SILVA, Sérgio Luiz Ferreira da. Mecanismos de proteção oxidativa contra estresses isolados e combinados de seca, salinidade e temperatura elevada em cajueiro. 2008. 174 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2008. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-07-28T13:44:41Z No. of bitstreams: 1 2008_tese_slfsilva.pdf: 1889228 bytes, checksum: 68be9e3049b90dcc357c42a9249cdbb7 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T17:36:30Z (GMT) No. of bitstreams: 1 2008_tese_slfsilva.pdf: 1889228 bytes, checksum: 68be9e3049b90dcc357c42a9249cdbb7 (MD5) / Made available in DSpace on 2016-08-02T17:36:30Z (GMT). No. of bitstreams: 1 2008_tese_slfsilva.pdf: 1889228 bytes, checksum: 68be9e3049b90dcc357c42a9249cdbb7 (MD5) Previous issue date: 2008 / In the present study, different biochemical and physiological mechanisms associated with oxidative protection were characterized in a semi-arid adapted species (cashew) submitted to drought, salinity and heat applied individually or in combination. The results demonstrate that cashew show high antioxidant capacity against the isolated effects of drought and salinity. This antioxidant protection is associated with the maintenance of the water status and the efficient interaction of the enzymatic and nonenzymatic antioxidant systems, avoiding H2O2 accumulation and lipid peroxidation. The activity of SOD and CAT, as the ASA and GSH antioxidants play a central role in oxidative protection in salt-treated plants, while the activity of SOD and APX associated with ASA and GSH are essential in plants exposed to drought. Oxidative stress is induced in cashew plants submitted to temperatures above 35 ºC, as indicated by H2O2 accumulation and lipid peroxidation, which may be due to enhanced photorespiration. The antioxidant enzymatic (SOD-CAT-APX) and nonenzymatic (ASA e GSH) systems are intensively modulated by heat stress. Salt-pretreated plants show higher stomatic restriction under heat stress than those previously exposed to drought. This results evidence that salt stress limits heat dissipation through transpiration more than drought when plants are exposed to high temperatures. APX activity is reduced in salt-pretreated plants under heat stress in comparison with drought-pretreated plants, suggesting that salinity could prominently affect the antioxidant role of this enzyme. Conversely, the antioxidant systems are dramatically restricted in drought-pretreated plants in relation to those initially exposed to salinity when these plants are subjected to high temperatures. This restriction may be associated with high oxidative injuries in plants exposed to drought followed by heat stress. According to the results of this work, high temperatures applied individually or in combination with drought enable oxidative stress more than salt stress associated with heat. In general, oxidative changes induced by drought and heat or salinity and heat are distinct from those triggered by these factors applied individually, as the metabolic alterations caused by combined stresses could not be estimated from the specific responses to drought, salinity or heat. / No presente estudo foram caracterizados diferentes mecanismos de proteção oxidativa do cajueiro, espécie adaptada ao semi-árido, frente aos efeitos isolados e combinados dos estresses salino, hídrico e temperatura elevada. Para tanto, foram realizados estudos para avaliar as alterações oxidativas induzidas pelos estresses salino, hídrico, temperatura elevada e pelas combinações dos estresses salino e hídrico com temperaturas elevadas na espécie. Os resultados demonstram que o cajueiro apresenta alta capacidade de proteção oxidativa frente os estresses salino e hídrico. Essa proteção está associada à restrição estomática, manutenção do status hídrico e eficiente interação dos sistemas antioxidantes enzimático e não enzimáticos, impedindo o acúmulo de H2O2 e a peroxidação de lipídios. Durante o estresse salino, as enzimas SOD, CAT e os antioxidantes ASA e GSH foram os principais responsáveis pela proteção oxidativa, enquanto sob condições de seca ocorreu predominância das enzimas SOD e APX, associadas aos sistemas ASA e GSH. Temperaturas acima de 35 ºC induzem estresse oxidativo na espécie, atribuído ao acúmulo de H2O2 e a peroxidação de lipídios, provavelmente associada à indução de fotorrespiração. O estresse térmico apresentou intensa modulação dos sistemas de proteção oxidativa enzimático (SOD-CAT-APX) e não enzimático (ASA e GSH), indicando o papel desses antioxidantes na proteção oxidativa durante temperaturas elevadas. As plântulas submetidas à combinação de salinidade e temperatura elevada apresentaram maior restrição estomática, comparadas àquelas expostas a combinação de seca e temperatura alta. Esse resultado indica que o estresse salino pode levar a maior limitação da dissipação de calor, via fluxo transpiratório, que o estresse hídrico, durante exposição de plantas a temperaturas elevadas. A salinidade limitou a atividade da APX nas plântulas submetidas ao estresse térmico, sugerindo que o estresse salino pode afetar o papel da APX na proteção oxidativa durante temperaturas elevadas. Durante a exposição das plântulas a temperaturas elevadas o estresse hídrico limitou mais a atividade dos sistemas antioxidantes SOD-CAT-APX e ASA e GSH, comparado ao estresse salino. Essa restrição ocorreu associada ao maior nível de injúrias oxidativas nas plântulas expostas a combinação de seca e calor. Os resultados demonstram que temperaturas elevadas é o principal estresse abiótico que causa dano oxidativo na espécie e que a combinação dos estresses hídrico e temperatura elevada está mais associada a dano oxidativo do que a combinação de salinidade e temperatura alta. No geral, os resultados mostram que as alterações oxidativas atribuídas à combinação de seca e calor ou salinidade e calor, são distintas daquelas associadas aos estresses isolados. Indicam ainda, que as mudanças induzidas pela combinação de seca e calor ou salinidade e calor não podem ser estimadas com base nos efeitos isolados dos respectivos estresses.

Why don’t crop plants grow as fast as they should? In optimal conditions, elite crop varieties routinely outperform those grown in the average field. The vast majority of this reduction in growth activity is due to abiotic stresses such as drought, heat, and nutrient limitation. Abiotic stress reduces plant growth by triggering a reduction of meristem size and causing premature differentiation of proliferating cells. Differentiated cells are no longer able to divide, and smaller meristems have a reduced capacity to restore growth when the abiotic stress passes. We have designed and evaluated a novel high-throughput screening system to identify compounds able to reduce or prevent this premature differentiation in order to retain modest growth capacity in stressful conditions and enable rapid recovery from stress. Such chemicals can be applied to crop plants using existing agricultural methods, and because there is no need for genetic modification, it is widely applicable to many different crop species. Using the novel technique of flow sorting followed by protoplast culture, we have developed a high-throughput automated confocal imaging method to screen chemicals for their effects upon cell differentiation. Meristem protoplasts isolated from the root tips of pROW1:GFP Arabidopsis plants were monitored for differentiation when exposed to different chemicals. To evaluate this system, a library of biologically active small molecules provided by Syngenta was screened against protoplasts and whole plants. Several compounds were identified with the ability to improve Arabidopsis root growth in in vitro growth conditions. Two subsets of these chemicals were identified: a subset of chemicals that improved stress tolerance through modulation of post-meristem differentiation, and a subset of chemicals that improve growth rate by increasing rates of cell division in the root apical meristem. This screening system is able to detect the subset of chemicals that was shown to affect postmeristem differentiation, but not the other subset. No false positives were detected. These results suggest that this single-cell screening system is a powerful, high-throughput method suitable for the detection of molecules for use in crop protection.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-03-26T14:57:06Z No. of bitstreams: 2 Dissertação - Thuana Marcolino Mota Rodrigues - 2018.pdf: 1461884 bytes, checksum: 1101273815dddbd06af73157343c0ccb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-03-26T14:57:32Z (GMT) No. of bitstreams: 2 Dissertação - Thuana Marcolino Mota Rodrigues - 2018.pdf: 1461884 bytes, checksum: 1101273815dddbd06af73157343c0ccb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-26T14:57:32Z (GMT). No. of bitstreams: 2 Dissertação - Thuana Marcolino Mota Rodrigues - 2018.pdf: 1461884 bytes, checksum: 1101273815dddbd06af73157343c0ccb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The genus Trichoderma ssp, is today the group of species most commonly studied and used to act in agriculture as biological control against phytopathogens. Its rapid mycelial growth, associated with high production of conidia, synthesis of several antibiotics and ability to live in different forms (saprotrophic, symbiont or mycoparasite) are characteristics that make it attractive for this purpose. The relation of mycoparasitism, as well as oscillations in environmental conditions, naturally causes cell stress and consequent response to the stressing agent. This response occurs through changes in cellular metabolism, activating its defense mechanisms that include the performance of heat shock proteins (Hsp). The objective of this work was to identify the Hsp70 family of Trichoderma asperellum and to analyze the expression of three genes encoding proteins of this family during mycoparasitism and in situations of thermal and ethanol stress. The identification of the T. asperellum Hsp70 proteins was possible from the database analysis (JGI) of the T. asperellum genome, available, but not annotated. We identified a total of 12 proteins from the Hsp70 family in T. asperellum, three of which were selected for gene expression assays. Paired cultivation was carried out between T. asperellum and phytopathogens: Sclerotinia sclerotiorum and Fusarium oxysporum in three phases of mycoparasitism: pre-contact, contact and post-contact. We verified that the expression of Tahsp70a, Tahsp70b and Tahsp70c genes varies according to the phases of the mycoparasitism and the phytopathogen studied. Expression of hsp70 under thermal stress was evaluated at the 38 °C condition for 30 minutes, 1, 2 and 4 hours and at the conditions of 4, 10 and 32 °C for 1 hour. Ethanol stress was also performed for 1 hour. During the thermal stress Tahsp70a and Tahsp70b presented higher induction and the Tahsp70c gene had its highest induction in the cold shock at 4 °C. In ethanol stress the analyzed genes did not present significant expression. / O gênero Trichoderma ssp, constitui hoje o grupo de espécies de fungos mais estudadas e comumente usadas para atuar na agricultura como controle biológico contra fitopatógenos. Seu rápido crescimento micelial, associado a alta produção de conídios, síntese de diversos antibióticos e capacidade de viver de diversas formas (saprotrófica, simbionte ou micoparasita) são características que o tornam atraente para esse fim. A relação de micoparasitismo, bem como as oscilações nas condições ambientais, geram naturalmente estresse sobre as células desse fungo e consequente resposta ante o agente estressante. Esta resposta ocorre através de mudanças no metabolismo celular, ativando seus mecanismos de defesa que incluem o desempenho de proteínas de choque térmico (Hsp). O objetivo deste trabalho foi identificar a família Hsp70 de Trichoderma asperellum e analisar a expressão de três genes codificando proteínas desta família durante o micoparasitismo e em situações de estresse térmico e estresse por etanol. A identificação das proteínas Hsp70 de T. asperellum foi possível a partir de análises em banco de dados (JGI) do genoma de Trichoderma asperellum, disponível, mas não anotado. Identificamos no total 12 proteínas da família Hsp70 em T. asperellum, sendo três selecionadas para ensaios de expressão gênica. Foi realizado cultivo pareado entre T. asperellum e os fitopatógenos: Sclerotinia sclerotiorum e Fusarium oxysporum em três fases do micoparasitismo: pré-contato, contato e pós-contato. Verificamos que a expressão dos genes Tahsp70a, Tahsp70b e Tahsp70c varia de acordo com as fases do micoparasitismo e do fitopatógeno estudado. A expressão dos genes hsp70 em estresse térmico, foi avaliada na condição de 38 °C durante 30 minutos, 1, 2 e 4 horas e nas condições de 4, 10 e 32 °C por 1 hora, O estresse por etanol também foi realizado por 1 hora. Durante o estresse térmico, Tahsp70a e Tahsp70b apresentaram maior indução sendo que o gene Tahsp70c teve sua maior indução no choque frio à 4 °C. No estresse por etanol os genes analisados não apresentaram expressão significativa.

Submitted by Mariana de Lara Campos Arcuri null (mariana.arcuri@aluno.ibb.unesp.br) on 2017-07-24T23:48:37Z No. of bitstreams: 1 Dissertação Final Ficha Catalográfica.pdf: 1462289 bytes, checksum: 6374b4edc59dd6c0ef4dab57c0176073 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-07-26T16:41:02Z (GMT) No. of bitstreams: 1 arcuri_mlc_me_bot.pdf: 1462289 bytes, checksum: 6374b4edc59dd6c0ef4dab57c0176073 (MD5) / Made available in DSpace on 2017-07-26T16:41:02Z (GMT). No. of bitstreams: 1 arcuri_mlc_me_bot.pdf: 1462289 bytes, checksum: 6374b4edc59dd6c0ef4dab57c0176073 (MD5) Previous issue date: 2017-05-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As lacases, p-diphenol-O2-oxidoredutases, são enzimas que desempenham papel fundamental na oxidação de monolignóis durante a biossíntese de lignina, estando, portanto, associadas a processos de crescimento e tolerância a alguns tipos de estresses abióticos. As lacases podem ser encontradas em bactérias, fungos, plantas e insetos. Estudos apontam que as lacases vegetais apresentam comportamento similar às de origem fúngica, atuando de formar complementar à rota de lignificação, em resposta ao estresse oxidativo, promovendo a detoxificação celular. As lacases são geralmente codificadas por famílias multigênicas. Através de análises in silico no genoma de eucalipto, o presente estudo identificou 54 genes codificadores de lacases (denominados EgLAC) que, filogeneticamente, se distribuem em seis diferentes subgrupos. Com base em dados de RNA-Seq, padrões distintos de expressão das lacases identificadas foram observados, sendo algumas enriquecidas em um dado órgão/tecido e outras com expressão não detectável pelo método. Análises de RT–qPCR de alguns genes selecionados com base em um banco de sequências expressas confirmaram, por exemplo, a expressão raiz-específica do gene EgLAC52 bem como as expressões preferenciais em raiz e folha dos genes EgLAC4 e EgLAC32, respetivamente. Em paralelo, a expressão de alguns destes genes em reposta a estresses foi investigado, e alterações na expressão relativa em resposta aos estresses oxidativo e osmótico foram constatadas, sugerindo a participação destas lacases em respostas a estresses abióticos. / Laccases are p-diphenol-O2-oxidoreductases encoded by multigene families widely distributed throughout the plant kingdom. They exhibit important roles in the oxidation of monolignols during lignin biosynthesis and are reported to be functionally involved in plant development, tolerance and response to stress. Apart from plants, laccases can be also found in bacteria, fungi and insects. Here, a genome-wide survey of the eucalyptus genome revealed the presence of 88 putative laccases genes. However, after meticulous analyzes using different approaches, the redundant sequences were discarded and 54 laccases genes (referred as EgLAC) were retrieved. These genes were phylogenetically distributed in six different subgroups. Based on RNA-Seq data, distinct organ/tissue expression patterns of the identified EgLAC genes were ascertained. The vast majority showed organ/tissue-enriched expression, while certain genes exhibited no detectable expression. RT-qPCR analyzes confirmed the organ/tissue expression patterns of a representative set of genes such as, for example, the rootspecific expression of EgLAC52 and the root and leaf preferential expressions of genes EgLAC4 and EgLAC32. Further expression profiling of selected EgLAC genes in response to oxidative and osmotic stresses revealed differences in their relative expression, with some genes being stress-induced. These results suggest that certain laccases might be implicated in abiotic stress responses.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / No presente estudo foram caracterizados diferentes mecanismos de proteÃÃo oxidativa do cajueiro, espÃcie adaptada ao semi-Ãrido, frente aos efeitos isolados e combinados dos estresses salino, hÃdrico e temperatura elevada. Para tanto, foram realizados estudos para avaliar as alteraÃÃes oxidativas induzidas pelos estresses salino, hÃdrico, temperatura elevada e pelas combinaÃÃes dos estresses salino e hÃdrico com temperaturas elevadas na espÃcie. Os resultados demonstram que o cajueiro apresenta alta capacidade de proteÃÃo oxidativa frente os estresses salino e hÃdrico. Essa proteÃÃo està associada à restriÃÃo estomÃtica, manutenÃÃo do status hÃdrico e eficiente interaÃÃo dos sistemas antioxidantes enzimÃtico e nÃo enzimÃticos, impedindo o acÃmulo de H2O2 e a peroxidaÃÃo de lipÃdios. Durante o estresse salino, as enzimas SOD, CAT e os antioxidantes ASA e GSH foram os principais responsÃveis pela proteÃÃo oxidativa, enquanto sob condiÃÃes de seca ocorreu predominÃncia das enzimas SOD e APX, associadas aos sistemas ASA e GSH. Temperaturas acima de 35 ÂC induzem estresse oxidativo na espÃcie, atribuÃdo ao acÃmulo de H2O2 e a peroxidaÃÃo de lipÃdios, provavelmente associada à induÃÃo de fotorrespiraÃÃo. O estresse tÃrmico apresentou intensa modulaÃÃo dos sistemas de proteÃÃo oxidativa enzimÃtico (SOD-CAT-APX) e nÃo enzimÃtico (ASA e GSH), indicando o papel desses antioxidantes na proteÃÃo oxidativa durante temperaturas elevadas. As plÃntulas submetidas à combinaÃÃo de salinidade e temperatura elevada apresentaram maior restriÃÃo estomÃtica, comparadas Ãquelas expostas a combinaÃÃo de seca e temperatura alta. Esse resultado indica que o estresse salino pode levar a maior limitaÃÃo da dissipaÃÃo de calor, via fluxo transpiratÃrio, que o estresse hÃdrico, durante exposiÃÃo de plantas a temperaturas elevadas. A salinidade limitou a atividade da APX nas plÃntulas submetidas ao estresse tÃrmico, sugerindo que o estresse salino pode afetar o papel da APX na proteÃÃo oxidativa durante temperaturas elevadas. Durante a exposiÃÃo das plÃntulas a temperaturas elevadas o estresse hÃdrico limitou mais a atividade dos sistemas antioxidantes SOD-CAT-APX e ASA e GSH, comparado ao estresse salino. Essa restriÃÃo ocorreu associada ao maior nÃvel de injÃrias oxidativas nas plÃntulas expostas a combinaÃÃo de seca e calor. Os resultados demonstram que temperaturas elevadas à o principal estresse abiÃtico que causa dano oxidativo na espÃcie e que a combinaÃÃo dos estresses hÃdrico e temperatura elevada està mais associada a dano oxidativo do que a combinaÃÃo de salinidade e temperatura alta. No geral, os resultados mostram que as alteraÃÃes oxidativas atribuÃdas à combinaÃÃo de seca e calor ou salinidade e calor, sÃo distintas daquelas associadas aos estresses isolados. Indicam ainda, que as mudanÃas induzidas pela combinaÃÃo de seca e calor ou salinidade e calor nÃo podem ser estimadas com base nos efeitos isolados dos respectivos estresses. / In the present study, different biochemical and physiological mechanisms associated with oxidative protection were characterized in a semi-arid adapted species (cashew) submitted to drought, salinity and heat applied individually or in combination. The results demonstrate that cashew show high antioxidant capacity against the isolated effects of drought and salinity. This antioxidant protection is associated with the maintenance of the water status and the efficient interaction of the enzymatic and nonenzymatic antioxidant systems, avoiding H2O2 accumulation and lipid peroxidation. The activity of SOD and CAT, as the ASA and GSH antioxidants play a central role in oxidative protection in salt-treated plants, while the activity of SOD and APX associated with ASA and GSH are essential in plants exposed to drought. Oxidative stress is induced in cashew plants submitted to temperatures above 35 ÂC, as indicated by H2O2 accumulation and lipid peroxidation, which may be due to enhanced photorespiration. The antioxidant enzymatic (SOD-CAT-APX) and nonenzymatic (ASA e GSH) systems are intensively modulated by heat stress. Salt-pretreated plants show higher stomatic restriction under heat stress than those previously exposed to drought. This results evidence that salt stress limits heat dissipation through transpiration more than drought when plants are exposed to high temperatures. APX activity is reduced in salt-pretreated plants under heat stress in comparison with drought-pretreated plants, suggesting that salinity could prominently affect the antioxidant role of this enzyme. Conversely, the antioxidant systems are dramatically restricted in drought-pretreated plants in relation to those initially exposed to salinity when these plants are subjected to high temperatures. This restriction may be associated with high oxidative injuries in plants exposed to drought followed by heat stress. According to the results of this work, high temperatures applied individually or in combination with drought enable oxidative stress more than salt stress associated with heat. In general, oxidative changes induced by drought and heat or salinity and heat are distinct from those triggered by these factors applied individually, as the metabolic alterations caused by combined stresses could not be estimated from the specific responses to drought, salinity or heat.

The occurrence of phenotypic and genotypic diversity is the key factor in crop improvement including abiotic stress tolerance. The focal objectives of this study were to evaluate and characterize 74 tropical indica rice breeding lines for phenotypic and genotypic diversity, screening for the most devastating abiotic stresses in rice; drought and salinity at the seedling stage at morpho-physiological and molecular levels. To fulfill these objectives, five studies were conducted in pots; first two experiments aimed at assessing phenotypic and yield variability at seedling and maturity stages respectively; based on several (more than 20) root and shoot traits which exploited a wide range of variability among genotypes for measured traits. Germplasm was then screened for drought stress at two moister regimes, 50%, and 100% moisture levels, under mini-hoop structures. Nine percent of the genotypes exhibited a high tolerance to drought stress, and genotypes IR86638 and IR49830 were identified as the most and least drought tolerant respectively. Germplasm was also screened for salinity tolerance in pure sand pot-culture (a simple, efficient and alternate screening method) at three levels; high salt stress (EC 12 dSm-1), moderate salt stress (EC 6 dSm-1), and control imposed one week after emergence. Thirteen genotypes (17.57%) were identified as highly salt tolerant; genotypes FED 473 and IR85427 were highly salt tolerant and salt sensitive, respectively. Root traits were found more crucial and best descriptors in identifying both salinity and drought tolerant genotypes. Genotypes were further used in Genome-wide Association Study (GWAS) to uncover important SNPs, QTLs or genes related to salinity tolerance. A higher number of significant SNPs were discovered for root traits, indicting the importance of root traits in identifying abiotic stress tolerance in rice. The knowledge gained from this investigation could be useful in breeding for better crop establishment, yield improvement, screening for any abiotic stress tolerance.

Abiotic stresses cause extensive loss to agriculture production worldwide. Cowpea is an important legume crop grown widely in tropical and subtropical regions where high temperature, ultraviolet-B (UVB) radiation and drought are the common stress factors limiting production. Various vegetative, physiological, biochemical and reproductive plant attributes were assessed under a range of UVB radiation levels in Experiment I and in a combination with two doses of each carbon dioxide concentration [CO2], temperature, and UVB radiation and their interactions in Experiment II by using six cowpea genotypes and sunlit plant growth chambers. The dynamics of photosynthesis and fluorescence processes were assessed in 15 cowpea genotypes under drought condition in Experiment III in pot-grown plants under sunlit conditions. A distinct response pattern was not observed in cowpea in response to UVB radiation form 0 to 15 kJ; however, plants grown under elevated UVB showed reduced photosynthesis resulting in shorter plants and produced smaller flowers and lower seed yield. Increased phenolic compounds appeared to be a defense response to UVB radiation. The growth enhancements observed by doubling of [CO2] were not observed when plants were grown in combination with elevated UVB or temperature which also showed the most detrimental effects on plant growth and seed yield. Results form Experiment I and II revealed that cowpea reproductive traits were highly sensitive to abiotic stresses compared to the vegetative growth and development. A total stress response index (TSRI) technique, derived from all vegetative and reproductive parameters, was used to screen genotypes for their stress tolerance to UVB or combination of stresses. An increase in water use efficiency while maintaining higher rate of photosynthesis was an important drought tolerance mechanism in tolerant cowpea genotypes. Using principal component analysis technique, four groups of the genotypes were identified for their drought tolerance. Evaluating same genotypes across stress conditions revealed that no single genotype has the absolute tolerance characters to all stress conditions. The identified diversity for abiotic stress tolerance among cowpea genotypes and associated traits can be used to develop tolerant genotypes suitable for an agro-ecological niche though traditional breeding or genetic engineering methods.

Le blé tendre, Triticum aestivum, est une des céréales les plus cultivées dans le monde. Le changement climatique qui se développe actuellement contraint fortement les cultures et altère leur rendement. La compréhension des mécanismes de réponse du blé tendre aux stress abiotiques est donc une problématique d’actualité. Plusieurs grandes familles de facteurs de transcription, dont la famille NAC,interviennent dans le développement de la plante et dans sa réponse aux stress environnementaux. Cette thèse, structurée en 3 volets, est ciblée sur l’étude de la famille NAC chez le blé tendre : les TaNAC. Dans un premier temps, nous avons étudié la structuration génomique et phylogénétique des 488 membres de la famille TaNAC, recensés à partir de la base de données la plus récente du blé tendre.Nous avons aussi étudié l’histoire évolutive de cette famille, qui a été marquée par des événements de duplication et de rétroposition. Enfin, une analyse de sa diversité allélique a permis d’identifier des gènes qui présentent des SNP montrant une forte association avec des paramètres d’accumulation des protéines de réserve dans le grain. Le deuxième chapitre de cette thèse a porté sur l’étude de l’expression de ces 488 gènes TaNAC dans plusieurs organes et en réponse aux stress thermique et sécheresse. Une analyse globale a été réalisée à partir de données bio-informatiques, suivie d’une étude in planta de l’expression d’une sélection de 23 gènes. Les profils d’expression obtenus ont révélé l’existence de 4 gènes TaNAC, encore jamais décrits dans la littérature et qui interviennent dans le développement du grain de blé tendre mais aussi dans sa réponse adaptative à plusieurs stress abiotiques. Le troisième volet de cette thèse a donc porté sur la caractérisation génétique, moléculaire et physiologique de ces 4 facteurs de transcription TaNAC. Ils appartiennent à un clade rassemblant des séquences présentant des similitudes génomique et structurale. De plus, ils sont localisés dans le noyau et leurs profils d’expression sont similaires, avec toutefois un niveau variable entre gènes et entre homéologues pour chaque gène. En réponse à un stress thermique modéré, ce profil d’expression est accéléré au cours du développement du grain ; le stade 120°Cj étant le stade clé qui montre la plus grande différence d’expression de ces gènes entre les conditions contrôle et stressée. Pour des raisons techniques, la production de plantes transgéniques sur- et sous-exprimant ces gènes n’a pas permis de valider l’implication de ces 4 TaNAC dans le développement du grain et en réponse à la température. Une analyse de génétique d’association a toutefois permis de mettre en évidence un lien entre des marqueurs moléculaires situés dans ces gènes et l’accumulation des protéines de réserve.Globalement, les résultats obtenus ont montré que des membres de la famille TaNAC sont impliqués dans le développement du blé tendre et dans sa réponse aux stress abiotiques. Plus particulièrement, 4 facteurs de transcription TaNAC semblent jouer un rôle clé dans l’accumulation des protéines dans le grain en réponse à un stress thermique modéré. / Bread wheat, Triticum aestivum, is one of the most cultivated cereal in the world. The climate change that is currently developing strongly constrains crops and impairs their yield. Understanding the wheat response mechanisms to abiotic stresses is therefore a current issue. Several major families of transcription factors, including the NAC family, are involved in the plant development and its response to environmental stresses. This thesis, structured in three parts, is focused on the study of the NAC family in bread wheat (TaNAC).First, we studied the genomic and phylogenetic structure of the 488 members of the TaNAC family identified from the latest database of bread wheat. We also studied the evolutionary history of this family, which was marked by duplication and retroposition events. Finally, an analysis of its allelic diversity allows us to identify genes with SNP showing a strong association with storage protein accumulation parameters in the grain. In a second part, we studied the expression of these 488 TaNAC genes in several organs and in response to heat and drought. An overall analysis was performed using bioinformatic data, followed by an in planta study of the expression of a selection of 23 genes. The expression profiles revealed that four TaNAC genes, never described in the literature, are involved in the wheat grain development but also in its adaptive response to several abiotic stresses. In a third part, we focused on the genetic, molecular and physiological characterization of these four TaNAC transcription factors. They belong to a clade gathering sequences with genomic and structural similarities. Moreover, they are localized in the nucleus and their expression profiles are similar, with a variable level between genes and between homeologs for each gene. In response to moderate heat stress, this expression profile is accelerated during grain development and a key stage at 120°Cj was identified, it shows the greatest difference in genes expression level between control and stressed conditions. For technical reasons, the production of transgenic plants over- and under-expressing these genes did not validate the involvement of these 4 TaNAC in grain development and in its temperature response. An association genetic analysis, however, showed a link between molecular markers located in these genes and the storage proteins accumulation. Overall, the results showed that members of the TaNAC family are involved in the bread wheat development and its response to abiotic stresses. In particular, four TaNAC transcription factors appear to play a key role in grain protein accumulation in response to a moderate heat stress.

Medicago is widely used as a forage crop. It is often susceptible to various pathogenic infections and exhibits low growth in drought and extreme climatic conditions. In the current study, a strategy was developed for over-expressing an “Osmotin-Chitinase” gene chimera in transgenic Medicago that could potentially confer resistance to different biotic and abiotic stresses. Seed germination of several cultivars of Medicago (M. sativa ssp. sativa, M. sativa ssp. falcata, M. sativa ssp. caerulea, M. truncatula, and M. Rugosa) was tested to determine the cultivars with good germination rates. Among these, M. sativa ssp. sativa showed an average of 80% germination over a period of one week and was subsequently selected for regeneration and transformation experiments. Different explants (cotyledons, hypocotyls, petioles) were tested for regeneration. Among these, hypocotyl explants showed highest (46.17 %) percent regeneration. Escherichia coli harboring Osmotin-Chitinase (OSM-CHI) gene chimera cloned into binary vector pBTEX with nptII as a selection marker was mobilized in Agrobacterium tumefaciens strain EHA105 which was employed in the transformation of hypocotyl explants of Medicago. Transformed calli were grown on callus inducing medium containing kanamycin for screening. Further screening of the positive transgenics was performed using PCR. Southern hybridization was carried out for further confirmation of successful transformation. Transformed shoots will be grown on the root inducing medium for developing into plantlets which would then be transferred to the green house and later tested for their degree of resistance to various biotic and abiotic stresses.

Du fait de sa croissance exceptionnelle combinée aux propriétés supérieures de son bois, l'Eucalyptus est devenu le feuillu le plus planté au monde et s'est imposé comme source de biomasse pour la production de papier et de biocarburants de seconde génération. Le bois est composé de parois secondaires lignifiées et sa formation est finement régulée par un réseau complexe, et globalement mal connu, de facteurs de transcription (FT). Les parois secondaires sont composées de 80% de polysaccharides, ciblés pour la plupart des bioproduits à haute valeur ajoutée, tandis que la lignine (20%) est responsable de la récalcitrance de la biomasse à la dégradation enzymatique mais augmente le potentiel énergétique du bois par combustion. Malgré son adaptabilité remarquable à différents sols et climats, la croissance de l'Eucalyptus varie fortement suivant ces facteurs. L'Eucalyptus est largement planté sur des sols lessivés dans les régions tropicales et subtropicales où les plantations industrielles font face à des épisodes de sécheresse de plus en plus fréquents, en combinaison avec des forts manques de nutriments, nécessitant de gros apports en fertilisants. Dans les région tempérées telles que l'Europe du Nord, la principale limitation à l'implantation de cet arbre dépourvu d'endodormance, est l'exposition au froid. Ces contraintes abiotiques sont aggravées par le changement climatique et leur impact sur la formation du bois et sa qualité restent peu documentés. Quelques données suggèrent que ces stress affectent le dépôt de la paroi secondaire ainsi que la structure du xylème. Cependant, ces résultats sont très hétérogènes entre différentes espèces et principalement focalisés sur des tissus différents du bois. La sélection de clones adaptés et le développement de pratiques culturales plus viables, sont essentiels pour améliorer la productivité et la qualité du bois, ce qui requiert une meilleure compréhension de la réponse des arbres au froid et au manque d'eau en interaction avec la nutrition. Dans le but de décrypter les régulations induites par le froid dans la différenciation du xylème, nous avons effectué une approche ciblée sur des Eucalyptus acclimatés au froid. Des analyses de biochimie, d'histochimie et de transcriptomique, ont révélé que le froid déclenche un dépôt de paroi secondaire précoce dans les cellules du xylème en développement, caractérisé par un fort dépôt de lignine. En parallèle, pour caractériser l'effet du manque d'eau combiné à différents régimes nutritifs, sur la formation et la qualité du bois, nous avons tiré profit d'un dispositif expérimental mis en place au champ avec un clone commercial d'Eucalyptus, soumis à une exclusion de pluie combinée à une fertilisation au potassium. Nous avons combiné des analyses globales du transcriptome et du métabolome, avec l'analyse des propriétés structurales et biochimiques du bois. L'approche intégrative de ces jeux de données a révélé que la fertilisation au potassium induit une répression de la biosynthèse de la paroi secondaire ainsi qu'une régulation de l'activité cambiale et la modification dans les propriétés du bois, avec une forte interaction avec l'exclusion d'eau. Ces deux approches ont permis l'identification de différents FT non caractérisés qui constituent des candidats prometteurs dans le contrôle de l'activité cambiale et du dépôt de paroi secondaire chez un ligneux. Leur caractérisation fonctionnelle chez le peuplier et l'Eucalyptus a révélé un nouveau régulateur clé de la biosynthèse de paroi secondaire, et plusieurs facteurs MYB potentiellement impliqués dans la balance entre formation de la paroi secondaire et croissance. / Due to its outstanding growth combined to superior wood properties, Eucalyptus genus has become the most planted hardwood on earth and emerged as the most appealing sources of renewable biomass feedstock for paper and second-generation biofuels. Wood is composed of lignified secondary cell walls (SCWs) and its formation is tightly regulated by a complex, partially unknown, transcription factors (TFs) network. SCWs are composed by 80% of polysaccharides targeted for most of value-added bioproducts, whereas lignin (20%) is responsible for biomass recalcitrance to enzymatic degradation but increase wood energetic potential for combustion. Despite its remarkable adaptability to various soils and climate environment Eucalyptus growth varies strongly according to these factors. Eucalyptus is extensively grown in highly weathered soils in tropical and subtropical regions where plantations are facing more frequent drought episodes in combination to nutrient starvation, requiring high amounts of expensive fertilizers. In temperate regions such as North of Europe, the main limitation for the expansion of this non-dormant tree is cold exposure, which reduces dramatically its growth. The effects of these stresses are emphasized in the actual context of climate change which induces sharp contrasting periods, and their impacts on wood formation and quality remain unknown. Scarce data from literature suggest that these stresses affect secondary cell wall (SCW) deposition as well as xylem cell patterning. However these results are highly heterogeneous among different species and mainly focused on non-woody tissues. The selection of adapted clones and the development of more sustainable cultural practices are crucial to improve wood productivity and quality, which require a better understanding of tree response to cold and water stress in interaction with nutrition. In order to unravel the regulation of xylem differentiation by low temperature, we performed a targeted approach on cold-acclimated Eucalyptus trees. By biochemical, histochemical and transcriptomic analyses, we revealed that low temperature trigger a precocious SCW deposition in developing xylem cells, characterized by a strong lignin deposition. In parallel, we aimed to characterize the effect of water stress combined to different mineral nutrition regimes, on wood formation and quality. To this end, we took advantage of an experimental design set up on field with a highly productive Eucalyptus commercial clone submitted to both rainfall exclusion combined to potassium fertilization. We combined large scale analyses of transcriptome and metabolome, with wood structural and biochemical properties analyses. The integrative approach with these datasets revealed that potassium fertilization induces a repression of SCW biosynthesis, together with regulation of cambial activity and modifications in wood properties, with a strong interaction with water exclusion. Both approaches allowed to point out several uncharacterized yet TFs which are highly promising candidates in the control of cambial activity and SCW deposition in a woody perennial. Characterization of their function in poplar and Eucalyptus revealed a new key regulator of SCW biosynthesis in wood, and several MYB TFs potentially involved in the trade-off between SCW biosynthesis and growth.