Non-destructive spatial analysis of phosphatase activity and total protein distribution in the rhizosphere using a root blotting method
Abstract
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. In this work 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 a SYPRO® Ruby gel 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 mappedmore »
- Authors:
-
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Michigan State Univ., East Lansing, MI (United States)
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Publication Date:
- Research Org.:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)
- OSTI Identifier:
- 1631594
- Alternate Identifier(s):
- OSTI ID: 1703307
- Report Number(s):
- PNNL-SA-149591
Journal ID: ISSN 0038-0717
- Grant/Contract Number:
- AC05-76RL01830
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Soil Biology and Biochemistry
- Additional Journal Information:
- Journal Volume: 146; Journal Issue: C; Journal ID: ISSN 0038-0717
- Publisher:
- Elsevier
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; rhizosphere; root exudates; phosphatase activity; protein localization
Citation Formats
Lin, Vivian S., Rosnow, Joshua J., McGrady, Monee Y., Smercina, Darian N., Nuñez, Jamie R., Renslow, Ryan S., and Moran, James J. Non-destructive spatial analysis of phosphatase activity and total protein distribution in the rhizosphere using a root blotting method. United States: N. p., 2020.
Web. doi:10.1016/j.soilbio.2020.107820.
Lin, Vivian S., Rosnow, Joshua J., McGrady, Monee Y., Smercina, Darian N., Nuñez, Jamie R., Renslow, Ryan S., & Moran, James J. Non-destructive spatial analysis of phosphatase activity and total protein distribution in the rhizosphere using a root blotting method. United States. https://doi.org/10.1016/j.soilbio.2020.107820
Lin, Vivian S., Rosnow, Joshua J., McGrady, Monee Y., Smercina, Darian N., Nuñez, Jamie R., Renslow, Ryan S., and Moran, James J. Thu .
"Non-destructive spatial analysis of phosphatase activity and total protein distribution in the rhizosphere using a root blotting method". United States. https://doi.org/10.1016/j.soilbio.2020.107820. https://www.osti.gov/servlets/purl/1631594.
@article{osti_1631594,
title = {Non-destructive spatial analysis of phosphatase activity and total protein distribution in the rhizosphere using a root blotting method},
author = {Lin, Vivian S. and Rosnow, Joshua J. and McGrady, Monee Y. and Smercina, Darian N. and Nuñez, Jamie R. and Renslow, Ryan S. and Moran, James J.},
abstractNote = {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. In this work 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 a SYPRO® Ruby gel 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.},
doi = {10.1016/j.soilbio.2020.107820},
journal = {Soil Biology and Biochemistry},
number = C,
volume = 146,
place = {United States},
year = {Thu Apr 16 00:00:00 EDT 2020},
month = {Thu Apr 16 00:00:00 EDT 2020}
}
Web of Science
Works referenced in this record:
Enzyme activity dot blots: A rapid and convenient assay for acetyltransferase or protein kinase activity immobilized on nitrocellulose
journal, November 1986
- Allis, C. David; Chicoine, Louis G.; Glover, Claiborne V. C.
- Analytical Biochemistry, Vol. 159, Issue 1
High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities
journal, January 2013
- Bell, Colin W.; Fricks, Barbara E.; Rocca, Jennifer D.
- Journal of Visualized Experiments, Issue 81
Root and Rhizosphere Bacterial Phosphatase Activity Varies with Tree Species and Soil Phosphorus Availability in Puerto Rico Tropical Forest
journal, October 2017
- Cabugao, Kristine G.; Timm, Collin M.; Carrell, Alyssa A.
- Frontiers in Plant Science, Vol. 8
Enzyme activities as a component of soil biodiversity: A review
journal, November 2005
- Caldwell, Bruce A.
- Pedobiologia, Vol. 49, Issue 6
The story of phosphorus: Global food security and food for thought
journal, May 2009
- Cordell, Dana; Drangert, Jan-Olof; White, Stuart
- Global Environmental Change, Vol. 19, Issue 2
Peak Phosphorus: Clarifying the Key Issues of a Vigorous Debate about Long-Term Phosphorus Security
journal, October 2011
- Cordell, Dana; White, Stuart
- Sustainability, Vol. 3, Issue 10
The influence of time, storage temperature, and substrate age on potential soil enzyme activity in acidic forest soils using MUB-linked substrates and l-DOPA
journal, June 2009
- DeForest, Jared L.
- Soil Biology and Biochemistry, Vol. 41, Issue 6
In vivo demonstration of acid phosphatase activity in the rhizosphere of soil-grown plants
journal, August 1992
- Dinkelaker, B.; Marschner, H.
- Plant and Soil, Vol. 144, Issue 2
The role of acid phosphatases in plant phosphorus metabolism
journal, April 1994
- Duff, Stephen M. G.; Sarath, Gautam; Plaxton, William C.
- Physiologia Plantarum, Vol. 90, Issue 4
Quantification of bacterial non-specific acid ( phoC) and alkaline ( phoD ) phosphatase genes in bulk and rhizosphere soil from organically managed soybean fields
journal, March 2017
- Fraser, Tandra D.; Lynch, Derek H.; Gaiero, Jonathan
- Applied Soil Ecology, Vol. 111
The use of fluorogenic substrates for measuring enzyme activity in peatlands
journal, August 1995
- Freeman, C.; Liska, G.; Ostle, N. J.
- Plant and Soil, Vol. 175, Issue 1
Organic phosphorus in the terrestrial environment: a perspective on the state of the art and future priorities
journal, October 2017
- George, T. S.; Giles, C. D.; Menezes-Blackburn, D.
- Plant and Soil, Vol. 427, Issue 1-2
Phosphorus in soils and plants – facing phosphorus scarcity
journal, March 2016
- George, Timothy S.; Hinsinger, Philippe; Turner, Benjamin L.
- Plant and Soil, Vol. 401, Issue 1-2
Calibration of 2-D soil zymography for correct analysis of enzyme distribution
journal, March 2019
- Guber, Andrey K.; Kravchenko, Alexandra N.; Razavi, Bahar S.
- European Journal of Soil Science, Vol. 70, Issue 4
Umbelliferone Phosphate as a Substrate for Acid and Alkaline Phosphatase
journal, January 1968
- Guilbault, G. G.; Sadar, S. H.; Glazer, R.
- Analytical Letters, Vol. 1, Issue 5
Genetic Responses to Phosphorus Deficiency
journal, August 2004
- Hammond, J. P.
- Annals of Botany, Vol. 94, Issue 3
Soil zymography as a powerful tool for exploring hotspots and substrate limitation in undisturbed subsoil
journal, September 2018
- Heitkötter, Julian; Marschner, Bernd
- Soil Biology and Biochemistry, Vol. 124
Matplotlib: A 2D Graphics Environment
journal, January 2007
- Hunter, John D.
- Computing in Science & Engineering, Vol. 9, Issue 3
Root traits and microbial community interactions in relation to phosphorus availability and acquisition, with particular reference to Brassica
journal, January 2014
- Hunter, Paul J.; Teakle, Grahams R.; Bending, Gary D.
- Frontiers in Plant Science, Vol. 5
High-resolution elemental mapping of the root-rhizosphere-soil continuum using laser-induced breakdown spectroscopy (LIBS)
journal, April 2019
- Ilhardt, Peter D.; Nuñez, Jamie R.; Denis, Elizabeth H.
- Soil Biology and Biochemistry, Vol. 131
Microbial hotspots and hot moments in soil: Concept & review
journal, April 2015
- Kuzyakov, Yakov; Blagodatskaya, Evgenia
- Soil Biology and Biochemistry, Vol. 83
Characterization of 9H-(1,3-dichlor-9,9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP)
journal, December 2000
- Leira, F.; Vieites, J. M.; Vieytes, M. R.
- Toxicon, Vol. 38, Issue 12
Spatial patterns of enzyme activities in the rhizosphere: Effects of root hairs and root radius
journal, March 2018
- Ma, Xiaomin; Zarebanadkouki, Mohsen; Kuzyakov, Yakov
- Soil Biology and Biochemistry, Vol. 118
Global patterns of phosphatase activity in natural soils
journal, May 2017
- Margalef, O.; Sardans, J.; Fernández-Martínez, M.
- Scientific Reports, Vol. 7, Issue 1
A microplate fluorimetric assay for the study of enzyme diversity in soils
journal, October 2001
- Marx, M. -C; Wood, M.; Jarvis, S. C.
- Soil Biology and Biochemistry, Vol. 33, Issue 12-13
Origin and expression of phosphatase activity in a semi-arid grassland soil
journal, January 1987
- Nakas, J. P.; Gould, W. D.; Klein, D. A.
- Soil Biology and Biochemistry, Vol. 19, Issue 1
Effects of phosphorus-mobilizing bacteria on tomato growth and soil microbial activity
journal, December 2017
- Nassal, Dinah; Spohn, Marie; Eltlbany, Namis
- Plant and Soil, Vol. 427, Issue 1-2
Use of commercial bio-inoculants to increase agricultural production through improved phosphrous acquisition
journal, February 2015
- Owen, D.; Williams, A. P.; Griffith, G. W.
- Applied Soil Ecology, Vol. 86
Root Architecture Responses: In Search of Phosphate
journal, October 2014
- Péret, Benjamin; Desnos, Thierry; Jost, Ricarda
- Plant Physiology, Vol. 166, Issue 4
Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms
journal, February 2009
- Richardson, Alan E.; Barea, José-Miguel; McNeill, Ann M.
- Plant and Soil, Vol. 321, Issue 1-2
Soil Microorganisms Mediating Phosphorus Availability Update on Microbial Phosphorus
journal, May 2011
- Richardson, Alan E.; Simpson, Richard J.
- Plant Physiology, Vol. 156, Issue 3
The effects of long term nitrogen deposition on extracellular enzyme activity in an Acer saccharum forest soil
journal, September 2002
- Saiya-Cork, K. R.; Sinsabaugh, R. L.; Zak, D. R.
- Soil Biology and Biochemistry, Vol. 34, Issue 9
Regulation and Characterization of Acid and Alkaline Phosphatase in Yeast
journal, March 1971
- Schurr, A.; Yagil, E.
- Journal of General Microbiology, Vol. 65, Issue 3
Managing Agricultural Phosphorus for Protection of Surface Waters: Issues and Options
journal, May 1994
- Sharpley, Andrew N.; Chapra, S. C.; Wedepohl, R.
- Journal of Environmental Quality, Vol. 23, Issue 3
Phosphorus Dynamics: From Soil to Plant
journal, May 2011
- Shen, Jianbo; Yuan, Lixing; Zhang, Junling
- Plant Physiology, Vol. 156, Issue 3
Soil zymography – A novel in situ method for mapping distribution of enzyme activity in soil
journal, March 2013
- Spohn, Marie; Carminati, Andrea; Kuzyakov, Yakov
- Soil Biology and Biochemistry, Vol. 58
Distribution of microbial- and root-derived phosphatase activities in the rhizosphere depending on P availability and C allocation – Coupling soil zymography with 14C imaging
journal, December 2013
- Spohn, Marie; Kuzyakov, Yakov
- Soil Biology and Biochemistry, Vol. 67
Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots—a soil zymography analysis
journal, February 2014
- Spohn, Marie; Kuzyakov, Yakov
- Plant and Soil, Vol. 379, Issue 1-2
Green/red dual fluorescence detection of total protein and alkaline phosphate-conjugated probes on blotting membranes
journal, April 2001
- Top, Karen Pretty On; Hatleberg, Gayle; Berggren, Kiera N.
- ELECTROPHORESIS, Vol. 22, Issue 5
Symbiotic Nitrogen Fixation and Phosphorus Acquisition. Plant Nutrition in a World of Declining Renewable Resources
journal, October 2001
- Vance, Carroll P.
- Plant Physiology, Vol. 127, Issue 2
Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource
journal, March 2003
- Vance, Carroll P.; Uhde-Stone, Claudia; Allan, Deborah L.
- New Phytologist, Vol. 157, Issue 3
The NumPy Array: A Structure for Efficient Numerical Computation
journal, March 2011
- van der Walt, Stéfan; Colbert, S. Chris; Varoquaux, Gaël
- Computing in Science & Engineering, Vol. 13, Issue 2