Plant Physiology and Biochemistry 45 (2007) 577e588www.elsevier.com/locate/plaphy

Research article

Differences in pigment composition, photosynthetic rates and chlorophyllfluorescence images of sun and shade leaves of four tree species

Hartmut K. Lichtenthaler a, Alexander A�c b, Michal V. Marek b, Jirı Kalina c, Otmar Urban b,*

a Botanical Institute (Molecular Biology and Biochemistry of Plants), University of Karlsruhe, Kaiserstrasse 12, D-76133 Karlsruhe, Germanyb Laboratory of Plants Ecological Physiology, Institute of Systems Biology and Ecology AS CR, Por�ıc�ı 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, Fagussylvatica, 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 thickersun leaves and needles in all tree species were characterized by a lower specific leaf area, lower water content, higher total chlorophyll (Chl) aþband 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 andneedles 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 andneedles exhibited higher rates of maximum net photosynthetic CO2 assimilation (PNmax) measured at saturating irradiance associated with highermaximum stomatal conductance for water vapor efflux. The differences in photosynthetic activity between sun and shade leaves and needlescould 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 additionpermitted detection and quantification of the large gradients in photosynthetic rates across the leaf area existing in sun and shade leaves.� 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

Sun and shade leaves of trees as well as high-light andlow-light plants can considerably differ in their relative com-position of photosynthetic pigments, electron carriers, their

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-

cence; Fs, steady-state Chl fluorescence; Fd, Chl fluorescence decrease from

Fm to Fs; Gs, stomatal conductance for water vapor; LAp, projected leaf

area; LHCII, light-harvesting complex II; PN, net photosynthetic CO2 assimi-

lation rate; PPFD, photosynthetic photon flux density; RFd, Chl fluorescence

decrease ratio; RWC, relative water content; SLA, specific leaf area; xþc,

xanthophylls þ carotenes.

* Corresponding author. Tel./fax: þ420 54 321 1560.

E-mail address: otmar@brno.cas.cz (O. Urban).

0981-9428/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserve

doi:10.1016/j.plaphy.2007.04.006

chloroplast ultrastructure and their photosynthetic rates[1,5,8,16,17,18,29,53]. Leaf and chloroplast adaptation to eitherhigh or low irradiance, to direct sun-light or shade proceed dur-ing leaf development and comprise special morphological andbiochemical adaptations. Sun leaves and high-light plantspossess sun-type chloroplasts that are adapted for high rates ofphotosynthetic quantum conversion, they possess a higher pho-tosynthetic capacity on a leaf area and chlorophyll (Chl) basis,exhibit higher values for the ratio Chl a/b, a much lower levelof light-harvesting Chl a/b proteins (LHCII), and a lower stack-ing degree of thylakoids than shade leaves and low-light plantswith their shade-type chloroplasts [23e25]. Major differencesin the chloroplast adaptation response to either high- or low-light quanta fluence rates have recently been summarized [21].This review, giving access to further literature in this field, shows

d.

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

that much work has been done with individual herbaceous plantsgrown at high or low quanta fluence rates or with selected decid-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 leaveshave been measured, whereas the question if sun and shadeleaves differ in their photosynthetic activity, also on a Chl basis,remained open because pigment determinations were notperformed.

So far the photosynthetic activities of leaves, and especiallythe differences in CO2 fixation rates between sun and shadeleaves, have primarily been performed by net CO2 measure-ments with CO2/H2O porometer gas-exchange systems. Theseprovide, however, only one integral PN rate per measurement ofa relatively large leaf area spot or of a certain quantity of nee-dles on a small conifer branch. Whether the photosynthetic ac-tivity is evenly distributed over the leaf area or whetherinhomogeneities or small local declines in photosynthetic ratesexist, cannot be detected using gas exchange measurements. Inrecent years it has been shown that the chlorophyll fluorescencedecrease ratio RFd is linearly correlated to the net photosyn-thetic CO2 assimilation rate PN and is an indicator of the pho-tosynthetic activity of leaves [21,27]. Moreover, a specialimaging technique has been developed [20,26] that allows im-aging the RFd ratio of whole leaves with at least several tens ofthousands, or more than 100,000 pixels per measurement. Thisshould principally be also possible with the new pulse ampli-tude fluorescence imaging system [32] when it is combinedwith an additional saturating light source.

Thus a major objective of this investigation was to prove bymeans of Chl fluorescence imaging, if the differences in photo-synthetic activity of sun and shade leaves would show up in theRFd images, and if there were differences between the two leaftypes in the distribution of photosynthetic activity across theleaf area. In order to better characterize the leaves, the relativeChl and carotenoid composition, as well as the photosyntheticactivity of sun and shade leaves were compared, and we alsochecked whether the higher CO2 assimilation rates of sunleaves/needles also exist on a Chl basis. Four common tree spe-cies of the temperate zone from the same location and climatewith different tolerance to shade conditions [49] were selectedfor this study: namely the strongly shade-tolerant linden (Tiliacordata Mill.) and fir (Abies alba Mill.) as well as the lessshade-tolerant beech (Fagus sylvatica L) and maple (Acerpseudoplatanus L.).

2. Methods

2.1. Site description

Experiments were carried out at the ecological research sta-tion in a natural stand of trees in a forest located in the Mora-vian-Silesian Beskydy Mountains (Bıly Krı�z, 49�330 N, 18�320

E, 908 m a.s.l., NE of the Czech Republic) in the middle of thegrowing season (mid to late July 2005). The climate of thearea is characterized by an annual mean temperature of5.5 �C, annual mean relative air humidity of 80%, and a totalrainfall of 1000e1400 mm. The geological bedrock is formed

by Mesozoic Godula sandstone (flysch type), with ferricpodzols. See detailed description in Kratochvılova et al. [12].

2.2. Plant material

Branches with fully developed sun and shade adapted leavesof four tree species were sampled: the deciduous species beech(Fagus sylvatica L.), maple (Acer pseudoplatanus L.), lindentree (Tilia cordata Mill.), and shoots of the coniferous fir (Abiesalba Mill.). All trees grew in the neighborhood of the ecologicalresearch station, either on the rim of a homogeneous 30-year-old Norway spruce forest, or on the transition to an openmeadow area and a small beech forest. Shade leaves at the innertree crown received up to 100 mmol(photons) m�2 s�1 on sunnydays, whereas the sun leaves were exposed to a maximum PPFDof 1200e1500 mmol(photons) m�2 s�1. A branch with the de-sired leaf or needles was cut from the tree and the cut endwas immediately re-cut under water to remove xylem embo-lisms. The branch end remained in the water during the mea-surements. The branches investigated here were derived fromthree to four trees per species, and from 30- to 40-year-old trees(Acer, Fagus, Tilia) and ca. 20- to 30-year-old trees (Abies). Thebranches were taken from completely healthy trees in mid-July2005 at rather cool temperatures (mid-day temperatures of 16 to20 �C), on partly cloudy days (maximum PPFD between 950and 1250 mmol(photons) m�2 s�1) with some rain, and theydid not exhibit signs of drought or photoinhibition that can,however, be experienced on hot, sunny and dry summer days.Under the experimental conditions there were no significant dif-ferences between attached and cut branches in the maximumCO2 assimilation rate (PNmax), stomatal conductance for watervapor (GSmax) or in the RFd values. In addition, samples for themeasurements were taken in the morning and afternoon, yet sig-nificant differences in these parameters during the course of theday, e.g. a mid-day depression, were not detected under thegiven climatic conditions.

The projected leaf area (LAp) of all investigated leaves wasestimated using a leaf area meter (LI-3000A, LI-COR, Lincoln,NE, USA), as well as their fresh weight (FW) and dry weight(DW) after drying at 80 �C for 48 h. Subsequently, the relativewater content (RWC [%]; RWC ¼ [(FW � DW)/FW] � 100)and specific leaf area SLA [cm2 g�1 DW] were determined,whereby the SLA ¼ LAp/DW was calculated in agreementwith Gilmore et al. [7]. In contrast to needles of other coniferoustrees, fir needles are flat, their needle area can easily be deter-mined, and thus their SLA can be compared to that of leavesof broadleaf trees. The leaf and needle thickness was determinedwith a digital micrometer system (Mitutoyo Corp., Japan).

2.3. Pigment analysis

Leaf and needle samples were frozen in liquid nitrogen (ca.100 mg of fresh weight) and later analyzed in the laboratory inOstrava. The photosynthetic plant pigments, Chls and Cars,were extracted with 80% acetone and a small amount ofMgCO3. The clear supernatant obtained after centrifugation at480 � g for 3 min was used for spectrophotometric (UV/VIS

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

550, Unicam, Cambridge, UK) estimation of Chl a, Chl b andtotal carotenoids in the same extract solution using the extinc-tion coefficients and equations redetermined by Lichtenthaler[19], and given in more detail in Lichtenthaler and Buschmann[22]. From the pigment levels the weight ratios of pigments, Chla/b and Chls/Cars (a þ b)/(x þ c) were determined that signif-icantly differ between sun and shade leaves.

2.4. Gas-exchange measurement

An infra-red gas analyzer (LI-6400, LI-COR) was used toestimate the maximum CO2 assimilation rate (PNmax). Thevalues were recorded after ca. 20 min of exposure to saturat-ing irradiance (1500 mmol(photons) m�2 s�1) when the sto-mata had fully opened (maximum stomatal conductance forwater vapor; GSmax). Leaf temperature, relative air humidity,and CO2 concentration inside the leaf chamber were keptconstant at 18e21 �C, 50e55%, and 375 mmol(CO2) mol�1,respectively.

2.5. Chlorophyll fluorescence imaging

The chlorophyll (Chl) fluorescence induction kinetics (Kaut-sky effect) of pre-darkened leaves (20 min) were measured atthe red Chl fluorescence band (near 690 nm) using the kineticimaging fluorometer (FluorCam, Photon System InstrumentsLtd., Brno, Czech Republic). The lens of the CCD camerawas ca. 7 cm perpendicular to the leaf surface. At first, the initialfluorescence level, Fo, was measured in 20 min dark-adaptedsamples by low-intensity measuring-pulses (10 ms pulses ofan intensity of ca. 0.003 mmol m�2 s�1). The latter were gener-ated in two panels of orange light-emitting diodes (HLMP-EH08, Agilent Technologies, Santa Clara, CA, USA). Theywere applied 2 s each and had no detectable actinic effect.Afterwards, in modifying the FluorCam program, continuousactinic saturating light (ca. 2000 mmol(photons) m�2 s�1), gen-erated by a separate Oriel 150 W xenon lamp (model 66056)with UV filters, was applied to obtain the maximum fluores-cence level, Fm. Continuous saturating light was also requiredto obtain the steady-state fluorescence level, Fs, that was esti-mated from the Chl fluorescence induction curve after 5 minof continuous illumination. See [32] for detailed descriptionof the fluorescence system and [27] for details of the measuringprotocol. The imaging instrument possesses a calibration devicecorrecting for non-uniform irradiation, yet when testing the dis-tribution of the irradiance in the leaf sample area, it was found tobe fairly homogeneous with a maximal deviation of 3%.

The Chl fluorescence decrease ratio (RFd) was determinedfrom the induction curve according to Lichtenthaler and Babani[20] and Lichtenthaler et al. [27]:

RFd ¼ Fd=Fs¼ ðFm�FsÞ=Fs ð1Þ

where Fm is the maximum fluorescence level, Fs is the steadystate fluorescence (5 min after onset of saturating irradiance),and Fd represents the chlorophyll fluorescence decline fromFm to Fs. The Fm, Fd and Fs values were calculated for

each individual leaf pixel. The applied irradiance of2000 mmol(photons) m�2 s�1 may not have been sufficient,e.g. in all sun leaf or sun needle samples, to reach the full max-imum level of Fm. Yet, in contrast to the absolute Chl fluores-cence signal, the RFd values as Chl fluorescence ratio are lessdependent on a further increase of the irradiance when an irra-diance of at least 2000 mmol(photons) m�2 s�1 has been ap-plied. We checked with a conventional PAM Chl fluorometerthat an increase of the irradiance up to saturating 3500 or4000 mmol(photons) m�2 s�1 increased the RFd values byless than 5%. Such a high PPFD given for 5 min is, however,not recommendable in order to avoid photoinhibition or dam-age to the photosynthetic apparatus. After the collection of theChl fluorescence images, the RFd images and the histogramswith the RFd frequency distribution were constructed usingthe imaging fluorometer software package. The histogramson the RFd frequency distribution in sun and shade leavesare based on 160,000 leaf pixels (Fagus), 90,000 (Acer, Tilia)and >40,000 needle pixels for each leaf type. For a bettercomparison of the RFd value distribution of the four trees thelatter is expressed in percentage units (Fig. 9).

2.6. Statistical analysis

The LSD test (belonging to ANOVA group of tests) was usedto evaluate the statistically significant differences between sunand shade leaves. Differences were tested at probability levelsp ¼ 0.05, 0.01 and 0.001. The homogeneity of the variance ofthe RFd frequency distribution was tested using the F test, andsubsequently, two sample t-test with uneven variances were ap-plied for testing the significant differences between sun andshaded RFd histograms of each tree species. All statistical testswere performed using STATISTICA software (StatSoft, Inc.,Tulsa, OK, USA).

3. Results

3.1. General leaf characteristics

The sun-exposed leaves from end-branches of the threebroadleaf trees investigated showed the typical, larger thick-ness of sun leaves (by ca. 40% to 65%) as compared to shadeleaves. Since this had not been clearly investigated in the caseof sun and shade fir needles, we have measured the thicknessof several needle age classes and found that sun needles were62% to 76% thicker than shade needles depending on the nee-dle age (Fig. 1). Sun leaves and needles had a smaller pro-jected leaf and needle area as compared to the thinner shadeleaves and needles that possess a higher relative water content.Thus, the relative water content (RWC) in sun leaves was sig-nificantly ( p < 0.01) lower (by 8e22%) as compared to shadeleaves in all tree species (Fig. 2A). In addition, there also ex-isted the expected statistically significant differences( p < 0.01) in the specific leaf area (SLA) with higher valuesfor shade leaves and needles as compared to sun leaves andsun needles (Fig. 2B).

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

3.2. Chlorophyll and carotenoid levels

The differences in chlorophyll (Chls) and total carotenoid(Cars) contents between sun and shade leaves are summarizedin Fig. 3. One has to differentiate between two reference sys-tems, the levels per leaf area unit and the levels per leaf dryweight, which yield contrasting results. Regarding photosyn-thetic processes that are controlled by incident light, it ismore appropriate to use the leaf area as a reference system;however, the dry weight often indicated in the literature isgiven here for comparative reasons as well. On a leaf area ba-sis the Chl (aþb) amounts were significantly higher in sunleaves of A. pseudoplatanus and T. cordata (Fig. 3A) as com-pared to shade leaves, but the differences were not statisticallysignificant ( p > 0.05) in F. sylvatica and A. alba. In contrast,on a dry matter basis the shade leaves and needles had a signif-icantly ( p < 0.01) higher Chl (aþb) content, up to 147% forall four tree species (Fig. 3B). The content of total Cars ona leaf area basis was significantly lower ( p < 0.01) in shadeleaves compared to sun leaves (Fig. 3C), except for A. albawhere the difference was not significant ( p > 0.05). Ona dry matter basis, however, the shade leaves and needleshad a significantly higher total Cars content (Fig. 3D).

3.2.1. Pigment ratiosThe typical differences between sun and shade leaves in the

pigment ratios Chl a/b and Chls/Cars were found to be as ex-pected (Fig. 4). The sun leaves of all investigated tree specieshad significantly higher values for the ratio Chl a/b (Fig. 4A)and lower values for the ratio of Chls/Cars (Fig. 4B), which isalso known as ratio (a þ b)/(x þ c) [2,42,54].

3.3. Measurements of photosynthetic rates PN

Maximum CO2 assimilation rates (PNmax) per leaf area unit(Fig. 5A) at saturating photosynthetic photon flux density

0

200

400

600

800

1st 2nd 3rd 4th 5thNeedle age class

Th

ic

kn

es

s (µ

m)

SunShade

Fig. 1. Differences in needle thickness (mm) between sun and shade needles of

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

each needle year and condition). Columns represent mean values and bars

standard deviations. The differences are highly significant ( p < 0.001).

(PPFD z 1500 mmol m�2 s�1) were significantly ( p < 0.01)higher in sun leaves and needles (range: from 97% to 168%)than in shade leaves. Even on a Chl (aþb) basis the sun leavesand needles exhibited a higher PNmax rate than shade leaves andneedles (Fig. 5B). However, the differences in PNmax rates ona Chl basis were not as high (range: from 76% to 128%) as ona leaf area basis. There was an additional difference when thePNmax rate on a leaf dry weight basis, reflecting the influenceof the leaves’ morphological structure, was taken as a referencesystem (Fig. 5C). As compared to sun leaves, higher PNmax rateson a leaf dry weight basis were detected in the shade leaves ofA. pseudoplatanus (19%) and F. sylvatica (48%), whereas thePNmax rates were lower in the shade leaves of T. cordata(34%) and A. alba (32%). These differences were statisticallysignificant ( p < 0.01) except for A. pseudoplatanus.

At saturating PPFD, PNmax linearly correlated (PNmax ¼0.0631 GSmax; R2 ¼ 0.78) with maximum stomatal conductance(GSmax) as shown in Fig. 6. The GSmax values of sun leaves andneedles were always significantly higher as compared to thoseof shade leaves. In all cases the latter remained below valuesof 100 mmol m�2 s�1, whereas sun leaves and needles exhibitedGSmax values of above100 mmol m�2 s�1 as indicated in Fig. 6by a broken line.

0

10

20

30

40

50

60

70

80

Acer Fagus Tilia Abies

RW

C (%

)

A

0

50

100

150

200

250

300

350

400

Acer Fagus Tilia Abies

SL

A (cm

2 g

-1)

SunShade

B

Fig. 2. A,B. Relative water content (RWC; A) and specific leaf area (SLA; B)

in sun (white columns) and shade leaves (black columns) of deciduous Acer

pseudoplatanus, Fagus sylvatica, Tilia cordata and coniferous Abies alba.

All differences between sun and shade leaves are statistically highly significant

( p < 0.01). Columns represent mean values and bars standard deviations.

n ¼ 8.

581H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Acer Fagus Tilia Abies

Ch

l (a+

b) (g

m

-2)

A**

**

0

2

4

6

8

10

12

14

Acer Fagus Tilia Abies

Ch

l (a+

b) (m

g g

-1)

B** **

**

**

0.0

0.5

1.0

1.5

2.0

2.5

Acer Fagus Tilia Abies

Cars (x

+c) (m

g g

-1)

SunShade

D** **

**

*

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Acer Fagus Tilia Abies

Cars (x+

c) (g

m

-2)

C**

****

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

The chlorophyll (Chl) fluorescence images (Fig. 7A,B)demonstrate that the red Chl fluorescence signal (band near690 nm) is unevenly distributed across the leaf area at boththe maximum Chl fluorescence at a saturating pulse (Fm),and at the steady state Chl fluorescence at a saturating photo-synthetic photon flux density (Fs). These images may subse-quently be applied to deduce the distribution of the RFd

values in leaves (Fig. 8C,D). The RFd images indicate signifi-cantly higher RFd values in sun leaves in comparison to shadeleaves, e.g. of the broadleaf F. sylvatica (Fig. 7C,D) and evenin the needles of the coniferous A. alba (Fig. 8).

The normalized frequency distribution of the RFd values ofall tree species studied demonstrated highly significant( p < 0.001) differences in the distribution of the RFd valuesbetween sun and shade leaves (Fig. 9AeD). The RFd value dis-tributions, shown here for better comparison as percentage dis-tribution, are based on more than 90,000 leaf pixels and onmore than 40,000 needle pixels per RFd image evaluated. Infact, the average RFd values of sun leaves were several timeshigher than those in shade leaves (Acer 2.0�, Fagus 1.7�,Tilia 2.7�, and Abies 3.7�). The Chl fluorescence imagesand the RFd frequency distributions, shown in Fig. 9, are ofone leaf and one needle shoot sample. Parallel images weretaken of additional leaves and needle shoots of all tree species,and the differences in images and RFd value distribution (datanot shown) were very similar and practically almost identicalto those shown.

When RFd values and PNmax rates, both measured at saturat-ing PPFD, were compared, a close linear correlation(RFd ¼ 0.27 PNmaxþ1.33; R2 ¼ 0.91) between both parametersshowed up (Fig. 10). The same linear relationship applies toboth sun and shade leaves and needles. Correspondingly, atzero net CO2 assimilation rate (PNmax ¼ 0 mmol m�2 s�1) anRFd value of ca. 1.33 is found.

4. Discussion

In this study all four common tree species of the temperatezone exhibited major differences between sun and shade leavesthat had developed at either sun exposed or fully shaded condi-tions. The lower RWC (Fig. 2A) and a lower SLA (Fig. 2B) insun leaves and sun needles of the four tree species investigatedas compared to shade leaves and needles, is in accordance withprevious studies showing a lower water content (51%), a lowerSLA (116 cm2 g�1) in beech sun leaves as compared to beechshade leaves (SLA: 335 cm2 g�1, water content 64%) [17].This was confirmed in an independent investigation indicatingthat sun leaves of F. sylvatica had a significantly lower SLA(118 cm2 g�1) and water content (42%) than shade leaves(SLA: 282 cm2 g�1; water content: 53%) [38]. Significantlylower SLA values were also described for the sunlit leaves ofthe upper canopy level as compared to leaves of the lower can-opy level in ash, hornbeam, maple and linden trees [10]. SLAvalues are considered a measurement of leaf structure, thick-ness, the amount of mechanical tissues in leaves, and a measure-ment of the morphological difference between sun and shade

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

leaves and needles [7]. In fact, beech sun leaves are thicker (144to 185 mm) than shade leaves (88e93 mm) [17,23], which wasconfirmed in the present investigation and extended for theother trees. Practically the same observations were made usingthe inverse SLA ratio, the leaf dry mass per area LMA, which, inseveral 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 andRWC was reported in groundnut (Arachis hypogaea) leaves[31], whereby the rate of reduction in leaf RWC during a pro-gressive moisture deficit was directly related to SLA and, asa consequence, affected the leaf carbon exchange.

Differences in PNmax on a leaf area unit between sun andshade leaves (Fig. 5A) were similar to those previously de-scribed for beech [17,23], several other broadleaf [13,26,28,36] and coniferous tree species [40,43], as well as variousother plants [5,20,21,35]. Also, in sunlit leaves of the upper can-opy level of four tree species higher photosynthetic rates per leafarea unit were found as compared to leaves of the lower canopylevel [10], whereby tree specific differences showed up. Higherlight saturated rates of photosynthesis in sun leaves are mainlyassociated with a greater amount of nitrogen per leaf area unitin sun leaves as compared to shade leaves [9,10,13,18], and

0

1

2

3

4

Acer Fagus Tilia Abies

Ch

l a/b

A

0

1

2

3

4

5

6

7

Acer Fagus Tilia Abies

Ch

ls/ C

ars

SunShade

B

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

leaves (black columns) of deciduous Acer pseudoplatanus, Fagus sylvatica,

Tilia cordata and coniferous Abies alba are presented. All differences between

sun and shade leaves are statistically highly significant ( p < 0.01). Columns

represent mean values and bars the standard deviations. n ¼ 8.

they usually have a higher level of Chls per leaf area unit(cf. Fig. 3A) as shown before for ginkgo, beech, hornbeam,and poplar [22,41,44]. Moreover, the higher nitrogen levelsper leaf area unit also result in a higher content of Rubisco en-zyme and subsequently in the stimulation of CO2 uptake athigh irradiances [13,18]. The level range of total Chls per leaf

0

2

4

6

8

10

12

14

PN

max (µ

mo

l m

-2 s

-1)

SunShade

SunShade

SunShade

A

0.0

0.2

0.4

0.6

0.8

1.0

Acer Fagus Tilia Abies

PN

max (µ

mo

l m

gD

W-1 h

-1)

C

0

20

40

60

80

100

120

140

Acer Fagus Tilia Abies

Acer Fagus Tilia Abies

PN

max (µ

mo

l m

gC

hl-1 h

-1)

B

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

(B), and on a leaf dry weight basis (C). Mean values (columns) and standard

deviations (bars) of sun (white columns) and shade leaves and needles (black

columns) of Acer pseudoplatanus, Fagus sylvatica, Tilia cordata, and Abies

alba are presented. All differences between sun and shade leaves are statisti-

cally highly significant ( p < 0.01), except PNmax expressed on a leaf dry

weight basis (C) in A. pseudoplatanus. n ¼ 8.

583H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588

area unit can partially overlap in sun and shade leaves. However,on a dry matter basis shade leaves always contain significantlyhigher amounts of total Chls as compared to sun leaves(Fig. 3B). This has been shown before for horse chestnut, oak,

0

2

4

6

8

10

12

14

16

0 50 100 150 200 250G

Smax (µmol m

-2 s

-2)

PN

max (µ

mo

l m

-2 s

-1)

Acer

Fagus

Tilia

Abies

y = 0.0631xR2 = 0.78

SunShade

Fig. 6. Relationship between maximum CO2 assimilation rate (PNmax) and

maximum stomatal conductance for water vapor (GSmax) at saturating photo-

synthetic photon flux density (PPFD z 1500 mmol m�2 s�1). Symbols repre-

sent the mean of five measurements of sun and shade leaves of Acerpseudoplatanus, Fagus sylvatica, Tilia cordata, and shoots of Abies alba.

All data were fitted using linear regression. The perpendicular broken line

was drawn to indicate the values of sun leaves (upper right part) and shade

leaves (lower left part).

hornbeam and ginkgo [17,22,41] and other plants [35]. Our ob-servation that sun leaves and sun needles possess much highervalues for the maximum stomatal conductance for water vapor(GSmax) (Fig. 6) as compared to shade leaves and shade needlesmay indicate that they are able to open their stomata much widerthan shade leaves and shade needles. This certainly appears to bean essential prerequisite for their higher photosynthetic rates.However, also a higher stomata density per leaf area unit ofsun leaves and upper canopy leaves as compared to shade leavesand lower canopy leaves must be taken into consideration in thisrespect [10,17,23].

The organization of the pigment apparatus and the relativelevels of Chl a and Chl b as well as the ratio of total Chls to totalCars is also an essential difference between sun and shadeleaves and needles. Sun leaves/needles possess sun-type chloro-plasts with higher values for the ratio Chl a/b and lower valuesfor the weight ratio Chls/Cars (Fig. 4A,B) than the shade-typechloroplasts of shade leaves and leaves of low light plants[2, 6, 18, 23]. The values for the ratio Chl a/b in sun (range:from 3.0 to 3.4) and shade leaves (range: from 2.4 to 2.7) aswell as the values for the ratio Chls/Cars (sun leaves 3.8 to4.4, shade leaves 4.8 to 5.7) were found in the normal rangeof physiologically active leaves [21,41]. Such differential pig-ment ratios are characteristic for sun-type and shade-type chlo-roplasts [21, 41]. Significantly higher values for the ratio Chl a/bwere also found in the sunlit leaves of the upper canopy levelof four broadleaf tree species as compared to leaves of the lowercanopy 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).

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, andhence lower values for the ratio Chls/Cars, is primarily causedby their higher level of the xanthophyll cycle carotenoids[6,10,45,47]. In addition, these differences in the pigment ratiosare also a result of the high irradiance adaptation response of thephotosynthetic pigment apparatus of sun leaves (sun chloro-plasts) with fewer light-harvesting Chl a/b proteins (LHCII)and a larger number of reaction center pigment proteins (e.g.CPa, CPI) on a total Chl basis as compared to shade chloroplasts[24], as well as a greater number of electron transport chains[21,23,53]. At the same time shade leaves and shade-type chlo-roplasts exhibit higher and broader grana thylakoid stacks andprimarily invest into the pigment antenna [24]. As a consequenceof the adaptation response of their chloroplasts to high irradi-ance, sun leaves of trees with their sun-type chloroplasts possessconsiderably higher PN rates on a leaf area basis than shadeleaves. One reason for the higher PNmax rates of sun leavesand needles as compared to shade leaves is their generally higherChl content per leaf area unit, but the major reason is the posses-sion of sun-type chloroplasts with their different ultrastructure,biochemical organization and a special arrangement of theirChls and Cars in the thylakoids. The possession of sun-typechloroplasts is also the major reason why sun leaves and needlesexhibit higher net CO2 assimilation rates PNmax, not only ona leaf area basis but also on a Chl basis (Fig. 5B). The lower dif-ferences in PN rates on a Chl basis between sun and shade leaves(Fig. 5B) indicate that one possible reason for the higher PN ratesin sun leaves/needles is their generally higher Chl content per

leaf area unit (Fig. 2A), as also found in the fan-shaped gymno-sperm leaves of Ginkgo [41]. The higher PNmax rates of sunleaves/needles are also well reflected in the higher values ofthe Chl fluorescence decrease ratio RFd (Fig. 10) that representsa non-destructive indicator of the in vivo photosynthetic rates ofleaves and is measured from near distance without any directcontact with the leaf [21,26,41].

Light-saturated rates of photosynthesis on leaf area basis(PNmax) depend not only on photosynthetic biochemistry butalso on the mesophyll structure of leaves. Since resistance toCO2 diffusion from the substomatal cavity to the stroma issubstantial, it is likely that the mesophyll structure affectsPNmax by affecting the diffusion of CO2 [40,46] and the pene-tration of light [51] in the leaf. In addition, the photosyntheticrate per leaf area unit represents the sum of assimilation ratesof individual cells; however, the thicker sun leaves contain sig-nificantly more cells as compared to the thinner shade leaves[17,18,37]. Therefore, the PNmax rates per unit leaf weight(Fig. 5C) reflect the influence of the morphological leaf struc-ture on CO2 uptake. We found significantly higher PNmax perleaf weight unit in shade leaves of A. pseudoplatanus and F.sylvatica as compared to sun leaves, whereas PNmax per leafweight unit of shade leaves was lower in the highly shade-toler-ant species A. alba and T. cordata as compared to sun leaves(Fig. 5C). Kubiske and Pregitzer [13] concluded that the shadeleaves of shade-intolerant species respond to shade primarilyby altering SLA, whereas shade-tolerant species respond largelyvia biochemical acclimation of the photosynthetic apparatus.

585H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588

0

5

10

15

20

25

RFd

(dimensionless)

Relat. freq

uen

cy d

istrib

utio

n (%

)

Sun

Shade

AAcer

0

5

10

15

20

25

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8R

Fd (dimensionless)

Relat. freq

uen

cy d

istrib

utio

n (%

)

Sun

Shade

BFagus

0

5

10

15

20

25

RFd

(dimensionless)

Relat. freq

uen

cy d

istrib

utio

n (%

)

Sun

Shade

Tilia C

0

5

10

15

20

25

0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8

RFd

(dimensionless)

Relat. freq

uen

cy d

istrib

utio

n (%

)

Sun

Shade

Abies D

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 havesmall stomata, however, a higher stomatal density as com-pared to shade leaves [21,23,37,55], and this also applies

0

1

2

3

4

5

6

0 2 4 6 8 10 12 14P

Nmax (µmol m

-2 s

-1)

RF

d

Acer

Fagus

Tilia

Abies

y = 0.27x + 1.33R2 = 0.91

Shade Sun

Fig. 10. Correlation between the red (band near 690 nm) chlorophyll fluores-

cence decrease ratio (RFd) and the maximum CO2 assimilation rate (PNmax) at

saturating photosynthetic photon flux density (PPFD z 1500 mmol m�2 s�1)

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-

pendicular broken line was drawn to indicate the values of the sun leaves (up-

per right part) and the shade leaves (lower left part).

to the leaves of high irradiance in comparison to low irradi-ance plants [52]. Hence, higher light saturated stomatal con-ductance (GSmax) is usually found in sun leaves (Fig. 6). Themuch higher stomata opening in sun leaves (GSmax range 100up to 300 mmol m�2 s�1; in shade leaves clearly below100 mmol m�2 s�1) contributes to the higher intercellularCO2 concentrations, and thus to the higher CO2 assimilationrates. Moreover, Fig. 6 shows that PNmax at saturating PPFDlinearly correlates with maximum stomatal conductance(GSmax). Similar relationships were found for Southern Beech(Nothofagus cunninghamii) [11], African savanna tree species[30] and for temperate grassland species [50]. The relation-ship between PNmax and GSmax indicates that stomatal controlof CO2 uptake at GSmax is smaller than about 200 mmolm�2 s�1. However, the response curves level off at high sto-matal conductance above 300 mmol m�2 s�1 [30].

Non-uniform opening of stomata was observed in leaves ofF. sylvatica [14], needles of A. alba [4] and other plants (re-viewed in [39]). The stomatal patchiness is a consequence ofthe heterogeneous water status in different parts of the leafand can be induced by all ambient factors causing such hetero-geneities [3]. It leads to a non-uniform distribution of intercel-lular CO2 concentration within the leaf and subsequently toa non-uniform distribution of photosynthetic activity acrossthe whole leaf area. However, the PNmax rate estimated viathe gas-exchange technique (Fig. 6) at the whole leaf level

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

represents the sum of assimilation rates of individual cells.The calculation of some photosynthetic characteristics (e.g.carboxylation efficiency) from such gas-exchange measure-ments is markedly affected by the patchy distribution of sto-matal apertures on the leaf/needle surface [4].

The close linear correlation between PNmax and Chl fluores-cence decrease ratio RFd at saturating PPFD (Fig. 10) provesthe usefulness of RFd values as indicators of photosyntheticactivity. Although the Chl fluorescence is primarily a trait ofphotosystem II, which in principle also applies to the ratioRFd, the latter is a good indicator of the photosynthetic rates,because high RFd values are only obtained when not only pho-tosystem II photochemistry works but the whole photosyn-thetic process including CO2 fixation. The relationshipbetween RFd values and CO2 assimilation is not only restrictedto sun and shade plants but also exists in non-stomatal crypto-gamic plants, such as mosses [48]. It has been underlined byour observation that at fully closed stomata, when no netCO2 exchange is measurable between the leaf and the atmo-sphere, the leaf photosynthetic activity persists making useof the intercellular leaf CO2 available from respiratory pro-cesses. The ratio RFd still exhibited a value of ca. 1.33 indicat-ing photosynthetic quantum conversion.

The uneven RFd distribution over the leaf (Fig. 7C,D) andshoot area (Fig. 8C,D) indicates the existence of a considerablevariability in photosynthetic activity within sun leaves/needlesand also within shade leaves/needles (Fig. 9AeD), as definedby significantly lower values of variance ( p < 0.05) in shadeleaves as compared to sun leaves. This had also been observed[21,26] using a different Chl fluorescence imaging method. Al-though we found gradients in the RFd values over the leaf andneedle area, we did not detect such small local areas or dotswithin the leaf or the leaf rim, which were virtually free of pho-tochemical activity (i.e. very low RFd values and no Chl fluores-cence induction kinetics), that can show up under stressconditions [15]. This, as well as the normal PN rates, indicatedthat the leaves investigated were physiologically active andwithout any recognizable stress constraints. Thus, the Chl fluo-rescence image technique represents a very useful approach inthe study of stomatal and photosynthetic heterogeneity acrossthe leaf area. We observed the distribution of the photosyn-thetic activity at saturating PPFD to be more uniform in shadeleaves (Fig. 6C,D) and needles (Fig. 7C,D) as compared to sunleaves/needles. These findings support the conclusion of [14]that the presence of stomatal patchiness does not necessarilyaffect the intercellular CO2 concentrations in shade and half-shade leaves. The hypothesis explaining the lower gradient ofphotosynthetic activity in shade leaves is that shade leavesare exposed to a more homogeneous environment duringmost of their existence, therefore enabling more homogeneousleaf properties.

5. Conclusion

The results of this investigation show that Chl fluorescenceimaging of RFd values of leaves, together with the RFd fre-quency distribution (Fig. 9), permits an extremely accurate

and highly statistically significant differentiation between thehigher photosynthetic activity of sun versus shade leaves andneedles. This method has also successfully been applied to de-tect a decrease in photosynthetic activity due to water stress inbean leaves [20], and it can be used to find out the effect ofother stress constraints on the photosynthetic performance ofleaves as well. Chl fluorescence imaging has the great advan-tage of the detection and location of smaller and larger gradi-ents in photosynthetic quantum conversion across the leaf andneedle areas as shown here for sun and shade leaves. Such gra-dients as well as small local disturbances and/or dots witha full decline of photosynthetic activity, e.g. on the leaf rimor at various isolated points across the leaf area, representingearly or very early stress symptoms, can be detected only viaChl fluorescence imaging, which simultaneously screens thephysiological information of many ten thousands leaf pixels.Such dots or gradients cannot be detected by the classicalPN measurements using gas-exchange systems that provideonly one integrated value of a larger leaf area, usually of sev-eral cm2, per measurement. This emphasizes the importance ofthe new technique of Chl fluorescence imaging for screeningdifferences in the physiological function of the photosyntheticapparatus, not only between leaves and plants of different ageand canopy light exposition, but also in the detection of earlystress symptoms and long before damage becomes visuallyevident.

Acknowledgments

The authors wish to thank Mr. Michal Popık for his excel-lent assistance with the Chl fluorescence imaging system in theBeskydy mountains and for collecting the appropriate leafbranches of the trees investigated. This project is part of theresearch supported by grant no. 522/05/P515 (Grant Agency ofthe Czech Republic), by CzechCarbo project VaV 640/18/03(Ministry of Environmental Protection), and by the ISBEresearch intention AV0Z60870520.

References

[1] J.M. Anderson, W.S. Chow, Y.-I. Park, The grand design of photosynthe-

sis: Acclimation of the photosynthetic apparatus to environmental cues,

Photosynth. Res. 46 (1995) 129e139.

[2] F. Babani, H.K. Lichtenthaler, Light-induced and age-dependent develo-

pment of chloroplasts in etiolated barley leaves as visualized by deter-

mination of photosynthetic pigments, CO2 assimilation rates and

different kinds of chlorophyll fluorescence ratios, J. Plant Physiol. 148

(1996) 555e566.

[3] W. Beyschlag, J. Eckstein, Towards a causal analysis of stomatal patch-

iness: the role of stomatal size variability and hydrological heterogeneity,

Acta Oecol. 22 (2001) 161e173.

[4] G.W. Beyschlag, F. Kresse, R.J. Ryel, H. Pfanz, Stomatal patchiness in

conifersdexperiments with Picea abies (L.) Karst and Abies alba Mill,

Trees Struct. Funct. 8 (1994) 132e138.

[5] N. Boardman, Comparative photosynthesis of sun and shade plants,

Annu. Rev. Plant Physiol. 28 (1977) 355e377.

[6] B. Demmig-Adams, Survey of thermal energy dissipation and pigment com-

position in sun and shade leaves, Plant Cell Physiol. 39 (1998) 474e482.

[7] D.W. Gilmore, R.S. Seymour, W.A. Halteman, M.S. Greenwood, Canopy

dynamics and the morphological development of Abies balsamea: effects

587H.K. Lichtenthaler et al. / Plant Physiology and Biochemistry 45 (2007) 577e588

of foliage age on specific leaf area and secondary vascular development,

Tree Physiol. 15 (1995) 47e55.

[8] T.J. Givnish, Adaptation to sun vs. shade: a whole plant perspective,

Aust. J. Plant Physiol. 15 (1988) 63e92.

[9] J.D. Herrick, R.B. Thomas, Effects of CO2 enrichment on the photosyn-

thetic light response of sun and shade leaves of canopy sweetgum trees

(Liquidambar styraciflua) in a forest ecosystem, Tree Physiol. 19

(1999) 779e786.

[10] D. Holscher, Leaf traits and photosynthetic parameters of saplings and

adult trees of co-existing species in a temperate broad-leaved forest,

Basic Appl. Ecol. 5 (2004) 163e172.

[11] M.J. Hovenden, T. Brodribb, Altitude of origin influences stomatal con-

ductance and therefore maximum assimilation rate in Southern Beech,

Nothofagus cunninghamii, Aust. J. Plant Physiol. 27 (2000) 451e456.

[12] I. Kratochvılova, D. Janous, M. Marek, M. Bartak, L. Rıha, Production

activity of mountain cultivated Norway spruce stands under the impact

of air pollution. I. General description of problems, Ecology (CSSR) 8

(1989) 407e419.

[13] M.E. Kubiske, K.S. Pregitzer, Ecophysiological responses to simulated

canopy gaps of two tree species of contrasting shade tolerance in elevated

CO2, Funct. Ecol. 11 (1997) 24e32.

[14] M. Kuppers, I. Heiland, H. Schneider, P.J. Neugebauer, Light-flecks

cause non-uniform stomatal openingdstudies with special emphasis on

Fagus sylvatica, L., Trees Struct, Funct. 14 (1999) 130e144.

[15] M. Lang, H.K. Lichtenthaler, M. Sowinska, F. Heisel, J.A. Miehe,

F. Tomasini, Fluorescence imaging of water and temperature stress in

plant leaves, J. Plant Physiol. 148 (1996) 613e621.

[16] O.L. Lange, E.-U. Schulze, Measurement of CO2 gas exchange and tran-

spiration in the beech (Fagus sylvatica, L.), in: H. Ellenberg (Ed.), Eco-

logical Studies, Analysis and Synthesis, Vol. 2, Springer, Berlin, 1971,

pp. 16e28.

[17] H.K. Lichtenthaler, Adaptation of leaves and chloroplasts to high quanta

fluence rates, in: G. Akoyunoglou (Ed.), Photosynthesis VI, Balaban

International Science Service, Philadelphia, 1981, pp. 273e285.

[18] H.K. Lichtenthaler, Differences in morphology and chemical composi-

tion of leaves grown at different light intensities and qualities, in:

N.R. Baker, W.J. Davies, C.K. Ong (Eds.), Control of Leaf Growth,

S.E.B. Seminar Series, vol. 27, Cambridge University Press, Cambridge,

1984, pp. 201e221.

[19] H.K. Lichtenthaler, Chlorophylls and carotenoidsdpigments of photo-

synthetic biomembranes, in: S.P. Colowick, N.O. Kaplan (Eds.), Methods

in Enzymology, vol. 148, Academic Press, San Diego, 1987, pp. 350e382.

[20] H.K. Lichtenthaler, F. Babani, Detection of photosynthetic activity and

water stress by imaging the red chlorophyll fluorescence, Plant Physiol.

Biochem. 38 (2000) 889e895.

[21] H.K. Lichtenthaler, F. Babani, Light adaptation and senescence of the

photosynthetic apparatus. Changes in pigment composition, chlorophyll fl-

uorescence parameters and photosynthetic activity, in: G.C. Papageorgiou,

Govindjee (Eds.), Chlorophyll Fluorescence: A Signature of Photosynthesis,

Springer, Dordrecht, 2004, pp. 713e736.

[22] H.K. Lichtenthaler, C. Buschmann, Chlorophylls and carotenoidsdMea-

surement and characterisation by UV-VIS, Current Protocols in Food An-

alytical Chemistry (CPFA), John Wiley, New York, 2001, Supplement 1

pp. F4.3.1eF4.3.8.

[23] H.K. Lichtenthaler, C. Buschmann, M. Doll, H.J. Fietz, T. Bach,

U. Kozel, D. Meier, U. Rahmsdorf, Photosynthetic activity, chloroplast

ultrastructure, and leaf characteristics of high-light and low-light plants

and of sun and shade leaves, Photosynth. Res. 2 (1981) 115e141.

[24] H.K. Lichtenthaler, G. Kuhn, U. Prenzel, D. Meier, Chlorophyll-protein

levels and stacking degree of thylakoids in radish chloroplasts from high-

light, low-light and bentazon-treated plants, Physiol. Plant. 56 (1982)

183e188.

[25] H.K. Lichtenthaler, D. Meier, C. Buschmann, Development of chloro-

plasts at high and low light quanta fluence rates, Israel, J. Bot. 33

(1984) 185e194.

[26] H.K. Lichtenthaler, F. Babani, G. Langsdorf, C. Buschmann, Measure-

ment of differences in red chlorophyll fluorescence and photosynthetic

activity between sun and shade leaves by fluorescence imaging, Photo-

synthetica 38 (2000) 521e529.

[27] H.K. Lichtenthaler, C. Buschmann, M. Knapp, How to correctly deter-

mine the different chlorophyll fluorescence parameters and the chloro-

phyll fluorescence decrease ratio RFd of leaves with the PAM

fluorometer, Photosynthetica 43 (2005) 379e393.

[28] M. Marek, E. Masarovi�cova, I. Kratochvılova, P. Elias, D. Janous, Stand

microclimate and physiological activity of tree leaves in an oak-horn-

beam forest. II. Leaf photosynthetic activity, Trees Struct. Funct. 4

(1989) 234e240.

[29] D. Meier, H.K. Lichtenthaler, Ultrastructural development of chloro-

plasts in radish seedlings grown at high and low light conditions and

in the presence of the herbicide bentazon, Protoplasma 107 (1981)

195e207.

[30] G.F. Midgley, J.N. Aranibar, K.B. Mantlana, S. Macko, Photosynthetic

and gas exchange characteristics of dominant woody plants on a mois-

ture gradient in an African savanna, Global Change Biol. 10 (2004)

309e317.

[31] P.C. Nautiyal, N.R. Rachaputi, Y.C. Joshi, Moisture-deficit-induced

changes in leaf-water content, leaf carbon exchange rate and biomass

production in groundnut cultivars differing in specific leaf area, Field

Crop Res. 74 (2002) 67e79.

[32] L. Nedbal, J. Soukupova, D. Kaftan, J. Whitmarsh, M. Trtılek, Kinetic

imaging of chlorophyll fluorescence using modulated light, Photosynth.

Res. 66 (2000) 3e12.

[33] U Niinemets, Role of foliar nitrogen in light harvesting and shade toler-

ance of four deciduous woody species, Funct. Ecol. 11 (1997) 518e531.

[34] U Niinemets, Global-scale climatic controls of leaf dry mass per area

density, and thickness in trees and shrubs, Ecology 82 (2001) 453e469.

[35] P.S. Nobel, Photosynthetic rates of sun versus shade leaves of Hyptisemoryi Torr, Plant Physiol. 58 (1976) 218e223.

[36] S. Pandey, S. Kumar, P.K. Nagar, Photosynthetic performance of Ginkgo

biloba, L. grown under high and low irradiance, Photosynthetica 41

(2003) 505e511.

[37] R.W. Pearcy, D.A. Sims, Photosynthetic acclimation to changing light

environments: scaling from the leaf to the whole plant, in:

M.M. Caldwell, R.W. Pearcy (Eds.), Exploitation of Environmental Het-

erogeneity by Plants, Academic Press, San Diego, 1994, pp. 145e174.

[38] K. Pilegaard, T.N. Mikkelsen, C. Beier, N.O. Jensen, P. Ambus,

H. Ro-Poulsen, Field measurements of atmosphere-biosphere interactions

in a Danish beech forest, Boreal Environ. Res. 8 (2003) 315e333.

[39] J. Pospısilova, J. Santru�cek, Stomatal patchiness, Biol. Plantarum 36

(1994) 481e510.

[40] T. Priwitzer, O. Urban, M. Sprtova, M.V. Marek, Chloroplastic carbon

dioxide concentration of Norway spruce (Picea abies [L.] Karst) needles

relates to the position within the crown, Photosynthetica 34 (1998)

109e112.

[41] G. Sarijeva, M. Knapp, H.K. Lichtenthaler, Differences in photosynthetic

activity, chlorophyll and carotenoid levels, and in chlorophyll fluores-

cence parameters in green sun and shade leaves of Ginkgo and Fagus,

J. Plant Physiol. (2006). doi:10.1016/jplph epub ahead of print.

[42] A. Seybold, K. Egle, Lichtfeld und Blattfarbstoffe I, Planta 26 (1937)

491e515.

[43] V. Spunda, M. �Cajanek, J. Kalina, I. Lachetova, M. Sprtova, M.V. Marek,

Mechanistic differences in utilization of absorbed excitation energy

within photosynthetic apparatus of Norway spruce induced by the verti-

cal distribution of photosynthetically active radiation through the tree

crown, Plant Sci. 133 (1998) 155e165.

[44] B. Sun, D.L. Dilcher, D.J. Beerling, C. Zhang, D. Yan, E. Kowalski, Var-

iation in Ginkgo biloba, L. leaf characters across a climate gradient in

China, Proc. Natl. Acad. Sci. USA 100 (2003) 7141e7146.

[45] M. Tausz, A.M. Gonzalez-Rodrıguez, A. Wonisch, J. Peters, D. Grill,

D. Morales, M.S. Jimenez, Photostress, photoprotection, and water solu-

ble antioxidants in the canopies of five Canarian laurel forest tree species

during a diurnal course, Flora 199 (2004) 110e119.

[46] I. Terashima, S.I. Miyazawa, Y.T. Hanba, Why are sun leaves thicker

than shade leaves? Consideration based on analyses of CO2 diffusion

in the leaf, J. Plant Res. 114 (2001) 93e105.

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

[47] S.S. Thayer, O. Bjorkman, Leaf xanthophyll content and composition in

sun and shade leaves, Photosyn. Res. 23 (1990) 331e343.

[48] Z. Tuba, Z. Csintalan, A. Badacsonyi, M.C.F. Proctor, Chlorophyll fluo-

rescence as an exploratory tool for ecophysiological studies on mosses

and other small poikilohydric plants, J. Bryol. 19 (1997) 401e407.

[49] L. Uradnı�cek, P. Madera, S. Koliba�cova, J. Koblı�zek, J. Sefl, Woody Spe-

cies in the Czech Republic, Matice Lesnicka, Pısek 2001.

[50] O. Urban, A. A�c, J. Kalina, T. Priwitzer, M. Sprtova, V. Spunda,

M.V. Marek, Temperature dependences of carbon assimilation processes

in four dominant species from mountain grassland ecosystem, Photosyn-

thetica 45 (2007) 392e399.

[51] T.C. Vogelmann, G. Martin, The functional-significance of palisade tis-

suedpenetration of directional versus diffuse light, Plant Cell Environ.

16 (1993) 65e72.

[52] A. Wild, G. Wolf, The effect of light intensities on the frequency and size

of stomata and on the number, size and the control of chloroplasts in the

mesophyll and guard cells during the development of the primary leaves

of Sinapis alba, Z. Pflanzenphys 97 (1980) 325e342.

[53] A. Wild, M. Hopfner, W. Ruhle, M. Richter, Changes in the stochiometry

of photosystem II components as an adaptive response to high-light and

low-light conditions during growth, Z, Naturforsch C 41 (1986)

597e603.

[54] R. Willstatter, A. Stoll, Untersuchungen uber Chlorophyll, Springer, Ber-

lin, 1913.

[55] H. Zhang, M.R. Sharifi, P.S. Nobel, Photosynthetic characteristics of sun

versus shade plants of Encelia farinosa as affected by photosynthetic

photon flux density. Intercellular CO2 concentration, leaf water potential,

and leaf temperature, Aust. J. Plant Physiol. 22 (1995) 833e841.