Name: quantifying plant soluble protein and digestible carbohydrate content, using corn (zea mays) as an exemplar
Uploaded: 2018-08-06T14:30:00
Description: Watch this Scientific Journal Video about Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar at JoVE.com

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&#38; 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&#38; 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).

1. Plant Collection and Processing
<ol>
	<li>Collect and process plant samples
	<ol>
		<li>After collecting plant samples, flash-freeze samples by dipping plant material into liquid nitrogen with forceps and store at -80 &#176;C. If the plant samples collected are too large to flash-freeze, quickly cool the samples using dry ice and transfer to a -80 &#176;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 ...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal,+genotypic,+and+environmental+effects+of+plant+soluble+protein+and+digestible+carbohydrate+content:+implications+for+insect+herbivores+with+cotton+as+an+exemplar.">Spatio-temporal, genotypic, and environmental effects of plant soluble protein and digestible carbohydrate content: implications for insect herbivores with cotton as an exemplar.</a> Journal of Chemical Ecology. 42 (11), 1151-1163 (2016).</li><li>Boisen, S., Bech-Andersen, S., Eggum, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+view+of+the+conversion+factor+6.25+from+total+nitrogen+to+protein.">A critical view of the conversion factor 6.25 from total nitrogen to protein.</a> Acta Agriculturae Scandinavica. 37, 299-304 (1987).</li><li>Ezeagu, I. E., Petzke, J. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Food carbohydrates: Chemistry, physical properties, and applications. , (2005).</li><li>Chow, P. S., Landh&#228;usser, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+routine+measurements+of+total+sugar+and+starch+content+in+woody+plant+tissues.">A method for routine measurements of total sugar and starch content in woody plant tissues.</a> Tree Physiology. 24 (10), 1129-1136 (2004).</li><li>Masuko, T., Minami, A., Iwasaki, N., Majima, T., Nishimura, S. I., Lee, Y. 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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...

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 &#34;macronutrient&#34; 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 macronutrients contained in plant tissues are broken down into their constituent parts and then used to drive the physiological processes of the consumer. In this way, plant macronutrients have a strong influence on consumer physiology along with important implications for higher order ecological interactions and food-web dynamics.
Across the animal kingdom, soluble protein and digestible carbohydrates are the macronutrients most closely tied to survival, reproduction, and performance1. Moreover, the majority of animals actively regulate their intake of these two macronutrients to meet their physiological demands1,2. This is particularly true for insect herbivores that detect the concentrations of sugars and amino acids in plant tissues, which in turn directs feeding behavior. As a result, plant soluble protein and digestible carbohydrate content has played a strong role in the evolution of plant-insect interactions.
While data on plant soluble protein and digestible carbohydrate content are relatively rare (but see6,7,8,9,10,11), there is a preponderance of data available on plant elemental content (carbon, nitrogen, and phosphorus). Largely this is because elements play a primary role in plant mineral nutrition3,4,5. Where elements are measured, correlations have been used to extrapolate the amount of soluble protein and digestible carbohydrate, but accurate calculations are often difficult to obtain. For instance, it is impossible to use carbon as an indicator of plant digestible carbohydrate content because carbon is ubiquitously present in all organic compounds. A stronger relationship exists between elemental nitrogen and plant soluble protein content, and generalized nitrogen-to-protein conversion factors are often utilized. However, there is strong evidence that nitrogen-to-protein conversions are highly species-specific12,13,14,15, making the use of generalized conversion likely inaccurate. Because of this, nitrogen-to-protein conversion factors often lack precision, particularly to the extent that is required for nutritional studies on herbivores. Also, the presence of N-containing plant allelochemicals,&#160;such as alkaloids and glucosinolates that are often toxic to herbivores, can confound these conversions.
Here, we offer two chemical assays for measuring the concentration of soluble proteins and digestible carbohydrates in plant tissues. These assays are presented separately, but it is suggested that they be used concurrently to analyze the same plant samples in order to achieve a more comprehensive analysis of plant macronutrients. Both employ similar methodologies, consisting of an extraction step, followed by quantification via absorbance. Plant sample prep is also identical for both protocols, making it easy to run both analyses in tandem. The utility of these assays do not stem from their novelty, as they rely on older, (Bradford, Jones, Dubois) well-established colorimetric assays16,17,18, but here we have organized a clear and easy-to-follow protocol that combines these methods with more obscure plant-specific extraction techniques17,19 in order to make the application of these assays more accessible to those in plant-relevant fields.
For both assays, plant macronutrients are first extracted physically by freezing, lyophilizing, and grinding the plant material. For the soluble protein assay, further chemical extraction is done17,19 through several rounds of vortexing and heating samples in NaOH solution. The well-known Bradford assay, utilizing Coomassie brilliant blue G-250, is then used to quantify soluble proteins and polypeptides between 3,000-5,000 Daltons16,17. This assay has a detection range between 1-20 &#181;g total proteins per microplate well or &#60;25 &#181;g/mL, but does not measure free amino acids. The extraction step of the digestible carbohydrate assay is based on the dilute acid method of Smith et al.20 and allows for the isolation of soluble sugars, starch, and fructosan &#8211; but not structural carbohydrates. A phenol-sulfuric acid quantification method is taken from Dubois et al.18 and measures all mono-, oligo-, and polysaccharides (as well as methyl derivatives). This assay is able to quantify specific sugars, but here we use it as an indicator of total digestible carbohydrate content (see Smith et al.20 for more detailed analysis). Together, these assays measure the two macronutrients that are strongly tied to plant eco-physiology and herbivore performance, providing important data on resource quality at the base of terrestrial food-webs. Presenting these protocols promotes the generation of plant macronutrient datasets in order to obtain a more thorough understanding of plant physiology, herbivore nutritional ecology, and plant-herbivore interactions.

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">microplate reader (spectrophotometer)</td>
			<td style="width:111px;">Bio-Rad</td>
			<td style="width:119px;">Model 680 XR</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Bio-Rad Protein Assay Dye Reagent concentrate</td>
			<td style="width:111px;">Bio-Rad</td>
			<td style="width:119px;">#5000006</td>
			<td>450mL</td>
		</tr>
	</tbody>
</table>

quantifying plant soluble protein and digestible carbohydrate content, using corn (zea mays) as an exemplar

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.

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<...

Watch this Scientific Journal Video about Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar at JoVE.com

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

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.

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

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

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