MOLECULAR BIOLOGY OF PLANTS

Author: SAMANIEGO GARCÍA RAFAEL.
Year: 2003.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA, UCM.

Summary: In this Doctoral Thesis characterized the two protein MFP1 (MAR-blinding Filament-like Proteín 1) nuclear Allum strain through inmunodetección and inmunoprecipitación two serums against proteins developed tomato and Arabidopsis. AcMFP1-78KDa is a protein constituent with a pl slightly acidic (5.5), and is distributed in different subdomains nuclear depending on the type of cell and tissue. By electron microscopy has shown that it does in small domains preferentially localized at the periphery of the masses of heterocromatina dense. It also has a dual subcellular localization, located in the chloroplasts with a distribution pattern similar to MFP1 in other species. The second sister, 90 Kda and basic (8,5-9,5), has different phosphorylation states of influencing binding to the nuclear matrix, having shown that casein kinase CK2 endogenous can regulate the union. AcMFP1-90KDa accumulates in nuclear forces whose number is increased by cell proliferation and the presence of light. It is also distributed by the reticle of the nuclear matrix, associated with the filaments of nucleoskeleton. Studies show that the co-location of proteins AcMFP1 not part of the complex replication, the processing co-transcripcional, granules intercromatínicos nor the Corps Cajal. The different characteristics and patterns of distribution and expression of both proteins suggest different roles, or at least different forms of regulation. The results suggest an interaction of AcMFP1-78KDa with eucromatina, and a structural role in the nuclear matrix for AcMFP1-90KDa.

Author: SALEH ABDELATY.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE FARMACIA.

Summary: Plants are exposed to many types of abiotic stress during His life cycle. Poor water resulting from the drought, low temperatures and high salt concentration in the soil is one of the most common environmental stress I affecting growth and development of plants, and leads to alterations in metabolism and gene expression . Drought: triggers a series of responses at the plant beginning with the perception of the signal, sigue.con transduce R signal and results in changes in gene expression. The loss of water from the cell triggers a way transduction [of the signal that converts a physical stress on a biochemical response. The ABA half a variety de.pRocesos physiological, which includes the response to drought and salt stress. Thus, the majority of genes inducible by drought, are also induced by 'ABA. It seems that water stress increases the production of the ABA, which consequently induces the expression of several genes. The functional analysis of genes rab, has revealed the existence of specific elements of sequence, specific elements in response to the ABA. ABREs (ABA responsive elements) and DREs (Drought responsive elements) are involved in the regulation of gene rab17 by I ABA and drought. The element cis DRE2 has shown its importance in the induction of gene expression rab17 by the ABA. Genes ZmDBFs corn were isolated using the technique of one-hybrid in yeast, with the element cis DRE2 promoter's rabl7 corn and a cDNA library prepared from leaves of seedlings of 5 days of corn previously treated for dehydration 3 hours at room temperature. Genes ZmDBFs corn are members of the family of transcription factors plant AP2/ERF. The gene ZmDBFl triggering during embryogenesis and post-treatment by dehydration, high salinity and treatment with ABA in maize seedlings. In the present work we studied the functional characterization of the gene ZmDBFl corn. Transgenic plants ZmDBFl showed some easing in the growth compared with control plants, and these plants were classified according to their phenotype into three groups:. ZmDBFlA, ZmDBFlB and ZmDBFlC. The Western and Northern blot analysis indicated that under normal conditions, stunting is well correlated with the level of expression of ZmDBFI. The overexpression of ZmDBFl resulted in transgenic plants were significantly more tolerant to drought and salinity control plants. In addition, Level I of drought tolerance correlated with the level of gene expression ZmDBFI. Transgenic plants ZmDBFl I showed significant hypersensitivity to ABA compared with the control plants during germination. Studies of the regulatory activity of the gene ZmDBFl showed the effect regulator ZmDBFl on promoters dependent ABA (rabl7 corn, rabl8 and rd29A Arabidopsis). Order to identify and characterize proteins that interact with ZmDBFl, we used the system twice - hybrid. Using this system, we have identified two novel proteins that interact with the ZmDBFI. These two proteins have been called ZmDIPl and ZmDIP2 (ZmDBFl interactor proteinl and 2). The protein ZmDIPl contains the domain conserved R3H belonging to the family R3H protein and form a new family of proteins R3H plant. While the ZmDIP2 contains the domain DUF614 and belongs to the family DUF614 that is not marked. According to our results, we propose the existence of a complex regulatory protein involved in the function of the protein ZmDBFl, in which proteins ZmDIPl and ZmDIP2. Furthermore, we have studied, the sub-cellular localization of protein ZmDBFl and interactor (ZmDBFl) using OFP and DsRED. The results showed that the subcellular localization of mergers of ZmDBFl with OUS, OFP and DsRED indicates that the ZmDBFl is a nuclear transcription factor. They also study the subcellular localization of ZmDBFl under treatment with ABA indicates that the location of ZmDBFl is independent of the ABA. Part C-termínal of protein ZmDBFl contains a consensus rich in lysine (KNSKAKSK), which could be a new NLS, possibly responsabl 8 and the 442 location nuclear protein ZmDBFI. , While the N-terminal part of the protein ZmDBFl contains a new NES (LPRNRTRL WL), which could be responsible for [cytoplasmic localization and the formation of vesicles citoplásmicas. The subcellular localization of the protein ZmDIPl using DsRED shows that ZmDIPl is a cytoplasmic protein. At the same time, the outcome of the co-expresión of ZmDBFl-OFP i with the ZmDIPI-DsRED confirmed interaction proteína-proteína observed in the yeast protein ZmDBFl yi ZmDIPI. These results indicated that the interaction between ZmDBFl and ZmDIPl may be necessary for nuclear localization 'of the protein ZmDIPI.

Author: BLANCO ALCALA OSCAR.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: The líguenes parmeliolides constitute a monophyletic group within the very broad family parmeliaceae.Dentro this group the concept of gender is not well establecido.Debido a result, there has been a revision of the concept based on genetic grounds for phylogenetic using molecular data and have reviewed different morphological characters. As a result of the study has been proposed synonymy of seven and gender segregation of two new.

Author: CASTILLO LOPEZ RAQUEL.
Year: 2004.
University: CASTILLA-LA MANCHA [www.uclm.es].
Place of defense: ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRONOMOS.
Place of preparation: ESCUELA TECNICA SUPERIOR DE INGENIEROS AGRONOMOS.

Summary: Given the economic importance of saffron stigmas are little studies on the corm, however this body depends reproduction as well as the quality of the entire plant. In our laboratory study of clones of a genoteca speech made from saffron corm revealed an abundance of defensive sequences for enzymes. Among these was identified gene of a quitinasa. Analysis of this sequence by comparison with cDNAs from other quitinasas plants deposited in databases (EMBL, NCBI) revealed that it was a quitinasa Class I truncated at the extreme 5 '. By TAIL PCR method we were able to obtain the complete sequence of quitinasa from clone truncated. The complete sequence the gene was cloned and named SafchiA. The EST obtained consists of 1078 nucleotides, with an ORF of 839 remains to codify a protein of 289 amino acids with a molecular weight of 31,768 KDa and a pl estimated at around 5. We also got a small portion upstream of the start codon of the transcript (non-coding region UTR), which revealed the presence of an element of induction by mechanical damage, namely that the gene expression of quitinasa be induced by mechanical damage. The analysis of the N-terminal end of the protein showed that the first 20 amino acid signal peptide characteristics of the following 35 amino acids fall under chitin binding domain that owns the eight cysteine residues typical of quitinasas plants that possess this type domain. The region of the substrate binding and catalytic are separated by 12 amino acids that make up a domain of low complexity or neck. At the other end of a small C-terminal sequence that appears at first thought it was a sign of transportation to vacuola, typical quitinasas class, however this little analysis revealed no sequence homology with this kind of signals . An analysis of the primary structure with those of other quitinasas of dicotyledons and monocotyledons allowed for certain phylogenetic relationships between different species. Analysis of gene expression showed as quitinasa saffron has a typical pattern of proteins defense constitutively expressed in a high groundwater in certain tissues of the plant (root and corm), making it weaker expression in the aerial parts (leaves) and very weak in floral fabrics; being inducible attack by pathogens and other situations as a wound, our study also shows that the wound by induction takes place via a route mediated acid jasmónico independent ethylene, this hormone also acting as an inhibitor the expression of the quitinasa. The recombinant protein expressed in E. coli showed activity quitinasa, antifungal against fungal pathogens of saffron and some ability to inhibit tumor cell growth of cancer cervix. The sequence analysis of the active site of the protein sample as quitinasa saffron has an active little changed as a result of vegetative reproduction of the species, lack of a waste indispensable for the development of the catalytic activity and possessing however activity . The expression of different versions mutadas the catalytic domain of the quitinasa ago hydrolysis predict a mechanism different from those described to date for quitinasas the family 19, the family that sequence homology of the quitinasa belongs but part of a new class of quitinasa not mentioned so far.

Author: BLAZQUEZ MARTIN SILVIA.
Year: 2004.
University: CASTILLA-LA MANCHA [www.uclm.es].
Place of defense: ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRONOMOS.
Place of preparation: ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRONOMOS.

Summary: In this paper we have studied the embryos of saffron at various levels. First was to optimize the development embryos until seedlings, analyzing the behavior of tissue in temporary immersion systems and subsequently characterized embryos. It has been made a division of the stages of development of embryos saffron according to a study from the morphological and histological induction (E0, proglobular) until maturity in seedlings (E4), passing through the stadium E2 monopolar and E3 , dipolar. It has been analyzed in each of these stages the response of antioxidant enzyme system, the degree of lipid peroxidation and variations in the levels of endogenous several polyamines (Dap, Put, Cad, Spd and Spm) to characterize the behavior of embryos this matter. Finally, tests were carried out preliminary processing systems of plant material; biolística, Agrobacterium-mediated transformation and transformation of protoplasts as possible avenues for genetic improvement in saffron.

Author: ARELLANO OSTOA GREGORIO.
Year: 2004.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: INSTITUTO DE INVESTIGACIONES AGROBIOLOGICAS DE GALICIA-CSIC.
Place of preparation: INSTITUTO DE INVESTIGACIONES AGROBIOLOGICAS DE GALICIA-CSIC.

Summary: The overall objective of this dissertation is to determine possible changes in biochemical and molecular level that may be related to the loss of the ability to rooting that occurs in adult material oak (Quercus robur L.). This is used in vitro production of outbreaks of two different lines of oak state ontogenético; a derivative of renuevos baseline, a youth and introducing high rates of rooting and other branches stemming from the cup, as an adult with low or no training capacity of adventitious roots. Both lines are the same genotype as coming from a single specimen of 300 years old. The analysis has been made: - The evaluation of the major morphological and physiological characteristics that distinguish the two types of material by the end of Phase multiplication. B - The quantification of endogenous levels of auxins (IAA acid indol-3-acético and AIA-Asp acid indol-3-acetil-aspártico) during the first 8 days of the process of rooting induced acid indole-3 - butyric (IBA), as well as assessing the impact of NPA (acid naftil-p-talámico) on the rooting and on the levels of endogenous auxins. C-Analysis of the levels of expression during the rooting of a cDNA (QRCPE) identified in oak and is preferentially expressed in cultured adult material. Study of the effect of NPA on levels of expression. Our results indicate that this clone can be used oak different morphological referring to the stem and leaves as markers associated with the phase shift. In addition, the line from renuevos baseline presents more outbreaks enraizables (greater 20mm), and high percentages of rooting while outbreaks from the branches of the cup not rooted. These physiological characteristics have remained stable for at least 10 years. On the other hand, when outbreaks renuevos baseline were treated with NPA during the first 5 days of the rooting process, show a rizogénica inhibiting their ability, as well as a slowdown in the kinetics of appearance of roots. As for concentrations of endogenous auxins in the basal area, in both outbreaks as in the cup of renuevos there is an elevation after treatment with AIB. Cultures of the cup presented high levels of auxins longer, but so does the renuevos baseline treated with NPA during the first 5 days of the process of induction. There is therefore both lines can capture the AIB's culture medium, as well as synthesizing IAA and AIA-Asp. The concentration of endogenous auxins is not the limiting factor for the differentiation of roots in outbreaks of the cup, but the lack of rooting might be related to alterations in the transport pile or the presence / absence of appropriate receptors. The molecular analysis of gene expression QRCPE indicates that this depends on the state of ontogenético outbreak, as the messenger RNA levels were generally higher in crops derived from the cup, which indicates that there is a negative correlation between the expression gene and rooting ability. In addition there is an inverse trend between the content of IAA and endogenous levels of expression of the gene QRCPE in both lines studied (cup and renuevos). Treatment with AIB generally inhibit gene expression during the first 8 os 8 days 509 s process rooted in both types of material. Furthermore, the pattern of expression of outbreaks renuevos baseline is more variables analyzed over time, especially in the materials treated with AIB, these changes may be related to the various events at the cellular level that take place prior to the formation of roots. The addition of NPA to outbreaks from renuevos treated with AIB increases markedly gene expression during the first 6 h of the process, reaching levels similar to those of the cup, which may be related to inhibition of NPA in training roots and the slowdown in rooting. The pattern of expression of outbreaks treated with AIB and NPA is similar to the outbreaks of the cup treated with AIB which could suggest a relationship between the gene QRCPE and transportation of auxins.

Author: BERDASCO MENENDEZ MARIA.
Year: 2004.
University: OVIEDO [www.uniovi.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: FACULTAD DE BIOLOGIA.

Summary: An understanding of the processes that govern aging and, in particular, the transition from state to state youth mature is essential for the development of new strategies to enhance the capacity for growth and spread of forest species. However, despite the advances that have taken place in recent years, the definition of mechanisms involved and further integration is at an early stage. With the exception of systems modeling in general are unknown genes that are expressed at a particular stage of development and, even more control mechanisms of gene expression proceso-dependientes. Among the potential mechanisms controlling gene expression for the growth and development of plants should outline the essential role played by epigenetic factors, in particular the methylation of DNA along with the acetylation of histones control chromatin structure and consequently the gene expression. Understanding the code epigenético in model systems, usually animal is more advanced than in plants. In long-lived woody species are virtually non-existent jobs to address the problems exposed, despite the fact that the only way to understand the loss of competition under age through a functional approximation. In its report to be presented, it becomes clear that the acquisition of reproductive competition in Pinus radiata D. Don is associated with the hipoacetilación of histone H4 in the four N-terminal residues, and simultaneously to the overall DNA hypermethylation. Status epigenético that affects so differential cells and differentiated cells meristemáticas elements. In states revigorizados is a reversal of the pattern of methylation and acetylation, which shows the dynamism of both processes and provides data to understand the plasticity of plants both in terms morfogénicos as adaptive. In addition, the overall DNA methylation patterns shows dynamic, independent of genotype, depending on the degree of differentiation of the organs analizados.Ante difficult undertaking in a period of Ph.D. Thesis, functionality gene in the species primary studies Genomics Funcionalse addressed in the model species Arabidopsis thaliana. Since the hypermethylation of tumor suppressor genes is one of the most important epigenetic mechanisms in the development of tumors in mammals states, the experimental system was selected so that it could study genes associated with cell proliferation processes in plants. The epigenetic regulation of DNA methylation in plants has been described only for genes associated with the processes of development and differentiation. This article demonstrates for the first time that aberrant methylation of specific promoters is also a common event in the growth of cell suspensions desdiferenciadas Arabidopsis thaliana. Through the use of agents demetilantes technology microarrays and sequencing of genomic regions that are bisulfide promoters of the genes TTG1, GSTF5 and SUVH8 are hipermetilados in cell suspensions with unchecked growth, while tissue from leaves are not metilados. It was confirmed that these promoters hypermethylation is associated with suppression gene, reversing the expression by applying the agent demetilante. Tests inmunoprecipitación of chromatin revealed that the hypermethylation of the promoters and suppression gene are associated with repressive histone modifications adjacent.

Author: Valderrama Rodríguez Raquel.
Year: 2004.
University: JAÉN [www.ujaen.es].
Place of defense: Universidad de Jaén.
Place of preparation: Universidad de Jaén.

Summary: The abiotic stress induced by salinity phenomena of oxidative stress and nitrosativo mediated oxygen species and reactive nitrogen (RONS), judging by the imbalances produced at non-enzymatic antioxidant levels and antioxidant enzyme activities, NADP-deshidrogenasas and systems producers nitric oxide. The enzyme induction secuestradoras hydrogen peroxide such as catalase, superoxide dismutase, ascorbate peroxidase, as well as monodeshidroascorbato reductase, glutathione reductase, deshidrogenasas dependent NADP, peroxirredoxinas and nitric oxide synthase protein type, indicates that plant olive these enzymes are involved in the mechanisms of tolerance of the plant versus stress (oxidative and nitrosativo) induced by salinity. The results obtained indicate that the thesis stress nitrosativo participates together with oxidative stress as a significant component in the cellular damage caused by abiotic stress by salinity in olive seedlings, demonstrating phenomena of cell and tissue communication between species of oxidative metabolism, nitrosativo and redox metabolism.

Author: MOLINA AZCONA ANDREA.
Year: 2004.
University: PÚBLICA DE NAVARRA [www.unavarra.es].
Place of defense: INST.DE AGROBIOTECNOLOGIA Y RECURSOS NATURALES.
Place of preparation: INSTITUTO DE AGROBIOTECNOLOGIA Y RECURSOS NATURALES.

Summary: The transformation of genome plastidial, instead of nuclear, has a number of advantages undoubtedly attractive for biotechnology application. The levels of expression of recombinant proteins are of the order of 10-1000 times higher than those obtained by nuclear transformation. It can integrate and express multiple genes in the form policistrónica under the control of a single promoter, and that the mechanisms of expression are similar to the bacteria. A major environmental advantage is that it avoids the transgene spreading to other floors, because of the absence of plastidios in pollen grains. Nor has described the silencing gene or there is a so-called effect of position due to placement of transgene integration because it is so directed by homologous recombination latter aspect reduces much workload to select lines transformadas.El This project aims to explore the feasibility of transforming plastidial for the production of vaccine antigens and test the immune response developed after their administration to laboratory animals. This was expressed in chloroplasts of snuff the peptide 2L21, which confers protection against the dog canine parvovirus (CPV), fused to an extreme carboxi-terminal of the B-subunit of the cholera toxin (CTB) or green fluorescent protein (GFP). We obtained high levels of expression of the fusion protein (up to 31% compared to the total soluble protein). A single plant is capable of producing 310 mg of antigen vaccine. The two fusion proteins were stable both in young leaves as old and the estimated average life, as determined by marking and pulso-caza was more than 48 hours in both cases. The freeze-dried powdered leaves was a simple method of post-harvest treatment without significant loss of the recombinant protein after 7 months of storage at room temperature ambiente.La immunization injecting mice and rabbits with protein extracts enriched leaf plants transformed CTB -2 L21 produced specific antibodies in serum with high qualifications. These antibodies were able to recognize the protein VP2 and in an in vitro test proved neutralizing the CPV. When mice received an oral suspension of pulverized tissue was induced antibody serum IgG and IgA anti-2L21. The data obtained with a combined immunization (a single parenteral injection followed by oral memories) show that the memories help keep the oral IgG anti-2L21 induced response after a single injection, while parenteral administration of antigen preparation of the subsequent response memories oral promote the induction of antibody IgA anti-2L21.El high level of expression of antigens in the chloroplast could reduce the amount of plant material required for vaccination (~ 100 mg for a dose of 500 mg of antigen) and allow the encapsulation lyophilized material or the formation of pills. These advantages suggest that vaccines based on transformation cloroplástica plant could be commercially viable.

Author: Becerra Baeza Cristian.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: Centre de Investigaciò y Desenv.IBMB-CSIC.
Place of preparation: Centre de Investigaciò y Desenv.IBMB-CSIC.

Author: LLUCH GOMEZ YOLANDA.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: INST DE AGROQUIMICA.
Place of preparation: INSTITUTO DE AGROQUIMICA Y TECNOLOGIA DE ALIMENTOS.

Author: ROSADO REY ABEL.
Year: 2005.
University: MÁLAGA [www.uma.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: This thesis discusses various aspects of tolerance to salt and water stress on plants using two models vegetable Arabaidopsis thaliana L., and tomato (Solanum lycopersicum L.). The results are presented in three separate chapters, although there is a general introduction to water stress and salinity. The first two chapters show the physiological characterization of two mutant tomato, hypersensitive to salt stress, which were previously isolated: tss1 and tss2. The study of mutant tss1 includes analysis of the role of CA2 + and NH4 + on the transport of K + and its implication on tolerance to NaCl. The results have already been published Plant Physiology (Rubio et al., 2004) and suggests that increased concentration external Ca2 + removes the weakness in the transport of K + from mutant tss1, observed both in the presence and in absence of NH4 +. The second chapter presents the study of the relationship between ethylene and ABA in osmotic stress tolerance using mutant tss2. The results obtained indicate that you mutant tss2 has affected the production of ethylene ABA dependent on osmotic stress situations. The last chapter contains the characterization and functional analysis of a family of Arabidopsis gene called TTL. This new gene family is involved in the regulation of osmotic response mediated through the ABA. The results show that genes encoding proteins that TTL function as positive regulators of the signaling cascade mediated by ABA during germination in a position to salt stress.

Author: ANTOLÍN LLOVERA MERITXEL.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: UNIVERSITAT DE BARCELONA.

Summary: The HMG-CoA reductase enzyme (HMGR) catalyzes the first step in limiting the synthesis of isoprenoides citosólicos. In plants, is a membrane protein and its subcellular first destination is the endoplasmic reticulum. Structurally consists of a N-terminal domain (which includes an N-terminal region cytosolic and two transmembrane fragments) and a catalytic domain highly conserved across the evolutionary scale. In all species of plants known so far, there isoforms of HMGR encoded by different genes. Specifically, A.thaliana gene hmg1 encodes for isoforms HMGR1S and HMGR1L, the gene hmg2 encodes for isoform HMGR2. At the level of primary structure HMGR1L differs from HMGR1S by the presence of a region aminoacídicos extra 50 residues at the end amino-terminal. The domain amino-terminal confers various destinations subcellular localization of the protein. In a previous job, identified three proteins that interact with isoforms arising from gene hmg1. Two of them, AtB "and AtB.", Interact specifically with the region amino-terminal of isoforms HMGR1S and HMGR1L. Proteins AtB "and AtB. Dela regulatory subunit isoforms are B complex protein phosphatase 2A (PP2A). Third AtKLC-1, interacts specifically with the N-terminal region of HMGR1L. Was observed by in vitro tests that PR65, structural protein of PP2A, interacts specifically AtKLC-1. This work has been marked a line in the mutant gene hmg1 of A.thaliana, which shows the absence of the crop sterile and manifest severe alterations of development when they are grown in soil. Adding mevalonato, product the reaction catalysed by HMGR, it reverses the phenotype. data indicate that genhmg1 performs a function nometabólica related to adaptation to the environment. has been shown that PP2A is a negative regulator of the enzyme HMGR. These data suggest that the regulation of HMGR by PP2A could be mediated subunits AtB "and / or AtKLC-1. Likewise, PP2A is involved in the subcellular localization of isoform HMGR1S and probably HMGR1L. The domino amino-terminal of HMGR1S is involved in the morphogenesis of vesicles derived from the endoplasmic reticulum. The protein PP2A is needed in this process. These vesicles could play a vital role in adaptation to the environment.

Author: MORALES RUIZ MARIA TERESA.
Year: 2005.
University: CÓRDOBA [www.uco.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The methylation of cytosine (5-meC) is a trademark of epigenetic DNA that promotes gene silencing and plays an important role in development and in maintaining the stability of the genome. The DNA methylation patterns remain stable during successive cell divisions but are also modified by desmitilación local or global genome at specific stages of development requiring an epigenetic reprogramming. In contrast to the property characterized mechanisms methylation, the nature of the enzymes responsible for DNA demethylation and the subsequent reactivation of silenced genes previously has not been able to be accurately determined, in part by the difficulty of conducting genetic analysis and molecular agencies with methylation patterns altered. While conducting these arguments have been identified and characterized two genes of the model plant Arabidopsis thaliana, called SMD and ROS1, coding for enzymes that initiate a process of active DNA demethylation. The results suggest that SMD and ROS1 are two DNA glicosilasas belonging to the superfamily HhH-GPD. Both proteins are much more voluminous that the DNA glicosilasas typical, and have a domain HhH-GPD approximately 240 amino acids coming to its end carboxilo-terminal. There are proteins with similar sequences in other plant species, but not in arqueobacterias, bacteria, fungi or animals. Therefore, SMD and ROS1 define a new subfamily of DNA glicosilasas specific plants. SMD and ROS1 are expressed in a range of tissues and plants deficient in either gene have a slower growth and a phenotype pleiotrópico with several alterations in its development, including changes in the morphology floral and fruit production with a smaller often contain seeds abortion. The biochemical characterization of the enzymatic activity of SMD and ROS1 has demonstrated that the two proteins are DNA glicosilasas dual-purpose catalyzing the removal of 5-meC, its replacement by a cytosine not metilada using a process similar to the excision repair bases. Besides processing 5-meC, SMD and ROS1 are able to recognize and eliminate DNA thymine residues erroneously matched with guanine, but no activity whatsoever on urcilo apareado incorrectly with guanine. However, both proteins showed a clear preference for 5-meC compared with thymine in the context CpG, where are most of the waste 5-meC in genomes of plants and animals. In addition, SCI and ROS1 also show activity in streams CpApG and CpNpN, which are additional sites of methylation in plants. Overall, the results of this thesis suggests that one of the functions of SMD and ROS1 on the ground is to activate the expression of genes silenced by initiating the deletion of 5-meC through a mechanism similar to the excision repair bases. Consequently, they are a strong experimental evidence for the existence of an active mechanism for DNA demethylation in plants.

Author: GILSANZ GONZALEZ SUSANA.
Year: 2005.
University: CÓRDOBA [www.uco.es].
Place of defense: ETSIAM.
Place of preparation: ETSIAM.

Summary: In order to analyze various strategies multiplication ex situ gene of Vicia faba, carried out a series of analysis, which includes: 1-Identifying the components of gene flow in fields multiplication germplasm V.faba in two locations, Cordoba (Spain) and Dijon (France), using molecular markers, isozymes and aloenzimas highly variable. 2 - The elucidation of the causes that can explain the variation of gene flow in fields multiplication germplasm in a position to open pollination, through a comparative analysis of the patterns of gene flow in the design of metapopulations, which examines the geographic variation, genotypic and the effect of different barriers (trap crops and spatial isolation) as a method for controlling gene flow. 3-A comparison of the temporal variation in two cycles of multiplication of two strategies multiplication: metapopulations and mass reservoir. 4 - The identification of floral characters predictors of gene flow with the potential for use in the design of strategies multiplication germplasm in a position to open pollination. The prediction of potential gene flow takes place through an approach that combines the analysis of paternity with molecular markers and analysis of the functionality of the flower by multivariate regressions. 5 - The study of the genetic diversity population using SSAP (Sequence-Specific Amplified Polymorphisms).

Author: VALLADARES LÓPEZ MARÍA SILVIA.
Year: 2005.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: INSTITUTO DE INVESTIGACIONES AGROBIOLÓGICAS DE GALICIA-CSIC.
Place of preparation: INSTITUTO DE INVESTIGACIONES AGROBIOLÓGICAS DE GALICA-CSIC.

Summary: The oak is a very important significance in forest ecosystems both in our country and in the rest of Europe. The somatic embryogenesis can be seen as a central element of gracious biotechnology, with a huge potential in improving forest species to be implicated in the mass clonal propagation, genetic transformation, and cryopreservation of material embryogenic. One of the most important constraints of this process is that it has made very few species in the formation of embryos in tissue from adult individuals, likely to be selected for their desirable characteristics. The establishment of lines of embryogenic oak sensitivity obtained by the induction of somatic embryogenesis from trees selected adults, leads to a demand for the maintenance of a large number of lines embryogenic selected. For ease of handling, and avoid the risks associated with maintaining prolonged culture conditions in vitro, imposes storage and long-term maintenance of such material by the method of cryopreservation, with the consequent creation of gene banks. Moreover, it is necessary to take into account, according to the genetics of plants regenerated in vitro is a requirement parala clonal propagation on a large scale and other applications dela somatic embryogenesis. In this thesis work we have obtained the following results: * has been obtained for the first time induction of somatic embryogenesis and regeneration of plants from adult material trees of Quercus robur, using as explantos initial sheets expanding isolated outbreaks epicórmicos forced into buckets of branches of trees selected dela cup. The induction embriogénica took place in an environment in addition to BA and ANA, the embryos developed as a means of expression provided with regulators growths. The frequency induction ranged between 0,3-4,1% and are heavily influenced by genotype. With this system it is possible to initiate crop at any time of year regardless sprouting in the countryside, although the best results were obtained with material collected in November. * It has assessed the effect of different carbohydrate on the proliferation, maturation and subsequent germination delos embryos. The addition of sucrose and maltose Middle favors keeping the proliferation and development of the embryos, whose conversion plant will be promoted in those embryos multiplied in a medium containing 3% maltose. The inclusion of a maturing stage of the embryos in culture medium added with sorbitol 6% + sucrose 3%, significantly improved the germination and development of plants. The size of the embryo, after being subjected to maturation, also influenced germination. * The ability of germination and plant conversion depends largely on genotype, obtaining values between 0-70% in six lines from 6 genotype adults. When evaluated the germination capacity of lines embryogenic juvenile home, it was found that there was no relationship between this ability and character juvenil-adulto of the starting material. The addition of BA to average germination improved results, while the GA3 not exercised any significant effect on the regeneration of plants. * Assessed by RAPD, genetic conformity of different lines embiogénicas and plants regenerated from embryos, with respect to the trees home. None of the 3 genotypes examined, have been detected signs of somaclonal variation within the limits of RAPD, suggesting that the systematic 8-of emb 774 riogénesis osmotic used maintains the genetic stability, and therefore it might be Fit for the clonal propagation of adult trees of Quercus robur. * We investigated the possibility of cryopreservation of embryos Quercus robur through the methods of drying and vitrification. In the first case was obtained 31-57% recovery embriogénica after subjecting explantos to precultivo in medium with high concentrations of sucrose, followed by drying chamber flow to a water content of 24-30% (on a fresh weight basis ) before immersion in liquid nitrogen (NL). It got better results (70% recovery embriogénica) with the vitrification method by applying a solution of vitrification (PVS2) for 60-90 min before immersion in the NL. * It defined a protocol cryopreservation of embryos Quercus namely through the process of vitrification, obtaining high levels of recovery embriogénica (88-93%) through precultivo amid added with sucrose 0.3 M and treatment of vitrification solution for 60 min prior to the immersion of explantaos in NL. The state of development of genetic embryos used as explantos to crioconservar influences recovery embriogénica post-congelación, determining which groups of 2-3 embryos in a globular (1,5-2,5 mm) are explantos more productive in the two lines embryogenic evaluated.

Author: Mansilla Mansilla María del Carmen.
Year: 2006.
University: POLITÉCNICA DE MADRID [www.upm.es].
Place of defense: Escuela Técnica Superior de Ingenieros Agrónomos.
Place of preparation: E.T.S. de Ingenieros Agrónomos.

Summary: Plant viruses are obligate intracellular parasites requiring the host to develop their replication cycle. In this thesis plant-virus interaction was studied, from both viral and host sides. The system used is based in the interaction of oilseed rape mosaic virus (ORMV) with some of its host plants. Aspects studied were: pathogenicity determinants in the viral genome; effects of the viral movement protein (MP) expressed in Arabidopsis thaliana transgenic plants; search for host factors in A. thaliana involved in the development of the viral infection, using a system based in the previously characterized transgenic plants; and, finally, the ability of an ORMV-based viral vector to silence transgenes in A. thaliana. A determinant associated to the necrotic spotting symptom produced by ORMV in Nicotiana tabacum cv. Samsun was localized in the polymerase domain (54k) of t 8 he viral 935 replicase gene. This is the first pathogenicity determinant localized in the tobamovirus 54k domain. In the Arabidopsis thaliana ORMV.PM transgenic lines, a genetic characterization showed that line TG.MP(+).7 presented the transgene in homozygosis in one locus placed at the terminal region of chromosome 1 right arm. Phenotypic characterization of this line showed that the movement of a phloem tracer to some flower organs was facilitated in the plant, in a selective way. No alterations were found in biomass partitioning or sugar availability. These plants were susceptible to ORMV infection, although with symptom attenuation. To search for host factors involved in the development of viral infection, an experimental specific system based on ORMV?PM+GFP and TG.MP(+).7 line was developed. ORMV?PM+GFP is a MP-defective chimeric virus, expressing GFP. This virus did not move from the initially inoculated cell and could be complemented in trans by viral or transgenic functional MP. The experimental system was based on reverting by mutagenesis the defective virus complementation by the transgenic plant. The phenotype could be established by GFP observation. Several mutants showing absence of complementation were obtained and characterized. These mutants were designated dotcom ('don't transgenically complement ORMV?PM movement'), or dcm in short. The mutant allele dcm-1, associated with an initial phenotype of individual fluorescent cells in ORMV?PM+GFP-inoculated leaves, was mapped to chromosome 4. The study of ORMV?PM+GFP ability to silence transgenes in A. thaliana showed the possibility to induce VIGS in GFP transgenic A. thaliana plants. Silencing efficiency was dependent of transgene dose.

Author: ORTIZ MASIA MARIA DOLORES.
Year: 2006.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: In plants, signaling via MAP kinase cascades resulting in a large number of cellular responses including cell division and differentiation, and stress response to biotic or abiotic origin (Mishra et al., 2006). The MAP kinase plant can be classified on the basis of the similarity between the amino acid sequence, into four groups (AD). The MAP kinase in each group have been classified turn into two subgroups (1 and 2). It has very little information about members of the subgroup C1 (Nakagami et al., 2005). Within this group are Ntf3 of snuff, PhMEK1 of petunia and PsMAPK2 of pea. It has been described changes in the levels of mRNA Ntf3 during the pollen (Wilson et al., 1993) and PhMEK1 during the development of ovarian cancer (Decroocq-Ferrant et al., 1995). In Arabidopsis, the subgroup C1 is made up of two genes of MAP kinases: AtMPK1 (At1g10210) and AtMPK2 (At1g59580). The function of these genes is not known, although there is some data suggesting a possible relationship between these genes and stress responses. It has been described that the levels of mRNA AtMPK1 and AtMPK2 increase slightly after a salinity (Mizoguchi et al., 1996), and decreasing to 24 hours after treatment at low temperature (Vogel et al., 2005). Moreover, the results obtained through analysis of microarrays indicate that the expression of AtMPK1 is lower in seedlings grown in darkness than in seedlings grown in the presence of light (Ma et al., 2005). The analysis of expression data stored in databases for public access show that the levels of expression of these genes are very low and there is no significant change after the various conditions tested (Zimmermann et al., 2004; Arabidopsis Functional Genomics Consortium (AFGC) Microarrays databases). There are no data on the regulation of the activity of the MAP kinase kinase subgroup C1. This thesis deals with the study of the role of PsMAPK2 (pea), AtMPK1 and AtMPK2 (Arabidopsis), genes encoding MAP kinases subgroup C1. To undertake such a study, in this paper we have made different approaches: 1 - has been analyzed the expression of these MAPKs in different plant tissues; 2-have been obtained transgenic Arabidopsis plants expressing various mutant versions of PsMAPK2; 3 - It has analyzed the kinase activity of these MAPKs in response to various signs of stress.

Author: COLLADOS COLLADOS RAQUEL.
Year: 2006.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: In this Doctoral Thesis have addressed two main scientific goals: on the one hand, the study of mechanisms of regulation of desaturasas of fatty acids in cell suspensions of soybeans, focusing on the effect of light and the influence of redox processes photosynthetic and, secondly, the study of the effect of the degree of unsaturated fatty acids of the membranes tilacoidales in the repair and assembly of fotosistema II and his involvement in the response to stress bright. On the first goal, our results showed that the light modulaba the fatty acid composition of cell suspensions of soybeans. Cells grown in darkness fewer polyunsaturated fatty acids (18:2 and 18:3) and more saturated and monounsaturated fatty acids to cells grown in light. These changes were related to regulation by the light of the expression of genes of desaturasas soy found the expression of genes FAD2, FAD3, FAD6, FAD8 and FAB2 regulated at least in the transcriptional level and the level of gene FAD7 to postranscripcional. This regulation by the light appeared to be dependent on the photosynthetic transport mail. Thus we find that the inhibition of transport mail photosynthetic mimetizó partly the effect of darkness descending levels of 18:3 and raising of 16:0, 18:0 and 18:1 and that transport mail photosynthetic influenced the expression of gene FAD3 and FAD6 at transcriptional and the FAD7 at postranscripcional. The expression of other genes of desaturasas (FAD2, FAD6, FAD8 and FAB2) did not appear to be sensitive to redox state of the chloroplast in the range of inhibition of transport electronic studied. The development of a specific antibody against the protein FAD7 allowed us to determine that the light might regulate gene expression FAD7 through a mechanism postraduccional additional involve activation / inactivation of protein FAD7 and that the protein FAD7 was mainly localized in membranes tilacoidales. Finally, the study's second goal of this Doctoral Thesis showed that the greatest sensitivity to fotoinhibición of mutant STR7 was not due to a deficiency in degradation processes, synthesis and integration of the protein Dl, but to the inability of join efficiently PS II. This inability is related with the highest percentage of saturated fatty acids which presents STR7 in their membranes tilacoidales, which makes these membranes are more rigid than those of the wild cell line, and not an effect of the mutation on the structure of Dl.

Author: SANCHEZ MARTINEZ ISIBENE.
Year: 2006.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE CIENCIA Y TECNOLOGÍA.
Place of preparation: FACULTAD DE CIENCIA Y TECNOLOGÍA.

Summary: The work developed in the different chapters of this thesis, has sought to build a functional map contributing in this way, the genetic study of the potato and improve the integration of information available in a reference map, the map of ultra-dense potato (map UHD). This has been made possible by the construction of the map transcriptoma, forming a total of 696 molecular markers that were integrated into the map UHD. Moreover, in this same map joined a total of 184 QTLs / genes for different characters published in potato as resistors, and other quality. The unification of all this information into a single map, the possible association of 73 QTLs of resistance against different pathogens with 144 molecular markers (TDFs) on the map ultra-dense potato. Of these 144 TDFs, analyzed the sequences of 36, 15 of them were potential candidate markers for the detection of resistance genes to different biotic factors and 9 TDFs did not show any homology in the analysis in the databases used may if possible resistance genes so far without describing. Moreover, new techniques were applied (BSA-cDNA-AFLP, cDNA-AFLP differential and analysis of microarrays) in the analysis of the expression of the genome of wild relatives of potatoes and resilient, with the aim of exploring the scope of these techniques and to associate genes with quantitative resistance to G.pallida. In addition to analyzing allelic variants of these genes candidate, also were integrated into the map UHD posiblitando this way, the relationship between genes and phenotypic expression and association as a result of functions to genes.