Avaliação funcional das proteínas desacopladoras mitocondriais em Arabidopsis thaliana utilizando mutantes de inserção
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Data
2022-04-08
Autores
Lima, Rômulo Pedro Macêdo
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Universidade Estadual Paulista (Unesp)
Resumo
As proteínas desacopladoras mitocondriais (UCPs) são proteínas especializadas no transporte mitocondrial capazes de dissipar o gradiente eletroquímico de prótons gerados na respiração independente da síntese de ATP. Essas proteínas fazem parte da família de carreadores aniônicos mitocondriais e desempenham um papel fundamental na manutenção da função mitocondrial. Em plantas, a importância das UCPs como componentes da tolerância celular ao estresse oxidativo já foi demonstrada em estudos prévios. Três genes codificando UCP já foram descritos em Arabidopsis thaliana (nomeados AtUCP1-3), sendo boa parte dos dados funcionais obtidos para a isoforma AtUCP1. Portanto, o papel desempenhado pelas demais isoformas é ainda pouco estudado. Diante disso, mutantes de inserção de arabidopsis foram empregados para investigar a existência de redundância funcional entre essas isoformas. Para tal, numa primeira abordagem, duplo-mutantes de inserção para diferentes combinações gênicas (atucp1/atucp2, atucp1/atucp3 e atucp2/atucp3) foram obtidos e analisados funcionalmente durante o desenvolvimento vegetativo e reprodutivo. Em uma segunda etapa foram empreendidas análises de metabólitos durante a fase vegetativa destes duplo-mutantes. Em paralelo, baseado em dados prévios da primeira abordagem que indicaram fenótipos mais evidentes na fase vegetativa do duplo-mutante atucp1/atucp3 em relação ao controle selvagem, o perfil global de expressão gênica deste duplo-mutante na fase vegetativa foi determinado usando RNA-Seq. Análises subsequentes de bioinformática com datasets encontrados na literatura foram empreendidas. Os dados obtidos na primeira etapa indicam que as três isoformas parecem atuar de forma complementar em diferentes processos associados ao crescimento e desenvolvimento de A. thaliana. As isoformas AtUCP1 e AtUCP2 atuariam de forma conjunta na manutenção da homeostase e função mitocondrial durante a fase reprodutiva. Por outro lado, as isoformas AtUCP2 e AtUCP3 teriam maior relevância funcional na atividade fotossintética, enquanto que a AtUCP1 atuaria na manutenção do aparato fotossintético. Além disso, as isoformas AtUCP2 e AtUCP3 atuariam em conjunto na manutenção do equilíbrio redox. Os dados indicam ainda que todas as três isoformas atuam em conjunto para a redução das espécies reativas de oxigênio (ERO) na mitocôndria. Cabe ressaltar que o duplo knockdown dos genes AtUCP1 e AtUCP3 foi o que promoveu as maiores alterações fenotípicas e de função mitocondrial na fase vegetativa. Curiosamente, uma maior atividade respiratória foi registrada no duplo-mutante atucp2/atucp3, o que pode ser justificado pela forte expressão compensatória do gene AtUCP1 sob condições normais. De maneira similar, uma forte expressão compensatória da isoforma não inativada foi detectada em todas as três combinações de duplo-mutantes crescidos em condições normais ou em presença de estresse (salino e osmótico), o sugerindo uma redundância parcial entre elas. As análises de metabólitos revelaram profundas alterações no teor de alguns metabólitos primários (açúcares, aminoácidos, ácidos orgânicos e componentes de ácidos nucléicos) e secundários nos três duplo-mutantes. Em consonância, diversos genes diferencialmente expressos associados ao cloroplasto, mitocôndria e à resposta aos estresses abióticos foram detectados no duplo-mutante atucp1/atucp3. As análises comparativas de RNA-Seq para validação com o dataset selecionado de A. thaliana tipo selvagem reforçam os impactos causados por toda a célula. Em conjunto, os resultados obtidos evidenciaram uma intensa reprogramação transcricional e metabólica em decorrência do duplo Knockdown de genes AtUCP1 and AtUCP3. As análises apontaram ainda para a existência de uma redundância funcional parcial entre as três isoformas, que atuariam ora de forma isolada, ora de maneira conjunta na manutenção da homeostase e função mitocondrial durante o crescimento e desenvolvimento de A. thaliana. Essas descobertas fornecem novos subsídios para fundamentar o papel desempenhado pelas diferentes isoformas e fornecem insights sobre a atuação in planta da isoforma AtUCP3 em associação com a AtUCP1.
Mitochondrial uncoupling proteins (UCP) are specialized proteins involved in mitochondrial transport, which are able to dissipate the proton electrochemical gradient generated by respiration independent of ATP synthesis. These proteins belong to the mitochondrial anionic carrier family and play a key role in the maintenance of the mitochondrial function. In plants, their relevance as components of cell tolerance to oxidative stress has already been shown in previous studies. Three genes encoding UCP have been described in Arabidopsis thaliana (named AtUCP1-3), with most functional data being obtained for the AtUCP1 isoform. Therefore, the role played by the other isoforms is still poorly understood. In view of this, we decided to investigate the occurrence of functional redundancy between these isoforms using arabidopsis insertion mutants. For this, in a first approach, double-insertion mutants for different gene combinations (atucp1/atucp2, atucp1/atucp3 and atucp2/atucp3) were obtained and functionally analyzed during vegetative and reproductive development. In a second step, metabolome analysis was carried out during the vegetative phase of these double mutants. In parallel, based on previous data from the first approach indicating more evident phenotypes in the vegetative phase of the atucp1/atucp3 mutant compared with the wild-type (WT) control, the global gene expression profile of this double mutant in the vegetative phase was determined using RNA-Seq. Subsequent bioinformatics analyzes with datasets found in the literature were carried out. The data obtained in the first approach indicate that the three isoforms act in a complementary way in different processes associated with A. thaliana growth and development. The concerted action of the AtUCP1 and AtUCP2 isoforms seems to be required for maintaining homeostasis and mitochondrial function during the reproductive phase. On the other hand, our data suggest that the AtUCP2 and AtUCP3 isoforms have higher functional relevance in the photosynthetic activity, while AtUCP1 would act in favor of photosynthetic apparatus maintenance. In addition, a concerted action of the AtUCP2 and AtUCP3 isoforms seems to be required for maintaining the redox balance. All three isoforms are necessary to reduce reactive oxygen species (ROS) in mitochondria. Of note, the double knockdown of the AtUCP1 and AtUCP3 promoted the highest phenotypical changes and alterations in mitochondrial function in the vegetative phase. Interestingly, the atucp2/atucp3 mutant showed a higher respiratory activity, which can be explained by the strong compensatory expression of AtUCP1 under normal conditions. Similarly, a strong compensatory expression of the non-inactivated isoform was detected in all three double mutants grown under normal and stressed conditions (saline and osmotic), thus suggesting a partial redundancy between the isoforms. Additionally, the metabolome analysis revealed profound changes in the content of some primary (sugars, amino acids, organic acids and nucleic acid components) and secondary metabolites in all three double mutants. Accordingly, several differentially expressed genes associated with chloroplast, mitochondria and response to abiotic stresses were detected in atucp1/atucp3 double mutant plants. Comparative analysis of RNA-Seq for validation with the selected dataset of wild-type A. thaliana reinforce the impacts caused throughout the cell. Overall, these results indicate that an intense transcriptional and metabolic reprogramming occurs as a consequence of the double knockdown of AtUCP1 and AtUCP3 genes. Our data revealed the existence of a partial functional redundancy between the three isoforms, which would act either alone or in a concerted way for the maintenance of homeostasis and mitochondrial function during A. thaliana growth and development. These findings provide clues for the functionality of different AtUCP isoforms and give insights for the role played by the AtUCP3 isoform in association with AtUCP1 in the entire plant level.
Mitochondrial uncoupling proteins (UCP) are specialized proteins involved in mitochondrial transport, which are able to dissipate the proton electrochemical gradient generated by respiration independent of ATP synthesis. These proteins belong to the mitochondrial anionic carrier family and play a key role in the maintenance of the mitochondrial function. In plants, their relevance as components of cell tolerance to oxidative stress has already been shown in previous studies. Three genes encoding UCP have been described in Arabidopsis thaliana (named AtUCP1-3), with most functional data being obtained for the AtUCP1 isoform. Therefore, the role played by the other isoforms is still poorly understood. In view of this, we decided to investigate the occurrence of functional redundancy between these isoforms using arabidopsis insertion mutants. For this, in a first approach, double-insertion mutants for different gene combinations (atucp1/atucp2, atucp1/atucp3 and atucp2/atucp3) were obtained and functionally analyzed during vegetative and reproductive development. In a second step, metabolome analysis was carried out during the vegetative phase of these double mutants. In parallel, based on previous data from the first approach indicating more evident phenotypes in the vegetative phase of the atucp1/atucp3 mutant compared with the wild-type (WT) control, the global gene expression profile of this double mutant in the vegetative phase was determined using RNA-Seq. Subsequent bioinformatics analyzes with datasets found in the literature were carried out. The data obtained in the first approach indicate that the three isoforms act in a complementary way in different processes associated with A. thaliana growth and development. The concerted action of the AtUCP1 and AtUCP2 isoforms seems to be required for maintaining homeostasis and mitochondrial function during the reproductive phase. On the other hand, our data suggest that the AtUCP2 and AtUCP3 isoforms have higher functional relevance in the photosynthetic activity, while AtUCP1 would act in favor of photosynthetic apparatus maintenance. In addition, a concerted action of the AtUCP2 and AtUCP3 isoforms seems to be required for maintaining the redox balance. All three isoforms are necessary to reduce reactive oxygen species (ROS) in mitochondria. Of note, the double knockdown of the AtUCP1 and AtUCP3 promoted the highest phenotypical changes and alterations in mitochondrial function in the vegetative phase. Interestingly, the atucp2/atucp3 mutant showed a higher respiratory activity, which can be explained by the strong compensatory expression of AtUCP1 under normal conditions. Similarly, a strong compensatory expression of the non-inactivated isoform was detected in all three double mutants grown under normal and stressed conditions (saline and osmotic), thus suggesting a partial redundancy between the isoforms. Additionally, the metabolome analysis revealed profound changes in the content of some primary (sugars, amino acids, organic acids and nucleic acid components) and secondary metabolites in all three double mutants. Accordingly, several differentially expressed genes associated with chloroplast, mitochondria and response to abiotic stresses were detected in atucp1/atucp3 double mutant plants. Comparative analysis of RNA-Seq for validation with the selected dataset of wild-type A. thaliana reinforce the impacts caused throughout the cell. Overall, these results indicate that an intense transcriptional and metabolic reprogramming occurs as a consequence of the double knockdown of AtUCP1 and AtUCP3 genes. Our data revealed the existence of a partial functional redundancy between the three isoforms, which would act either alone or in a concerted way for the maintenance of homeostasis and mitochondrial function during A. thaliana growth and development. These findings provide clues for the functionality of different AtUCP isoforms and give insights for the role played by the AtUCP3 isoform in association with AtUCP1 in the entire plant level.
Descrição
Palavras-chave
Proteína desacopladora, Mitocôndrias, Expressão gênica, Mutagênese insercional, Metaboloma, Proteínas de desacoplamento mitocondrial, Uncoupling protein, Mitochondria, Double mutant, Gene expression and metabolome