RESUMEN
Aquaporins are thought to be associated with water transport and play important roles in cotton (Gossypium hirsutum) fibre elongation. Among aquaporins, plasma membrane intrinsic proteins (PIPs) constitute a plasma-membrane-specific subfamily and are further subdivided into PIP1 and PIP2 groups. In this study, four fibre-preferential GhPIP2 genes were functionally characterized. The selective interactions among GhPIP2s and their interaction proteins were studied in detail to elucidate the molecular mechanism of cotton fibre development. GhPIP2;3 interacted with GhPIP2;4 and GhPIP2;6, but GhPIP2;6 did not interact with GhPIP2;4. Coexpression of GhPIP2;3/2;4 or GhPIP2;3/2;6 resulted in a positive cooperative effect which increased the permeability coefficient of oocytes, while GhPIP2;4/2;6 did not. GhBCP2 (a blue copper-binding protein) inhibited GhPIP2;6 water channel activity through their interaction. Overexpression of GhPIP2 genes in yeast induced longitudinal growth of the host cells. By contrast, knockdown of expression of GhPIP2 genes in cotton by RNA interference markedly hindered fibre elongation. In conclusion, GhPIP2 proteins are the primary aquaporin isoforms in fibres. They selectively form hetero-oligomers in order to regulate their activities to meet the requirements for rapid fibre elongation.
Asunto(s)
Acuaporinas/metabolismo , Fibra de Algodón , Gossypium/metabolismo , Proteínas de Plantas/metabolismo , Animales , Acuaporinas/genética , Membrana Celular/metabolismo , Regulación hacia Abajo , Fluorescencia , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Genes de Plantas/genética , Gossypium/genética , Oocitos/metabolismo , Proteínas de Plantas/genética , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schizosaccharomyces/genética , Fracciones Subcelulares/metabolismo , Técnicas del Sistema de Dos Híbridos , Xenopus laevis/metabolismoRESUMEN
Copper is vitally required for plants at low concentrations but extremely toxic for plants at elevated concentrations. Plants have evolved a series of mechanisms to prevent the consequences of the excess or deficit of copper. These mechanisms require copper-interacting proteins involved in copper trafficking. Blue copper-binding proteins (BCPs) are a class of copper proteins containing one blue copper-binding domain binding a single type I copper. To investigate the role of BCPs in plant development and in response to stresses, we isolated nine cDNAs encoding the putative blue copper-binding proteins (GhBCPs) from cotton (Gossypium hirsutum). Meanwhile, four corresponding genes (including GhBCP1-GhBCP4), which contain a single intron inserted in their conserved position, were isolated from cotton genome. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that the nine GhBCP genes are differentially expressed in cotton tissues. Among them, GhBCP1 and GhBCP4 were predominantly expressed in fibers, while the transcripts of GhBCP2 and GhBCP3 were accumulated at relatively high levels in fibers. These four genes were strongly expressed in early fiber elongation, but dramatically declined with further fiber development. In addition, these GhBCP genes were upregulated in fibers by Cu(2+) , Zn(2+) , high-salinity and drought stresses, but downregulated in fibers by Al(3+) treatment. Overexpression of GhBCP1 and GhBCP4 in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu(2+) , Zn(2+) and high-salinity stresses. These results suggested that these GhBCPs may participate in the regulation of fiber development and in response to high-salinity and heavy metal stresses in cotton.
Asunto(s)
Proteínas Portadoras/genética , Fibra de Algodón , Regulación de la Expresión Génica de las Plantas , Gossypium/genética , Metales Pesados/toxicidad , Salinidad , Estrés Fisiológico/genética , Secuencia de Aminoácidos , Proteínas Portadoras/química , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas/genética , Gossypium/efectos de los fármacos , Gossypium/crecimiento & desarrollo , Manitol/farmacología , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/metabolismo , Análisis de Secuencia de Proteína , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacosRESUMEN
Cotton (Gossypium hirsutum), the most important textile crop worldwide, often encounters water stress such as drought or waterlog during its growth season (Summer). To investigate molecular mechanism of water regulation in cotton plants, three cDNAs encoding the plasma membrane intrinsic protein (PIP) were isolated from cotton root cDNA library, and designated GhPIP1;1, GhPIP2;1 and GhPIP2;2, respectively. All of the three PIP proteins displayed water channel activity in Xenopus laevis oocytes. GhPIP2;1 and GhPIP2;2 proteins, however, showed much higher water transport activity than that of the GhPIP1;1 protein. Northern blot analysis revealed that all of the three genes were preferentially expressed in young roots. Further analysis by Real-time quantitative RT-PCR revealed that the transcripts of all the three genes were accumulated at high levels in 3-day-old young roots, but dramatically declined to much lower levels in 6-14 days old roots during seedling development, suggesting that expressions of the isolated GhPIP genes are developmentally regulated in roots. Additionally, expressions of the three genes were remarkably up-regulated or down-regulated under different stresses such as NaCl, cold, PEG (polyethylene glycol) treatments. Collectively, the results suggest that these genes may be involved in root development and in response to stresses.