Tree Physiology 00, 1–18
https://doi.org/10.1093/treephys/tpad010
Research paper
Red pigments in autumn leaves of Norway maple do not offer
significant photoprotection but coincide with stress symptoms
Heta Mattila 1,2 and Esa Tyystjärvi 1,3
1Department of Life Technologies/Molecular Plant Biology, University of Turku, 20014 Turku, Finland; 2Present address: Centre for Environmental and Marine Studies
(CESAM), Department of Biology, University of Aveiro, Portugal; 3Corresponding author (esatyy@utu.fi)
Received June 10, 2022; accepted January 25, 2023; handling Editor Molly Cavaleri
The reasons behind autumn colors, a striking manifestation of anthocyanin synthesis in plants, are poorly understood.
Usually, not all leaves of an anthocyanic plant turn red or only a part of the leaf blade turns red. In the present
study, we compared green, red and yellow sections of senescing Norway maple leaves, asking if red pigments offer
photoprotection, and if so, whether the protection benefits the senescing tree. Green and senescing maple leaves were
illuminated with strong white, green or red light in the absence or presence of lincomycin which blocks photosystem II
(PSII) repair. Irrespective of the presence of anthocyanins, senescing leaves showed weaker capacity to repair PSII than
green leaves. Furthermore, the rate of photoinhibition of PSII did not significantly differ between red and yellow sections
of senescing maple leaves. We also followed pigment contents and photosynthetic reactions in individual leaves, from
the end of summer until abscission of the leaf. In maple, red pigments accumulated only during late senescence, but
light reactions stayed active until most of the chlorophyll had been degraded. PSII activity was found to be lower and
non-photochemical quenching higher in red leaf sections, compared with yellow sections of senescing leaves. Red leaf
sections were also thicker. We suggest that the primary function of anthocyanin synthesis is not to protect senescing
leaves from excess light but to dispose of carbohydrates. This would relieve photosynthetic control, allowing the light
reactions to produce energy for nutrient translocation at the last phase of autumn senescence when carbon skeletons
are no longer needed.
Keywords: Acer platanoides, autumnal, chlorophyll fluorescence, deciduous, nutrient resorption.
Introduction
During autumn senescence, deciduous plants degrade pho-
tosynthetic protein complexes and other macromolecules in
senescing leaves, to remobilize nutrients for winter storage. Pro-
tective mechanisms, such as accumulation of tocopherols and
xanthophylls, are often upregulated (e.g., Junker and Ensminger
2016), possibly to enable efficient nutrient resorption while
photosynthesis declines (Krieger-Liszkay et al. 2019, Mattila
et al. 2021). Many plant species synthetize anthocyanins during
autumn, and it has been proposed that anthocyanins protect
senescing leaves from (excess) light (Hoch et al. 2003, Renner
and Zohner 2019).
In non-senescing plants, anthocyanin synthesis is often
upregulated in high light (e.g., Viola et al. 2016), possibly via
chloroplast retrograde signaling (Richter et al. 2020). Also, in
senescing leaves of, e.g., red-osier dogwood (Cornus sericea L.)
and maple (Acer), anthocyanin accumulation further increases
in high light (Feild et al. 2001, Lee et al. 2003, Duan et al.
2014). Although it has often been concluded that anthocyanins
protect photosystem II (PSII) of young or mature leaves from
light-induced damage (photoinhibition) (see the summary of
Gould et al. 2018 and the review of Agati et al. 2021), only a
small number of these studies (Feild et al. 2001, Hoch et al.
2003, Manetas and Buschmann 2011) have been conducted
on senescing autumn leaves. In addition, several investigations
on senescing leaves have failed to find a protective effect
or have found only a minor effect (Lee et al. 2003, van
den Berg and Perkins 2007, van den Berg et al. 2009,
© The Author(s) 2023. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
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2 Mattila and Tyystjärvi
Duan et al. 2014, Lo Piccolo et al. 2018; see also the literature
analysis by Gould et al. 2018). Whether the distribution
of yellow and redsenescing species in northern America
and Europe supports the photoprotection hypothesis is also
disputed (Renner and Zohner 2019, 2020, Pena-Novas and
Archetti, 2020a, 2020b). On the other hand, photoprotection
may not be the (primary) function of anthocyanins (see
Manetas, 2006, Renner and Zohner 2019, Pena-Novas and
Archetti, 2021b). A competing (but not mutually exclusive)
hypothesis, suggesting that anthocyanins are involved in
defense against insect herbivores (the co-evolution hypothesis),
is also commonly used to explain autumn colors (Furuta, 1986;
for a review, see Lev-Yadun and Gould 2007).
Anthocyanins could protect leaves by screening light and/or
by functioning as antioxidants. Anthocyanin accumulation has
been shown to attenuate light in senescing leaves of red-osier
dogwood and Norway maple (Acer platanoides L.) (Feild et al.
2001, Merzlyak et al. 2008). In vitro, anthocyanins are able
to quench superoxide, hydrogen peroxide and singlet oxygen
(Sondheimer and Kertesz 1952, Neill and Gould 2003, De
Rosso et al. 2008). Some studies have suggested that this is the
case also in vivo (e.g., Gould et al. 2002); however, evidence
from senescing leaves is scarce (van den Berg and Perkins
2007). The efficacy of anthocyanins as antioxidants depends
on their localization within leaves (Kytridis and Manetas 2006).
In senescing maple leaves, anthocyanins are mainly localized in
the vacuoles of palisade mesophyll cells (Merzlyak et al. 2008,
Junker and Ensminger 2016) and therefore their impact on
chloroplast-localized reactive oxygen species is expected to be
low. Screening of light by anthocyanins, however, may indirectly
decrease the production of reactive oxygen species (e.g., Zhang
et al. 2016).
Anthocyanins absorb green light efficiently. Indeed, vacuole-
located anthocyanins protected PSII in green but not in red light
and only when the light was shone on the anthocyanic adaxial
(upper) side of beetleweed (Galax urceolata (Poir.) Brummitt)
leaves (Hughes et al. 2005). It is often mentioned that green
light is not well absorbed by chlorophylls and therefore does
not necessarily contribute to ‘excess’ light (Manetas, 2006,
Pena-Novas and Archetti, 2021b). However, the contribution
of green light to photosynthetic processes is not insignificant
(e.g., Nichelmann and Bilger 2017, Hughes et al. 2022).
The extent of PSII photoinhibition is also not (at least not
solely; see Tyystjärvi, 2013) determined by the absorption
profiles of chlorophylls, and green light has a relatively high
capacity to damage PSII (Hakala et al. 2005). In addition, some
anthocyanins absorb blue (or even red) light (e.g., Feild et al.
2001, Merzlyak et al. 2008; see also Agati et al. 2021); blue
light is highly efficient in damaging PSII (Jones and Kok 1966,
Hakala et al. 2005, Zavafer et al. 2015).
The benefit (or lack of it) obtained by protecting senesc-
ing leaves should also be considered. Nutrient resorption
efficiencies greatly vary among species and environmental
conditions; a high resorption efficiency may improve plant
fitness, especially if the nutrient content of the soil is low
(May and Killingbeck 1992; for a review, see Brant and
Chen 2015). It has been suggested that plants suffering from
nitrogen deficiency benefit more from anthocyanins than well
fertilized plants (Hughes et al. 2022). In fact, anthocyanin
accumulation may be a common response to nitrogen limitation
(Schaberg et al. 2003, Hughes et al. 2022). Earlier research
has also shown that high leaf sugar content induces anthocyanin
accumulation in maple and other species (Murakami et al. 2008,
K.M. Zhang et al. 2013, Schaberg et al. 2017). In aspen,
during late phases of senescence, starch content decreases
and glucose and fructose become the dominant soluble sugars
(Keskitalo et al. 2005).
Nutrient resorption efficiencies of anthocyanic and non-
anthocyanic leaves have rarely been compared, and the results
are contradictory (Feild et al. 2001, Hoch et al. 2003, Duan
et al. 2014, Pena-Novas and Archetti, 2021a). Schaberg et al.
(2008) observed that in sugar maple (Acer saccharum Marsh.)
red senescing leaves contained slightly more chlorophyll and
were more firmly attached, due to a less formed abscission
layer, than yellow senescing leaves. The authors suggested
that delayed senescence in red leaves could promote nutrient
resorption. However, the duration of senescence in individual
leaves was not evaluated. On the other hand, Y.J. Zhang et al.
(2013) suggested that in Lyonia ovalifolia Drude, which retains
red senescing leaves for several months during winter, delayed
senescence offers a competitive benefit by increasing carbon
gain.
Some leaves of a senescing Norway maple tree turn red
(mostly due to anthocyanin accumulation) while other leaves
remain yellow. Furthermore, a senescing maple leaf may con-
tain dark green, pale green, yellow and red sections at the
same time. Therefore, maple is an excellent model plant for
studying the roles of anthocyanins in autumn senescence. In
the present study, we addressed the question of whether red
pigments protect senescing leaves and whether such protection
offers a fitness advantage for the senescing tree. To test the
photoprotection hypothesis, green, yellow and red sections of
senescing leaves of Norway maple were exposed to high light;
a pair of red and yellow sections was always obtained from
a single senescing leaf, to minimize differences other than
anthocyanin content. To accurately probe the damaging reaction
of photoinhibition of PSII, we repeated the treatments in the
presence of lincomycin, an antibiotic that inhibits PSII repair,
because in the absence of lincomycin, damage and repair occur
simultaneously (for reviews, see Nath et al. 2013, Tyystjärvi,
2013). To our knowledge, lincomycin has not been previously
tested with senescing leaves. In addition, pigment contents
and photosynthetic reactions were followed in individual leaves
thorough the autumn, until abscission. The question whether
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Red pigments in autumn leaves of Norway maple 3
photoprotection offers fitness advantage was approached by
measurements of leaf thickness and protein content to find
out whether anthocyanins enhance dislocation of valuable sub-
stances from the senescing leaf. Leaf protein content also
correlates well with leaf nitrogen content in senescing leaves
(Yasumura et al. 2007). The protein measurements were done
on aspen leaves because an aspen tree typically has leaves that
turn red and others that remain yellow, which allows testing of
the relationship between redness and transport of nutrients at
a leaf level.
Materials and methods
Plant material
Norway maple (A. platanoides) and European aspen (Populus
tremula L.) leaves were collected during the autumn of 2020
and 2021 from trees growing in small city parks (Turku, Fin-
land). Laboratory measurements were conducted on the same
day as the leaves were collected, if not otherwise mentioned.
The course of senescence in vivo was followed in attached
leaves, in their natural environment. For that reason, four to eight
leaves per tree across four maple trees were selected. All the
trees, growing at 60◦26′31.7′′N 22◦14′38.7′′E, had started to
senesce at the beginning of the measurements but only leaves
that did not yet show any visible signs of senescence were
selected for the measurements. Due to a different timing of
senescence in each tree, measurements were started between
3 September 2021 and 2 October 2021; the last measurement
was conducted on 26 October 2021. Several parameters were
recorded from the selected leaves, almost on a daily basis, with
a handheld device (MultispeQ v1, PhotosynQ Inc., East Lansing,
MI, USA; see below) from six sites on each leaf, until the leaf
dropped. The measurements were conducted between 9:00 and
12:00h.
MultispeQ measurements
A leaf was gently placed between the measuring heads
of the MultispeQ. The MultispeQ measurements covered
(i) absorbance at several wavelengths, (ii) pulse amplitude
modulated chlorophyll a fluorescence, (iii) oxidation of the
primary donor (P700) of photosystem I (PSI), assayed by
near-infrared absorbance changes, (iv) electrochromic shift,
assayed by 530-nm absorbance changes during a light-to-dark
transition, (v) leaf thickness, (vi) leaf temperature, (vii) ambient
light, (viii) temperature, (ix) pressure and (x) humidity. For the
analysis, the last temperature recording of the day was used, as
this choice allowed the device temperature to equilibrate with
ambient temperature. The measuring protocol (Photosynthesis
RIDES) illuminated the leaves with photosynthetic photon
flux density (PPFD) matching that of the incident light.
The fluorescence parameter F0′ was measured during far-
red illumination, FS during a period of simulated incident
light and FM′ during a saturating pulse. Calculations of non-
photochemical quenching (NPQt and NPQ) and non-regulated
quenching (NO) of fluorescence are based on Tietz et al.
(2017) and Kuhlgert et al. (2016). qL was calculated as (FM′
− FS)/(FM′ − F0′)×(F0′/FS), NPQt as (4.88/((FM′/F0′) − 1))
− 1 and NO as 1/(NPQt +1 + qL×4.88). The PSI parameters
are described by Kanazawa et al. (2017). Parameters (vH+,
gH+) derived from the measurements of electrochromic shift
are based on Cruz et al. (2001), Kanazawa and Kramer
(2002) and Avenson et al. (2005). Chlorophyll content was
estimated from the absorbance of leaves at 650 and 940 nm,
employing the so-called Minolta SPAD method (see e.g., Hunt
and Daughtry 2014).
Calibration of pigment measurements
Amounts of red pigments were estimated by measuring
absorbance at 530 nm with MultispeQ. Also chlorophylls absorb
at 530 nm and therefore absorbance at 530 nm was measured
from dark and pale green maple leaves (with no visually
detectable redness), collected on 17 September 2021 and
21 September 2021. 530-nm absorbance increased linearly
with chlorophyll values measured with the same device (Figure
S1a available as Supplementary data at Tree Physiology Online)
and thus the ‘excess’ (chlorophyll-related) 530-nm absorbance
can be subtracted from the absorbance signal (A530) to get
a redness index (R) = A530 − (0.0113 × SPAD+0.495),
where SPAD refers to the chlorophyll content measured with
MultispeQ (Figure S1a available as Supplementary data at
Tree Physiology Online). Leaf sections with R < 0.05 were
considered to contain low amounts of red pigments, those
with R between 0.05 and 0.1 medium amounts and those
with R > 0.1 high amounts. In some cases, leaf classes with
very high (R > 0.2) or very low (R < 0.025) amounts
of red pigments were additionally used. In photoinhibition
experiments (see below), leaves were considered ‘red’ if the
index was higher than 0.1. The redness index correlated well
with visual estimation when leaves were collected from trees
(personal observation). However, redness indexes of fallen
leaves (colored yellow or brown, collected on 26 October
2021) were unrealistically high (R = 0.34 ± 0.02). Therefore,
the method should be used with caution if dead leaves or dead
leaf parts are used.
To verify that a high concentration of red pigments did
not disturb the optical chlorophyll measurements, chlorophyll
contents of selected maple leaves (collected 12–14 October
2020, 15 October 2020, 20–21 October 2020 and 21
September 2021) were measured with MultispeQ, after which
chlorophylls a and b were extracted by incubating the leaf discs
in dimethylformamide in darkness at 4 ◦C for 4–10 days, and
quantified spectroscopically (Porra et al. 1989). Carotenoids
were quantified according to Wellburn (1994) from maple
leaves collected on 12 October 2020, 15 October 2020,
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4 Mattila and Tyystjärvi
20 October 2020 and 21 September 2021. The optical SPAD
method utilized by MultispeQ responded non-linearly to actual
leaf chlorophyll content but seemed indifferent to the presence
of red pigments (Figure S1b available as Supplementary data
at Tree Physiology Online). However, MultispeQ measurements
on brown, fallen leaves produced unrealistically high chlorophyll
readings (Figure S1b available as Supplementary data at Tree
Physiology Online). As parts of senescing maple leaves may
appear brown and dead while the leaf is still attached to the
tree, leaves with very low chlorophyll content (<0.5 μg cm−2)
were excluded from some analyses, as indicated.
Chlorophyll values measured with MultispeQ were converted
according to the following empirical equation, obtained from the
calibration:
Chlorophyll content (a + b) in maple leaves, μg cm−2 = Mul-
tispeQ value2 × 0.0226 + MultispeQ value × 0.0345.
Absorptance of green, yellow and red pieces (2 cm ×
2 cm) of maple leaves, collected on 13 September 2021, was
measured with an integrating sphere (Labsphere, North Sutton,
NH, USA) coupled with an STS-VIS spectrometer (Ocean Insight,
Orlando, FL, USA), as previously described (Mattila et al. 2021).
Photoinhibition treatments
Maple leaves were collected on 15 September 2021, 22
September 2021, 29 September 2021 and 13 October
2021. Leaves were incubated over-night under low light
(PPFD 5–10 μmol m−2 s−1) with petioles in lincomycin
solution (0.6 mgml−1) prepared in tap water. When lincomycin
was not used, illumination treatments were conducted on
the collection day. Leaves were cut to pieces (∼2 cm ×
3 cm); fully green and senescing leaves were collected from
the same trees. Pieces with pale green color (‘yellow’) and
reddish color (‘red’) were cut from a single senescing leaf.
Leaf pieces were placed on a wet paper and dark-acclimated
for at least 30 min. The fluorescence parameter (FM −
F0)/FM = FV/FM was measured with a FluorPen (Photon
Systems Instruments, Brno, Czech Republic), after which
pigments were measured with MultispeQ. Leaf pieces were
illuminated for 45 min to 2 h with white (sunlight simulator
SLHolland; for the spectrum, see Havurinne et al. 2022),
green (500–600 nm) or red (λ > 600 nm) light, PPFD
2000 μmol m−2 s−1, or incubated in darkness, as indicated.
Green and red light were defined with long-pass and short-
pass edge filters (LL-500 and LS-600 for green and LL600 for
red; Corion, USA). To observe clear photoinhibition but avoid
a situation where the FV/FM values would decrease to zero,
different illumination times were used for different treatments.
During illumination, leaf pieces were kept on wet paper
on a temperature-controlled metal block set to 20 ◦C. After
illumination and subsequent 30-min dark-acclimation, FV/FM
and MultispeQ measurements were repeated, and leaf pieces
were let to recover over-night on a wet paper in low light (PPFD
5–10 μmol m−2 s−1). After the recovery, leaves were dark-
acclimated for 30 min and FV/FM and MultispeQ measurements
were repeated. Photoinhibition was quantified via its rate
constant kPI, calculated from the lincomycin-treated samples
as kPI = ln(FV/FM_control/FV/FM_illuminated)/(duration of
illumination, min).
Protein content
To test the efficiency of nutrient resorption, protein content was
quantified from senescing red and yellow aspen leaves, collected
on 12–22 October 2021. When attached leaves were used,
leaves that were loose (about to fall) were selected. Recently
fallen leaves (distinguishable by their bright color, as opposed
to the brown color of leaves fallen a long time ago) were
also used, as indicated. Pigments (with MultispeQ) and fresh
weight were measured, after which leaf pieces were ground
to fine powder in liquid nitrogen and dissolved in a buffer
(50-mM Tris–HCl (pH 8), 2% sodium dodecyl sulfate, 10-mM
ethylenediaminetetra-acetic acid) and stored at −20 ◦C. Protein
content was measured from the supernatant after centrifugation
with the DCTM Protein assay kit (Bio-Rad, Hercules, CA, USA),
which is based on the Lowry method.
Weather data
Global radiation, temperature and precipitation data were pro-
vided by Finnish meteorological institute; all other variables
except UV-radiation were measured at a weather station located
at about 8 km from the measurement sites (Artukainen, Turku);
UV-radiation was measured in Jokioinen, 85 km from the mea-
surement sites.
Statistical analyses
Statistical models Linear models were constructed for FV/FM,
for the rate constant of photoinhibition (kPI) and for recovery
from photoinhibition; linear mixed effect models for gH+, vH+,
leaf thickness, NPQt and for relative number of active PSI
centers; and a beta regression model was used for FV′/FM′.
Variable transformations A logit transformation was applied
to vH+ data, the leaf thickness was raised to the power 0.85,
kPI to the power 0.4, recovery from photoinhibition to the power
0.9 (the model for data obtained in the presence and absence of
lincomycin) and to the power 0.8 (the model for data obtained in
the absence of lincomycin). The exponent was chosen by testing
different power transformations and by selecting the exponent
so that the transformation led to as normal distribution as
possible, as judged by plotting the quantiles of the transformed
distribution against theoretical quantiles and by investigating
histograms of the transformed distributions. A Yeo–Johnson
transformation (Weisberg, 2001) was applied to gH+, FV/FM,
FV′/FM′, NPQt and active PSI centers, with the respective values
of the parameter λ of 0.6, 6.5, 1.8, 2.9 and 8.3. In addition,
linear and log transformations were applied to independent
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Red pigments in autumn leaves of Norway maple 5
variables to get them on a similar range. See Summary of
statistics available as Supplementary data at Tree Physiology
Online for detailed information.
Filtering The data obtained by direct measurements from
attached leaves were filtered so that only records with
chlorophyll content above 0.5 μg cm−2 were used. Further
filtering to records with chlorophyll content above 5.0 μg cm−2
was applied in the models for FV’/FM’ and NPQt. Random
effects by sample ID, leaf, tree or date was added to models
when their inclusion increased the explanatory power of the
model. Physiologically impossible values occasionally produced
by the handheld device were removed by limiting values of
gH+ between 2.0 and 160.0, vH+ values between 0.005
and 0.2, NPQt values between 0 and 25 and active PSI
centers between 0 and 5.0. In addition, records with PPFD
less than 1 μmol m−2 s−1 were considered too noisy for the
gH+ model, and only records with the FM’ value between 500
and 4000 were used in the NPQt model. In modeling recovery
from photoinhibition, eight records were discarded because the
absolute value of the recovery parameter, defined as fraction
of the photoinhibitory loss of FV/FM recovered, was larger than
2.0. Data used for the analysis of FV/FM were not filtered.
Justification of model parameters The redness index and
chlorophyll content were included in all models, as these param-
eters are the main topics of the study. PPFD was included as a
fixed effect in models of the functional photosynthetic param-
eters qH+, vH+, FV’/FM’ and NPQt. Chlorophyll content and
the number of days passed after the start of the measurements
on 2 September 2021, describing the advancement of autumn
senescence, were included in all mixed effect models. Boxplots
of the data also suggested that FV’/FM’, NPQt, and the number
of active PSI centers would change during the progress of the
autumn (Figures S1.5, S1.7 and S1.8 in Summary of statistics
available as Supplementary data at Tree Physiology Online).
Model of gH+ The linear mixed effect model constructed for
the analysis of gH+ tested the effects of PPFD, chlorophyll
content, redness and number of days after 2 September 2021
as fixed effects, and used the sample ID, tree and days after 2
September 2021 as random effects.
Model of vH+ In the linear mixed effect model for vH+, the
gH+ parameter was included as a fixed effect because thylakoid
proton conductivity affects proton flux. Leaf temperature was
also included as a fixed effect, as temperature may obviously
affect a flux rate value. Thus, the model for vH+ had PPFD,
gH+, chlorophyll content, redness, leaf temperature, number of
days after 2 September 2021, and interaction between gH+
and PPFD, between gH+ and chlorophyll content, between
gH+ and leaf temperature, and between gH+ and redness
as fixed effects, and sample ID, tree, leaf and date as random
effects. Leaf temperature was included, as it was clear that leaf
temperature varied in the course of the autumn with only a weak
decreasing trend and no clear connection to PPFD (Figures S1.1
and S1.2 in Summary of statistics available as Supplementary
data at Tree Physiology Online).
Model for FV′/FM′ In the beta regression model for FV′/FM′,
the lowest temperature of the previous night was included as
a variable because inspection of the data suggested that this
factor affects FV′/FM′. Effects of leaf temperature and total
radiation of the day were tested because these environmental
factors, alone or together with PPFD, may lower FV′/FM′ by
causing photoinhibition. PSI may be important for the repair of
PSII, and therefore the relative number of open PSI centers was
tested in the model of FV′/FM′. Fluorescence measurements
may depend on abiotic environmental factors, and therefore
leaf thickness and humidity were also included in the model
of FV′/FM′. The model for FV′/FM′ thus tested the effects of
PPFD, chlorophyll content, redness, leaf temperature, lowest
temperature of previous night, number of days after 2 Septem-
ber 2021, total radiation, leaf thickness, humidity, relative
number of active PSI centers and interaction between PPFD
and chlorophyll content, between leaf temperature and lowest
temperature of previous night, and between total radiation and
lowest temperature of the previous night.
Model for leaf thickness According to descriptive statistics
(Figure S1.9 of the Summary of statistics available as Supple-
mentary data at Tree Physiology Online), average leaf thickness
weakly decreased during the autumn. The linear mixed effect
model for leaf thickness tested the effects of leaf temperature
and humidity as additional potential fixed effects, as these
factors might exert purely physical effects on leaf thickness.
Thus, the model for leaf thickness tested chlorophyll content,
redness, number of days after 2 September 2021, humidity and
leaf temperature as fixed effects and tree, leaf and measurement
site on leaf as random effects.
Model for NPQt The linear mixed effect model for NPQt
tested the effect of the lowest temperature of the previous
night, and leaf temperature, as these factors that seemed to
affect FV′/FM′ might also affect other fluorescence parameters.
Also, like for FV′/FM′, leaf thickness and humidity were tested.
Thus, the model for NPQt tested chlorophyll content, redness,
PPFD, number of days after 2 September 2021, the lowest
temperature of the previous night, humidity, leaf thickness and
leaf temperature as fixed effects and tree, leaf and date as
random effects.
Model for the relative number of active PSI centers The
linear mixed effect model for the relative number of active PSI
centers tested the effect of leaf thickness, as the number of
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6 Mattila and Tyystjärvi
stacked cells and cell size may obviously affect the number of
PSI centers. Leaf temperature was included because it might
affect the measurement. The model for the number of active
PSI centers thus tested the number of days after 2 September
2021, chlorophyll content, redness, leaf thickness and leaf
temperature as fixed effects, and date as a random effect.
Model for kPI The dependent variable, kPI, was calculated as
ln(C/I)/t, where C and I are the FV/FM values measured before
and after the illumination treatment, respectively, and t is the
duration of the treatment (Tyystjärvi and Aro 1996). The linear
model for kPI tested the effects of leaf type (green, green
senescing, yellow, red), redness index, chlorophyll content, light
color of the treatment, interactions between redness and light
color and redness and chlorophyll content, as well as the
interaction between all three.
Models for recovery from photoinhibition The dependent
variable, recovery, was calculated as (R − I)/(C − I), where C,
I and R are FV/FM values measured before the photoinhibition
treatment, after the treatment and after the subsequent recovery
period, respectively. Recovery from photoinhibition was tested
with two different models. First, a linear model was constructed
to test whether lincomycin actually prevents photoinhibition in
senescent leaves. The model thus tested the effects of light
color, chlorophyll content, redness, severity of photoinhibition
(defined as I/C, where C and I are FV/FM measured before
and after the photoinhibition treatments), and the presence of
lincomycin. The second model tested recovery from photoin-
hibition in the absence of lincomycin, with leaf type (green,
green senescent, yellow, red), color of light, chlorophyll content
and redness of the leaf, severity of photoinhibition and the
interaction between chlorophyll content and redness.
A detailed description of each model is shown in Summary
of statistics available as Supplementary data at Tree Physiology
Online.
Modeling In the case of the linear effect mixed models,
statistical significance was tested a posteriori with a likelihood
ratio test by using analysis of variance to compare a reduced
model with the full model, both fitted without applying the
residualized maximum likelihood criterion. The linear and linear
mixed effect models were constructed in R version 4.2.2. (R
Core Team, 2021), using the lme4 library (Bates et al. 2015)
and the beta regression model was constructed with the betareg
library (Cribari-Neto and Zeileis 2010). The Yeo–Johnson trans-
formation was done with the VGAM library (Yee, 2020) and
the optimization of the λ parameter of the transformation was
done by testing the quantiles of the transformed variable with
the EnvStats library (Millard, 2013).
Student’s t-tests (two-tailed, heteroscedastic) for protein data
(aspen) and data fitting for pigment calibrations were performed
in Microsoft Excel.
Figure 1. Absorptance of fully green and senescing (yellow or red)
maple leaves. Average chlorophyll (a + b) contents were 31.6 ± 9.1,
8.1 ± 5.0 and 2.3 ± 1.6 μg cm−2, for green, yellow and red leaf pieces,
respectively. The redness index R was 0.096 ± 0.07, 0.12 ± 0.03 and
0.78 ± 0.7 for green, yellow and red leaf pieces, respectively (see
Materials and methods for details). Each line represents an average of
four biological replicates, and error bars show the standard deviation
(SD).
Figures were prepared with SigmaPlot (Systat Software Inc.,
USA) or with R. For the number of individual trees, leaves and
leaf sections used in each figure and for descriptive statistics,
see Table S1 Summary of statistics available as Supplementary
data at Tree Physiology Online.
Results
Photoinhibition did not differ significantly between red and
yellow senescing leaf pieces
Absorptance was measured from fully green and senescing
maple leaves, collected during the autumn from outdoors-grown
trees. Red pieces (from senescing leaves) absorbed more green
light (∼500–600 nm) than yellow leaf pieces, or green pieces
obtained from non-senescing leaves (Figure 1). The position
of the red edge of the absorptance, in turn, decreased with
chlorophyll content (Figure 1).
To test if red pigments (quantified by their 530-nm
absorbance; see Figure S1 available as Supplementary data at
Tree Physiology Online and Materials and methods for details)
protect senescing maple leaves from photoinhibition of PSII,
pieces of green and senescing leaves were illuminated with
strong light (PPFD 2000 μmol m−2 s−1). As the absorptance
measurements (Figure 1) suggest that maple anthocyanins did
not significantly absorb red light, the effects of white, green
(500–600 nm) and red (λ > 600 nm) light were compared.
In all leaf types, illumination with white light damaged PSII
most efficiently and illumination with green light least efficiently
(Figure 2a; for the original values, see Figure S2 and Table S2
available as Supplementary data at Tree Physiology Online). A
linear statistical model for the rate constant of photoinhibition
(kPI) was highly significant (F(8,95) = 50.3, P < 2.2 × 10−16)
and showed that kPI was significantly affected by light color
(coefficient 0.70 ± 0.0095, P = 6.75 × 10−11) and leaf
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Red pigments in autumn leaves of Norway maple 7
Figure 2. Photoinhibition of PSII in green and senescing maple leaves. (a)
Leaf pieces were illuminated (PPFD 2000 μmol m−2 s−1) with green
(500–600 nm), red (λ > 600 nm) or white light in the presence of
lincomycin. Rate constants of photoinhibition were then calculated by
assuming that the light-induced loss in the FV/FM parameter followed
first-order reaction kinetics. (b) Leaf pieces were illuminated in the
absence of lincomycin and let to recover over-night under low light and
the recovery in the FV/FM parameter value was quantified. Leaf pieces
were cut from fully green leaves (green), or from senescing (S) leaves
containing green, yellow or pale green (yellow) and red sections (red).
The thick line shows the median and the box represents data between
the 25th and the 75th percentiles, and the error bars extend either to
±1.5 times the interquantile range or to the maximum or minimum of the
data, whichever produces a shorter bar. For the original data, see Figure
S2 available as Supplementary data at Tree Physiology Online, and for the
average chlorophyll contents, see Table S3 available as Supplementary
data at Tree Physiology Online. (c) An example of a set of leaf pieces
used.
group (coefficient 0.016 ± 0.0069, P = 0.0256) but not
by the other variables (see Summary of statistics available as
Supplementary data at Tree Physiology Online). In particular,
illumination with green and white light (where anthocyanins
can contribute to light absorption) caused a similar decrease
in FV/FM in yellow and red leaf pieces (Figure 2a; Figure S2
available as Supplementary data at Tree Physiology Online). The
data show that redness did not protect against the damaging
reaction of photoinhibition.
When the leaf pieces were allowed to recover in low light
after the photoinhibition treatment, the FV/FM value showed
some increase even in lincomycin-treated leaves. The efficiency
of lincomycin in preventing the recovery from photoinhibition
was therefore tested statistically by measuring the extent of
recovery as (R − I)/(C − I), where C, I and R are the FV/FM
values measured in the control, after the inhibition treatment and
after the recovery, respectively. A linear statistical model of this
parameter was highly significant (F(6190) = 36.62; P < 2.2
× 10−16) and showed that lincomycin inhibited the increase in
FV/FM during the recovery period strongly but not completely
(effect size−0.32± 0.03, P < 2× 10−16). Senescence-related
parameters including chlorophyll content, redness and their
interaction had additional significant effects, and the severity
of photoinhibition affected recovery (see Summary of statistics
available as Supplementary data at Tree Physiology Online for
details).
Next, recovery from photoinhibition was studied in samples
treated without lincomycin. When the PSII repair cycle was not
blocked, green leaf pieces were able to completely restore their
PSII activity if allowed to recover overnight under low light,
but recovery was incomplete in senescent leaves (Figure 2b;
Figure S2 and Table S2 available as Supplementary data at Tree
Physiology Online). A linear statistical model testing the recovery
from photoinhibition was highly significant (F(6,88) = 10.54;
P = 8.65 × 10−9) and showed that recovery from photoinhibi-
tion was negatively affected by the leaf type, from green non-
senescent to red-senescent leaves (effect size −0.11 ± 0.05,
P = 0.033). The severity of the photoinhibition treatment also
affected, with more relative recovery in samples that were less
severely inhibited (effect size 0.23 ± 0.11, P = 0.0459).
The effect of the severity of photoinhibition might be related
to the finding that in the absence of lincomycin, similar light
treatments caused more severe photoinhibition in senescent
than in green leaves (Figure S2 available as Supplementary data
at Tree Physiology Online). However, removing the severity of
photoinhibition from the model did not significantly affect the
effect of leaf type (data not shown).
No significant change in the FV/FM parameter was observed
in the dark (Figure S2g available as Supplementary data at Tree
Physiology Online), indicating that the changes observed after
light treatments and low-light recovery were caused by the high
light in all leaf types.
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Figure 3. Chlorophyll contents (green symbols, solid line) and the redness index (orange symbols, dashed line), based on 530-nm absorbance, in
four maple leaves (a–d) during autumn 2021, until abscission. Three measurement sites per leaf are marked with different symbols (see Figure S4h
available as Supplementary data at Tree Physiology Online for the positions on the leaf blade). The vertical dashed line highlights the time point at
which all chlorophyll was degraded in one of the measurement sites of the leaf. The vertical yellow bars indicate days with high irradiance (daily
irradiance >3000 Wm−2, except on 16–17 October ∼2000 Wm−2) and the blue-gray bars indicate days when the previous night had been cold
(temperature below 0 ◦C). The photographs show the leaf after natural abscission. Each leaf belonged to a different tree. For the whole data, see
Figures S4–S7 available as Supplementary data at Tree Physiology Online.
Red pigments increased during the last stages of senescence
Next, physiological parameters of several maple leaves were fol-
lowed in vivo during the course of natural autumn senescence,
until abscission (for a summary of the data, see Figure S3 and
Boxplots in Summary of statistics available as Supplementary
data at Tree Physiology Online). In most cases, maple leaves
did not senesce uniformly, but a part of the leaf started to
lose chlorophyll much, even a week, earlier than other parts
(Figure 3; for the whole data, see Figures S4–S7 available
as Supplementary data at Tree Physiology Online). Also, red
pigments appeared unevenly, in both time and space. In gen-
eral, red pigments appeared when little chlorophyll was left
(Figure 3). In a few cases, small amounts of red pigments were
present already in the beginning of senescence (see Figures
S6 and S7 and Figure S1.4 in Summary of statistics available
as Supplementary data at Tree Physiology Online), but also in
these cases the strongest increase in 530-nm absorbance was
observed when most of the chlorophyll had been degraded.
In some cases, 530-nm absorbance increased after complete
loss of chlorophyll, however, the observation should be treated
with caution, as senescing leaves may develop brown (dead)
areas while still attached, which may affect the absorbance
measurements (Figure S1b available as Supplementary data at
Tree Physiology Online).
In some cases, a combination of high irradiance with low (pre-
vious) night temperature (for the complete weather data, see
Figure S8a available as Supplementary data at Tree Physiology
Online) appeared to coincide with an increase in red pigments
(on 21–22 September in Figure 3a and c). However, when
all changes in red pigments in senescing leaves were plotted
against daily irradiances or night temperatures, no general trend
could be seen (Figure S9a and b available as Supplementary
data at Tree Physiology Online). On the other hand, leaves
most shadowed by their neighbors (Figures S4c and f and
S7h available as Supplementary data at Tree Physiology Online)
stayed yellow until all chlorophyll was degraded.
The rate of chlorophyll degradation was initially slow but then
increased and all chlorophyll was lost within a week (Figure 3).
Although the data obtained from individual leaves shows a
biphasic loss of chlorophyll, a boxplot of the dependence of
chlorophyll content as a function of Julian day fails to catch
the biphasic nature of chlorophyll degradation (Figure 1.3 in
Summary of statistics available as Supplementary data at Tree
Physiology Online). Once initiated at a particular section of
the leaf blade, the rate of chlorophyll degradation appeared
not to correlate with the presence of red pigments (Figure 3;
Figure S9c available as Supplementary data at Tree Physiology
Online).
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Red pigments in autumn leaves of Norway maple 9
Figure 4. The fluorescence parameter (FM′ − F0′)/FM′ = FV′/FM′ (green symbols, solid line), reflecting functional PSII units, and oxidizable P700,
measured during a saturating pulse, reflecting active PSI centers (gray symbols; arbitrary units), in four maple leaves (a–d) during autumn 2021, until
abscission. Three measurement sites per leaf are marked with different symbols (see Figure S4h available as Supplementary data at Tree Physiology
Online for the positions on the leaf blade). The vertical dashed line highlights the time point at which all chlorophyll was degraded in one of the
measurement sites of the leaf. The vertical yellow bars indicate days with high irradiance (daily irradiance >3000 Wm−2, except on 16–17 October
∼2000 Wm−2) and the blue-gray bars indicate days when the previous night had been cold (temperature below 0 ◦C). The parameters have been
measured from the same leaves as shown in Figure 3. For the whole data, see Figures S10–S13 available as Supplementary data at Tree Physiology
Online.
PSII activity was low after cold nights in maple leaves
In maple leaves the PSII parameter FV′/FM′ stayed high through-
out the autumn until almost all chlorophyll was degraded (Fig-
ures S3a and S10–S13 available as Supplementary data at Tree
Physiology Online). However, transient decreases in the FV′/FM′
value were observed; large drops almost always occurred after
cold nights, in both senescing and green leaves (Figure 4; Figure
S1.5 in Summary of statistics available as Supplementary data at
Tree Physiology Online). A combination of low temperature and
light was not needed for the decrease in FV′/FM′, as on these
days, the temperature had already risen above zero by the time
the sun rose; only on 19 October did sub-zero temperatures
coincide with some amount of sunlight (Figure S8b–e available
as Supplementary data at Tree Physiology Online). In most cases,
FV′/FM′ fully recovered within a day (a notable exception was on
21–22 September when two consecutive cold nights occurred,
after which the recovery of FV′/FM′ took several days).
On the other hand, a parameter reflecting active PSI units did
not seem respond to weather conditions but showed a general
decrease with leaf chlorophyll content (Figure 4; Figure S3b
available as Supplementary data at Tree Physiology Online). A
linear mixed effect model confirmed the significant dependence
of the number of active PSI centers on chlorophyll content
(effect size 15.12, P < 2.2 × 10−16, χ 2 = 71.1).
In maple, photochemical activity remained high almost until
abscission
In maple leaves, the qL parameter, reflecting photochemical
quenching of chlorophyll fluorescence, stayed remarkably high
until almost all chlorophyll was degraded (Figure 5; Figure S3d
available as Supplementary data at Tree Physiology Online). In
fact, qL stayed clearly higher than FV′/FM′ or (FM′ − FS)/FM′
(Figure S3a and c available as Supplementary data at Tree
Physiology Online). Unsurprisingly, qL strongly depended on the
incident PPFD (Figure S3d available as Supplementary data at
Tree Physiology Online), but correlation with the total irradiance
of the day was poor (Figure 5; Figures S14–S17 available as
Supplementary data at Tree Physiology Online).
Two measured parameters of regulated non-photochemical
quenching (NPQ) of chlorophyll fluorescence, NPQt (an NPQ
parameter that can be measured without dark-acclimation; Tietz
et al. 2017) and NPQ yield (NPQ), increased in senescing
leaves, especially during bright days (Figure 5; Figure S3e and
f available as Supplementary data at Tree Physiology Online).
A linear mixed effect model confirmed that NPQt significantly
increased with decreasing chlorophyll content (size of the
effect of chlorophyll content was −0.062, P < 2.2 × 10−16,
χ 2 = 454.93) and increased with redness (effect size 0.053,
P = 5.3 × 10−5, χ 2 = 16.4). The effect of PPFD was highly
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Figure 5. Photochemical quenching (qL; gray symbols, solid line), the yield of unregulated non-photochemical quenching (NO; red symbols, dashed
line) and regulated non-photochemical quenching (NPQt; dark red symbols, dotted line) of chlorophyll fluorescence in four maple leaves (a–d) during
autumn 2021. Three measurements per leaf were conducted, marked with different symbols (see Figure S4h available as Supplementary data at Tree
Physiology Online for the positions on the leaf blade). The vertical yellow bars indicate days with high irradiance (daily irradiance >3000 Wm−2,
except on 16–17 October ∼2000 Wm−2), and the blue-gray bars indicate days when the previous night had been cold (temperature below 0 ◦C).
Measurements conducted on leaf areas with (almost) no chlorophyll (chlorophyll content less than 0.5 μg cm−2) have been excluded. The parameters
have been measured from the same leaves, as shown in Figure 3. For the whole data, see Figures S14–S17 available as Supplementary data at Tree
Physiology Online.
significant but relatively small (effect size 0.0044, P < 2.2
× 10−16, χ 2 = 352.0). In addition, leaf temperature had a
significant effect (effect size 0.0012, P = 0.0012, χ 2 = 10.5).
The yield of unregulated fluorescence quenching (NO), on
the other hand, stayed relatively constant during the whole
measurement period, though a decrease was observed when
only little chlorophyll was left (Figure 5; Figure S3g available
as Supplementary data at Tree Physiology Online). A pecu-
liar peak in NO, observed after the two cold days (21–22
Sept), may be related to low PSII activity during that time
(Figures 4 and 5).
In line with the high qL values, senescing leaves were able to
form a proton gradient over the thylakoid membrane; values for
the parameters reflecting proton conductivity (gH+) and steady
state proton flux (vH+) were in senescing leaves close to those
measured from green leaves, though slightly smaller at low light
intensities (Figure 6). Linear mixed effect models for gH+ and
vH+ showed that both parameters were positively affected by
PPFD (effect size 0.50, P = 4.8 × 10−12, χ 2 = 47.76 for gH+
and effect size 1.80, P < 2.2 × 10−16, χ 2 = 1516.2 for vH+).
Proton conductivity decreased with the number of days after 2
September 2021 (effect size −3.41, P = 0.0044, χ 2 = 8.11).
As expected, vH+ depended on gH+ (effect size 0.53, P < 2.2
× 10−16, χ 2 = 622.6) but the effects of interaction between
gH+ and PPFD as well as the interaction between gH+ and
chlorophyll content were negative (effect sizes −0.91, P < 2.2
× 10–16, χ 2 = 183.8 and − 0.49, P = 8.4 × 10−7, χ 2 = 24.3,
respectively). vH+ also depended on chlorophyll content (effect
size 0.77, P < 2.2 × 10−16, χ 2 = 112.3). Redness had a major
positive effect on gH+ (effect size 6.75), but the effect was only
barely significant (P = 0.047, χ 2 = 3.96), whereas the effect
of redness on vH+ was negative (effect size −0.52, P = 7.98
× 10−11, χ 2 = 46.5).
Lastly, the difference between ambient temperature and leaf
temperature, reflecting functionality of vasculature and stomata,
was found to be comparable in green and senescing leaves
(Figure S3h available as Supplementary data at Tree Physiology
Online).
In maple leaves, high 530-nm absorbance coincided with low
PSII efficiency
Next, the amounts of red pigments, chlorophyll contents and PSII
values of green and senescing maple leaves were compared in
more detail. Leaf sections with high redness indexes tended
to have low FV/FM values (measured after 30-min dark-
acclimation from detached non-stressed leaves; Figure 7a).
As PSII activity decreases with decreasing chlorophyll content
of the leaf, average FV/FM values of leaves with different
amounts of red pigments were calculated for senescing
leaves only (Figure 7b); in this comparison, the average
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Red pigments in autumn leaves of Norway maple 11
Figure 6. The effect of ambient light PPFD on thylakoid membrane proton conductivity (gH+; a) and steady-state proton flux (vH+; b), measured
in vivo from maple leaves in their natural environment, in leaf areas with low (less than 10 μg cm−2; olive circles), medium (10–25 μg cm−2, light
green upward triangles) or high (over 25 μg cm−2; green diamonds) chlorophyll (Chl) content. Measurements conducted on leaf areas with very
low chlorophyll content (<0.5 μg cm−2) have been excluded. The insets show the complete data (excluding fewer than 10 outliers). Each symbol
represents an individual measurement.
chlorophyll contents of the groups were close to each other
(Table S4 available as Supplementary data at Tree Physiology
Online). A decreasing trend in FV/FM as a function of
530-nm absorbance was found. A linear model of FV/FM was
highly significant (F(3252) = 113.8; P < 2.2 × 10−16) and
showed that redness lowered the Yeo–Johnson-transformed
FV/FM value strongly (coefficient 7.36 ± 0.92), whereas
chlorophyll content had a smaller positive effect (5.2 ± 0.69).
See the Summary of statistics available as Supplementary data
at Tree Physiology Online for the full results of the model.
We also investigated the relationship of chlorophyll con-
tent and 530-nm absorbance with FV′/FM′ values, measured
from attached maple leaves during ambient light conditions
(Figures 3 and 4; Figures S4–S7 and S10–S13 available as
Supplementary data at Tree Physiology Online). In maple leaves,
as noted already above, red pigments preferentially appeared
only during late senescence; in general, when the chlorophyll
content had decreased to ∼10 μg cm−2, a clear increase
in red pigments was observed (Figure 7c; Figure S1.4 in the
Summary of statistics available as Supplementary data at Tree
Physiology Online). The few exceptions, where a leaf section
with high chlorophyll content showed also a high redness index
(Figure 7c; Figures S4a, S6b and S7b available as Supplemen-
tary data at Tree Physiology Online), seemed to coincide with
low FV′/FM′ values (Figures S10a, S12b and S13b available as
Supplementary data at Tree Physiology Online).
Expectedly, the FV′/FM′ values were slightly lower than the
FV/FM values; however, the responses of both parameters
to chlorophyll content were very similar (Figure 7a and d).
Furthermore, when average FV′/FM′ values were calculated
for leaf sections with different amounts of red pigments,
red senescing leaf sections had lower FV′/FM′ values than
yellow (low 530-nm absorbance) senescing leaf sections
(Figure 7e). In contrast, FV′/FM′ values of non-senescing
(green) leaves were high regardless the amount of red pigments
(Figure 7e). The beta regression model for factors affecting the
FV′/FM′ value showed that redness index strongly lowered
FV′/FM′ (coefficient −1.21 ± 0.24). The effect of PPFD was
smaller (coefficient − 0.52 ± 0.02), whereas chlorophyll
content strongly increased FV′/FM′ (coefficient 3.23 ± 0.14),
thus confirming the findings based on descriptive statistics
(Figure 7). The effect of the lowest temperature of the previous
night was large (coefficient 4.05 ± 2.4) but not statistically
significant (P = 0.09). Furthermore, the number of days
passed after 2 September 2021 had a strong negative effect
(−2.433 ± 0.20), and weak but significant effects by leaf
thickness, humidity, number of open PSI centers, and by
interaction between PPFD and chlorophyll content, between leaf
temperature and time after 2 September 2021 and between
total radiation and the lowest temperature of the previous night
were also observed (see Summary of statistics available as
Supplementary data at Tree Physiology Online).
Next, to test if low PSII efficiency could induce synthesis of
red pigments, changes in the amounts of red pigments between
two subsequent measurement days were calculated, separately
for leaves with high or low FV′/FM′ (on the first day). Leaf
sections with low FV′/FM′ values, regardless of whether they
were senescing or not, did not, on the average, synthetize
more red pigments than leaf sections with high FV′/FM′ values
(Figure 7f). To conclude, a causal relationship between low PSII
efficiency and red pigments could not be observed. Curiously,
leaf sections with high chlorophyll content showed negative
values for the change in red pigments (Figure 7c and f; see also
Figure S4c available as Supplementary data at Tree Physiology
Online).
Red leaves were thicker than yellow ones, and red aspen
leaves contained more protein than yellow ones
Senescing maple leaf sections were found to be thinner than leaf
sections with high chlorophyll content (Figure 8a). In addition,
leaf sections with low amounts of red pigments were thinner
than red sections (Figure 8b; see also Figures S6.4 and S6.5 in
Summary of statistics available as Supplementary data at Tree
Physiology Online). The correlation between leaf thickness and
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Figure 7. PSII activity in maple leaves with different amounts of red pigments and chlorophylls. (a) FV/FM values of leaf pieces with different (low,
medium, high or very high) redness indexes (R; estimated based on 530-nm absorbance) plotted against their chlorophyll contents. (b) Average
FV/FM values of senescing leaf pieces (chlorophyll (a + b) content <10 μg cm−2) with different redness indexes. The data (a–b) are from Figure 2.
(c) Redness index and chlorophyll content in leaves of four different maple trees. (d) FV′/FM′ values of leaf sections with different (very low, low,
medium, high or very high) redness indexes plotted against their chlorophyll contents. (e) Average FV′/FM′ values of senescing leaf sections (non-
hatched bars; chlorophyll (a + b) content 3–10 μg cm−2) and non-senescing leaf sections (hatched bars; chlorophyll (a + b) content >30 μg cm−2)
with different redness indexes. (f) Average changes in redness index (between two subsequent measurement days) in senescing leaf sections (non-
hatched bars; chlorophyll (a + b) content 5–20 μg cm−2) and non-senescing leaf sections (hatched bars; chlorophyll (a + b) content >30 μg cm−2)
with different FV′/FM′ values (on the first measurement day). The data (c–f) are from Figures S4–S7 and S10–S13 available as Supplementary data
at Tree Physiology Online. Error bars show SD. In (a), (c) and (d), each symbol represents an individual measurement. See Table S4 available as
Supplementary data at Tree Physiology Online for the chlorophyll contents.
redness was unclear in leaf sections with very low chlorophyll
content (≤ 10 μg cm−2) but pronounced in sections with high
chlorophyll content (Figure 8b). The red senescing leaf sections
contained slightly less chlorophyll than yellow sections, and red
sections with high chlorophyll content contained more chloro-
phyll than their non-red counterparts (Table S4 available as
Supplementary data at Tree Physiology Online). Thus, correlation
with chlorophyll content (Figure 8a) may have flattened and
enhanced the correlation between redness and thickness in
senescing and green leaves, respectively. A linear mixed effect
model of leaf thickness revealed that both chlorophyll content
and redness index increased thickness but the effect of redness
was much stronger (effect size 1.08 ± 0.06 on the transformed
thickness variable) than that of chlorophyll content (effect size
0.70 ± 0.08). Leaf temperature had a very small effect (effect
size −0.013 ± 0.002). See the Summary of statistics available
as Supplementary data at Tree Physiology Online for a full
summary of the results of the model.
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Red pigments in autumn leaves of Norway maple 13
Figure 8. Thickness of green and senescing maple leaves. (a) Average thickness of leaf sections with low (0.5–10 μg cm−2), medium (10–
25 μg cm−2) or high (over 25 μg cm−2) chlorophyll (a + b) contents. (b) Average thickness of leaf sections with low, medium or high chlorophyll
content, and low (open bars), medium (hatched bars) or high (cross-hatched bars) redness index (R; estimated from 530-nm absorbance). See
Table S4 available as Supplementary data at Tree Physiology Online for the average chlorophyll values. The error bars show the SD.
Finally, to estimate the effect of red pigments on nutrient
resorption, recently fallen aspen leaves, and leaves that were
about to fall, were collected. As in maple, red aspen leaves
were on the average thicker than yellow aspen leaves (Figure 9),
even though the difference was not statistically significant.
Red senescing aspen leaves were also heavier than yellow
senescing leaves (Figure 9). Accordingly, red leaves contained
more proteins than yellow leaves, both on an area and a fresh
weight basis (Figure 9).
Discussion
Based on the previous literature, it seems clear that antho-
cyanins (or red pigments, as the actual chemical compositions
causing the red colors have rarely been determined) can protect
young or mature leaves from photoinhibition, at least in some
species and in harsh environmental conditions (Gould et al.
2018, Agati et al. 2021, Hughes et al. 2022). The question,
whether photoprotection is the (primary) role of anthocyanins
also in senescing leaves, has recently been a source for active
discussion (Renner and Zohner 2019, 2020, Agati et al. 2021,
Pena-Novas and Archetti, 2021a, Hughes et al. 2022). A
reason for the debate is that results regarding a photoprotective
function of red pigments in senescing leaves are contradictory
and scarce. To our knowledge, only one study has shown
a clear protective effect by anthocyanins in senescing leaves
(Hoch et al. 2003; though, the site(s) of the mutation causing
the anthocyanin deficiencies and flavonoid compositions of the
mutants are not known to us).
A reason for contrasting results (Feild et al. 2001, Hoch et al.
2003, Duan et al. 2014, Pena-Novas and Archetti, 2021b) may
be that comparisons between anthocyanic and non-anthocyanic
leaves are not simple. For example, if the compared leaves
belong to different species or varieties or have grown under
different light conditions (e.g., Feild et al. 2001, Lee et al.
2003, Duan et al. 2014), it is probable that the anthocyanin
content is not the sole difference between the compared leaves.
In addition, leaves or species that do not synthesize antho-
cyanins may have compensatory protection mechanisms; lack
of anthocyanins can coincide with a large xanthophyll pool
(Logan et al. 2015), high amount of antioxidant enzymes (Landi
et al. 2013) or different gene regulation during senescence
(Vangelisti et al. 2020). It has been hypothesized that different
senescence strategies or environmental conditions would favor
different protection mechanisms (Moy et al. 2015, Hughes et al.
2022). A protective effect by anthocyanins may thus remain
unnoticed. On the other hand, a growth condition inducing
anthocyanin synthesis (e.g., high light) may also induce other
protective mechanisms and an observed (protective) effect may
not be related to anthocyanins. In the present study, we have
tried to minimize the above-mentioned difficulties by comparing
yellow and red sections within one and the same leaf (Figure 2),
or yellow and red leaves collected from one and the same tree
(Figure 9).
Another important factor to consider is chlorophyll content;
leaves with low chlorophyll content (such as senescing leaves)
are more vulnerable to PSII damage than leaves with high chloro-
phyll content (Pätsikkä et al. 2002, Manetas and Buschmann
2011, Havurinne et al. 2022, Mattila et al. 2021, Serôdio
and Campbell 2021) because the amount of light absorbed
per chloroplast decreases with increasing chlorophyll content.
This is especially important in the case of senescing leaves,
as anthocyanins often gradually increase while chlorophyll is
degraded (e.g., Hoch et al. 2003; Figure 7). In addition, the
chlorophyll content of a (senescing) leaf is a strong determinant
of also other parameters, such as PSII activity, PSI amount,
NPQ induction, carotenoid amounts, leaf thickness and even
absorption of green (530 nm) light (Figures 7 and 8; Figures
S1, S3 and S18 available as Supplementary data at Tree
Physiology Online; for statistical evidence, see Summary of
statistics available as Supplementary data at Tree Physiology
Online). Therefore, chlorophyll content should be taken into
account when yellow and red senescing leaves are compared.
Here, chlorophyll content was always measured and taken
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14 Mattila and Tyystjärvi
Figure 9. Fresh weight, leaf thickness and protein content of senescent yellow and red aspen leaves. The protein amount was calculated on fresh
weight (FW) and leaf area (A) basis. Leaves were collected when they were still weakly attached to a tree (a–f), when the tree was still mostly green
(a–c) or when the tree was mostly yellow/red (d–f), or from the ground (g–i). The mean 530-nm absorbance (chlorophyll effect was not corrected
for) values of yellow aspen leaves were 0.58–0.62 and those of red leaves 0.82–1.04. Average chlorophyll (SPAD) values of yellow leaves were
between −2.55 and −1.21, and those of red leaves were between −0.83 and −0.45. Visually, no chlorophyll could be detected, except for very
low amounts in some red leaves. Each bar represents an average calculated from nine leaves, collected from at least three individual trees, and the
error bars show the SD. The differences between red and yellow senescing leaves in fresh weight and protein amounts were significant when all the
measurements (a–c) were pooled (P < 0.026; t-test).
into account in statistical modeling; for descriptive statistics
we aimed at comparing only leaves with similar chlorophyll
contents (Tables S1–S4 available as Supplementary data at Tree
Physiology Online).
Can anthocyanins enhance carbohydrate disposal in
senescing maple leaves?
White light caused more photoinhibition than red or green light,
in both green and senescing leaves (Figure 2). The result agrees
with previously measured action spectra of photoinhibition,
obtained with several laboratory-grown plant species (for a
review, see Zavafer et al. 2015), considering that the white
light source used here (a sunlight simulator) emits blue light
and also some UV-A radiation (Havurinne et al. 2022). Green
light caused very little photoinhibition in all leaf types, indicating
that if anthocyanins are to protect PSII, then the crucial factor is
their capacity to absorb blue light (Manetas, 2006, Agati et al.
2021). Here, absorptance of blue wavelengths (λ < 500 nm)
was close to 100% in both green and senescing maple leaves
(Figure 1) and, thus, the present data do not allow us to
judge whether red pigments significantly absorbed blue light.
Cyanidin-3-glucoside and cyanidin-3-rutinoside, two common
anthocyanins in maple (Ji et al. 1992, Schmitzer et al. 2009),
show the highest absorbance (in a mixture of water, formic acid
and acetonitrile) at ∼520 nm but the spectra extend to 350 nm
(Vergara et al. 2009).
We did not find significant differences in photoinhibition
between red and yellow senescing leaf sections of Norway
maple (Figure 2; see also Summary of statistics available as
Supplementary data at Tree Physiology Online). Furthermore,
red senescing leaves showed lower FV/FM and FV′/FM′ values
(Figure 7), an observation reported also earlier (e.g., Kytridis
et al. 2008, Nikiforou and Manetas 2010, Nikiforou et al. 2011).
Indeed, in Norway maple, red pigments often appeared only
when a large part of chlorophyll had already been degraded
(Figure 7c). However, if the primary function of anthocyanins
were to protect senescing leaves, one would expect that
anthocyanins would start to accumulate at the beginning of
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Red pigments in autumn leaves of Norway maple 15
chlorophyll degradation. Thus, if anthocyanins protect, they
protect only at a late stage of senescence. Furthermore, the
finding that the repair of PSII during and after a strong light
treatment was equally inefficient in red, green and yellow
sections of senescent maple leaves (Figure 2b; Figure S2, Table
S2 available as Supplementary data at Tree Physiology Online;
see also Summary of statistics available as Supplementary data
at Tree Physiology Online) indicates that anthocyanins do not
enhance repair of PSII. The results are in agreement with the
previous observation that anthocyanin accumulation does not
significantly lower light absorption by chlorophylls in sugar
maple (van den Berg et al. 2009).
As discussed by Hughes et al. (2022) and above, a small
(or non-existing) difference in photoinhibition susceptibility
between red and yellow senescing leaves cannot be taken
as an absolute proof for the inefficiency of anthocyanins to
protect, as yellow leaves may have different, compensatory
protection mechanisms. For example, senescing peduncles
of common elderberry (Sambucus canadensis) rely either
on NPQ or anthocyanins, depending on their growth light
history (Cooney et al. 2018). Young leaves of Castanopsis
chinensis and Acmena acuminatissima also induce either NPQ
or anthocyanin synthesis depending on the season, but in a
contrasting way (Yu et al. 2021). In addition, while mutant
plants lacking anthocyanins were susceptible to high light stress,
yellow senescing birch was as tolerant as the anthocyanin-
containing wild types of C. sericea, Vaccinium elliottii Chapmn.
and Viburnum sargentii Koehne (Hoch et al. 2003). We
did not, however, observe an inverse relationship between
NPQ induction and anthocyanin accumulation in maple but,
instead, red leaf sections induced more NPQ than yellow
sections (Figure S18a and b available as Supplementary data
at Tree Physiology Online; see also Summary of statistics
available as Supplementary data at Tree Physiology Online).
Besides NPQ, other photoprotection mechanisms exist and,
for example, the carotenoid to chlorophyll ratio commonly
increases during senescence (e.g., Junker and Ensminger
2016). We also observed the increase, occurring because
chlorophylls were degraded more rapidly than carotenoids
(Figure S18c available as Supplementary data at Tree Physiology
Online). Again, however, red leaves contained rather more
carotenoids than yellow leaves, although the difference was not
statistically significant (Figure S18c available as Supplementary
data at Tree Physiology Online). To summarize, anthocyanin
accumulation did not render other protective mechanisms
unnecessary.
Regarding NPQ, Junker and Ensminger (2016) observed
opposite results: a decrease in NPQ and an increase in NO in
senescing sugar maple leaves. The discrepancy with our results
may reflect a difference in the method of estimation of NPQ or
the fact that the senescing leaves used by Junker and Ensminger
(2016) had lost almost all chlorophyll.
Red senescing maple leaf sections also did not live longer
than yellow sections (Figure S9c available as Supplementary
data at Tree Physiology Online). In aspen, protein amount at
abscission was higher in red than in yellow leaves (Figure 9),
suggesting that red leaves had problems with nitrogen resorp-
tion. Therefore, in the present study, anthocyanin accumulation
did not increase the fitness of the tree by increasing carbon
gain or nutrient resorption. On the contrary, high NPQ and high
amount of carotenoids suggest that red leaf sections were under
stronger stress than yellow leaf sections. Indeed, red senescing
leaf sections exhibited lower PSII activities than yellow sections
(Figure 7; see also Summary of statistics available as Sup-
plementary data at Tree Physiology Online). A higher proton
conductivity of thylakoid membranes of red leaves, measured
with the gH+ parameter, may suggest that redness is associated
with membrane leakiness (see Summary of statistics available
as Supplementary data at Tree Physiology Online). Red light
caused a similar amount of PSII damage in red and yellow
sections of senescing maple leaves, whereas in white light,
yellow sections were, on average, damaged slightly faster than
red sections (Figure 2). This finding may suggest that the PSII
units in the red sections were intrinsically more vulnerable to
PSII damage but anthocyanins offered some photoprotection so
that the resulting rate of photodamage was similar when white
light was used to induce photoinhibition.
We attempted to test whether low PSII activity induces
anthocyanin synthesis. However, a low FV′/FM′ value did not
predict high anthocyanin synthesis during the following day
(Figure 7f) or 2 days (data not shown). The result suggests
that the same conditions cause both low PSII activity and
anthocyanin accumulation. The findings that anthocyanic leaf
sections (Figure 8b) and leaves (Figure 9; see also Summary
of statistics available as Supplementary data at Tree Physiology
Online) are thicker and that anthocyanic leaves contain more
protein than non-anthocyanic leaves (Figure 9) suggest that
problems in translocation of photosynthates induces antho-
cyanin synthesis. Here, increased leaf thickness and leaf mass
(per area), common responses to nitrogen deficiency, correlated
with redness (Figures 8 and 9) and have earlier been found
to correlate with high amounts of anthocyanins (Hughes et al.
2022). Problems in translocation would also induce NPQ and
contribute to the stress symptoms found in red leaf sections
(Figure 7; Figure S18 available as Supplementary data at Tree
Physiology Online).
We suggest that anthocyanin accumulation is a stress reaction
and propose that during senescence, when carbon skeletons
are no longer required, nitrogen is actively transported from
the leaves and chloroplasts are being degraded, anthocyanin
synthesis may replace starch synthesis as a carbohydrate stor-
age mechanism. As anthocyanins contain only carbon, oxygen
and hydrogen, their synthesis would help, by disposing of
carbohydrates, the light reactions to continue to produce energy
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16 Mattila and Tyystjärvi
required for nutrient resorption right before abscission. The
carbohydrate disposal hypothesis is also supported by the
fact that red leaves are often found in exposed parts of the
Norway maple canopy (personal experience) or under high light
(Duan et al. 2014), where carbohydrates would be produced
in abundance. High light could also contribute to the observed
stress symptoms in red senescing leaves, independently of
the proposed translocation problems. However, disruption of
phloem export by stem girdling or by cooling induces antho-
cyanin synthesis (Murakami et al. 2008, Schaberg et al. 2017),
indicating that high sugar content may induce anthocyanin syn-
thesis even in the absence of high light. If carbohydrate disposal
is the main function of anthocyanin synthesis, then leaf-to-leaf
and within-leaf variation in anthocyanin synthesis would be
caused by variation in sink source relationships or in differences
in the functionality of vasculature in a senescing leaf.
Our suggestion about the role of anthocyanin synthesis in
senescence is related to an earlier suggestion of anthocyanin
synthesis functioning as an energy escape valve that would
also protect the photosynthetic apparatus from light stress, by
enabling fast electron transfer via prevention of over-reduction
of the photosynthetic electron transport chain when sinks dimin-
ish (Lo Piccolo et al. 2018, Hernández and Van Breusegem
2010, Soubeyrand et al. 2018). Protection against light stress
was not observed here, suggesting that photoprotection is not
the primary function of anthocyanins in senescing maple leaves.
It is also possible that anthocyanins serve multiple functions
in senescing leaves. More research is needed with deciduous
plants, especially at the molecular level, to fully understand
the roles of anthocyanins in senescence. For example, possible
within-leaf differences in nitrogen content of senescing leaf have
not been, to our knowledge, investigated.
Supplementary data
Supplementary data for this article are available at Tree Physiol-
ogy Online.
Acknowledgments
Riina Aitokari is acknowledged for helping to optimize lincomycin
treatments.
Funding
The authors thank the Ella and Georg Ehrnrooth Foundation, the
Emil Aaltonen Foundation, the Osk. Huttunen Foundation and
Academy of Finland (grant 333421) for financial support.
Conflict of interest statement
None declared.
Authors’ contributions
H.M. and E.T. designed the study. H.M. performed the experi-
ments and wrote the first version of the manuscript. H.M. and
E.T. analysed the results and wrote the final version of the
manuscript.
Data availability statement
Data are at https://data.mendeley.com/datasets/thjgzhv928/1.
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