas-fir LEC1 homologue, PmLEC1, via the polymerase chain reaction (PCR) with degenerate primers based on the Arabidopsis conserved domain, and the full-length cDNA sequence was obtained by rapid amplification of cDNA ends-PCR (RACE-PCR). The putative protein sequence shared high sequence identity with Arabidopsis LEC1. Northern analysis and quantitative real-time PCR indicate that this is an embryo-specific gene, expressed with the highest abundance during early embryogenesis. Antibodies were raised against a synthetic 18-amino acid PmLEC1 peptide, and in contrast to mRNA expression, Western blotting shows that PmLEC1 protein expression persists until the seedling stage. To gain insight into modulation of PmLEC1 expression and its inducibility in mature tissues, stress and hormone treatments were performed on mature seed and the promoter sequence was isolated by genome walking. Sorbitol, mannitol and 2,4-epibrassinolide were found to significantly up-regulate PmLEC1 expression. The PmLEC1 promoter contains a 5’ UTR intron with numerous enhancer elements, and factors that bind to these elements mediate responses to auxin, UV light and developmental cues, osmotic stress, biotic stress, and tissue culture. Some of the regulatory elements are binding sites for seed-specific transcription factors that are well known from angiosperms, providing new evidence that AGL15, ABI3 and VP1 proteins have a direct role on LEC1 expression. In investigating the embryogenic capacity of PmLEC1, ectopic expression of PmLEC1 in the embryo lethal Arabidopsis lec1-1 null mutant complemented the mutation and permitted the production of viable, desiccation tolerant seeds. In addition, transgenic seedlings produced embryo-like structures from vegetative organs and expressed seed-specific genes. In wild type plants, ectopic expression of PmLEC1 resulted in a bushy phenotype but expression of seed-specific genes was not observed. Taken together, these results show that PmLEC1 is an embryo-specific gene with an essential role throughout embryogenesis, and PmLEC1 expression may be induced in mature seeds by stress and hormone treatments. Because mature seeds show only trace amounts of PmLEC1 transcripts and Douglas-fir somatic embryogenesis can only be induced from immature embryos, this information provides useful insight into initiation of embryogenesis from vegetative tissues. The identification of binding sites for transcription factors known from angiosperms in the promoter region of PmLEC1 has revealed the identity of several genes which are expected to play pivotal roles in conifer embryogenesis.

Brassinosteroids and ecdysteroid are naturally occuring chemical compounds in plants. The aim of this study was to show whether the application of exogenous steroids such as brassinosteroids (24-epibrassinolide, 28-homobrassinolide, 24-epicastasterone) and ecdysteroid (20-hydroxyecdysone) can affect the morphological parameters and primary photosyntetic processes of selected monocotyledons that was barley, wheat, maize and sorghum. Non-destructive method for measuring of fast kinetics of chlorophyll fluorescence in investigated plants was used. Analysis of primary photosynthetic processes was realized in five time periods from the application of exogenous steroids on the two different old leaves. The response to the treatment by exogenous steroid was different species by plant species. One week after the application of exogenous steroids plants showed differences in their morphological parameters but most of them were not signifficant. The most steroid-sensitive plant was wheat morphological parameters of were significantly different from untreated control plants. No change in maximum quantum yield of photosystem II to the application of exogenous steroid in plants of Sorghum bicolor L. was detected. In the other examined plant species changes in photosynthesis parameters were detected which...