Aluminum tolerance in Secale species: genetic diversity, molecular mechanisms and gene characterization
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2018-10-04
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O alumínio (Al) é um dos principais constituintes do solo que apenas solubiliza quando o pH se torna ácido (≤ 5.5), convertendo-se numa das formas mais fitotóxicas (Al3+). A toxicidade deste metal, nos abundantes solos ácidos, é uma das principais limitações da produtividade agrícola em grande parte do mundo. O centeio (Secale cereale L.) é uma cultura importante pela sua capacidade de resistir a stresses bióticos e abióticos. Dentro dos cereais, o centeio é o que mais tolera a exposição ao Al tendo, por isso, um fundo genético valioso para estudos de melhoramento genético. Pouco ou nada se sabe sobre o comportamento das espécies silvestres de centeio e, torna-se importante o seu estudo a fim de garantir novos recursos genéticos para contornar esta restrição no desenvolvimento das plantas, respondendo assim às necessidades crescentes da alimentação Mundial. Neste trabalho, foram estudadas seis espécies/subespécies silvestres assim como sete cultivares de centeio em diferentes contextos. Numa primeira abordagem (Capítulo II), usaram-se os marcadores moleculares dominantes RAPDs (Random Amplified Polymorphic DNA) e ISSRs (Inter Simple Sequence Repeats) para estudos de diversidade molecular, de relações genéticas e de associação quanto à característica da tolerância ao Al. Estes marcadores revelaram-se bastante informativos, obtendo um elevado grau de polimorfismo tanto no estudo de bulks como de genótipos individuais, o que permitiu aferir com sucesso a vasta variabilidade genética existente nos centeios em estudo. Foi possível determinar relações genéticas fiáveis com o recurso destes marcadores, uma vez que se obtiveram um grande número de fragmentos de DNA. A taxonomia do género Secale ainda é alvo de muita controvérsia e com estes dados contribuímos para aumentar o conhecimento adquirido e melhorar a classificação, uma vez que é o primeiro estudo publicado de relações genéticas dentro do género Secale com o uso de ambos os marcadores (RAPDs e ISSRs). Também se fizeram estudos de filogenias com os genes ScMATE1 e ScMATE3 (Multidrug and Toxic Compound Extrusion) que se mostraram similares. Todos estes dados levaram-nos a reconhecer três espécies de centeio (S. cereale, S. strictum e S. sylvestre) sendo possivelmente os ancestrais o S. strictum e o S. sylvestre. Os centeios foram caracterizados quanto à sua tolerância ao Al através do método de hidroponia, que se revelou bem-sucedido. A maioria dos centeios (cultivados e silvestres) testados mostrou uma grande capacidade de resistir ao choque provocado pelo Al e de recuperar a elevadas concentrações deste metal tóxico. Para além disso, encontrou-se uma vasta variabilidade quanto ao comportamento das plantas ao stress do Al quer intra- e interespecífica como intra- e intercultivar. A característica altamente polimórfica dos RAPDs e dos ISSRs atribui-lhes potencial na identificação de marcadores genéticos ligados à tolerância ao Al. De facto, foram encontrados 34 fragmentos de DNA com ambas as técnicas com associação a este traço genético que podem no futuro ser convertidos em SCARs (Sequence-characterized amplified region). Ambos os sistemas de marcadores se revelaram eficazes em todas as análises efetuadas, com especial destaque para os ISSRs, fornecendo recursos importantes para a seleção assistida por marcadores moleculares (MAS) quanto à tolerância ao Al. Numa segunda abordagem (Capítulo III), estudaram-se genes possivelmente envolvidos na tolerância ao Al assim como distúrbios relacionados com a toxicidade do Al, de modo a melhor entender o controlo genético e os mecanismos de resistência ao Al envolvidos em cada centeio. Pela primeira vez em centeios silvestres, foram estudados oito genes candidatos (ScALMT1, ScMATE1, ScMATE2, ScSTOP1, ScMDH1, ScMDH2, ScCu/ZnSOD e ScMnSOD) possivelmente envolvidos na tolerância ao Al. Todos eles parecem contribuir com o aumento da tolerância ao Al de, pelo menos, um centeio silvestre, com os genes ScALMT1 e ScMATE2 a ter um papel fundamental. Foi observado na maioria das raízes de centeio um padrão de exsudação de ácidos orgânicos induzível, com dois tempos distintos de libertação, enquanto uma minoria apresentou um padrão constitutivo, mas em quantidade abundante. Quatro perturbações celulares (acúmulo de Al, deteção de H2O2 e visualização de peroxidação lipídica e de morte celular) relacionadas com a toxicidade do Al em raízes mostraram estar correlacionados entre si e também, com os recrescimentos radiculares. As estratégias dos centeios tolerantes ao Al parecem basear-se nos mecanismos de exclusão e de desintoxicação. Encontrámos um gene candidato para controlar o locus Alt1 situado no cromossoma 6RS, que denominámos de ScMATE3. Os estudos de expressão e de filogenias, tal como, a existência de três SNPs (single-nucleotide polymorphism) exclusivos a centeios tolerantes, sugerem que este gene está relacionado com a tolerância ao Al. A localização subcelular prevista indica que pode ser um MATE tonoplástico e que podemos estar perante um mecanismo interno de tolerância. Obtiveram-se várias sequências codificantes dos genes ScMATE1 e ScMATE3, publicadas no GenBank, que mostraram variabilidade genética tanto dentro como entre as espécies e cultivares de centeio. Encontraram-se duas cópias do gene ScMATE1 associadas à tolerância/sensibilidade ao Al. Pela primeira vez, os mecanismos de resistência ao Al foram elucidados nos centeios silvestres. No género Secale, a tolerância ao Al parece ser um traço evolutivo e geneticamente complexo onde mecanismos diferentes parecem coexistir. Vários genes aparentam estar implicados na resposta ao stress provocado pelo Al, cujo efeito cumulativo poderá ser a chave para a elevada capacidade que os centeios possuem para resistir à toxicidade do Al.
Aluminum (Al) is one of the main constituents of the soil that only solubilizes when the pH becomes acidic (≤ 5.5), being converted into its most phytotoxic form (Al3+). Acid soils are abundant and Al toxicity is the major limitation for plant productivity worldwide. Rye (Secale cereale L.) is an important crop due to its ability to resist biotic and abiotic stresses. Within cereals, rye is the one that most tolerates Al exposure and, therefore, has a valuable genetic background for Al tolerance genetic improvement. Little or nothing is known about the behavior of wild rye relatives, and its study is crucial in order to guarantee new genetic resources to circumvent this constraint on plant development, thus keeping up with the rising world food demands. In this work, six wild species/subspecies and seven rye cultivars were studied in different contexts. In a first approach (Chapter II), the dominant molecular markers RAPDs (Random Amplified Polymorphic DNAs) and ISSRs (Inter Simple Sequence Repeats) were used for molecular diversity, genetic relationships and Al tolerance association studies. These markers were very informative, reaching high polymorphism values with both bulks and individual genotypes studies. Moreover, they successfully revealed the vast genetic variability present in the ryes at study. Since a large number of DNA fragments were obtained, reliable genetic relationships were determined with both markers. The taxonomy of the genus Secale is still a matter of much controversy. This is the first published work in genetic relationships within this genus with both RAPD and ISSR markers, which allows us to contribute for the increment of the acquired knowledge improving the classification of the Secale genus. Similar phylogenetic relationships were obtained with the genes ScMATE1 and ScMATE3 (Multidrug and Toxic Compound Extrusion). All data led us to recognize three rye species (S. cereale, S. strictum and S. sylvestre) with S. strictum and S. sylvestre being possibly the ancestors. Screening of Al tolerance was carried out through hydroponic methodologies which proved to be successful. Most of the cultivated and wild ryes showed great skills to resist and to recover Al shock at huge concentrations. In addition, a wide intra- and interspecific as intra- and intercultivar variability for Al tolerance was found. The highly polymorphic feature of ISSRs and RAPDs gives them potential for the identification of genetic markers linked to Al tolerance. Indeed, 34 loci associated with this genetic trait were found with both techniques that may be converted into SCARs (Sequence-characterized amplified region) in the future. Both marker systems proved to be effective in all analyzes with particular emphasis on ISSRs, so providing important resources for molecular markerassisted selection (MAS) of Al tolerance. In a second approach (Chapter III), genes possibly involved in Al tolerance as well as cell disturbances related to Al toxicity were studied in order to better understand the genetic control and the Al resistance mechanisms involved in each rye. Eight candidate genes (ScALMT1, ScMATE1, ScMATE2, ScSTOP1, ScMDH1, ScMDH2, ScCu/ZnSOD and ScMnSOD) possibly involved in Al tolerance were studied for the first time in wild ryes. All candidate genes seem to have an active contribution on enhanced Al-tolerance of, at least, one wild rye where ScALMT1 and ScMATE2 have a key role. An inducible organic acid exudation pattern was observed in most rye roots with two distinct release time whereas a minority presented a constitutive pattern but in abundant quantity. Four cell disorders (Al accumulation, H2O2 detection and visualization of lipid peroxidation and cell death) related to Al toxicity in roots showed a positive correlation with each other’s and with root regrowth measurement. Al-tolerant ryes strategies seem to be based on Al-exclusion and Al-detoxification mechanisms. We found a candidate gene to control the Altl locus located on chromosome 6RS that we named ScMATE3. Expression and phylogeny studies suggest that this gene is related to Al tolerance, such as the existence of three single-nucleotide polymorphisms (SNPs) exclusive to tolerant ryes. The predicted subcellular localization indicates a possible tonoplast MATE suggesting an internal Al tolerance mechanism. Several coding sequences of ScMATE1 and ScMATE3 genes were obtained and published on GenBank who’s showed a great genetic variability both within and between rye species/subspecies and cultivars. Two copies of ScMATE1 gene associated with Al tolerance/sensitivity were found in the species S. sylvestre and S. vavilovii. Al-resistance mechanisms were clarified for the first time in wild ryes. In Secale genus, Al tolerance appears to be an evolutionary and genetically complex trait where different mechanisms coexist. Several genes seem to be implicated in the Al-stress response of rye, whose cumulative effect may be the key to its high ability to resist Al toxicity.
Aluminum (Al) is one of the main constituents of the soil that only solubilizes when the pH becomes acidic (≤ 5.5), being converted into its most phytotoxic form (Al3+). Acid soils are abundant and Al toxicity is the major limitation for plant productivity worldwide. Rye (Secale cereale L.) is an important crop due to its ability to resist biotic and abiotic stresses. Within cereals, rye is the one that most tolerates Al exposure and, therefore, has a valuable genetic background for Al tolerance genetic improvement. Little or nothing is known about the behavior of wild rye relatives, and its study is crucial in order to guarantee new genetic resources to circumvent this constraint on plant development, thus keeping up with the rising world food demands. In this work, six wild species/subspecies and seven rye cultivars were studied in different contexts. In a first approach (Chapter II), the dominant molecular markers RAPDs (Random Amplified Polymorphic DNAs) and ISSRs (Inter Simple Sequence Repeats) were used for molecular diversity, genetic relationships and Al tolerance association studies. These markers were very informative, reaching high polymorphism values with both bulks and individual genotypes studies. Moreover, they successfully revealed the vast genetic variability present in the ryes at study. Since a large number of DNA fragments were obtained, reliable genetic relationships were determined with both markers. The taxonomy of the genus Secale is still a matter of much controversy. This is the first published work in genetic relationships within this genus with both RAPD and ISSR markers, which allows us to contribute for the increment of the acquired knowledge improving the classification of the Secale genus. Similar phylogenetic relationships were obtained with the genes ScMATE1 and ScMATE3 (Multidrug and Toxic Compound Extrusion). All data led us to recognize three rye species (S. cereale, S. strictum and S. sylvestre) with S. strictum and S. sylvestre being possibly the ancestors. Screening of Al tolerance was carried out through hydroponic methodologies which proved to be successful. Most of the cultivated and wild ryes showed great skills to resist and to recover Al shock at huge concentrations. In addition, a wide intra- and interspecific as intra- and intercultivar variability for Al tolerance was found. The highly polymorphic feature of ISSRs and RAPDs gives them potential for the identification of genetic markers linked to Al tolerance. Indeed, 34 loci associated with this genetic trait were found with both techniques that may be converted into SCARs (Sequence-characterized amplified region) in the future. Both marker systems proved to be effective in all analyzes with particular emphasis on ISSRs, so providing important resources for molecular markerassisted selection (MAS) of Al tolerance. In a second approach (Chapter III), genes possibly involved in Al tolerance as well as cell disturbances related to Al toxicity were studied in order to better understand the genetic control and the Al resistance mechanisms involved in each rye. Eight candidate genes (ScALMT1, ScMATE1, ScMATE2, ScSTOP1, ScMDH1, ScMDH2, ScCu/ZnSOD and ScMnSOD) possibly involved in Al tolerance were studied for the first time in wild ryes. All candidate genes seem to have an active contribution on enhanced Al-tolerance of, at least, one wild rye where ScALMT1 and ScMATE2 have a key role. An inducible organic acid exudation pattern was observed in most rye roots with two distinct release time whereas a minority presented a constitutive pattern but in abundant quantity. Four cell disorders (Al accumulation, H2O2 detection and visualization of lipid peroxidation and cell death) related to Al toxicity in roots showed a positive correlation with each other’s and with root regrowth measurement. Al-tolerant ryes strategies seem to be based on Al-exclusion and Al-detoxification mechanisms. We found a candidate gene to control the Altl locus located on chromosome 6RS that we named ScMATE3. Expression and phylogeny studies suggest that this gene is related to Al tolerance, such as the existence of three single-nucleotide polymorphisms (SNPs) exclusive to tolerant ryes. The predicted subcellular localization indicates a possible tonoplast MATE suggesting an internal Al tolerance mechanism. Several coding sequences of ScMATE1 and ScMATE3 genes were obtained and published on GenBank who’s showed a great genetic variability both within and between rye species/subspecies and cultivars. Two copies of ScMATE1 gene associated with Al tolerance/sensitivity were found in the species S. sylvestre and S. vavilovii. Al-resistance mechanisms were clarified for the first time in wild ryes. In Secale genus, Al tolerance appears to be an evolutionary and genetically complex trait where different mechanisms coexist. Several genes seem to be implicated in the Al-stress response of rye, whose cumulative effect may be the key to its high ability to resist Al toxicity.
Descrição
Tese de Doutoramento em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Secale spp. , solos ácidos , alumínio (Al) , fitotoxicidade