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.

磷（P）是植物生长必不可少的大量营养素，但由于pH和地球化学相互作用导致的固定化，土壤中的生物利用磷通常受到限制。了解土壤中磷的动态并阐明植物从环境中获取磷的机制对于评估生产力（特别是在营养不良的环境中）至关重要。有机质中的磷可作为土壤系统中生物的主要P来源。磷酸酶是从有机源释放出无机磷的酶，由植物和微生物共同产生，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在根际空间成像磷酸酶活性。来自根际的蛋白质被转移到硝酸纤维素膜上，同时保持其酶活性和二维空间分布。随后应用荧光磷酸酶指示剂DDAO磷酸盐，可以使样品中磷酸酶活性的分布可视化。然后可以将蛋白质固定在膜上，并用SYPRO®Ruby蛋白质印迹染色剂（荧光的总蛋白染色剂）进行处理，以实现总蛋白分布的可视化。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，并提供深入了解影响根际酶活性和蛋白积累的空间因素。值得注意的是 该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）的根际箱中生长的植物，并且由于这种方法的非破坏性，因此可以随时间进行以跟踪变化。我们预计，这种基于根印迹的荧光指示剂成像技术可用于多种植物-微生物-土壤系统中，以更好地了解磷酸酶在磷获取和土壤磷循环中的作用。