Photosynth Res (2007) 93:235–244
DOI 10.1007/s11120-007-9174-0
RESEARCH ARTICLE
Chlorophyll fluorescence imaging of photosynthetic activity in sun
and shade leaves of trees
Hartmut Karl Lichtenthaler Æ Fatbardha Babani Æ
Gabriele Langsdorf
Received: 21 August 2006 / Accepted: 11 April 2007 / Published online: 8 May 2007
Springer Science+Business Media B.V. 2007
Abstract The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease
ratio RFd (as indicator of photosynthetic rates) of green sun
and shade leaves of three broadleaf trees (Platanus acerifolia
Willd., Populus alba L., Tilia cordata Mill.) were compared.
Sun leaves were characterized by higher levels of total
chlorophylls a + b and total carotenoids x + c as well as
higher values for the weight ratio chlorophyll (Chl) a/b (sun
leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c)
(with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves).
Sun leaves exhibited higher photosynthetic rates PN on a leaf
area basis (mean of 9.1–10.1 lmol CO2 m–2 s–1) and Chl
basis, which correlated well with the higher values of stomatal conductance Gs (range 105–180 mmol m–2 s–1), as
compared to shade leaves (Gs range 25–77 mmol m–2 s–1;
PN: 3.2–3.7 lmol CO2 m–2 s–1). The higher photosynthetic
rates could also be detected via imaging the Chl fluorescence
decrease ratio RFd, which possessed higher values in sun
leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In
addition, via RFd images it was shown that the photosynthetic
activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in
sun leaves than in shade leaves.
adaptation CO2 assimilation PN rates Stomatal
conductance Gs
Abbreviations
a+b
a/b
(a + b)/(x + c)
c
Chl
Fp, Fo, and Fs
Fd
Fp
Fv/Fm and Fv/Fo
Gs
PN
RFD
Keywords Carotenoids Chlorophylls Chl a/b ratio Chlorophyll fluorescence decrease ratio RFd Chloroplast
x+c
x
Total chlorophylls
Ratio of chlorophyll a to b
Weight ratio of chlorophylls to
carotenoids
Carotenes
Chlorophyll
Maximum, initial, and steady Chl
fluorescence
Fluorescence decrease from Fp to Fs
Maximum Chl fluorescence at nonsaturating light conditions
Maximum quantum yield of
photosystem II photochemistry
measured in the dark adapted, nonfunctional state 2 of the photosynthetic
apparatus
Stomatal conductance measured at
light saturation
Net photosynthetic CO2 assimilation
measured at light saturation
Chl fluorescence decrease ratio
measured in the red band near 690 nm
Total carotenoids
Xanthophylls
H. K. Lichtenthaler (&) F. Babani G. Langsdorf
Botanisches Institut, University of Karlsruhe, Kaiserstr.
12, 76133 Karlsruhe, Germany
e-mail: [email protected]
Introduction
F. Babani
Biological Research Institute, Academy of Sciences, Tirana,
Albania
Sun and shade leaves of trees as well as high- and low-light
plants considerably differ in their relative composition of
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photosynthetic pigments, electron carriers, their chloroplast
ultrastructure, and their photosynthetic rates (Boardman
1977; Lichtenthaler 1981; Meier and Lichtenthaler 1981;
Wild et al. 1986; Givnish 1988; Anderson et al. 1995).
Leaves that develop under high irradiance (sun leaves and
high-light leaves) possess sun-type chloroplasts that are
adapted to high rates of photosynthetic quantum conversion in comparison to shade leaves or leaves from low-light
plants. Sun leaves possess a higher photosynthetic capacity
on a leaf area basis, exhibit higher values for the ratio Chl
a/b, a much lower level of light-harvesting Chl a/b proteins
(LHCII), and a lower stacking degree of thylakoids than
shade leaves and low-light plants with their low-irradiance
shade-type chloroplasts (Lichtenthaler et al. 1981, 1982,
1984). The chloroplasts’ adaptation response to high- and
low-light quanta fluence rates and the major differences
between both chloroplast types have recently been summarized by Lichtenthaler and Babani (2004), a review that
gives access to additional literature in this field.
The differences in photosynthetic capacity of sun leaves
and shade leaves have usually been determined by measuring the CO2 fixation rates PN of leaves or of leaf parts at
light saturation. This yields one integrated value of PN per
measurement, however, gradients or stress-induced inhomogeneities in photosynthetic activity across the leaf area
cannot be detected in this way. The photosynthetic function
can also be judged via various Chl fluorescence ratios that
are determined by the Chl fluorescence induction kinetics
of dark-adapted leaves (Lichtenthaler 1988; Krause and
Weis 1991; Govindjee 1995). Both ratios, Fv/Fm (Kitajima
and Butler 1975) and its related more sensitive form Fv/Fo
(Babani and Lichtenthaler 1996), are a measure of the
potential photosystem II efficiency of dark-adapted leaves.
They only reflect a small portion of the leaf chloroplasts, in
particular those of the illuminated upper leaf side, but they
do not reflect the photochemical activity of all leaf chloroplasts (Lichtenthaler et al. 2005a). In contrast, the Chl
fluorescence decrease ratio RFd (Lichtenthaler 1988),
determined at light saturation, is an indicator of the photosynthetic quantum conversion capacity of light-adapted
leaves at steady-state conditions (Tuba et al. 1994; Lichtenthaler and Miehé 1997) and is directly correlated to the
net CO2 fixation rates of leaves (Lichtenthaler and Babani
2004). Moreover, it has been shown that via imaging of the
RFd values of leaves one can see gradients in photosynthetic activity across the leaf area, detect water stress
conditions (Lichtenthaler and Babani 2000), and recognize
differences in photosynthetic activity between sun leaves
and shade leaves of beech (Lichtenthaler et al. 2000).
In the present study, sun and shade leaves of three different broadleaf trees (Platanus, Populus, Tilia) were
chosen to comparatively investigate the differences in the
photosynthetic characteristics (pigment content, PN rates,
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Photosynth Res (2007) 93:235–244
photochemical activity) in fully developed leaves during
early July before summer stress events (heat and sun
exposure combined with water deficit) reduce the physiological state of leaves. Particular emphasis was placed on
the imaging of the photosynthetic activity of leaves via the
Chl fluorescence decrease ratio RFd in order to determine
not only the differences between sun leaves and shade
leaves, but, in addition, to evaluate if the photosynthetic
activity is evenly distributed across the leaf area, if possible
gradients or local inhomogeneities exist, and to find out if
such gradients or inhomogeneities occur both in sun leaves
and in shade leaves.
Materials and methods
Plant material and growth conditions
For our investigations fully developed sun leaves and shade
leaves were taken from 30- to 60-year-old trees of platanus
(Platanus acerifolia Willd.), linden (Tilia cordata Mill.)
and poplar (Populus alba L.) at the Karlsruhe University
campus that is part of the Karlsruhe Palace Gardens. At this
particular location in the Rhine valley plains all investigated trees had the same soil and water conditions and were
exposed to the same climate. On sunny days the shade
leaves in the inner tree part received ca. 80 lmol photons m–2 s–1 PAR, whereas sun leaves were exposed to a
maximum PPFD from 1700 lmol m–2 s–1 to 2000 lmol
m–2 s–1. The measurements were either performed with
samples from three different trees (pigments, imaging), or
two different trees (porometer) for each tree species.
Pigment determination
The photosynthetic pigments, chlorophylls a and b as well
as total carotenoids x + c, were extracted with 100% acetone. Their levels were determined spectrophotometrically
(with a Shimadzu UV–VIS scanning spectrophotometer
UV-2001 PC) using the extinction coefficients and equations redetermined by Lichtenthaler (1987), see also
Lichtenthaler and Buschmann (2001) for details. From the
pigment levels the weight ratios of pigments, Chl a/b and
Chls/carotenoids (a + b)/(x + c), were determined. They
significantly differ for sun leaves and shade leaves. The
pigment values are the mean of at least six determinations
from three trees.
Porometer gas exchange measurements
A branch with the desired leaves was cut from the plant,
and the cut end was immediately re-cut under water to
remove and prevent xylem embolisms. The light-induced
Photosynth Res (2007) 93:235–244
photosynthetic CO2 fixation rates PN (lmol CO2 m–2 s–1)
and stomatal conductance (Gs also termed gH20) were
measured in pre-darkened (20 min) leaves using a CO2/
H2O-porometer system (Walz, Effeltrich, Germany). The
leaves were irradiated by white light with
1500 lmol photons m–2 s–1 PAR that saturated with
respect to the PN-rates. The latter ranged from ca. 300 to
400 (shade leaves) and ca. 700 to 900 lmol photons m–2 s–
1
(sun leaves). Stable maximum PN-rates were usually
reached between 24 min and 30 min after onset of
illumination.
Chlorophyll fluorescence imaging
The chlorophyll (Chl) fluorescence induction kinetics
(Kautsky effect) of pre-darkened leaves (30 min) were
measured at the red Chl fluorescence band (k = 690 nm)
using the Karlsruhe flash-lamp fluorescence imaging system (FL-FIS) as described by Lichtenthaler and Babani
(2000), and Lichtenthaler et al. (2000). A Xenon flashlamp (300 W, Cermax, Perkin Elmer Optoelectronics,
Cambridge, UK) and a blue filter (Corning No. 9782; range
370–600 nm; kmax 465 nm) were applied to induce the red
Chl fluorescence. The Chl fluorescence was excited and
sensed at the upper (adaxial) leaf side. Via computer-aided
data-processing false color images of the measured Chl
fluorescence intensity were obtained, whereby blue was the
lowest (zero) and red the highest fluorescence. We applied
an uniformity correction to eliminate the effect of inhomogeneous radiation distribution by the xenon lamp. For
the uniformity correction the UV-A excited fluorescence at
440 nm of a white sheet of paper was determined, and the
software corrected the leaves’ fluorescence by means of
this uniformity image (for further details see Lichtenthaler
et al. 2005b). This fluorescence imaging system was
applied in the present investigation to determine the images
of the Chl fluorescence decrease ratio RFd (see below).
In contrast to the PAM-type chlorophyll fluorometers
that work with pulsed modulated light and a non-saturating
actinic light (Schreiber 1986; Schreiber et al. 1986; Genty
et al. 1989), the chlorophyll fluorescence decrease ratio RFd
is determined from the Chl fluorescence induction kinetics
measured at saturating continuous white light (Lichtenthaler and Miehé 1997; Lichtenthaler et al. 2005a). Due to
the application of saturating excitation light some typical
Chl fluorescence parameters of the PAM fluorometer, such
as Fo, Fv¢, Fm¢, the ratio DF/Fm¢, or the quenching coefficients qP and qN, cannot be measured when determining
the RFd values due to the fact that the excitation light is
saturating, and additional saturating light pulses (as given
in the PAM fluorometer) do not increase the Chl fluorescence. An advantage of using either saturating or almost
saturating excitation light when measuring the Chl
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fluorescence induction kinetics of leaves is the fact that the
Chl fluorescence signals determined are representative of
the signals of the total leaf chloroplasts. On the other hand,
the PAM-type Chl fluorescence parameters and ratios are,
in most cases (fully green leaves), representative only for
the chloroplasts of the upper outer leaf-half where the
illumination is applied, as has recently been demonstrated
in detail (Lichtenthaler et al. 2005a). The RFd values, in
turn, linearily correlate with the net CO2 assimilation rates
PN (Lichtenthaler and Babani 2004; Lichtenthaler et al.
2005b).
The Chl fluorescence decrease ratio (RFd) was determined by imaging and based on the equation: RFd = Fd/
Fs = (Fm – Fs)/Fs, where Fm is the maximum Chl fluorescence level. Fs is the steady-state Chl fluorescence
(5 min after onset of saturating irradiance), and Fd
represents the Chl fluorescence decline from Fm to Fs. In
the present case the excitation light of ca. 1300–
1400 lmol m–2 s–1 has almost but not completely been
saturating. According to the proposal of Van Kooten and
Snell (1990), the Fm level is then called Fp (and not Fm).
Hence, the RFd ratio determined here is the Chl fluorescence decrease ratio Fp/Fs. Chl fluorescence images were
taken during the induction kinetics at Fp (reached after ca.
200 ms) and Fs (after 5 min), and in one case also at other
time intervals according to Lichtenthaler and Babani
(2000), and Lichtenthaler et al. (2000, 2005b). The images
of the Chl fluorescence decrease ratio RFd were obtained by
a pixel to pixel division procedure and their individual
values were also expressed in false colors from red (highest
values) to blue (zero value). The histograms on the RFd
frequency distribution in sun leaves and shade leaves are
based on 100,000 (Populus, Tilia) and 150,000 (Platanus)
pixels per leaf, which means that for the statistical significance calculations the number of individual RFd values n is
100,000 and 150,000, respectively.
Unfortunately a commercial Chl fluorescence imaging
system, that allows the measurement of RFd images, is
presently not available. The other Chl fluorescence imaging
systems represent PAM-type imaging systems (Genty and
Meyer 1994; Nedbal et al. 2000; Nedbal and Whitmarsh
2004; Ralph et al. 2005) work with non-saturating modulated light. However, recently, it has been shown that
one can determine RFd ratios also with the classical
PAM-fluorometer (Lichtenthaler et al. 2005a) when an
additional light source with saturating light is applied. This
principally also applies to the PAM-type Chl fluorescence
imaging systems.
Statistical analysis
The differences in pigment parameters and in the RFd
values between sun leaves and shade leaves (Tables 1, 2;
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Photosynth Res (2007) 93:235–244
Figs. 3–6) were checked for significance using the Student’s t-test. The correlation between the Gs values and the
PN rates of sun leaves and shade leaves (Fig. 1) was
assessed by the analysis of variance one-way ANOVA.
Significant differences were considered at P < 0.01 and
P < 0.001.
values for the ratio Chl a/b (range 3.23–3.45) than shade
leaves (Chl a/b range 2.74–2.81). In addition, they exhibited significantly lower values for the ratio of total Chls to
total carotenoids, i.e., (a + b)/(x + c), ranging from 4.44 to
4.70 for sun leaves and 5.04 to 5.72 for shade leaves.
Measurements of photosynthetic rates PN
Results
Chlorophyll and carotenoid levels
In a preliminary test we found that the total pigment content of sun leaves can considerably vary (up to 20%)
depending on the part of the tree (height and north, south,
or west orientation, etc.) where the samples are taken (data
not shown). In contrast, the variation of pigment levels in
shade leaves from the inner tree shade of the same tree or
other trees of the same tree species was very low and
clearly less than 3%. However, when the sun leaf samples
were taken at the south-exposed part of the tree at nearly
the same height, e.g., 3–5 m above the ground as in this
investigation, the pigment levels between sun leaves of the
same tree and the same tree species showed very little
variation <3%.
The differences in chlorophyll (Chl) and total carotenoid
levels on a leaf area basis between sun leaves and shade
leaves are summarized in Table 1. The total Chl (a + b)
amounts were significantly higher in sun leaves of all three
tree species as compared to the corresponding shade leaves.
This also applied to the content of total carotenoids (x + c).
We also detected the expected typical differences in the
pigment ratios Chl a/b and Chl/carotenoids between sun
leaves and shade leaves (Table 1). The sun leaves of all
three investigated tree species had significantly higher
The maximum CO2 assimilation rates (PN) per leaf area
unit (Table 2) at saturating photosynthetic photon flux
density (PPFD 1500 lmol m–2 s–1) were significantly
(P < 0.01) higher in sun leaves: in Platanus 2.68·, in
Populus 2.94· and in Tilia 2.73· higher than in the corresponding shade leaves. Also on a Chl (a + b) basis the
sun leaves exhibited a significantly higher PN rate than the
shade leaves. Yet, the differences in PN rates on a Chl basis
were not as high (between 2.01· and 2.40· higher) as on a
leaf area basis (Table 2). There also existed large differences in the mean values and the range of stomatal conductance Gs (expressed in mmol m–2 s–1) between sun
leaves and shade leaves. Thus, the mean values of Gs were
much higher in sun leaves (>100 up to 177 in Populus) than
in shade leaves, where the mean Gs values were found to be
43 (Populus), 53 (Platanus), and 54 (Tilia) as shown in
Table 2. In fact, the ranges for Gs values of sun leaves and
shade leaves (Table 2) never overlapped in the physiologically active leaves analyzed in this investigation.
The differences in PN and Gs values between sun and
shade leaves of two trees of the same tree species were in
the same range as those found in individual leaves of the
same tree as has been indicated by the joint mean values
shown in Table 2. The deviation for the PN rates per leaf
area unit and per mg Chl basis from the mean ranged from
15% to 18% (Platanus), 20% to 25% (Populus) and 16% to
20% (Tilia). The Gs means showed a somewhat larger
Table 1 Chlorophyll (a + b) and total carotenoid content (x + c) in mg m–2 leaf area, and pigment weight ratios Chl a/b and Chls/carotenoids
(a + b)/(x + c) in fully developed green sun leaves and shade leaves of platanus (Platanus), poplar (Populus), and linden tree (Tilia) in July
Chlorophylls
Carotenoids
Pigment ratios
(a + b)
(x + c)
a/b
(a + b)/(x + c)
Platanus
Sun leaf
451
96
3.23
4.70
Shade leaf
Populus
338
67
2.81
5.04
Sun leaf
417
94
3.29
4.44
Shade leaf
341
66
2.76
5.17
Sun leaf
492
110
3.45
4.47
Shade leaf
406
71
2.74
5.72
Tilia
Mean of seven determinations from four leaves of three trees of each species, maximal standard deviation <3% (pigment levels) and <1.2%
(pigment ratios). The differences between sun leaves and shade leaves are highly significant (P < 0.001)
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Table 2 Photosynthetic net CO2 assimilation rates PN expressed on a
projected leaf area basis (lmol m–2 s–1) and a chlorophyll a + b basis
(lmol mg Chl–1 h–1), as well as mean and range of stomatal
conductance Gs (values in mmol m–2 s–1) in sun and shade leaves
of the three tree species platanus, poplar, and linden
Sun leaf
Shade leaf
Ratio sun/shade
Platanus
PN per leaf area
9.1 ± 1.4
3.4 ± 0.6
2.68·
PN per mg Chl
72.6 ± 12
36.2 ± 6.4
2.01·
Gs mean
123 ± 31
53 ± 19
2.32·
Gs range
105 – 170
25 – 74
PN per leaf area
PN per mg Chl
9.4 ± 2.2
81.2 ± 19
3.2 ± 0.8
33.8 ± 8.5
2.94·
2.40·
Gs mean
177 ± 41
43 ± 10
4.12·
Gs range
135 – 245
29– 61
PN per leaf area
10.1 ± 1.8
3.7 ± 0.6
2.73·
PN per mg Chl
73.9 ± 13
32.8 ± 5.3
2.25·
Gs mean
154 ± 20
54 ± 14
2.85·
Gs range
123 – 180
33 – 77
Populus
Tilia
Mean of six determinations from three leaves of two trees for each tree species. The mean values are shown in bold print to contrast them against
the standard deviation. The differences between sun leaves and shade leaves of all tree species were highly significant (P < 0.01)
variation: 25% (sun leaves) and 36% (shade leaves) in
Platanus, 23% in Populus, and 13% (sun leaves), and 26%
(shade leaves) in Tilia.
When the Gs values of sun leaves and shade leaves of all
three trees are plotted against the photosynthetic PN rates, a
close linear correlation shows up between Gs and PN
(Fig. 1). The broken line in Fig. 1 clearly separates sun
leaves from shade leaves, which exhibit significantly different photosynthetic rates and stomatal conductance as
summarized in Table 2. Half-shade leaves and sunfleck
leaves, which receive sun light only for 1 or 3 h per day,
range with their PN rates (4.3–6.0 lmol CO2 m–2 s–1) and
their Gs values (65–100 mol m–2 s–1) in between those of
sun leaves and full-shade leaves and also follow the correlation shown in Fig. 1.
Imaging of the chlorophyll fluorescence decrease ratio
RFd
Fig. 1 Correlation between light-saturated photosynthetic CO2
assimilation rates PN and the maximum stomatal conductance Gs in
sun leaves and shade leaves from two trees each of Platanus, Populus,
and Tilia. All data were fitted using the linear regression analysis of
Micosoft excel with the analysis of variance one-way ANOVA
(r = 0.965; P < 0.001; significance factor F = 465.45; significance of
the correlation = 2.03 ·10–21). The perpendicular, broken line separates the values of sun leaves (upper right part) and shade leaves
(lower left part)
The red Chl fluorescence images of pre-darkened Platanus
leaves showed a high Chl fluorescence yield when measured at the maximum Fp in both sun leaves and shade
leaves (Fig. 2A, C). Upon continuous illumination and at
the onset of photosynthesis, the Chl fluorescence yield
steadily decreased and declined after 5 min to the very low
steady-state Chl fluorescence level Fs (Fig. 2B, D). From
the images at Fp and Fs the RFd images were processed,
which demonstrated much higher RFd values for sun leaves
than for shade leaves (Fig. 2E, F). Moreover, the RFd
images show that the photosynthetic activity, as indicated
by the RFd values, is unevenly distributed across the leaf
area. Major leaf veins possessed significantly lower RFd
values than the intercostal leaf parts. In the case of shade
leaves the major veins had practically almost zero RFd
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Photosynth Res (2007) 93:235–244
Fig. 3 Histogram of the frequency distribution of the Chl fluorescence decrease ratio RFd in a sun leaf and a shade leaf of Platanus.
The RFd value distributions are based on more than 150,000 pixels per
leaf and the differences are highly significant (P < 0.001)
Fig. 2 Images of the maximum Chl fluorescence at a high light pulse
(Fp; A, C), and at steady-state Chl fluorescence after 5 min of
continuous illumination (Fs; B, D), and the Chl fluorescence decrease
ratio (RFd) in sun leaves (E) and shade leaves (F) of Platanus · acerifolia. The differences in the relative Chl fluorescence intensity (A–D)
of the different leaf parts are shown by false colors, whereby red
stands for high and blue for zero Chl fluorescence as indicated in the
scale (0–2 K), and ‘‘K’’ represents 1,000 fluorescence units. In case
of the RFd ratio images (E, F) the colors indicate the absolute values
of the ratio from zero up to a maximum RFd value of 4
values (Fig. 2F) indicating no photosynthetic activity
although they possessed chlorophyll.
Forming the histograms using the frequency distribution
of the RFd values of all leaf pixels allows the quantification
of the differences in RFd values between sun leaves and
shade leaves (Fig. 3). The RFd values of sun leaves and
shade leaves partly overlap, but the mean values are significantly different and exhibit maxima at ca. 3.0 (sun leaf)
and ca. 1.8 (shade leaf). Similar results were also obtained
with sun leaves and shade leaves of poplar and linden tree.
The RFd images and the RFd frequency distribution
(histograms) also indicated significantly higher RFd values
in sun leaves of Populus (Fig. 4) and Tilia (Fig. 5) as
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Fig. 4 Images of the Chl fluorescence decrease ratio RFd in a sun leaf
and a shade leaf of Populus and the histogram of the RFd ratio
frequency distribution. Please note that the RFd scales are different for
sun leaves (A) and shade leaves (B). The RFd value distributions are
based on more than 100,000 pixels per leaf and the differences are
highly significant (P < 0.001)
compared to shade leaves. In poplar leaves the RFd values
had a mean at ca. 2.8 (sun leaf) and ca. 1.4 (shade leaf),
whereas those of Tilia exhibited mean values of ca. 2.9
(sun leaf), and the shade leaf a double peak at ca. 1.7 and
1.0 (Fig. 5). Due to the high number of leaf pixels measured (>100,000 in each case) the differences in the RFd
values between sun leaves and shade leaves are highly
significant (P < 0.001).
Photosynth Res (2007) 93:235–244
241
Fig. 6, demonstrate again the differences in RFd values
between sun leaves and shade leaves of all three trees
species. In shade leaves the maximal RFd values were
reached after 5 min, whereas those in Populus and Tilia
still increased slightly within 5–8 min after onset of the
illumination (Fig. 6B, C).
Discussion
The results of this investigation demonstrate that the sun
leaves of the three tree species, as compared to shade
leaves, are characterized by higher levels of total Chl a + b
and total carotenoids, as well as by higher values of the
weight ratio Chl a/b and by lower values for the weight
ratio Chls/carotenoids (a + b)/(x + c). Such differential
pigment ratios are characteristic for sun-type and shadetype chloroplasts of trees and are found in leaves from
high-light and low-light plants as well (Lichtenthaler 1981;
Lichtenthaler et al. 1981; and review Lichtenthaler and
Babani 2004). These differences in the pigment ratios,
Fig. 5 Images of the Chl fluorescence decrease ratio RFd in a sun leaf
and a shade leaf of Tilia and the histogram of the RFd value frequency
distribution. Please note that the RFd scales are different for sun leaves
(A) and shade leaves (B). The RFd value distributions are based on
more than 100,000 pixels per leaf and the differences are highly
significant (P < 0.001)
The data shown in Figs. 3–5 are based on one typical
sun leaf and shade leaf of each tree species. In order to test
the overall validity of our results, Chl fluorescence imaging
of additional sun leaves and shade leaves of the same tree
and of two other trees of the same tree species was performed (data not shown). After leaves had been taken from
the sun-exposed south part and the inner shade of the tree
crown of the three tree species, we obtained the same
results for each tree species with similar, highly significant
differences in the RFd values of sun leaves versus shade
leaves. In fact, the range of RFd values, as shown in the
histograms of Figs. 3–5, was the same for different trees of
each tree species, and the mean RFd values varied by <5%
(sun leaves) and <7% (shade leaves) from leaf to leaf and
from tree to tree of each tree species investigated.
In a separate investigation we checked the development
of the RFd values in the course of the Chl fluorescence
induction kinetics during the continuous illumination of
dark-adapted leaves. We determined Chl fluorescence
images at 0, 1, 5, and 8 min after the onset of continuous
light (PPFD 1300 lmol m–2 s–1). The results, shown in
Fig. 6 Development of the RFd ratio (with standard deviation) upon
illumination of dark-adapted sun leaves and shade leaves of the three
tree species platanus, poplar, and linden. The values shown are based
on RFd ratio images with more than 150,000 leaf pixels per image.
The differences between sun leaves and shade leaves after 5 and
8 min of illumination, and in Platanus also after 1 min of
illumination, are highly significant (P < 0.001)
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higher values for the ratio Chl a/b and lower values for the
weight ratio Chls/carotenoids, are caused by the high
irradiance adaptation response of the photosynthetic pigment apparatus of sun leaves (sun chloroplasts). The latter
possess, on a Chl basis, much lower amounts of lightharvesting Chl a/b proteins (LHCII), more reaction center
pigment proteins (e.g. CPa, CPI) (Lichtenthaler et al.
1982), and also a greater number of electron transport
chains as compared to shade chloroplasts (Lichtenthaler
et al. 1981; Wild et al. 1986). In contrast, shade leaves and
shade-type chloroplasts possess higher and broader grana
thylakoid stacks and primarily invest in the pigment antenna (Boardman 1977; Lichtenthaler et al. 1982, 1984;
Meier and Lichtenthaler 1981).
Due to the adaptation response to high irradiance, sun
leaves of trees with their sun-type chloroplasts possess
considerably higher photosynthetic net CO2 assimilation
rates PN on a leaf area basis in comparison to shade leaves
as shown in Table 2. Sun leaves are thicker and usually
have a higher total amount of Chl per leaf area unit. One
could assume that the higher Chl level is mainly responsible for the higher PN rates in sun leaves; however, this is
not the case. Even on a Chl basis they exhibit higher net
CO2 assimilation rates PN (Table 2). This shows that the
reason for the higher PN rates of sun leaves as compared to
shade leaves is only partly due to their generally higher Chl
content per leaf area unit, whereas the most essential factor
is their possession of sun-type chloroplasts exhibiting a
different structural and functional organization of their
relative Chl and carotenoid levels. The fact that the differences between sun leaves and shade leaves in PN rates
on a Chl basis are not as high as the PN rates on a leaf area
unit indicates that, to a minor extent, also the generally
higher Chl content of sun leaves per leaf area unit contributes to their higher PN rates.
The higher photosynthetic rates of sun leaves are supported by considerably higher values for the stomatal
conductance Gs. The latter can be up to several times
higher (range 105–275) than the rather low Gs values (21–
77) of shade leaves (Table 2), indicating that the stomata
opening is apparently larger in sun leaves than in shade
leaves (Schulze et al. 1975; Farquhar and Sharkey 1982).
This contributes to higher intercellular CO2 concentrations
and thus to the higher CO2 assimilation rates of sun leaves.
This is further supported by a higher stomata density of sun
leaves as compared to shade leaves (Lichtenthaler et al.
1981; Pearcy and Sims 1994; Zangh et al. 1995), and this
also applies to high irradiance leaves in comparison to low
irradiance plants (Wild and Wolf 1980). These data show
that the average stomata density in sun leaves was ca.
1.4–1.9 times higher as compared to shade leaves, whereas
the values for stomatal conductance were 2.4–4 times
higher. This underlines that the higher Gs values in sun
123
Photosynth Res (2007) 93:235–244
leaves are only partially caused by an increase in stomata
density and to a major part by a larger stomata aperture.
Our observation, that at saturating light conditions the PN
rates linearly correlate with the stomatal conductance Gs
(Fig. 1), demonstrates a stomatal control of CO2 uptake.
Such correlations had already been observed for the same
leaf types of plants kept under different physiological
conditions (Pereira et al. 1987; Schulze et al. 1975;
Tenhunen et al. 1984; Wong et al. 1979; Cornic 2000). In
addition, also the mesophyll structure of leaves influences
the PN rates through affecting the diffusion of CO2
(Terashima 1992), and the penetration of light (Vogelmann
and Martin 1993) into the leaf. Thus, the thicker sun leaves
contain significantly more cells per leaf area and section
unit as compared to the thinner shade leaves (Lichtenthaler
1981; Pearcy and Sims 1994).
The much higher PN rates of sun leaves as compared to
shade leaves are also well reflected in the significantly
higher values of the Chl fluorescence decrease ratio RFd
(Figs. 3–5), which represents a non-destructive indicator of
the in vivo photosynthetic rates of leaves (Lichtenthaler
and Babani 2004). The photosynthetic activity, as indicated
by the height of the RFd values is, however, not uniformly
distributed across the leaf area. It shows a certain heterogeneity and patchiness with higher and lower values in
both sun and shade leaves (Figs. 2, 4, 5). A possible
explanation is that this may be caused by or is related to the
non-uniform distribution of stomata opening, as described
for beech leaves, fir needles (Küppers et al. 1999; Beyschlag et al. 1994), and various other plants (as reviewed by
Pospı́šilová and Šantrucek 1994), however, further research
is required. The same patchiness in the distribution of the
RFd values across the leaf area, as described here, has also
been observed via RFd images in beech leaves (Lichtenthaler et al. 2000). Stomatal patchiness is apparently the
result of a heterogeneous water supply in different parts of
the leaf (Cheeseman 1991), which may cause local differences in CO2 levels and thus affect the photosynthetic
rates. In fact, it has been shown via RFd images in bean
leaves that a water deficit reduces the RFd values (Lichtenthaler and Babani 2000). Thus, the assumption of local
differences in the leaf water level across several adjacent
leaf pixels might be a reasonable explanation for the nonuniform distribution of RFd values and the photosynthetic
activity across the leaf area. A local photoinhibition or
damage of the photochemical pigment apparatus, which
may show up under stress conditions, is less likely, since
the leaves were fully physiologically active and did not
show any stress symptoms or strain. In any case, the results
show that imaging the photosynthetic activity of leaves via
the non-invasive method of imaging the RFd values is a
valuable and powerful technique for ecophysiological plant
research.
Photosynth Res (2007) 93:235–244
Acknowledgments We are grateful to Ms Sabine Zeiler for the
excellent implementation of pigment determinations, and to Ms
Gabrielle Johnson for English language assistance.
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