JoVE Logo
Faculty Resource Center

Sign In





Representative Results






Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Published: August 6th, 2018



1Department of Entomology, Texas A&M University, 2Department of Entomology, University of Minnesota, 3Department of Entomology, University of Kentucky

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Plant biomass forms the foundation of virtually all terrestrial food-webs. Plants acquire nutritional elements from the soil through their roots systems and utilize sunlight in their foliar tissues to synthesize biomolecules. In particular, carbon and nitrogen are used to create carbohydrates, proteins (comprised of amino acids), and lipids that are needed to build plant biomass (it should be noted that in plant physiology the term "macronutrient" often refers to soil elements, such as N, P, K, and S, however, throughout this paper this term will refer to biomolecules, such as proteins, carbohydrates, and lipids). When herbivores consume plant material, the ma....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Plant Collection and Processing

  1. Collect and process plant samples
    1. After collecting plant samples, flash-freeze samples by dipping plant material into liquid nitrogen with forceps and store at -80 °C. If the plant samples collected are too large to flash-freeze, quickly cool the samples using dry ice and transfer to a -80 °C freezer as soon as possible. The macronutrient content of plant material can change after tissues are separated from the plant, so it is important to freeze plant .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

To show the usefulness of these methods, we analyzed the soluble protein and digestible carbohydrate content of four different field and sweetcorn tissues that serve as distinct potential nutritional resources for insect herbivores. We collected ears of corn from three agricultural regions in the United States (Minnesota, North Carolina, and Texas), encompassing five different varieties of sweet corn (i.e., genotypes) and one variety of field corn as an outgroup. Table 3<.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

By combining well-established colorimetric assays with effective plant-specific extraction protocols, the assays demonstrated here provide a reasonable and accurate method for measuring plant soluble protein and digestible carbohydrate content. Our results using corn as an exemplar illustrates how these protocols can be used to obtain precise measurements across different biologically-relevant spatial scales. For example, we were able to detect differences in plant soluble protein and digestible carbohydrate content betw.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Thanks to all of our collaborators who have assisted with sweet corn field collections, including Dominic Reisig and Dan Mott at North Carolina State University, and Pat Porter at Texas A& M University in Lubbock, TX. Thanks to Fiona Clissold for helping to optimize the protocols and for providing edits to this manuscript. This work was supported in part by the Texas A& M C. Everette Salyer Fellowship (Department of Entomology) and the Biotechnology Risk Assessment Grant Program competitive grant no. 2015-33522-24099 from the U.S. Department of Agriculture (awarded to GAS and STB).


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
microplate reader (spectrophotometer) Bio-Rad Model 680 XR
Bio-Rad Protein Assay Dye Reagent concentrate Bio-Rad #5000006 450mL

  1. Simpson, S. J., Raubenheimer, D. . The Nature of Nutrition: A Unifying Framework from Animal Adapation to Human Obesity. , (2012).
  2. Behmer, S. T. Insect herbivore nutrient regulation. Annual Review of Entomology. 54, 165-187 (2009).
  3. Epstein, E. Mineral nutrition of plants: mechanisms of uptake and transport. Annual Review of Plant Physiology. 7 (1), 1-24 (1956).
  4. Chapin, F. S. The mineral nutrition of wild plants. Annual Review of Ecology, Evolution, and Systematics. 11 (1), 233-260 (1980).
  5. Marschner, H. . Marschner's Mineral Nutrition of Higher Plants. , (1956).
  6. Stieger, P. A., Feller, U. Senescence and protein remobilization in leaves of maturing wheat plants grown on waterlogged soil. Plant and Soil. 166, 173-179 (1994).
  7. Li, R., Volenec, J. J., Joern, B. C., Cunningham, S. M. Seasonal changes in nonstructural carbohydrates, protein, and macronutrients in roots of alfalfa, red clover, sweetclover, and birdsfoot trefoil. Crop Science. 36, 617-623 (1996).
  8. Sánchez, E., Rivero, R. M., Ruiz, J. M., Romero, L. Changes in biomass, enzymatic activity and protein concentration in roots and leaves of green bean plants (Phaseolus vulgaris L. cv. Strike) under high NH4NO3 application rates. Scientia Horticulturae. 99, 237-248 (2004).
  9. Lenhart, P. A., Eubanks, M. D., Behmer, S. T. Water stress in grasslands: Dynamic responses of plants and insect herbivores. Oikos. 124, 381-390 (2015).
  10. Machado, A. R., Arce, C. C. M., Ferrieri, A. P., Baldwin, I. T., Erb, M. Jasmonate-dependent depletion of soluble sugars compromises plant resistance to Manduca sexta. New Phytologist. 207, 91-105 (2015).
  11. Deans, C. A., Behmer, S. T., Fiene, J., Sword, G. A. Spatio-temporal, genotypic, and environmental effects of plant soluble protein and digestible carbohydrate content: implications for insect herbivores with cotton as an exemplar. Journal of Chemical Ecology. 42 (11), 1151-1163 (2016).
  12. Boisen, S., Bech-Andersen, S., Eggum, B. O. A critical view of the conversion factor 6.25 from total nitrogen to protein. Acta Agriculturae Scandinavica. 37, 299-304 (1987).
  13. Ezeagu, I. E., Petzke, J. K., Metges, C. C., Akinsoyinu, A. O., Ologhobo, A. D. Seed protein contents and nitrogen-to-protein conversion factors for some uncultivated tropical plant seeds. Food Chemistry. 78, 105-109 (2002).
  14. Izhaki, I. Influence of nonprotein nitrogen on estimation of protein from total nitrogen in fleshy fruits. Journal of Chemical Ecology. 19, 2605-2615 (1993).
  15. Mossé, J. Nitrogen to protein conversion factor for ten cereals and six legume or oilseeds. A reappraisal of its definition and determination. Variation according to species and seed protein content. Journal of Agricultural and Food Chemistry. 38, 18-24 (1990).
  16. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72 (1-2), 248-254 (1976).
  17. Jones, C. G., Hare, J. D., Compton, S. J. Measuring plant protein with the Bradford assay. Journal of Chemical Ecology. 15 (3), 979-992 (1989).
  18. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., Smith, F. Colormetric method for determination of sugars and related substances. Analytical Biochemistry. 28, 350-358 (1956).
  19. Clissold, F. J., Sanson, G. D., Read, J. The paradoxical effects of nutrient ratios and supply rates on an outbreaking insect herbivore, the Australian plague locust. Journal of Animal Ecology. 75, 1000-1013 (2006).
  20. Smith, D., Paulsen, G. M., Raguse, C. A. Extraction of total available carbohydrates from grass and legume tissue. Plant Physiology. 39 (6), 960-962 (1964).
  21. Cui, S. W. . Food carbohydrates: Chemistry, physical properties, and applications. , (2005).
  22. Chow, P. S., Landhäusser, S. M. A method for routine measurements of total sugar and starch content in woody plant tissues. Tree Physiology. 24 (10), 1129-1136 (2004).
  23. Masuko, T., Minami, A., Iwasaki, N., Majima, T., Nishimura, S. I., Lee, Y. C. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Analytical Biochemistry. 339 (1), 69-72 (2005).
  24. Foley, W. J., McIlwee, A., Lawler, I., Aragones, L., Woolnough, A. P., Berding, N. Ecological applications of near infrared reflectance spectroscopy- a tool for rapid, cost-effective prediction of the composition of plant and animal tissues and aspects of animal performance. Oecologia. 116 (3), 292-305 (1998).
  25. Kokaly, R. F. Investigating a physical basis for spectroscopic estimates of leaf nitrogen concentration. Remote Sensing of Environment. 75 (2), 153-161 (2001).
  26. Schulz, H., Baranska, M. Identification and quantification of valuable plant substances by IR and Raman spectroscopy. Vibrational Spectroscopy. 43 (1), 13-25 (2007).
  27. Cozzolino, D., Morón, A. The potential of near-infrared reflectance spectroscopy to analyse soil chemical and physical characteristics. The Journal of Agricultural Science. 140, 65-71 (2003).
  28. Simpson, S. J., Sword, G. A., Lorch, P. D., Couzin, I. D. Cannibal crickets on a forced march for protein and salt. Proceedings of the National Academy of Sciences of the United States of America. 103 (11), 4152-4156 (2006).
  29. Lihoreau, M., Buhl, J., Sword, G. A., Raubenheimer, D., Simpson, S. J. Nutritional ecology beyond the individual: a conceptual framework for integrating nutrition and social interactions. Ecology Letters. 18 (3), 273-286 (2015).
  30. Deans, C. A., Behmer, S. T., Tessnow, A., Tamez-Guerra, P., Pusztai-Carey, M., Sword, G. A. Nutrition affects insect susceptibility to Bt. Scientific Reports. 7, 39705 (2017).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved