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Plant Physiology and Biochemistry 45 (2007) 577e588

www.elsevier.com/locate/plaphy

Research article

Differences in pigment composition, photosynthetic rates and chlorophyll

fluorescence images of sun and shade leaves of four tree species
Hartmut K. Lichtenthaler a, Alexander Ac b, Michal V. Marek b, Jiřı́ Kalina c, Otmar Urban b,*
a
Botanical Institute (Molecular Biology and Biochemistry of Plants), University of Karlsruhe, Kaiserstrasse 12, D-76133 Karlsruhe, Germany
b
Laboratory of Plants Ecological Physiology, Institute of Systems Biology and Ecology AS CR, Porı́cı́ 3b, CZ-60300 Brno, Czech Republic
c
Department of Physics, Faculty of Science, Ostrava University, 30. dubna 22, CZ-70103 Ostrava, Czech Republic
Received 25 January 2007; accepted 25 April 2007
Available online 1 May 2007

Abstract

The differential pigment composition and photosynthetic activity of sun and shade leaves of deciduous (Acer pseudoplatanus, Fagus
sylvatica, Tilia cordata) and coniferous (Abies alba) trees was comparatively determined by studying the photosynthetic rates via CO2 measure-
ments and also by imaging the Chl fluorescence decrease ratio (RFd), which is an in vivo indicator of the net CO2 assimilation rates. The thicker
sun leaves and needles in all tree species were characterized by a lower specific leaf area, lower water content, higher total chlorophyll (Chl) aþb
and total carotenoid (Cars) content per leaf area unit, as well as higher values for the ratio Chl a/b compared to the much thinner shade leaves and
needles that possess a higher Chl aþb and Cars content on a dry matter basis and higher values for the weight ratio Chls/Cars. Sun leaves and
needles exhibited higher rates of maximum net photosynthetic CO2 assimilation (PNmax) measured at saturating irradiance associated with higher
maximum stomatal conductance for water vapor efflux. The differences in photosynthetic activity between sun and shade leaves and needles
could also be sensed via imaging the Chl fluorescence decrease ratio RFd, since it linearly correlated to the PNmax rates at saturating irradiance.
Chl fluorescence imaging not only provided the possibility to screen the differences in PN rates between sun and shade leaves, but in addition
permitted detection and quantification of the large gradients in photosynthetic rates across the leaf area existing in sun and shade leaves.
Ó 2007 Elsevier Masson SAS. All rights reserved.

Keywords: Carbon dioxide assimilation; Carotenoid level; Chlorophyll fluorescence decrease ratio; Chlorophyll a/b ratio; Chlorophyll content; Fluorescence
imaging; Stomatal conductance

1. Introduction chloroplast ultrastructure and their photosynthetic rates

[1,5,8,16,17,18,29,53]. Leaf and chloroplast adaptation to either
Sun and shade leaves of trees as well as high-light and high or low irradiance, to direct sun-light or shade proceed dur-
low-light plants can considerably differ in their relative com- ing leaf development and comprise special morphological and
position of photosynthetic pigments, electron carriers, their biochemical adaptations. Sun leaves and high-light plants
possess sun-type chloroplasts that are adapted for high rates of
photosynthetic quantum conversion, they possess a higher pho-
Abbreviations: Cars, total carotenoids; Chl, chlorophyll; Chl a/b, ratio Chl
a to Chl b; DW, dry weight; FW, fresh weight; Fm, maximum Chl fluores-
tosynthetic capacity on a leaf area and chlorophyll (Chl) basis,
cence; Fs, steady-state Chl fluorescence; Fd, Chl fluorescence decrease from exhibit higher values for the ratio Chl a/b, a much lower level
Fm to Fs; Gs, stomatal conductance for water vapor; LAp, projected leaf of light-harvesting Chl a/b proteins (LHCII), and a lower stack-
area; LHCII, light-harvesting complex II; PN, net photosynthetic CO2 assimi- ing degree of thylakoids than shade leaves and low-light plants
lation rate; PPFD, photosynthetic photon flux density; RFd, Chl fluorescence with their shade-type chloroplasts [23e25]. Major differences
decrease ratio; RWC, relative water content; SLA, specific leaf area; xþc,
xanthophylls þ carotenes.
in the chloroplast adaptation response to either high- or low-
* Corresponding author. Tel./fax: þ420 54 321 1560. light quanta fluence rates have recently been summarized [21].
E-mail address: [email protected] (O. Urban). This review, giving access to further literature in this field, shows

0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.plaphy.2007.04.006
 Author's personal copy

578 H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588

that much work has been done with individual herbaceous plants by Mesozoic Godula sandstone (flysch type), with ferric
grown at high or low quanta fluence rates or with selected decid- podzols. See detailed description in Kratochvı́lová et al. [12].
uous broad-leaf trees [1,5,8,10,35]. In general, only the net CO2
assimilation rates PN per leaf area unit of sun and shade leaves 2.2. Plant material
have been measured, whereas the question if sun and shade
leaves differ in their photosynthetic activity, also on a Chl basis, Branches with fully developed sun and shade adapted leaves
remained open because pigment determinations were not of four tree species were sampled: the deciduous species beech
performed. (Fagus sylvatica L.), maple (Acer pseudoplatanus L.), linden
So far the photosynthetic activities of leaves, and especially tree (Tilia cordata Mill.), and shoots of the coniferous fir (Abies
the differences in CO2 fixation rates between sun and shade alba Mill.). All trees grew in the neighborhood of the ecological
leaves, have primarily been performed by net CO2 measure- research station, either on the rim of a homogeneous 30-year-
ments with CO2/H2O porometer gas-exchange systems. These old Norway spruce forest, or on the transition to an open
provide, however, only one integral PN rate per measurement of meadow area and a small beech forest. Shade leaves at the inner
a relatively large leaf area spot or of a certain quantity of nee- tree crown received up to 100 mmol(photons) m2 s1 on sunny
dles on a small conifer branch. Whether the photosynthetic ac- days, whereas the sun leaves were exposed to a maximum PPFD
tivity is evenly distributed over the leaf area or whether of 1200e1500 mmol(photons) m2 s1. A branch with the de-
inhomogeneities or small local declines in photosynthetic rates sired leaf or needles was cut from the tree and the cut end
exist, cannot be detected using gas exchange measurements. In was immediately re-cut under water to remove xylem embo-
recent years it has been shown that the chlorophyll fluorescence lisms. The branch end remained in the water during the mea-
decrease ratio RFd is linearly correlated to the net photosyn- surements. The branches investigated here were derived from
thetic CO2 assimilation rate PN and is an indicator of the pho- three to four trees per species, and from 30- to 40-year-old trees
tosynthetic activity of leaves [21,27]. Moreover, a special (Acer, Fagus, Tilia) and ca. 20- to 30-year-old trees (Abies). The
imaging technique has been developed [20,26] that allows im- branches were taken from completely healthy trees in mid-July
aging the RFd ratio of whole leaves with at least several tens of 2005 at rather cool temperatures (mid-day temperatures of 16 to
thousands, or more than 100,000 pixels per measurement. This 20  C), on partly cloudy days (maximum PPFD between 950
should principally be also possible with the new pulse ampli- and 1250 mmol(photons) m2 s1) with some rain, and they
tude fluorescence imaging system [32] when it is combined did not exhibit signs of drought or photoinhibition that can,
with an additional saturating light source. however, be experienced on hot, sunny and dry summer days.
Thus a major objective of this investigation was to prove by Under the experimental conditions there were no significant dif-
means of Chl fluorescence imaging, if the differences in photo- ferences between attached and cut branches in the maximum
synthetic activity of sun and shade leaves would show up in the CO2 assimilation rate (PNmax), stomatal conductance for water
RFd images, and if there were differences between the two leaf vapor (GSmax) or in the RFd values. In addition, samples for the
types in the distribution of photosynthetic activity across the measurements were taken in the morning and afternoon, yet sig-
leaf area. In order to better characterize the leaves, the relative nificant differences in these parameters during the course of the
Chl and carotenoid composition, as well as the photosynthetic day, e.g. a mid-day depression, were not detected under the
activity of sun and shade leaves were compared, and we also given climatic conditions.
checked whether the higher CO2 assimilation rates of sun The projected leaf area (LAp) of all investigated leaves was
leaves/needles also exist on a Chl basis. Four common tree spe- estimated using a leaf area meter (LI-3000A, LI-COR, Lincoln,
cies of the temperate zone from the same location and climate NE, USA), as well as their fresh weight (FW) and dry weight
with different tolerance to shade conditions [49] were selected (DW) after drying at 80  C for 48 h. Subsequently, the relative
for this study: namely the strongly shade-tolerant linden (Tilia water content (RWC [%]; RWC ¼ [(FW  DW)/FW]  100)
cordata Mill.) and fir (Abies alba Mill.) as well as the less and specific leaf area SLA [cm2 g1 DW] were determined,
shade-tolerant beech (Fagus sylvatica L) and maple (Acer whereby the SLA ¼ LAp/DW was calculated in agreement
pseudoplatanus L.). with Gilmore et al. [7]. In contrast to needles of other coniferous
trees, fir needles are flat, their needle area can easily be deter-
2. Methods mined, and thus their SLA can be compared to that of leaves
of broadleaf trees. The leaf and needle thickness was determined
2.1. Site description with a digital micrometer system (Mitutoyo Corp., Japan).

Experiments were carried out at the ecological research sta- 2.3. Pigment analysis
tion in a natural stand of trees in a forest located in the Mora-
vian-Silesian Beskydy Mountains (Bı́lý Křı́z, 49 330 N, 18 320 Leaf and needle samples were frozen in liquid nitrogen (ca.
E, 908 m a.s.l., NE of the Czech Republic) in the middle of the 100 mg of fresh weight) and later analyzed in the laboratory in
growing season (mid to late July 2005). The climate of the Ostrava. The photosynthetic plant pigments, Chls and Cars,
area is characterized by an annual mean temperature of were extracted with 80% acetone and a small amount of
5.5  C, annual mean relative air humidity of 80%, and a total MgCO3. The clear supernatant obtained after centrifugation at
rainfall of 1000e1400 mm. The geological bedrock is formed 480  g for 3 min was used for spectrophotometric (UV/VIS
 Author's personal copy

H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588 579

550, Unicam, Cambridge, UK) estimation of Chl a, Chl b and each individual leaf pixel. The applied irradiance of
total carotenoids in the same extract solution using the extinc- 2000 mmol(photons) m2 s1 may not have been sufficient,
tion coefficients and equations redetermined by Lichtenthaler e.g. in all sun leaf or sun needle samples, to reach the full max-
[19], and given in more detail in Lichtenthaler and Buschmann imum level of Fm. Yet, in contrast to the absolute Chl fluores-
[22]. From the pigment levels the weight ratios of pigments, Chl cence signal, the RFd values as Chl fluorescence ratio are less
a/b and Chls/Cars (a þ b)/(x þ c) were determined that signif- dependent on a further increase of the irradiance when an irra-
icantly differ between sun and shade leaves. diance of at least 2000 mmol(photons) m2 s1 has been ap-
plied. We checked with a conventional PAM Chl fluorometer
2.4. Gas-exchange measurement that an increase of the irradiance up to saturating 3500 or
4000 mmol(photons) m2 s1 increased the RFd values by
An infra-red gas analyzer (LI-6400, LI-COR) was used to less than 5%. Such a high PPFD given for 5 min is, however,
estimate the maximum CO2 assimilation rate (PNmax). The not recommendable in order to avoid photoinhibition or dam-
values were recorded after ca. 20 min of exposure to saturat- age to the photosynthetic apparatus. After the collection of the
ing irradiance (1500 mmol(photons) m2 s1) when the sto- Chl fluorescence images, the RFd images and the histograms
mata had fully opened (maximum stomatal conductance for with the RFd frequency distribution were constructed using
water vapor; GSmax). Leaf temperature, relative air humidity, the imaging fluorometer software package. The histograms
and CO2 concentration inside the leaf chamber were kept on the RFd frequency distribution in sun and shade leaves
constant at 18e21  C, 50e55%, and 375 mmol(CO2) mol1, are based on 160,000 leaf pixels (Fagus), 90,000 (Acer, Tilia)
respectively. and >40,000 needle pixels for each leaf type. For a better
comparison of the RFd value distribution of the four trees the
2.5. Chlorophyll fluorescence imaging latter is expressed in percentage units (Fig. 9).

The chlorophyll (Chl) fluorescence induction kinetics (Kaut-

2.6. Statistical analysis
sky effect) of pre-darkened leaves (20 min) were measured at
the red Chl fluorescence band (near 690 nm) using the kinetic
The LSD test (belonging to ANOVA group of tests) was used
imaging fluorometer (FluorCam, Photon System Instruments
to evaluate the statistically significant differences between sun
Ltd., Brno, Czech Republic). The lens of the CCD camera
and shade leaves. Differences were tested at probability levels
was ca. 7 cm perpendicular to the leaf surface. At first, the initial
p ¼ 0.05, 0.01 and 0.001. The homogeneity of the variance of
fluorescence level, Fo, was measured in 20 min dark-adapted
the RFd frequency distribution was tested using the F test, and
samples by low-intensity measuring-pulses (10 ms pulses of
subsequently, two sample t-test with uneven variances were ap-
an intensity of ca. 0.003 mmol m2 s1). The latter were gener-
plied for testing the significant differences between sun and
ated in two panels of orange light-emitting diodes (HLMP-
shaded RFd histograms of each tree species. All statistical tests
EH08, Agilent Technologies, Santa Clara, CA, USA). They
were performed using STATISTICA software (StatSoft, Inc.,
were applied 2 s each and had no detectable actinic effect.
Tulsa, OK, USA).
Afterwards, in modifying the FluorCam program, continuous
actinic saturating light (ca. 2000 mmol(photons) m2 s1), gen-
erated by a separate Oriel 150 W xenon lamp (model 66056) 3. Results
with UV filters, was applied to obtain the maximum fluores-
cence level, Fm. Continuous saturating light was also required 3.1. General leaf characteristics
to obtain the steady-state fluorescence level, Fs, that was esti-
mated from the Chl fluorescence induction curve after 5 min The sun-exposed leaves from end-branches of the three
of continuous illumination. See [32] for detailed description broadleaf trees investigated showed the typical, larger thick-
of the fluorescence system and [27] for details of the measuring ness of sun leaves (by ca. 40% to 65%) as compared to shade
protocol. The imaging instrument possesses a calibration device leaves. Since this had not been clearly investigated in the case
correcting for non-uniform irradiation, yet when testing the dis- of sun and shade fir needles, we have measured the thickness
tribution of the irradiance in the leaf sample area, it was found to of several needle age classes and found that sun needles were
be fairly homogeneous with a maximal deviation of 3%. 62% to 76% thicker than shade needles depending on the nee-
The Chl fluorescence decrease ratio (RFd) was determined dle age (Fig. 1). Sun leaves and needles had a smaller pro-
from the induction curve according to Lichtenthaler and Babani jected leaf and needle area as compared to the thinner shade
[20] and Lichtenthaler et al. [27]: leaves and needles that possess a higher relative water content.
Thus, the relative water content (RWC) in sun leaves was sig-
RFd ¼ Fd=Fs ¼ ðFm  FsÞ=Fs ð1Þ nificantly ( p < 0.01) lower (by 8e22%) as compared to shade
leaves in all tree species (Fig. 2A). In addition, there also ex-
where Fm is the maximum fluorescence level, Fs is the steady isted the expected statistically significant differences
state fluorescence (5 min after onset of saturating irradiance), ( p < 0.01) in the specific leaf area (SLA) with higher values
and Fd represents the chlorophyll fluorescence decline from for shade leaves and needles as compared to sun leaves and
Fm to Fs. The Fm, Fd and Fs values were calculated for sun needles (Fig. 2B).
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580 H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588

80
800 Sun
Shade
A
70

60
600
Thickness (µm)

50

RWC (%)
40
400
30

20
200
10

0
0 Acer Fagus Tilia Abies
1st 2nd 3rd 4th 5th
Needle age class 400
Sun B
Fig. 1. Differences in needle thickness (mm) between sun and shade needles of 350 Shade
fir (Abies alba) of different needle years. The values are given for 1st, 2nd, 3rd,
4th and 5th year needles. Needles from three branches of two trees (n ¼ 18 for 300
each needle year and condition). Columns represent mean values and bars
SLA (cm2 g-1) 250
standard deviations. The differences are highly significant ( p < 0.001).
200
3.2. Chlorophyll and carotenoid levels
150

The differences in chlorophyll (Chls) and total carotenoid 100

(Cars) contents between sun and shade leaves are summarized
50
in Fig. 3. One has to differentiate between two reference sys-
tems, the levels per leaf area unit and the levels per leaf dry 0
Acer Fagus Tilia Abies
weight, which yield contrasting results. Regarding photosyn-
thetic processes that are controlled by incident light, it is Fig. 2. A,B. Relative water content (RWC; A) and specific leaf area (SLA; B)
more appropriate to use the leaf area as a reference system; in sun (white columns) and shade leaves (black columns) of deciduous Acer
however, the dry weight often indicated in the literature is pseudoplatanus, Fagus sylvatica, Tilia cordata and coniferous Abies alba.
All differences between sun and shade leaves are statistically highly significant
given here for comparative reasons as well. On a leaf area ba- ( p < 0.01). Columns represent mean values and bars standard deviations.
sis the Chl (aþb) amounts were significantly higher in sun n ¼ 8.
leaves of A. pseudoplatanus and T. cordata (Fig. 3A) as com-
pared to shade leaves, but the differences were not statistically
(PPFD z 1500 mmol m2 s1) were significantly ( p < 0.01)
significant ( p > 0.05) in F. sylvatica and A. alba. In contrast,
higher in sun leaves and needles (range: from 97% to 168%)
on a dry matter basis the shade leaves and needles had a signif-
than in shade leaves. Even on a Chl (aþb) basis the sun leaves
icantly ( p < 0.01) higher Chl (aþb) content, up to 147% for
and needles exhibited a higher PNmax rate than shade leaves and
all four tree species (Fig. 3B). The content of total Cars on
needles (Fig. 5B). However, the differences in PNmax rates on
a leaf area basis was significantly lower ( p < 0.01) in shade
a Chl basis were not as high (range: from 76% to 128%) as on
leaves compared to sun leaves (Fig. 3C), except for A. alba
a leaf area basis. There was an additional difference when the
where the difference was not significant ( p > 0.05). On
PNmax rate on a leaf dry weight basis, reflecting the influence
a dry matter basis, however, the shade leaves and needles
of the leaves’ morphological structure, was taken as a reference
had a significantly higher total Cars content (Fig. 3D).
system (Fig. 5C). As compared to sun leaves, higher PNmax rates
on a leaf dry weight basis were detected in the shade leaves of
3.2.1. Pigment ratios A. pseudoplatanus (19%) and F. sylvatica (48%), whereas the
The typical differences between sun and shade leaves in the PNmax rates were lower in the shade leaves of T. cordata
pigment ratios Chl a/b and Chls/Cars were found to be as ex- (34%) and A. alba (32%). These differences were statistically
pected (Fig. 4). The sun leaves of all investigated tree species significant ( p < 0.01) except for A. pseudoplatanus.
had significantly higher values for the ratio Chl a/b (Fig. 4A) At saturating PPFD, PNmax linearly correlated (PNmax ¼
and lower values for the ratio of Chls/Cars (Fig. 4B), which is 0.0631 GSmax; R2 ¼ 0.78) with maximum stomatal conductance
also known as ratio (a þ b)/(x þ c) [2,42,54]. (GSmax) as shown in Fig. 6. The GSmax values of sun leaves and
needles were always significantly higher as compared to those
3.3. Measurements of photosynthetic rates PN of shade leaves. In all cases the latter remained below values
of 100 mmol m2 s1, whereas sun leaves and needles exhibited
Maximum CO2 assimilation rates (PNmax) per leaf area unit GSmax values of above100 mmol m2 s1 as indicated in Fig. 6
(Fig. 5A) at saturating photosynthetic photon flux density by a broken line.
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H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588 581

0.7 14 **
** A ** B
0.6 12

Chl (a+b) (mg g-1)

Chl (a+b) (g m-2)

0.5 ** 10 **
0.4 8

0.3 6 **
0.2 4

0.1 2

0 0
Acer Fagus Tilia Abies Acer Fagus Tilia Abies

0.14 2.5
** ** **
0.12 ** C Sun
D
** Shade
2.0

Cars (x+c) (mg g-1)

Cars (x+c) (g m-2)

0.10
**
0.08 1.5

0.06
1.0 *
0.04
0.5
0.02

0.00 0.0
Acer Fagus Tilia Abies Acer Fagus Tilia Abies

Fig. 3. A-D. Total chlorophyll (Chl (aþb); A, B), and carotenoid content (Cars (xþc); C, D) expressed per leaf area unit (A, C) and dry weight unit (B, D). Sun
(white columns) and shade leaves (black columns) of deciduous Acer pseudoplatanus, Fagus sylvatica, Tilia cordata and coniferous Abies alba are presented.
Columns represent mean values and bars standard deviations. ** and * represent the statistically significant differences at p < 0.01 and p < 0.05 level, respectively.
n ¼ 8.

3.4. Chlorophyll fluorescence imaging When RFd values and PNmax rates, both measured at saturat-
ing PPFD, were compared, a close linear correlation
The chlorophyll (Chl) fluorescence images (Fig. 7A,B) (RFd ¼ 0.27 PNmax þ1.33; R2 ¼ 0.91) between both parameters
demonstrate that the red Chl fluorescence signal (band near showed up (Fig. 10). The same linear relationship applies to
690 nm) is unevenly distributed across the leaf area at both both sun and shade leaves and needles. Correspondingly, at
the maximum Chl fluorescence at a saturating pulse (Fm), zero net CO2 assimilation rate (PNmax ¼ 0 mmol m2 s1) an
and at the steady state Chl fluorescence at a saturating photo- RFd value of ca. 1.33 is found.
synthetic photon flux density (Fs). These images may subse-
quently be applied to deduce the distribution of the RFd 4. Discussion
values in leaves (Fig. 8C,D). The RFd images indicate signifi-
cantly higher RFd values in sun leaves in comparison to shade In this study all four common tree species of the temperate
leaves, e.g. of the broadleaf F. sylvatica (Fig. 7C,D) and even zone exhibited major differences between sun and shade leaves
in the needles of the coniferous A. alba (Fig. 8). that had developed at either sun exposed or fully shaded condi-
The normalized frequency distribution of the RFd values of tions. The lower RWC (Fig. 2A) and a lower SLA (Fig. 2B) in
all tree species studied demonstrated highly significant sun leaves and sun needles of the four tree species investigated
( p < 0.001) differences in the distribution of the RFd values as compared to shade leaves and needles, is in accordance with
between sun and shade leaves (Fig. 9AeD). The RFd value dis- previous studies showing a lower water content (51%), a lower
tributions, shown here for better comparison as percentage dis- SLA (116 cm2 g1) in beech sun leaves as compared to beech
tribution, are based on more than 90,000 leaf pixels and on shade leaves (SLA: 335 cm2 g1, water content 64%) [17].
more than 40,000 needle pixels per RFd image evaluated. In This was confirmed in an independent investigation indicating
fact, the average RFd values of sun leaves were several times that sun leaves of F. sylvatica had a significantly lower SLA
higher than those in shade leaves (Acer 2.0, Fagus 1.7, (118 cm2 g1) and water content (42%) than shade leaves
Tilia 2.7, and Abies 3.7). The Chl fluorescence images (SLA: 282 cm2 g1; water content: 53%) [38]. Significantly
and the RFd frequency distributions, shown in Fig. 9, are of lower SLA values were also described for the sunlit leaves of
one leaf and one needle shoot sample. Parallel images were the upper canopy level as compared to leaves of the lower can-
taken of additional leaves and needle shoots of all tree species, opy level in ash, hornbeam, maple and linden trees [10]. SLA
and the differences in images and RFd value distribution (data values are considered a measurement of leaf structure, thick-
not shown) were very similar and practically almost identical ness, the amount of mechanical tissues in leaves, and a measure-
to those shown. ment of the morphological difference between sun and shade
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582 H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588

4
A
14 Sun A
Shade
12
3

PNmax (µmol m-2 s-1)

10
Chl a/b

8
2
6

1 4

0 0
Acer Fagus Tilia Abies Acer Fagus Tilia Abies

7
Sun B 140 Sun B
6 Shade Shade
120

PNmax (µmol mgChl-1 h-1)

5
100
Chls/ Cars

4
80
3
60
2
40
1
20
0
Acer Fagus Tilia Abies 0
Acer Fagus Tilia Abies
Fig. 4. A,B. Ratio of Chl a to Chl b (Chl a/b; A) and weight ratio of total chlo-
rophylls to total carotenoids (Chls/Cars; B) in sun (white columns) and shade 1.0
leaves (black columns) of deciduous Acer pseudoplatanus, Fagus sylvatica, Sun C
Tilia cordata and coniferous Abies alba are presented. All differences between Shade
0.8
PNmax (µmol mgDW-1 h-1)

sun and shade leaves are statistically highly significant ( p < 0.01). Columns
represent mean values and bars the standard deviations. n ¼ 8.

0.6
leaves and needles [7]. In fact, beech sun leaves are thicker (144
to 185 mm) than shade leaves (88e93 mm) [17,23], which was
confirmed in the present investigation and extended for the 0.4
other trees. Practically the same observations were made using
the inverse SLA ratio, the leaf dry mass per area LMA, which, in 0.2
several hundred broad-leaf trees and woody species, linearly in-
creased with increasing irradiance together with leaf thickness
[33,34]. Moreover, a close relationship between SLA and 0.0
Acer Fagus Tilia Abies
RWC was reported in groundnut (Arachis hypogaea) leaves
[31], whereby the rate of reduction in leaf RWC during a pro- Fig. 5. A-C. Maximum of light-saturated CO2 assimilation rate (PNmax) ex-
pressed on a projected leaf area basis (A), on a chlorophyll (aþb) basis
gressive moisture deficit was directly related to SLA and, as
(B), and on a leaf dry weight basis (C). Mean values (columns) and standard
a consequence, affected the leaf carbon exchange. deviations (bars) of sun (white columns) and shade leaves and needles (black
Differences in PNmax on a leaf area unit between sun and columns) of Acer pseudoplatanus, Fagus sylvatica, Tilia cordata, and Abies
shade leaves (Fig. 5A) were similar to those previously de- alba are presented. All differences between sun and shade leaves are statisti-
scribed for beech [17,23], several other broadleaf [13,26, cally highly significant ( p < 0.01), except PNmax expressed on a leaf dry
weight basis (C) in A. pseudoplatanus. n ¼ 8.
28,36] and coniferous tree species [40,43], as well as various
other plants [5,20,21,35]. Also, in sunlit leaves of the upper can-
opy level of four tree species higher photosynthetic rates per leaf they usually have a higher level of Chls per leaf area unit
area unit were found as compared to leaves of the lower canopy (cf. Fig. 3A) as shown before for ginkgo, beech, hornbeam,
level [10], whereby tree specific differences showed up. Higher and poplar [22,41,44]. Moreover, the higher nitrogen levels
light saturated rates of photosynthesis in sun leaves are mainly per leaf area unit also result in a higher content of Rubisco en-
associated with a greater amount of nitrogen per leaf area unit zyme and subsequently in the stimulation of CO2 uptake at
in sun leaves as compared to shade leaves [9,10,13,18], and high irradiances [13,18]. The level range of total Chls per leaf
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H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588 583

16 hornbeam and ginkgo [17,22,41] and other plants [35]. Our ob-
Shade Sun
servation that sun leaves and sun needles possess much higher
14
values for the maximum stomatal conductance for water vapor
12 (GSmax) (Fig. 6) as compared to shade leaves and shade needles
PNmax (µmol m-2 s-1)

may indicate that they are able to open their stomata much wider
10 y = 0.0631x
than shade leaves and shade needles. This certainly appears to be
R2 = 0.78
8 an essential prerequisite for their higher photosynthetic rates.
However, also a higher stomata density per leaf area unit of
6 sun leaves and upper canopy leaves as compared to shade leaves
4 Acer and lower canopy leaves must be taken into consideration in this
Fagus respect [10,17,23].
2 Tilia The organization of the pigment apparatus and the relative
Abies
levels of Chl a and Chl b as well as the ratio of total Chls to total
0
0 50 100 150 200 250 Cars is also an essential difference between sun and shade
GSmax (µmol m-2 s-2) leaves and needles. Sun leaves/needles possess sun-type chloro-
plasts with higher values for the ratio Chl a/b and lower values
Fig. 6. Relationship between maximum CO2 assimilation rate (PNmax) and
maximum stomatal conductance for water vapor (GSmax) at saturating photo-
for the weight ratio Chls/Cars (Fig. 4A,B) than the shade-type
synthetic photon flux density (PPFD z 1500 mmol m2 s1). Symbols repre- chloroplasts of shade leaves and leaves of low light plants
sent the mean of five measurements of sun and shade leaves of Acer [2, 6, 18, 23]. The values for the ratio Chl a/b in sun (range:
pseudoplatanus, Fagus sylvatica, Tilia cordata, and shoots of Abies alba. from 3.0 to 3.4) and shade leaves (range: from 2.4 to 2.7) as
All data were fitted using linear regression. The perpendicular broken line well as the values for the ratio Chls/Cars (sun leaves 3.8 to
was drawn to indicate the values of sun leaves (upper right part) and shade
leaves (lower left part).
4.4, shade leaves 4.8 to 5.7) were found in the normal range
of physiologically active leaves [21,41]. Such differential pig-
ment ratios are characteristic for sun-type and shade-type chlo-
area unit can partially overlap in sun and shade leaves. However, roplasts [21, 41]. Significantly higher values for the ratio Chl a/b
on a dry matter basis shade leaves always contain significantly were also found in the sunlit leaves of the upper canopy level
higher amounts of total Chls as compared to sun leaves of four broadleaf tree species as compared to leaves of the lower
(Fig. 3B). This has been shown before for horse chestnut, oak, canopy level [10]. The considerably higher level of carotenoids

Fig. 7. A-D. Images of the maximum Chl fluorescence at a saturating light pulse (Fm; A), and at steady state Chl fluorescence after 5 min of continuous saturating
light (Fs; B), and the Chl fluorescence decrease ratio (RFd) in sun (C) and shade leaves (B) of Fagus sylvatica. The differences in the relative Chl fluorescence yield
of the different leaf parts are indicated by false colors with red for high and blue for low Chl fluorescence. In case of the RFd ratio images (C, D) the colors indicate
the absolute values of the ratio. See scale in (D).
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584 H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588

Fig. 8. A-D. Images of the maximum Chl fluorescence (red band near 690 nm) at a saturating pulse (Fm; A), the steady state Chl fluorescence after 5 min of
continuous saturating light (Fs; B), and the Chl fluorescence decrease ratio (RFd) in first-year sun (C) and shade needles (B) of Abies alba. The differences in
the relative Chl fluorescence yield of the different needle parts are indicated by false colors with red for high and blue for low Chl fluorescence. The fluorescence
images of the RFd ratio are given in false colors that state the absolute values of the ratio. See scale in (D).

on a Chl reference basis in sun leaves versus shade leaves, and leaf area unit (Fig. 2A), as also found in the fan-shaped gymno-
hence lower values for the ratio Chls/Cars, is primarily caused sperm leaves of Ginkgo [41]. The higher PNmax rates of sun
by their higher level of the xanthophyll cycle carotenoids leaves/needles are also well reflected in the higher values of
[6,10,45,47]. In addition, these differences in the pigment ratios the Chl fluorescence decrease ratio RFd (Fig. 10) that represents
are also a result of the high irradiance adaptation response of the a non-destructive indicator of the in vivo photosynthetic rates of
photosynthetic pigment apparatus of sun leaves (sun chloro- leaves and is measured from near distance without any direct
plasts) with fewer light-harvesting Chl a/b proteins (LHCII) contact with the leaf [21,26,41].
and a larger number of reaction center pigment proteins (e.g. Light-saturated rates of photosynthesis on leaf area basis
CPa, CPI) on a total Chl basis as compared to shade chloroplasts (PNmax) depend not only on photosynthetic biochemistry but
[24], as well as a greater number of electron transport chains also on the mesophyll structure of leaves. Since resistance to
[21,23,53]. At the same time shade leaves and shade-type chlo- CO2 diffusion from the substomatal cavity to the stroma is
roplasts exhibit higher and broader grana thylakoid stacks and substantial, it is likely that the mesophyll structure affects
primarily invest into the pigment antenna [24]. As a consequence PNmax by affecting the diffusion of CO2 [40,46] and the pene-
of the adaptation response of their chloroplasts to high irradi- tration of light [51] in the leaf. In addition, the photosynthetic
ance, sun leaves of trees with their sun-type chloroplasts possess rate per leaf area unit represents the sum of assimilation rates
considerably higher PN rates on a leaf area basis than shade of individual cells; however, the thicker sun leaves contain sig-
leaves. One reason for the higher PNmax rates of sun leaves nificantly more cells as compared to the thinner shade leaves
and needles as compared to shade leaves is their generally higher [17,18,37]. Therefore, the PNmax rates per unit leaf weight
Chl content per leaf area unit, but the major reason is the posses- (Fig. 5C) reflect the influence of the morphological leaf struc-
sion of sun-type chloroplasts with their different ultrastructure, ture on CO2 uptake. We found significantly higher PNmax per
biochemical organization and a special arrangement of their leaf weight unit in shade leaves of A. pseudoplatanus and F.
Chls and Cars in the thylakoids. The possession of sun-type sylvatica as compared to sun leaves, whereas PNmax per leaf
chloroplasts is also the major reason why sun leaves and needles weight unit of shade leaves was lower in the highly shade-toler-
exhibit higher net CO2 assimilation rates PNmax, not only on ant species A. alba and T. cordata as compared to sun leaves
a leaf area basis but also on a Chl basis (Fig. 5B). The lower dif- (Fig. 5C). Kubiske and Pregitzer [13] concluded that the shade
ferences in PN rates on a Chl basis between sun and shade leaves leaves of shade-intolerant species respond to shade primarily
(Fig. 5B) indicate that one possible reason for the higher PN rates by altering SLA, whereas shade-tolerant species respond largely
in sun leaves/needles is their generally higher Chl content per via biochemical acclimation of the photosynthetic apparatus.
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H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588 585

25 25
Relat. frequency distribution (%)

Relat. frequency distribution (%)

Acer A Fagus B
20 20

15 Sun 15 Sun
Shade Shade
10 10

5 5

0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
RFd (dimensionless) RFd (dimensionless)

25 25
Relat. frequency distribution (%)

Relat. frequency distribution (%)

Tilia C Abies D
20 20

15 15 Sun
Sun
Shade
Shade
10 10

5 5

0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
RFd (dimensionless) RFd (dimensionless)

Fig. 9. A-D. Histograms of the relative frequency distribution of the Chl fluorescence decrease ratio (RFd) measured in sun (empty symbols) and shade leaves and
needles (filled symbols) of Acer pseudoplatanus (A), Fagus sylvatica (B), Tilia cordata (C), and Abies alba (D). The RFd value distributions are based on several ten
thousand leaf and needle pixels as indicated in Section 2. The relative frequency is indicated here as percentage, which allows to better compare the results of the
four trees, since the absolute pixel numbers were not the same for all leaves.

Sun leaves and sun adapted plants are known to have to the leaves of high irradiance in comparison to low irradi-
small stomata, however, a higher stomatal density as com- ance plants [52]. Hence, higher light saturated stomatal con-
pared to shade leaves [21,23,37,55], and this also applies ductance (GSmax) is usually found in sun leaves (Fig. 6). The
much higher stomata opening in sun leaves (GSmax range 100
6 up to 300 mmol m2 s1; in shade leaves clearly below
Shade Sun 100 mmol m2 s1) contributes to the higher intercellular
5 CO2 concentrations, and thus to the higher CO2 assimilation
rates. Moreover, Fig. 6 shows that PNmax at saturating PPFD
4
y = 0.27x + 1.33 linearly correlates with maximum stomatal conductance
R2 = 0.91 (GSmax). Similar relationships were found for Southern Beech
(Nothofagus cunninghamii) [11], African savanna tree species
RFd

3
[30] and for temperate grassland species [50]. The relation-
ship between PNmax and GSmax indicates that stomatal control
2
of CO2 uptake at GSmax is smaller than about 200 mmol
Acer
Fagus
m2 s1. However, the response curves level off at high sto-
1
Tilia matal conductance above 300 mmol m2 s1 [30].
Abies Non-uniform opening of stomata was observed in leaves of
0 F. sylvatica [14], needles of A. alba [4] and other plants (re-
0 2 4 6 8 10 12 14
viewed in [39]). The stomatal patchiness is a consequence of
PNmax (µmol m-2 s-1)
the heterogeneous water status in different parts of the leaf
Fig. 10. Correlation between the red (band near 690 nm) chlorophyll fluores- and can be induced by all ambient factors causing such hetero-
cence decrease ratio (RFd) and the maximum CO2 assimilation rate (PNmax) at geneities [3]. It leads to a non-uniform distribution of intercel-
saturating photosynthetic photon flux density (PPFD z 1500 mmol m2 s1) lular CO2 concentration within the leaf and subsequently to
of sun and shade leaves/needles of Acer pseudoplatanus, Fagus sylvatica, Tilia
cordata, and Abies alba. All data were fitted using linear regression. The per- a non-uniform distribution of photosynthetic activity across
pendicular broken line was drawn to indicate the values of the sun leaves (up- the whole leaf area. However, the PNmax rate estimated via
per right part) and the shade leaves (lower left part). the gas-exchange technique (Fig. 6) at the whole leaf level
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586 H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588

represents the sum of assimilation rates of individual cells. and highly statistically significant differentiation between the
The calculation of some photosynthetic characteristics (e.g. higher photosynthetic activity of sun versus shade leaves and
carboxylation efficiency) from such gas-exchange measure- needles. This method has also successfully been applied to de-
ments is markedly affected by the patchy distribution of sto- tect a decrease in photosynthetic activity due to water stress in
matal apertures on the leaf/needle surface [4]. bean leaves [20], and it can be used to find out the effect of
The close linear correlation between PNmax and Chl fluores- other stress constraints on the photosynthetic performance of
cence decrease ratio RFd at saturating PPFD (Fig. 10) proves leaves as well. Chl fluorescence imaging has the great advan-
the usefulness of RFd values as indicators of photosynthetic tage of the detection and location of smaller and larger gradi-
activity. Although the Chl fluorescence is primarily a trait of ents in photosynthetic quantum conversion across the leaf and
photosystem II, which in principle also applies to the ratio needle areas as shown here for sun and shade leaves. Such gra-
RFd, the latter is a good indicator of the photosynthetic rates, dients as well as small local disturbances and/or dots with
because high RFd values are only obtained when not only pho- a full decline of photosynthetic activity, e.g. on the leaf rim
tosystem II photochemistry works but the whole photosyn- or at various isolated points across the leaf area, representing
thetic process including CO2 fixation. The relationship early or very early stress symptoms, can be detected only via
between RFd values and CO2 assimilation is not only restricted Chl fluorescence imaging, which simultaneously screens the
to sun and shade plants but also exists in non-stomatal crypto- physiological information of many ten thousands leaf pixels.
gamic plants, such as mosses [48]. It has been underlined by Such dots or gradients cannot be detected by the classical
our observation that at fully closed stomata, when no net PN measurements using gas-exchange systems that provide
CO2 exchange is measurable between the leaf and the atmo- only one integrated value of a larger leaf area, usually of sev-
sphere, the leaf photosynthetic activity persists making use eral cm2, per measurement. This emphasizes the importance of
of the intercellular leaf CO2 available from respiratory pro- the new technique of Chl fluorescence imaging for screening
cesses. The ratio RFd still exhibited a value of ca. 1.33 indicat- differences in the physiological function of the photosynthetic
ing photosynthetic quantum conversion. apparatus, not only between leaves and plants of different age
The uneven RFd distribution over the leaf (Fig. 7C,D) and and canopy light exposition, but also in the detection of early
shoot area (Fig. 8C,D) indicates the existence of a considerable stress symptoms and long before damage becomes visually
variability in photosynthetic activity within sun leaves/needles evident.
and also within shade leaves/needles (Fig. 9AeD), as defined
by significantly lower values of variance ( p < 0.05) in shade Acknowledgments
leaves as compared to sun leaves. This had also been observed
[21,26] using a different Chl fluorescence imaging method. Al- The authors wish to thank Mr. Michal Popı́k for his excel-
though we found gradients in the RFd values over the leaf and lent assistance with the Chl fluorescence imaging system in the
needle area, we did not detect such small local areas or dots Beskydy mountains and for collecting the appropriate leaf
within the leaf or the leaf rim, which were virtually free of pho- branches of the trees investigated. This project is part of the
tochemical activity (i.e. very low RFd values and no Chl fluores- research supported by grant no. 522/05/P515 (Grant Agency of
cence induction kinetics), that can show up under stress the Czech Republic), by CzechCarbo project VaV 640/18/03
conditions [15]. This, as well as the normal PN rates, indicated (Ministry of Environmental Protection), and by the ISBE
that the leaves investigated were physiologically active and research intention AV0Z60870520.
without any recognizable stress constraints. Thus, the Chl fluo-
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