A macieira (Malus x domestica Borkh.), uma das frutíferas mais importantes das regiões de clima temperado, é caracterizada pela cessação de crescimento visível durante o inverno, processo este chamado de dormência. A dormência de gemas permite que a planta sobreviva às baixas temperaturas e é determinante para a eficiência na produção de maçãs. Entender o processo de dormência, assim como seus mecanismos de controle, tornou-se fundamental para contornar as perdas na produção, seja por meio de técnicas de manejo ou pela geração de variedades comerciais melhor adaptadas às regiões de cultivo. A enzima galactinol sintase (GolS) catalisa a primeira etapa da via de síntese de oligossacarídeos da família da rafinose, cujo acúmulo em resposta a estresses abióticos é fato bem conhecido. Em trabalho anterior, nosso grupo mostrou um acúmulo de transcritos constrastante de quatro genes MdGolS durante o inverno, sugerindo-se uma possível função adaptativa desses genes durante a dormência de gemas em macieira. Pelo presente trabalho, tem-se como objetivo quantificar o acúmulo de quatro proteínas MdGolS em gemas apicais da macieira Fuji Standard visando confirmar os perfis transcricionais previamente obtidos. Anticorpos policlonais capazes de reconhecer as quatro MdGolS foram separadamente produzidos para emprego na técnica de Western blot Análise por Dot blot permitiu-nos mostrar que os quatro conjuntos de anticorpos são específicos para o seu respectivo peptídeo, sem reações cruzadas. Cinco protocolos de extração de proteínas foram testados e otimizados para a obtenção de maiores quantidades de proteínas de gemas fechadas de macieira. Dois desses extratos foram escolhidos para realizar a técnica de Western blot. Proteínas totais foram quantificadas pelo método de Bradford e 20 μg foi considerada a quantidade mais adequada para os ensaios. O anticorpo MdGolS1 permitiu a detecção de sinais em ambos os extratos, mas nenhum no tamanho esperado para a respectiva proteína, isto é, com aproximadamente 38 kDa. Os anticorpos MdGolS2, MdGolS3 e MdGolS4 não permitiram detectar quaisquer sinais ou, ainda, sinais fracos que não puderam ser relacionados com o perfil transcricional previamente obtido. Estratégias alternativas, em nível proteico, para validar os níveis aumentados de transcritos de GolS anteriormente observados são discutidas. / Apple tree (Malus x domestica Borkh), one of the world’s most important fruit crops in temperate regions, is characterized by the cessation of visible growth during winter, a process called dormancy. Bud dormancy allows plant survival under low temperatures and it is crucial for the efficiency of apple production. The knowledge about the dormancy process, as well as its control mechanisms, became an essential approach to circumvent production losses either through management techniques or by the generation of new commercial varieties better adapted to each regional cultivation environment. The galactinol synthase (GolS) enzyme catalyzes the first step in the synthesis pathway of the raffinose family of oligosaccharides, whose accumulation in response to abiotic stress is well known. A previous work of our group allowed us to show contrasting transcript levels of four MdGolS during winter, suggesting a possible adaptative role for some of these genes during bud dormancy. The present work aims to quantify MdGolS protein accumulation in apical buds of the Fuji Standard apple tree cultivar in order to verify and associate with transcriptional profiles previously obtained. Polyclonal antibodies that recognize the four MdGolS produced to perform Western blot assays. Dot blot analysis revealed that the four sets of antibodies were specific to their respective peptides, without cross-reactions. Five protein extraction protocols were tested and optimized for obtaining greater amounts of proteins from apple apical buds. Two of these extracts were chosen to perform Western blot assays. Total protein was quantified using the Bradford method and 20 μg was found to be the most adequate for the assay. MdGolS1 antibody allowed the detection of signals in both extracts, but none in the expected protein mass, i.e., approximately 38 kDa. MdGolS2, MdGolS3 and MdGolS4 antibodies allowed us to detect only weak or no signals, that could not be correlated to the transcript profile previously obtained. Alternative strategies to validate the previously observed increased GolS transcript levels at the protein level are discussed.

A macieira (Malus x domestica Borkh.), uma das frutíferas mais importantes das regiões de clima temperado, é caracterizada pela cessação de crescimento visível durante o inverno, processo este chamado de dormência. A dormência de gemas permite que a planta sobreviva às baixas temperaturas e é determinante para a eficiência na produção de maçãs. Entender o processo de dormência, assim como seus mecanismos de controle, tornou-se fundamental para contornar as perdas na produção, seja por meio de técnicas de manejo ou pela geração de variedades comerciais melhor adaptadas às regiões de cultivo. A enzima galactinol sintase (GolS) catalisa a primeira etapa da via de síntese de oligossacarídeos da família da rafinose, cujo acúmulo em resposta a estresses abióticos é fato bem conhecido. Em trabalho anterior, nosso grupo mostrou um acúmulo de transcritos constrastante de quatro genes MdGolS durante o inverno, sugerindo-se uma possível função adaptativa desses genes durante a dormência de gemas em macieira. Pelo presente trabalho, tem-se como objetivo quantificar o acúmulo de quatro proteínas MdGolS em gemas apicais da macieira Fuji Standard visando confirmar os perfis transcricionais previamente obtidos. Anticorpos policlonais capazes de reconhecer as quatro MdGolS foram separadamente produzidos para emprego na técnica de Western blot Análise por Dot blot permitiu-nos mostrar que os quatro conjuntos de anticorpos são específicos para o seu respectivo peptídeo, sem reações cruzadas. Cinco protocolos de extração de proteínas foram testados e otimizados para a obtenção de maiores quantidades de proteínas de gemas fechadas de macieira. Dois desses extratos foram escolhidos para realizar a técnica de Western blot. Proteínas totais foram quantificadas pelo método de Bradford e 20 μg foi considerada a quantidade mais adequada para os ensaios. O anticorpo MdGolS1 permitiu a detecção de sinais em ambos os extratos, mas nenhum no tamanho esperado para a respectiva proteína, isto é, com aproximadamente 38 kDa. Os anticorpos MdGolS2, MdGolS3 e MdGolS4 não permitiram detectar quaisquer sinais ou, ainda, sinais fracos que não puderam ser relacionados com o perfil transcricional previamente obtido. Estratégias alternativas, em nível proteico, para validar os níveis aumentados de transcritos de GolS anteriormente observados são discutidas. / Apple tree (Malus x domestica Borkh), one of the world’s most important fruit crops in temperate regions, is characterized by the cessation of visible growth during winter, a process called dormancy. Bud dormancy allows plant survival under low temperatures and it is crucial for the efficiency of apple production. The knowledge about the dormancy process, as well as its control mechanisms, became an essential approach to circumvent production losses either through management techniques or by the generation of new commercial varieties better adapted to each regional cultivation environment. The galactinol synthase (GolS) enzyme catalyzes the first step in the synthesis pathway of the raffinose family of oligosaccharides, whose accumulation in response to abiotic stress is well known. A previous work of our group allowed us to show contrasting transcript levels of four MdGolS during winter, suggesting a possible adaptative role for some of these genes during bud dormancy. The present work aims to quantify MdGolS protein accumulation in apical buds of the Fuji Standard apple tree cultivar in order to verify and associate with transcriptional profiles previously obtained. Polyclonal antibodies that recognize the four MdGolS produced to perform Western blot assays. Dot blot analysis revealed that the four sets of antibodies were specific to their respective peptides, without cross-reactions. Five protein extraction protocols were tested and optimized for obtaining greater amounts of proteins from apple apical buds. Two of these extracts were chosen to perform Western blot assays. Total protein was quantified using the Bradford method and 20 μg was found to be the most adequate for the assay. MdGolS1 antibody allowed the detection of signals in both extracts, but none in the expected protein mass, i.e., approximately 38 kDa. MdGolS2, MdGolS3 and MdGolS4 antibodies allowed us to detect only weak or no signals, that could not be correlated to the transcript profile previously obtained. Alternative strategies to validate the previously observed increased GolS transcript levels at the protein level are discussed.

A macieira (Malus x domestica Borkh.), uma das frutíferas mais importantes das regiões de clima temperado, é caracterizada pela cessação de crescimento visível durante o inverno, processo este chamado de dormência. A dormência de gemas permite que a planta sobreviva às baixas temperaturas e é determinante para a eficiência na produção de maçãs. Entender o processo de dormência, assim como seus mecanismos de controle, tornou-se fundamental para contornar as perdas na produção, seja por meio de técnicas de manejo ou pela geração de variedades comerciais melhor adaptadas às regiões de cultivo. A enzima galactinol sintase (GolS) catalisa a primeira etapa da via de síntese de oligossacarídeos da família da rafinose, cujo acúmulo em resposta a estresses abióticos é fato bem conhecido. Em trabalho anterior, nosso grupo mostrou um acúmulo de transcritos constrastante de quatro genes MdGolS durante o inverno, sugerindo-se uma possível função adaptativa desses genes durante a dormência de gemas em macieira. Pelo presente trabalho, tem-se como objetivo quantificar o acúmulo de quatro proteínas MdGolS em gemas apicais da macieira Fuji Standard visando confirmar os perfis transcricionais previamente obtidos. Anticorpos policlonais capazes de reconhecer as quatro MdGolS foram separadamente produzidos para emprego na técnica de Western blot Análise por Dot blot permitiu-nos mostrar que os quatro conjuntos de anticorpos são específicos para o seu respectivo peptídeo, sem reações cruzadas. Cinco protocolos de extração de proteínas foram testados e otimizados para a obtenção de maiores quantidades de proteínas de gemas fechadas de macieira. Dois desses extratos foram escolhidos para realizar a técnica de Western blot. Proteínas totais foram quantificadas pelo método de Bradford e 20 μg foi considerada a quantidade mais adequada para os ensaios. O anticorpo MdGolS1 permitiu a detecção de sinais em ambos os extratos, mas nenhum no tamanho esperado para a respectiva proteína, isto é, com aproximadamente 38 kDa. Os anticorpos MdGolS2, MdGolS3 e MdGolS4 não permitiram detectar quaisquer sinais ou, ainda, sinais fracos que não puderam ser relacionados com o perfil transcricional previamente obtido. Estratégias alternativas, em nível proteico, para validar os níveis aumentados de transcritos de GolS anteriormente observados são discutidas. / Apple tree (Malus x domestica Borkh), one of the world’s most important fruit crops in temperate regions, is characterized by the cessation of visible growth during winter, a process called dormancy. Bud dormancy allows plant survival under low temperatures and it is crucial for the efficiency of apple production. The knowledge about the dormancy process, as well as its control mechanisms, became an essential approach to circumvent production losses either through management techniques or by the generation of new commercial varieties better adapted to each regional cultivation environment. The galactinol synthase (GolS) enzyme catalyzes the first step in the synthesis pathway of the raffinose family of oligosaccharides, whose accumulation in response to abiotic stress is well known. A previous work of our group allowed us to show contrasting transcript levels of four MdGolS during winter, suggesting a possible adaptative role for some of these genes during bud dormancy. The present work aims to quantify MdGolS protein accumulation in apical buds of the Fuji Standard apple tree cultivar in order to verify and associate with transcriptional profiles previously obtained. Polyclonal antibodies that recognize the four MdGolS produced to perform Western blot assays. Dot blot analysis revealed that the four sets of antibodies were specific to their respective peptides, without cross-reactions. Five protein extraction protocols were tested and optimized for obtaining greater amounts of proteins from apple apical buds. Two of these extracts were chosen to perform Western blot assays. Total protein was quantified using the Bradford method and 20 μg was found to be the most adequate for the assay. MdGolS1 antibody allowed the detection of signals in both extracts, but none in the expected protein mass, i.e., approximately 38 kDa. MdGolS2, MdGolS3 and MdGolS4 antibodies allowed us to detect only weak or no signals, that could not be correlated to the transcript profile previously obtained. Alternative strategies to validate the previously observed increased GolS transcript levels at the protein level are discussed.

A macieira (Malus x domestica Borkh.) é uma frutífera de clima temperado de grande importância econômica, e sua produtividade está diretamente relacionada à dormência. Além dos genes responsáveis pelo controle molecular, uma série de proteínas e metabólitos também é recrutada para proteger a integridade da gema dormente, destacando-se as desidrinas (DHN) e as enzimas galactinol sintases (GolS). As DHNs são proteínas que atuam na resposta adaptativa vegetal a estresses abióticos, enquanto que GolS são enzimas responsáveis pela síntese de galactinol, essencial à síntese de oligossacarídeos da família da rafinose (RFOs), os quais se acumulam em resposta a estresses abióticos. O objetivo do presente trabalho foi explorar a adaptação das gemas a condições de estresse a que são submetidas na dormência, visando identificar genes com potencial uso biotecnológico. Para tal, foram identificados e caracterizados os genes codificadores de DHNs e GolS no genoma da macieira por meio da utilização de ferramentas in silico para estudar a evolução, experimentos a campo e sob condições controladas, análises de expressão, localização subcelular, e geração de plantas transgênicas. As análises evolutivas sugerem que eventos de duplicação do genoma inteiro (WGD) foram responsáveis por moldar a evolução e diversificação dos genes GolS em macieira, enquanto que no caso das DHN eventos de duplicação em tandem e WGD nortearam a sua evolução. Nossos resultados sugerem que DHNs, galactinol e rafinose integram uma série de mecanismos que agem em conjunto durante a dormência a fim de proteger a integridade da gema, além dos carboidratos constituírem uma fonte de energia para a brotação. Ao longo da evolução, o aparecimento de novas estruturas e programas de desenvolvimento, tais como a gema e a dormência, necessitaram de adaptação de vias moleculares já estabelecidas, o que ajuda a explicar por que as dormências de gemas e de sementes compartilham rotas moleculares comuns. Finalmente, o gene MdDHN11 foi funcionalmente caracterizado e nossos resultados fornecem evidências de que MdDHN11 desempenha importantes papéis durante o desenvolvimento da semente de maçã, protegendo o embrião e o endosperma de alterações no status da água. Além disso, apenas a planta superexpressando MdDHN11 sobreviveu ao ensaio de simulação de seca, confirmando o potencial uso biotecnológico de DHNs de macieira no aumento da tolerância ao déficit hídrico. / Apple tree (Malus x domestica Borkh.) is a temperate fruit crop of great economic importance worldwide and its productivity is related to bud dormancy. Besides genes responsible for the molecular control of the process, a number of proteins and metabolites are also recruited to protect bud integrity, such as dehydrins (DHN) and galactinol synthases (GolS). DHNs are proteins that act on plant adaptive responses to abiotic stresses, while GolS are enzymes that catalyze for the synthesis of galactinol, an essential carbohydrate in the synthesis of raffinose family oligosaccharides (RFOs), which also accumulate in response to abiotic stresses. The objective of this work was to explore bud adaptation to stress conditions that occur during dormancy to identify genes with potential biotechnological applications. DHN and GolS genes were identified and characterized in the apple genome employing in silico tools, experiments under field and controlled conditions, expression analysis, subcellular localization assays, and the generation of transgenic plants. Evolutionary analyses suggest that whole genome duplication (WGD) events were responsible for shaping the evolution and diversification of GolS genes in apple, whereas WGD and tandem duplication events could be held accountable for DHN evolution. Our results suggest that DHNs, galactinol and raffinose integrate a series of mechanisms that act together during dormancy in order to protect bud integrity, besides the carbohydrates being an energy source for budbreak. During evolution, the appearance of new structures and developmental programs, such as buds and dormancy, required the adaptation of already established molecular pathways, partially explaining why bud and seed dormancy share common pathways. Finally, the MdDHN11 gene has been functionally characterized and our results provide evidences that MdDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from water deficit. Moreover, only the plant overexpressing MdDHN11 survived the water withholding assay, confirming the potential biotechnological use of apple DHNs in increasing tolerance to drought.

Raffinose family oligosaccharides (RFO) are major soluble carbohydrates of lentil (Lens culinaris Medik) seeds. When consumed by humans, RFO pass indigested through upper digestive tract as ¦Á-galactosidase enzyme required for RFO breakdown is not produced in humans. Consumption of lentils with high concentrations of RFO result in stomach discomfort, bloating and diarrhea due to bacterial fermentation of RFO in large intestine. This has lead to a relatively low use of lentils for human consumption. RFO are therefore considered anti-nutritional factors and development of lentil cultivars with reduced RFO concentrations is desired to improve lentil quality and consumption. To explore the possibility to develop lentil cultivars for low-RFO concentration, heritability of RFO trait and influence of environmental conditions on RFO must be known. In addition, RFO biosynthesis and accumulation in lentil seeds must be understood. However, very limited information is available on the above mentioned aspects of RFO in general and in lentil in particular. Therefore, the objectives of this study were: (1) to evaluate natural variation in RFO concentration and composition in commonly grown lentil cultivars and to determine the correlation between RFO concentration and other important seed constituents, (2) to investigate heritability and effect of environment on concentration and composition of lentil seed soluble carbohydrates, (3) to assess natural variation and diversity in RFO concentration in the genus Lens, (4) and to evaluate the association between galactinol synthase activity and accumulation of RFO in lentil seeds. Analysis of 22 lentil genotypes revealed significant (P ¡Ü 0.05) variation in total starch, amyolse, protein, total RFO and seed weight and seed colour. Stachyose was the major RFO in all lentil genotypes followed by raffinose and verbascose. A significant (P ¡Ü 0.05) inverse correlation was found between RFO and amylose concentration (r = -0.34); whereas RFO concentration and thousand seed weight correlated positively (r = 0.35). The analyses of variance of eleven cultivars grown at ten different environments showed that cultivar, environment and their interaction had significant effects on sugar concentration in lentil seeds. The high broad sense heritability of RFO (h2= 0.85) indicated that RFO concentration in lentil seeds is highly heritable and thus amenable to genetic improvement. An extensive evaluation of domesticated and wild species and subspecies of the genus Lens revealed significant (P ¡Ü 0.05) variation and diversity in RFO concentration and composition of individual oligosaccharides. Higher Shannon-Weaver diversity indices (SDI) for total RFO, raffinose and verbascose traits were observed in wild lentils compared to domesticated genotypes. Lens ervoides genotypes and some wild genotypes contained almost half the RFO concentration of cultivated lentils and therefore, wild genotypes may be useful for developing low-RFO lines. Higher verbascose and lower stachyose concentration was found in Lens ervoides genotypes, whereas higher raffinose and lower verbascose concentration is found in Lens nigricans genotypes. Study of galactinol synthase activity in developing seeds with varying RFO concentration showed no clear association between galactinol synthase activity and RFO concentration. The sucrose and galactinol concentration of developing seeds were also not associated with total RFO concentration of lentil seeds. This finding suggests a non-regulatory role of galactinol synthase in RFO biosynthetic pathway in lentil seeds. Together, all these findings are not only significant to devise strategies to develop lentil cultivars with reduced RFO concentration but also for understanding RFO biosynthesis in lentil seeds.

Raffinose family oligosaccharides (RFO) are major soluble carbohydrates of lentil (Lens culinaris Medik) seeds. When consumed by humans, RFO pass indigested through upper digestive tract as ¦Á-galactosidase enzyme required for RFO breakdown is not produced in humans. Consumption of lentils with high concentrations of RFO result in stomach discomfort, bloating and diarrhea due to bacterial fermentation of RFO in large intestine. This has lead to a relatively low use of lentils for human consumption. RFO are therefore considered anti-nutritional factors and development of lentil cultivars with reduced RFO concentrations is desired to improve lentil quality and consumption. To explore the possibility to develop lentil cultivars for low-RFO concentration, heritability of RFO trait and influence of environmental conditions on RFO must be known. In addition, RFO biosynthesis and accumulation in lentil seeds must be understood. However, very limited information is available on the above mentioned aspects of RFO in general and in lentil in particular. Therefore, the objectives of this study were: (1) to evaluate natural variation in RFO concentration and composition in commonly grown lentil cultivars and to determine the correlation between RFO concentration and other important seed constituents, (2) to investigate heritability and effect of environment on concentration and composition of lentil seed soluble carbohydrates, (3) to assess natural variation and diversity in RFO concentration in the genus Lens, (4) and to evaluate the association between galactinol synthase activity and accumulation of RFO in lentil seeds. Analysis of 22 lentil genotypes revealed significant (P ¡Ü 0.05) variation in total starch, amyolse, protein, total RFO and seed weight and seed colour. Stachyose was the major RFO in all lentil genotypes followed by raffinose and verbascose. A significant (P ¡Ü 0.05) inverse correlation was found between RFO and amylose concentration (r = -0.34); whereas RFO concentration and thousand seed weight correlated positively (r = 0.35). The analyses of variance of eleven cultivars grown at ten different environments showed that cultivar, environment and their interaction had significant effects on sugar concentration in lentil seeds. The high broad sense heritability of RFO (h2= 0.85) indicated that RFO concentration in lentil seeds is highly heritable and thus amenable to genetic improvement. An extensive evaluation of domesticated and wild species and subspecies of the genus Lens revealed significant (P ¡Ü 0.05) variation and diversity in RFO concentration and composition of individual oligosaccharides. Higher Shannon-Weaver diversity indices (SDI) for total RFO, raffinose and verbascose traits were observed in wild lentils compared to domesticated genotypes. Lens ervoides genotypes and some wild genotypes contained almost half the RFO concentration of cultivated lentils and therefore, wild genotypes may be useful for developing low-RFO lines. Higher verbascose and lower stachyose concentration was found in Lens ervoides genotypes, whereas higher raffinose and lower verbascose concentration is found in Lens nigricans genotypes. Study of galactinol synthase activity in developing seeds with varying RFO concentration showed no clear association between galactinol synthase activity and RFO concentration. The sucrose and galactinol concentration of developing seeds were also not associated with total RFO concentration of lentil seeds. This finding suggests a non-regulatory role of galactinol synthase in RFO biosynthetic pathway in lentil seeds. Together, all these findings are not only significant to devise strategies to develop lentil cultivars with reduced RFO concentration but also for understanding RFO biosynthesis in lentil seeds.

A macieira (Malus x domestica Borkh.) é uma frutífera de clima temperado de grande importância econômica, e sua produtividade está diretamente relacionada à dormência. Além dos genes responsáveis pelo controle molecular, uma série de proteínas e metabólitos também é recrutada para proteger a integridade da gema dormente, destacando-se as desidrinas (DHN) e as enzimas galactinol sintases (GolS). As DHNs são proteínas que atuam na resposta adaptativa vegetal a estresses abióticos, enquanto que GolS são enzimas responsáveis pela síntese de galactinol, essencial à síntese de oligossacarídeos da família da rafinose (RFOs), os quais se acumulam em resposta a estresses abióticos. O objetivo do presente trabalho foi explorar a adaptação das gemas a condições de estresse a que são submetidas na dormência, visando identificar genes com potencial uso biotecnológico. Para tal, foram identificados e caracterizados os genes codificadores de DHNs e GolS no genoma da macieira por meio da utilização de ferramentas in silico para estudar a evolução, experimentos a campo e sob condições controladas, análises de expressão, localização subcelular, e geração de plantas transgênicas. As análises evolutivas sugerem que eventos de duplicação do genoma inteiro (WGD) foram responsáveis por moldar a evolução e diversificação dos genes GolS em macieira, enquanto que no caso das DHN eventos de duplicação em tandem e WGD nortearam a sua evolução. Nossos resultados sugerem que DHNs, galactinol e rafinose integram uma série de mecanismos que agem em conjunto durante a dormência a fim de proteger a integridade da gema, além dos carboidratos constituírem uma fonte de energia para a brotação. Ao longo da evolução, o aparecimento de novas estruturas e programas de desenvolvimento, tais como a gema e a dormência, necessitaram de adaptação de vias moleculares já estabelecidas, o que ajuda a explicar por que as dormências de gemas e de sementes compartilham rotas moleculares comuns. Finalmente, o gene MdDHN11 foi funcionalmente caracterizado e nossos resultados fornecem evidências de que MdDHN11 desempenha importantes papéis durante o desenvolvimento da semente de maçã, protegendo o embrião e o endosperma de alterações no status da água. Além disso, apenas a planta superexpressando MdDHN11 sobreviveu ao ensaio de simulação de seca, confirmando o potencial uso biotecnológico de DHNs de macieira no aumento da tolerância ao déficit hídrico. / Apple tree (Malus x domestica Borkh.) is a temperate fruit crop of great economic importance worldwide and its productivity is related to bud dormancy. Besides genes responsible for the molecular control of the process, a number of proteins and metabolites are also recruited to protect bud integrity, such as dehydrins (DHN) and galactinol synthases (GolS). DHNs are proteins that act on plant adaptive responses to abiotic stresses, while GolS are enzymes that catalyze for the synthesis of galactinol, an essential carbohydrate in the synthesis of raffinose family oligosaccharides (RFOs), which also accumulate in response to abiotic stresses. The objective of this work was to explore bud adaptation to stress conditions that occur during dormancy to identify genes with potential biotechnological applications. DHN and GolS genes were identified and characterized in the apple genome employing in silico tools, experiments under field and controlled conditions, expression analysis, subcellular localization assays, and the generation of transgenic plants. Evolutionary analyses suggest that whole genome duplication (WGD) events were responsible for shaping the evolution and diversification of GolS genes in apple, whereas WGD and tandem duplication events could be held accountable for DHN evolution. Our results suggest that DHNs, galactinol and raffinose integrate a series of mechanisms that act together during dormancy in order to protect bud integrity, besides the carbohydrates being an energy source for budbreak. During evolution, the appearance of new structures and developmental programs, such as buds and dormancy, required the adaptation of already established molecular pathways, partially explaining why bud and seed dormancy share common pathways. Finally, the MdDHN11 gene has been functionally characterized and our results provide evidences that MdDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from water deficit. Moreover, only the plant overexpressing MdDHN11 survived the water withholding assay, confirming the potential biotechnological use of apple DHNs in increasing tolerance to drought.

A macieira (Malus x domestica Borkh.) é uma frutífera de clima temperado de grande importância econômica, e sua produtividade está diretamente relacionada à dormência. Além dos genes responsáveis pelo controle molecular, uma série de proteínas e metabólitos também é recrutada para proteger a integridade da gema dormente, destacando-se as desidrinas (DHN) e as enzimas galactinol sintases (GolS). As DHNs são proteínas que atuam na resposta adaptativa vegetal a estresses abióticos, enquanto que GolS são enzimas responsáveis pela síntese de galactinol, essencial à síntese de oligossacarídeos da família da rafinose (RFOs), os quais se acumulam em resposta a estresses abióticos. O objetivo do presente trabalho foi explorar a adaptação das gemas a condições de estresse a que são submetidas na dormência, visando identificar genes com potencial uso biotecnológico. Para tal, foram identificados e caracterizados os genes codificadores de DHNs e GolS no genoma da macieira por meio da utilização de ferramentas in silico para estudar a evolução, experimentos a campo e sob condições controladas, análises de expressão, localização subcelular, e geração de plantas transgênicas. As análises evolutivas sugerem que eventos de duplicação do genoma inteiro (WGD) foram responsáveis por moldar a evolução e diversificação dos genes GolS em macieira, enquanto que no caso das DHN eventos de duplicação em tandem e WGD nortearam a sua evolução. Nossos resultados sugerem que DHNs, galactinol e rafinose integram uma série de mecanismos que agem em conjunto durante a dormência a fim de proteger a integridade da gema, além dos carboidratos constituírem uma fonte de energia para a brotação. Ao longo da evolução, o aparecimento de novas estruturas e programas de desenvolvimento, tais como a gema e a dormência, necessitaram de adaptação de vias moleculares já estabelecidas, o que ajuda a explicar por que as dormências de gemas e de sementes compartilham rotas moleculares comuns. Finalmente, o gene MdDHN11 foi funcionalmente caracterizado e nossos resultados fornecem evidências de que MdDHN11 desempenha importantes papéis durante o desenvolvimento da semente de maçã, protegendo o embrião e o endosperma de alterações no status da água. Além disso, apenas a planta superexpressando MdDHN11 sobreviveu ao ensaio de simulação de seca, confirmando o potencial uso biotecnológico de DHNs de macieira no aumento da tolerância ao déficit hídrico. / Apple tree (Malus x domestica Borkh.) is a temperate fruit crop of great economic importance worldwide and its productivity is related to bud dormancy. Besides genes responsible for the molecular control of the process, a number of proteins and metabolites are also recruited to protect bud integrity, such as dehydrins (DHN) and galactinol synthases (GolS). DHNs are proteins that act on plant adaptive responses to abiotic stresses, while GolS are enzymes that catalyze for the synthesis of galactinol, an essential carbohydrate in the synthesis of raffinose family oligosaccharides (RFOs), which also accumulate in response to abiotic stresses. The objective of this work was to explore bud adaptation to stress conditions that occur during dormancy to identify genes with potential biotechnological applications. DHN and GolS genes were identified and characterized in the apple genome employing in silico tools, experiments under field and controlled conditions, expression analysis, subcellular localization assays, and the generation of transgenic plants. Evolutionary analyses suggest that whole genome duplication (WGD) events were responsible for shaping the evolution and diversification of GolS genes in apple, whereas WGD and tandem duplication events could be held accountable for DHN evolution. Our results suggest that DHNs, galactinol and raffinose integrate a series of mechanisms that act together during dormancy in order to protect bud integrity, besides the carbohydrates being an energy source for budbreak. During evolution, the appearance of new structures and developmental programs, such as buds and dormancy, required the adaptation of already established molecular pathways, partially explaining why bud and seed dormancy share common pathways. Finally, the MdDHN11 gene has been functionally characterized and our results provide evidences that MdDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from water deficit. Moreover, only the plant overexpressing MdDHN11 survived the water withholding assay, confirming the potential biotechnological use of apple DHNs in increasing tolerance to drought.

Le réchauffement climatique planétaire annoncé ne sera pas sans conséquence sur le métabolisme de la baie et en particulier sur la teneur en composés phénoliques. Les objectifs de cette thèse visent à mieux comprendre les processus de régulation associant le microclimat des baies et la synthèse des composés phénoliques. Par des approches moléculaires et biochimiques, ce travail a permis de mieux décrire les réponses spécifiques des baies de raisin (cultivar Cabernet-Sauvignon) aux facteurs rayonnement et température.L'analyse du transcriptome des baies exposées soit à un stress thermique soit à un stress lumineux a mis en lumière deux processus, une réponse rapide ayant lieu dès les premières heures de traitement et une réponse apparaissant au bout de plusieurs jours d’exposition aux stress. Cette étude a également permis de valider le système expérimental de découplage des effets du rayonnement et de la température.L'analyse des profils d’expression d’une vingtaine de gènes intervenant dans la voie de biosynthèse des flavonoïdes révèle des différences dans la réponse transcriptionnelle des gènes en fonction du stress et du stade développement auquel il a été appliqué. Néanmoins cette réponse n'est pas ou peu corrélée avec les variations des teneurs en anthocyanes et flavonols observées. Les teneurs en anthocyanes sont fortement réduites par l’action de la chaleur alors que les teneurs de certains flavonols augmentent sous l’influence du rayonnement. Au niveau des acides présents dans la pulpe des baies, l’acide malique voit sa teneur réduite sous l’effet de la chaleur ainsi que d’une forte intensité lumineuse. Les analyses montrent un impact important du stress thermique sur la teneur de la phénylalanine, de la tyrosine et de la lysine dans la pellicule.Parallèlement, l'initiation d’une étude du métabolome des pellicules de baies de raisin a été entreprise par UPLC-ESI-LTQ-Orbitrap™ et a permis d'acquérir des bases solides pour optimiser le protocole utilisé en vue d'analyses futures. Cette étude permet de dresser une liste préliminaire de métabolites d’intérêts.Enfin, le gène VvGOLS1 (Galactinol synthase 1) voit son expression stimulée dans les baies exposées au stress thermique, ce qui se traduit par une accumulation de galactinol, précurseur de la voie des RFOs. Un rôle de molécule «signal» est envisagé pour le galactinol. Un régulateur de la transcription de VvGOLS1 a également été identifié par des expériences d'expression transitoire en protoplastes. Il s'agit du facteur de transcription VvHsfA2, dont l'expression est également stimulée par le stress thermique. Dans ce contexte, la caractérisation des gènes de la famille des Hsfs (Heat Stress Factors) a été initée. / Global warming will affect berry metabolism, and especially phenylpropanoïd contents. This PhD work aimed to acquire a better understanding on the cellular processus linking the microclimate and the phenolic synthesis. By molecular and biochemical approaches, we extended this study to detail specific responses taking place in berries under heat and light stress.Transcriptomic analysis of heat-stressed and light-stressed berries showed the existence of two processes that occur in exposed berries. The first one triggers a rapid and transient expression of genes within the first hours of treatment. The second one mobilizes a set of genes showing increase in their expression after several days of stress exposure. Furthermore, this study validated the experimental set used to discriminate the effects of light and temperature, respectively.Expression analysis of 20 genes involved in the flavonoid biosynthetic pathway revealed strong differences among the transcriptional responses, depending on the nature of stress and the developmental stage of the berry. However, expression patterns of genes involved in the biosynthesis of flavonoid could not fully explain the changes in anthocyanin and flavonol contents. This suggests that additional regulation processes such as post-traductional modifications of enzymes or metabolite degradation might take place in berries under abiotic stress. Anthocyanin content decreases under heat stress whereas flavonol content increases under high light. Malic acid increases in berry exposed to heat stress and high light. Moreover, heat-stressed berries showed an accumulation of phenylalanine, tyrosine and lysine in skin but not in pulp.In parallel, a metabolomic analysis was initiated on stress exposed berry skins by using UPLC-ESI-LTQ-Orbitrap™ technology. The first experiments revealed contrasted metabolite contents in berries according to the stress applied, and highlighted several metabolites of interest. The preliminary assays will help optimize this powerful tool for futures analysis.Finally, expression of VvGOLS1 (Galactinol synthase 1) was strongly induced in grape berries exposed to heat stress, in good agreement with the observed galactinol accumulation. Role of galactinol as a signaling molecule is discussed. Transient expression experiments revealed that VvGOLS1 expression is regulated at the transcriptional level through VvHsfA2 action. VvHsfA2 expression is also stimulated under heat stress. In this context, characterization of the grapevine heat stress factors was initiated.

The barley scald pathogen (Rhynchosporium secalis) causes extensive economic losses, not only through lost product and quality, but also due to costs associated with chemical control. Economic and environmental impacts and the emerging resistance to fungicides and dominant resistance genes are reasons to understand molecular defence responses in order to develop new strategies to increase resistance of barley to this pathogen. In most pathosystems, defence gene expression in susceptible or resistant genotypes commonly differs quantitatively. Thus, differentially expressed genes between genotypes contrasting for response to infection by pathogens are considered candidate genes that have a role in resistance. This thesis presents functional analysis of a subset of genes isolated from a Suppression Subtractive Hybridisation library. The library was previously established and enriched for differentially expressed genes in epidermis of resistant and susceptible near-isogenic barley cultivars inoculated with R. secalis. Functional characterisation involved both investigating their putitative biochemical function as well as the genes‟ role(s) in biotic and abiotic stress responses. Three cDNA clones from the library were selected based on the putative function of the encoded proteins and the full length of the clones and their homologues were isolated from cDNA and genomic DNA. One of the clones represented a member of the pathogenesis-related protein family 17 (PR-17). Southern hybridisation showed that a small multigene family encodes the barley PR-17 proteins. Three members were cloned with two of them being novel. The second clone was homologous to galactinol synthases (GolS) and Southern blot analysis indicated existence of two GolS genes in the barley genome and subsequently two HvGolS members were isolated. The last clone (a single gene) showed similarity to very long chain fatty acid elongases, which indicates its involvement in synthesis of cuticular waxes. A characterised Arabidopsis mutant named fiddlehead (Atfdh) was highly similar to this gene and it was named HvFdh. Detailed expression analysis using Q-PCR, Northern blot analysis and publically available microarray data revealed that the isolated genes are regulated in response to a variety of abiotic and biotic stresses as well as different tissues during barley development. Under some treatments expression patterns were consistent with their putative roles and in agreement with results of other studies. Nevertheless, in other treatments expression profiles were not in agreement with previous findings in other plants indicating potentially different stress adaptation mechanisms between species. Further insight into the function of the encoded proteins was gained by their subcellular localisation using transient expression as GFP fusion proteins followed by confocal laser scanning microscopy. The results were in agreement with in silico predictions and their putative cellular function. In addition, a comprehensive list of homologous genes from other species was compiled for each gene by using public EST databases. Analyses of phylogenetic relationship and multiple sequence alignment of the homologues provided further clues to their function and conserved regions of the proteins. HvPR-17 anti-fungal properties were investigated by heterologous protein expression in E. coli and subsequent in vitro bioassays using purified protein under different conditions against a number of phytopathogenic fungi. However, no anti-fungal activity was observed. A construct with the AtFdh promoter driving the coding region of barley Fiddlehead was used for complementation of the Arabidopsis fiddlehead mutant to investigate functional orthology between these genes from dicots and monocots. The Arabidopsis fiddlehead mutant phenotype that shows contact-mediated organ fusion, germination of spore on epidermis and reduced number of trichomes was completely reverted by HvFdh. Finally, more than fifty transgenic barley lines were regenerated over-expressing or suppressing one of the three genes. The analyses of the transgenic progeny exhibited some interesting developmental phenotypes and resistance to scald and drought tolerance. These lines are awaiting further experiments to investigate the effect of altered expression in conferring resistance to other pathogens and abiotic stress tolerance as well as biochemical analysis. Collectively, in this work six barley genes were cloned and characterised by a variety of in silico techniques, temporal and transient expression analyses, subcellular localisation, in vitro bioassays and mutant complementation in Arabidopsis and loss- and gain-of-function transgenic barley plants. This work has provided insight into the function of these gene families in barley. Furthermore, the data suggest that they are regulated by the defence response to pathogenic fungi as well as drought, salinity and frost in barley. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375755 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2009