PHOTOSYNTHETICA 47 (2): 223-231, 2009

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Evidence for light-independent and steeply decreasing PSII efficiency along twig depth in four tree species C. YIOTIS, Y. PETROPOULOU and Y. MANETAS* Laboratory of Plant Physiology, Section of Plant Biology, Department of Biology, University of Patras, GR−265 00 Patras, Greece Abstract Recent reports have indicated a considerably inactivated PSII in twig cortices, in spite of the low light transmittance of overlying periderms. Corresponding information for more deeply located and less illuminated tissues like xylem rays and pith are lacking. In this investigation we aimed to characterize the efficiency of PSII and its light sensitivity along twig depth, in conjunction with the prevailing light quantity and quality. To that aim, optical methods (spectral reflectance and transmittance, chlorophyll fluorescence imaging, low temperature fluorescence spectra) and photo-inhibitory treatments were applied in cut twig sections of four tree species, while corresponding leaves served as controls. Compared to leaves, twig tissues displayed lower chlorophyll (Chl) levels and dark-adapted PSII efficiency, with strong decreasing gradients towards the twig center. The low PSII efficiencies in the inner stem were not an artifact due to an actinic effect of measuring beam or to an enhanced contribution of PSI fluorescence. In fact, the PSII/PSI ratios in cortices were higher and those in the xylem rays similar to that of leaves. Inner twig tissues were quite resistant to photoinhibitory treatments, tolerating irradiation levels several-fold higher than those encountered in their microenvironment. Moreover, the extent of high light tolerance was similar in naturally exposed and shaded twig sides. The results indicate an increasing, inherent and light-independent inactivation of PSII along twig depth. The findings are discussed on the basis of a recently proposed model for photosynthetic electron flow in twigs, taking into account the specific atmospheric and light microenvironment as well as the possible metabolic needs of such bulky organs. Additional key words: chlorophyll fluorescence imaging, cortex, Eleagnus angustifolia L., light transmittance, Nerium oleander L., photoinhibition, pith, Platanus orientalis L., Quercus coccifera L., twig photosynthesis, xylem rays. Introduction

Leaves are the organs optimized for photosynthesis, yet chloroplasts are also present in organs designed for other functions (Aschan and Pfanz 2003). Although the presence of chlorophyll (Chl) in such organs is mani-fested by a green color, in some cases the green color is masked by overlying tissues. In stems with a well developed periderm, for example, chloroplasts are hidden in inner tissues not only adjacent to periderm (i.e. cortex) but in light remote sites like xylem rays and pith as well (Pfanz et al. 2002, Dima et al. 2006). Photosynthesis in such stems is believed to re-cycle internal CO2 and participate in the total tree carbon balance (Pfanz et al. 2002). The very low gross photosynthetic rates in such organs have been explained on the basis of a shade acclimation syndrome (Wittmann et al. 2001). Indeed, transmittance of periderms is low (cca 10-50 %

depending on species and age of twigs or stems, see Pfanz et al. 2002). Moreover, the shade imposed by the periderm is wavelength selective, with very low penetration of blue and gradually increasing penetration of green and red light (Kauppi 1991, Manetas 2004a, Manetas and Pfanz 2005). Apart from this peculiarity, photosynthesis in twigs takes place in an internal atmosphere completely different from that of leaves. Periderms are resistant to gas diffusion and, in conjunction with the high heterotrophic/photosynthetic cell ratios and the ascent of CO2 with the transpiration stream, CO2 concentrations in stems are at least an order of magnitude higher, while oxygen can fall to hypoxic levels (Pfanz et al. 2002). Hence, chloroplasts of twig internal tissues function in a light and atmospheric microenvironment very different from that of leaves.

——— Received 27 June 2008, accepted 4 April 2009. *Author for correspondence; fax: + 30 2610 997411, e-mail: y.manetas@upatras.gr Abbreviations: A − absorptivity; Chl − chlorophyll; F0 and Fm − minimal and maximum fluorescence in dark-adapted state, i.e. the fluorescence yields with open and closed PSII reaction centers, respectively; Fv/Fm − maximum quantum yield of PSII; MB − measuring beam; PAR − photosynthetically active radiation; PS − photosystem.

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Photosynthesis in the cortex (i.e. the most well lit site of a stem) has been probed from the twig surface with Chl fluorescence methods. A common finding is that both dark- and light-adapted PSII yields are, to a more or less extent, lower compared to corresponding leaves (Damesin 2003, Manetas 2004b, Alessio et al. 2005, Manetas and Pfanz 2005, Tausz et al. 2005, Filippou et al. 2007). Within this context, Kotakis et al. (2006) reported that twig cortices of Eleagnus angustifolia maintained a higher population of non-QB-reducing PSII centers obstructing linear electron flow, while cyclic flow around PSI was quite active. Hence, an adaptive significance was ascribed to PSII inactivation as a means of adjusting the ATP/NADPH ratio and replenishing the ATP pool from losses due to hypoxia. Data on adenylate pool sizes and PSI activity of barks and needles of Pinus sylvestris corroborated this hypothesis (Ivanov et al. 2006). Adaptive or not, dysfunction of PSII could be the result of photoinhibition. Towards that point, a field study by Solhaug and Haugen (1998) reported that the

lowest values of corticular PSII yield were found during the leafless winter period and on exposed rather than on shaded twig sides, inferring a sensitivity of corticular photosynthesis to light and cold. However, PSII efficien-cies of cortices were lower than those of the corresponding leaves even during the favoring period of the year. Moreover, the assumed extreme light sensitiv-ity of PSII in the inner stem tissues could be further questioned on the basis of the very low light levels expected in deeper stem, especially in the regions of xylem rays and pith. We argued that a photoinhibitory hypothesis would entail higher dark-adapted PSII efficiencies as well as higher photoinhibitory risk along twig depth. Alternatively, a light-independent PSII inactivation could be assumed. Towards this aim, mapping of PSII efficiency in cut stem sections of four tree species was accomplished through imaging Chl fluorescence and the internal light environment was characterized by conventional optical methods.

Materials and methods Plants and sampling: Plant material was selected on the basis of previous reports for the presence of chloroplasts in internal stem tissues including xylem rays and pith (Dima et al. 2006). An additional criterion was the easiness of isolation of intact periderm and bark strips for optical measurements. Four woody species were used, Eleagnus angustifolia L., Platanus orientalis L., Nerium oleander L. and Quercus coccifera L.. Experiments were performed during the favorable growth period for these species in spring, early summer and autumn. Healthy, south facing leafy twigs of the previous growth season (1–2 years old), having well developed periderms were cut during afternoon, wrapped in air-tight plastic bags lined with moistened filter paper and kept at room temperature in darkness for 12 hours.

Chl fluorescence imaging: A pulse-amplitude modulated system (Imaging PAM, Walz, Effeltrich, Germany) equipped with a bank of red (λ = 650 nm) LEDs and a CCD camera capturing fluorescence in the 670-800 nm band was used. The red LEDs are used for both measuring beam, actinic light and saturating pulses. In addition to Chl fluorescence parameters, the system is also capable of estimating tissue Chl absorptivity (A) by means of reflectance measurements after illuminating the sample with pulses of red (λ = 650 nm) and infra-red (λ = 780 nm) irradiation. A build-in equation transforms reflectance measurements to A values, giving a relative estimation of sample Chl levels. During measurements, leaf discs or longitudinal twig sections were kept in Petri dishes on moistened filter paper to avoid tissue desicca-tion. With this system, small mirror images of LEDs due to reflectance by the cover glass of Petri dishes appear at some edges of the working area, causing energy overflow

in the camera. To avoid the problem, the disturbing areas were covered with non-reflecting paper. After deter-mination of the areas of interest (i.e. cortex, xylem rays and pith, see Fig. 2A), the fully dark-adapted (overnight) samples were given a saturating (ca 3500 μmol m–2 s–1) pulse to assess the maximum PSII photochemical efficiency as Fv/Fm = (Fm–F0)/Fm, where F0 and Fm are the fluorescence yields with open and closed PSII reaction centers, respectively.

Photoinhibitory treatments: The samples were illuminated with actinic irradiances of various levels (up to 2000 μmol m–2 s–1) with the instrument’s source for 20 min. PSII photochemical efficiencies (as Fv/Fm = (Fm–F0)/Fm) in predetermined areas of interest were probed before illumination and after 20 min of dark recovery. Areas of interest encompassed the cortex, xylem rays and pith regions. When needed (see Results), a distinction between the “exposed” and the “shaded” twig side was made. The “exposed” side is that pointing to the direction of the sun (south exposure), which receives solar radiation filtered by leaves and occasional sunflecks. Photosynthetically active radiation (PAR) received by sampled twigs at midday of clear days was ca 800, 100 and 50 μmol m–2 s–1 for “exposed” side with foliage removed, “exposed” side with foliage in place and “shaded” side, respectively. Transmittance of isolated periderms and barks: Strips of intact periderms and intact cortices with periderms attached having enough size to completely cover the mini-quantum sensor of the leaf clip of a Mini-PAM fluorimeter (Walz, Effeltrich, Germany) were manually removed. The quantum sensor was placed perpendicular

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to the sun rays and PAR was read before and after interposing the samples. Transmittance was computed as the ratio of the two readings.

Spectral transmittance of periderm and bark: Only in one species (E. angustifolia) intact, cracks-free and wide enough periderm and bark samples that could cover the entrance port of an integrating sphere (Optronic1S 1000) could be isolated. Spectral transmittance was measured with a spectroradiometer (OL 752, Optronic, Orlando, FL, USA) equipped with a stabilized light source (OL 752–104). Monochromator slit width was set at 1 nm.

Room temperature fluorescence spectra: For the same reasons described above, room temperature fluorescence spectra of various twig tissues were only measured in E. angustifolia. Wide enough, isolated intact cortex strips (with periderms removed), xylem cylinders (i.e. after removal of both periderm, cortex and phloem layers) and whole leaves were fastened in the solid sample attachment of a Hitachi (Tokyo, Japan) F-2500 spectrofluorimeter. Since the aim of these measurements was to assess the relative contribution of fluorescence from the two photosystems (PSII and PSI) in the various tissues and with excitation conditions roughly similar to those applied with the Imaging-PAM, the excitation wavelength was set at 630 nm and spectral fluorescence was measured in the 660–800 nm band after 4 min of sample equilibration. Both excitation and emission slit widths were set at 5 nm. Hence, the fluorescence spectra obtained are characteristic of the steady state (Fs) fluorescence. Since only comparisons between leaves and the various twig tissues were sought, spectra were not corrected for photomultiplier sensitivity at the various wavelengths.

Low temperature (77 K) fluorescence spectra are used for the calculation of the F686/F736 ratio, as a relative indication of the PSII/PSI ratio, provided that Chl concentration of the probed sample is low enough (within the region of 1–2 μg cm–2 as judged from preliminary trials), to avoid self-absorption of Chl fluorescence (Weis 1985). To that aim, attempts were made to isolate thylakoids from stem tissues, yet this was only feasible

with cortex of E. angustifolia. Hence, an adequate number of leaves (without the middle vein) or cortex strips (after periderm removal) were cut in small segments, put in a mortar and frozen with a small volume of liquid N2 to facilitate grinding. Thylakoids were then isolated according to Šiffel et al. (2000), in ‘buffer A’ (0.4 M sorbitol, 5 mM MgCl2, 10 mM KCl, 1 mM MnCl2, 1 mM sodium ascorbate, 0.5 % bovine serum albumine, 50 mM Tricine/KOH, pH 7.5) by two sequen-tial centrifugations at 2000 × g for 2 min and 8000 × g for 10 min. The pellet of the second centrifugation was resuspended in ‘buffer B’ (0.33 M sorbitol, 5 mM MgCl2, 10 mM KCl, 1 mM MnCl2, 50 mM Tricine/KOH, pH 7.5) and, after two consecutive resuspensions-centrifugations at 8000 × g for 10 min, the final pellet was resuspended in 2 ml of ‘buffer B’. The final thylakoid preparation was homogeneously absorbed in pre-cut Whatman No 1 filter papers. The paper was cylindrized and inserted in glass Pasteur pipettes of appropriate diameter fitting the Dewar cuvette of the spectrofluorimeter low temperature attachment. The cuvette was filled with liquid nitrogen while a water vapor-free air stream prevented conden-sation. Excitation was set at 490 nm (slit width 10 nm). After scanning between 660-800 nm (slit width 2.5 nm), the filter paper was immersed in pure methanol and Chl of the cleared sample was measured with a Shimadzu (Duisburg, Germany) UV-160A double beam spectro-photometer (slit width 2 nm). Pigment concentration was estimated by the equations of Lichtenthaler and Wellburn 1983. Only samples containing the appropriate Chl levels were used for assessing the F686/F736 ratio. Although thylakoids could not be isolated from the tough xylem of E. angustifolia, intact xylem cylinders could also be measured, since their Chl concentrations are at the appropriate low range (Dima et al. 2006). For this purpose, xylem cylinders (i.e. after removal of periderm, cortex and phloem layers) were inserted in the Pasteur pipettes and directly into the liquid nitrogen cuvette.

Statistics: When needed, significance of differences in the measured parameters between various tissue types for each species was assessed by one-way analysis of variance (ANOVA, SPSS Inc., Chicago, IL, USA).

Results Characterization of light quantity and quality pene-trating into twig tissues: Table 1 shows the transmit-tances of isolated periderms and periderms plus cortices to solar incident PAR. In all four tested plants, the top of the cortex layer receives ca 30–40 % and the top of the xylem cylinder ca 3–5 % of incident light, depending on plant species. Apparently, lower photon flux rates are expected in the central pith tissues. We may note that the inner stem tissues are not only shaded by overlying tissues of the stems per se, but by the canopy leaves as

well, at least during the leafy periods of the year. Concerning spectral transmittance in twigs of

E. angustifolia, Fig. 1 indicates a strong attenuation of blue light by isolated periderms, with transmittance increasing gradually in the green and further in the red and infra-red bands. Accordingly, the light incident on the cortex is red- and far red-enriched but green- and, especially, blue-depleted. The light incident in the periphery of the xylem cylinder is composed mainly of far-red and infra-red photons (Fig. 1).

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Table 1. Percent transmittance of PAR through periderm and periderm plus cortex strips in the indicated species. Data are means ± SD from 6–13 independent preparations depending on species.

Transmittance [%] Plant species Periderm Bark

E. angustifolia 36.6 ± 1.2 3.1 ± 0.3 N. oleander 40.0 ± 1.0 4.4 ± 0. 5 P. orientalis 37.5 ± 1.6 5.1 ± 1.7 Q. coccifera 30.4 ± 1.0 3.0 ± 0.6

Fig. 1. Spectral transmittance of isolated periderms (open circles) and cortices with attached periderms (closed circles) of E. angustifolia in the visible and near infra-red band. Data are means from 4 independent preparations displaying similar values. Apparent dark-adapted PSII yield in twigs is low and decreases with twig depth: Fig. 2A shows a false-color representation of E. angustifolia inner twig absorptivity due to Chl. Although absolute Chl levels cannot be easily assessed, a decreasing gradient from outer cortex to central pith is evident. The corresponding graphs of Fig. 2 display the gradients in absorptivity (A), minimal fluorescence yield (F0) and dark-adapted PSII yield (Fv/Fm) in 16 areas of interest (diameter ca 500 μm,) indicated in Fig. 2A. As shown, F0 increases from the outer part of the cortex and up to the xylem/pith borders, to decrease again in the pith (Fig. 2B). F0 in leaves is lower than in twigs (Table 2). Paradoxically, Fv/Fm decreases with twig depth, in spite of the extremely low photon fluence rates experienced by internal twig tissues. As shown in Fig. 2C, the maximum PSII yield decreases gradually from 0.80 in the outermost cortex area receiving ca 37 % of PAR incident on a twig to 0.74 at the cortex/xylem border receiving only ca 3 % of the corresponding PAR, and further to 0.647 at the most deeply shaded center of the twig (pith area).

Table 2 indicates that a similar pattern in A, F0 and Fv/Fm was evident in the twig internal tissues of all plant

Fig. 2. A false-color picture of chlorophyll absorptivity probed from a longitudinal twig section of E. angustifolia. The color code bar shows absorptivity values ranging from 0 to 1. The three main areas shown from the twig periphery towards the twig center correspond roughly to cortex, xylem rays and pith. Numerical values of absorptivity (A), minimal fluorescence (F0) and dark-adapted PSII yield (Fv/Fm) for the areas of interest shown along the twig section are given as graphs in A, B and C, respectively. The right side of the section corresponds to the shaded (i.e. facing the inner part of the tree canopy) side of the twig. species examined. The values shown are means for the whole cortex, xylem rays and pith areas for each species, not discriminating between sub-areas within each twig tissue, as it was the case in Fig. 2. Since it is difficult to ascribe these low maximum PSII yields to

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Table 2. Values of absorptivity (A), minimal fluorescence (F0) and dark-adapted PS II yield (Fv/Fm) obtained from stem internal tissues and corresponding leaves (upper surface) of all plants tested. Values are means ± SD from 16–20 (leaves) and 15–44 (twigs, depending on plant) independent preparations. Measured areas in twig sections refer to the whole cortex, xylem rays and pith areas. Different letters within each column and for each species indicate statistically significant differences (p<0.05) in the measured parameter.

A F0 Fv/Fm

E. angustifolia leaf 0.841 ± 0.026a 0.070 ± 0.006a 0.810 ± 0.013a cortex 0.769 ± 0.037b 0.092 ± 0.015b 0.778 ± 0.017b xylem rays 0.530 ± 0.019c 0.144 ± 0.013c 0.749 ± 0.015c pith 0.324 ± 0.029d 0.097 ± 0.019b 0.682 ± 0.032d N. oleander leaf 0.899 ± 0.011a 0.074 ± 0.009a 0.806 ± 0.054a cortex 0.679 ± 0.033b 0.090 ± 0.014b 0.726 ± 0.032b xylem rays 0.444 ± 0.056c 0.129 ± 0.027c 0.621 ± 0.055c pith 0.243 ± 0.030d 0.128 ± 0.036c 0.391 ± 0.098d P. orientalis leaf 0.889 ± 0.013a 0.077 ± 0.009a 0.834 ± 0.008a cortex 0.746 ± 0.022b 0.095 ± 0.016b 0.740 ± 0.029b xylem rays 0.514 ± 0.042c 0.160 ± 0.036c 0.657 ± 0.054c pith 0.562 ± 0.046d 0.222 ± 0.047d 0.620 ± 0.070c Q. coccifera leaf 0.883 ± 0.022a 0.066 ± 0.009a 0.830 ± 0.010a cortex 0.757 ± 0.024b 0.073 ± 0.011b 0.766 ± 0.015b xylem rays 0.423 ± 0.033c 0.114 ± 0.016c 0.678 ± 0.027c pith 0.565 ± 0.033d 0.144 ± 0.032d 0.700 ± 0.032c

Fig. 3. Room temperature fluorescence emission spectra of intact leaves (solid line), isolated cortex strips (with periderm removed, dashed line) and isolated xylem cylinders (with cortex removed, dotted line) of E. angustifolia. Excitation at 630 nm. Fluorescence values of cortex and xylem were normalized to those of leaf at λ = 736 nm.

photoinhibition occurring at just a few μmol m–2 s–1 PAR, we examined the possibility of an artifact due to an overestimation of F0.

Tests pertaining to a possibly artifactual over-estimation of F0 in twig internal tissues: Increasing the intensity of the measuring beam (MB) of the Imaging- PAM fluorimeter from 0.25 μmol m–2 s–1 to the highest available level (roughly 1 μmol m–2 s–1) had no appreciable effect on the measured Fv/Fm (not shown). In addition, using a Mini-PAM fluorimeter, affording a very weak (<0.1 μmol m–2 s–1) measuring beam with twig sections (i.e. probing collectively all tissues) gave the same results. Hence, an actinic effect of MB is unlikely.

We further argued that the low Fv/Fm values could be due to a higher contribution of PSI fluorescence, in case that the PSII/PSI ratios are lower in twig tissues. Yet, room temperature fluorescence spectra obtained from leaf, cortex and xylem surfaces of E. angustifolia displayed a higher red/far-red fluorescence ratio in xylem rays followed by cortex, while the lowest ratios were obtained from leaves (Fig. 3). Thus, the contribution of PSI fluorescence is higher in leaves than twigs, further weakening the possibility of a significant PSI contribution to an artifactually low Fv/Fm in twigs. We may note at this point that our Imaging-PAM uses excitation light at 650 nm and detects fluorescence along the whole 670–780 nm region.

Fig. 4 shows low temperature fluorescence spectra from dilute thylakoid preparations from leaves and cortices, as well as from intact xylem cylinders isolated from E. angustifolia. It is known that low temperatures

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Fig. 4. 77 K fluorescence spectra of thylakoid preparations from leaves (solid line) and cortices (dashed line) as well as from isolated xylem cylinders (with cortex removed, dotted line) of E. angustifolia. Chlorophyll (Chl) levels at ca 1–2 μg cm–2. Values for cortex and xylem were normalized to those of leaf at λ = 736 nm. The insert shows means ± SD of F686 / F736 ratios obtained from 4 independent preparations. Different letters above error bars indicate statistically significant differences (p<0.05).

Fig. 5. Recovery [%] of PSII efficiency (Fv/Fm) in longitudinal twig section of E. angustifolia after 20 min dark adaptation following 15 min of illumination with 500 μmol m–2 s–1 red light. Positions of areas probed are denoted in the horizontal bar. Values are means ± SD from 4 independent trials. The asterisk denotes corresponding recovery in leaves treated similarly. enhance fluorescence yield from PSI displaying a maximum at 736 nm, while PSII fluoresces maximally at 686 nm (Papageorgiou and Govindjee 2006). As shown, the F686/F736 ratio (which is indicative of the relative PSII/PSI ratio) is similar in leaves and xylem rays, while it is considerably increased in the cortex. Hence, the hypothesis for an artifactual underestimation of PSII yield in twigs due to a high PSI content is further weakened.

Light sensitivity of PSII yield in twig internal tissues: Fig. 5 shows the extent of photoinhibition of PSII yield after exposing stem sections of E. angustifolia for 15 min at 500 μmol m–2 s–1 red light followed by 20 min of dark recovery. Compared to similarly treated leaves, internal stem tissues recovered to a lower extent. Moreover, the extent of photoinhibition displayed an increasing gradient from the peripheral to the more central and, accordingly, shade adapted tissues. It is also noteworthy that the extent of photoinhibition was similar in the naturally exposed and shaded sides of twig segments. Although the results generally indicate a higher light sensitivity in stems compared to leaves, we have to note that the fixed, applied photon fluence rate of 500 μmol m–2 s–1 is quite higher than that experienced by stem internal tissues but quite lower for an exposed leaf. Accordingly, the dependence of PSII photoinhibition on incident light intensity was further examined for each tissue at various PAR levels in order to compare on a realistic photon fluence rate basis for each tissue.

Fig. 6. Recovery [%] of PSII efficiency (Fv/Fm) in leaves (○) and internal twig tissues (cortex: ●, xylem rays: □, pith: ∆) versus photon fluence rates during a photoinhibitory treatment. The samples were dark-adapted for 20 min after a 15 min illumination at the indicated PAR. Values are means ± SD from 4 independent trials. The arrows indicate the maximum PAR likely to reach a fully exposed leaf or penetrating at the most peripheral point of the corresponding twig tissue during a clear day. An arrow for pith is absent since penetrating light in this tissue was not measured. For twigs, data from naturally exposed and shaded sides were similar.

In Fig. 6, the light intensity versus PSII photo-inhibitory damage is shown. At the maximum realistic photon fluence rate received by each tissue (shown by arrows and assumed to be at 2000, 730 and 60 μmol m–2 s–1 for leaves and the exposed side of cortex and

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xylem rays respectively, see also Table 1), the light sensi-tivity of leaves and cortex was similar, while the most tolerant seem to be the xylem rays and pith. An arrow for pith is lacking since the penetrating light in this twig area could not be measured. The exposed and shaded sides of twigs display similar light sensitivity (Fig. 5). Moreover, internal twig tissues seem to tolerate short term exposures

to extremely high (unrealistic) photon fluence rates. For example, xylem rays just below the cortex of the exposed side of a twig, receiving in planta less than 60 μmol m–2 s–1 PAR (Table 1), require a 20-fold higher PAR (i.e. 1200 μmol m–2 s–1) given for 15 min to display a cca. 60 % recovery (Fig. 6). Similar results were obtained with the three other test plants (not shown).

Discussion A reasonable argument against the finding of a low PSII yield in twigs is that the MB intensity is not sufficiently low for such deeply shaded tissues, inducing an actinic effect which is manifested in an artifactually high F0. This was clearly rejected by manipulating MB intensity. Another argument concerns the possibility of an enhanced contribution of PSI fluorescence to the measured F0. It is known that at room temperatures, PSII emits with peaks in the red (maximum at 686 nm) and far-red (maximum at 736 nm) region of the spectrum, while PSI displays a much lower fluorescence yield with peak at 720 nm (Franck et al. 2002). Since PSI presents no variable fluorescence, its effect is more pronounced in F0 than in Fmax. Thus, at normal PSII/PSI ratios the final influence on Fv/Fm may be negligible and may become appreciable only at low PSII/PSI ratios (Pfundel 1998). Yet, as shown by both room temperature and 77K fluorescence spectra (Figs. 3,4), a PSI-linked over-estimation of F0 is not likely. F0 is a function of PSII antenna size (Anderson 1986) being higher in shade adapted tissues (Fork and Govindjee 1980, Lichtenthaler et al. 1981). However, F0 is also a complex function of Chl density, since both emission and self-absorption of fluorescence increase with Chl concentration. Accordin-gly, the increase in F0 with twig depth (Fig. 2B) cannot be fully ascribed to an increasing shade acclimation gradient, since Chl absorptivity (inserted graph in Fig. 2A) decreases considerably with depth. Hence, we assume that the F0 increase with depth is linked both to shade adaptation and to the gradual inactivation of PSII towards the twig center.

In any case, we conclude that the low apparent PSII yield in twigs is not an artifact. Given that, the results of this investigation do not confirm the hypothesis of an extreme light sensitivity of internal stem photosynthetic tissues, which could lead to a sustained photoinhibition. The evidence towards this point is the relative tolerance of PSII to light levels far higher than those found in the stem interior (Fig. 6), the similarity in photoinhibitory risk between the naturally exposed and shaded twig sides (Fig. 5) and the antiparallel gradients of PSII inactivation and light penetration along twig depth (Fig. 2 and Table 1). We may also note that the analysis of light-dependent photoinhibitory damage in various twig internal tissues (Figs. 5,6) is based on incident light, yet the absorbed light is also important. As shown in Fig. 2 and Table 2, tissue absorptivity is considerably reduced

towards the twig center, further decreasing the photo-inhibitory pressure of penetrating light. Given also that the apparent low PSII yield is not an artifact (at least of the kinds tested in this study), we may conclude for an inherent, light-independent PSII inactivation. One may argue that the sudden exposure of twig tissues to a normal atmosphere after periderm removal and sample sectioning would affect negatively the PSII efficiency. However, Chl fluorescence probed from intact twigs in the field gave similarly low PSII yields in various plants (Manetas 2004ab, Levizou and Manetas 2008, Filippou et al. 2007). Moreover, removal of the periderm and, hence, restoration of atmospheric O2 and CO2 partial pressures in the cortex did not negatively affect the PSII yield (Manetas 2004b). Yet, one may further argue that the extremely low light levels, especially in deeper twig sites, would keep chloroplasts in a partly etiolated state. Indeed, Fv/Fm during de-etiolation of dark grown leaves remains low (Lebkuecher et al. 1999) until the full development of PSII. Symptoms of partial etiolation in normally lit plants have been reported in cotyledons of Helianthus annuus covered by pericarps (Solymosi et al. 2007), internal leaves of white cabbage (Solymosi et al. 2004) and leaf primordial covered by bud scales (Solymosi and Boddi 2006). In covered sunflower cotyle-dons a low Fv/Fm was also reported. All the above cases have similarities to our twigs concerning the internal light environment, both quantitatively and qualitatively. Thus, shade is deep and permanent, while light is depleted in blue/green and gradually enriched in red and far red photons (Kauppi 1991, Manetas and Pfanz 2005, Solymosi and Boddi 2006). However, the syndrome of partial etiolation includes together a prolamellar body, some protochlorophyllide pigments and a high Chl a/b ratio (Solymosi et al. 2007, Ryberg et al. 1980, Solymosi et al. 2004). No such attributes have been reported for twigs. Thus, a prolamellar body was absent even in the most deeply shaded pith (Buns et al. 1993, Ivanov et al. 1990, Larcher et al. 1988), Chl a/b ratios are always low in the stem interior (Pfanz et al. 2002), especially in pith (Pilarski 1999) and protochlorophyllide was not detected in chromatograms of whole twig extracts from five species (not shown, see also Levizou et al. 2004). Hence, we have to admit that the low dark-adapted PSII efficiency of the inner stem tissues is inherent and not related to any photoinhibitory damage under natural conditions or to a sustained etiolated state.

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A dysfunctional, partly inactivated PSII does not necessarily imply a dysfunctional chloroplast. Since stem tissues may suffer from hypoxia (Pfanz et al. 2002, Eklund 2000), it was proposed that cortex and wood photosynthesis could replenish the missing oxygen (Pfanz et al. 2002). However, such a function could be limited by light availability. Apart from deep shading, the spectral distribution of light penetrating to the cortex and further to the twig center is shifted towards the red and far red band due to selective transmittance of overlying tissues (Sun et al. 2003, Manetas and Pfanz 2005, see also Fig. 1). Since photosynthetic O2 evolution is driven by PSII which better absorbs at wavelengths shorter than 680 nm, the relative scarcity of appropriate photons in the cortex and further onto the twig interior may render an O2 replenishing function questionable. In fact, the observed (Fig. 4) increase of PSII/PSI ratio in cortices may reflect an adjustment of photosystem stoichiometry to the uneven spectral distribution of penetrating light (Chow et al. 1990). Hence, a photosynthetic alleviation of hypoxia may be valid for cortices but questionable for wood and pith. Even in the case of cortex, however, the very low linear electron transport rates may not allow an ample O2 production (Manetas 2004b). Although the unavoidable hypoxia may not be relieved due to the highly inactivated PSII, its indirect effects can be alleviated by cyclic flow around PSI. At sub-optimal O2 tensions respiration and ATP production are restricted (Geigenberger 2003). A chloroplast deficient in linear but sufficient in cyclic electron flow would compensate for ATP losses, since

linear flow contributes both to ATP and NADPH production, but cyclic flow delivers only ATP (Bukhov and Carpentier 2004). Indeed, Kotakis et al. (2006) reported that cortices of E. angustifolia maintain effective cyclic flow, potentially high PSI activities and high levels of intermediate electron carriers, in the presence of an extremely deficient linear flow. In addition, Ivanov et al. (2006) found high adenylate pools and ATP/ADP ratios in the bark compared to needles of Pinus sylvestris. According to the proposed model (Kotakis et al. 2006) chloroplasts in twig cortices may function as regulators of ATP/NAD(P)H ratios which are potentially perturbed by low internal O2 tensions. An additional reason demanding a high adenylate/reducing power ratio deals with the report for a high C4-acid decarboxylating activity in twigs (Ivanov et al. 2006) and petioles (Hibberd and Quick 2002), comparable to that found in bundle sheaths of C4 plants (Edwards and Walker 1983). The permanent, light-independent inactivation of PSII found in the present study may relieve the competition between linear and cyclic flow, hence guaranteeing an unhindered, PSI driven ATP production. Moreover, the finding that PSII efficiency diminishes drastically with twig depth (Fig. 2 and Table 2) may be indicative of an enhancement in the corresponding metabolic demands and/or stresses in deeper stem tissues. Within this context, the reported decrease of O2 levels towards the stem interior (Spicer and Holbrook 2005, Sorz and Hietz 2006) may not be irrelevant.

References Alessio, G.A., Pietrini, F., Brilli F., Loreto, F.: Characteristics

of CO2 exchange between peach stems and the atmosphere. – Funct. Plant Biol. 32: 787-795, 2005.

Anderson, J.M.: Photoregulation of the composition, function, and structure of thylakoid membranes. – Ann. Rev. Plant Physiol. 37: 93-136, 1986.

Aschan G, Pfanz H.: Non-foliar photosynthesis - a strategy of additional carbon acquisition. – Flora 198: 81-97, 2003.

Bukhov N., Carpentier R.: Alternative Photosystem I-driven electron transport routes: mechanisms and functions. – Photosynth. Res. 82: 17-33, 2004.

Buns, R., Acker, G., Beck, E.: The plastids of the yew tree (Taxus baccata L.): ultrastructure and immunocytochemical examination of chloroplastic enzymes. – Bot. Acta 106: 32-41, 1993.

Chow, W.S., Melis, A., Anderson, J.M.: Adjustments of photosystem stoichiometry in chloroplasts improve the quantum efficiency of photosynthesis. – Proc. Natl. Acad. Sci. 87: 7502-7506, 1990.

Damesin, C.: Respiration and photosynthesis characteristics of current-year stems of Fagus sylvatica: from the seasonal pattern to an annual balance. – New Phytol. 158: 465-475, 2003.

Dima, E, Manetas Y., Psaras, G.K.: Chlorophyll distribution pattern in inner stem tissues: evidence from epifluorescence microscopy and reflectance measurements in 20 woody

species. – Trees-Struct. Funct. 20: 515-521, 2006. Edwards, G.E., Walker, D.A.: C3, C4 Mechanisms and

Environmental Regulation of Photosynthesis. – Blackwell, Oxford 1983.

Eklund, L.: Internal oxygen levels decrease during the growing season and with increasing stem height. – Trees-Struct. Funct. 14: 177-180, 2000.

Filippou, M., Fasseas, C., Karabourniotis, G.: Photosynthetic characteristics of olive tree (Olea europaea) bark. – Tree Physiol. 27: 977-984, 2007.

Fork, D.C., Govindjee: Chlorophyll a fluorescence transients from sun and shade plants. – Naturwissenshaften 67: 510-511, 1980.

Franck, F., Juneau, P., Popovic, R.: Resolution of the Photosystem I and Photosystem II contributions to chlorophyll fluorescence of intact leaves at room temperature. – Biochim. Biophys. Acta - Bioenerg. 1556: 239-246, 2002.

Geigenberger, P.: Response of plant metabolism to too little oxygen. – Curr. Opin. Plant Biol. 6: 247-256, 2003.

Hibberd, J.M., Quick, W.P.: Characteristics of C4 photosynthesis in stems and petioles of C3 flowering plants. – Nature 415: 451-454, 2002.

Ivanov, A.G., Ignatova, N.S., Christov, A.M.: Comparative ultrastructural and fluorescence studies of grapevine (Vitis vinifera L.) chloroplasts isolated from stem and leaf tissues. – Plant Sci. 67: 253-257, 1990.

INCREASING INACTIVATION OF PSII ALONG TWIG DEPTH

231

Ivanov, A.G., Krol, M., Sveshnikov, D., Malmberg, G., Gardeström, P., Hurry, V., Öquist, G., Huner, N.P.A.: Characterization of the photosynthetic apparatus in cortical bark chlorenchyma of Scots pine. – Planta 223: 1165-1177, 2006.

Kauppi, A.: Seasonal fluctuations in chlorophyll content in birch stems with special reference to bark thickness and light transmission, a comparison between sprouts and seedlings. – Flora 185: 107-125, 1991.

Kotakis, C., Petropoulou, Y., Stamatakis, K., Yiotis, Ch., Manetas, Y.: Evidence for active cyclic electron flow in twig chlorenchyma in the presence of an extremely deficient linear electron transport activity. – Planta 225: 245-253, 2006.

Larcher, W., Lutz, C., Nagele, M., Bodner, M.: Photosynthetic functioning and ultrastructure of chloroplasts in stem tissues of Fagus sylvatica. – J. Plant Physiol. 132: 731-737, 1988.

Lebkuecher, J.G., Haldeman, K.A., Harris, C.E., Holz, S.L., Joudah, S.A., Minton, D.A.: Development of photosystem-II activity during irradiance of etiolated Helianthus (Asteraceae) seedlings. – Am. J. Bot. 86: 1087-1092, 1999.

Levizou, E. Manetas, Y.: Maximum and effective PSII yields in the cortex of the main stem of young Prunus cerasus trees: effects of seasons and exposure. – Trees-Struct. Funct. 22: 159-164, 2008.

Levizou, E., Petropoulou, Y., Manetas, Y.: Total carotenoid amount in crude twig extracts may be overestimated due to interference by high contents of co-extracted phenolics. – Photosynthetica 42: 295-297, 2004.

Lichtenthaler, H.K., Buschmann, C., Döll, M., Fietz, H-J., Bach, T., Kozel, U., Meier, D., Rahmsdorf, U.: Photosynthetic activity, chloroplast ultrastructure, and leaf characteristics of high-light and low-light plants and of sun and shade leaves. – Photosynth. Res. 2: 115-141, 1981.

Lichtenthaler, H.K., Wellburn, A.R.: Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. – Biochem. Soc. Trans. 11: 591-592, 1983.

Manetas, Y.: Photosynthesizing in the rain: beneficial effects of twig wetting on corticular photosynthesis through changes in the periderm optical properties. – Flora 199: 334-341, 2004a.

Manetas, Y.: Probing corticular photosynthesis through in vivo chlorophyll fluorescence measurements: evidence that high internal CO2 levels suppress electron flow and increase the risk of photoinhibition. – Physiol. Plant. 120: 509-517, 2004b.

Manetas, Y., Pfanz, H.: Spatial heterogeneity of light penetration through periderm and lenticels and concomitant patchy acclimation of corticular photosynthesis. – Trees-Struct. Funct. 19: 409-414, 2005.

Papageorgiou, G.C. Govindjee: Chlorophyll a Fluorescence: A Signature of Photosynthesis. – Springer, Dordrecht 2004.

Pfanz, H., Aschan, G., Langanfeld-Heyser, R., Wittmann, C.,

Loose, M.: Ecology and ecophysiology of tree stems: corticular and wood photosynthesis. – Naturwissenschaften 89: 147-162, 2002.

Pfundel, E.: Estimating the contribution of Photosystem I to total leaf chlorophyll fluorescence. – Photosynth. Res. 56: 185-195, 1998.

Pilarski, J.: Gradient of photosynthetic pigments in the bark and leaves of lilac (Syringa vulgaris L.). – Acta Physiol. Plant. 21: 365-373, 1999.

Ryberg, H., Axelsson, L., Widell, K.O., Virgin, H.I.: Chlorophyll b accumulation and grana formation in low intensities of red light. – Physiol. Plant. 49: 431-436, 1980.

Šiffel, P., Hunalová, I., Roháček, K.: Light-induced quenching of chlorophyll fluorescence at 77 K in leaves, chloroplasts and Photosystem II particles. – Photosynth. Res. 65: 219-229, 2000.

Solhaug, K.A., Haugen, V: Seasonal variation of photoinhibition of photosynthesis in bark from Populus tremula L. – Photosynthetica 35: 411-417, 1998.

Solymosi, K., Boddi, B.: Optical properties of bud scales and protochlorophyll(ide) forms in leaf primordia of closed and opened buds. – Tree Physiol. 26: 1075-1085, 2006.

Solymosi, K., Martinez, K., Kristof, Z., Sundqvist, C., Boddi, B.: Plastid differentiation and chlorophyll biosynthesis in different leaf layers of white cabbage (Brassica oleracea cv. capitata). – Physiol. Plant. 121: 520-529, 2004.

Solymosi, K., Vitanyi, B., Hideg, E., Boddi, B.: Etiolation symptoms in sunflower (Helianthus annuus) cotyledons partially covered by the pericarp of the achene. – Ann. Bot. 99: 857-867, 2007.

Sorz, J., Hietz, P.: Gas diffusion through wood: implications for oxygen supply. – Trees-Struct. Funct. 20: 34-41, 2006.

Spicer, R., Holbrook, N.M. Within-stem oxygen concentration and sap flow in four temperate tree species: does long-lived xylem parenchyma experience hypoxia? – Plant Cell Environ. 28: 192–201, 2005.

Sun, Q., Yoda, K., Suzuki, M., Suzuki, H.: Vascular tissue in the stem and roots of woody plants can conduct light. – J. Exp. Bot. 54: 1627-1635, 2003.

Tausz, M., Warren, C.R., Adams, M.A.: Is the bark of shining gum (Eucalyptus nitens) a sun or a shade leaf? – Trees-Struct. Funct. 19: 415-421, 2005.

Weis, E.: Chlorophyll fluorescence at 77 K in intact leaves: characterization of a technique to eliminate artifacts related to self-absorption. – Photosynth. Res. 6: 73-86, 1985.

Wittmann, C., Aschan, G., Pfanz, H.: Leaf and twig photosynthesis of young beech (Fagus sylvatica) and aspen (Populus tremula) trees grown under different light regimes. – Basic Appl. Ecol. 2: 145–154, 2001.