Protein typically represents the largest input of organic nitrogen (N) into soil. Proteases subsequently make this protein available for use by both plants and microorganisms, however, the factors that regulate protein breakdown in the rhizosphere remain limited. Root exudation of carbon (C) and N into soil promotes microbial growth and thus enzyme production, which is further enhanced by root morphological traits such as root hairs. However, it is not clear how inputs of protein from external sources (e.g. necromass) affect enzyme activity in the rhizosphere. Insight into the interaction between protein addition and root morphology will enhance our knowledge of plant and microbial strategies for promoting N acquisition. Using soil zymography, we investigated the spatial distribution of leucine aminopeptidase activity in the rhizosphere of Hordeum vulgare L. (barley) with and without root hairs subject to localised protein addition. Seedlings of barley were grown for two weeks in rhizoboxes and soluble protein was applied 48 h before analysis of leucine aminopeptidase activity. In situ zymography was used to quantitatively visualise leucine aminopeptidase activity while ex situ sampling was used to determine its enzyme kinetics. In the zymograms, we found that mean and maximal leucine aminopeptidase activity was highest in the barley genotype with root hairs and in the presence of soil protein hotspots. This suggests that microorganisms and plant roots in the rhizosphere of genotypes with root hairs have a greater advantage in accessing protein hotspots in the soil. Leucine aminopeptidase activity did not follow the same trends when analysed by in situ zymography and ex situ sampling methods. Therefore, we recommend the use of in situ zymography to detect the spatial distribution of enzymatic hotspots and rhizosphere extent followed by ex situ sampling for assessing enzyme kinetics in the hotspot areas detected by in situ sampling. However, sampling biases must be considered to ensure enzyme activities are being interpreted as the true rhizosphere.

蛋白质通常代表土壤中最大的有机氮（N）输入。蛋白酶随后使该蛋白质可用于植物和微生物，但是，调节根际中蛋白质分解的因素仍然有限。碳（C）和氮的根系渗入土壤促进了微生物的生长，从而促进了酶的产生，而根系形态特征（例如根毛）则进一步促进了这种增长。然而，尚不清楚来自外部来源（例如坏死质）的蛋白质输入如何影响根际中的酶活性。深入了解蛋白质添加与根形态之间的相互作用将增强我们对植物和微生物促进氮素吸收策略的了解。使用土壤酶谱学，我们调查了亮氨酸氨基肽酶活性在根际中的空间分布大麦（大麦），有或没有根毛，都需要局部添加蛋白质。在分析亮氨酸氨基肽酶活性之前，将大麦的幼苗在根瘤菌中生长两周，并应用可溶性蛋白48 h。原位酶谱被用来定量形象化亮氨酸氨而活动易地取样用于确定其酶动力学。在酶谱图中，我们发现，在有根毛的大麦基因型中和存在土壤蛋白热点的情况下，平均和最大亮氨酸氨肽酶活性最高。这表明具有根毛的基因型根际中的微生物和植物根系在接近土壤中的蛋白质热点方面具有更大的优势。当通过原位酶谱分析和非原位采样方法分析时，亮氨酸氨肽酶活性没有遵循相同的趋势。因此，我们建议使用原位酶谱法来检测酶促热点的空间分布和根际范围，然后再进行异位采样以评估通过现场采样检测到的热点区域中的酶动力学。但是，必须考虑采样偏差以确保将酶活性解释为真正的根际。