Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees

Transcription

1 Photosynth Res (2007) 93: DOI /s RESEARCH ARTICLE Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees Hartmut Karl Lichtenthaler Æ Fatbardha Babani Æ Gabriele Langsdorf Received: 21 August 2006 / Accepted: 11 April 2007 / Published online: 8 May 2007 Ó Springer Science+Business Media B.V Abstract The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves ; shade leaves: ), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with in sun leaves and in shade leaves). Sun leaves exhibited higher photosynthetic rates P N on a leaf area basis (mean of lmol CO 2 m 2 s 1 ) and Chl basis, which correlated well with the higher values of stomatal conductance G s (range mmol m 2 s 1 ), as compared to shade leaves (G s range mmol m 2 s 1 ; P N : lmol CO 2 m 2 s 1 ). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R Fd, which possessed higher values in sun leaves ( ) as compared to shade leaves ( ). In addition, via R Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves. Keywords Carotenoids Chlorophylls Chl a/b ratio Chlorophyll fluorescence decrease ratio R Fd Chloroplast H. K. Lichtenthaler (&) F. Babani G. Langsdorf Botanisches Institut, University of Karlsruhe, Kaiserstr. 12, Karlsruhe, Germany hartmut.lichtenthaler@bio.uka.de F. Babani Biological Research Institute, Academy of Sciences, Tirana, Albania adaptation CO 2 assimilation PN rates Stomatal conductance G s Abbreviations a + b Total chlorophylls a/b Ratio of chlorophyll a to b (a + b)/(x + c) Weight ratio of chlorophylls to carotenoids c Carotenes Chl Chlorophyll Fp, Fo, and Fs Maximum, initial, and steady Chl fluorescence Fd Fluorescence decrease from Fp to Fs Fp Maximum Chl fluorescence at nonsaturating light conditions Fv/Fm and Fv/Fo Maximum quantum yield of photosystem II photochemistry measured in the dark adapted, nonfunctional state 2 of the photosynthetic apparatus G s Stomatal conductance measured at light saturation P N Net photosynthetic CO 2 assimilation measured at light saturation R FD Chl fluorescence decrease ratio measured in the red band near 690 nm x + c Total carotenoids x Xanthophylls Introduction Sun and shade leaves of trees as well as high- and low-light plants considerably differ in their relative composition of

2 236 Photosynth Res (2007) 93: photosynthetic pigments, electron carriers, their chloroplast ultrastructure, and their photosynthetic rates (Boardman 1977; Lichtenthaler 1981; Meier and Lichtenthaler 1981; Wild et al. 1986; Givnish 1988; Anderson et al. 1995). Leaves that develop under high irradiance (sun leaves and high-light leaves) possess sun-type chloroplasts that are adapted to high rates of photosynthetic quantum conversion in comparison to shade leaves or leaves from low-light plants. Sun leaves possess a higher photosynthetic capacity on a leaf area basis, exhibit higher values for the ratio Chl a/b, a much lower level of light-harvesting Chl a/b proteins (LHCII), and a lower stacking degree of thylakoids than shade leaves and low-light plants with their low-irradiance shade-type chloroplasts (Lichtenthaler et al. 1981, 1982, 1984). The chloroplasts adaptation response to high- and low-light quanta fluence rates and the major differences between both chloroplast types have recently been summarized by Lichtenthaler and Babani (2004), a review that gives access to additional literature in this field. The differences in photosynthetic capacity of sun leaves and shade leaves have usually been determined by measuring the CO 2 fixation rates P N of leaves or of leaf parts at light saturation. This yields one integrated value of P N per measurement, however, gradients or stress-induced inhomogeneities in photosynthetic activity across the leaf area cannot be detected in this way. The photosynthetic function can also be judged via various Chl fluorescence ratios that are determined by the Chl fluorescence induction kinetics of dark-adapted leaves (Lichtenthaler 1988; Krause and Weis 1991; Govindjee 1995). Both ratios, Fv/Fm (Kitajima and Butler 1975) and its related more sensitive form Fv/Fo (Babani and Lichtenthaler 1996), are a measure of the potential photosystem II efficiency of dark-adapted leaves. They only reflect a small portion of the leaf chloroplasts, in particular those of the illuminated upper leaf side, but they do not reflect the photochemical activity of all leaf chloroplasts (Lichtenthaler et al. 2005a). In contrast, the Chl fluorescence decrease ratio R Fd (Lichtenthaler 1988), determined at light saturation, is an indicator of the photosynthetic quantum conversion capacity of light-adapted leaves at steady-state conditions (Tuba et al. 1994; Lichtenthaler and Miehé 1997) and is directly correlated to the net CO 2 fixation rates of leaves (Lichtenthaler and Babani 2004). Moreover, it has been shown that via imaging of the R Fd values of leaves one can see gradients in photosynthetic activity across the leaf area, detect water stress conditions (Lichtenthaler and Babani 2000), and recognize differences in photosynthetic activity between sun leaves and shade leaves of beech (Lichtenthaler et al. 2000). In the present study, sun and shade leaves of three different broadleaf trees (Platanus, Populus, Tilia) were chosen to comparatively investigate the differences in the photosynthetic characteristics (pigment content, P N rates, photochemical activity) in fully developed leaves during early July before summer stress events (heat and sun exposure combined with water deficit) reduce the physiological state of leaves. Particular emphasis was placed on the imaging of the photosynthetic activity of leaves via the Chl fluorescence decrease ratio R Fd in order to determine not only the differences between sun leaves and shade leaves, but, in addition, to evaluate if the photosynthetic activity is evenly distributed across the leaf area, if possible gradients or local inhomogeneities exist, and to find out if such gradients or inhomogeneities occur both in sun leaves and in shade leaves. Materials and methods Plant material and growth conditions For our investigations fully developed sun leaves and shade leaves were taken from 30- to 60-year-old trees of platanus (Platanus acerifolia Willd.), linden (Tilia cordata Mill.) and poplar (Populus alba L.) at the Karlsruhe University campus that is part of the Karlsruhe Palace Gardens. At this particular location in the Rhine valley plains all investigated trees had the same soil and water conditions and were exposed to the same climate. On sunny days the shade leaves in the inner tree part received ca. 80 lmol photons m 2 s 1 PAR, whereas sun leaves were exposed to a maximum PPFD from 1700 lmol m 2 s 1 to 2000 lmol m 2 s 1. The measurements were either performed with samples from three different trees (pigments, imaging), or two different trees (porometer) for each tree species. Pigment determination The photosynthetic pigments, chlorophylls a and b as well as total carotenoids x + c, were extracted with 100% acetone. Their levels were determined spectrophotometrically (with a Shimadzu UV VIS scanning spectrophotometer UV-2001 PC) using the extinction coefficients and equations redetermined by Lichtenthaler (1987), see also Lichtenthaler and Buschmann (2001) for details. From the pigment levels the weight ratios of pigments, Chl a/b and Chls/carotenoids (a + b)/(x + c), were determined. They significantly differ for sun leaves and shade leaves. The pigment values are the mean of at least six determinations from three trees. Porometer gas exchange measurements A branch with the desired leaves was cut from the plant, and the cut end was immediately re-cut under water to remove and prevent xylem embolisms. The light-induced

3 Photosynth Res (2007) 93: photosynthetic CO 2 fixation rates P N (lmol CO 2 m 2 s 1 ) and stomatal conductance (G s also termed gh 2 0) were measured in pre-darkened (20 min) leaves using a CO 2 / H 2 O-porometer system (Walz, Effeltrich, Germany). The leaves were irradiated by white light with 1500 lmol photons m 2 s 1 PAR that saturated with respect to the P N -rates. The latter ranged from ca. 300 to 400 (shade leaves) and ca. 700 to 900 lmol photons m 2 s 1 (sun leaves). Stable maximum P N -rates were usually reached between 24 min and 30 min after onset of illumination. Chlorophyll fluorescence imaging The chlorophyll (Chl) fluorescence induction kinetics (Kautsky effect) of pre-darkened leaves (30 min) were measured at the red Chl fluorescence band (k = 690 nm) using the Karlsruhe flash-lamp fluorescence imaging system (FL-FIS) as described by Lichtenthaler and Babani (2000), and Lichtenthaler et al. (2000). A Xenon flashlamp (300 W, Cermax, Perkin Elmer Optoelectronics, Cambridge, UK) and a blue filter (Corning No. 9782; range nm; kmax 465 nm) were applied to induce the red Chl fluorescence. The Chl fluorescence was excited and sensed at the upper (adaxial) leaf side. Via computer-aided data-processing false color images of the measured Chl fluorescence intensity were obtained, whereby blue was the lowest (zero) and red the highest fluorescence. We applied an uniformity correction to eliminate the effect of inhomogeneous radiation distribution by the xenon lamp. For the uniformity correction the UV-A excited fluorescence at 440 nm of a white sheet of paper was determined, and the software corrected the leaves fluorescence by means of this uniformity image (for further details see Lichtenthaler et al. 2005b). This fluorescence imaging system was applied in the present investigation to determine the images of the Chl fluorescence decrease ratio R Fd (see below). In contrast to the PAM-type chlorophyll fluorometers that work with pulsed modulated light and a non-saturating actinic light (Schreiber 1986; Schreiber et al. 1986; Genty et al. 1989), the chlorophyll fluorescence decrease ratio R Fd is determined from the Chl fluorescence induction kinetics measured at saturating continuous white light (Lichtenthaler and Miehé 1997; Lichtenthaler et al. 2005a). Due to the application of saturating excitation light some typical Chl fluorescence parameters of the PAM fluorometer, such as Fo, Fv, Fm, the ratio DF/Fm, or the quenching coefficients qp and qn, cannot be measured when determining the R Fd values due to the fact that the excitation light is saturating, and additional saturating light pulses (as given in the PAM fluorometer) do not increase the Chl fluorescence. An advantage of using either saturating or almost saturating excitation light when measuring the Chl fluorescence induction kinetics of leaves is the fact that the Chl fluorescence signals determined are representative of the signals of the total leaf chloroplasts. On the other hand, the PAM-type Chl fluorescence parameters and ratios are, in most cases (fully green leaves), representative only for the chloroplasts of the upper outer leaf-half where the illumination is applied, as has recently been demonstrated in detail (Lichtenthaler et al. 2005a). The R Fd values, in turn, linearily correlate with the net CO 2 assimilation rates P N (Lichtenthaler and Babani 2004; Lichtenthaler et al. 2005b). The Chl fluorescence decrease ratio (R Fd ) was determined by imaging and based on the equation: R Fd = Fd/ Fs = (Fm Fs)/Fs, where Fm is the maximum Chl fluorescence level. Fs is the steady-state Chl fluorescence (5 min after onset of saturating irradiance), and Fd represents the Chl fluorescence decline from Fm to Fs. In the present case the excitation light of ca lmol m 2 s 1 has almost but not completely been saturating. According to the proposal of Van Kooten and Snell (1990), the Fm level is then called Fp (and not Fm). Hence, the R Fd ratio determined here is the Chl fluorescence decrease ratio Fp/Fs. Chl fluorescence images were taken during the induction kinetics at Fp (reached after ca. 200 ms) and Fs (after 5 min), and in one case also at other time intervals according to Lichtenthaler and Babani (2000), and Lichtenthaler et al. (2000, 2005b). The images of the Chl fluorescence decrease ratio R Fd were obtained by a pixel to pixel division procedure and their individual values were also expressed in false colors from red (highest values) to blue (zero value). The histograms on the R Fd frequency distribution in sun leaves and shade leaves are based on 100,000 (Populus, Tilia) and 150,000 (Platanus) pixels per leaf, which means that for the statistical significance calculations the number of individual R Fd values n is 100,000 and 150,000, respectively. Unfortunately a commercial Chl fluorescence imaging system, that allows the measurement of R Fd images, is presently not available. The other Chl fluorescence imaging systems represent PAM-type imaging systems (Genty and Meyer 1994; Nedbal et al. 2000; Nedbal and Whitmarsh 2004; Ralph et al. 2005) work with non-saturating modulated light. However, recently, it has been shown that one can determine R Fd ratios also with the classical PAM-fluorometer (Lichtenthaler et al. 2005a) when an additional light source with saturating light is applied. This principally also applies to the PAM-type Chl fluorescence imaging systems. Statistical analysis The differences in pigment parameters and in the R Fd values between sun leaves and shade leaves (Tables 1, 2;

4 238 Photosynth Res (2007) 93: Figs. 3 6) were checked for significance using the Student s t-test. The correlation between the G s values and the P N rates of sun leaves and shade leaves (Fig. 1) was assessed by the analysis of variance one-way ANOVA. Significant differences were considered at P < 0.01 and P < Results Chlorophyll and carotenoid levels In a preliminary test we found that the total pigment content of sun leaves can considerably vary (up to 20%) depending on the part of the tree (height and north, south, or west orientation, etc.) where the samples are taken (data not shown). In contrast, the variation of pigment levels in shade leaves from the inner tree shade of the same tree or other trees of the same tree species was very low and clearly less than 3%. However, when the sun leaf samples were taken at the south-exposed part of the tree at nearly the same height, e.g., 3 5 m above the ground as in this investigation, the pigment levels between sun leaves of the same tree and the same tree species showed very little variation <3%. The differences in chlorophyll (Chl) and total carotenoid levels on a leaf area basis between sun leaves and shade leaves are summarized in Table 1. The total Chl (a + b) amounts were significantly higher in sun leaves of all three tree species as compared to the corresponding shade leaves. This also applied to the content of total carotenoids (x + c). We also detected the expected typical differences in the pigment ratios Chl a/b and Chl/carotenoids between sun leaves and shade leaves (Table 1). The sun leaves of all three investigated tree species had significantly higher values for the ratio Chl a/b (range ) than shade leaves (Chl a/b range ). In addition, they exhibited significantly lower values for the ratio of total Chls to total carotenoids, i.e., (a + b)/(x + c), ranging from 4.44 to 4.70 for sun leaves and 5.04 to 5.72 for shade leaves. Measurements of photosynthetic rates P N The maximum CO 2 assimilation rates (P N ) per leaf area unit (Table 2) at saturating photosynthetic photon flux density (PPFD 1500 lmol m 2 s 1 ) were significantly (P < 0.01) higher in sun leaves: in Platanus 2.68, in Populus 2.94 and in Tilia 2.73 higher than in the corresponding shade leaves. Also on a Chl (a + b) basis the sun leaves exhibited a significantly higher P N rate than the shade leaves. Yet, the differences in P N rates on a Chl basis were not as high (between 2.01 and 2.40 higher) as on a leaf area basis (Table 2). There also existed large differences in the mean values and the range of stomatal conductance G s (expressed in mmol m 2 s 1 ) between sun leaves and shade leaves. Thus, the mean values of G s were much higher in sun leaves (>100 up to 177 in Populus) than in shade leaves, where the mean G s values were found to be 43 (Populus), 53 (Platanus), and 54 (Tilia) as shown in Table 2. In fact, the ranges for G s values of sun leaves and shade leaves (Table 2) never overlapped in the physiologically active leaves analyzed in this investigation. The differences in P N and G s values between sun and shade leaves of two trees of the same tree species were in the same range as those found in individual leaves of the same tree as has been indicated by the joint mean values shown in Table 2. The deviation for the P N rates per leaf area unit and per mg Chl basis from the mean ranged from 15% to 18% (Platanus), 20% to 25% (Populus) and 16% to 20% (Tilia). The G s means showed a somewhat larger Table 1 Chlorophyll (a + b) and total carotenoid content (x + c) inmgm 2 leaf area, and pigment weight ratios Chl a/b and Chls/carotenoids (a + b)/(x + c) in fully developed green sun leaves and shade leaves of platanus (Platanus), poplar (Populus), and linden tree (Tilia) in July Chlorophylls Carotenoids Pigment ratios (a + b) (x + c) a/b (a + b)/(x + c) Platanus Sun leaf Shade leaf Populus Sun leaf Shade leaf Tilia Sun leaf Shade leaf Mean of seven determinations from four leaves of three trees of each species, maximal standard deviation <3% (pigment levels) and <1.2% (pigment ratios). The differences between sun leaves and shade leaves are highly significant (P < 0.001)

5 Photosynth Res (2007) 93: Table 2 Photosynthetic net CO 2 assimilation rates P N expressed on a projected leaf area basis (lmol m 2 s 1 ) and a chlorophyll a + b basis (lmol mg Chl 1 h 1 ), as well as mean and range of stomatal conductance G s (values in mmol m 2 s 1 ) in sun and shade leaves of the three tree species platanus, poplar, and linden Sun leaf Shade leaf Ratio sun/shade Platanus P N per leaf area 9.1 ± ± P N per mg Chl 72.6 ± ± G s mean ±31 53 ± G s range Populus P N per leaf area 9.4 ± ± P N per mg Chl 81.2 ± ± G s mean 177 ±41 43 ± G s range Tilia P N per leaf area 10.1 ± ± P N per mg Chl 73.9 ± ± G s mean 154 ± ± G s range Mean of six determinations from three leaves of two trees for each tree species. The mean values are shown in bold print to contrast them against the standard deviation. The differences between sun leaves and shade leaves of all tree species were highly significant (P < 0.01) variation: 25% (sun leaves) and 36% (shade leaves) in Platanus, 23% in Populus, and 13% (sun leaves), and 26% (shade leaves) in Tilia. When the G s values of sun leaves and shade leaves of all three trees are plotted against the photosynthetic P N rates, a close linear correlation shows up between G s and P N (Fig. 1). The broken line in Fig. 1 clearly separates sun leaves from shade leaves, which exhibit significantly different photosynthetic rates and stomatal conductance as summarized in Table 2. Half-shade leaves and sunfleck leaves, which receive sun light only for 1 or 3 h per day, range with their P N rates ( lmol CO 2 m 2 s 1 ) and their G s values ( mol m 2 s 1 ) in between those of sun leaves and full-shade leaves and also follow the correlation shown in Fig. 1. Imaging of the chlorophyll fluorescence decrease ratio R Fd Fig. 1 Correlation between light-saturated photosynthetic CO 2 assimilation rates P N and the maximum stomatal conductance G s in sun leaves and shade leaves from two trees each of Platanus, Populus, and Tilia. All data were fitted using the linear regression analysis of Micosoft excel with the analysis of variance one-way ANOVA (r = 0.965; P < 0.001; significance factor F = ; significance of the correlation = ). The perpendicular, broken line separates the values of sun leaves (upper right part) and shade leaves (lower left part) The red Chl fluorescence images of pre-darkened Platanus leaves showed a high Chl fluorescence yield when measured at the maximum Fp in both sun leaves and shade leaves (Fig. 2A, C). Upon continuous illumination and at the onset of photosynthesis, the Chl fluorescence yield steadily decreased and declined after 5 min to the very low steady-state Chl fluorescence level Fs (Fig. 2B, D). From the images at Fp and Fs the R Fd images were processed, which demonstrated much higher R Fd values for sun leaves than for shade leaves (Fig. 2E, F). Moreover, the R Fd images show that the photosynthetic activity, as indicated by the R Fd values, is unevenly distributed across the leaf area. Major leaf veins possessed significantly lower R Fd values than the intercostal leaf parts. In the case of shade leaves the major veins had practically almost zero R Fd

6 240 Photosynth Res (2007) 93: Fig. 3 Histogram of the frequency distribution of the Chl fluorescence decrease ratio R Fd in a sun leaf and a shade leaf of Platanus. The R Fd value distributions are based on more than 150,000 pixels per leaf and the differences are highly significant (P < 0.001) Fig. 2 Images of the maximum Chl fluorescence at a high light pulse (Fp; A, C), and at steady-state Chl fluorescence after 5 min of continuous illumination (Fs; B, D), and the Chl fluorescence decrease ratio (R Fd ) in sun leaves (E) and shade leaves (F)ofPlatanus acerifolia. The differences in the relative Chl fluorescence intensity (A D) of the different leaf parts are shown by false colors, whereby red stands for high and blue for zero Chl fluorescence as indicated in the scale (0 2 K), and K represents 1,000 fluorescence units. In case of the R Fd ratio images (E, F) the colors indicate the absolute values of the ratio from zero up to a maximum R Fd value of 4 values (Fig. 2F) indicating no photosynthetic activity although they possessed chlorophyll. Forming the histograms using the frequency distribution of the R Fd values of all leaf pixels allows the quantification of the differences in R Fd values between sun leaves and shade leaves (Fig. 3). The R Fd values of sun leaves and shade leaves partly overlap, but the mean values are significantly different and exhibit maxima at ca. 3.0 (sun leaf) and ca. 1.8 (shade leaf). Similar results were also obtained with sun leaves and shade leaves of poplar and linden tree. The R Fd images and the R Fd frequency distribution (histograms) also indicated significantly higher R Fd values in sun leaves of Populus (Fig. 4) and Tilia (Fig. 5) as Fig. 4 Images of the Chl fluorescence decrease ratio R Fd in a sun leaf and a shade leaf of Populus and the histogram of the R Fd ratio frequency distribution. Please note that the R Fd scales are different for sun leaves (A) and shade leaves (B). The R Fd value distributions are based on more than 100,000 pixels per leaf and the differences are highly significant (P < 0.001) compared to shade leaves. In poplar leaves the R Fd values had a mean at ca. 2.8 (sun leaf) and ca. 1.4 (shade leaf), whereas those of Tilia exhibited mean values of ca. 2.9 (sun leaf), and the shade leaf a double peak at ca. 1.7 and 1.0 (Fig. 5). Due to the high number of leaf pixels measured (>100,000 in each case) the differences in the R Fd values between sun leaves and shade leaves are highly significant (P < 0.001).

7 Photosynth Res (2007) 93: Fig. 6, demonstrate again the differences in R Fd values between sun leaves and shade leaves of all three trees species. In shade leaves the maximal R Fd values were reached after 5 min, whereas those in Populus and Tilia still increased slightly within 5 8 min after onset of the illumination (Fig. 6B, C). Discussion The results of this investigation demonstrate that the sun leaves of the three tree species, as compared to shade leaves, are characterized by higher levels of total Chl a + b and total carotenoids, as well as by higher values of the weight ratio Chl a/b and by lower values for the weight ratio Chls/carotenoids (a + b)/(x + c). Such differential pigment ratios are characteristic for sun-type and shadetype chloroplasts of trees and are found in leaves from high-light and low-light plants as well (Lichtenthaler 1981; Lichtenthaler et al. 1981; and review Lichtenthaler and Babani 2004). These differences in the pigment ratios, Fig. 5 Images of the Chl fluorescence decrease ratio R Fd in a sun leaf and a shade leaf of Tilia and the histogram of the R Fd value frequency distribution. Please note that the R Fd scales are different for sun leaves (A) and shade leaves (B). The R Fd value distributions are based on more than 100,000 pixels per leaf and the differences are highly significant (P < 0.001) The data shown in Figs. 3 5 are based on one typical sun leaf and shade leaf of each tree species. In order to test the overall validity of our results, Chl fluorescence imaging of additional sun leaves and shade leaves of the same tree and of two other trees of the same tree species was performed (data not shown). After leaves had been taken from the sun-exposed south part and the inner shade of the tree crown of the three tree species, we obtained the same results for each tree species with similar, highly significant differences in the R Fd values of sun leaves versus shade leaves. In fact, the range of R Fd values, as shown in the histograms of Figs. 3 5, was the same for different trees of each tree species, and the mean R Fd values varied by <5% (sun leaves) and <7% (shade leaves) from leaf to leaf and from tree to tree of each tree species investigated. In a separate investigation we checked the development of the R Fd values in the course of the Chl fluorescence induction kinetics during the continuous illumination of dark-adapted leaves. We determined Chl fluorescence images at 0, 1, 5, and 8 min after the onset of continuous light (PPFD 1300 lmol m 2 s 1 ). The results, shown in Fig. 6 Development of the R Fd ratio (with standard deviation) upon illumination of dark-adapted sun leaves and shade leaves of the three tree species platanus, poplar, and linden. The values shown are based on R Fd ratio images with more than 150,000 leaf pixels per image. The differences between sun leaves and shade leaves after 5 and 8 min of illumination, and in Platanus also after 1 min of illumination, are highly significant (P < 0.001)

8 242 Photosynth Res (2007) 93: higher values for the ratio Chl a/b and lower values for the weight ratio Chls/carotenoids, are caused by the high irradiance adaptation response of the photosynthetic pigment apparatus of sun leaves (sun chloroplasts). The latter possess, on a Chl basis, much lower amounts of lightharvesting Chl a/b proteins (LHCII), more reaction center pigment proteins (e.g. CPa, CPI) (Lichtenthaler et al. 1982), and also a greater number of electron transport chains as compared to shade chloroplasts (Lichtenthaler et al. 1981; Wild et al. 1986). In contrast, shade leaves and shade-type chloroplasts possess higher and broader grana thylakoid stacks and primarily invest in the pigment antenna (Boardman 1977; Lichtenthaler et al. 1982, 1984; Meier and Lichtenthaler 1981). Due to the adaptation response to high irradiance, sun leaves of trees with their sun-type chloroplasts possess considerably higher photosynthetic net CO 2 assimilation rates P N on a leaf area basis in comparison to shade leaves as shown in Table 2. Sun leaves are thicker and usually have a higher total amount of Chl per leaf area unit. One could assume that the higher Chl level is mainly responsible for the higher P N rates in sun leaves; however, this is not the case. Even on a Chl basis they exhibit higher net CO 2 assimilation rates P N (Table 2). This shows that the reason for the higher P N rates of sun leaves as compared to shade leaves is only partly due to their generally higher Chl content per leaf area unit, whereas the most essential factor is their possession of sun-type chloroplasts exhibiting a different structural and functional organization of their relative Chl and carotenoid levels. The fact that the differences between sun leaves and shade leaves in P N rates on a Chl basis are not as high as the P N rates on a leaf area unit indicates that, to a minor extent, also the generally higher Chl content of sun leaves per leaf area unit contributes to their higher P N rates. The higher photosynthetic rates of sun leaves are supported by considerably higher values for the stomatal conductance G s. The latter can be up to several times higher (range ) than the rather low G s values (21 77) of shade leaves (Table 2), indicating that the stomata opening is apparently larger in sun leaves than in shade leaves (Schulze et al. 1975; Farquhar and Sharkey 1982). This contributes to higher intercellular CO 2 concentrations and thus to the higher CO 2 assimilation rates of sun leaves. This is further supported by a higher stomata density of sun leaves as compared to shade leaves (Lichtenthaler et al. 1981; Pearcy and Sims 1994; Zangh et al. 1995), and this also applies to high irradiance leaves in comparison to low irradiance plants (Wild and Wolf 1980). These data show that the average stomata density in sun leaves was ca times higher as compared to shade leaves, whereas the values for stomatal conductance were times higher. This underlines that the higher G s values in sun leaves are only partially caused by an increase in stomata density and to a major part by a larger stomata aperture. Our observation, that at saturating light conditions the P N rates linearly correlate with the stomatal conductance G s (Fig. 1), demonstrates a stomatal control of CO 2 uptake. Such correlations had already been observed for the same leaf types of plants kept under different physiological conditions (Pereira et al. 1987; Schulze et al. 1975; Tenhunen et al. 1984; Wong et al. 1979; Cornic 2000). In addition, also the mesophyll structure of leaves influences the P N rates through affecting the diffusion of CO 2 (Terashima 1992), and the penetration of light (Vogelmann and Martin 1993) into the leaf. Thus, the thicker sun leaves contain significantly more cells per leaf area and section unit as compared to the thinner shade leaves (Lichtenthaler 1981; Pearcy and Sims 1994). The much higher P N rates of sun leaves as compared to shade leaves are also well reflected in the significantly higher values of the Chl fluorescence decrease ratio R Fd (Figs. 3 5), which represents a non-destructive indicator of the in vivo photosynthetic rates of leaves (Lichtenthaler and Babani 2004). The photosynthetic activity, as indicated by the height of the R Fd values is, however, not uniformly distributed across the leaf area. It shows a certain heterogeneity and patchiness with higher and lower values in both sun and shade leaves (Figs. 2, 4, 5). A possible explanation is that this may be caused by or is related to the non-uniform distribution of stomata opening, as described for beech leaves, fir needles (Küppers et al. 1999; Beyschlag et al. 1994), and various other plants (as reviewed by Pospíšilová and Šantrucek 1994), however, further research is required. The same patchiness in the distribution of the R Fd values across the leaf area, as described here, has also been observed via R Fd images in beech leaves (Lichtenthaler et al. 2000). Stomatal patchiness is apparently the result of a heterogeneous water supply in different parts of the leaf (Cheeseman 1991), which may cause local differences in CO 2 levels and thus affect the photosynthetic rates. In fact, it has been shown via R Fd images in bean leaves that a water deficit reduces the R Fd values (Lichtenthaler and Babani 2000). Thus, the assumption of local differences in the leaf water level across several adjacent leaf pixels might be a reasonable explanation for the nonuniform distribution of R Fd values and the photosynthetic activity across the leaf area. A local photoinhibition or damage of the photochemical pigment apparatus, which may show up under stress conditions, is less likely, since the leaves were fully physiologically active and did not show any stress symptoms or strain. In any case, the results show that imaging the photosynthetic activity of leaves via the non-invasive method of imaging the R Fd values is a valuable and powerful technique for ecophysiological plant research.

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