Soluble Sugar and Starch Extraction and Quantification from Maize (Zea mays) Leaves

Kristen A. Leach1, David M. Braun1

1 Division of Biological Sciences, Interdisciplinary Plant Group, Missouri Maize Center, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20018
Online Posting Date:  May, 2016
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Abstract

An easy‐to‐perform protocol for isolating and quantifying soluble sugars (sucrose, glucose, and fructose) and starch from maize (Zea mays) leaf tissue is described. The method has been optimized to extract non‐structural carbohydrates (NSC) from frozen, finely ground tissue in a methanol:chloroform:water solution. Three rounds of tissue extraction for 30 min each at 50°C provide quantitative recovery of soluble sugars. The use of alternative extraction solvents is discussed, as well as the advantages and disadvantages of these solvents. Additionally, we provide two support protocols. The first quantifies the isolated NSC via commercially available enzymatic kits that couple the amount of each specific sugar to the production of NADPH, which is detected using equipment readily available to most laboratories. The second describes the preparation of a purification column to remove strongly charged or hydrophobic molecules from the extracted sugar solution, which is required prior to quantification with high‐pressure liquid chromatography or high‐performance anion‐exchange chromatography. The protocols are robust and easily adapted for use in measuring NSC extracted from other plant species or tissues, making them ideal for new users. © 2016 by John Wiley & Sons, Inc.

Keywords: CPA column; fructose; glucose; maize leaf; MCW; non‐structural carbohydrate; NSC; soluble sugars; starch; sucrose

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Soluble Sugar Extraction
  • Basic Protocol 2: Starch Extraction and Enzymatic Degradation to Glucose
  • Support Protocol 1: Using a Commercially Available Enzymatic Kit and a Spectrophotometer to Measure the Concentrations of Soluble Sugars and Starch
  • Support Protocol 2: Collecting the Neutral Fraction Containing Soluble Sugar by Utilizing a Cation:PVPP:anion (CPA) Purification Column to Remove Strongly Charged Molecules
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Soluble Sugar Extraction

  Materials
  • Maize leaf tissue
  • Liquid nitrogen
  • 12:5:3 (v/v/v) MCW extraction buffer containing lactose (see recipe)
  • 12:5:3 (v/v/v) MCW extraction buffer (no lactose; see recipe)
  • Lactose (10 μg/μl)
  • Mortars and pestles
  • Spatulas
  • 1.7‐ml microcentrifuge tubes
  • Microcentrifuge and centrifuge
  • 50°C water bath
  • 15‐ml polypropylene conical tubes

Basic Protocol 2: Starch Extraction and Enzymatic Degradation to Glucose

  Materials
  • Tissue pellet from step 14 of protocol 1
  • Dimethyl sulfoxide (DMSO)
  • 100 mM sodium acetate, pH 5.0 (see recipe)
  • α‐Amylase working solution (see recipe)
  • Amyloglucosidase (Megazyme total starch kit, bottle 2, Megazyme, cat. no. K‐TSTA)
  • Boiling water bath
  • 50°C water bath
  • Microcentrifuge
  • −20°C freezer

Support Protocol 1: Using a Commercially Available Enzymatic Kit and a Spectrophotometer to Measure the Concentrations of Soluble Sugars and Starch

  Materials
  • Soluble sugar extract (from step 20 in protocol 1)
  • Sucrose/D‐Glucose/D‐Fructose assay kit (R‐Biopharm, cat. no. E0716260):
  • Prepare solution 1 by adding 10 ml H 2O to bottle 1
  • Prepare solution 2 by adding 45 ml H 2O to bottle 2
  • Use solution 3 as provided by the manufacturer
  • Use solution 4 as provided by the manufacturer
  • Post‐enzymatic starch digestion containing liberated glucose collected in step 12 of protocol 2
  • Starch assay kit (R‐Biopharm, cat. no. E0207748)
  • Prepare solution 2 by adding 27 ml of H 2O to bottle 2
  • Use solution 3 as provided by the manufacturer
  • 1.5‐ml disposable spectrophotometer cuvettes (Fisher Scientific, cat. no. S759085D)
  • UV spectrophotometer
  • Parafilm (Fisher Scientific, cat. no. S37440)
  • 1.7‐ml microcentrifuge tubes
NOTE: All absorbance measurements are made at 340 nm.

Support Protocol 2: Collecting the Neutral Fraction Containing Soluble Sugar by Utilizing a Cation:PVPP:anion (CPA) Purification Column to Remove Strongly Charged Molecules

  Materials
  • Cation exchange resin (AG50W‐X4; BioRad, cat. no. 142‐1351)
  • 18.2 mΩ filtered, deionized water
  • 20% methanol
  • Polyvinylpyrrolidone (PVPP) matrix (Acros Organics, cat. no. 227481000)
  • Anion exchange resin (Amberlite IRA‐67 free base, Sigma cat. no. A9960‐1KG or AG1‐X8; BioRad, cat. no. 140‐1441)
  • 1 M sodium carbonate (Na 2CO 3)
  • pH strips, pH 0 to 14 (Fisher Scientific, cat. no. M1095350001)
  • Soluble sugar extract (from step 20 in protocol 1)
  • Beakers
  • Flat‐bottomed disposable paper coffee filters (2¾ in. × 3 in.)
  • Magnetic stir bar
  • Magnetic stir plate
  • 50‐ml polypropylene conical tubes
  • Bio‐Spin Disposable Chromatography Columns (BioRad, cat. no. 732‐6008)
  • 3‐ml (Fisher Scientific, cat. no. 22‐040‐116) and 6‐ml (Fisher Scientific, cat. no. 22‐040‐163) Vacuette tubes (Fisher Scientific, cat. no. 22‐040‐163)
  • 1‐ml syringe filter without needle (Fisher Scientific cat. no. 14‐823‐261)
  • 0.2 μm PVDF syringe filter (Whatman, 13 mm, cat. no. 6779‐1302)
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Figures

Videos

Literature Cited

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