Aflatoxin is a highly carcinogenic secondary metabolite produced by Aspergillus flavus contributing to significant loss of corn production. There are several breeding techniques to make the corn resistant to aflatoxin accumulation with the genotype having desirable characters. Corn- resistant inbred line Mp313E provides resistance against A. flavus. Near-isogenic lines containing QTLs were developed by backcrossing Mp313E (resistant parent) and Va35 (susceptible parent). WRKY transcription factors play an important role in the growth and development of plants. Searching DNA markers associated with these WRKY genes is a considerable approach for the plant breeders. In this study, PCR and agarose gel electrophoresis methods were used to determine the polymorphic WRKY gene DNA markers associated with aflatoxin reduction quantitative trait loci in maize. The analysis revealed 26 polymorphic maize WRKY genes. Computational 3D models of these WRKY genes were predicted. Virtual 2D plot was made for these WRKY proteins to determine their position.

Orientadores: Marcelo Menossi Teixeira, Augustina Gentile / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T18:33:40Z (GMT). No. of bitstreams: 1 Feltrim_Daniela_M.pdf: 2255232 bytes, checksum: c1f34d09fda3a2de4f7c62d0c7b9ec71 (MD5) Previous issue date: 2013 / Resumo: O presente trabalho possui como objetivo caracterizar fisiológica e funcionalmente um gene de milho que têm expressão relacionada com a tolerância ao cultivo em solo ácido contendo níveis tóxicos de alumínio (Al). Este gene, denominado Zmwrky69, foi identificado na linhagem de milho Cat100-6, tolerante ao Al (Mattiello et al., 2010). A análise da sequência da proteína ZmWRKY69 indica que ela pertence ao grupo III dessa família de fatores de transcrição, apresentando o motivo WRKYGKQ na região amino terminal e o motivo dedo de zinco Cx7Cx23HxC na região carboxi terminal. A proteína ZmWRKY69 está localizada no núcleo, conforme inferido por ensaio de localização subcelular em epitélio de cebola empregando uma fusão da proteína de milho com a proteína verde fluorescente (GFP). Ensaios de duplo híbrido em levedura indicam que a proteína ZmWRKY69 interage com um receptor de giberelina, uma proteína envolvida com a regulação por auxinas e uma proteína com função desconhecida. Plantas transgênicas de tabaco superexpressando Zmwrky69 apresentaram menor inibição do crescimento radicular na presença de Al, comparativamente às plantas selvagens. Esses resultados indicam que o gene Zmwrky69 codifica um fator de transcrição que apresenta um papel importante na tolerância ao Al em milho / Abstract: The objective of the present work is to characterize a gene differentially expressed in the maize inbred line Cat100-6 (aluminum tolerant) grown in acidic soil containing toxic levels of aluminum. This gene called Zmwrky69 was identified in the aluminum tolerant Cat100-6 inbred line (Mattiello et al., 2010). The sequence analysis of the ZmWRKY69 protein indicated that it belongs to the group III of this transcription factor family classification. The protein has the motif WRKYGKQ in the amino terminal region and a zinc finger motif Cx7Cx23HxC in the carboxi terminal region. This protein is localized in the nucleus as inferred by subcellular localization in onion epidermis using a maize protein fused to the Green Fluorescent Protein (GFP). Yeast two-hybrid essays indicate that the protein ZmWRKY69 interacts with a gibberellin receptor, a protein involved in auxin regulation and a protein with an unknown function. Transgenic tobacco plants overexpressing Zmwrky69 presented a lower degree of root growth inhibition in the presence of Al compared to wild type plants. These results indicate that Zmwrky69 gene encodes a transcription factor that presents an important role in Al tolerance in maize / Mestrado / Genetica Vegetal e Melhoramento / Mestra em Genética e Biologia Molecular

Le lin cultivé (Linum usitatissimum L.) est l’une des principales sources de lignanes, faisant de cette plante un modèle d’étude de cette voie du métabolisme spécialisé. Les principaux lignanes de lin dérivent de composés optiquement actifs, en particuliers les stéréoisomères du sécoisolaricirésinol qui sont synthétisés à partir de pinorésinol via laricirésinol. Les principales enzymes impliquées dans la synthèse de ces stéréoisomères sont deux isoformes de pinorésinol-laricirésinol réductases (PLR) déjà caractérisées et possédant des énantiospécificitées opposées. Néanmoins l'action de ces deux réductases bifonctionnelles ne permet pas d'expliquer les profils d'accumulation complexes notamment de laricirésinol et ses dérivés observés dans les graines, tiges et suspensions cellulaires de lin. Afin de mieux comprendre les mécanismes mis en oeuvre menant à ces profils d’accumulation de lignanes chez cette plante, la recherche de nouvelles PLRs a révélé l’existence de deux nouvelles isoformes. L’analyse de l'expression des gènes ainsi que l'activité enzymatique in vitro de ces deux nouvelles PLR putatives, LuPLR3 et LuPLR4 ont été élucidées. LuPLR4, in vitro, présente une activité réductase uniquement du pinorésinol. Ce type d’activité est ici décrit pour la première fois en dehors de la famille Brassicées et permet d’expliquer en partie les profils d’accumulation complexes observés chez le lin. De par leurs propriétés biocides, les lignanes sont suspectés jouer un rôle dans les mécanismes de défense des plantes. Dans le cadre de ce travail, suite à une élicitation fongique à l’aide d’extraits de Fusarium oxysporum spp. linii, un agent pathogène commun du lin, l’analyse de l’expression des différents gènes codant les isoformes de PLRs a révélées une induction globale et coordonnée. En particulier, dans le cas de l’isoforme LuPLR1, des délétions et mutations dans la région promotrice de son gène ont permis de mettre en évidence une région impliquée dans la régulation de la réponse à l’élicitation par F. oxysporum. Cette région contient plusieurs boîtes W, sites de liaison putatifs pour des facteurs de transcription de type WRKY. Les facteurs de transcription WRKY jouent un rôle dans les réponses aux stress biotique et abiotique. Un facteur de transcription candidat LuWRKY36 a été isolé à partir de suspensions cellulaires traitées avec des éliciteurs de F. oxysporum ou de l'acide abscissique. En particulier, les expériences de gel-retard et DPI-ELISA ont montré la capacité de liaison de LuWRKY36 à la boîte W3 présente du promoteur du gène LuPLR1. Cette régulation a ensuite été confirmée in vivo. Nous rapportons également l'impact différentiel de l'élicitation par des extraits de F. oxysporum sur l’expression des gènes LuWRKY36 et LuPLR1 ainsi que la production de sécoisolaricirésinol dans les variétés de lin sensible (Barbara) et résistante (Baïkal) à la fusariose. Enfin, la pleine exploitation des nombreux effets bénéfiques (en santé humaine ou cosmétique notamment) du sécoisolaricirésinol et des autres composés phénoliques accumulés dans les graines de lin nécessitent la mise au point de procédés d’extraction “verts”, efficaces voir sélectifs. Nous rapportons ici que l’utilisation de solvants eutectiques de type NADES (Natural Deep Eutectic Solvent) qui couplée à une extraction assistée par ultrasons, dans le cadre d’un procédé de type cracking, utilisant comme matériel de départ un coproduit d’extraction de l’huile de lin produit de manière innovante, permet d’obtenir des rendements d’extraction élevés et sélectifs de ces différents composés d’intérêt dans le cadre d’une démarche d’éco-extraction. / L. usitatissimum is one of the richest sources of lignans. Main flax lignan is optically active secoisolariciresinol that is synthesized from pinoresinol via lariciresinol. Key enzymes involved in the synthesis of this lignans are two isoforms of pinoresinol-lariciresinol reductases with opposite enantiospecificity. The action of bifunctional reductase does not allow for an explanation for the accumulation of lariciresinol and its derivates in seeds, stem and cell suspension. To try and better understand complex lignan profile we report expression and activity of two new putative PLRs, LuPLR3 and LuPLR4. LuPLR4 in vitro acts only as pinoresinol reductase what has only been seen in Brassicaceae family until now. Lignans play a role in plant defense. All PLRs are upregulated following Fusarium oxysporum attack, a common flax pathogen. Promoter deletions and mutation evidenced region involved in regulation of LuPLR1 gene response to Fusarium. The region contains several W boxes, putative binding sites for WRKY transcription factors. WRKY transcription factors play a role in response to biotic and abiotic stress. We have isolated LuWRKY36 from two cell suspension treated with Fusarium oxysporum or abscisic acid. Gel-shift assay and DPI-ELISA showed binding of LuWRKY36 to W box present in the LuPLR1 gene promoter. This regulation was also confirmed in vivo. We also report the differential impact of F. oxysporum elicitation on LuWRKY36 and LuPLR1 gene expression and secoisolariciresinol production in flax cultivars Barbara (Fusarium sensitive) and Baikal (Fusarium tolerant). Many beneficial effects of secoisolariciresinol and other phenolic compounds found in flax require “green” extraction and sometimes targeted purification of a specific compound. We report here that natural deep eutectic solvents using ultrasound assisted extraction can extract phenolic compounds from flax seed coat and that results indicate that by tuning different parameters of extraction we can target purification of desired plant product.

Wheat plays an important role in ensuring the global food security. Salinity of soil and water poses a major threat to its production and it affects both growth and development of wheat in a negative way. Wheat plants uses certain molecular mechanisms to adapt themselves under the salt stress.Transcription factor proteins are the proteins that control the response of the wheat towards abiotic stress like salinity.There are 56 transcription factor protein families in the wheat genome. However these transcription factor protein families are not classified into subfamilies.The main goal of this research study is to understand how machine learning algorithm can be used to identify and cluster the transcription factor proteins into sub families that can help in associating them with specific biological processes like salt stress. In this project K Mean Clustering method is used to cluster the WRKY transcription factor family into subfamilies. WRKY is identified and clustered into three distinct clusters. Cluster validation is performed using external validation and resulted in 90% validation score. This method can be applied to other transcription factor families also. This can ultimately be helpful in producing salt-tolerant varieties of the wheat that are resistant to abiotic stress like salinity and this can help to improve crop yield.

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semi-synthetic anticancer drugs. Biosynthesis of TIAs is tissue-specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. This study reports the isolation and characterization of the G10H promoter and two WRKY transcription factors regulating TIA biosynthesis. Geraniol 10-hydroxylase (G10H) controls the first committed step in the biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a PCR-based genome walking method. Sequence analysis revealed that the G10H promoter contains several potential eukaryotic regulatory elements involved in regulation of gene expression. For functional characterization, fusion constructs of G10H promoter fragments with the GUS reporter gene were generated and expression was analyzed in a tobacco protoplast transient expression assay. Gain-of-function experiments revealed the presence of three potential transcriptional enhancers located in regions between -191 and -147, -266 and -188, and -318 and -266, respectively. The G10H promoter was capable of conferring stable GUS expression in transgenic tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings, GUS expression was tissue-specific, restricted to the leaf and actively growing cells around the root tip. GUS expression was not detected in the hypocotyls, root cap and older developing areas of the root. The GUS expression in both transgenic C. roseus hairy roots and tobacco seedlings were responsive to fungal elicitors and methyljasmonate. Compared to other known promoters of TIA pathway genes, the G10H promoter contains unique binding sites for several transcription factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade. The majority of TIA pathway gene promoters contain typical W-box elements, which are frequently found to be the binding sites of WRKY transcription factors. CrWRKY1 and CrWRKY2 transcription factors were isolated using a degenerate PCR method. The C. roseus WRKY transcription factor, CrWRKY1 is preferentially expressed in roots and induced by phytohormones, jasmonate, gibberellic acid and ethylene. Overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially tryptophan decarboxylase (TDC), as well as transcriptional repressors ZCT1, ZCT2 and ZCT3. In contrast, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3 and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX-repressor domain to CrWRKY1, resulted in down-regulation of TDC and ZCTs but up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W-box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid and C. roseus protoplast assays. In CrWRKY1 hairy roots, up-regulation of TDC increased TDC activity, tryptamine concentration and resistance to 4-methyl tryptophan inhibition. Compared to control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors, including ORCA3, CrMYC2 and ZCTs, may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants. CrWRKY2 is induced by methyljasmonate induction. In plant, CrWRKY2 expression is mainly found in young leaves and the stem. The stable transformation of CrWRKY2 in C. roseus hairy roots up-regulated many pathway genes, especially the genes in vindoline biosynthesis. The accumulation of vindoline was observed in CrWRKY2 hairy roots.

Les alcaloïdes indoliques monoterpéniques (AIM) sont des molécules à propriétés anti-tumorales extraites de Catharanthus roseus. Leur coût de production et les besoins encore importants de ces médicaments en chimiothérapie, en font des cibles majeures pour la recherche de stratégies de production plus efficaces. L’objectif de ce travail vise à identifier de nouveaux facteurs de transcription (FT) régulateurs de la production des AIM. Cette étude porte plus particulièrement sur la caractérisation fonctionnelle et l’implication dans la régulation de la biosynthèse des AIM, de protéines de la famille des WRKY (CrWRKYs) et des protéines de type doigt de zinc (ZCTs) précédemment isolées au sein de l’EA2106 « Biomolécules et Biotechnologies Végétales ». Nos expériences ont révélé que, parmi ces protéines, CrWRKY22, CrWRKY32, ZCT1 et ZCT2 agissent en tant que facteurs de transcription et plus spécifiquement interagissent avec le promoteur du gène Crhds. Ce dernier code une enzyme de la voie du méthyl érythritol phosphate (MEP) considérée limitante pour la production des AIM. Ces travaux ont permis d’identifier de nouveaux FT ciblant la voie du MEP dont la régulation via les FT est encore peu élucidée à ce jour et d’envisager leur utilisation en ingénierie métabolique pour augmenter la production d’AIM par la modulation du flux terpénique chez C. roseus. / Monoterpene indole alkaloids (MIA) are molecules with anti-cancer properties from Catharanthus roseus. Their production cost and the important need in chemiotherapy make them major targets for the research of more efficient production strategies. The aim of this work is to identify new transcription factors (TF) that regulate MIA production. This study focuses especially on the functional characterization and the involvement in the MIA biosynthesis regulation, of proteins previously isolated in the EA2106 “Plant Biocompounds and Biotechnology” laboratory: 3 proteins that belong to the WRKY family (CrWRKYs) and 3 zinc finger proteins named ZCTs. Our experiments revealed that among them, CrWRKY22, CrWRKY32, ZCT1 and ZCT2 act as transcription factors and more specifically interact with the promoter of Crhds gene. Crhds encodes an enzyme of the methyl erythritol phosphate (MEP) pathway that is considered as limiting for MIA production. Our work allowed identifying new TFs targeting the MEP pathway those regulation through TFs is mostly unknown. Using such transcription factors in metabolic engineering could be now considered increasing MIA production by the modulation of terpenoid flux in C. roseus.