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Quercus suber L. (sobreiro) é um importante recurso florestal natural com relevância ecológica (sequestro de carbono, ciclo da água, reservatório de biodiversidade, proteção do solo) e impacto socioeconómico em Portugal. Compreender as causas do declínio do sobreiro e obter um conhecimento molecular mais profundo dos transportadores que intervêm nos processos que lidam com as respostas do ecossistema a fatores externos, emergiu como uma questão importante a abordar. As aquaporinas desempenham, provavelmente, um papel importante na forma como esta espécie lida com o stresse hídrico. A análise genómica revelou a presença de vários genes putativos de aquaporinas, mas o estudo do papel fisiológico de cada aquaporina é frequentemente impossibilitado pela sua expressão a nível tecidular. Assim, este estudo visou a caracterização molecular e fisiológica de duas aquaporinas de Quercus suber L., analisando também a sua regulação. Para esse efeito, selecionaram-se dois genes que codificam aquaporinas, que foram clonados e expressos numa estirpe aqy-null de Saccharomyces cerevisiae, a fim de se caracterizar individualmente cada uma das aquaporinas (QsTIP2;1 e QsPIP2;4) em relação ao transporte de água e de substratos atípicos. A caracterização funcional destas aquaporinas permitiu verificar que ambas as aquaporinas são funcionais como transportadores de água e de substratos atípicos, nomeadamente peróxido de hidrogénio e boro, sendo o resíduo Thr70 da aquaporina QsPIP2;4 determinante para o seu funcionamento enquanto transportador de água. Em relação aos aspectos relacionados com a regulação do transporte de água, verificou-se que nenhuma das aquaporinas estudadas é regulada por fosforilação mas são ambas reguladas pelo pH extracelular, sendo a sua atividade maior a pH 6.8. Para além disso, e contrariamente ao esperado, a atividade de QsPIP2;4 A aumenta em presença de cloreto de mercúrio 54%. Este aumento é revertido pela adição de β-mercaptoethanol em 14%.
Quercus suber L. (cork oak) is an important natural forest resource with ecological relevance (carbon sequestration, water cycle, biodiversity reservoir, soil protection) and socioeconomic impact in Portugal. Understanding the causes of cork oak decline and gaining deeper molecular knowledge of transporters involved in processes that deal with ecosystem responses to external factors, has emerged as an important issue to be addressed. Aquaporins probably play an important role in how this species copes with water stress. Genomic analysis has revealed several putative aquaporin-genes, but the study of the physiological role of each aquaporin is often difficult due to its tissue level expression. The present study aimed to perform the molecular and physiological characterization of two Quercus suber L. aquaporins, as well as to study their regulation. For this purpose, two genes encoding aquaporins were selected, which were cloned and expressed in a Saccharomyces cerevisiae aqy-null strain, in order to characterize individually each of the aquaporins (QsTIP2;1 and QsPIP2;4) in relation to transport water and atypical substrates. The functional characterization of these aquaporins allowed us to verify that both aquaporins are functional as water channels and transport of atypical substrates, namely hydrogen peroxide and boron. The Thr70residue of aquaporin QsPIP2;4 was considered determinant for its functioning as a water transporter. Regarding the aspects related to the regulation of water transport, it was verified that none of the studied aquaporins are regulated by phosphorylation but are both regulated by extracellular pH, and their activity is higher at pH 6.8. In addition, contrary to what was expected, the activity of QsPIP2;4 A increases in the presence of mercury chloride by 54%. This increase is reversed by the addition of β-mercaptoethanol by 14%.
Quercus suber L. (cork oak) is an important natural forest resource with ecological relevance (carbon sequestration, water cycle, biodiversity reservoir, soil protection) and socioeconomic impact in Portugal. Understanding the causes of cork oak decline and gaining deeper molecular knowledge of transporters involved in processes that deal with ecosystem responses to external factors, has emerged as an important issue to be addressed. Aquaporins probably play an important role in how this species copes with water stress. Genomic analysis has revealed several putative aquaporin-genes, but the study of the physiological role of each aquaporin is often difficult due to its tissue level expression. The present study aimed to perform the molecular and physiological characterization of two Quercus suber L. aquaporins, as well as to study their regulation. For this purpose, two genes encoding aquaporins were selected, which were cloned and expressed in a Saccharomyces cerevisiae aqy-null strain, in order to characterize individually each of the aquaporins (QsTIP2;1 and QsPIP2;4) in relation to transport water and atypical substrates. The functional characterization of these aquaporins allowed us to verify that both aquaporins are functional as water channels and transport of atypical substrates, namely hydrogen peroxide and boron. The Thr70residue of aquaporin QsPIP2;4 was considered determinant for its functioning as a water transporter. Regarding the aspects related to the regulation of water transport, it was verified that none of the studied aquaporins are regulated by phosphorylation but are both regulated by extracellular pH, and their activity is higher at pH 6.8. In addition, contrary to what was expected, the activity of QsPIP2;4 A increases in the presence of mercury chloride by 54%. This increase is reversed by the addition of β-mercaptoethanol by 14%.
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Aquaporinas Levedura Regulação Sobreiro Transporte de água e substratos atípicos Aquaporins Cork oak Regulation Transport of water and atypical substrates Yeast