A method for routine measurements of total sugar and starch content in woody plant tissues

Abstract

gested that running parallel assays for each sample with and without a color developer could also provide a correction of the absorbance readings. This approach has yet to be tested in sugar assays using phenol-sulfuric acid. To estimate starch content in tissue samples, most methods hydrolyze the starch to glucose, which is subsequently assayed. Among these methods are perchloric acid hydrolysis (MacRae et al. 1974, Rose et al. 1991), which is considered dangerous because of the instability of perchloric acid; sulfu-ric acid hydrolysis (Smith et al. 1964), which is considered the simplest and most rapid; and enzyme digestion with amylase and amyloglucosidase (Smith 1969, Haissig and Dickson 1979, Rose et al. 1991, Hendrix 1993), which is considered the most accurate but the most labor intensive. Grotelueschen and Smith (1967), Greub and Wedin (1969) and Kozloski et al. (1999) compared the sulfuric acid and enzymatic methods on legume roots, grasses and cereals; however, they determined the total nonstructural carbohydrate content without separating the soluble sugars from starch. In addition, the authors used sulfuric acid at concentrations of 0.2 N or higher, which could break down structural carbohydrates. So far, few data are available on the use of more dilute acid for the exclusive estimation of starch in woody plant tissues. Rose et al. (1991) described methods for digesting starch with enzyme mixtures of α-amy-lase and amyloglucosidase, but the completeness of digestion was not tested in their studies. Currently, the removal of the ethanol after extraction is among the most time-consuming steps in the determination of sugar and starch in tissue samples. Buysse and Merckx (1993) showed that, in pure sugar solutions, absorbance readings increased for glucose but decreased for fructose and sucrose, with increasing ethanol content when analyzed by the phenol -sulfuric acid method. However, the effects of residual eth-anol on the analysis of total sugar in plant extracts and on the subsequent analysis of starch have not been investigated. The overall objective of this paper was to review selected methodologies for total sugar and starch analyses in plant tissues , and to shorten the existing procedures to establish a protocol suitable for rapid routine measurements of total sugar and starch reserves in plant tissues. Materials and methods Plant materials and preparation Leaf, stem and root samples of aspen (Populus tremuloides Michx.), black spruce (Picea mariana (Mill.) BSP.) and lodge-pole pine (Pinus contorta Loudon) seedlings and saplings were oven-dried at 68 °C for 2-3 days, ground in a Wiley mill to pass 40-mesh and stored in airtight containers at room temperature , in the dark, until analysis. Tissue samples analyzed in the different studies varied by type and collection. Comparison of sugar extraction methods Two methods of soluble sugar extraction, hot ethanol and methanol:chloroform:water (MCW, 12:5:3, v/v/v), were compared on dried samples of five different woody plant tissues (leaves of aspen, spruce and pine, aspen roots and pine shoots). For each tissue sample, five subsamples (50 mg) were extracted three times with 5 ml of 80% ethanol, by boiling the samples in glass tubes capped with glass marbles in a 95 °C water bath for 10 min each. After each extraction, the tubes were centrifuged at 2500 rpm for 5 min, and the supernatants of the three extractions combined for sugar analysis. A fourth ethanol extraction yielded less than 0.5% of the total sugar in the first three extracts. The residues remaining in the tubes were stored wet at-20 °C for starch analysis. Whether residues were oven-dried or wet had no effect on the subsequent starch analysis (see below). A second set of five subsamples was extracted three times with 5 ml of MCW solution. Sample tubes were loosely capped , placed in a sonic bath for 5 s, and left at room temperature for 10 min. Samples were then centrifuged at 2500 rpm for 10 min and the supernatants of the three extractions were combined (Rose et al. 1991). An earlier study had shown that three extractions with MCW removed about 98% of the water-alcohol soluble compounds from leaf material (Dickson 1979). A 5-ml sample was taken from each combined extract, mixed with 3 ml of deionized water (dH 2 O) and separated into two phases by centrifuging at 2500 rpm for 5 min. The chloroform phase was discarded and the methanol:water phase was analyzed for sugar. The residues were oven-dried at 50 °C overnight to remove the residual solvent, and stored at-20 °C for starch analysis. For both extraction methods, sugar concentrations in extracts were determined by the phenol-sulfuric acid method without removing the aqueous ethanol or methanol solvent. Optimization of phenol dosage and absorbance wavelength for sugar mixtures Oligosaccharides are hydrolyzed by concentrated sulfuric acid during the phenol-sulfuric assay and form monomers, namely glucose, fructose and galactose (Sturgeon 1990). Therefore, in the analysis of mono-and oligosaccharides in a plant extract, the intermediate product after acid hydrolysis is mostly a mixture of glucose, fructose and galactose, which are thus the principal compounds measured in the sugar assay. Thus optimizing the phenol concentration and absorbance wavelength for these three monomers should increase the accuracy of total sugar estimation in plant extracts based on a single measurement. For the optimization, five 0.5 ml solutions of glucose, fruc-tose, or galactose (50 µg ml-1) were mixed with 1 ml of a phenol solution at one of five concentrations (1, 1.5, 2, 3 and 4% phenol) followed by the rapid addition of 2.5 ml of concentrated sulfuric acid (H 2 SO 4). After 10 min of color development in the dark and an additional 30 min of cooling in a water bath at 22 °C, absorbance was measured at wavelengths ranging from 465 to 505 nm in 5 nm increments. Each sugar and phenol combination was replicated five times. After optimization of the phenol concentration and the absorption wavelength, the absorption coefficient of the sugar mixture (1:1:1, glucose, fructose, galactose; GFG) was compared with the absorption coefficients of the single sugars: glucose , fructose, galactose, sucrose, maltose, melibiose, raffi-nose and stachyose, to verify that the GFG mixture can be used 1130 CHOW AND LANDHA¨USSERLANDHA¨LANDHA¨USSER