Standardized protocols and procedures can precisely and accurately quantify non-structural carbohydrates

Abstract

Non-structural carbohydrates (NSCs), the stored products of photosynthesis, building blocks for growth and fuel for respiration, are central to plant metabolism, but their measurement is challenging. Differences in methods and procedures among laboratories can cause results to vary widely, limiting our ability to integrate and generalize patterns in plant carbon balance among studies. A recent assessment found that NSC concentrations measured for a common set of samples can vary by an order of magnitude, but sources for this variability were unclear. We measured a common set of nine plant material types, and two synthetic samples with known NSC concentrations, using a common protocol for sugar extraction and starch digestion, and three different sugar quantification methods (ion chromatography, enzyme, acid) in six laboratories. We also tested how sample handling, extraction solvent and centralizing parts of the procedure in one laboratory affected results. Non-structural carbohydrate concentrations measured for synthetic samples were within about 11.5% of known values for all three methods. However, differences among quantification methods were the largest source of variation in NSC measurements for natural plant samples because the three methods quantify different NSCs. The enzyme method quantified only glucose, fructose and sucrose, with ion chromatography we additionally quantified galactose, while the acid method quantified a large range of mono- and oligosaccharides. For some natural samples, sugars quantified with the acid method were two to five times higher than with other methods, demonstrating that trees allocate carbon to a range of sugar molecules. Sample handling had little effect on measurements, while ethanol sugar extraction improved accuracy over water extraction. Our results demonstrate that reasonable accuracy of NSC measurements can be achieved when different methods are used, as long as protocols are robust and standardized. Thus, we provide detailed protocols for the extraction, digestion and quantification of NSCs in plant samples, which should improve the comparability of NSC measurements among laboratories.

Figure 1.

The effect of sample handling and storage on the concentrations of sugar, starch and total NSC for three plant materials. Samples were freeze-dried, microwaved prior to oven drying or placed in a drying oven without microwaving, but not stored prior to drying (left panels). A comparison was performed of samples that were either microwaved or not microwaved, then stored at 4 or 20 °C for 8 h before oven drying (right panels). Significant differences among treatments for each sample material in each panel are shown with letters (P < 0.05). All data shown here were measured with the phenol-sulfuric acid method, and means of three replicates are shown for each bar. Error bars are one standard deviation.

Figure 2.

Concentrations of sugar, starch and total NSC for two synthetic samples (s1 and s2). Expected values (i.e., the known concentrations of sugars and starch) and measurement means (actual) are shown for three NSC quantification methods: IC, enzyme and acid. Significant differences between measured results and expected values (P < 0.05) from a one-sample t-test are indicated with an asterisk. Expected values for sugar and total NSC differ among quantification methods for sample s1 as they show only the portion of sugars specifically measured by the corresponding methods (see Table S1 available as Supplementary Data at Tree Physiology Online). The expected value of sugars did not differ for s2, because this sample was constructed with sugars that all three quantification methods can detect (see Table S1 available as Supplementary Data at Tree Physiology Online). Expected values of starch for s1 and s2 are the same for all three quantification methods. Error bars above means are one standard deviation.

Figure 3.

Mean differences between measured values and expected (known) concentrations of sugar, starch and total NSC for the two constructed synthetic samples (s1 and s2). Differences are quantified as percent of the expected values (Figure 2) for each carbohydrate component of three extraction methods: IC, enzyme and acid. Significant differences from expected values (P < 0.05) are indicated with an asterisk, as determined from a one-sample t-test of measured concentrations and expected concentrations (same analysis as Figure 2). Error bars are one standard deviation.

Figure 4.

Concentrations of sugar, starch and total NSC in nine plant material types from three different tree species measured with three quantification methods (IC, enzyme and acid). Letters indicate significant differences among quantification methods within each material type for each carbohydrate component (P < 0.05). Error bars are one standard deviation.

Figure 5.

Concentrations of sugar, starch, and total NSC measured in two plant materials. ‘Independent’ refers to samples that were extracted and quantified in six individual labs using one of three quantification methods (IC, enzyme or acid); ‘Central extraction’ refers to samples that were centrally extracted in one lab, but quantified in six individual labs using the same quantification method (IC, enzyme or acid) which that lab used for independent samples. Asterisks indicate significant differences between independent and central extractions within each material type for each carbohydrate component (P < 0.05). Error bars are one standard deviation.

Figure 6.

Concentrations of sugar, starch and total NSC in plant samples that were extracted in six laboratories, but quantified centrally in one lab using the acid method. Letters indicate significant differences among labs for each plant material type for each carbohydrate pool (P < 0.05). Error bars are one standard deviation.