Tree Physiology 33, 202–210
doi:10.1093/treephys/tps112
Research paper
Electron transport efficiency at opposite leaf sides: effect of
vertical distribution of leaf angle, structure, chlorophyll content
and species in a forest canopy
Pille Mänd1,5, Lea Hallik2, Josep Peñuelas3,4 and Olevi Kull1
1Department of Botany, Institute of Ecology and Earth Sciences, Tartu University, Lai 40, Tartu 51005, Estonia; 2Department of Plant Physiology, Institute of Agricultural and
Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 5, Tartu 51014, Estonia; 3CSIC, Global Ecology Unit CREAF-CEAB-CSIC-UAB, Cerdanyola del Vallès
08913, Catalonia, Spain; 4CREAF, Cerdanyola del Vallès 08913, Catalonia, Spain; 5Corresponding author ([email protected])
Received March 22, 2012; accepted October 10, 2012; published online November 25, 2012; handling Editor Jörg-Peter Schnitzler
We investigated changes in chlorophyll a fluorescence from alternate leaf surfaces to assess the intraleaf light acclimation
patterns in combination with natural variations in radiation, leaf angles, leaf mass per area (LMA), chlorophyll content (Chl)
and leaf optical parameters. Measurements were conducted on bottom- and top-layer leaves of Tilia cordata Mill. (a shadetolerant sub-canopy species, sampled at heights of 11 and 16 m) and Populus tremula L. (a light-demanding upper canopy
species, sampled at canopy heights of 19 and 26 m). The upper canopy species P. tremula had a six times higher PSII quantum yield (ΦII) and ratio of open reaction centres (qP), and a two times higher LMA than T. cordata. These species-specific
differences were also present when the leaves of both species were in similar light conditions. Leaf adaxial/abaxial fluorescence ratio was significantly larger in the case of more horizontal leaves. Populus tremula (more vertical leaves), had smaller
differences in fluorescence parameters between alternate leaf sides compared with T. cordata (more horizontal leaves).
However, optical properties on alternate leaf sides showed a larger difference for P. tremula. Intraspecifically, the measured
optical parameters were better correlated with LMA than with leaf Chl. Species-specific differences in leaf anatomy appear to
enhance the photosynthetic potential of leaf biochemistry by decreasing the interception of excess light in P. tremula and
increasing the light absorptance in T. cordata. Our results indicate that intraleaf light absorption gradient, described here as
leaf adaxial/abaxial side ratio of chlorophyll a fluorescence, varies significantly with changes in leaf light environment in a
multi-layer multi-species tree canopy. However, this variation cannot be described merely as a simple function of radiation,
leaf angle, Chl or LMA, and species-specific differences in light acclimation strategies should also be considered.
Keywords: chlorophyll fluorescence, chloroplast acclimation, leaf inclination angle distribution, leaf optics, photochemical
quenching, reflectance, transmittance, within-canopy variability.
Introduction
Large heterogeneity in light capture strategies has been demonstrated for different elements of the forest canopy (Turnbull
1991, Rijkers et al. 2000, Souza and Válio 2003, Delagrange
et al. 2004), nevertheless process-based plant models mainly
use two parameters according to the Beer–Lambert law (Monsi
and Saeki 1953, Pierce and Running 1988)—leaf area index
and extinction coefficient, for the calculation of light interception (Lai et al. 2000, Krinner et al. 2005, Verbeeck et al. 2011).
This simplification of photosynthesis modelling should be
revised and a number of important architectural phenotypic
plant characteristics, especially leaf angle, should be incorporated in functional–structural plant models due to the significant vertical variation of photosynthesis in the canopy (Sarlikioti
© The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Chlorophyll fluorescence, leaf angle and optics 203
et al. 2011). Most plant models use the assumption of spherical leaf angle distribution for the leaf canopy, but this approach
may cause an underestimation of vertically transmitted light
(Stadt and Lieffers 2000), meaning that the calculations for
the daily amount of photosynthesis are also biased (Sarlikioti
et al. 2011).
Another assumption that is occasionally used in modelling
exercises for estimating the photosynthetic performance of
plants is that the non-destructive and timesaving fluorescence measurements of electron transport rate in the
absence of photorespiration relate well to the quantum efficiency of photosynthesis measured gasometrically (Genty
et al. 1989, Edwards and Baker 1993). However, numerous
factors can change the shape of this relationship (Seaton
and Walker 1990, Edwards and Baker 1993, Evans 1993,
Maxwell and Johnson 2000, Tsuyama et al. 2003, van der
Tol et al. 2009). Tsuyama et al. (2003) showed that the relationship between assimilation of CO2 and fluorescencebased measurements of electron transport rate is affected
the most by chlorophyll content and leaf structure. They proposed that the measurements of gas exchange and chlorophyll fluorescence detect signals from different populations
of chloroplasts in a leaf. Already, Evans et al. (1993) proposed that fluorescence-based photosynthesis estimates
from leaf adaxial side possibly provide underestimations,
since the measuring beam does not penetrate deep enough
into the leaf. Tsuyama et al. (2003), on the other hand,
pointed out that the mere spatial restriction of the origin of
the fluorescence measure as such is not enough to cause
deviations from linearity in plots of quantum efficiency measured by fluorescence versus quantum efficiency of CO2, but
the non-linearity clearly depends on the chloroplasts at the
illuminated surface.
Instead of using the common assumptions of spherical leaf
angle distribution and homologous internal morphology of
leaves, we investigate the natural variation in leaf angles,
chlorophyll content, structure and optical traits of leaves
resulting from the different light environments in a canopy
vertical gradient and the corresponding leaf fluorescence
from alternate leaf surfaces. Since ordinary chlorophyll fluorescence equipment measures photosynthetic properties
within a relatively thin leaf layer, measurements from alternate
leaf surfaces should reflect the intraleaf gradient of acclimation. We wanted to check a previously raised hypothesis, that
the difference in fluorescence parameters measured from
alternative surfaces of the leaf depends on the difference in
average photon flux density at the two surfaces and should
therefore be a function of leaf angle (Myers et al. 1997,
Tsuyama et al. 2003). We emphasize the effects of two
light gradients: (i) vertical light gradient within a canopy and
(ii) light gradient inside a leaf between the abaxial and adaxial surfaces.
Materials and methods
Study area and canopy heights
Material for the study was collected from a deciduous mixed
stand at Järvselja (58°22′N, 27°20′E, elevation 38–40 m),
Estonia in July to August 1999. The canopy, with a total height
of 27 m, had two distinct layers: the upper layer consisting
mainly of dominating Populus tremula L. foliage (19–27 m) and
a subcanopy layer of Tilia cordata Mill. (7–17 m). Leaves were
mature and did not show visible signs of senescence. Trees
were reached from permanent scaffoldings (height 25 m).
Measurements were carried out for the top and bottom layers
of both species (at heights of approximately 11 and 16 m for
Tilia and 19 and 26 m for Populus ±1 m variation inside all
height classes depending on branches accessible from the
scaffolding).
Light conditions
Long-term light conditions at these four heights of canopy (11
and 16 m in Tilia, and 19 and 26 m in Populus foliage) were
estimated by hemispherical photographic technique using
Nikon Coolpix 900 digital camera equipped with a Nikon
Fisheye Converter and analysis software Winscanopy 2.0
(Quebec, Canada). Light conditions were estimated using photographs taken at one time from above 10 leaves from each
measured canopy layer (the same leaves that were used for
fluorescence and pigment analysis). Direct radiation was calculated using analysis software from canopy gap fractions along
solar tracks during 2-month intervals beginning from the summer solstice. Daily diffuse radiation was estimated from the
proportion of visible sky, assuming a standard overcast sky
model (Anderson 1966). As daily diffuse radiation was in good
relation with direct radiation (r = 0.85, P < 0.001), canopy light
conditions in present study were characterized by estimations
of diffuse radiation, which showed less spatial and temporal
variability than estimations of direct radiation.
Leaf angle distribution
The zenith angles of leaf blade to normal were measured
using a protractor. One hundred leaves were measured in
each canopy layer. For each foliage height, leaf inclination
angle distribution was fitted using an ellipsoidal function,
which is based on the assumption that the distribution of leaf
angles in a canopy is identical to the angles of normal to small
area elements on the surface of an ellipsoid (Campbell 1986).
A single parameter, x = b/a, is required to describe the shape
of the distribution; b is the horizontal semi-axis of the ellipsoid, and a is the vertical semi-axis. When x = 1, the ellipsoidal
distribution becomes a spherical distribution, when x > 1, the
ellipsoidal distribution becomes an oblate spheroid. First,
theo­retical leaf angle distributions were calculated for various
x values. To find the best theoretical approximation (and
Tree Physiology Online at http://www.treephys.oxfordjournals.org
204 Mänd et al.
­respective x) the experimental data were fitted by minimizing
the χ2 parameter. Inclination angles were measured also in the
set of leaves used later in chlorophyll fluorescence and pigment measurements.
Chlorophyll fluorescence
Fluorescence yield from both leaf sides of at least 10 leaves
from each canopy layer was measured in situ using a portable
pulse-modulated fluorometer (PAM-2000, Heinz Walz GmbH,
Effeltrich, Germany). Minimum fluorescence yield (F0) and
maximal fluorescence yield (Fm) were measured after a darkening period of 10 min. PAM-2000 internal halogen actinic
light source was switched on for 5 min on an intensity level of
1000 µmol m−2 s−1. Then fluorescence yield Ft, maximal (F′m)
and minimum fluorescence (F′0) were measured.
Quantum yield of dark-adapted leaves (Fv/Fm) and lightadapted leaves (ΦII) was calculated from the equations of
Genty et al. (1989). Photochemical quenching (qP) and nonphotochemical quenching (NPQ) were calculated according to
Bilger and Björkman (1990). All fluorescence measurements
were carried out between 9 AM and 7 PM (measuring period
shortened with the shortening of natural daylight period).
Measurements were randomized so that leaves from each
layer were measured at various times of the day and on various days of the measurement period. The same protocol was
used for measuring both leaf adaxial and abaxial sides. The
time dependency of measured parameters was checked and
temperature adjustment was applied to the fluorescence data
according to Niinemets et al. (1999). As no significant correlations of fluorescence parameters with time or temperature
were found (data not shown), the time and temperature
dependency of fluorescence parameters was neglected in this
study.
Leaf mass per area
After field measurements were finished, all leaves measured in
situ were harvested and the area of each leaf was measured
using a desk-scanner (ScanJet 4c, Hewlett Packard, Palo Alto,
CA, USA) and an in-house computer program. Each leaf was
dried to constant mass at 80 °C and leaf mass was measured
for the determination of leaf mass per area (LMA) (g m−2).
Pigment analysis
Leaf-discs (1 cm2) were cut from every harvested leaf (at least
10 leaves of fluorescence measurements and 5 leaves of
optics measurements from each canopy layer) and stored at
−18 °C. Leaf chlorophyll was extracted in 80% aqueous acetone buffered with sodium phosphate (2.5 mM, pH 7.8), measured spectrophotometrically (S2000, Ocean Optics, Dunedin,
FL, USA) and calculated according to the equation of Porra
et al. (1989). A phosphate buffer was used to avoid the transformation of chlorophylls into pheophytins.
Tree Physiology Volume 33, 2013
Leaf optics
Due to technical limitations, leaf optics were measured in
another set of leaves in the year 2000. Five leaves at the same
heights as the measurements of the previous year were collected. Leaf transmittance and reflectance spectra were measured with an integrating sphere (ISP-80-8-R, Ocean Optics)
and fibre optic spectrophotometer (S2000, Ocean Optics). We
used white reflectance standard (WS-2, Top Sensor Systems,
Eerbeek, The Netherlands) for reflectance measurements.
Although a wider range of spectra was measured and analysed,
we have only shown reflectance parameters which were averaged from the wavelength range of 655–665 nm (red spectral
region—the range of the strongest absorption by chlorophyll)
and 550–560 nm (green spectral region) for the sake of clarity.
Sample absorption at the same wavelength ranges was calculated as absorptance = (1 − reflectance − transmittance). Leaf
spectral properties were measured from both leaf sides. For
each leaf LMA and pigments content were determined as
described above. Leaf mass per area and pigments content did
not differ significantly between years (data not shown).
Results
Light distribution and leaf characteristics
In a joined canopy of T. cordata (shade tolerant) and P. tremula
(light-demanding), relative diffuse radiation increased with
increasing height ranging from 32% in the lower layer of T.
cordata measured at 11 m to 68% in the upper layer of P.
tremula, measured at 26 m (Figure 1a). However, light conditions between the two intermediate layers—the top layer of T.
cordata at a height of 16 m and the bottom layer of P. tremula
at 19 m—did not differ significantly (Figure 1a), probably due
to the forest canopy structure. At a height of 16–19 m there
was a leafless gap between the canopies of P. tremula and T.
cordata.
Leaf mass per area and chlorophyll content per leaf area
(Chls) were highest in the top layer of P. tremula and lowest in
the bottom layer of T. cordata; however, within-species differences in LMA and Chls between the two height levels were not
statistically significant (Figure 1b, c). Populus tremula had two
times higher LMA than T. cordata (Figure 1b).
When the leaf angle distribution of a wider selection of leaves
(N = 100 in each canopy layer) was approximated with an ellipsoidal function, we found parameter x decreasing with height in
both species (Figure 2). In the upper layer of P. tremula, leaves
were more pendant (x = 0.72), while in the lower layer of P.
tremula, an almost spherical distribution of leaf inclination angles
was detected (x = 0.96). Leaves of shade-tolerant T. cordata
were in general more horizontal (x = 4.8 in the top layer and
x = 5.1 in the bottom layer of T. cordata). The inclination angle of
the smaller set of leaves used for ­chlorophyll fluorescence and
Chlorophyll fluorescence, leaf angle and optics 205
Figure 1. ​(a) Relative diffuse radiation, (b) LMA, (c) leaf chlorophyll
per area; (d) quantum yield of light-adapted leaves, (e) photochemical
and (f) non-photochemical quenching plotted against height from
ground (mean with SD, N = 10). Chlorophyll fluorescence parameters
(d–f) are measured from leaf adaxial sides. Data of T. cordata were
gathered at heights of 11 and 16 m and that of P. tremula at heights 19
and 26 m. Height groups with the same letter were not significantly
different (Kruskal–Wallis ANOVA by ranks: P > 0.05).
pigment measurements, was highest in the upper layer of P.
tremula and lowest in the upper layer of T. cordata (Figure 2),
similar significant difference between leaf angles of different
species was found also in wider set of leaves (Kruskal–Wallis
analysis of variance (ANOVA): P < 0.05), but in either of the
datasets differences between two heights of single species
were not statistically significant. One could expect the smallest
leaf inclination angles in the bottom layer of T. cordata, as was
the case in the larger set of leaves used for angle distribution
calculations (fluorescence measurements were performed on a
smaller set of leaves, explanation in Materials and methods,
Figure 2). However, it appeared that the smaller random selection of leaves in the lowest canopy layer had slightly higher average inclination angles than those in the upper layer of T. cordata
(Figure 2), but the difference was not statistically significant.
Chlorophyll fluorescence
Chlorophyll fluorescence parameters measured from the adaxial side of the leaf revealed significant differences between
species. Quantum yield of light-adapted leaves (ΦII) and photochemical quenching coefficient (qP) were six times higher in
the P. tremula than in the T. cordata canopy (Figure 1d–f). It is
also important to note that these fluorescence parameters differed between canopy layers at a height of 16 m (the top layer
Figure 2. ​Leaf inclination angle distribution in top and base layers of T.
cordata and P. tremula. Average inclination angles of the leaves that
were used for physiology and LMA measurements (N = 10) are shown
as αs. Leaf average inclination angle if a larger set of leaves were measured (N = 100) are shown as α . x is the parameter of fitted ellipsoidal
distribution.
of T. cordata) and at a height of 19 m (the bottom layer of P.
tremula) (Figure 1d–f), where light conditions did not differ
(Figure 1a), indicating that differences in ΦII and qP are species specific. Non-photochemical quenching had a maximal
value in the top layer of T. cordata and a lowest value in the top
layer of P. tremula (Figure 1f). Non-photochemical quenching
also differed between top and bottom layer of the T. cordata
canopy, but not in the canopy of P. tremula. Maximum photochemical efficiency of PSII (Fv/Fm) did not differ between species or different canopy layers.
Leaf adaxial/abaxial side fluorescence
We looked for differences between leaf adaxial and abaxial side
measurements of chlorophyll fluorescence. We found that ΦII
measured from leaf adaxial side was 1.6 times higher in
Tree Physiology Online at http://www.treephys.oxfordjournals.org
206 Mänd et al.
P. tremula and two times higher in T. cordata compared with
abaxial side measurements, but the difference between species
was not significant (Figure 3), nor did we find significant differences in adaxial/abaxial ΦII between measured canopy layers of
either species. However, ΦII ratio of different leaf sides had a
significant negative correlation with leaf angle when data from
leaves of both species were pooled (Figure 3). The correlation
remained significant for the P. tremula canopy, but was not significant in the T. cordata canopy (Figure 3). qP measured from
leaf adaxial side was significantly larger than that measured
from the abaxial side in both species and, like the ΦII ratio of
different leaf sides, qP ratio was also n­ egatively correlated with
leaf angle (Figure 3). There was a significant species-specific
difference, since the value of qP ratio was 1.5 times higher in
T. cordata than in P. tremula (Figure 3). Non-photochemical
quenching measured from leaf adaxial side was significantly
larger than that measured from the abaxial side in the canopy of
T. cordata, while in the canopy of P. tremula NPQ did not differ
between alternate leaf sides (Figure 3). However, NPQ ratio of
different leaf sides was clearly negatively correlated to leaf
angle (Figure 3). If we looked at different species separately,
the correlation remained significant in the canopy of P. tremula,
but not in T. cordata (Figure 3). In the canopy of T. cordata NPQ
ratio had a significant positive correlation with relative diffuse
radiation (r = 0.62, P < 0.001) and LMA (r = 0.52, P < 0.05). No
significant differences were found in adaxial/abaxial Fv/Fm when
comparing different species. Our data did not reveal significant
differences between different canopy layers of a single species
when we looked at the adaxial/abaxial ratios of any fluorescence parameters. It was apparent that these fluorescence
parameters (qP and ΦII) that had the strongest differences
between species (Figure 1), also had the largest average adaxial/abaxial ratio (Figure 3).
Leaf optics
Figure 3. ​Relationships between ratio of leaf adaxial and abaxial side
chlorophyll fluorescence parameters and leaf angles. Black dots mark
data at two different heights of T. cordata canopy and empty dots are
for P. tremula. Correlation coefficients are shown. Mean and SE
(N = 20) of respective chlorophyll fluorescence ratios of both species
are shown in figures on the right. Asterisks denote a significant difference between leaf sides (Wilcoxon signed rank test: P < 0.001).
Species with the same letter were not significantly different (Kruskal–
Wallis ANOVA by ranks: P > 0.05).
Tree Physiology Volume 33, 2013
Spectral measurements showed that leaves of T. cordata
absorbed more light than the leaves of P. tremula (Table 1),
despite having a lower chlorophyll content (Figure 1c). When
illuminated from the abaxial side, leaf absorptance was lower
and reflectance was higher compared with the adaxial side in
both species (Table 1). Differences between optical properties
of adaxial and abaxial leaf surfaces were larger in P. tremula
compared with T. cordata (Table 1).
Intraspecifically reflectance and absorptance were better
related to LMA than Chls for both species and both wavelength
regions (Table 2, Figure 4). However, chlorophyll content was
clearly a better predictor for leaf optical properties when both
species were pooled (Table 2, Figure 4). Chls and LMA were
strongly correlated in both species (P. tremula r = 0.79, P < 0.05;
T. cordata r = 0.65, P < 0.05). When both Chls and LMA were
incorporated into multiple regression models, LMA still had a
significant (P < 0.05) positive effect on leaf adaxial side absorptance in both wavelength regions (red and green) and within
both species. Due to the strong effect of species on the relationship between leaf absorptance and LMA (Type III general
linear model on leaf adaxial absorptance: species × LMA interaction P < 0.005), LMA, which was a good estimate for leaf
optical parameters of individual species, was not suitable when
species were pooled (Figure 4).
Discussion
Leaf adaxial/abaxial side fluorescence
Our results showed significant differences in leaf adaxial/­
abaxial side ratio of fluorescence parameters, referring to
Chlorophyll fluorescence, leaf angle and optics 207
Table 1. ​Reflectance and absorptance measured from alternate leaf surfaces and the ratio between leaf adaxial and abaxial side reflectance and
absorptance averaged from wavelength range of 655–665 nm (red spectral region) and 550–560 nm (green spectral region). Mean and SD are
shown (N = 5). Asterisks denote a significant difference between leaf sides (Wilcoxon signed rank test: P < 0.001). Species with the same letter
were not significantly different (Kruskal–Wallis ANOVA by ranks: P > 0.05).
Leaf adaxial side
Green reflectance
Red reflectance
Green absorptance
Red absorptance
Leaf abaxial side
Adaxial/abaxial
Tilia cordata
Populus tremula
Tilia cordata
Populus tremula
Tilia cordata
Populus tremula
0.12 ± 0.008a
0.05 ± 0.004a
0.73 ± 0.04a
0.92 ± 0.01a
0.13 ± 0.02b
0.05 ± 0.006a
0.72 ± 0.03a
0.90 ± 0.009b
0.18 ± 0.01a
0.12 ± 0.01a
0.65 ± 0.04a
0.86 ± 0.02a
0.24 ± 0.01b
0.10 ± 0.01b
0.61 ± 0.02b
0.83 ± 0.01b
0.63 ± 0.05a ***
0.48 ± 0.05a ***
1.12 ± 0.02a ***
1.07 ± 0.01a ***
0.54 ± 0.06b ***
0.43 ± 0.07b ***
1.18 ± 0.04b ***
1.08 ± 0.02b ***
Table 2. ​Correlations between leaf optical parameters measured from leaf adaxial side (reflectance and absorptance averaged from wavelength
range of 655–665 nm—red spectral region, and 550–560 nm—green spectral region) and LMA and chlorophyll content per leaf area (Chls).
Asterisks denote significant correlations where ***means P < 0.001, **P < 0.01 and *P < 0.05, ns means not significant values of P.
Tilia cordata
Green reflectance
Red reflectance
Green absorptance
Red absorptance
Populus tremula
Both species
LMA
Chls
LMA
Chls
LMA
Chls
−0.80***
−0.65**
0.94***
0.86***
−0.75***
ns
0.80***
0.64**
−0.74***
−0.47*
0.80***
0.72***
−0.59**
ns
0.66**
0.55*
ns
ns
ns
−0.43**
−0.37*
ns
0.68**
ns
Figure 4. ​Relationships between leaf absorptance measured from leaf adaxial and abaxial sides (absorptance averaged from wavelength range of
655–665 nm—red spectral region, and 550–560 nm—green spectral region) and LMA and chlorophyll content per leaf area (Chl). Black dots
representing T. cordata, empty dots P. tremula. Corresponding statistical parameters (mean, SD and correlation coefficients) are shown in Tables 1
and 2.
v­ arying intraleaf light acclimation profiles of chloroplasts at different canopy positions while the differences depended significantly on species. Similarly light adaption profile along canopy
light availability gradient appeared to be strongly species
dependent, since chlorophyll content, LMA and chlorophyll a
fluorescence (ΦII and qP) measured from leaf adaxial side
appeared to differ mainly between species.
Our findings, that ΦII and qP measured from leaf abaxial side
were lower than for the adaxial side, indicated that chloroplasts
near the abaxial surface were more shade adapted and had
lower photosynthetic capacities than chloroplasts near the
adaxial leaf surface of both species. Furthermore, as the ratio
of adaxial/abaxial ΦII and qP of leaves varied through the forest
canopy, the interpretation of chlorophyll a fluorescence signals
that are usually measured on the adaxial sides of leaves
becomes complicated. The origin of the chlorophyll fluorescence signal depends both on the depth of penetration and on
the absorption of irradiance within the leaf and also on the
emission and re-absorption of fluorescent light, and it changes
with differences in the excitation wavelength (Vogelmann and
Han 2000, Buschmann 2007, Peguero-Pina et al. 2009), chlorophyll content (Evans 1999, Vogelmann and Evans 2002) and
leaf structure (Cui et al. 1991). As a result, chlorophyll fluorescence signals are derived mainly from the surface layers, since
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208 Mänd et al.
even high-light-grown leaves accumulate a significant proportion of low-light-acclimated chloroplasts due to leaf internal
light gradient. When we further investigated the natural variation in acclimation profiles of chloroplasts (by measuring leaf
adaxial/abaxial ratios of fluorescence) we found that differences in ΦII and qP measured from alternate leaf sides were
smaller in more vertically oriented leaves. As leaf inclination
angle controls the proportion of irradiance that strikes the
upper and lower leaf surfaces, reducing excess light on the
adaxial leaf surface and allowing more light to be utilized by
chloroplasts located near the abaxial surface, more vertical leaf
inclination angle indeed should reduce the adaxial/abaxial ratio
of fluorescence parameters.
Traditionally, the leaf has been taken as a photosynthesizing
unit and in several ecological plant or community models the
fact that a leaf consists of a population of chloroplasts with
variable properties resulting from intraleaf light gradient has
been ignored (de Pury and Farquhar 1997, Kull 2002). Our
results suggest the need for more extensive investigations on
actual intraleaf light adaptation profiles of chloroplasts and
their relationship to fluorescence parameters measured from
adaxial side of leaf in order to avoid generating systematic
errors where different correlative relationships are extrapolated over whole canopies or whole ecosystems. This is
because differences in the light conditions of different subpopulations of chloroplasts can change the correlation between
whole-leaf photosynthetic properties and the photosynthetic
properties of individual chloroplasts near the leaf surface
(Peguero-Pina et al. 2009). Thus, when comparing fluorescence signals derived from the surface layers with data that are
not derived from the same sub-population of chloroplasts, for
example, the whole-leaf gas-exchange measurements, some
caution is needed. The origin of fluorescence signal becomes
especially important in the light of recent attempts that are
made with fluorescence imaging of whole plant (Baker 2008)
or assessing terrestrial photosynthesis from space (Grace et al.
2007). Our results also indicate that the population of chloroplasts which are represented by fluorescence measurements
might vary in multi-species systems, not merely because of
different light intensity histories of single leaves, but also
because of different within-leaf light-acclimation strategies of
species (Uemura et al. 2006), which affect the steepness of
leaf-internal light absorption gradient of leaves of different
species.
Not all fluorescence parameters presented similar variations
in adaxial/abaxial ratios within the forest canopy. Nonphotochemical quenching measured from adaxial and abaxial
surface did not differ in leaves of P. tremula, which was in
accordance with our results that NPQ was also not related to
vertical canopy light gradient in P. tremula. In the canopy of
T. cordata, NPQ increased with increasing irradiance in the
canopy light gradient, and we found a similar response to leaf
Tree Physiology Volume 33, 2013
internal light gradient. In the leaves of T. cordata, NPQ measured from the adaxial leaf surface was 1.5 times higher than
that from the abaxial surface. Dark-adapted maximum quantum
yield (Fv/Fm) did not differ between alternate leaf sides (data
not shown), indicating that chloroplasts near adaxial and abaxial leaf surfaces were adequately acclimated to their respective
light conditions. Our measurements suggest that, if we use
fluorescence-based measurements of leaf surface for estimations of whole-leaf performance, more attention should be paid
to possible errors in the fluorescence parameters that also
have the strongest gradients in the plant canopy, or largest differences between species.
Leaf optics
Leaf optical properties are often used to describe the combined impact of leaf anatomy and biochemistry on light penetration through the leaf (Govaerts et al. 1996). Our
measurements showed that both species had higher reflectance and lower absorptance when illuminated from leaf abaxial side, compared with adaxial side in both wavelengths: the
difference between leaf sides was greater for P. tremula, which
means that the change in leaf angle that allows a greater proportion of light to strike the abaxial surface results in a greater
reduction in excess light through enhanced reflectance from
the abaxial surface in P. tremula. In their multi-species synthesis Falster and Westoby (2003) showed that steeper leaf
angles function more to reduce exposure to excess light, rather
than to maximize carbon gain. Similarly, our fluorescence measurements revealed that the large differences in reflectance of
alternate sides of P. tremula leaves helped to counterbalance
the effect of asymmetric internal anatomy of dorsiventral leaves
by reducing the inhibitive effect of excess light on the abaxial
side of the leaf and resulted in smaller differences in fluorescence parameters measured from leaf adaxial and abaxial surfaces. For different species the means of enhancing leaf
reflectance may vary from changed epidermal structures at the
leaf surface, such as leaf hairs (Ehleringer et al. 1976) or
waxes (Cameron 1970), to different anatomical features, like a
separation between the epidermis and mesophyll (Vogelmann
1993).
Light absorption gradient within a leaf depends strongly on
leaf chlorophyll content and leaf anatomy, including the
arrangement of chloroplasts (Brodersen and Vogelmann
2010). Based on the chlorophyll content, it would be expected
that the leaves of P. tremula and T. cordata should have relatively similar absorptance, since, although the leaves of
T. ­cordata had a slightly lower chlorophyll content per leaf area,
the difference was minimal. Indeed, we found that P. tremula
and T. cordata had similar absorptances in the green spectral
region when illuminated on the adaxial leaf surface. However,
at a strongly absorbing wavelength (red spectral region)
absorptance is unlikely to vary much with chlorophyll, which is
Chlorophyll fluorescence, leaf angle and optics 209
c­ onsistent with our findings of a better correlation of chlorophyll with absorptance measured for the green spectral region.
As we found a significant individual effect of LMA, and
LMA × species interaction on leaf absorptance, we assumed
that leaf structural parameters (for example, the arrangement of
chloroplasts), which determine the light profile inside the leaf,
can be best estimated by LMA for individual species (also
reported by Souza and Válio 2003). As P. tremula had somewhat lower absorptance, but higher LMA, when compared with
T. cordata, this means that leaf internal light gradient had to be
less steep in leaves of P. tremula. This could influence the depth
within the leaf, where the fluorescence signal is retrieved. The
finding that P. tremula leaves somehow managed to more efficiently homogenize within-leaf light environment can be attributed to the sieve effect (Das et al. 1967), since the pigment
distribution in leaves of P. tremula may be somewhat different
from T. cordata. However, our optical measurements revealed
that the differences between leaf sides were larger for P.
tremula, while fluorescence parameters differed more between
the alternate leaf sides of T. cordata. Therefore, the difference in
optical properties between leaf sides did not cause all of the
difference in fluorescence. It is possible that a fraction of the
differences in leaf adaxial/abaxial fluorescence ratios can
account for the specifics of fluorescence measurements, since,
a less steep internal light gradient in P. tremula means that a
larger fraction of fluorescence light is also reabsorbed on its
way back to the measuring instruments. Buschmann (2007)
has shown that chlorophyll fluorescence, which is generated
deep within the mesophyll, would be partially re-absorbed
(especially measurements made at 680 nm). This re-absorption
is caused by the overlapping of the short-wavelength range of
the chlorophyll fluorescence emission spectrum with the longwavelength range of the chlorophyll absorption spectrum. As a
result, our study indicates the need for more complex multispecies research on factors that could cause variations in the
origin of the fluorescence signal in a dorsiventral leaf.
Acknowledgements
Dedicated to the memory of Professor Olevi Kull (22 June
1955 to 31 January 2007). We are grateful to Ingmar Tulva for
his valuable help in providing us with data on leaf inclination
angle distribution.
Conflict of interest
None declared.
Funding
The study has been supported by the Estonian Ministry of
Education and Science (grant SF0180025s12), the European
Commission through the European Regional Fund (the Center of
Excellence in Environmental Adaptation), Estonian Science
Foundation (grant ETF9186) and the European Social Fund
(programme Mobilitas grant MJD 122). J.P.’s research was
­supported by Spanish Government grants CGL2010-17172/BOS
and Consolider-Ingenio Montes CSD2008-00040, and by
Catalan Government grant SGR 2009-458.
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