Faculty of Science and Technology

Environmental Agrobiology

Improving The DMSO Method For Measuring Chlorophyll

Content In Natural Tree Canopies

By: Melissa Llerena Zambrano

Submitted to the Plant Ecophysiology and Contamination Research Group in partial

fulfillment of the requirements for the degree of Master of Environmental Agrobiology

Supervisor: Jose Ignacio García Plazaloa

Co-supervisor: Marina López Pozo

A thesis presented in Spanish.

September-2023

Leioa-Spain
 Improving The DMSO Method For Measuring Chlorophyll Content In
Natural Tree Canopies

Abstract

Active chlorophyll extraction methods using acetone or methanol as extractants are

expensive, time-consuming, and could represent a danger to human health. The aim of this
thesis was to improve the passive and non-toxic method for extracting chlorophyll content in
tree leaves using Dimethyl-sulfoxide (DMSO) as an extractant. First, we compared the
effectiveness of different temperatures (20°C and 75°C) in extracting chlorophyll using DMSO
as an extractant in soft and tough leaves. We also examined the feasibility of using DMSO
for chlorophyll extraction across 47 species from the Basque Country University (UPV/EHU)
Arboretum. Additionally, we compared the chlorophyll content of sunlit leaves from the basal
and top canopy in UPV/EHU Arboretum and compared our results in a natural beech (Fagus
sylvatica) forest. Our results showed that in higher temperatures (75°C) chlorophyll extraction
was completed in one hour, compared to room temperature (20°C). Furthermore, our
investigation revealed no significant differences in chlorophyll content between leaves from
basal canopy and top canopy in both the UPV/EHU Arboretum and Monte Santiago Natural
Monument, as they experience similar sun exposure levels. Since pigment concentrations
between basal canopy and top canopy leaves are similar, our approach provides an easily
applicable tool for chlorophyll quantification, even at the school level. However, it's important
to consider that the method may not be effective for all species, Therefore, preliminary testing
is necessary before proceeding with the extraction.

Keywords: Chlorophyll, Dimethyl-sulfoxide (DMSO), high precision liquid chromatography

(HPLC), passive chlorophyll extraction.
Introduction

Photosynthetic pigments and their importance in plant physiology

In photosynthetic organisms, chlorophylls constitute the essential pigments responsible of

harvesting light energy transforming it into chemical energy through photosynthesis (Stirbet
et al., 2020). Located in chloroplasts, chlorophylls are green-colored pigments with two forms
in plants, chlorophyll a and b, that typically occur in a ratio (a/b) of approximately 3 to 1.5 and
constitute a fundamental parameter of both photosynthesis and the physiological status of
plants. Chlorophyll a, being the predominant pigment plays a central role in photosynthesis,
while chlorophyll b acts as an accessory pigment, enhancing light absorption. (Lichtenthaler,
1987).
The green color of chlorophyll and its hues are mainly attributed to the structural differences
between chlorophylls a and b and their ability to absorb light in the red and blue regions of
the visible spectrum (Roca & Pérez-Gálvez, 2021). Chlorophyll a absorbs energy more
efficiently in blue-violet (~430 nm) and red regions (~662 nm), while chlorophyll b effectively
absorbs light in the blue (~453) and red-orange (~642) regions of the visible spectrum (Figure
1). These distinctions in absorption wavelengths are the main reasons why chlorophyll a has
a bluish-green color while chlorophyll b has a yellow-green color, which contributes to the
distinct characteristic hues of plants (Gross, 2012; Zhang et al., 2022).
Carotenoids, on the other hand, are primarily yellow, red, and orange pigments (occurring in
less concentration than chlorophylls) chemically derived from isoprenoids (Pogson & Rissler,
2000; Stahl & Sies, 2005). Divided into two main categories: oxygenated xanthophylls
(neoxanthin, lutein, zeaxanthin, and violaxanthin) and hydrocarbon carotenes (α-carotene, β-
carotene, and lycopene), they are considered essential metabolites due to their role in
photosynthesis, photoprotection, and key metabolic pathways (Gross, 2012; Zielewicz et al.,
2020).
When photosynthesis occurs, carotenoids transfer harvested light energy to chlorophylls
inside the chloroplasts (Demmig-Adams et al., 1996). Furthermore, they protect the
photosynthesis apparatus from oxidative damage caused for reactive oxygen species (ROS)
inevitably produced by photosynthesis (McElroy & Kopsell, 2009; Pogson & Rissler, 2000).
All this through processes such as quenching, active non-photochemical quenching, and heat
dissipation, activated when plants are under excessive light exposure conditions (Demmig-
Adams et al., 1996; Pogson & Rissler, 2000; Wurtzel, 2019; Yabuzaki, 2017).
In situations where plants are exposed to high light levels, oxygenated xanthophylls initiate
the xanthophyll cycle (Demmig-Adams et al., 1996; Gross, 2012; Roca & Pérez-Gálvez,
2021). This photoprotection mechanism involves the reversible de-epoxidation of
violaxanthin to zeaxanthin. In contrast, when light levels are reduced zeaxanthin is reversibly
converted to violaxanthin, which potentially functions as a light-harvesting antennae pigment
(Pogson & Rissler, 2000; Sandmann, 2001).

Chlorophyll extraction and quantification methods

Accurate quantification of photosynthetic pigments and carotenoids allows researchers to

assess the efficiency of photosynthesis and gain deeper knowledge of plants response to
environmental changes and stressors (Gross, 2012; McElroy & Kopsell, 2009; Roca & Pérez-
Gálvez, 2021; Stahl & Sies, 2005). However, most of the methodologies used to extract and
quantify pigments are expensive, time-consuming, or involve toxic reagents. Among these,
methanol (Arnon, 1949), and acetone (López-Pozo et al., 2019; Sánchez-Moreiras &
Reigosa, 2018) are the most traditionally used for this purpose.

Using acetone or methanol as chlorophyll extractants is classified as an active extraction

process in which leaf tissue needs to be homogenized with a mill in order to get the highest
pigment content (Arnon, 1949; Sánchez-Moreiras & Reigosa, 2018). This is why they are
considered the most traditional techniques for chlorophyll extraction, as they yield the highest
pigment extraction efficiency. However, these steps are noticeably time-consuming when
sample numbers are large, and extractants may cause potential harm during extraction as
they are toxic to human health.

For instance, methanol is a highly toxic alcohol, and its toxicity may occur via ingestion,
dermal absorption, or inhalation. According to Tephly (1991), exposure to methanol can
cause central nervous system depression, digestive system failure, blindness, and even
death in extreme cases. In comparison, acetone is less toxic, but it can still be harmful.
Excessive exposure to this solvent can cause skin, eyes, and respiratory tract irritation,
dizziness, headache, and nausea (Umeh et al., 2021).
Alternatively, extracting chlorophyll using dimethyl sulfoxide (DMSO) is considered a passive
extraction. DMSO efficiently penetrates leave cell membranes, accessing the interior of plant
cells to extract chlorophyll pigments. DMSO toxicity is lower than methanol as it is not
metabolically converted into harmful compounds (Ritchie et al., 2021). Moreover, is not very
volatile and it reaches its flash point at 87°C, which allows DMSO to be subjected to higher
temperatures (ThermoFisher Scientific, 2018), the main reason why it has been used by
several authors to extract chlorophyll from higher plants (Hiscox & Israelstam, 1979; Parry et
al., 2014; Wellburn, 1994). Tait & Hik (2003) found that chlorophyll extraction with DMSO
involves fewer steps, and a large number of samples may be prepared and analyzed quickly.

Non-destructive chlorophyll quantification

Other authors (Gitelson & Merzlyak, 1997; Parry et al., 2014; Percival et al., 1997) , have explored non-
destructive methodologies to evaluate pigment concentrations and the physiological status
in higher plants by using indices associated with spectral reflectance and fluorescence. Leaf
spectral reflectance is the percentage of incident light reflected from its surface and is mainly
determined by pigment concentrations (Percival et al., 1997), while chlorophyll fluorescence
is an indicator of the fate of excitation energy in the leaf photosynthetic apparatus and
provides a rapid diagnostic system for quantifying physiologic injury in higher plants in the
field.

Objectives

General objective

In this sense, the main purpose of our study was to develop a simple, straightforward, and
non-toxic protocol for the estimation of chlorophyll content in natural tree canopies that can
be used by non-trained users in wide-field campaigns.

In addition, this study aimed to test whether the pigment composition of the non-easily
accessible upper canopy can be estimated from measurements performed at the canopy
bottom.
Specific objectives
 To determine the effectiveness of temperature (20°C and 75°C) in extracting
chlorophyll from different plant tissues using DMSO as an extractant and to
compare the extraction yield and quantification of total chlorophyll by
spectrophotometry with chlorophyll quantification using HPLC.
 To test the feasibility of chlorophyll extraction using DMSO across various
species.
 To determine the differences in total chlorophyll content from top canopy and
basal canopy leaves using an HPLC method extraction method using acetone
as an extractant.

Hypothesis

High-temperature conditions will promote a faster and more comprehensive extraction of

chlorophyll, which will result on extraction yields similar to those obtained with the HPLC
technique.

In addition to the methodological developments mentioned above, we tested whether pigment

composition of the upper forest canopy can be estimated from measurements performed in
sunlit leaves from the basal crown. In this sense, we expect that the total chlorophyll extracted
from the top canopy leaves will be similar to that extracted from the sunlit basal canopy due
to their shared exposure to direct sunlight. This main hypothesis was tested first in a
representation of tree species growing in the arboretum of the UPV/EHU campus, and later
in a natural beech forest in Monte Santiago Natural Monument taking advantage of the use
of a truck-mounted aerial platform.

Materials and methods

Sampling sites.
Leaves from two different locations were collected. The Basque Country University
Arboretum (UPV/EHU Arboretum, from now on), located on the south-southeast slope of the
Leioa Campus, was the first study site. This educational park spans 13 ha of 2200 trees and
shrubs from 420 species and varieties. These plants belong to a botanical collection that
represents the flora of the five continents, reflecting Mediterranean, North American,
Southern Hemisphere, Asian, and native (Basque Country) plant species diversity. This study
site is characterized by mild winters and occasional cold waves, a maximum temperature
fluctuation of 11ºC, an average temperature of 14ºC, rainfall ranging between 1000-1200
mm, and prevailing moist winds from the northwest (Ayuntamiento de Leioa, 2023).

The second study site, Monte Santiago Natural Monument, is located in the northeastern part
of the Burgos province within the autonomous community of Castilla y León, Spain. Situated
at 900 m above sea level (a.s.l), the main habitat of the study area is a natural beech (Fagus
sylvatica) forest on a calcareous substrate. With a mean annual rainfall of 1116 mm and
mean temperatures ranging from 16 °C in August to 2.7 °C in February this area has a typical
temperate oceanic climate (Fernández-Marín et al., 2015).

Monte Santiago Natural Monument is characterized by diverse vegetation composition. In the

middle and lower slopes, populations of evergreen oak forests can be found, while the higher
areas are dominated by beech and coniferous forests (Red Natura 2000, 2022).

Experiment 1.- Efficiency of temperature on extracting chlorophyll from different leaf

tissue using DMSO as a solvent.
To determine the optimal time and temperature for extracting total chlorophyll from different
plant tissues using DMSO as a solvent, fully expanded leaves of two plant species: holm oak
(Quercus ilex) and dandelion (Taraxacum officinale) were collected from the UPV/EHU
Arboretum. The mentioned species were chosen as representatives of two extremes of leaf
toughness, showing clear differences in terms of their structure and leaf morphology; While
Quercus ilex consists of coriaceous, hard, and not very flexible leaves, Taraxacum officinale
is located at the other extreme, showing membranous or papery leaves, weak and with little
cuticle. The study and comparison of these two types of leaves allow the optimization of the
method for any kind of species, regardless of the hardness of the leaf.

Chlorophyll extracts were prepared by cutting leaf tissue (two discs of 3mm of diameter) in
half and placed in 2 mL Eppendorf microtubes with 1 mL DMSO. Three replicates from specie
(n=3) were then incubated for 8 hours in darkness at 75°C in an oven and at room
temperature (20°C). The spectrophotometric analysis of chlorophylls was conducted using a
Shimadzu Europe – UV spectrophotometer mini-1240 (wavelength precision = 0.1 nm) at 665
and 649 wavelengths every hour (8 times) using 1.5 mL Polymethyl Methacrylate (PMMA)
cuvettes, calibrated against a blank containing pure DMSO. Turbidity was checked at 750
nm.
Chlorophyll a+b content (total chlorophyll henceforth) was calculated using Wellburn’s (1994)
equations for a low-resolution spectrophotometer. For DMSO, Chla = 12.19A665–3.45A649 and
Chlb = 21.99A649–5.32A665. Chlorophyll content was expressed in micromoles per leaf area
(µmol/m2).

Additionally, chlorophyll was extracted from Quercus ilex leaves using 100% acetone and
then analyzed by HPLC method to determine their concentration. This approach provided a
reference value for comparison with the concentration obtained through DMSO extraction.

Chlorophyll extracts were obtained as follows. Two discs of 3mm of plant tissue per replicate
(n=3) were milled with liquid nitrogen in 2 Eppendorf microtubes, 0.5 mL of 100% acetone
buffered with CaCO3 was added and centrifuged at 15000 g for 10 minutes. After
centrifugation, the supernatant was carefully separated from the pellet. An additional 0.5 mL
of 100 % acetone was added to ensure complete chlorophyll extraction, and the mixture was
centrifuged again at 15000 g for an extra time of 10 minutes. Polytetrafluoroethylene filters
(0.2 µm) were used to filter both supernatants before high-performance liquid
chromatography (HPLC) was performed.

Following the method of García-Plazaola & Becerril (1999) 15 µl of the extracts were injected
into a reverse-phase Waters (Milford, MA, USA) HPLC system. Photosynthetic pigments
were measured using a Photodiode array detector (Waters model 996) in the range of 250–
700 nm and peaks were detected and integrated at 445 nm for photosynthetic pigments
content. Finally, retention times and conversion factors for pigments were consistent with
García-Plazaola and Becerril's reports from 1999 and (2001).

Experiment 2.- Potential interference in chlorophyll extracts using DMSO.

To test for the chemical stability of chlorophylls during the extraction with DMSO and potential
interference with plant metabolites, a set of 47 species (all of them angiosperms) from the
UPV/EHU Arboretum were analyzed. For each species, three fully expanded leaves were
collected, and two discs of 3mm were cut in half and placed in 2 mL Eppendorf microtubes
with 1 mL DMSO to prepare chlorophyll extracts. In accordance with the Experiment 1
conditions, samples were maintained at 75°C in darkness for one hour during incubation.

Chlorophyll content was measured spectrophotometrically as described in experiment 1.

Experiment 3.- Differences in total top canopy and basal canopy in UPV/EHU
Arboretum.
Subsequently, we selected five out of the fifty species previously analyzed (those in which
chlorophyll did not show any sign of interference or degradation) to assess the differences in
total chlorophyll content in top canopy and basal canopy of the forest. The chosen species
include Cydonia oblonga, Diospyros kaki, Fagus sylvatica, Quercus ilex, and Rhododendron
arboreum.

As replicates from each canopy layer, we selected three individuals with ≤5 m in height for
each species, and a sample of five fully expanded, sunlit leaves was taken per each canopy
layer (n=15). All collected leaves had a south orientation position, ensuring they were
exposed to direct sunlight and leaves had the least amount of herbivory or other damage
(fungal attack, necrosis, or diseases).

Leaf tissue (two discs of 3mm of diameter) was cut in half and placed in 2 mL Eppendorf
microtubes with 1 mL DMSO to prepare chlorophyll extracts. Ten replicates per individual,
five per canopy layer, were then incubated in darkness at 75°C for 1 hour and total chlorophyll
content was measured spectrophotometrically as described in experiment 1.

Experiment 4.- Chlorophyll content variation across canopy layers in a natural forest
Furthermore, to test in a natural forest whether the approach of using leaves from the basal
canopy as equivalents to the top canopy was effective, a truck-mounted aerial platform was
used to collect fully expanded sun leaves from top (15 m) and basal canopy (2 m), using
shade leaves as a comparison of six adult Fagus sylvatica trees in Monte Santiago Natural
Monument.

Three leaves per canopy layer were collected for each individual, subsequently, four discs of
3mm of leaf tissue were taken from each leave and immediately immersed in liquid nitrogen
to preserve their physiological features and finally stored at -80ºC. Following the protocol
previously detailed in experiment 1, chlorophyll was extracted using acetone and analyzed
by HPLC.

Fluorescence
Chlorophyll fluorescence was measured in leaves from experiment 4 using the Opti-Sciences’
Y(II) meter. All leaves were dark-adapted for at least 30 minutes using dark clips before
minimum chlorophyll fluorescence (F 0) was recorded, and Fm was measured as the maximum
fluorescence induced with a saturating pulse (7000 μmol m -²s-1). The variable fluorescence
(Fv) value was then calculated as Fm-Fo and the photochemical efficiency of PSII was
calculated as the Fv/Fm.

Spectral analysis methods

Spectral reflectance was measured by using a SpectraPen SP 110 in leaves from

experiments 2 and 3. Normalized Difference Vegetation Index (NDVI) was then calculated
with the formula: NDVI = (NIR - R) / (NIR + R). Where NIR is the Reflectance in the near-
infrared band and R corresponds to the Reflectance in the red band. Photochemical
Reflectance Index (PRI) was calculated as PRI = (R 570 - R531) / (R570 + R531). Where R570 is
the Reflectance in the red edge band (570 nm) and R 531 corresponds to the Reflectance in
the green band (531 nm).

Leaf morphology
Leaf mass per area (LMA) was determined in leaves from experiments 3 and 4. Four discs
of 4mm diameter of leaf tissue were taken and oven-dried at 70°C for 72 hours until. Dry
weight was obtained using a high-precision analytical balance (± 0.002, OHAUS PA153).
Disc area was determined by digital image analysis using ImageJ software (version 1.50i,
NIH, USA).

Finally, LMA (g cm-2) was calculated as the leaf dry mass (g) divided by its leaf area (cm2).

LMA= dry weight/area

Data analyzes
The effect of temperature (75°C and 20°C) in DMSO chlorophyll extraction was determined
using a student t-test. Additionally, we observed the percentage of chlorophyll extracted
relative to the acetone extraction after an 8-hour incubation period at different temperatures,
using DMSO as extractant.

Subsequently, we categorized the forty-seven analyzed species into two primary groups
based on their chlorophyll a/b ratio values. Species exhibiting a chlorophyll a/b ratio value
lower than 2, as well as those surpassing 5, were identified as candidates associated with
chlorophyll degradation. Moreover, we considered a phylogenetic tree where all species
collected were grouped following the major groups presented in (Soltis & Soltis, 2004) for
angiosperms.
We also conducted a Kruskall-Wallis test in parameters from experiments 3 and 4.
Homoscedasticity of the data using a Levene test and data normality using a Shapiro–Wilk
test (Sokal and Rohlf 2011). All the analysis was carried out in JMP 17.0 (SAS 2023).

Results

Efficiency of temperature on extracting chlorophyll from different leaf tissue using

DMSO as a solvent
The student t-test showed significant differences in the chlorophyll extraction at both
temperatures, (Figure 1). In tough leaves (Quercus ilex leaves), the total chlorophyll extracted
after 1h of incubation at 75°C equals the amount of chlorophyll extracted with acetone and
analyzed by HPLC, (where extraction efficiency is considered complete) (Figure 1A).
Besides, chlorophyll content remains constant in the following 7 hours of extraction at 75ºC
with DMSO. This value significantly differs from the total chlorophyll extracted at room
temperature (20°C) in the first hour of extraction which accounts for a mere 39% (Figure 1B).
Further, total chlorophyll values at room temperature reach 87,5% only after 8 hours of
incubation.

Figure 1.- Efficiency of temperature in extracting chlorophyll from different leaves tissues. Extracts prepared
from leaves of two different species. A) Quercus ilex and B) Taraxacum officinale. Black lines represent the
standard error for each measurement. T1 represents the total chlorophyll content (µmol m-2) extracted at each
time interval at room temperature (20°C). T2 represents the total chlorophyll content (µmol m-2) extracted at
75°C and, T3 illustrates the maximum chlorophyll value in Quercus ilex leaves extracted using acetone as
extractant and analyzed through the HPLC method. Samples: n=3. Levels of significance for p-values are
indicated as follows: ** (p = 0.01) and *** (p = 0.001).

Conversely, in soft leaves (Taraxacum officinale leaves) 100% of the total chlorophyll content
was extracted at 75°C in the first hour of extraction (Figure 2B). We didn't use acetone to
show 100% of the total chlorophyll content due to the high efficiency of DMSO in extracting
chlorophyll in soft tissues; after one hour of extraction, leaf tissue was totally cleared. In
addition, the student t-test showed that the total chlorophyll extracted at 75°C differs
significantly from the total chlorophyll content extracted at 20°C.

Potential interference in chlorophyll extracts using DMSO.

When classifying the set of 47 species into the main subclasses according to Soltis & Soltis,
(2004) for angiosperms (Figure 2), 81% (38 species) turned out to be eudicots. Within this
group, 74% of the species did not show chlorophyll degradation when using the DMSO
extraction method, the remaining 26% (9 species) were distributed among the magnoliids,
monocots, and other non-eudicot clades, in which 55% of the extracts displayed chlorophyll
degradation (Table S1).

Figure 2. Phylogenetic classification according to (Soltis & Soltis, 2004) for angiosperms of a set of 47
species selected to determine interference in chlorophyll extracts. Green color bars represent all species in
which chlorophyll extracts with DMSO did not show degradation. Bars in red show species in which
chlorophyll extracts showed degradation. Species belonging to each subclass are indicated by the number
next to each bar.

Experiment 3.- Differences in total top canopy and basal canopy in UPV/EHU
Arboretum.
Results from the Kruskal-Wallis test showed that in all species studied in experiment 3 total
chlorophyll content (µmol m-2), NDVI and PRI indices, and LMA (g m-2) between top canopy
and basal canopy exhibited no statistically significant differences in all species studied
(p>0.05) (Figure 3). This absence of divergence in the p-values implies a scenario in which
environmental factors, specifically light, could be uniformly influencing both strata within the
study area.
Figure 3.- Box plots illustrating the distribution of five leaf parameters measured across five species within two
different forest layers: BC (basal canopy) and TC (top canopy) at the UPV/EHU Arboretum. Parameters
analyzed encompass Chl a+b (µmol m -2), NDVI, PRI, and LMA (g m -2). Species analyzed were A) Cydonia
oblonga, B) Diosperos kaki, C) Fagus sylvatica, D) Quercus ilex, and E) Rhodendron arboreum. The horizontal
line inside the box represents the median value for each parameter. Outliers, represented as individual data
points beyond the whiskers, are displayed as circles. Additionally, letters on top of the whiskers denote
significant differences between groups.

Experiment 4.- Chlorophyll content variation across three canopy layers in a natural
forest.
Based on the p-values of the Kruskal-Wallis there were no significant differences in Chl a+b
(Figure 4A), Neo/Chl a+b (Figure 4C), Viola/Chl a+b (Figure 4D), and Fv/Fm (Figure 4O)
across all canopy layers studied (p>0.05). However, significant differences were found in
Lut/Chl a+b (Figure 5G), Zea/Chl a+b (Figure 5H), α-carot/Chl a+b (Figure 5I), β-carot/Chl
a+b (Figure 5J), Total-carot/Chl a+b (Figure 5K), Vaz/Chl a+b (Figure 5L), PRI (Figure 5N),
and LMA (Figure 5P) for shade leaves, while in basal canopy and top canopy there were no
significant differences.
Figure 4.- Box plots illustrating the distribution of sixteen leaf parameters measured within three different forest
layers: BC (basal canopy), TC (top canopy), and S (Shadow layer) in beech (Fagus sylvatica) collected at Monte
Santiago Natural Monument. Parameters analyzed involve: A) Chl a+b (mmol/m 2), B) Chl a/b (mmol/m 2), C)
Neo/Chl (a+b), D) Violax/Chl (a+b), E) Lx/Chl (a+b), F) Anterax/Chl (a+b), G) Lut/Chl (a+b), H) Zeax/Chl (a+b),
I) α-carot/Chl (a+b), J) β-carot/Chl (a+b), K) Total_carot (a+b), L)Vaz/Chl (a+b), M) NDVI, N) PRI, O) Fv/Fm,
and P) LMA (g/m2). The horizontal line inside the box represents the median value for each parameter. Outliers,
represented as individual data points beyond the whiskers, are displayed as circles. Additionally, letters on top
of the whiskers denote significant differences between groups.

Discussion

In our study, we assessed the effectiveness of temperature and DMSO in extracting

chlorophyll passively from different plant tissues. By incorporating two plant species with
contrasting leaf textures, we sought to examine the effectiveness of DMSO in extracting
chlorophyll even from tough sclerophyllous leaves (Quercus ilex). It is worth noting that after
chlorophyll extraction using DMSO, foliar tissue of Taraxacum officinale was completely
cleared, in contrast to Quercus ilex which maintained its green coloration, highlighting the
challenging nature of chlorophyll extraction from these tough tissues. To address this, we
compared the yield of passive chlorophyll extraction with DMSO with a traditional active
chlorophyll extraction with 100% acetone as extractant for Quercus ilex leaves and excluding
Taraxacum officinale leaves from this last analysis.

We expected that higher temperatures (75°C) would accelerate the chlorophyll extraction
process using DMSO as an extractant. Consistent with our first hypothesis, chlorophyll
extraction at 75°C was complete after one hour of incubation with DMSO for Quercus ilex
and Taraxacum officinale tissues. In contrast, chlorophyll extraction at room temperature
(20°C) for Quercus ilex leaves was completed only 8 hours after incubation. Additionally, the
total chlorophyll extracted passively at 75°C with DMSO for one hour was similar to the total
chlorophyll extracted after homogenization using acetone and analyzed by the HPLC method.
These results agree with previous studies that established temperatures below 40°C as
suboptimal for chlorophyll extraction from tough, coriaceous leaves (Tait & Hik, 2003).

To assess the effectiveness of our method to extract chlorophyll in various species we

classified our set of 47 species from UPV/EHU Arboretum according to Soltis and Soltis
(2004). Of the 47 chlorophyll, 38 species (81%) were identified as eudicots, within this group
more than half 50% exhibit interference in the chlorophyll extracts when subjected to the
DMSO extraction at higher temperatures (75°C). Furthermore, the remaining 20% of species,
belonging to various non-eudicot subclasses including magnoliids and monocots, showed the
same trend (Chlorophyll a/b values less than 2 or more than 5 and yellowish extracts). This
finding suggests that chlorophyll degradation or interference with other metabolites is
independent of phylogeny. Pennington et al. (1964) have demonstrated that in prolonged
exposure to temperatures above 37°C chlorophylls a and b are converted to pheophytin a
and b, which leads to chlorophyll degradation; thus, chlorophyll extracts could change color
from bright green to brown-yellow.

We expected that sunlight leaves from the top canopy and leaves from the basal canopy of
the UPV/EHU Arboretum exhibit similar profiles concerning photosynthetic pigments
(Chlorophyll a+b (µmol m-2)), plant health (NDVI and PRI indices), and structure
characteristics (LMA (g m-2)) in all species studied. Consistent with our hypothesis leaves
from both canopy layers exhibit the same physiology showing that sunlight in UPV/EHU
Arboretum is equally distributed in all canopy layers. This finding gives empirical support to
those studies aiming to characterize the pigment composition of tree canopies using sun
leaves as models. This could be of importance for the correct interpretation of the remote
sensing signals retrieved by airborne platforms.

The study conducted by Lichtenthaler et al. (2007) in four species of trees suggests that sunlit
leaves possess sun-type chloroplasts adapted for high rates of photosynthetic quantum
conversion. These sun leaves exhibited a higher photosynthetic capacity on a leaf area and
chlorophyll basis, along with higher values for the Chl a/b ratio than shade leaves with their
shade-type chloroplasts. Specifically, Fagus sylvatica showed a great capacity to adjust the
biochemical and physiological traits through the entire canopy.

We corroborated these results with the findings in the natural beech (Fagus sylvatica) we
found that leaves from the basal and top canopies exhibited similar concentrations of
photosynthetic pigments, while shade leaves displayed comparable total chlorophyll
concentrations. However, significant disparities were noted in the Chl a/b ratio, and certain
carotenoid concentrations were observed in shaded leaves. This finding confirms that light
environment is the main, if not the only factor, determining pigment composition in leaves
(Niinemets et al., 2003), being in comparison tree height negligible.

According to Demmig-Adams & Adams III (1992), the concentration of carotenoids is higher
in sunlit leaves than in shade. Particularly, carotenoids related to the xanthophyll cycle, which
serve a photoprotective function in plants when exposed to varying light intensities.

In our study, we observed that the total carotenoid concentration, β-carotene, carotenoids
associated with the xanthophyll cycle (VAZ/Chl), zeaxanthin, and lutein, are significantly
lower in shade leaves compared to sunlit leaves. These carotenoids are involved in
dissipating luminous energy as heat when light intensity is high, and therefore their
concentration is reduced in shade leaves where light availability is reduced, and there is less
demand for protection against excessive light (Gitelson et al., 2006; Lichtenthaler et al.,
2007).

According to Filella et al., (2009), The Photochemical Reflectance Index (PRI) is correlated
with xanthophyll interconversion and chlorophyll content. Thus, we expected that PRI values
in shaded leaves would be lower than those in sunlit leaves, reflecting the reduced levels of
chlorophyll and carotenoids observed. However, our findings showed significantly higher
values in shaded leaves. In low-light conditions, plants may undergo pigment redistribution
to enhance light capture in wavelengths more conducive to photosynthesis, enhancing light
use efficiency.

Lastly, we evaluated Leaf Mass Area (LMA) as a leaf functional trait linked to leaf morphology
and density differences between sunlit and shaded leaves. In Fagus sylvatica, it has been
reported that sun leaves are thicker (144 to 185 µm) than shade leaves (88-93 µm)
(Lichtenthaler et al., 2007). This morphological trait is typically expected to decrease in sunlit
leaves compared to shaded leaves due to increasing irradiance together with leave thickness
(Hölscher, 2004). Surprisingly, in our study, we found lower values of LMA was found in
shadow leaves.

Our study offers a simple, analytical, and logistic protocol to determine plant physiology that
opens doors to a world of possibilities in interpreting remote sensing signals. This approach
can be extended to large-scale geographic studies and, even more importantly, involve civil
society or students in data collection and understanding of our environment. By doing so, we
not only advance the science of remote sensing but also promote active community
participation in understanding our planet.

Conclusions

Based on our findings, extracting chlorophyll at 75°C using DMSO as extractant is highly
efficient, requiring only one hour to extract all pigments from both sclerophyllus (Quercus ilex)
and soft leaves (Taraxacum officinale). In this sense our study offers an improved approach
to extract chlorophyll pigments from tree leaves that could be used by anyone interested in
extracting photosynthetic pigments from leaves, including students, due to its simplicity and
safety. However, it is important to understand that the method is not universally applicable
for all species as certain species may contain additional metabolites that could potentially
interfere with the quality of the extracts and, consequently, affect chlorophyll quantification.

Furthermore, we have demonstrated that leaves from bottom canopy and top canopy are
similar in photosynthetic pigments (Chlorophyll a+b (µmol m-2)), plant health (NDVI and PRI
indices), structure characteristics (LMA (g m-2), and carotenoids content, as long as the
collected leaves are those that are exposed to direct sunlight.

With these results, our study contributes to an advancement to ecophysiology studies, since
basal leaves have similar physiology as canopy leaves the need for cranes or special
equipment to reach higher leaves is eliminated. This, in turn, could lead to cost reductions in
the field leaf collection process.
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Supplementary material

Table S1. Mean values and standard deviations of Chlorophyll a/b ratio in all 47 species
analyzed in experiment 3.

Leaves Chl a/b (µmol

CLADE Specie_Name
(n) m-2)
Commelinids Phyllostachys vivax 3 1.752±0.904
Eurasterids I Fraxinus excelsior 3 1.734±0.096
Eurasterids I Ehretia rigida 3 2.685±1.03
Eurasterids I Fraxinus angustifolia 3 2.779±0.657
Eurasterids I Forsythia x. intermedia 3 2.904±0.598
Eurasterids I Olea europea 3 2.914±1.014
Eurasterids I Paulownia tomentosa 3 4.056±0.23
Eurasterids II Arbutus unedo 3 0.704±0.402
Eurasterids II Vivurnum tinus 3 2.266±0.086
Eurasterids II Rhododendrum arboreum 3 2.765±0.335
Eurasterids II Taraxacum officinale 3 3.668±1.065
Eurosids I Alnus glutinosa 3 1.478±0.095
Eurosids I Crataegus monogyna 3 1.773±0.338
Eurosids I Juglans regia 3 1.832±0.378
Eurosids I Salix atrocinerea 3 2.069±1.558
Eurosids I Ceratonia siliqua 3 2.122±0.495
Eurosids I Quercus myrsinifolia 3 2.549±0.76
Eurosids I Photinia fraseri 3 2.606±0.391
Eurosids I Quercus ithaburensis 3 2.964±0.513
Eurosids I Carpinus caroliana 3 3.038±1.084
Eurosids I Quercus rubur 3 3.161±0.881
Eurosids I Diospyros kaki 3 3.34±1.054
Eurosids I Quercus ilex 3 3.476±0.68
Eurosids I Castanea sativa 3 3.543±0.199
Eurosids I Quercus castaneiforlia 3 3.701±0.73
Eurosids I Quercus suber 3 3.948±0.26
Eurosids I Celtis australis 3 4.303±1.819
Eurosids I Ficus carica 3 4.326±1.008
Eurosids I Cydonia oblonga 3 4.416±0.213
Eurosids I Betula pendula 3 5.168±0.88
Eurosids I Pyrus calleryana 3 5.492±2.487
Eurosids I Prunus acium 3 6.318±0.691
Eurosids I Zelkova serrata 3 6.636±3.884
Eurosids II Metrosideros excelsa 3 1.731±0.107
Eurosids II Eucalypstus globulus 3 2.023±0.282
Eurosids II Brachychiton populneus 3 2.181±0.279
Eurosids II Pistacia lentiscus 3 2.548±0.427
Eurosids II Acer rubrum 3 2.652±0.231
Eurosids II Callistemon viminalis 3 2.732±1.364
Magnoliidae Laurus nobilis 3 2.25±0.185
Magnoliidae Magnolia lilliflora 3 2.736±0.372
Magnoliidae Magnolia kobus 3 9.316±1.733
other clade Liquindambar stryraciflua 3 1.211±0.165
other clade Banksia serrata 3 1.854±0.325
other clade Parrotia persica 3 2.223±1.013
other clade Venus protea 3 2.258±0.31
other clade Leucadendron argenteum 3 2.88±0.798