Non-destructive spatial analysis of phosphatase activity and total protein distribution in the rhizosphere using a root blotting method
Non-destructive spatial analysis of phosphatase activity and total protein distribution in the rhizosphere using a root blotting method
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DOI：
10.1016/j.soilbio.2020.107820
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发表时间：
2020-07
影响因子：
9.7
通讯作者：
Vivian S. Lin;J. Rosnow;Monee Y. McGrady;Darian N. Smercina;Jamie R. Nuñez;R. Renslow;J. Moran
中科院分区：
文献类型：
--
作者：
Vivian S. Lin;J. Rosnow;Monee Y. McGrady;Darian N. Smercina;Jamie R. Nuñez;R. Renslow;J. Moran
磷(P)是植物生长必需的常量养分，但由于pH和地球化学相互作用造成的固定化，土壤中的生物可利用磷往往受到限制。了解土壤中磷的动态和阐明植物从其环境中获取磷的机制对评估生产力至关重要，特别是在营养贫乏的环境中。来自有机质的磷可以作为土壤系统中生物磷的主要来源。磷酸酶是一种从有机来源中释放无机磷的酶，由植物和微生物产生，被认为是土壤中最活跃的酶之一。我们开发了一种根印迹法来对根际磷酸酶活性进行空间成像。来自根际的蛋白质被转移到硝化纤维素膜上，同时保留其酶活性和二维空间分布。随后应用荧光磷酸酶指示剂DDAO磷酸，使磷酸酶活性在样品中的分布可视化。然后可以将蛋白质固定在膜上，并用SYPRO®Ruby蛋白印迹染色处理，这是一种荧光总蛋白染色，可以可视化总蛋白的分布。综上所述，磷酸酶活性和总蛋白定位的图像可以映射回根结构，并提供影响根际酶活性和蛋白质积累空间分布的因素。值得注意的是，这种方法可以应用于在含有土壤或无土生长混合物（例如，沙子或各种盆栽混合物）的根箱中生长的植物，并且由于这种方法的非破坏性，可以随时间进行跟踪变化。我们期望这种根印迹荧光指示剂成像技术可以用于不同的植物-微生物-土壤系统，以更好地了解磷酸酶在磷获取和土壤磷循环中的作用。
Phosphorus (P) is an essential macronutrient for plant growth, but bioavailable P in soils is often limited due to immobilization resulting from pH and geochemical interactions. Understanding the dynamics of P in soils and elucidating the mechanisms by which plants access P from their environment are critical to evaluating productivity, particularly in nutrient poor environments. Phosphorus from organic matter can act as a major source of P for organisms in soil systems. Phosphatases, enzymes that liberate inorganic P from organic sources, are produced by both plants and microbes and are considered one of the most active classes of enzymes in soil. We developed a root blotting method to spatially image phosphatase activity in the rhizosphere. Proteins from the rhizosphere are transferred to a nitrocellulose membrane while retaining their enzymatic activity and two-dimensional spatial distribution. Subsequent application of a fluorogenic phosphatase indicator, DDAO phosphate, enables visualization of the distribution of phosphatase activity in the sample. The proteins can then be fixed to the membrane and treated with SYPRO® Ruby Protein Blot Stain, a fluorescent total protein stain, allowing for visualization of total protein distribution. Taken together, the images of phosphatase activity and total protein localization can be mapped back to the root architecture and provide insight into factors affecting the spatial distribution of enzymatic activity and protein accumulation in the rhizosphere. Notably, this method can be applied to plants growing in rhizoboxes containing soil or soilless growth mixtures (e.g., sand or various potting mixes) and, because of the non-destructive nature of this approach, be performed over time to track changes. We anticipate that this fluorescent indicator imaging technique on root blots can be used in diverse plant-microbe-soil systems to better understand the role of phosphatases in P acquisition and soil P cycling.