期刊
SOIL BIOLOGY & BIOCHEMISTRY
卷 116, 期 -, 页码 158-166出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.soilbio.2017.08.011
关键词
Barley; Image analysis; 4-Methylumbelliferone; Phosphatase; Rhizosphere; Soil zymography
类别
资金
- Biotechnology and Biological Research Council UK [BBK0170471]
- Brazilian Ministry of Education (MEC) through the CAPES agency (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior) [10359/13-3]
- BBSRC [BBS/E/C/000I0310, BBS/E/C/00005197, BBS/E/C/000I0320, BB/K018167/1, BB/K017047/1, BB/K018795/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BBS/E/C/000I0310, BB/K017047/1, BBS/E/C/00005197, BB/K018795/1, BBS/E/C/000I0320, BB/K018167/1] Funding Source: researchfish
Zymographic methods for the 2D distribution of phosphatase activity in soils have markedly advanced our understanding of root-soil-microbiota interactions. Robust quantitative approaches for 2D assays, which use 4-methylubelliferyl phosphate (4-MUP), are needed to advance a mechanistic understanding of enzyme behaviour and distribution in soils. We present improvements to the method for phosphatase zymography in rhizobox studies, involving (1) a systematic evaluation of 4-methylumbelliferone (4-MU) based calibration functions in relation to image exposure time and (2) the development of advanced image analysis tools for lateral and longitudinal distributions of phosphatase activity along barley roots (Hordeum vulgare L., cv Optic). Exposure time (<1-32 s) affected the slope and intercept of 4-MU calibration equations by 4.4- and 5.8-fold, respectively. In lateral root profiles, a linear relationship was found between phosphatase activity and root hair length at 0 cm (7 nKat mm(-2)), 0.2 cm (48 nKat mm(-2)), and 2 cm (234 nKat mm(-2)) distance from the root tips (r = 0.9795, p < 0.0001); an algorithm designed to optimise estimates of phosphatase activity longitudinally confirmed this relationship (r = 0.9462, p < 0.0001). To improve the precision and accuracy of fluorescence-based soil zymography, careful control of calibration and imaging conditions and further development of advanced image analysis techniques are recommended. (C) 2017 Published by Elsevier Ltd.
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