Root development impacts on the distribution of phosphatase activity: Improvements in quantification using soil zymography

A - Papers appearing in refereed journals

Giles, C. D., Dupuy, L., Boitt, G., Brown, L. K., Condron, L. M., Darch, T., Blackwell, M. S. A., Menezes-Blackburn, D., Shand, C. A., Stutter, M. I., Lumsdon, D. G., Wendler, R., Cooper, P., Wearing, C., Zhang, H., Haygarth, P. M. and George, T. S. 2018. Root development impacts on the distribution of phosphatase activity: Improvements in quantification using soil zymography. Soil Biology and Biochemistry. 116, pp. 158-166.

AuthorsGiles, C. D., Dupuy, L., Boitt, G., Brown, L. K., Condron, L. M., Darch, T., Blackwell, M. S. A., Menezes-Blackburn, D., Shand, C. A., Stutter, M. I., Lumsdon, D. G., Wendler, R., Cooper, P., Wearing, C., Zhang, H., Haygarth, P. M. and George, T. S.
Abstract

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.

KeywordsBarley; Image analysis; 4-Methylumbelliferone; Phosphatase; Rhizosphere; Soil zymography
Year of Publication2018
JournalSoil Biology and Biochemistry
Journal citation116, pp. 158-166
Digital Object Identifier (DOI)doi:10.1016/j.soilbio.2017.08.011
Open accessPublished as non-open access
FunderBiotechnology and Biological Sciences Research Council
Funder project or codeOrganic P utilization in soils (OPUS)
Output statusPublished
Publication dates
Online21 Oct 2017
Publication process dates
Accepted12 Aug 2017
PublisherElsevier
Copyright licenseCC BY
ISSN0038-0717

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