Title: 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 » 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.« less

Authors:

Lin, Vivian S. ^[1]; Rosnow, Joshua J. ^[1]; McGrady, Monee Y. ^[1]; Smercina, Darian N. ^[2]; Nuñez, Jamie R. ^[1]; Renslow, Ryan S. ^[1]; Moran, James J. ^[3]

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+ Show Author Affiliations

1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
2. Michigan State Univ., East Lansing, MI (United States)
3. Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)

Publication Date:

Thu Apr 16 00:00:00 EDT 2020

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