Aqueous-acetone extraction improves the drawbacks of using dimethylsulfoxide as solvent for photometric pigment quantification in Quercus ilex leaves

Aim of study: We evaluated the use of dimethylsulfoxide (DMSO) for the photometric determination of chlorophyll (Chla and Chlb) and carotenoids in Quercus ilex L. leaves by comparative analysis using aqueous-acetone extraction. Area of study: a Q. ilex dehesa in Las Majadas del Tietar, Cáceres, Spain Material and methods: Q. ilex leaves were sampled during two vegetative periods. Field SPAD-502 Chlorophyll measurements and photometric chlorophyll determinations were performed. Two procedures were used: extraction of intact foliar discs at 65oC with DMSO and fine foliar powder with cold aqueous-acetone. Main results: DMSO produced Chlb overestimation and different fitting performance for SPAD vs pigment calibrations (R 2=0.64, RMSE=0.20, p<0.0001 for Chla (μg/cm 2); R2=0.33, RMSE=0.23, p<0.0001 for Chlb (μg/cm 2) and R2=0.50, RMSE=0.23, p<0.0001 for carotenoids (μg/cm2)). Aqueous-acetone provided more accurate predictions (R2=0.90, RMSE=0.16, p<0.0001 for Chla and R 2=0.91, RMSE=0.16, p<0.0001 for Chlb, R 2=0.90, RMSE=0.02, p<0.0001 for carotenoids) and mean ratio Chla/Chlb=3.6 inside the range for sun exposed leaves. Research highlights: Oxidizing conditions and polyphenol concentrations in Q. ilex leaves generated brown colorations in the DMSO extraction procedure, interfering with the photometric measurements in the red-orange region. Aqueous-acetone extraction was free from interference. DMSO should be avoided for pigment determination in Q. ilex leaves or when comparing different tree species. Additional keywords: chlorophyll; carotenoids; DMSO; SPAD-502; phenolic compounds. Funding: Ministry of Economy and Competitiveness, Spain (Project CGL-2015-69095-R). Competing interests: The authors have declared that no competing interests exist. Copyright © 2017 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License. Citation: Gonzalez-Cascon, R.; Jiménez-Fenoy, L.; Verdú-Fillola, I.; Martín, M. P. (2017). Short communication: Aqueous-acetone extraction improves the drawbacks of using dimethylsulfoxide as solvent for photometric pigment quantification in Quercus ilex leaves. Forest Systems, Volume 26, Issue 2, eSC04. https://doi.org/10.5424/fs/2017262-11099 Received: 01 Feb 2017 Accepted: 05 Jul 2017. 26 (2), eSC04, 5 pages (2017) eISSN: 2171-9845 https://doi.org/10.5424/fs/2017262-11099


Introduction
Laboratory analysis of pigments in tree leaves has traditionally been conducted by extraction methods using several water-soluble organic solvents followed by spectrometric determination based on the maxima absorption spectra of Chlorophylls (Chls) and carotenoids in the red and blue spectral regions (Minocha et al., 2009).Quantification is routinely made by applying various equations developed for different solvents and spectrophotometer resolutions (Barnes et al., 1992;Wellburn, 1994).The solvent DMSO was introduced for pigment extractions as it avoids the need for grinding and centrifuging foliar samples required by other solvents, allowing a large amount of samples to be analysed and a higher stability of the extracted Chls with time (Hiscox & Israelstam, 1979).The Chl b peak in the red region is much less intense than the Chl a and is superposed by the later in fresh leaves extracts.For this reason, Chl b quantification is very sensitive to extract turbidity or high pigments concentration (Lichtenthaler & Buschmann, 2001).F 2 We investigated the drawbacks in the use of DMSO as solvent for pigment quantification in Quercus ilex L. leaves used for SPAD calibrations.An alternative extraction method with aqueous-acetone was studied.

Study site and sampling
Nine sampling campaigns were conducted in five mature Q.ilex trees along two vegetative periods.Field SPAD (SPAD-502, Konica Minolta) measurements were obtained from 12 leaves per tree (6 current and 6 from the previous year).Immediately, each leaf was individually packed in black polyethylene bags and transported in cooled containers to the laboratory.Leaves were frozen at -20ºC within a maximum of 3 h after sampling until required.

Pigment analysis
DMSO extraction.One (two for new sprouted leaves) 6 mm diameter leaf disc was extracted with 5 mL of DMSO PRS (Panreac Quimica S.A.U.) in a 10 mL glass reagent tube wrapped in aluminium foil at 65ºC for 4 h (to ensure complete disc decolouration) and centrifuged (3000 rpm).Pigment concentrations were measured spectrophotometrically immediately after extraction (Beckman DU 530 UV/Vis) at 665.1, 649.1 and 480 nm and quantified using Wellburn's equations (1994).
Aqueous-acetone extraction.The solvent (80% acetone) was prepared immediately before analysis using acetone Chromasolv® HPLC 99.8% (Sigma-Aldrich) and deionized water, both previously stored overnight at 3-4ºC.Three foliar discs per leaf (5 mm diameter) were ground in a 2 mL eppendorf tube with 1 mL of aqueous-acetone (Techtnica Millmix 20).The grinding time was adapted to the grade of foliar cutinisation and leaf thickness that increases during the leaf aging process.Samples were transferred to a 10 mL reagent glass followed by washing the Eppendorf tube three times with 1 mL aqueous-acetone and completing to a final volume of 8 mL.Pigments were extracted for 2 h in a cooled isolated black box and centrifuged at 3000 rpm (Tables S1 and S2 [suppl]).Absorbance was immediately measured at 663.2, 646.8 and 470 nm and pigment concentration calculated by Wellburn's equations (1994).
A fourth measurement at 750 nm was performed in both methods as an extract turbidity control.Comparative extractions in the same leaf with DMSO and aqueous-acetone were performed in the current and one year old leaves.For this comparison, the extractionsurface/solvent-volume ratio was equally maintained in both methods.Half of each leaf side was extracted with one of the two solvents.Ten leaves per solvent type and age were analysed.

Results and discussion
Pigment concentrations analysed in the current year leaves using DMSO solvent were in the range of 70-190 µg/cm 2 and exhibited low Chla/Chlb ratios (0.8 to 2.3).The SPAD regression performed with the DMSO method produced a significant correlation with the Chla concentrations analysed (R 2 =0.64,RMSE=0.20,p<0.0001) (Fig. 1A).However the same regression with Chlb concentrations was much weaker (R 2 =0.33;RMSE=0.23;p<0.0001) (Fig. 1B).Gonsiorkiewicz Rigon et al. (2012) found a similar dispersion only for Chlb calibration by DMSO extractions using a ClorofiLOG®-1030.We measured high and variable absorptions readings at 750 nm that should be close to zero in all the DMSO extracts.A strong significant relation between the absorption at 750 nm and Chlb was found (R 2 =0.83;RMSE=0.007;p<0.0001) (Fig. 1D).The ratio between Chla/Chlb decreased significantly as absorption at 750 nm increased (R 2 = 0.57; RMSE=0.01;p<0.0001) (Fig. 1E).Likewise, a relationship between absorption at 750 nm and extract weight was observed (R 2 =0.38;RMSE=0.02;p<0.0001) (Fig. 1F).DMSO seemed to co-extract compounds that absorb especially in the 649.1 nm peak used for Chl b quantification, increasing 2-3 fold its value and narrowing the Chl a /Chl b ratio.
Extraction with DMSO increased significantly (20%) the Chla concentration in the younger leaves with lower pigment concentration and in 5% in the previous year leaves compared with the aqueous-acetone (Table 1).The differences were much greater and significant, in the case of Chlb concentrations with an increase of 115% and 110% in the DMSO extracts in current and previous year leaves respectively compared with the aqueous-acetone.Minocha et al. (2009) detected difficulties in using DMSO for pigment quantification in tree leaves when compared with other solvents due to brown colorations.They attributed the colour interference to the tannins and phenolic compounds co-extracted by this solvent.We detected the same brown colorations only in the DMSO extractions (Figs. S1 and S2 [suppl]).However phenolics and tannins should be present in both extracts, since DMSO and aqueous-acetone are able to dissolve them (Dai & Mumper, 2010;Alves et al., 2013) and their absorption maxima in the UV was distant from the orange-red spectral region (Fig. 2A).
Phenolic compounds are easily oxidized and degraded at high temperatures and during long extraction times (Dai & Mumper, 2010) forming stable polymerized brown coloured anthocyanintannin complexes (Giusti & Wrolstad, 2001).The main spectral differences between the fresh pigment extracts obtained with DMSO and acetone from the same Q.ilex leaf are located in the 500-650 nm region (Fig. 2A).Brownish DMSO extracts showed higher absorbance in this region overlapping the absorption at 649.1 nm and leading to a Chl b overestimation.Subtraction of absorbance at 750 nm (Table 1) did not achieve reliable Chl a / Chl b ratios because the absorbance addition effect in the red region was not constant and higher as in the IR region (Fig. 2A).The oxidation process increased notably three days after extraction only in the DMSO extracts (Fig. 2A).Under the stronger oxidizing effect of DMSO (Clark et al., 2008), enhanced by the higher temperature and longer extraction time of this procedure, the coextracted phenolic compounds are easily oxidized producing the brown colorations that interfere during the photometric measurements.
In the alternative extraction with aqueousacetone, absorption at 750 nm was negligible.Chl a /Chl b ratios obtained for current and previous year leaves were in the range of 2.5-4.4A B characteristics of sun-exposed leaves (Lichtenthaler, 1987).Chls concentration during the vegetative period varied in the range 18-119 µg/cm 2 (Table S3 [suppl]).High SPAD-pigment calibration correlations (µg/cm 2 ) were obtained for all pigments: R 2 =0.90, RMSE=0.16,p<0.0001 for Chl a ; R 2 =0.91,RMSE=0.16,p<0.0001 for Chl b and R 2 =0.90, RMSE=0.02,p<0.0001 for carotenoids (N=167).The calibration equations obtained for the sum of Chl a+b and carotenoids in µg/cm 2 were: Chl a+b =exp(1.8244+0.0408*Spad)and C x+c =(0.2907-0.0039*Spad) - .Filtered tissue powder after extraction was completely decoloured (Fig. S1 [suppl]).The weaker oxidizing properties of aqueous-acetone, the low temperature and the shorter extraction time prevented the oxidation of co-extracted polyphenolic compounds when this extraction method was used.DMSO extracts of several perennial forest and shrub species were additionally analysed to confirm our results (Fig. 2B).All species had absorbance at 750 nm under 0.010 with the exception of Q. ilex that exhibited the maximum values (0.050).Furthermore, the ratio Chl a / Chl b was under 2 only in the Q. ilex samples.The differences in foliar phenolic concentration between species (Hermes & Hedges, 2004) could explain the differences in the rapid development of colored interferences under the same oxidizing conditions.Analysis of total polyphenols using the Folin-Ciocalteu procedure in forest and shrub species extracted by DMSO were performed (mg gallic acid (GA) per gram dry leaf tissue).The Q. ilex leaves had the highest total polyphenol contents of all samples analyzed: 18.8 mg/g GA while all other leaf species were in the range 2.4 mg/g GA (Laurus nobilis) to 7.2 mg/g GA (Arbutus unedo).The development of colored interferences in photometric pigment determinations by DMSO could be also possible in leaves of other forest species with high polyphenol contents.
As conclusions, the use of the DMSO solvent should be avoided for photometric pigment analysis in Quercus ilex leaves.Cold extraction of finely homogenized foliar material with aqueous-acetone, free of interferences is recommended as we have proved that it provides more reliable pigment concentrations.

Figure 1 .
Figure 1.SPAD values versus chlorophyll a (A) and b (B) concentrations in µg/cm 2 .Chlorophyll a (C), chlorophyll b (D) concentrations (µg/mL), Chl a /Chl b ratio (E) and fresh extraction weight (F) vs absorption readings at 750 nm.All extractions for pigment quantification performed with the DMSO procedure (N=82).

Figure 2 .
Figure 2. (A) VIS spectra of two pigment extracts of the same Quercus ilex leaf performed by the two extraction procedures: 4 h extraction of a 6 mm foliar disc in 5 mL DMSO at 65ºC (continuous red line); 2 h extraction of a 6 mm foliar disc in 5 mL of 80% acetone at 3ºC (continuous blue line).Spectrum of the same extracts 3 days after extraction (red and blue doted lines); DMSO extract preserved in the dark at room temperature (13-15ºC) and aqueous-acetone extract preserved at -20ºC.Tannin acid VIS-spectrum in DMSO (continuous green line).(B) Absorption readings at 750 nm of DMSO extracts (4 h extraction at 65ºC) in leaves of different forest and shrub species (Laurus nobilis, Buddleja davidii, Arbutus unedo, Magnolia grandiflora, Quercus agrifolia and Quercus ilex).Nine leaves extracted per species.

4 Table 1 .
Average ± standard deviation of pigment concentration in current and previous year leaves.Same leaf extracted with acetone and DMSO simultaneously.N=10 for each age class and solvent type.Results from ANOVA test, different letters in the same column indicate significant differences (α<0.01).Absorbance at 665.1, 649.1 and 480 nm subtracted by the 750 nm absorbance readings