DOI: 10.5061/dryad.4b8gtht87
title: Does microwaving or freezing reduce the losses of non-structural carbohydrates during plant sample processing?

description: Context: Non-structural carbohydrates (NSC) mediate tree survival, but
 precise determinations of NSC concentrations remain challenging. An
 unclear aspect is whether carbohydrate losses during sample processing can
 be reduced by different sample treatments.  Aims: We postulated
 that due to higher metabolic rates, leaves should be more responsive to
 sample treatments than branch sapwood, and resource-acquisitive species
 should be more responsive to sample treatment than resource-conservative
 species.  Methods: In leaves and branch sapwood of six tree
 species, we compared the effects of three sample treatments on the
 concentrations of NSC, starch, and low-molecular weight sugars: sample
 microwaving before drying (microwave), sample freezing at -20ºC before
 drying (freezing), and only drying (stove). Results: We found that across
 species and tissues, freezing led to significantly higher NSC and sugar
 concentrations than microwave and stove. This effect was, however,
 entirely driven by the leaves. Although the effect of sample treatments on
 NSC, starch, and sugar concentrations differed among species,
 resource-acquisitive species were not necessarily more affected by sample
 treatments than resource-conservative species.  Conclusion:
 Results suggest that either freezing or microwaving does not reduce
 carbohydrate losses in branch sapwood during sample handling and
 processing; however, freezing reduces leaf sugar and NSC
 losses when compared to microwave or direct stove-drying.
description: Sample collection was performed in May 2017 within the Coyhaique
 National Reserve (45° 59′ S and 71° 52′ W, 650 m above sea level
 (a.s.l.)), Coyhaique Province, Patagonia (Chile). Six woody species were
 selected to represent a range of leaf and wood economies. Species sampled
 were <i>Berberis mycrophylla</i> G. Forst., <i>Embothrium coccineum </i>J.R.Forst. & G.Forst<i>.</i>, <i>Nothofagus dombeyi</i> Blume, <i>Nothofagus antarctica </i>Oerst., <i>Nothofagus pumilio </i>Krasser, and <i>Ribes magellanicum</i> Poir. Six individuals of similar size were selected per species. One terminal, ~1 m long, fully expanded, sun exposed branch was cut at breast height. From each of these branches, we collected sun-exposed leaves of the two last cohorts (current and previous growing season) and 4-year-old branch segments (with bark and phloem removed in the field with a knife) for NSC determination. Samples were immediately transported to the lab, where they were assigned to each of three treatments. The first treatment consisted of the application of a microwave shock (3 cycles x 30 seconds each at 700 W) (Fancy 2000, Somela), followed by 72 h of drying in a forced-air stove at 70°C (Memmert GmbH, Schwabach, Germany). In the second treatment, samples were put inside Ziploc bags, frozen at -20°C for 7 days, and then dried for 72 h at 70°C in the forced-air stove. Finally, in the third treatment, samples were directly placed into the forced-air stove at 70°C for 72 h (<i>i.e.</i> no pre-treatment was applied). Samples from the three treatments were ground into a fine powder and stored in dry and cool conditions until chemical analyses were conducted. NSC concentrations were determined as the sum of the three most abundant low-molecular weight soluble sugars (glucose, fructose and sucrose) and starch. About 13 mg of dried powder were extracted with 1.6 ml of distilled water at 100 °C for 60 min. An aliquot of the extract was used to determine low-molecular weight soluble sugars after enzymatic conversion (invertase and phosphoglucose isomerase from <i>Saccharomyces cerevisiae</i>, Sigma Aldrich I4504 and P5381, respectively, St Louis, MO, USA) of sucrose and fructose to glucose. The concentration of free glucose was determined photometrically after the enzymatic conversion of glucose to gluconate-6-phosphate (Glucose Assay Reagent, G3293 Sigma Aldrich) on a 96-well multiplate reader. Following the degradation of starch to glucose using a purified fungal amylase (‘amiloglucosydase’ from <i>Aspergillus niger</i>, Sigma Aldrich 10115) at 45 °C overnight, NSC was determined in a separate analysis. The starch concentration was calculated as NSC minus the sum of free sugars. Total low-molecular weight soluble sugars, starch and NSC concentrations are presented as percent of dry matter. The influence of species, plant tissue (branch sapwood, foliage) and sample treatment (microwave, freezer, stove), and the interactions among them, on NSC, starch and sugar concentrations were analyzed with linear mixed-effects models, considering the individuals as the random factor.