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Photoinhibition of photosynthesis in mature and young leaves of grapevine (Vitis vinifera L.)
Plant Science 164 (2003) 635 /644 www.elsevier.com/locate/plantsci
Photoinhibition of photosynthesis in mature and young leaves of grapevine (Vitis vinifera L.) Massimo Bertamini, Namachevayam Nedunchezhian * Istituto Agrario di San Michele all’ Adige, 38010 San Michele all’ Adige, Italy Received 15 November 2002; received in revised form 29 December 2002; accepted 7 January 2003
Abstract Photoinhibition of photosynthesis was studied in young (fully expanded) and mature sun leaves of grapevine (Vitis vinifera L.), under controlled conditions (irradiation of detached leaves to about 1900 mE m 2 s 1). The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (Fv/Fm) and electron transport measurements. Compared with the mature leaves, the young leaves, containing about half the amount of Chl a/b per unit area, exhibited a higher proportion of total carotenoids as xanthophyll cycle pigments and had an increased ratio of total carotenoids to Chl a/b. The potential efficiency of PS II, Fv/Fm, markedly declined in high light irradiated young leaves without significant increase of Fo level. In contrast, Fv/Fm ratio declined with significant increase of Fo level in mature leaves. When various photosynthetic activities were followed on isolated thylakoids, the rate of whole chain and PS II activity were markedly decreased in high light irradiated young leaves than mature leaves. A smaller inhibition of PS I activity was also observed in both leaves. In the subsequent dark incubation, fast recovery was observed in both leaves and reached maximum PSII efficiencies similar to those observed in non-photoinhibited leaves. The artificial exogenous electron donors DPC, NH2OH and Mn2 failed to restore the high light induced loss of PS II activity in mature leaves, while DPC and NH2OH significantly restored in young leaves. It is concluded that high light inactivates on the donor side of PS II and acceptor side of PS II in young and mature leaves, respectively. Quantification of the PS II reaction center protein D1 and 33 kDa protein of water splitting complex following high light exposure of leaves showed pronounced differences between young and mature leaves. The marked loss of PS II activity in high light irradiated leaves was due to the marked loss of D1 protein of the PS II reaction center and 33 kDa protein of the water splitting complex in mature and young leaves, respectively. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chlorophyll fluorescence; Donor side; D1 protein; Electron transport; Photoinhibition
1. Introduction
Abbreviations: A, antheraxanthin; Car, carotenoids; Chl, chlorophyll; DCBQ, 2,6-dichloro-p -benzoquinone; DCPIP, 2,6dichlorophenol indophenol; DPC, diphenyl carbazide; Fo, minimal fluorescence; Fm, maximum fluorescence; kDa, kilodalton; MV, methyl viologen; PPFD, photosynthetic photon flux density; PS, photosystem; SDS-PAGE, sodium dodecylsulphate-polyacrylamide gel electrophoresis; SiMo, silicomolybdate; V, violaxanthin; Z, zeaxanthin. * Corresponding author. Present address: Government Higher Secondary School, Vellimedupettai-604 207, Tindivanam (T.K), India. Tel.: /91-4147-22512. E-mail address: [email protected] (N. Nedunchezhian).
Light is necessary to drive the process of photosynthesis; however, absorbed irradiance in excess of that required for the saturation of photosynthesis may cause photoinhibition. The photoinhibition occurs at thylakoid level, particularly at PS II [1 /3]. Several investigators view photoinhibition of photosynthesis as a process of stress-induced damage to PS II. This is based on the fact that, as a consequence of photoinhibition, the D1 protein of PS II reaction center becomes degraded [1,4,5]. But some recent reports suggest that photoinhibition, first of all, results from the formation of photochemically inactive PS II centers, which convert the excitation energy into heat. This down regulation of PS II and thermal dissipation is considered as a
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00018-9
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protective mechanism against high light stress [2,6,7]. The photoinactivation and impairment of electron transport occurs at the acceptor and donor sides of PS II, although inactivation of the acceptor side may be the main mechanism for the impairment of electron transport [3,6]. Studies of canopy sun leaves of several tree species of the tropical forest [8] showed a higher susceptibility to photoinhibition of young, light green leaves, as compared with corresponding mature, dark green leaves. Apparently, in the young leaves, a given irradiance results in stronger excitation of chlorophylls due to their lower chlorophyll content per unit leaf area. Thus, young leaves experience more severe light stress than mature ones. In the young tropical canopy leaves, recovery from photoinhibition was characterized by a dominant fast phase, which apparently was related to epoxidation of zeaxanthin [9]. The effect of photoinhibition, alone or interacting with other stresses, on the long-term consequences of carbon balance or productivity, is little known. This, as Powles [10] noted, is in part because the recovery of plants from photoinhibition has received very little attention. A limited number of studies have shown that recovery takes at least several hours to occur. In fresh-water phytoplankton, for instance, recovery of photosynthesis occurred over 4/20 h, depending on the extent of the inhibition [11]. Similarly, the recovery of the photon yield of leaves of Phaseolus vulgaris took between 4 and 8 h [12]. The D1 reaction center protein of PS II is a target of light-induced damage to the PS II complex; turnover of the D1 protein is accelerated by increasing irradiance [6]. The hypothesis that degradation of D1 protein may regulate the functioning of the PS II repair cycle under photoinhibitory conditions has arisen from experiments with higher plants acclimated to different growth irradiances. It has been shown that low light grown or shade plants are more susceptible to photoinhibition than high light or sun species [6,10,13]. This higher susceptibility is accompanied by slow degradation of D1 protein [6,14]. In addition to the proteolysis of damaged D1 protein and de novo synthesis of a new copy of D1, the repair cycle of PS II involves several other reactions, including post-translational processing and modification of the protein, and ligation of the electron-transfer components [6]. It was suggested that, like in coldacclimated spinach, the D1 protein is stabilized in young canopy leaves and in D1 inactivation, and that turnover takes little part in photoinhibition and recovery. The object of our work was to compare the susceptibility to photoinhibition and the process of recovery in young and mature leaves of Vitis vinifera L. The effect of photoinhibition was analyzed with respect to photosynthetic oxygen evolution and potential PS II function by fluorescence. The amount of D1 and 33 kDa proteins
were also analyzed in relation to the functional properties of PS II after photoinhibition. The significance of the capacity of the PS II repair cycle for the protection against photoinhibition was analyzed by establishing rates of recovery from photoinhibition.
2. Materials and methods 2.1. Plant material Young and mature leaves of V. vinifera L. were collected from selected 10-year-old seedlings grown under field condition on training system with upright growing shoots condition in Istituto Agrario di San Michele all’ Adige, Italy. Leaf samples were harvested early in the morning. Sun-exposed light-green, almost fully expanded young leaves and dark-green, mature leaves were chosen for the study. 2.2. Pigments Photosynthetic pigments in leaf sections were analyzed by HPLC according to the method of Krause et al. [8]. The amount of Chl a/b per unit leaf area was determined according to Arnon [15] after extraction of leaf segments with 80% acetone in the presence of Na2CO3. 2.3. Photoinhibition and recovery under controlled conditions Detached leaves were placed into a controlled-environment chamber equipped with a 24 V/250 W metal / halide lamp (H. Walz, Effeltrich, FRG). The upper leaf surface was exposed to a photon flux density (PFD) of 1900 mE m 2 s 1. Air temperature was 20 8C and relative humidity 669/5%. The PPFD was measured with a quantum sensor (LI-Cor, Lincoln, Neb. USA). Leaf temperatures, recorded with thermocouple attached to the lower surface were between 27 and 29 8C. Discs of 1.6 cm 2 area were punched from the leaf blades after specified times of high-light exposure and placed on moist filter paper in petri dishes (temperature 25/27 8C). The leaf discs were darkened for 5 min before the degree of photoinhibition was determined by fluorescence measurement. For recovery from photoinhibition the leaf discs were kept in complete dark for 60 min. 2.4. Modulated Chl fluorescence in leaves Chl fluorescence was measured on leaf discs using a PAM 2000 fluorometer (H. Walz, Effeltrich, FRG). Before the measurements, the leaves were dark adapted for 30 min. Fo was measured by switching on the
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modulated light 0.6 kHz; PPFD was less than 0.1 mE m 2 s 1 at the leaf surface. Fm was measured at 20 kHz with a 1s pulse of 6000 mE m 2 s 1 of white light.
2.5. Activities of electron transport Thylakoid membranes were isolated from the leaves as described by Berthhold et al. [16]. Whole chain electron transport (H2O 0/MV) and partial reactions of photosynthetic electron transport mediated by PS II (H2O 0/DCBQ; H2O 0/SiMo) and PS I (DCPIPH2 0/ MV) were measured as described by Nedunchezhian et al. [17]. Thylakoids were suspended at 10 mg Chl ml 1 in the assay medium containing 20 mM Tris /HCl, pH 7.5, 10 mM NaCl, 5 mM MgCl2, 5 mM NH4Cl and 100 mM sucrose supplemented with 0.5 mM DCBQ and 0.2 mM SiMo.
2.6. DCPIP photoreduction The rate of DCPIP photoreduction was determined as the decrease in absorbance at 590 nm using a Hitachi 557 spectrophotometer. The reaction mixture (3 cm3) contained 20 mM Tris /HCl, pH 7.5, 5 mM MgCl2, 10 mM NaCl, 100 mM sucrose, 0.1 mM DCPIP and thylakoid membranes equivalent to 20 mg of Chl. Where mentioned, the concentration of MnCl2, DPC and NH2OH were 5, 0.5 and 5 mM, respectively.
2.7. Immunological determination of thylakoid proteins The relative contents of certain thylakoid proteins per mg Chl were determined immunologically by western blotting. Thylakoids were solubilized in 5% SDS, 15% glycerine, 50 mM Tris /HCl (pH 6.8) and 2% mercaptoethanol at room temperature for 30 min. The polypeptides were separated by SDS-PAGE as described by Laemmli [18] and proteins were then transferred to nitrocellulose by electroblotting for 3 h at 0.4 A. After saturation with 10% milk powder in TBS buffer (pH 7.5). The first antibody in 1% gelatine was allowed to react overnight at room temperature. After washing with TBS containing 0.05% Tween-20, the secondary antibody [Anti-Rabbit IgG (whole molecule) Biotin Conjugate, Sigma] was allowed to react in 1% gelatine for 2 h. For detection of D1 protein a polyclonal antiserum against spinach D1 protein was used (kindly provided by Professor I. Ohad, Jerusalum, Israel), and the antibody against the 33 kDa protein of the watersplitting system was a gift from Dr Barbato, Padova, Italy. The densitometry analysis of western blots was performed with a Bio-Image analyzer (Millipore Corporation, Michigan, USA).
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3. Results 3.1. Changes in pigments Detailed analyses of photosynthetic pigments in mature and young leaves of grapevine are given in Table 1. The Chl a/b content per unit area in young leaves was about 51% of that in mature leaves, but the Chl a/b ratio was not significantly different between the two leaf types (Table 1), indicating that in the young leaves the photosynthetic apparatus fully developed. In contrast, the young leaves also exhibited an increased ratio of total carotenoids and of xanthophyll cycle pigments to Chl a/b (Table 1). The young leaves absorbed about 9% less of the incident light than mature leaves (Table 1). 3.2. Changes in Chl fluorescence In order to compare the susceptibility to photoinhibition between mature and young leaves, leaf samples were subjected to high light exposure in a controlledenvironment chamber, followed by a recovery incubated in dark for 60 min. Fig. 1 illustrates that young leaves responded more sensitively to high light than mature leaves, as indicated by the more pronounced decrease in Fv/Fm ratios of young leaves. Recovery during dark incubation was remarkably fast, so that after 45 min only small differences in Fv/Fm ratios between mature and young leaves remained (Fig. 2). The treatment with high light for 60 min lead to a decline of about 40, 65% in Fv/Fm and elevation of about 21, 12% in Fo in mature and young leaves, respectively. In the subsequent dark incubation, Fo decayed with increase in Fv/Fm, and both of them could largely recover after 60 min dark (Fig. 2). 3.3. Changes in photosynthetic activities Photosynthetic electron transport activities were measured in thylakoids isolated from high light irradiated mature and young leaves (Fig. 3). The rate of PS II activity was decreased with increase of the time of high
Table 1 Photosynthetic pigments and fractional light absorbance in young and mature leaves of V. vinifera L. (mean9/S.E.; n /5) Young leaves Mature leaves Chl a/b (mmol m 2) Chl a/b ratio Total carotenoids/Chl (mmol mol 1) (V/A/Z)/Chl (mmol mol 1) Absorbance
2989/15 3.59/0.4 6019/34 1519/8.5 0.7459/0.002
5909/31 3.49/0.3 4819/28 789/4.1 0.8159/0.005
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was used instead of DCBQ in young leaves. A smaller inhibition of PS I activity was also observed in both mature and young leaves (Fig. 3). In the subsequent dark incubation, the leaves reached maximum rate of PS II activity similar to those observed in non-photoinhibited leaves (Fig. 4). 3.4. Changes in DCPIP photoreduction measurements To locate the possible site (s) of inhibition in the PS II reaction, we followed the DCPIP photoreduction supported by various exogenous electron donors used in thylakoids isolated from 60 min high light irradiated mature and young leaves. Wydrzynski and Govindjee [19] have shown that MnCl2, DPC, NH2OH and HQ could donate electrons in the PS II reaction. Fig. 5 shows the electron transport activity of PS II in the presence and absence of three of the above compounds. The PS II activity was reduced to about 35 and 66% in mature and young leaves, when water served as electron donor. A similar trend was also found using MnCl2 as donor in both leaves. In contrast, a significant restoration of PS II mediated DCPIP reduction was observed when NH2OH and DPC were used as electron donors in young leaves, while the PS II activity was not restored using either DPC or NH2OH in mature leaves (Fig. 5). 3.5. Changes in D1 and 33 kDa proteins by immunoblot
Fig. 1. Changes in the relative levels of fluorescence emitted as minimal fluorescence (Fo), maximum fluorescence (Fm) and the ratio of variable to maximum fluorescence (Fv/Fm) of mature and young leaves of V. vinifera L. at different time intervals. Data are given in % of untreated controls. Control values for Fo, Fm and Fv/Fm were 2.7, 12.8, 0.789 and 2.7, 14.2, 0.809 in mature and young leaves, respectively (mean9/S.E.; n /5).
light in both mature and young leaves. After 60 min, photosynthetic electron transport from H2O 0/DCBQ and H2O 0/SiMo was reduced by about 44, 13% in mature and 26, 59% in young leaves, respectively. A significant reduction of PS II activity was noticed when DCBQ was used electron acceptor in mature leaves but marginally inhibited when SiMo was used electron acceptor (Fig. 3). In contrast, a marked reduction of PS II activity was noticed when electron acceptor SiMo
Photoinhibition of PS II is known to induce breakdown of the D1 protein [4,20]. In systems without protein biosynthesis this can be seen directly as a loss in D1 protein content. In intact plant the correlation between D1 protein content and activity of PS II is more complex [21,22]. Photoinhibition induced inhibition of PS II activity in thylakoids of mature and young leaves was compared with changes in the relative contents of D1 and 33 kDa proteins as determined by western blotting (Fig. 6) followed by quantification by the Bio-Image apparatus (Fig. 6). The relative content of D1 and 33 kDa proteins decreased to 62, 9% in mature and 8, 82% in young leaves irradiated with 60 min high light, respectively. In the subsequent dark recovery, the leaves reached the original D1 in mature and 33 kDa protein content in young leaves, similar to those observed in non-photoinhibited leaves (Fig. 6). In Fig. 7, relative D1 protein contents and Fv/Fm ratios are compared after photoinhibitory treatments of mature and young leaves. In the young leaves, no significant D1 degradation could be attributed to the action of photoinhibitory light, even when Fv/Fm ratios had decreased to 62 /65% of the controls. It should be noted that the D1 protein was quantified after extended periods in dark incubation so that all D1 protein that had been inactivated during high light period could be degraded. The mature leaves showed a strong decrease
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Fig. 2. Photoinhibition and subsequent dark recovery of mature and young leaves under controlled conditions as indicated by Fo, Fm and Fv/Fm ratios. Control values for Fo, Fm and Fv/Fm were 2.7, 10.0, 0.787 and 2.7, 11.1, 0.804 in mature and young leaves, respectively (mean9/S.E.; n /5).
in D1 protein content together with the decline of Fv/ Fm ratio about 40% (Fig. 7).
4. Discussion The present results indicate that exposure of young and mature leaves to high light produces, differential loss of photosynthetic activity and potential efficiency of PS II (Fv/Fm) where the former are more sensitive to high light than the latter. In situ, the mature and young sun leaves of grapevine experienced similar exposure to full sunlight. However, the higher carotenoid content per mol Chl a/b and xanthophylls cycle pigments (Table 1) indicate a strengthened acclimation of the young leaves to excess light [23]. This probably resulted from higher light absorbance per mol Chl in the young leaves. Young leaves are similarly or even more exposed to sun than mature leaves and should not be less acclimated to high light. The characteristics of pigment composition, light absorbance and photosynthetic performance of the
grapevine leaves may at least in part explain the differences in susceptibility to photoinhibition: (a) the young leaves contained around 50% less Chl per unit leaf area than the mature ones, but light absorbance was only slightly lower in the young leaves. The Chl a/b ratio, however, was the same in the two leaf types, indicating similarly developed antenna systems but fewer photosynthetic units per unit leaf area (data not shown) in young than mature leaves, (b) young leaves exhibited much lower capacities of photosynthetic O2 evolution, which roughly corresponds to lower Chl content. Due to these two factors, the same light exposure would result in a much higher fraction of excess light and higher average Chl excitation in the young leaves. The decline in Fv/Fm, used here as a convenient measure of photoinhibition, indicates a reduction in potential PS II efficiency. In many studies, a close correlation of the Fv/Fm ratio with the quantum yield of photosynthetic O2 evolution or CO2 assimilation under light limiting conditions has been reported [8,24,25]. The reduction of Fv/Fm in high light irradiated young and
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Fig. 3. Changes in the rates of whole chain (H2O0/MV), PS II (H2O0/DCBQ; H2O0/SiMo) and PS I (DCPIPH2 0/MV) electron transport activities in thylakoids isolated from high light irradiated mature and young leaves of V. vinifera L. at different time intervals. The 100% values are [mmol (O2) mg 1 Chl h 1]: DCPIPH2 0/MV 396, 390; H2O0/DCBQ 168, 144; H2O0/SiMo 102, 70; H2O 0/MV 139, 96 for thylakoids isolated from mature and young leaves, respectively (mean9/S.E.; n /5).
mature leaves was mainly caused by a decline of Fm and increase of Fo, respectively. It has been proposed that an increase of Fo may be induced by the inactivation of part of PS II reaction centers [26 /28]. Our experimental results from mature leaves are in accordance with this idea. When Fo increased in mature leaves under high light, some PS II reaction centers lost their photochemical activity as indicated by a marked decline in the photochemical efficiency of PS II (Fv/Fm). BolharNordenkampf et al. [29] observed relatively low Fv/Fm ratios; even small changes of Fo or Fm would result in considerable changes in the Fv/Fm ratio. In the subsequent dark incubation, both young and mature leaves reached maximum PS II photochemistry efficiencies similar to those observed in non-photoinhibited leaves.
The rate of recovery agrees with other reports on photoinhibition in higher plants [8,13,30]. From analysis of electron transport activities in thylakoids isolated from high light irradiated mature leaves, the oxygen evolution was inhibited markedly when the electron acceptor used were DCBQ, but not SiMo. This is mainly due to high light induced changes on the reducing side of PS II and is due to photoinhibition. This is also supported by our Chl fluorescence studies where Fo was markedly increased [31,32]. In contrast, thylakoids isolated from young leaves, the rate of PS II activity observed with SiMo is lower than the one observed with that DCBQ. This is due to donor side being more impaired than the acceptor side of PS II. The extent of variable fluorescence (Fv) was reduced markedly with slight increase of Fo level. This is characteristic for inhibition of donor side of PS II. If the acceptor side of PS II is photoinhibited the Fo level is significantly increased [33,34]. A relationship between Fv/Fm and PS II electron transport activity in thylakoids isolated from photoinhibited leaves has also been shown [35,36]. To locate the possible site of inhibition in the PS II reaction, we followed the DCPIP photoreduction supported by various exogenous electron donors in thylakoids isolated from 60 min high light irradiated both mature and young leaves. Among the artificial electron donors tested DPC and NH2OH donates electrons directly to the reaction center of PS II [19]. Addition of DPC and NH2OH markedly restored the high light induced loss of PSII activity in young leaves. This is due to water-oxidizing system is sensitive to high light in grapevine young leaves. In contrast, the loss of PS II activity was not restored by using neither DPC nor NH2OH in mature leaves. It is clear that high light induced changes on the acceptor side of PS II in mature leaves [3,6,31,37]. Similar observations were found for field grown Schefflera arboricola leaves adapted to different light environments [38]. The loss of PS II activity could only partially be ascribed to functional inhibition of PS II since Fv/Fm was reduced by about 65 and 45% in 60 min high light irradiated young and mature leaves, respectively. We, therefore, assume that it was mainly due to loss of PS II centers on a Chl basis. This could be confirmed by the immunological determination of the PS II reaction center protein of D1 and 33 kDa protein of watersplitting complex. It is often thought that photoinhibition is a result of marked loss of D1 protein in mature leaves. So it occurs only when the rate of damage to D1 protein exceeds the rate of its repair [1,39,40]. Moreover, the fluorescence parameter Fv/Fm is considered to be a good measure of photoinhibition, and a decrease in Fv/ Fm under photoinhibitory conditions is often attributed to the loss of D1 protein. Furthermore, after high light treatment, inactive PS II reaction centers were also
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Fig. 4. Photoinhibition and subsequent dark recovery of mature and young leaves under controlled conditions as indicated by PS II, PS I and whole chain electron transport activities at different time intervals. The 100% values are [mmol (O2) mg 1 Chl h 1]: DCPIPH2 0/MV 396, 390; H2O0/ DCBQ 168, 144; H2O0/SiMo 102, 70; H2O0/MV 139, 96 for thylakoids isolated from mature and young leaves, respectively (mean9/S.E.; n /5).
Fig. 5. Effect of various exogenous electron donors on PS II activity (H2O0/DCPIP) in thylakoids isolated from 60 min high light irradiated mature and young leaves of V. vinifera L. The 100% values are [mmol (DCPIP red.) mg 1 Chl h 1]: H2O0/DCPIP 176, 130; DPC0/DCPIP 179, 158; NH2OH0/DCPIP 180, 154; MnCl2 0/ DCPIP 177, 132 for thylakoids isolated from mature and young leaves, respectively (mean9/S.E.; n /5).
found to be accumulate in mature leaves or in other leaves [41]. However, as shown by D1 protein quantification, even strong photoinhibition of young leaves does not seem to be related to loss of the D1 protein in the PS II reaction center. In contrast, in mature leaves (Fig. 7), substantial D1 degradation appeared to be coincident with photoinhibition. This confirms our hypothesis that in young leaves, similar to cold-acclimated spinach [42], the treatment with excess light the D1 protein is not susceptible to photoinactivation. We found no correlation between photoinhibition of photosynthesis and loss of the D1 protein when young leaves irradiated with high light were compared. In previous studies on the relationship between photoinhibition and loss of the D1 protein, there are reports showing both good [43] and poor [44] correlations. Studies of photoinhibition at low temperatures have also shown that photoinhibition is
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Fig. 6. Relative content of D1 and 33 kDa proteins of thylakoids isolated from high light irradiation followed by dark incubation in young and mature leaves of V. vinifera L. at different time intervals. Lane a, 0 min; lane b, 30 min high light; lane c, 60 min high light; lane d, 30 min dark; lane e, 60 min dark.
not correlated with D1 protein degradation either in vivo [45] or in vitro [46]. The extrinsic protein of 33 kDa associated with the lumenal surface of the thylakoid membranes are required for optimal functioning of the oxygen evolving machinery [47 /49]. Our immunological results indicate that the significant loss of 33 kDa protein could be one of the reasons for significant loss of O2 evolution capacity in young leaves. From the results we suggested that the high degree of photoinhibition in the young leaves indicated by a strong decrease in the Fv/Fm ratio probably reflects a dynamic regulator response of the photosynthetic system to excess absorbed light energy. The observed photoinhibition is possibly associate with some loss of productivity but might protect photosynthetic pigments and electron transport apparatus from severe destruction. The fast recovery probably does not require D1 protein synthesis and may be related to xanthophylls cycle activity, which is increased in the young leaves.
Our results also suggest that photoinactivation of PS II is not correlated at all with net loss of D1, and photoinhibition represents the formation of inactive centers [21,50,51]. In addition, we concluded that high light induced changes not only in the acceptor side of PS II (mature leaves) but also on the donor side of PS II (young leaves). Depending on the leaf age, high light acclimation probably results in differing degrees inhibition of PS II.
Acknowledgements This work was in part supported by a grant from Provincia Autonoma of Trento and National Council of Research (CNR): project ‘Analisi e Ricerche per il sistema Agri-Industriale’ sub-project ‘Prometavit’. The authors gratefully acknowledge the two anonymous reviewers for improving the manuscript.
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Fig. 7. Quantification of D1 protein and degree of photoinhibition in V. vinifera L. leaves under controlled conditions. The Fv/Fm ratios were determined as a measure of photoinhibition. Data are given in % of untreated controls (morning samples) (mean9/S.E.; n /5).
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[31] K. Asada, U. Heber, U. Schreiber, Pool size of electrons that can be donated to P700 , as determined in intact leaves: donation to P700/ from stromal components via the intersystem chain, Plant Cell Physiol. 33 (1992) 927 /932. [32] T. Endo, T. Shikanai, F. Sata, K. Asada, NAD(P)H dehydrogenase-dependent, antimycin A-sensitive electron donation to plastoquinone in tabacco chloroplasts, Plant Cell Physiol. 39 (1998) 1226 /1231. [33] S.I. Allakhverdiev, A. Setlikova, V.V. Klimov, I. Setlik, In photoinhibited photosystem II particles pheophytin photoreduction remains unimpaired, FEBS Lett. 226 (1987) 186 /190. [34] I. Setlik, S.I. Allakhverdiev, L. Nedbal, E. Setlikova, V.V. Klimov, Three types of photosystem II photoinactivation. 1. Damaging processes on the acceptor side, Photosynth. Res. 23 (1990) 39 /48. [35] S. Somersalo, G.H. Krause, Reversible photoinhibition of unhardened and cold-acclimated spinach leaves at chilling temperatures, Planta 180 (1990) 181 /187. [36] B. Schnettger, C. Critchley, U.J. Santore, M. Graf, G.H. Krause, Relationship between photoinhibition of photosynthesis, D1 protein turnover and chloroplast structure: effects of protein synthesis inhibitors, Plant Cell Physiol. 17 (1994) 55 /64. [37] S.-S. Hong, D.-Q. Xu, Light induced increase in initial chlorophyll fluorescence Fo level and the reversible inactivation of PS II reaction centers in soybean leaves, Photosynth. Res. 61 (1999) 269 /280. [38] U. Schiefthaler, A.W. Russell, H.R. Bolhar-Nordenkampf, C. Critchley, Photoregulation and photodamage in Scheflera arboricola leaves adapted to different light environments, Aust. J. Plant Physiol. 26 (1999) 485 /494. [39] J. Barber, Molecular basis of photoinhibition, in: P. Mathis (Ed.), Photosynthesis, from Light to Biosphere, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1995, pp. 159 /164. [40] R. Carpentier, Influence of high light intensity on photosynthesis: photoinhibition and energy dissipation, in: M. Pessarakli (Ed.), Handbook of Photosynthesis, Marcel Dekker, New York, 1995, pp. 443 /450. [41] J.M. Anderson, E.-M. Aro, Grana stacking and protection of photosystem II in thylakoid membranes of higher plant leaves
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Photoinhibition of photosynthesis in mature and young leaves of grapevine (Vitis vinifera L.) Massimo Bertamini, Namachevayam Nedunchezhian * Istituto Agrario di San Michele all’ Adige, 38010 San Michele all’ Adige, Italy Received 15 November 2002; received in revised form 29 December 2002; accepted 7 January 2003
Abstract Photoinhibition of photosynthesis was studied in young (fully expanded) and mature sun leaves of grapevine (Vitis vinifera L.), under controlled conditions (irradiation of detached leaves to about 1900 mE m 2 s 1). The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (Fv/Fm) and electron transport measurements. Compared with the mature leaves, the young leaves, containing about half the amount of Chl a/b per unit area, exhibited a higher proportion of total carotenoids as xanthophyll cycle pigments and had an increased ratio of total carotenoids to Chl a/b. The potential efficiency of PS II, Fv/Fm, markedly declined in high light irradiated young leaves without significant increase of Fo level. In contrast, Fv/Fm ratio declined with significant increase of Fo level in mature leaves. When various photosynthetic activities were followed on isolated thylakoids, the rate of whole chain and PS II activity were markedly decreased in high light irradiated young leaves than mature leaves. A smaller inhibition of PS I activity was also observed in both leaves. In the subsequent dark incubation, fast recovery was observed in both leaves and reached maximum PSII efficiencies similar to those observed in non-photoinhibited leaves. The artificial exogenous electron donors DPC, NH2OH and Mn2 failed to restore the high light induced loss of PS II activity in mature leaves, while DPC and NH2OH significantly restored in young leaves. It is concluded that high light inactivates on the donor side of PS II and acceptor side of PS II in young and mature leaves, respectively. Quantification of the PS II reaction center protein D1 and 33 kDa protein of water splitting complex following high light exposure of leaves showed pronounced differences between young and mature leaves. The marked loss of PS II activity in high light irradiated leaves was due to the marked loss of D1 protein of the PS II reaction center and 33 kDa protein of the water splitting complex in mature and young leaves, respectively. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chlorophyll fluorescence; Donor side; D1 protein; Electron transport; Photoinhibition
1. Introduction
Abbreviations: A, antheraxanthin; Car, carotenoids; Chl, chlorophyll; DCBQ, 2,6-dichloro-p -benzoquinone; DCPIP, 2,6dichlorophenol indophenol; DPC, diphenyl carbazide; Fo, minimal fluorescence; Fm, maximum fluorescence; kDa, kilodalton; MV, methyl viologen; PPFD, photosynthetic photon flux density; PS, photosystem; SDS-PAGE, sodium dodecylsulphate-polyacrylamide gel electrophoresis; SiMo, silicomolybdate; V, violaxanthin; Z, zeaxanthin. * Corresponding author. Present address: Government Higher Secondary School, Vellimedupettai-604 207, Tindivanam (T.K), India. Tel.: /91-4147-22512. E-mail address: [email protected] (N. Nedunchezhian).
Light is necessary to drive the process of photosynthesis; however, absorbed irradiance in excess of that required for the saturation of photosynthesis may cause photoinhibition. The photoinhibition occurs at thylakoid level, particularly at PS II [1 /3]. Several investigators view photoinhibition of photosynthesis as a process of stress-induced damage to PS II. This is based on the fact that, as a consequence of photoinhibition, the D1 protein of PS II reaction center becomes degraded [1,4,5]. But some recent reports suggest that photoinhibition, first of all, results from the formation of photochemically inactive PS II centers, which convert the excitation energy into heat. This down regulation of PS II and thermal dissipation is considered as a
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00018-9
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protective mechanism against high light stress [2,6,7]. The photoinactivation and impairment of electron transport occurs at the acceptor and donor sides of PS II, although inactivation of the acceptor side may be the main mechanism for the impairment of electron transport [3,6]. Studies of canopy sun leaves of several tree species of the tropical forest [8] showed a higher susceptibility to photoinhibition of young, light green leaves, as compared with corresponding mature, dark green leaves. Apparently, in the young leaves, a given irradiance results in stronger excitation of chlorophylls due to their lower chlorophyll content per unit leaf area. Thus, young leaves experience more severe light stress than mature ones. In the young tropical canopy leaves, recovery from photoinhibition was characterized by a dominant fast phase, which apparently was related to epoxidation of zeaxanthin [9]. The effect of photoinhibition, alone or interacting with other stresses, on the long-term consequences of carbon balance or productivity, is little known. This, as Powles [10] noted, is in part because the recovery of plants from photoinhibition has received very little attention. A limited number of studies have shown that recovery takes at least several hours to occur. In fresh-water phytoplankton, for instance, recovery of photosynthesis occurred over 4/20 h, depending on the extent of the inhibition [11]. Similarly, the recovery of the photon yield of leaves of Phaseolus vulgaris took between 4 and 8 h [12]. The D1 reaction center protein of PS II is a target of light-induced damage to the PS II complex; turnover of the D1 protein is accelerated by increasing irradiance [6]. The hypothesis that degradation of D1 protein may regulate the functioning of the PS II repair cycle under photoinhibitory conditions has arisen from experiments with higher plants acclimated to different growth irradiances. It has been shown that low light grown or shade plants are more susceptible to photoinhibition than high light or sun species [6,10,13]. This higher susceptibility is accompanied by slow degradation of D1 protein [6,14]. In addition to the proteolysis of damaged D1 protein and de novo synthesis of a new copy of D1, the repair cycle of PS II involves several other reactions, including post-translational processing and modification of the protein, and ligation of the electron-transfer components [6]. It was suggested that, like in coldacclimated spinach, the D1 protein is stabilized in young canopy leaves and in D1 inactivation, and that turnover takes little part in photoinhibition and recovery. The object of our work was to compare the susceptibility to photoinhibition and the process of recovery in young and mature leaves of Vitis vinifera L. The effect of photoinhibition was analyzed with respect to photosynthetic oxygen evolution and potential PS II function by fluorescence. The amount of D1 and 33 kDa proteins
were also analyzed in relation to the functional properties of PS II after photoinhibition. The significance of the capacity of the PS II repair cycle for the protection against photoinhibition was analyzed by establishing rates of recovery from photoinhibition.
2. Materials and methods 2.1. Plant material Young and mature leaves of V. vinifera L. were collected from selected 10-year-old seedlings grown under field condition on training system with upright growing shoots condition in Istituto Agrario di San Michele all’ Adige, Italy. Leaf samples were harvested early in the morning. Sun-exposed light-green, almost fully expanded young leaves and dark-green, mature leaves were chosen for the study. 2.2. Pigments Photosynthetic pigments in leaf sections were analyzed by HPLC according to the method of Krause et al. [8]. The amount of Chl a/b per unit leaf area was determined according to Arnon [15] after extraction of leaf segments with 80% acetone in the presence of Na2CO3. 2.3. Photoinhibition and recovery under controlled conditions Detached leaves were placed into a controlled-environment chamber equipped with a 24 V/250 W metal / halide lamp (H. Walz, Effeltrich, FRG). The upper leaf surface was exposed to a photon flux density (PFD) of 1900 mE m 2 s 1. Air temperature was 20 8C and relative humidity 669/5%. The PPFD was measured with a quantum sensor (LI-Cor, Lincoln, Neb. USA). Leaf temperatures, recorded with thermocouple attached to the lower surface were between 27 and 29 8C. Discs of 1.6 cm 2 area were punched from the leaf blades after specified times of high-light exposure and placed on moist filter paper in petri dishes (temperature 25/27 8C). The leaf discs were darkened for 5 min before the degree of photoinhibition was determined by fluorescence measurement. For recovery from photoinhibition the leaf discs were kept in complete dark for 60 min. 2.4. Modulated Chl fluorescence in leaves Chl fluorescence was measured on leaf discs using a PAM 2000 fluorometer (H. Walz, Effeltrich, FRG). Before the measurements, the leaves were dark adapted for 30 min. Fo was measured by switching on the
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modulated light 0.6 kHz; PPFD was less than 0.1 mE m 2 s 1 at the leaf surface. Fm was measured at 20 kHz with a 1s pulse of 6000 mE m 2 s 1 of white light.
2.5. Activities of electron transport Thylakoid membranes were isolated from the leaves as described by Berthhold et al. [16]. Whole chain electron transport (H2O 0/MV) and partial reactions of photosynthetic electron transport mediated by PS II (H2O 0/DCBQ; H2O 0/SiMo) and PS I (DCPIPH2 0/ MV) were measured as described by Nedunchezhian et al. [17]. Thylakoids were suspended at 10 mg Chl ml 1 in the assay medium containing 20 mM Tris /HCl, pH 7.5, 10 mM NaCl, 5 mM MgCl2, 5 mM NH4Cl and 100 mM sucrose supplemented with 0.5 mM DCBQ and 0.2 mM SiMo.
2.6. DCPIP photoreduction The rate of DCPIP photoreduction was determined as the decrease in absorbance at 590 nm using a Hitachi 557 spectrophotometer. The reaction mixture (3 cm3) contained 20 mM Tris /HCl, pH 7.5, 5 mM MgCl2, 10 mM NaCl, 100 mM sucrose, 0.1 mM DCPIP and thylakoid membranes equivalent to 20 mg of Chl. Where mentioned, the concentration of MnCl2, DPC and NH2OH were 5, 0.5 and 5 mM, respectively.
2.7. Immunological determination of thylakoid proteins The relative contents of certain thylakoid proteins per mg Chl were determined immunologically by western blotting. Thylakoids were solubilized in 5% SDS, 15% glycerine, 50 mM Tris /HCl (pH 6.8) and 2% mercaptoethanol at room temperature for 30 min. The polypeptides were separated by SDS-PAGE as described by Laemmli [18] and proteins were then transferred to nitrocellulose by electroblotting for 3 h at 0.4 A. After saturation with 10% milk powder in TBS buffer (pH 7.5). The first antibody in 1% gelatine was allowed to react overnight at room temperature. After washing with TBS containing 0.05% Tween-20, the secondary antibody [Anti-Rabbit IgG (whole molecule) Biotin Conjugate, Sigma] was allowed to react in 1% gelatine for 2 h. For detection of D1 protein a polyclonal antiserum against spinach D1 protein was used (kindly provided by Professor I. Ohad, Jerusalum, Israel), and the antibody against the 33 kDa protein of the watersplitting system was a gift from Dr Barbato, Padova, Italy. The densitometry analysis of western blots was performed with a Bio-Image analyzer (Millipore Corporation, Michigan, USA).
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3. Results 3.1. Changes in pigments Detailed analyses of photosynthetic pigments in mature and young leaves of grapevine are given in Table 1. The Chl a/b content per unit area in young leaves was about 51% of that in mature leaves, but the Chl a/b ratio was not significantly different between the two leaf types (Table 1), indicating that in the young leaves the photosynthetic apparatus fully developed. In contrast, the young leaves also exhibited an increased ratio of total carotenoids and of xanthophyll cycle pigments to Chl a/b (Table 1). The young leaves absorbed about 9% less of the incident light than mature leaves (Table 1). 3.2. Changes in Chl fluorescence In order to compare the susceptibility to photoinhibition between mature and young leaves, leaf samples were subjected to high light exposure in a controlledenvironment chamber, followed by a recovery incubated in dark for 60 min. Fig. 1 illustrates that young leaves responded more sensitively to high light than mature leaves, as indicated by the more pronounced decrease in Fv/Fm ratios of young leaves. Recovery during dark incubation was remarkably fast, so that after 45 min only small differences in Fv/Fm ratios between mature and young leaves remained (Fig. 2). The treatment with high light for 60 min lead to a decline of about 40, 65% in Fv/Fm and elevation of about 21, 12% in Fo in mature and young leaves, respectively. In the subsequent dark incubation, Fo decayed with increase in Fv/Fm, and both of them could largely recover after 60 min dark (Fig. 2). 3.3. Changes in photosynthetic activities Photosynthetic electron transport activities were measured in thylakoids isolated from high light irradiated mature and young leaves (Fig. 3). The rate of PS II activity was decreased with increase of the time of high
Table 1 Photosynthetic pigments and fractional light absorbance in young and mature leaves of V. vinifera L. (mean9/S.E.; n /5) Young leaves Mature leaves Chl a/b (mmol m 2) Chl a/b ratio Total carotenoids/Chl (mmol mol 1) (V/A/Z)/Chl (mmol mol 1) Absorbance
2989/15 3.59/0.4 6019/34 1519/8.5 0.7459/0.002
5909/31 3.49/0.3 4819/28 789/4.1 0.8159/0.005
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was used instead of DCBQ in young leaves. A smaller inhibition of PS I activity was also observed in both mature and young leaves (Fig. 3). In the subsequent dark incubation, the leaves reached maximum rate of PS II activity similar to those observed in non-photoinhibited leaves (Fig. 4). 3.4. Changes in DCPIP photoreduction measurements To locate the possible site (s) of inhibition in the PS II reaction, we followed the DCPIP photoreduction supported by various exogenous electron donors used in thylakoids isolated from 60 min high light irradiated mature and young leaves. Wydrzynski and Govindjee [19] have shown that MnCl2, DPC, NH2OH and HQ could donate electrons in the PS II reaction. Fig. 5 shows the electron transport activity of PS II in the presence and absence of three of the above compounds. The PS II activity was reduced to about 35 and 66% in mature and young leaves, when water served as electron donor. A similar trend was also found using MnCl2 as donor in both leaves. In contrast, a significant restoration of PS II mediated DCPIP reduction was observed when NH2OH and DPC were used as electron donors in young leaves, while the PS II activity was not restored using either DPC or NH2OH in mature leaves (Fig. 5). 3.5. Changes in D1 and 33 kDa proteins by immunoblot
Fig. 1. Changes in the relative levels of fluorescence emitted as minimal fluorescence (Fo), maximum fluorescence (Fm) and the ratio of variable to maximum fluorescence (Fv/Fm) of mature and young leaves of V. vinifera L. at different time intervals. Data are given in % of untreated controls. Control values for Fo, Fm and Fv/Fm were 2.7, 12.8, 0.789 and 2.7, 14.2, 0.809 in mature and young leaves, respectively (mean9/S.E.; n /5).
light in both mature and young leaves. After 60 min, photosynthetic electron transport from H2O 0/DCBQ and H2O 0/SiMo was reduced by about 44, 13% in mature and 26, 59% in young leaves, respectively. A significant reduction of PS II activity was noticed when DCBQ was used electron acceptor in mature leaves but marginally inhibited when SiMo was used electron acceptor (Fig. 3). In contrast, a marked reduction of PS II activity was noticed when electron acceptor SiMo
Photoinhibition of PS II is known to induce breakdown of the D1 protein [4,20]. In systems without protein biosynthesis this can be seen directly as a loss in D1 protein content. In intact plant the correlation between D1 protein content and activity of PS II is more complex [21,22]. Photoinhibition induced inhibition of PS II activity in thylakoids of mature and young leaves was compared with changes in the relative contents of D1 and 33 kDa proteins as determined by western blotting (Fig. 6) followed by quantification by the Bio-Image apparatus (Fig. 6). The relative content of D1 and 33 kDa proteins decreased to 62, 9% in mature and 8, 82% in young leaves irradiated with 60 min high light, respectively. In the subsequent dark recovery, the leaves reached the original D1 in mature and 33 kDa protein content in young leaves, similar to those observed in non-photoinhibited leaves (Fig. 6). In Fig. 7, relative D1 protein contents and Fv/Fm ratios are compared after photoinhibitory treatments of mature and young leaves. In the young leaves, no significant D1 degradation could be attributed to the action of photoinhibitory light, even when Fv/Fm ratios had decreased to 62 /65% of the controls. It should be noted that the D1 protein was quantified after extended periods in dark incubation so that all D1 protein that had been inactivated during high light period could be degraded. The mature leaves showed a strong decrease
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Fig. 2. Photoinhibition and subsequent dark recovery of mature and young leaves under controlled conditions as indicated by Fo, Fm and Fv/Fm ratios. Control values for Fo, Fm and Fv/Fm were 2.7, 10.0, 0.787 and 2.7, 11.1, 0.804 in mature and young leaves, respectively (mean9/S.E.; n /5).
in D1 protein content together with the decline of Fv/ Fm ratio about 40% (Fig. 7).
4. Discussion The present results indicate that exposure of young and mature leaves to high light produces, differential loss of photosynthetic activity and potential efficiency of PS II (Fv/Fm) where the former are more sensitive to high light than the latter. In situ, the mature and young sun leaves of grapevine experienced similar exposure to full sunlight. However, the higher carotenoid content per mol Chl a/b and xanthophylls cycle pigments (Table 1) indicate a strengthened acclimation of the young leaves to excess light [23]. This probably resulted from higher light absorbance per mol Chl in the young leaves. Young leaves are similarly or even more exposed to sun than mature leaves and should not be less acclimated to high light. The characteristics of pigment composition, light absorbance and photosynthetic performance of the
grapevine leaves may at least in part explain the differences in susceptibility to photoinhibition: (a) the young leaves contained around 50% less Chl per unit leaf area than the mature ones, but light absorbance was only slightly lower in the young leaves. The Chl a/b ratio, however, was the same in the two leaf types, indicating similarly developed antenna systems but fewer photosynthetic units per unit leaf area (data not shown) in young than mature leaves, (b) young leaves exhibited much lower capacities of photosynthetic O2 evolution, which roughly corresponds to lower Chl content. Due to these two factors, the same light exposure would result in a much higher fraction of excess light and higher average Chl excitation in the young leaves. The decline in Fv/Fm, used here as a convenient measure of photoinhibition, indicates a reduction in potential PS II efficiency. In many studies, a close correlation of the Fv/Fm ratio with the quantum yield of photosynthetic O2 evolution or CO2 assimilation under light limiting conditions has been reported [8,24,25]. The reduction of Fv/Fm in high light irradiated young and
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Fig. 3. Changes in the rates of whole chain (H2O0/MV), PS II (H2O0/DCBQ; H2O0/SiMo) and PS I (DCPIPH2 0/MV) electron transport activities in thylakoids isolated from high light irradiated mature and young leaves of V. vinifera L. at different time intervals. The 100% values are [mmol (O2) mg 1 Chl h 1]: DCPIPH2 0/MV 396, 390; H2O0/DCBQ 168, 144; H2O0/SiMo 102, 70; H2O 0/MV 139, 96 for thylakoids isolated from mature and young leaves, respectively (mean9/S.E.; n /5).
mature leaves was mainly caused by a decline of Fm and increase of Fo, respectively. It has been proposed that an increase of Fo may be induced by the inactivation of part of PS II reaction centers [26 /28]. Our experimental results from mature leaves are in accordance with this idea. When Fo increased in mature leaves under high light, some PS II reaction centers lost their photochemical activity as indicated by a marked decline in the photochemical efficiency of PS II (Fv/Fm). BolharNordenkampf et al. [29] observed relatively low Fv/Fm ratios; even small changes of Fo or Fm would result in considerable changes in the Fv/Fm ratio. In the subsequent dark incubation, both young and mature leaves reached maximum PS II photochemistry efficiencies similar to those observed in non-photoinhibited leaves.
The rate of recovery agrees with other reports on photoinhibition in higher plants [8,13,30]. From analysis of electron transport activities in thylakoids isolated from high light irradiated mature leaves, the oxygen evolution was inhibited markedly when the electron acceptor used were DCBQ, but not SiMo. This is mainly due to high light induced changes on the reducing side of PS II and is due to photoinhibition. This is also supported by our Chl fluorescence studies where Fo was markedly increased [31,32]. In contrast, thylakoids isolated from young leaves, the rate of PS II activity observed with SiMo is lower than the one observed with that DCBQ. This is due to donor side being more impaired than the acceptor side of PS II. The extent of variable fluorescence (Fv) was reduced markedly with slight increase of Fo level. This is characteristic for inhibition of donor side of PS II. If the acceptor side of PS II is photoinhibited the Fo level is significantly increased [33,34]. A relationship between Fv/Fm and PS II electron transport activity in thylakoids isolated from photoinhibited leaves has also been shown [35,36]. To locate the possible site of inhibition in the PS II reaction, we followed the DCPIP photoreduction supported by various exogenous electron donors in thylakoids isolated from 60 min high light irradiated both mature and young leaves. Among the artificial electron donors tested DPC and NH2OH donates electrons directly to the reaction center of PS II [19]. Addition of DPC and NH2OH markedly restored the high light induced loss of PSII activity in young leaves. This is due to water-oxidizing system is sensitive to high light in grapevine young leaves. In contrast, the loss of PS II activity was not restored by using neither DPC nor NH2OH in mature leaves. It is clear that high light induced changes on the acceptor side of PS II in mature leaves [3,6,31,37]. Similar observations were found for field grown Schefflera arboricola leaves adapted to different light environments [38]. The loss of PS II activity could only partially be ascribed to functional inhibition of PS II since Fv/Fm was reduced by about 65 and 45% in 60 min high light irradiated young and mature leaves, respectively. We, therefore, assume that it was mainly due to loss of PS II centers on a Chl basis. This could be confirmed by the immunological determination of the PS II reaction center protein of D1 and 33 kDa protein of watersplitting complex. It is often thought that photoinhibition is a result of marked loss of D1 protein in mature leaves. So it occurs only when the rate of damage to D1 protein exceeds the rate of its repair [1,39,40]. Moreover, the fluorescence parameter Fv/Fm is considered to be a good measure of photoinhibition, and a decrease in Fv/ Fm under photoinhibitory conditions is often attributed to the loss of D1 protein. Furthermore, after high light treatment, inactive PS II reaction centers were also
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Fig. 4. Photoinhibition and subsequent dark recovery of mature and young leaves under controlled conditions as indicated by PS II, PS I and whole chain electron transport activities at different time intervals. The 100% values are [mmol (O2) mg 1 Chl h 1]: DCPIPH2 0/MV 396, 390; H2O0/ DCBQ 168, 144; H2O0/SiMo 102, 70; H2O0/MV 139, 96 for thylakoids isolated from mature and young leaves, respectively (mean9/S.E.; n /5).
Fig. 5. Effect of various exogenous electron donors on PS II activity (H2O0/DCPIP) in thylakoids isolated from 60 min high light irradiated mature and young leaves of V. vinifera L. The 100% values are [mmol (DCPIP red.) mg 1 Chl h 1]: H2O0/DCPIP 176, 130; DPC0/DCPIP 179, 158; NH2OH0/DCPIP 180, 154; MnCl2 0/ DCPIP 177, 132 for thylakoids isolated from mature and young leaves, respectively (mean9/S.E.; n /5).
found to be accumulate in mature leaves or in other leaves [41]. However, as shown by D1 protein quantification, even strong photoinhibition of young leaves does not seem to be related to loss of the D1 protein in the PS II reaction center. In contrast, in mature leaves (Fig. 7), substantial D1 degradation appeared to be coincident with photoinhibition. This confirms our hypothesis that in young leaves, similar to cold-acclimated spinach [42], the treatment with excess light the D1 protein is not susceptible to photoinactivation. We found no correlation between photoinhibition of photosynthesis and loss of the D1 protein when young leaves irradiated with high light were compared. In previous studies on the relationship between photoinhibition and loss of the D1 protein, there are reports showing both good [43] and poor [44] correlations. Studies of photoinhibition at low temperatures have also shown that photoinhibition is
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Fig. 6. Relative content of D1 and 33 kDa proteins of thylakoids isolated from high light irradiation followed by dark incubation in young and mature leaves of V. vinifera L. at different time intervals. Lane a, 0 min; lane b, 30 min high light; lane c, 60 min high light; lane d, 30 min dark; lane e, 60 min dark.
not correlated with D1 protein degradation either in vivo [45] or in vitro [46]. The extrinsic protein of 33 kDa associated with the lumenal surface of the thylakoid membranes are required for optimal functioning of the oxygen evolving machinery [47 /49]. Our immunological results indicate that the significant loss of 33 kDa protein could be one of the reasons for significant loss of O2 evolution capacity in young leaves. From the results we suggested that the high degree of photoinhibition in the young leaves indicated by a strong decrease in the Fv/Fm ratio probably reflects a dynamic regulator response of the photosynthetic system to excess absorbed light energy. The observed photoinhibition is possibly associate with some loss of productivity but might protect photosynthetic pigments and electron transport apparatus from severe destruction. The fast recovery probably does not require D1 protein synthesis and may be related to xanthophylls cycle activity, which is increased in the young leaves.
Our results also suggest that photoinactivation of PS II is not correlated at all with net loss of D1, and photoinhibition represents the formation of inactive centers [21,50,51]. In addition, we concluded that high light induced changes not only in the acceptor side of PS II (mature leaves) but also on the donor side of PS II (young leaves). Depending on the leaf age, high light acclimation probably results in differing degrees inhibition of PS II.
Acknowledgements This work was in part supported by a grant from Provincia Autonoma of Trento and National Council of Research (CNR): project ‘Analisi e Ricerche per il sistema Agri-Industriale’ sub-project ‘Prometavit’. The authors gratefully acknowledge the two anonymous reviewers for improving the manuscript.
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Fig. 7. Quantification of D1 protein and degree of photoinhibition in V. vinifera L. leaves under controlled conditions. The Fv/Fm ratios were determined as a measure of photoinhibition. Data are given in % of untreated controls (morning samples) (mean9/S.E.; n /5).
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