Aquaporins are important transmembrane water transport proteins which transport water and several neutral molecules. However, how aquaporins are involved in the synergistic transport of Mg2+and water remains poorly understood. Here, we found that the cassava aquaporin Me PIP2;7 was involved in Mg2+transport through interaction with Me MGT9, a lower affinity magnesium transporter protein. Knockdown of Me PIP2;7 in cassava led to magnesium deficiency in basal mature leaves with chlorosis and necrotic spots on their edges and starch over-accumulation. Mg2+content was significantly decreased in leaves and roots of Me PIP2;7-RNA interference(PIP-Ri) plants grown in both field and Mg2+-free hydroponic solution. Xenopus oocyte injection analysis verified that Me PIP2;7 possessed the ability to transport water only and Me MGT9 was responsible for Mg^(2+)effux.More importantly, Me PIP2;7 improved the transportability of Mg^(2+)via Me MGT9 as verified using the CM66 mutant complementation assay and Xenopus oocytes expressing system. Yeast twohybrid, bimolecular fuorescence complementation,co-localization, and co-immunoprecipitation assays demonstrated the direct protein–protein interaction between Me PIP2;7 and Me MGT9 in vivo. Mg2+fux was significantly elevated in Me PIP2;7-overexpressing lines in hydroponic solution through non-invasive micro-test technique analysis. Under Mg^(2+)-free condition, the retarded growth of PIP-Ri transgenic plants could be recovered with Mg^(2+)supplementation. Taken together, our results demonstrated the synergistic effect of the Me PIP2;7 and Me MGT9 interaction in regulating water and Mg2+absorption and transport in cassava.
Water uptake is crucial for crop growth and development and drought stress tolerance. The water channel aquaporins(AQP) play important roles in plant water uptake. Here, we discovered that a jasmonic acid analog, coronatine(COR), enhanced maize(Zea mays) root water uptake capacity under artificial water deficiency conditions. COR treatment induced the expression of the AQP gene Plasma membrane intrinsic protein 2;5(ZmPIP2;5).In vivo and in vitro experiments indicated that COR also directly acts on ZmPIP2;5 to improve water uptake in maize and Xenopus oocytes. The leaf water potential and hydraulic conductivity of roots growing under hyperosmotic conditions were higher in ZmPIP2;5-overexpression lines and lower in the zmpip2;5 knockout mutant, compared to wild-type plants. Based on a comparison between ZmPIP2;5 and other PIP2s, we predicted that COR may bind to the functional site in loop E of ZmPIP2;5. We confirmed this prediction by surface plasmon resonance technology and a microscale thermophoresis assay, and showed that deleting the binding motif greatly reduced COR binding. We identified the N241 residue as the COR-specific binding site, which may activate the channel of the AQP tetramer and increase water transport activity,which may facilitate water uptake under hyperosmotic stress.
相较于传统的抗体检测,适配体更易于大量快速合成,且可和多种检测技术相结合,在蛋白检测方面具有巨大的潜力.水孔蛋白作为生物体内水分跨膜运输的主要途径,了解其表达量的变化在植物水代谢研究中有着重要意义.利用传统的混合列分法构建了8个C端恒定半胱氨酸残基的类肽适配体文库,结合表面等离激元共振成像技术,筛选得到能特异性结合高等植物水孔蛋白PIP2的类肽适配体PPA7,其亲和力 K D高达2.52×10 -9 mol/L.利用PPA7检测了石竹玻璃化和正常植株的水孔蛋白表达量,结果表明,石竹玻璃化植株的水孔蛋白表达量显著高于正常植株.研究提供了一种新的植物蛋白定量检测策略,也为进一步明确水孔蛋白在组培苗玻璃化发生中的作用奠定了基础.
Objective: To investigate the effects of electroacupuncture(EA) on endolymphatic hydrops(EH) and the regulation of arginine vasopressin(AVP)-aquaporin-2(AQP2) pathway in guinea pigs. Methods: EH was induced in male guinea pigs by an intraperitoneal injection of AVP. For the treatment, EA was delivered to Baihui(GV 20) and Tinggong(SI 19) acupoints, once per day for 10 consecutive days. In histomorphological studies, cochlear hydrops degree was evaluated by hematoxylin-eosin(HE) staining, and then the ratio of scala media(SM) area to SM + scala vestibuli(SV) area(R value) was calculated. In mechanical studies, a comparison of plasma AVP(p-AVP) concentrations, cyclic adenosine monophosphate(cAMP) levels, vasopressin type 2 receptor(V2R) and AQP2 mRNA expressions in the cochlea were compared among groups. Results: EA significantly reduced cochlear hydrops in guinea pigs(P=0.001). EA significantly attenuated the AVPinduced up-regulation of p-AVP concentrations(P=0.006), cochlear c AMP levels(P=0.003) and AQP2 mRNA expression(P=0.016), and up-regulated the expression of V2R mRNA(P=0.004) in the cochlea. Conclusion: The dehydrating effect of EA might be associated with its inhibition of AVP-AQP2 pathway activation.
