Supporting Information Article title: Non-structural ... · extraction and quantification of...

Quentin et al. Supporting Information -‐ 1

Supporting Information

Article title: Non-structural carbohydrates in woody plants cannot be quantitatively compared among laboratories Authors: Audrey G. Quentin, Elizabeth A. Pinkard, Michael G. Ryan, David T. Tissue, L. Scott Baggett, Henry D. Adams, Pascale Maillard, Jacqueline Marchand, Simon M. Landhäusser, André Lacointe, Yves Gibon, William R.L. Anderegg, Shinichi Asao, Owen K. Atkin, Marc Bonhomme, Caroline Claye, Pak S. Chow, Anne Clément-Vidal, Noel W. Davies, L. Turin Dickman, Rita Dumbur, Kristen Falk, Lucía Galiano, José M. Grünzweig, Henrik Hartmann, Günter Hoch, Joanna E. Jones, Takayoshi Koike, Iris Kuhlmann, Francisco Lloret, Melchor Maestro, Shawn D. Mansfield, Jordi Martínez-Vilalta, Mickael Maucourt, Nathan G. McDowell, Annick Moing, Bertrand Muller, Sergio G. Nebauer, Ülo Niinemets, Sara Palacio, Frida Piper, Eran Raveh, Andreas Richter, Gaëlle Rolland, Teresa Rosas, Brigitte Saint Joanis, Anna Sala, Renee A. Smith, Frank Sterck, Joseph R. Stinziano, Mari Tobias, Faride Unda, Makoto Watanabe, Danielle A. Way, Lasantha K. Weerasinghe, Birgit Wild, David R. Woodruff

List of Supporting Information

Method S1. Preparation of standard samples.

Method S2. Summary of extraction and quantification procedures for soluble sugars and starch.

Method S3. Summary of extraction procedures for soluble sugars used in for experiment where different extractions were applied in the same laboratory.

Method S4. Enzyme de-activation treatments using microwave and effect on non-structural carbohydrate in foliar and twig samples of Pinus edulis.

Method S5. Analyses of non-structural carbohydrates in Pinus banksiana samples in an experiment on grinding particle size.

Table S1. Summary of main procedures of soluble sugars and starch measurements, and non-structural carbohydrate concentrations reported in the literature for well-watered and fertilised plants of Eucalyptus globulus and Prunus persica, and for Pinus edulis.

Table S2. Main procedures of extraction and quantification for soluble sugars measurements used by participating laboratories.

Table S3. Main procedures of gelatinisation, extraction and quantification for starch measurements.

Note S1. “Matrix effect”.


Note S2. Standardisation of sample preparation.

Figure S1A, S1B. Estimates of total soluble sugar, starch and total NSC by method of extraction and quantification of soluble sugars and starch for the two samples with the highest and the lowest variability among methods and laboratories.

Figure S2. Principal component analysis on total soluble sugar values for each laboratory as a function the extraction method.

Figure S3. Principal component analysis on starch values for each laboratory as a function the starch extraction methods.

Figure S4. Effect of microwaving on amount of glucose + fructose, sucrose, starch and total non-structural carbohydrate for foliar and twig samples of Pinus edulis.

Figure S5. Particle size slightly affected the starch concentration of stem and root tissues of Pinus banksiana samples (Method S4).

Figure S6. Suggested flow chart for collecting, handling and preparing plant tissue before analysis.


Method S1. Preparation of standard samples

Ten plants of Eucalyptus globulus were selected for destructive biomass harvesting in March

2013 from the CSIRO Ecosystem Sciences glasshouse facilities (42° 55' 0" S / 147° 20' 0" E).

The seedlings were obtained from the Forestry Tasmania Nursery near Perth, Tasmania.

Plants were grown for 12 months in 75 L plastic bags with potting mix consisting of eight

parts composted pine bark to three parts coarse river sand, and amended with a low

phosphorus premium controlled-release fertiliser (N:P:K – 17.9:0.8:7.3). Plants were

irrigated twice daily for 30 minutes using drip irrigation at a rate of 12 L per hour to maintain

soil at field capacity. Harvested plant biomass was divided into leaves (EGL), stem (EGS)

and coarse roots (>5 mm) (EGR). Immediately after harvesting, each tissue component was

placed overnight into a -4ºC freezer until processing. All samples were freeze-dried at -80ºC

for 24 to 48 hours (LY-5-FM, Breda Scientific, Netherlands). Dried samples were ground

into a fine powder. Leaves were ground and homogenised by passing through a cyclone mill

(18 mesh, 1 mm; Foss Cyclotec 1093, Denmark) and a Micro Ball Mill “Lab Wizz” 320

(Laarmann Group B.V., Op het Schoor 6, 6041 AV Roermond, Netherlands) at 30 Hz for 45

seconds (270mesh, 53 µm). Woody tissues (stem and roots) were ground and homogenised

by passing through a Wiley Mill (40 mesh, 400 µm), then with a Mini Wiley Mill (60 mesh,

250 µm). Following grinding, each sample was well homogenised by thorough mixing.

Foliage for the pine standard was sampled from a mature Pinus edulis (PEN) at mid-day on

26/7/2010 at Mesita del Buey, Los Alamos, USA (35° 51' 0" N / 106° 17' 12" W). The

sample was collected, stored on dry ice, oven-dried at 70°C until constant weight, then

ground and homogenised to a fine powder by passing it twice through a cyclone mill (Udy

Corporation, Fort Collins, CO, USA). Following homogenization, the sample was stored in a

plastic bag in a cool dry cabinet.

Standard foliar samples of Prunus persica (PPL) (SRM1547) were commercially purchased.

The plant material was collected from healthy peach trees in Peach County, Georgia, USA.

The leaves were dried and ground in a stainless steel mill past a 1 mm screen, and then jet

milled and air classified to a particle size of ~75 µm (200 mesh). Ground material was then

homogenised by mixing in a large blender.


All standard materials were irradiated at 27.8 kGy for microbiological control to meet

international quarantine requirements, homogenised, bottled, and stored at room temperature

until shipment to the analytical laboratories.

Method S2. Summary of extraction and quantification procedures for soluble sugars and starch

For soluble sugars three types of extraction were performed: alcohol using either ethanol or

methanol, water, and methanol:chloroform:water (MCW, 12:5:3, v:v:v). Quantification of

soluble sugars were done on supernatant solution, with one of five methods: high-

performance liquid chromatography (HPLC), High Performance Anion Exchange

Chromatography with Pulsed Amperometric Detection (HPAEC-PAD), enzymatic assay

based on NAD-linked enzymatic reactions and monitoring reduction of NAD+ to NADH at

340 nm, anthrone-sulfuric acid method with a colorimetric determination at 620 nm, and

phenol-sulfuric acid method with a colorimetric determination at 490 nm. HPLC and

HPAEC-PAD quantified individual sugars: glucose, fructose and sucrose. H-NMR profiling

of ethanolic extracts was also used for targeted quantification of selected soluble sugars. The

enzymatic assay quantified glucose+fructose, and sucrose as glucose equivalents. The

anthrone and phenol methods measured only total soluble sugars. Total soluble sugars issued

from chromatography-based and enzymatic methods were expressed as the sum of glucose,

fructose and sucrose. More detailed protocols are summarised in Table S2.

Estimation of starch was performed on the pellets remaining after the sugar extraction or on a

second aliquot of the same sample. Prior to the starch extraction procedure, samples were

treated with buffers, an ultrasonic bath or acid to solubilise granules of starch and to better

allow the extraction of starch and conversion into glucose by enzymes (see Table 1; Table

S3). Two main methods were used to hydrolyse starch into glucose: acid or enzymatic.

Within the enzymatic digestion, we distinguished two main methods: 1. amyloglucosidase

(AMG) from Aspergillus niger used solely, and 2. AMG used in association with α-amylase

(AA+AMG). Quantification of starch content in the material was determined as a glucose

equivalent (when based on two separate aliquots: minus the glucose and half of the fructose

concentration of the in soluble sugar extraction). In addition to the quantification methods

previously used for determination of soluble sugars, namely HPLC and colorimetric methods

using anthrone (620 nm) or phenol (490 nm), a total starch assay kit (Megazyme International


Ireland Ltd, Wicklow, Ireland) was also used which was then colorimetrically assayed by

using a mixture of glucose oxidase/peroxidase-o-dianisidine (GOPOD ) with a determination

at 510 nm (Table 1; Table S3). More detailed protocols are summarised in Table S3. Total

non-structural carbohydrates (NSC) were the sum of total soluble sugars and starch.

Method S3. Summary of extraction procedures for soluble sugars used in for experiment

where different extractions were applied in the same laboratory

Method 1- 80%EtOH: Samples were placed in 500µl of 80% aqueous ethanol (EtOH)

solution in a conical tube and mixed well. The mixture was placed in an incubator (Digital

Dry Bath D1100, Labtech Inc., Woodbridge, NJ, USA) at 80°C for 20 minutes, and then

centrifuged at 10 000 g for 5 minutes at 5°C. The supernatant was collected and the

procedure was repeated and supernatant from both extractions was combined for analysis.

Method 2 – 70%MeOH: Samples were placed in 650µl of 70% aqueous methanol (MeOH)

solution in a conical tube and mixed well. The solution was incubated and mixed at room

temperature for 10 minutes, and then centrifuged for 5 minutes at 17000 g. The supernatant

was collected, and the procedure was repeated twice more, and supernatant from all

extractions was combined for analysis.

Method 3 – MCW80: Samples were placed in 350µl of MCW solution (12/5/3) into each

conical tube, and mixed well. The mixture was incubated at 80°C (Digital Dry Bath D1100,

Labtech Inc., Woodbridge, NJ, USA) and mixed for 30 minutes, and then centrifuged for 3

minutes at 11 400 g at room temperature (derived from Dickson and Larson 1975). The

supernatant was collected, and the procedure was repeated twice, and supernatant from both

extractions was combined for analysis.

Method 4 – MCWamb: Samples were placed in 400µl of MCW solution (12/5/3) into each

conical tube, and mixed well. The solution was incubated and mixed at room temperature

(~20°C) for 30 minutes, and then centrifuged for 10 minutes at 2000 g at 15ºC. The

supernatant was collected, and the procedure was repeated, and supernatants from both

extractions were combined for analysis.


In each method and sample, the supernatants were filtered prior to quantification. Soluble

sugars from the supernatants were quantified by anthrone-sulfuric acid method with a

colorimetric determination at 620 nm (Spectrophotometer UV-visible DU 640 B, Beckman

Coulter, USA) as described by Hansen and Møller (1975) with glucose as standard.

Method S4. Enzyme de-activation treatments using microwave and effect on non-structural carbohydrate in foliar and twig samples of Pinus edulis

Four foliar and twig samples were collected from six Pinus edulis trees at Mesita Del Buey,

Los Alamos, USA (35° 51' 0" N / 106° 17' 12" W) in June 2012. Samples were collected in

liquid nitrogen and stored at -70 °C. Prior to drying, one sample from each tree was subject

to one of four enzyme deactivation treatment times using a microwave at 800 Watts: 1) 0

seconds, 2) 90 seconds, 3) 180 seconds, and 4) 300 seconds. Samples were then oven-dried

at 65 °C to constant weight. Leaf tissues were ball milled to a fine powder (high throughput

homogenizer; VWR). Woody tissues were milled to 40 mesh (400 µm) prior to ball milling

(Wiley Mini Mill; Thomas Scientific).

Samples were analysed following the protocol described by Hoch et al. (2002) with minor

modifications. Approximately 12 mg of fine ground plant material was extracted in a 2 mL

deep-well plate with 1.6 mL distilled water for 60 min in a 100 °C water bath (Isotemp 105;

Fisher Scientific). Following extraction, an NAD-linked enzymatic assay was used to

evaluate NSC content. All sugars were hydrolysed to glucose, linked to the reduction of

NAD+ to NADH, and monitored at 340 nm with a spectrophotometer (Cary® 50 UV-Vis).

Data were analysed using one-way analysis of variance (ANOVA) in R. Tukey's Honest

Significant Difference was used for pairwise comparison of significant effects (α < 0.05).

Method S5. Analyses of non-structural carbohydrates in Pinus banksiana samples in the experiment on grinding particle size, fine (< 105 µm) versus coarse (> 400 µm)

The samples were collected from leaves, stem and roots of ten jack pine (Pinus

banksiana Lamb.) seedlings in 2013. Samples were immediately oven-dried at 70ºC until

constant weight, and either ground with a Wiley Mini-Mill (Thomas Scientific, Swedesboro,

NJ, USA) to pass a 40 mesh (400 µm) or with a Micro Ball Mill “Lab Wizz” 320 (Laarmann

Group B.V., Op het Schoor 6, 6041 AV Roermond, Netherlands) at 30 Hz for 2 minutes.


Root and stem samples from the Ball Mill had particles <105 µm (140 mesh), and particles

from the needle sample were < 53 µm (270 mesh).

Total soluble sugars were extracted from 50 mg of dried plant tissue in 4.7 ml 80%

aqueous ethanol in a glass test tube. The mixture was placed in a water bath at 90ºC for 10

mins, and then centrifuged at 2500 rpm for 4 minutes. The supernatant was collected, and the

procedure was repeated two more times. Total soluble sugars were determined on the

supernatant combined using a phenol–sulphuric acid colorimetric assay, and absorbance was

read at 490 nm, consistent with methods described by Chow and Landhäusser (2004). Starch

was determined on the remaining pellets and assayed enzymatically using a combination of

α-amylase and amyloglucosidase. Prior to digestion, pellets were solubilised using a 0.1 M

of NaOH solution. Starch was determined using GOPOD assay, and absorbance was read at

525 nm. A Student t-test determined the effect of particle sizes on total soluble sugars, starch

and NSC contents for the different tissues of P. banksiana (α = 0.05).


Notes:

Note S1. “Matrix effect”

Signals from many metabolites present in plant tissue can be influenced by other interfering

metabolites through several different mechanisms, collectively known as matrix effects. For

example, sulphuric acid used in colorimetric assays can break down structural carbohydrates

such as cellulose and hemicelluloses and could potential be converted into glucose. The

gravity of this problem is easily realized when considering a situation where the signal of a

given metabolite is affected by the concentration of a different metabolite with fluctuating

concentration. In this scenario, the first metabolite will appear to change even if its

concentration is stable. In this study, our samples had a priori different levels of NSCs and

different compounds that may interfere with analysis. P. persica leaves have a high level of

sugar-alcohol sorbitol (Loescher 1987), which may not be assessed as sugar by colorimetric

methods, but identified in HPLC (Fig. 3F). In this study, HPLC and enzymatic methods only

targeted glucose, fructose and sucrose, which are supposed to be the major sugars present in

plant material, and could be easily targeted by those methods, whereas colorimetric assays

were non-specific. Foliage of E. globulus contains phenolics (Macauley and Fox 1980,

Rapley et al. 2008), and foliage in P. edulis has terpenes (Cobb et al. 1997). These secondary

compounds may interfere with extraction and/or quantification (Ashwell 1957, Hendrix and

Peelen 1987), which may explain the high variability of the EGL results (Fig. 3). Purification

treatment using charcoal can increase the recovery rate of soluble sugars from tissues

containing phenolics (Hendrix and Peelen 1987). The presence of matrix effects is also an

important reason why quantification of an authentic standard is very different from

untargeted analysis of metabolites in an actual biological sample.

Matrix components present in biological samples, such as ligneous versus non-ligneous

components, can also affect the response of the analyte of interest. These phenomena, termed

generally as matrix effects, can lead to inaccurate quantification (Silva et al. 2012), and

therefore are important to be addressed in analytical method development and validation

(Thompson and Ellison 2005). Here, ligneous or woody samples (i.e. EGR and EGS) showed

least variability in soluble sugar results compared to starch results following acid hydrolysis

(Fig. 3A) or colorimetric assays (Fig. 3F). In woody tissues, starch exists in small

concentrations that are locked within a matrix of structural polysaccharides (e.g. cellulose and

hemicellulose) and organic polymers (e.g. lignin). Acid hydrolysis may not be suitable for


wood matrix because not all acid methods have a selective step, and can result in low

selectivity because both structural carbohydrates and starch are hydrolysed by concentrated

acids into monomeric sugars which could result in high interference (Ebell 1969, MacRae et

al. 1974, Marshall 1984, Rose et al. 1991). In contrast, enzyme methods rely on the inherent

specificity of the catalyst, which is superior to the precipitation selectivity (Rose et al. 1991).

To the best of our knowledge, no research has been devoted to measuring the matrix effect on

extraction of NSC in woody plants. Appropriate matrix management could gear toward

minimizing or correcting these effects. Therefore, in order to fully extract NSC from tissues,

it is necessary to use solvents that readily dissolve the NSC, but also overcome the

interactions between the NSC and the tissue matrix.

Note S2. Standardisation of sample preparation

The validity and usefulness of a plant analysis hinges on the care and method used to obtain

the required plant material (Westerman 1990). If the sample taken is not representative of the

general population, all the careful and costly work put into the subsequent analysis will be

wasted because the results will be invalid. Unfortunately, less research has been devoted to

sampling and sample preparation procedures than to other aspects of plant analysis

techniques. Yet sampling and sample preparation, if not properly done, can contribute to

errors which may have a cumulative effect on the final reported results.

The elemental content of a plant is not a fixed entity, yet it is crucial to obtain a

representative sample from a particular plant species. Plant part selected and time of

sampling must correspond to the best relationship between element concentration and

physical appearance of the plant (Westerman 1990). In establishing sampling procedures for

plant analysis, the researcher and user must be aware that the concentrations of non-structural

carbohydrates, as well as other elements, change rather rapidly with time, particularly in

leaves, and physiological maturity, and may also vary more greatly between organs (Palacio

et al. 2007).

The most difficult logistical problem facing plant analysis is the preservation of fresh

material during transport from field to the laboratory. Delays and adverse conditions during

transport can cause substantial respiratory losses in weight or enhanced enzymatic activity

(Handreck 1972). Deinum and Maasen (1994) showed that pre-freezing treatment prior to


drying did not influence the outcome, suggesting that snap-freezing using liquid nitrogen or

dry ice may keep the attributes of the samples.

Oven drying is often used as sample preparation before sugar and starch analyses, even

though results have shown that considerable losses may occur (Deinum and Maassen 1994,

Pelletier et al. 2010). With oven drying, thick specimens (e.g., big roots) or large sample

volumes can be problematic because the complete drying process may last for many hours

and rapid removal of moisture is necessary (Udén 2010). Evidence implicates respiratory

loss, metabolic interconversions, and deleterious effects of heat as factors that may alter plant

carbohydrate composition during conventional sampling and drying procedures (Deinum and

Maassen 1994). Low temperatures (below 50°C) allow time for enzymatic conversions and

respiratory losses, whereas high temperatures (above 80°C) can cause thermochemical

degradation (Smith 1973). For those reasons, sampling and drying procedures that rapidly

inhibit enzyme activity while preserving chemical attributes are preferred. Freeze-drying is

generally recognized as the best method for preserving the labile metabolites, including NSC

(Pelletier et al. 2010, Raessler et al. 2010). Pre-treatment with a microwave before drying at

55ºC gave similar values as for freeze drying by denaturing proteins that cause enzymatic

conversions and respiratory losses (Pelletier et al. 2010). However, sucrose concentration

were consistently higher in microwave pre-treated samples than in freeze-dried samples due

to rapid deactivation of all plant enzyme, thereby minimising respiratory weight losses and

interconversions between carbohydrate fractions (see Fig. S4; Popp et al. 1996, Pelletier et al.

2010, Raessler et al. 2010). The drawbacks of freeze-drying include access to expensive

equipment and difficulty in application to large samples under field conditions.

Dried samples are customarily ground to facilitate the preparation of homogeneous samples

for chemical analysis, and should be ground to an appropriate and consistent fineness (Greub

and Wedin 1969). There is a paucity of information in the literature regarding the effect of

particle size on NSC values and the impact of the grinding technique, which is accomplished

in a variety of mills. In our study, there was consistently higher starch concentration in finely

ground root and stem samples compared with coarser ground samples, presumably due to

more efficient breakdown of xylem structure and exposure of more parenchyma cells to

enzymes (Fig. 7). The lack of differences in NSC between fine- and coarse-ground foliar

samples of P. banksiana indicates that particle size of ground material does not impede

extraction of NSC in these leaf tissues, most likely due to the lack of woody tissues. It


appears that a standardized sample preparation would be advisable to allow for comparisons

of absolute values and that consistent sample grinding methods are necessary to evaluate

NSC patterns in plant tissues as samples may pulverise at different rates and segregate

differently which could have an impact on starch concentrations. Finally, dried tissue should

be stored under conditions that allow minimal changes (Smith 1973). The longer the storage

time, the larger the changes are likely to be, therefore carbohydrate analyses should be done

as soon as possible after tissue-drying (Steyn 1959).

Standardisation of handling and preparation procedures of samples could be achievable (see

Fig. S6). Each of these preparatory procedures may provide opportunities to enhance the

accuracy and reliability of the analytical results.


Tables:

Table S1. Summary of main procedures of soluble sugars and starch measurements, and non-structural carbohydrate (NSC) concentrations reported in the literature for well-watered and fertilised plants of Eucalyptus globulus (A) and Prunus persica (B) and for Pinus edulis (C).

References Age Tissue Sample weight (mg)

Soluble sugars Starch Concentration (mg g-1) in the literature

Extr. Quant. (assay) Dig. Quant.

(assay) GLUC FRUC SUC TSS St Total NSC

A. Eucalyptus globulus

Shvaleva et al. (2005) ~12 mo L 20

EtOH Spec. (anthrone) HCl Spec. 620 72-83 49-56 115-117

R 50 32-45 29-32 78-88

Eyles et al. (2009a) 11 mo L

50 EtOHx1 Spec. 490 (phenol) Amylo. Spec. 490

(phenol) 105 94 199

S 40 79 118 R 33 100 132

Eyles et al. (2009b) ~16 mo L 50 EtOHx1 Spec. 490 (phenol) Amylo. Spec. 490

(phenol) 46 93 140

Merchant et al. (2010) ~12 mo L 40 MCW GC 5 4 2 12

O'Grady et al. (2010) >6yo L (at 7m high)


(phenol) 56 64 120

L (at 15m high) 19 37 56

Quentin et al. (2010) ~8 mo L 50 EtOHx1 Spec. 490 (phenol) Amylo. Spec. 490

(phenol) 93-106 37-39 130-145

Pinkard et al. (2011) ~3-4 mo L 50 EtOHx1 Spec. 490 (phenol) Amylo. Spec. 490

(phenol) 142 93 187

Quentin et al. (2011) > 6yo L 50

EtOHx1 Spec. 490 (phenol) Amylo. Spec. 490

(phenol) 145

S 60 R 63

Barry et al. (2012) 18 mo L


(phenol) 60 16 76

S 24 9 32


R 28 40 67

Drake et al. (2013) ? S

100 EtOHx2 Spec 630 (anthrone)

6-14 R (tap) 7-16

Duan et al. (2013) 8 mo L

20 EtOHx2+W Spec 620 (anthrone)

ΑΑ + amylo. Spec. 515

83-90 33-140 117-224 S 32-60 2-8 35-62 R 10-24 1-2 12-25

Eyles et al. (2013) 7 mo L 50 EtOHx1 UPLC Amylo. Spec. 490 (phenol) 18 22 1 54 92 146

Mitchell et al. (2013) 6 mo L

20 EtOHx2+W Spec 620 (anthrone)

AA + amylo.

Spec. 515 (GOPOD)

85 120 206 S 20 13 33 R 46 30 76

(Gauthier et al. 2014)x <6 mo L 5 EtOHx3 Enz. Amylo. Spec 515 (GOPOD) 7 10 17

B. Prunus persica

Moing et al. (1992) 2 mo L ? EtOHx2 HPLC Amylo. HPLC 11.1 5.69 36.7 95 89 184

Nii (1997) 37-38 yo L ? EtOH Spec (anthrone) 78 77 155

Tworkoski et al. (1997) 5-6 yo L

200 EtOH HPLC Amylo. Spec. 38-158 33-48 86-191 S 44-77 39-45 83-122

Escobar-Gutiérrez et al. (1997) 2.5 mo L ? EtOHx2 HPLC Amylo. 39 10 53 215 135 350

Inglese et al. (2002) 3 yo R 150 EtOH Enz. Amylo. Enz. 6 - 9

Graham (2002) 2 mo R

50 MCW HPLC Amylo. Spec. 450 16 9 9 57 52 109

S 11 3 7 54 33 88 L 20 7 15 106 26 132

Leite et al. (2004) 11 yo S (Oct)

10 EtOHx2 HPLC Amylo. Enz. 5 27 69 65.5 134

S (Feb) 14 35 74 16 90 Bonhomme et al. (2005) 4 yo S 10 EtOHx2 HPLC Amylo. Enz. 15 28 72 12 84


Gordon et al. (2006) 2 yo R ? ? HPLC Amylo. ? 150

Dichio et al. (2007) >3 yo L

? EtOH Spec. 625 (anthrone) Amylo. Spec. 425

(GOPOD) 120 5 125

S 100 10 110 R 240 60 300

Li et al. (2007)y 5 yo L 15000 EtOHx3 HPLC Amylo. Spec. (GOPOD) 4 2 9 51 25 76

Cheng et al. (2009)y 8 yo L 15000

EtOHx3 HPLC Amylo. Spec. (GOPOD) 4 4 11 43 23 65

Weibel et al. (2008) 4-5 yo R

EtOH Spec. (anthrone)

260 S

? 160 R 190

C. Pinus edulis

Adams et al. (2013)z 15-25 yo L 12 W Enz. Amylo. Enz. 10-56 0-185 19-216

Anderegg and Anderegg (2013) 10-15 yo

L ? EtOH+MCW Spec 595 BA +

amylo. Spec. 595 5-10 0-30 30-60

R 10-28 8 -21 45-95

Dickmann et al. (2014) z ? L (2007)

12 W Enz. Amylo. Enz. 5 1 6 10 16

L (2008) 4 1 5 3 8 L (2009) 4 0 4 12 16

Sevanto et al. (2014) z 15-25 yo L 12 W Enz. Amylo. Enz. 13-27 1-19 27-36 6-31 36-59 x values reported in g m-2. y estimations were made on fresh weight. z no fertiliser used. AA: α-amylase; Amylo : amyloglucosidase ; BA.: β-amylase; DMSO : dimethylsulfoxide ; Dig.: digestion; Enz: enzymatic; EtOH: ethanol; Extr.: extraction; FRUC: fructose; GLUC: glucose; GOPOD: glucose oxidase/peroxidase-o-dianisidine; HCl: hydrochloride acid; L: leaf; MCW: methanol:chloroform:water; mo: month-old; Quant.: quantification; R: roots; spec: spectrophotometry; S: stem; St: starch; SUC: sucrose; TSS: total soluble sugars; W: water; yo: year-old.


Table S2. Main procedures of extraction and quantification for soluble sugars measurements used by participating laboratories. Lab ID Extraction Quantification (assay; standards used) References

O 70% MeOH (room temperaure; 10 mins) (x3) Spec. 620 (anthrone-sulfuric assay; GLUC) Hansen and Møller (1975)

U 70% EtOH (55-65°C; 30 mins) Spec. 620 (anthrone-sulfuric assay; GLUC) Yemm and Willis (1954); Hansen and Møller (1975)

S 80% EtOH +30% EtOH + W (each 90°C; 15 mins) Enz. (340 nm; GLUC, FRUC, SUC) Blunden and Wilson (1985)

V 80% EtOH (60°C; 30 mins) Spec. 490 (phenol-sulfuric assay; GLUC) Dubois et al. (1956); Buysse and Merckx (1993); Palacio et al. (2007)

CC 80% EtOH (80°C; 20 mins) HPAEC-PAD Van Meeteren et al. (1995)

Q 80% EtOH + 50% EtOH + W (each 80°C; 15 mins) xH-NMR (GLUC, FRUC, SUC) Adapted from Moing et al. (2004)

C 80% EtOH (x2) + W (each 95°C; 30 mins) Spec. 620 (anthrone-sulfuric assay; GLUC) Ebell (1969)

D 80% EtOH (x2) + 50% EtOH (each 80°C; 20 mins) Enz. (340 nm; GLUC, FRUC, SUC) Jelitto et al. (1992)

L

80% EtOH (80°C; 30 mins) + 80% EtOH (80°C; 15 mins) + 50% EtOH (80°C; 15 mins) HPLC (mannitol)y

Moing et al. (1992) 80% EtOH (80°C; 30 mins) + 80% EtOH (80°C; 15 mins) + 50% EtOH (80°C; 15 mins) HPLC (mannitol)z

B 80% EtOH (75°C; 30 mins) (x3) HPAEC-PAD (GLUC, FRUC, SUC) Mialet-Serra et al. (2005)

E 80% EtOH (100°C; 2 mins) + 80% EtOH (90°C; 2 mins) (x2) HPLC (trehalose) Lucas et al. (1993)

J 80% EtOH (60°C; 60 mins) (x3) HPLC (trehalose) Lambrechts et al. (1994); Blake (1999)

M 80% EtOH (90°C; 10 mins) (x3) Spec. 490 (phenol-sulfuric assay; GLUC, FRUC, GALAC) Chow and Landhäusser (2004)

T 80% EtOH (100°C; 10 mins) (x3) Spec. 630 (anthrone-sulfuric assay; GLUC) McCready et al. (1950)

X 80% EtOH (60°C; 60 mins) (x3) HPLC (trehalose) Lambrechts et al. (1994); Blake (1999)

DD 80% EtOH (80°C; 20 mins) (x3) Enz. (340 nm; GLUC, FRUC, SUC) Hendrix (1993)


A 80% EtOH (80°C; 10 mins) (x2) + (room temperature) (x2) Enz. (340 nm; GLUC, FRUC, SUC) Sigma®

K 80% EtOH (room temperature; 3 mins) (x4) Spec. 490 (phenol-sulfuric assay; GLUC) Kabeya and Sakai (2003)

Y 80% EtOH (80°C; 20 mins) (x5) Spec. 620 (anthrone-sulfuric assay; GLUC) Schaffer et al. (1985)

P MCW (4°C; overnight) + MCW (x2) HPLC (galactitol) Coleman et al. (2009)

W MCW (20°C; 5 mins) (x2) Spec. 490 (phenol-sulfuric assay; SUC) Haissing and Dickson (1979); Rose et al. (1991); Chow and Landhäusser (2004)

Z2 MCW (60°C; 30 mins) HPAEC-PAD (GALAC, GLUC, FRUC, SUC) Wanek et al. (2001)

F W (65°C; 10 mins) (x3) HPLC Raessler et al. (2010)

Z1 W (85°C; 30 mins) HPAEC-PAD (GALAC, GLUC, FRUC, SUC) Richter et al. (2009)

G, N, R W (steam; 60 mins)

Enz. (340 nm; GLUC, FRUC, SUC)

Wong (1979); Hoch et al. (2002) AA, BB

& EE Enz: enzymatic; EtOH: ethanol; FRUC: fructose; GALAC: galactitol; GLUC: glucose; MCW: methanol:chloroform:water; spec: spectrophotometry; SUC: sucrose; W: water. x H-NMR profiling of ethanolic extracts was also used for targeted quantification of selected soluble sugars. y HPLC using Metrosep Carb1 250 x 4.6 mm column (Metrohm ltd, CH-9101 Herisau, Switzerland) z HPLC using CARBOSep COREGEL 87C column (Transgenomic Inc., NE 68164, Omaha, USA)


Table S3. Main procedures of gelatinisation, extraction and quantification for starch measurements.

Lab ID Gelatinisation or Solubilisation Extraction/Digestion Quantification (assay; standards used) References

F Ultrasound 52% HClO4 (23°C; 20 hrs) (x2) HPLC Raessler et al. (2010); Hartmann et al. (2013)

P - H2SO4+ (120°C; 3.5 mins) HPLC (GLUC) Coleman et al. (2009)

T - 35% HClO4 (23°C; 16 hrs) Spec. 630 (anthrone-sulfuric assay; GLUC) McCready et al. (1950)

U Vigorous shaking 1% HCl (100°C; 30 mins) Spec. 620 (anthrone-sulfuric assay; GLUC)

Yemm and Willis (1954); Hansen and Møller (1975)

C AA (100°C; 6 mins) Amylo. (50°C; 30 mins) Spec. 515 (GOPOD assay; GLUC) Megazyme ®; McCleary et al. (1997)

D 0.1 M NaOH (95°C; 30 mins) AA + amylo. (37°C; 10-16 hrs) Enz. (340 nm; GLUC) Hendriks et al. (2003)

J DMSO (100°C; 5 mins) AA (100°C; 6 mins) + amylo. (50°C; 30 mins) Spec. 515 (GOPOD assay; GLUC) Megazyme ®;

M 0.1 M NaOH (50°C ; 30 mins) AA + amylo. (50°C; 20-24 hours) Spec. 525 (GOPOD assay; GLUC) Chow and Landhäusser (2004)

DD DMSO (100°C; 5 mins) AA (100°C; 16 mins) + amylo. (50°C; 30 mins) Spec. 510 (with GOPOD assay; GLUC) Megazyme ®

A AA (100°C; 9 mins) Amylo. (50°C; 30 mins) Spec. 515 (with GOPOD assay; starch) Megazyme ®

CC AA (90°C; 30 mins) Amylo. (60°C; 15 mins) HPAEC Van Meeteren et al. (1995) Z1 AA (85°C; 30 mins) Amylo. (55°C; 30 min) HPLC (GLUC) Göttlicher et al. (2006) Z2 K 0.2 M KOH (95°C; 30 mins) Amylo. (55°C; 30 mins) Spec. 490 (phenol-sulfuric assay; GLUC) Komatsu et al. (2013)

L 0.02N NaOH (120°C; 60 mins) BA (52°C; 90 mins) Enz. (340 nm; GLUC) Moing et al. (1992)

B 0.02N NaOH (95°C; 90 mins) Amylo. (50°C; 60 mins) HPAEC (GLUC) Mialet-Serra et al. (2005)

E Water (100ºC; 60 mins) Amylo. (55°C; 3 hrs) Spec 340 (GOPOD assay; GLUC) Lucas et al. (1993)

Q S Autoclave (120°C; 90 mins) Amylo. (56°C; 90 mins + 100°C; 5 mins) Enz. (340 nm; GLUC) Thivend et al. (1965); Gomez et

al. (2003b); Gomez et al.


(2003a)

V 0.1 M NaOH (100°C; 60 mins) Amylo. (50°C; 16 hours) Spec. 490 (phenol-sulfuric assay; GLUC) Dubois et al. (1956); Buysse and Merckx (1993); (Palacio et al. 2007)

X 0.1 M NaOH (100°C; 60 mins) Amylo. (50°C; 16 hours) Spec. 490 (phenol-sulfuric assay; GLUC) Dubois et al. (1956); Buysse and Merckx (1993); Palacio et al. (2007)

Y W + autoclave (120°C; 60 mins) Amylo. (55°C; 22 hrs) Spec. 620 (anthrone-sulfuric assay; GLUC) Schaffer et al. (1985)

O 0.02 N NaOH (100°C; 60 mins) Amylo. (50°C; 30 mins) Spec. 620 (anthrone-sulfuric assay; GLUC) Hansen and Møller (1975)

W 47.5% EtOH (100°C; 30 mins) Amylo. (45°C; overnight) Spec. 490 (phenol-sulfuric assay; GLUC) Rose et al. (1991); Marquis et al. (1997)

G, N, R, AA, BB & EE

0.1 M NaOH Amylo. (48°C; overnight) Enz (340 nm; GLUC) Wong (1979); Hoch et al. (2002)

AA: α-amylase; Amylo.: amyloglucosidase; BA.: β-amylase; DMSO : diméthylsulfoxide; Enz: enzymatic; EtOH: ethanol; GLUC: glucose; GOPOD: glucose oxidase/peroxidase-o-dianisidine; H2SO4: Sulfuric acid ; HClO4: Perchloric acid ; HPAEC: High Performance Anion Exchange Chromatography; HPLC: High-performance liquid chromatography; KOH: Potassium hydroxide ; NaOH: Sodium hydroxide; Spec: spectrophotometry; W: water.


Figures


K J X L1 L2 D B CC E A Y V T U O DD M Q S C P Z1 W AA N BB R G F EE Z2

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Z F P T U L S N B K AA EE R G BB E W O Y V X C D M CC A JDD

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Z F CC B P D L S N AA EE R G BB E K W V X T U O Y C M A JDD

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K J D L1 L2 B CC A E T U M Y V DD O X S C Z1 P W N AA Z2 R F EE BB G

020406080

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EtOH EtOH+WMCWW

Z1 Z2 F P T U K N AA R L1 EE L2 B BB G S E Y V W O X J D CC A C M DD

020406080

100120140160

AcidAmylo.AA+amylo.

J Z1 Z2 F L1 L2 P B CC E X N AA R D EE BB G S A DD K M V W T C U Y O

020406080

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HPLCEnz.Spec. 490Spec. 620

Z1 Z2 F P B CC N AA R D L1 EE L2 BB G S E K V W X T U Y O J A C M DD

020406080

100120140160

Spec. 510

Legend:

Total NSCTotal soluble sugars StarchExtraction methods

Quantification methods

Figure S1. A on this page, B on next. Estimates of total soluble sugar, starch and total NSC by method of extraction and quantification of soluble sugars and starch for the two samples with the highest and the lowest variability among methods and laboratories: (A) Prunus persica leaves (PPL, high variability), and (B) Eucalyptus globulus stem (EGS, lower variability). Total soluble sugars results are shown by sugar extraction and quantification methods. Starch results are shown by sugar extraction method and starch extraction methods. Total NSC results are shown for sugar and starch extraction methods, and for sugar and starch quantification methods. The linear mixed model analysis showed some differences among methods (Fig. 3), but these differences were lower than the 90-percentile range of the data (Fig. 4).


K J A Y E VC

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D

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Legend:

Extraction methods

Quantification methods

Figure S1B.


A

-5

-4

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EtOH EtOH+WMCW W

B

Dim 1 (53%)-2 -1 0 1 2 3 4

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2 (2

0%)

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HPLC Enz.Spec. 490 Spec. 620

Figure S2. Principal component analysis on total soluble sugar values for each laboratory as a function the extraction method (A), and the quantification methods (B). Panel A shows that the water (W, red) extraction method had the most homogeneous results. Ethanol (EtOH, blue) and EtOH+W (green) extraction yields highly variable results. Panel B showed that total soluble sugar quantified using colorimetric Spec 490 (pink) and Spec 620 (grey) methods were more variable than using HPLC (turquoise) and Enz. (purple) methods. Principal component analysis (PCA) was performed on centred and scaled data using the R Software (http://www.r-project.org/) and the package FactomineR (Lê et al. 2008).


A

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2 (2

0%)

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B

Dim 1 (71%)-2 -1 0 1 2 3 4 5 6

-5

-4

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-2

-1

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1

HPLC Enz.Spec. 490Spec. 620 Spec. 510

Figure S3. Principal component analysis on starch values for each laboratory as a function the starch extraction methods (A) and quantification methods (B). Panel A shows that the AA+ amylo. extraction method (light red) yields less variable results than the other methods. Panel B shows that all methods mostly yielded similar results. Principal component analysis (PCA) was performed on centred and scaled data using the R Software (http://www.r-project.org/) and the package FactomineR (Lê et al. 2008).


A

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(mg

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cb

a ac

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abca

Figure S4. Effect of microwaving on amount of glucose + fructose (Gluc+Fruc), sucrose (Suc), starch and total non-structural carbohydrate (NSC) for foliar (A) and twig (B) samples of Pinus edulis. See Method S4 for details on the method. At low microwaving time (0 and 90 sec.), sucrose hydrolyzing and starch debranching enzymes are still active, leading to lower sucrose levels, higher glucose + fructose levels, and higher starch levels because debranching enzyme make starch more accessible to the enzymatic assay. At 180 sec. and above, enzymes are deactivated, yielding consistent sucrose and glucose + fructose. At 300 sec., starch starts to gelatinize, again making it more accessible to the assay.


Figure S5. Particle size slightly affected the starch concentration of stem and root tissues of Pinus banksiana samples (Method S4). Different letters within the same tissue type indicate significant difference at a=0.05 using the Student t-test.


Store samples in freezer (at -20ºC) before drying

Store samples in controlled air

temperature room

Snap-freezing following collection of samples

Sample transfer from field to laboratory in tight

cooled container

Thoroughly mix whole sample and put samples

in airtight storage containers

Redry samples at 60ºC prior to analysis

Laboratory analysis

Drying: Large samples: 70-90ºC in

forced-draft oven* Small samples: freeze-

drying

Grinding preferably using a ball mill

(<0.4 mm particle size)

Figure S6. Suggested flow chart for collecting, handling and preparing plant tissue before analysis. *Microwave pre-treatment required to deactivate enzyme activity.


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