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

Giles, Courtney D.; Dupuy, Lionel; Boitt, Gustavo; Brown, Lawrie; Condron, Leo M.; Darch, Tegan; Blackwell, Martin; Shand, C. A.; Stutter, Marc; Lumsdon, David G.; Wendler, Renate; Cooper, Pat; Wearing, Catherine; Zhang, H.; Haygarth, P. M.; George, Timothy S.

doi:10.1016/j.soilbio.2017.08.011

Cited by 46 publications

(32 citation statements)

References 24 publications

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“…The SNR gradually increased with MUF concentration until saturation of the zymography membranes, but it decreased under oversaturation, indicating that application of the Bouguer–Lambert–Beer law (the signal is proportional to the concentration) is restricted at large values of G . The deviation from linearity with increasing MUF concentration was also reported by Giles et al (). These authors calibrated zymography membranes for phosphatase activity using standard MUF solutions at varying exposure times.…”

Section: Discussionsupporting

confidence: 79%

“…Published information pertains mostly to the type of camera (Liu et al, ; Ma, Razavi, Holz, Blagodatskaya, & Kuzyakov, ; Razavi et al, , ) and to the combination of camera type and source of the UV light (Spohn et al, ). We found only one detailed analysis of exposure times for 2‐D zymography of phosphatase activity in a recent publication by Giles et al (). Lack of understanding of the effects exerted by camera and light choices limits the accuracy in calibrating fluorescence patterns observed on zymograms and, thus, in estimating enzyme activities.…”

Section: Introductionmentioning

confidence: 99%

“…A review of recent publications showed that there is no general agreement on the precision of image calculations. An 8‐bit format was used by Spohn & Kuzyakov (, ) and Giles et al (), 16‐bit was used by Razavi et al (), Sanaullah et al () and Liu et al (), and 32‐bit images were used by Ma et al (). Original colour images taken by camera are 32‐bit, and their conversion to a smaller bit format may considerably reduce the accuracy of image calculations.…”

Section: Introductionmentioning

confidence: 99%

“…Some studies subtracted the grey values from zymograms corresponding to zero MUF concentration obtained during the calibration (Ma et al, ; Razavi et al, ; Sanaullah et al, ), whereas others set the intercept in the calibration curve to zero (Spohn & Kuzyakov, , ). Some studies mentioned the importance of background correction; however, they did not describe whether or how this was done (Giles et al, ). Consistency in the image processing is needed for comparison of results of various studies and further developments.…”

Section: Introductionmentioning

confidence: 99%

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Calibration of 2‐D soil zymography for correct analysis of enzyme distribution

Guber

Kravchenko

Razavi

et al. 2019

European J Soil Science

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Soil zymography is a new technique developed to visualize two‐dimensional distributions of enzyme activities. The method consists of incubating a membrane saturated with an enzyme‐specific fluorogenic substrate on a surface of the soil sample, followed by recording the membrane image generated by a fluorescent product (e.g. MUF: methylumbelliferone) in ultraviolet light. Despite its relative ease of use, performing zymography involves multiple user‐made decisions that might affect the accuracy of enzyme activity estimates. Therefore, unification of the zymography methodology is required for correct estimations and comparisons of various studies. We evaluated the following methodological aspects of the implementation of zymography: (a) camera settings and image processing, (b) effects of evaporation and (c) calibration procedures. Camera settings (shutter speeds or exposure time) affected the intensity of background fluorescence and signal‐to‐noise ratios (SNR). However, because their combined effects varied depending on MUF concentrations, light and camera setting need to be optimized for the expected range of MUF concentrations prior to zymography. Evaporation of MUF solution from the membrane had no effect on fluorescence. Relations between MUF concentration and intensity of fluorescence during calibrations demonstrated a saturated pattern and were strongly affected by image noise outside the optimal range (e.g. 8–14 μm MUF pixel−1). We developed a new calibration approach that is based on a piecewise linear regression. The new approach accounted for specific ranges of MUF concentration and uses nonuniformly saturated membranes, reflecting the real distribution of enzyme activities in soil. The new calibration algorithm eliminated biases of the standard calibration and resulted in greater accuracy in predicting MUF concentrations. Highlights We developed a new approach to calibration for 2‐D soil zymography. The approach accounted for spatial nonuniformity of soil zymograms. Standard calibration resulted in systematic underestimation of enzyme activity. Soil zymography requires pixel‐based calibration with nonuniformly saturated membranes.

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Section: Discussionsupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Calibration of 2‐D soil zymography for correct analysis of enzyme distribution

Guber

Kravchenko

Razavi

et al. 2019

European J Soil Science

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“…It is therefore likely that root exudates such as phosphatases contribute to plant phosphorus acquisition and that root hairs release phosphatases into soil. In fact, several studies observed that high phosphatase activity is associated with root hair production ( Gahoonia et al., ; Giles et al., ; Ma et al., ). However, Pausch et al.…”

Section: Discussionmentioning

confidence: 99%

The effect of root hairs on rhizosphere phosphatase activity

Holz

Zarebanadkouki

Carminati

et al. 2020

J. Plant Nutr. Soil Sci.

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Background: Phosphatases in soil are of great importance for plant P acquisition. It is hypothesized that root hairs increase rhizosphere phosphatase activity as they release enzymes into soil and stimulate microbial activity. Methods: To test the effect of root hairs on soil phosphatase activity, we grew barley (Hordeum vulgare 'Pallas') wild type and its root-hairless mutant in rhizoboxes and determined phosphatase activity using soil zymography. Measurements were done at three moisture levels (30, 15, and 5% VWC). Rhizosphere phosphatase activity was estimated for the two genotypes and two locations along the root [root tip region (0-4 cm behind tip) and mature roots (> 7 cm behind tip)]. Results: Rhizosphere phosphatase activity was similar in the two locations along the root (root tip region vs. mature root parts). In contrast, rhizosphere phosphatase extension was two times larger for the root tip region of the wild type than for the mutant at 30% and 15% VWC. However, as phosphatase activities at the root surface of tips and mature root parts were slightly higher for the mutant than for the wild type, average enzyme activities were unaffected by the genotype. Conclusions: We conclude that the mutant seems to compensate for the lack of root hairs by increased phosphatase activity close to the root surface. However, the increased rhizosphere phosphatase extension for the wild type may be equally efficient as it allows P mobilization and uptake from large soil volumes.

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