No description available.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O feijÃo-de-corda (Vigna unguiculata) Ã uma leguminosa bastante utilizada na alimentaÃÃo de famÃlias de baixa renda da regiÃo nordeste do Brasil. Embora suas sementes sejam ricas em proteÃnas, estas sÃo deficientes em aminoÃcidos sulfurados na sua composiÃÃo. Dessa forma, um aumento na qualidade nutricional dessas sementes tem sido um dos principais objetivos dos programas de melhoramento genÃtico para essa espÃcie. AlÃm de determinar o padrÃo de deposiÃÃo de proteÃnas durante o desenvolvimento de embriÃes zigÃticos e somÃticos, nÃs pretendemos identificar genes com padrÃes especÃficos de expressÃo durante a embriogÃnese com a finalidade de utilizÃ-los em programas de melhoramento genÃtico. Utilizando a tÃcnica de eletroforese bidimensional (2D-PAGE) foi comparado o padrÃo protÃico de sementes em desenvolvimento com 10 dias apÃs a antese (DAA), sementes maduras e de suspensÃes celulares embriogÃnicas (SCE) obtidas de calos embriogÃnicos friÃveis (CEF) de feijÃo-de-corda. Para cada estÃgio de desenvolvimento da semente e para as SCE foram obtidos mapas de referÃncia proteÃmicos altamente reprodutÃveis numa faixa de pH de 3-10 e 4-7. VÃrios âspotsâ foram selecionados baseando-se na quantidade ou volume relativo de cada âspotâ e na taxa de expressÃo. Cerca de 800 (para sementes em desenvolvimento) e 130 (para SCE) âspotsâ protÃicos regulados para cima, para baixo, que se mantÃm constantes ou que sÃo especÃficos durante o desenvolvimento foram retirados dos gÃis 2D para anÃlise por espectrometria de massa. Algumas estratÃgias foram utilizadas para a identificaÃÃo das proteÃnas, como: PMF (peptide mass fingerprinting) e busca atravÃs de dados nÃo processados (MS/MS ion search). Dessa forma, cerca de 400 proteÃnas foram identificadas em sementes e cerca de 70 proteÃnas foram identificadas para SCE. A maioria das proteÃnas fram classificadas como proteÃnas do metabolismo primÃrio, energÃtico, proteÃnas de destinaÃÃo/proteÃnas de reserva e proteÃnas de defesa ou relacionadas a algum tipo de estresse. / Cowpea (Vigna unguiculata) is a leguminous plant highly utilized by low-income earners of Northeastern Brazil. However, proteins found in the crop are highly deficient in sulfur-containing amino acids. In line with this, improvement of nutritional quality of cowpea seeds has been one of the major goals of breeding programs of the crop. A starting point in this respect is a concerted effort to study the basic biochemical and physiological aspects of the development of cowpea seed. Besides determining the protein deposition pattern during the development of zygotic embryos and somatic embryos of the species, we set out to identify genes with specific pattern of expression during the two processes which will be of immense importance in cowpea breeding. Using the technique of two-dimensional electrophoresis (2D-PAGE), we compared the protein deposition pattern of developing cowpea seeds with 10 days after anthesis (DAA) and mature seeds and embryogenic cell suspension (ECS). From each developmental stage and for ECS, we obtained proteomic reference maps that were highly reproducible within the pH of 3-10 and 4-7. Various spots were selected based on quantity or relative volume and rate of expression. About 800 (for seeds development) and 130 (for ECS) proteins spots up- or down-regulated, remained constantly expressed and were specific for each developmental stage. The spots were removed from the 2-D gels, processed, and subjected to mass spectrometry. Some strategies like peptide mass fingerprinting (PMF) and non-processed data search (MS/MS ion search) were employed for protein identification. Over 400 proteins in seeds and over 70 proteins in ECS were identified. A large number of these proteins were found to be primary metabolism proteins, energy, protein destination and storage and defense proteins and others related to stress.

TOOTH/MIR160A regulates leaf margin outgrowth in Arabidopsis thaliana Unlike animals, a striking aspect of the plant development is that they have evolved a flexible pattern of post embryonic development. This exposes them to the challenges of many biotic and abiotic signals throughout their life. So, plants have to evolve/regulate various mechanisms to modulate their growth and development for accomplishing a successful life cycle in the prevailing environmental conditions. Auxin is involved in the initiation of lateral organs at the meristem and serration development along the leaf margin (Bilsborough et al., 2011, Hay et al., 2006). These two developmental mechanisms share common molecular players. For example, CUC2 is required for the boundary formation at the SAM and also is shown to be essential for serration formation at the leaf margin. Similarly, tth shows increased leaf serration phenotype as well as defects in the positioning of flowers at the meristem. This demonstrates the functional significance of TTH-regulated ARFs in controlling auxin mediated developmental pathways. Leaves originate as small lumps of undifferentiated cells at the flanks of the shoot apical meristem which undergo several rounds division and expansion to generate the mature leaf with characteristic size, shape and leaf margin. Both, endogenous as well as environmental factors modulate the growth and development of a leaf. This is evident from the plasticity in leaf form, observed during the life time of a single plant, as well as from the diversity among closely related species living in different habitats. It is well known that pathways controlling leaf form are subjected to the effects of selection and adaptation. Leaf margin is a key feature of the final leaf shape and it contributes to the abundant diversity in leaf form. Leaf margin architecture varies quite significantly from smooth or entire margin to margins with large outgrowths (lobed margins). The evolution and ecological advantages of this diversity is a subject of intense investigation. It also provides a wonderful system to study the mechanistic details of iterative generation of repeated units, which is a common feature in producing many biological shapes. Recent advances in molecular technologies and the availability of genomic resources ushered the identification of new factors involved in leaf margin development. Our current knowledge of this developmental programme is that CUC2 establishes auxin maxima at the leaf margin by reorienting an auxin efflux carrier PIN1 which ultimately results in serration outgrowth (Bilsborough et al., 2011, Hay et al., 2006). A few missing links in this pathway are the mechanistic details of CUC2 function in reorienting PIN1 and the molecular details of auxin mediated serration outgrowth. Forward genetic screens have been valuable in characterizing a genetic pathway even in the post genomic era. An EMS mutagenesis screen was performed in this context to identify novel factors that can improve our understanding of this intricate mechanism. tooth was identified in the M2 population based on its increased leaf serration phenotype. Genetic analysis showed that tth phenotype is due to a monogenic recessive mutation. Along with increased leaf serration, tth also shows various developmental defects such as aberrant phyllotaxy, narrower cotyledons and narrower leaves. Positional cloning and sequencing analysis showed a G to A transition at the AT2G39175 locus which codes for MIR160A. The mutation is at the 7th base position of the mature miRNA sequence. Functional characterization of miRNAs by isolating mutations is hampered by their small genomic sizes. Till now, only a few miRNAs have been characterized by mutational analysis in plants (Allen et al., 2007, Baker et al., 2005, Cartolano et al., 2007, Chuck et al., 2007, Knauer et al., 2013, Nag et al., 2009, Nikovics et al., 2006). miR160-ARF10 regulatory module is shown to be required for leaf blade out growth and serration, but not leaf complexity in tomato (Hendelman et al., 2012). miR160 is coded by 3 loci in Arabidopsis, MIR160A, B and C. All three loci encode identical mature miRNA that targets 3 Auxin response factors, ARF10, 16 and 17. ARFs are the effector molecules of auxin mediated developmental programmes. Genetic analysis showed that enhanced serration outgrowth in tth is due to the up-regulation of its target genes. Here, we have identified a miRNA that negatively regulates serration outgrowth by repressing ARF10, 16 and 17 whose functional significance in regulating leaf margin development was not known previously. Extensive genetic interaction studies have shown that TTH acts in parallel to SAW-BP and MIR164-CUC pathways in regulating leaf margin development. We have also shown that CUC2 and PIN1 are absolutely essential for serration development in tth. CUC2 establishes a pattern required for the expression of ARF10 at the leaf margin. In the absence of CUC2, downstream effector molecules such as ARFs can not perform their function. arf10-2 arf16-2 could reduce, but not suppress serration outgrowth in various mutants suggesting their functional redundancy with other ARF family members. CUC2 establishes auxin maxima at the leaf margin that triggers the degradation of AUX/IAA repressors thereby relieving ARF proteins which mediate serration outgrowth. Whereas, TTH acts at the post transcriptional level for maintaining normal ARF transcript levels Role of SPYINDLY in Arabidopsis leaf margin development SPYNDLY encodes an O-linked N-acetyl glucosamine transferase that acts as a negative regulator of GA response. Consistent with its role in GA response, spy mutants show several GA dependent phenotypes such as early flowering and hyper branched trichomes. spy mutants also show several GA independent phenotypes such as aberrant phyllotaxy and smooth leaf margin. We have studied its role in regulating Arabidopsis leaf serration development. Reporter analysis of ARF10::GUS and CUC2::GUS in spy-3 revealed that SPY is not involved in establishing serration pattern. The spy-3 leaves did not show any defects during the early stages of serration development, but the mature leaves display smooth leaf margin indicating that SPY function is required for serration outgrowth. As shown in the present study, TTH regulated ARFs are also involved in serration outgrowth. Analysis of leaf margin phenotype in tth spy-3 showed that SPY activity is not required for ARF mediated serration outgrowth. Similar genetic interaction studies with SAW-BP pathway mutants showed that leaf margin out growth mediated by meristematic genes is not dependent on SPY function. Genetic interaction studies with MIR164-CUC pathway genes showed that SPY is required for serration outgrowth in these mutants. Interestingly, the cuc2-3 mutant is defective at both patterning and outgrowth of serration. The spy-3 could suppress serration out growth in cuc2-D suggesting that CUC2 mediated serration out growth is dependent on SPY activity. Protein-protein interaction studies between SPY and CUC2 are in progress to demonstrate whether SPY directly interacts with CUC2 or CUC2 derived signal to regulate serration out outgrowth. It is interesting to examine how mutations at SPY locus can abolish serration out growth mediated by CUC2, but does not affect the serration pattern, even though CUC2 is reported to be essential for both the patterning and outgrowth of serration.

[ES] El calcio (Ca2+) es un catión esencial que juega un papel fundamental en todos los organismos vivos. Desde el punto de vista funcional, el Ca2+ actúa como un segundo mensajero que regula distintos procesos celulares. Trabajos anteriores indican que la señalización mediante Ca2+ podría estar implicada en las primeras etapas de la inducción de la embriogénesis in vitro de las plantas, pero el verdadero papel del Ca2+ en este proceso es aún desconocido. Por eso, el principal objetivo de la presente Tesis es el estudio del papel del Ca2+ en la embriogénesis in vitro mediante dos sistemas in vitro: la embriogénesis somática y la embriogénesis de microsporas. Para determinar la importancia de la homeostasis del Ca2+ en la inducción de la embriogénesis y las dinámicas de los niveles de Ca2+ durante la inducción y el establecimiento de embriones somáticos y derivados de microsporas, se utilizaron tratamientos químicos y se detectaron los niveles de Ca2+ mediante sondas fluorescentes y sensores cameleon codificados genéticamente, visualizados con microscopía fluorescente y confocal. Observamos que el aumento de Ca2+ es un marcador temprano en la inducción de la embriogénesis in vitro y que los niveles de Ca2+ durante la embriogénesis in vitro son dinámicos en todos los sistemas estudiados. Además, las oscilaciones en los niveles de Ca2+ podrían estar relacionadas con los procesos de diferenciación que ocurren en las células inducidas una vez une el Ca2+ a la calmodulina. Mostramos que un aumento de Ca2+ dentro de un rango definido de concentración tiene un efecto positivo, dependiendo del sistema, en la producción de embriones, siendo más sensibles aquellos sistemas basados en suspensiones de células aisladas que aquellos que usan tejidos como explantes. Finalmente, estudiamos el papel de la calosa durante la embriogénesis somática, observando que la inhibición de la deposición de calosa impide el desarrollo embrionario, lo que sugiere una relación entre la formación de una barrera de calosa y el establecimiento de la identidad embrionaria en las células somáticas. / [CAT] El calci (Ca2+) és un catió essencial que juga un paper fonamental en tots els organismes vius. Des del punt de vista funcional, el Ca2+ actua com a un segon missatger que regula diferents processos cel·lulars. Treballs anteriors indiquen que la senyalització mitjançant el Ca2+ podria estar implicada en les primeres etapes de la inducció de l'embriogènesi in vitro de les plantes, però el paper real del Ca2+ en aquest procés encara és desconegut. Per això, el principal objectiu de la present Tesi és l'estudi del paper del Ca2+ en l'embriogènesi in vitro mitjançant dos sistemes in vitro: l'embriogènesi somàtica i l'embriogènesi de micròspores. Per tal de determinar la importància de l'homeòstasi del Ca2+ en la inducció de l'embriogènesi i les dinàmiques dels nivells de Ca2+ durant la inducció i l'establiment d'embrions somàtics i derivats de micròspores, es van utilitzar tractaments químics i es van detectar els nivells de Ca2+ mitjançant sondes fluorescents i sensors de cameleon codificats genèticament, visualitzats amb microscòpia fluorescent i confocal. Vam observar que l'augment de Ca2+ és un marcador primerenc en la inducció de l'embriogènesi in vitro i que els nivells de Ca2+ durant l'embriogènesi in vitro són dinàmics en tots els sistemes estudiats. A més, les oscil·lacions en els nivells de Ca2+ podrien estar relacionades amb els processos de diferenciació que tenen lloc en les cèl·lules induïdes una vegada uneix el Ca2+ a la calmodulina. Vam mostrar que un augment de Ca2+ dins d'un rang definit de concentració té un efecte positiu, depenent del sistema, en la producció d'embrions, essent més sensibles aquells sistemes basats en suspensions de cèl·lules aïllades que aquells que usen teixits com a explants. Finalment, vam estudiar el paper de la cal·losa durant l'embriogènesi somàtica, i vam observar que la inhibició de la deposició de cal·losa impedeix el desenvolupament embrionari, la qual cosa suggereix una relació entre la formació d'una barrera de cal·losa i l'establiment de la identitat embrionària en les cèl·lules somàtiques. / [EN] Calcium (Ca2+) is an essential cation that plays fundamental roles in all living organisms. From a functional point of view, Ca2+ acts as a second messenger that regulates different cellular processes. Previous works point to the fact that Ca2+ signaling may be involved in the early stages of induction of in vitro plant embryogenesis, but the actual role of Ca2+ in this process remained unveiled. Thus, the main goal of the present Thesis is to study the role of Ca2+ in in vitro embryogenesis using two in vitro systems: somatic embryogenesis and microspore embryogenesis. Chemical treatments and detection of Ca2+ with fluorescent probes and genetically-encoded cameleon sensors imaged by fluorescence and confocal microscopy were performed to determine the importance of Ca2+ homeostasis for induction of embryogenesis and the dynamics of Ca2+ levels during the induction and establishment of somatic and microspore-derived embryos. We observed that Ca2+ increase is an early marker of induction of in vitro embryogenesis and Ca2+ levels during in vitro embryogenesis are dynamic in all the systems we studied. Moreover, Ca2+ oscillations might be related to the differentiation processes that take place in the induced cells upon binding to calmodulin. We showed that Ca2+ increase within a defined range has system-specific positive effects in embryo yield, being more sensitive those systems using isolated cell suspensions rather than those using tissues as explants. Finally, we studied the role of callose during somatic embryogenesis, and we observed that inhibiting callose deposition prevents embryo development, which suggests a relationship between the formation of a callose barrier and the establishment of embryo identity in somatic cells. / Calabuig Serna, A. (2023). Multidisciplinary study of the role of calcium in plant in vitro embryogenesis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/196022