Cypress canker induced inhibition of photosynthesis in field grown cypress (Cupressus sempervirens L.) needles

Physiological and Molecular Plant Pathology 67 (2005) 33–39 www.elsevier.com/locate/pmpp

Cypress canker induced inhibition of photosynthesis in field grown cypress (Cupressus sempervirens L.) needles K. Muthuchelian a,b,*, N. La Porta a, M. Bertamini a, N. Nedunchezhian b b

a Istituto Agrario di San Michele all’ Adige, 38010 San Michele all’ Adige, Italy School of Energy, Centre for Biodiversity and Forest Studies, Environment and Natural Resources, Madurai Kamaraj University, Madurai 625 021, India

Accepted 3 August 2005

Abstract Investigations were carried out to envisage the effect of the cypress canker infection on some features of the thylakoids from field grown cypress (Cupressus sempervirens) needles. Changes in photosynthetic pigments, soluble proteins, soluble starch, starch, ribulose-1,5-bisphosphate carboxylase, nitrate reductase, photosynthetic activities and thylakoid membrane proteins were investigated. The level of total chlorophyll and carotenoids were markedly reduced in cypress canker-infected needles. Similar results were also observed for soluble proteins, nitrate reductase and ribulose-1,5-bisphosphate carboxylase activity. In contrast, the content of soluble starch and sugar were increased in infected needles. In isolated thylakoids, cypress canker infection caused marked inhibition of whole chain and photosystem II activity while the inhibition of photosystem I activity was only marginal. The artificial exogenous electron donors, diphenyl carbazide and hydroxylamine significantly restored the loss of photosystem II activity in infected needles. The same results were obtained when Fv/Fm was evaluated by chlorophyll fluorescence measurements. The marked loss of photosystem II activity in infected needles could be due to the loss of 47, 43, 33, 28, 25, 23, 17 and 15 kDa polypeptides. It is concluded that cypress canker infection inactivates the donor side of photosystem II. This conclusion was confirmed by immunological studies showing that the content of the 33 kDa protein of the water-splitting complex was diminished significantly in infected needles. q 2005 Elsevier Ltd. All rights reserved. Keywords: Donor side; Electron transport; Fluorescence; Nitrate reductase

1. Introduction Canker disease is now occurring as serious epidemics in the Mediterranean area. Seiridium cardinale is presently associated with canker diseases of cypress trees. The disease continued to

Abbreviations CC, cypress canker; DCBQ, 2,6-dichloro-p-benzoquinone; DCMU, K3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCPIP, 2,6-dichlorophenol indophenol; DPC, diphenyl carbazide; DTT, dithiothreitol; EDTA, ethylene diamine tetra aceticacid; Fo, minimal fluorescence; Fm, maximum fluorescence; kDa, kiloDalton; LHCP, light-harvesting chlorophyll protein; MV, methyl viologen; PMSF, phenylmethylsulfonyl fluoride; PPFD, photosynthetic photon flux density; PS, photosystem; SDS-PAGE, sodium dodecylsulphate– polyacrylamide gel electrophoresis; SiMo, silicomolybdate; TBS, Tris-buffered saline.. * Corresponding author. Address: Centre for Biodiversity and Forest Studies, School of Energy, Environment and Natural Resources, Madurai Kamaraj University, Madurai 625 021, India. Tel.: C91 452 2458020; fax: C91 452 2459139. E-mail address: [email protected] (K. Muthuchelian). 0885-5765/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2005.08.007

spread epidemically causing serious and visible damage in forests, nurseries and ornamental plantations in all the Mediterranean countries. The most damaged areas resulted in Tuscany (Italy), Rhone Valley (France), Peloponnese and Euboia Island (Greece), with a disease incidence ranking from 50 to 90% [10]. Infection by cypress canker of susceptible host trees induces both local and systemic symptoms. The first type of symptoms is represented by lesions on the bark of branches or stem around the site of infection, which progressively evolve in a canker formation. Commonly, sectors of the tree on the side of the canker decline and die. The inner tissues of the lesion show a cardinal red discoloration and a necrotic browning. Often a flow of resin bleeds from cracks or fissures formed on the cankers. Outgrowths of bark tissues, histological abnormalities and plant cell necrosis may occur in the adjacencies of the tissues invaded by the pathogen [30]. These phenomena are generally interpreted as host defense reactions intended to limit the pathogens advance [31]. It has been demonstrated that low concentrations of certain secondary metabolites of Seiridium (seiridin and sericardines B and C) stimulate growth of cypress

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tissues and cells in vitro and produce hypertrophic reactions if injected into the stem of susceptible cypress seedlings [34]; however, no information on the effect of such metabolites on tissues of naturally infected hosts is available. It is most likely that phytotoxins secreted by the pathogen are involved in the development of symptoms produced at a distance from the site of infection. Actually, foliar symptoms may develop independently from a girdling effect of the cankers in parts of the foliage distal to where the fungus can be isolated. Injection of a few millilitres of a weak solution of seiridin and other minor fungal metabolites into the stem of cypress seedlings caused symptoms very reminiscent of those shown by Seiridium-infected seedlings, i.e., chlorosis or reddening of foliage followed by die-back of twigs, branches or apical parts. However, although traces of seiridin have been detected in cankered tissues of cypress trees naturally infected by S. cardinale, no evidence is available so far for the occurrence of proper concentrations of fungal toxins at the initial stages of the disease, and neither a translocation of toxins from cankers to symptomatic parts of the tree has been monitored [34]. More is known about the involvement of phytotoxins in pathogenesis of S. cupressi canker disease, where a major toxin, cyclopaldic acid [14], not only has been shown to reproduce the systemic symptoms of the disease, but it has been detected in physiological concentrations in tissues of susceptible host seedlings artificially inoculated with the pathogen [34,35] using a polyclonal antibody developed by Del Sorbo [11]. Systemic infection of plant pathogens changes the physiology of photosynthesis, chloroplast number, ultra structure, chlorophyll metabolism and PSII [2,5,6,15–17,24,33]. During the last 10 years a severe outbreak of the disease has been observed in several cypress-growing areas of Tuskany and Trentino Alto Adige, Italy. It is largely unknown how S. cardinale interacts with its host plants and a lack of the knowledge of its physiology, biochemistry and molecular biology effects in host plants [10]. To our knowledge, the importance of S. cardinale infection in cypress needles has not been evaluated to date in the field. The aim of the present investigation was to test the hypothesis that the cypress canker infection induced effects on photosynthesis could be related to potential quantum yield, electron transport activities, RuBP case and thylakoid membrane polypeptides. For this purpose, the effect of cypress canker infection on PSII efficiency and thylakoid membrane proteins was studied in field-grown cypress canker infected cypress needles.

2. Materials and methods 2.1. Plant materials The cypress (Cupressus sempervirens L.) needles used in this study were taken from naturally cypress canker (S. cardinale) infected field grown plants located in Istituto

Agrario di San Michele all’Adige, San Michele all’Adige, Italy. In order to simplify the experimental procedure, we proceeded to classify control and infected needle samples into two groups according to their cardinal red discoloration and their chlorophyll content. Needles under infection (infected) consisted of samples with a Chl concentration below 10 nmol Chl cmK2; leaves above 35 nmol Chl cmK2 and unaffected leaves were classified as control (healthy control). 2.2. Pigment, starch and sugar analysis Chlorophyll (Chl) concentration was estimated using the SPAD-502, Minolta system, which was calibrated against total chlorophyll measured by extraction. Chl was extracted with 100% acetone from liquid N2 frozen needles and stored at K208C. Chl and carotenoids (Car) were analyzed spectrophotometrically according to the method of Lichtenthaler [21]. Total sugar was thoroughly extracted with boiling 80% ethanol and estimated by the anthrone reagent method [12]. Soluble starch was extracted and its concentration determined following the method of McCready [25]. 2.3. Modulated Chl fluorescence All measurements of Chl fluorescence were performed with a portable PAM-210 fluorometer (Walz, Effeltrich, Germany). Before each measurement, the sample leaf was dark-adapted for 30 min with leaf-clips provided by Walz. The angle and distance from the leaf surface to the end of the optic fiber cable were kept constant during the experiments. To determine the initial fluorescence Fo in control or in S. cardinale infected needles, the weak measuring light was turned on and Fo was recorded. Then the leaf sample was exposed to a 0.1 s saturated flash of approximately 6000 mmol mK2 sK1 to obtain the maximal fluorescence yield, Fm. The ratio of variable to maximal fluorescence, Fv/Fm, was calculated automatically according to Fo and Fm measured [Fv/FmZ(FmKFo)/Fm]. All measurements of Fm were performed with the measuring beam set to a frequency of 600 Hz, whereas, all measurements of Fm were performed with saturating flash automatically switching to 20 kHz. 2.4. Activities of electron transport Thylakoid membranes were isolated from the needles as described Berthhold [8]. Oxygen evolution [H2O/DCBQ; H2O/SiMo (PSII activity)] or uptake [DCPIPH2/MV (PS1 activity)] was measured following the method of Nedunchezhian [29] with a Clark-type electrode (Hansatech, UK) fitted with a circulating water jacket at 278C. Actinic light from a slide projector placed on the side of the electrode chamber was filtered through 9.5 cm of water. The light intensity was 1100 mmol mK2 sK1 at the surface of the water bath cell. Thylakoid membranes were suspended at 10 mg Chl mlK1 in the assay medium containing 20 mM Tris–HCl (pH 7.5), 10 mM NaCl, 5 mM MgCl2, 5 mM NH4Cl and 100 mM

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sucrose supplemented with 500 mM DCBQ and 200 mM SiMo for oxygen evolution (PS II) and 1 mM MV, 2 mM ascorbate, 5 mM DCMU, 1 mM sodium azide and 100 mM DCPIP for uptake (PSI). The rate of whole chain electron transport (H2O/MV) in isolated thylakoids was measured as described by Armond [3]. Thylakoid membranes were suspended at 10 mg Chl mlK1 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 1 mM MV and 1 mM sodium azide.

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Tris–HCl, pH 7.8 containing 15 mM MgCl2, 1 mM EDTA, 10 mM 2-mercaptoethanol, 10 mM PMSF in the presence of liquid nitrogen. Homogenates were filtered through nylon cloth. The extract was clarified by centrifugation at 11,000 rpm for 10 min. The clear supernatant was decanted slowly and used as the soluble proteins. The Bio-rad protein assay (BioRad Laboratories, Richmond, CA) based on Coomassie Blue was used to determine total soluble protein [9] using bovine serum albumin as the standard. 2.9. SDS-PAGE

2.5. DCPIP photoreduction The rate of DCPIP photoreduction was determined by following the decrease in absorbance at 590 nm using a Hitachi 557 spectrophotometer. The reaction mixture contained 20 mM Tris–HCl, pH 7.5, 5 mm MgCl2, 10 mM NaCl, 100 mM sucrose, 100 mm DCPIP and thylakoid membranes equivalent to 20 mg of Chl. Electron donation to the oxidizing side of PSII was measured in the presence 5 mM MnCl2, 0.5 mM DPC and 5 mM NH2OH as electron donors. 2.6. Extraction and assay of RuBP case activity Needles were cut into small pieces and homogenized in a grinding medium of 50 mM Tris–HCl, pH 7.8, 10 mM MgCl2, 5 mM DTT and 0.25 mM EDTA. The extract was clarified by centrifugation at 10,000 rpm for 10 min. The clear supernatant was decanted slowly and used as the source of RuBP case. All these steps are from the method of Nedunchezhian and Kulandaivelu [28]. The incubation mixture contained 50 mM DTT and 10 mM NaH14CO2 (9.25 kBq/mmol) in a total volume of 2.0 ml. The reaction mixture was placed in pyrex tubes and flushing with N2 for 3 min, the tubes were sealed with serum caps and gently shaken in a water bath at 328C for 3 min. Aliquots of 0.2 ml of the enzyme extract were then injected through the serum cap into the mixture to initiate the reaction. After 3 min at 328C injecting 0.2 ml of 6 M glacial acetic acid stopped the reaction. The known aliquots were transferred to Whatman No. 3 filter discs, dried under infrared lamp, and the radioactivity was determined using a Packard model 2425 liquid scintillation counter. 2.7. Nitrate reductase activity Needles (100 mg) was suspended in a glass vial containing 5 ml of the assay medium consisting of 100 mM KH2PO4– KOH, pH 7.0, 100 mM KNO3, 1% (v/v) n-propanol. The vial was sealed and incubated in the dark at room temperature at 278C for 60 min. Suitable aliquots of the assay medium were removed for nitrate analysis. The amount of nitrate formed was expressed as mmol NOK 2 formed/g fresh mass/h [18]. 2.8. Soluble proteins content Total soluble proteins was extracted by grinding needles (0.3–0.5 g fresh weight) in a mortar with 6 ml of 100 mM

Thylakoids membrane proteins were separated using the polyacrylamide gel system of Laemmli [20], with the following modifications. Gels consisted of a 12–18% gradient of polyacrylamide containing 4 M urea. Samples were solubilized at 208C for 5 min in 2% (w/v) SDS, 60 mM DTT and 8% sucrose using SDS–Chl ratio of 20:1. Electrophoresis was performed at 208C with constant current of 5 mA. Gels were stained in methanol–acetic acid–water (4:1:5, v/v/v) containing 0.1% (w/v) coomassie brilliant blue R and destained in methanol–acetic acid–water (4:1:5, v/v/v). Thylakoid membrane protein was estimated according to the method of Lowry et al. [22]. 2.10. Immunological determination of thylakoid membrane proteins The relative contents of certain thylakoid proteins per milligram 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 above 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 [AntiRabbit 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 (kindly provided by Prof. I. Ohad, Jerusalum, Israel) and the antibody against the 33 kDa protein of the water-splitting system was a gift from Dr Barbato, Padova, Italy. The densitometry analysis of western blots was performed with a Bio-Image analyser (Millipore Corporation, Michigan, USA). 3. Results 3.1. Changes in pigments and metabolic contents The changes in the level of total chlorophyll (Chl) and carotenoids (Car) in healthy control and cypress canker infected needles are shown in Table 1. When determined on the basis of unit fresh weight, the total Chl and Car concentrations in infected needles were significantly reduced

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Table 1 Changes of photosynthetic pigments, soluble proteins, RuBP case and nitrate reductase activity in healthy control and cypress canker infected cypress needles Parameters Chl a (mg g fresh weight) Chl b (mg gK1 fresh weight) Total Chl (aCb) (mg gK1 fresh weight) Carotenoids (mg gK1 fresh weight) Chl a/b ratio Car/Chl ratio Soluble proteins (mg gK1 fresh weight) Soluble sugars (mg gK1 fresh weight) Soluble starch (mg gK1 fresh weight) CO2 fixation (mmol (CO2) mgK1 (pro.) hK1) RuBP case activity (mmol (CO2) mgK1 (pro.) hK1) Nitrate reductase (mmol (NO2) mgK1 (pro.) hK1) K1

Healthy control

Infected

1.354G0.12 0.653G0.02 2.007G0.15 0.600G0.02 2.07G0.11 0.30G0.01 43.93G3.9 28.40G1.8 14.18G1.2 52.38G3.7 44.8G3.4 0.456G0.02

0.621G0.03 0.361G0.01 0.983G0.04 (K51) 0.428G0.02 (K29) 1.72G0.06 0.44G0.01 29.48G1.7 (K32) 38.34G2.3 (C35) 19.28G1.7 (C36) 31.43G2.5 (K40) 15.36G1.1 (K66) 0.137G0.01 (K69)

Values in parentheses are percent reduction and increase with reference to healthy controls. MeanGSE (nZ5).

by 51 and 29%, respectively. The Chl a/b ratio was markedly decreased in infected needles. In contrast to this, Car/Chl ratio was increased in infected needles. The content of starch and sugar increases in cypress canker infected needles (Table 1). 3.2. Changes in Chl fluorescence and photosynthetic activities Photosynthesis in cypress canker infected needles was examined by carrying out in vivo studies of Chl fluorescence. The photochemical efficiency measured as the Fv/Fm ratio is 0.832 in control and 0.471 in infected needles (Table 2). The level of Fv was three times higher in healthy control needles compared to infected leaves and Fv/Fm ratio was significantly decreased in infected needles without changing the level of Fo. Decrease in Fv/Fm ratio in needles usually reflects altered rates of electron transport through PSII and/or PSI. To confirm such alterations, an in vitro analysis of electron transport in these thylakoid membranes was undertaken. Photosynthetic electron transport from DCPIPH2/MV (PSI) was reduced by 9% in infected needles (Fig. 1). The PSII mediated electron transport activity was measured in infected needles, photosynthetic electron transport from H2O/DCBQ and H2O/SiMo was reduced by about 11 and 61% in infected needles, respectively (Fig. 1). A similar trend was also noticed for whole chain electron transport (H2O/MV) activity (Fig. 1).

3.3. Changes in DCPIP photoreduction measurements To locate the possible site of inhibition in the PSII reaction, we followed the DCPIP reduction supported by using various exogenous electron donors in thylakoids of healthy control and infected needles. Wydrzynski and Govindjee [39] have shown that MnCl2, DPC, NH2OH and HQ could donate the electrons to the PSII reaction. Fig. 2 shows the electron transport activity of PSII in the presence and absence of three of the above compounds. PSII activity was reduced to about 60% in infected needles, when water served as electron donor (Fig. 2). A similar trend was also found using MnCl2 as donor. In contrast, a significant restoration of PSII mediated DCPIP reduction was observed when NH2OH and DPC were used as electron donor in infected needles (Fig. 2). 3.4. Changes in thylakoid membrane proteins Since the changes in photosynthetic electron transport activities could be caused primarily by the changes or reorganization of thylakoid components, the thylakoid polypeptide profiles of healthy control and infected needles were analyzed by SDS-PAGE. A comparison of thylakoid polypeptides of cypress canker infected needles with those of

Table 2 Changes in the relative levels of fluorescence emitted as minimal fluorescence (Fo), variable fluorescence (Fv) and the ratio of variable to maximum fluorescence (Fv/Fm) in healthy control and cypress canker infected cypress needles

F0 Fv Fm Fv/Fm Fv/F0

Healthy control

Infected

0.242G0.01 1.202G0.06 1.444G0.08 0.832G0.04 4.96G0.16

0.245G0.01 0.217G0.01 (81) 0.460G0.02 0.471G0.02 (43) 0.89G0.04 (82)

Values in parentheses are percent reduction with reference to healthy controls. MeanGSE (nZ5)

Fig. 1. Changes in the rates of whole chain (H2O/MV), PSII (H2O/DCBQ; H2O/SiMo) and PSI (DCPIPH2/MV) electron transport activities in thylakoids isolated from healthy control and cypress canker infected needles. MeanGSE (nZ5).

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Fig. 2. Effect of various exogenous electron donors on PSII activity (H2O/ DCPIP) in thylakoids isolated from healthy control and cypress canker infected needles. MeanGSE (nZ5).

the respective healthy control indicates a decrease in the amount of 47, 43, 33, 28-25, 23, 17 and 15 kDa polypeptides (Fig. 3)

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Fig. 4. Degradation of the D1 and 33 kDa proteins in thylakoids of healthy control and infected needles. Lane A, healthy control; lane B, cypress canker infected. Each lane was loaded to equal amounts of Chl (5 mg). Histogram: BioImage densitometrical evaluation. Inset: Western-blot.

D1 protein was negligible (6%), that of 33 kDa protein was significant (90%) in infected needles. 3.6. Changes in RuBP case activity, nitrate reductase activity, soluble proteins, sugar and starch

3.5. Changes in D1 and 33 kDa proteins by immunoblot Cypress canker induced inhibition of PSII activity in cypress thylakoids was compared with changes in the relative contents of D1 and 33 kDa proteins as determined by western blotting (Fig. 4) followed by quantification by the Bio-Image apparatus (Fig. 4). While the decrease in the relative content of

Changes in the level of CO2 fixation, RuBP case and nitrate reductase activities in healthy control and infected needles are shown in Table 1. When the enzyme activity was expressed on a protein basis, a marked reduction of CO2 fixation, RuBP case and nitrate reductase activities were observed in infected needles. As much as 40, 66 and 69% reduction was noticed in infected needles (Table 1). Similar trend was also noticed for total soluble proteins. On contrary, soluble starch (36%) and sugar (35%) were accumulated more in the infected needles than in the control (Table 1). 4. Discussion

Fig. 3. Coomassie blue stained polypeptide profiles of thylakoid membranes isolated from healthy control and infected needles. Gel lanes were loaded with equal amount of protein (100 mg). A, healthy control; B, cypress canker infected.

Cypress trees affected by cypress canker showed discolored depression on stem or branches, cracking browning and necrosis of the bark, often accompanied by resin bleeding and canker formation. Affected trees showed first chlorotic or necrotic leaves and subsequently die back of twig and branches. S. cardinale associated with canker diseases of cypress, produces several phytotoxins. These compounds were identified as four :ab butenolides (sieridins), four cyclic sesquiterpenes (siericardines), one 14-marolide (seiricuprolide) and one aromatic ortho-dialdehyde (cyclopaldic acid). Sieridin and cyclopaldic acid caused losses of electrolytes from shoot tissues and to a greater extent from shoots of C. sempervirens. The type of damage caused by individual toxins suggested that their mechanisms of action are not clearly known. Possible factors most often investigated are the effects of cypress canker infection on phloem function, carbohydrate translocation, and carbohydrate levels in various tissues [10, 34]. Our result shows that the contents of Chl and Car were significantly decreased in cypress canker infected needles. The decrease in both Chl a and Chl b contents in infected needles

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was probably cypress canker infection enhanced the chlorophyllase activity in needles. We found an increase in Chl/Car ratio and a decrease in Chl a/b ratio in cypress canker infected needles could be due to the relatively faster decrease in Chl than Car. The content of starch and sugar increases in cypress canker infected needles. Due to infection cypress canker significantly affect the phloem and xylem tissues of the cypress trees and this could be the main reason for the poor translocation of above metabolites to the other regions of the trees. Thus sugars probably function as a compatible solute in cystosol, where they can very significantly contribute to the osmoticum adjustment. Canker induced inhibition of photosynthesis may be a consequence of an altered source–sink relationship inhibiting certain reactions of photosynthesis, rather than of ion excess directly affecting photosynthetic metabolism. The healthy control needles showed a good PSII activity, measured as the Fv/Fm ratio. The lowest Fv/Fm ratio observed in the infected needles was mainly due to decrease in variable fluorescence (Fv) without increasing the Fo level. This is characteristic for inhibition of donor side of PSII [1,38]. Similar results were observed Reinero and Beachy [33] in TMV-infected tobacco leaves. Analysis of electron transport in thylakoids isolated from infected needles showed that O2 evolution was markedly inhibited when the electron acceptor was used SiMo but not inhibited when the electron acceptor used DCBQ. This indicates that the donor side is more impaired than the acceptor side of PSII. Similar changes were observed in virus and cypress canker infection induced PSII inactivation in tobacco and grapevine plants [7,32]. Measurement of PSII mediated DCPIP reduction in the presence of various artificial exogenous electron donors acting at the oxidizing side of PSII were made to locate the possible site of cypress canker induced inhibition. Among the various electron donors DPC and NH2OH were found to be more effective in restoring PSII activity in infected needles. These results clearly indicate that cypress canker induced changes only on the oxidizing side of PSII, perhaps close to the DPC donation side. The most likely explanation for the inactivation of electron transport PSII activity is that the related protein(s) is (are) exposed at the thylakoid surface [37]. Such reduction was associated with a major decrease in the level of 33, 23 and 17 kDa polypeptides. The extrinsic proteins of 33, 23 and 17 kDa associated with the lumenal surface of the thylakoid membranes are required for optimal functioning of the oxygen evolving machinary. The three proteins are present in equimolar amounts [13,27], but it is still disputed whether one copy or two copies of each of the proteins are associated with the PSII unit [26,27]. Our results indicate that the significant loss of 33, 23 and 17 kDa polypeptides could be one of the reasons for significant loss of O2 evolution capacity in cypress canker infected needles. From the results we here concluded that cypress canker inactivates on the donor side of PSII. As shown by the corresponding western blots, a marginal decrease in the D1 protein occurs in infected needles.

The decrease in the D1 protein was accompanied by a significant decrease in the amount of 33 kDa protein of the water-splitting system, showing that the whole PSII rapidly degraded under prolonged infected conditions. A similar phenomenon has already been observed by Schuster et al. [36] under photoinhibitory conditions. These data also confirm assumptions that PSII is especially vulnerable to stress conditions [4,19]. Cypress canker infection induced not only the loss of extrinsic proteins but also a marked loss of 47 and 28– 25 kDa polypeptides in thylakoid membranes, which may be due to greater disruption of the PSII complex. Light harvesting complexes play important role in light absorption, thylakoid stacking and energy distribution and any damage to these complexes will have multiple effects on the photosynthetic system. In our experiment, a significant loss of LHCPII (28–25 kDa) polypeptides was observed. This could be a reason for the observed marked loss of PSII activity in infected needles. The content of soluble proteins was reduced markedly in infected needles. This might have been due to the decrease in synthesis of RuBP case, the major soluble protein of the leaf. The loss of leaf soluble protein in infected needles would be partially accounted for damaged chloroplasts or would be the result of inhibition of protein synthesis. The reduction in the overall photosynthetic rate correlated well with the decrease RuBP case activity in infected needles. A marked reduction of RuBP case activity was observed in infected needles. Such a reduction was due to inhibition of protein synthesis induced by cypress canker. We showed that the loss of RuBP case activity was due to a decline in the amount of available functioning RuBP case protein that was regulated at the level of transcription. The low photosynthetic rate in infected needles may also be attributed to the lower activity of photosynthetic enzymes, which include mainly RuBP case of the reductive pentose pathway [23]. Cypress canker infected needles had a relatively low nitrate reductase activity. The reduction of nitrate reductase activity in S. cardinale-infected needles might reflect a balance between synthesis and activation on one hand and degradation or inactivation on the other. The change in the intercellular pH levels due to cypress canker infection might decrease the transfer of nitrate (substrate) from a vacuolar pool to active cytoplasmic pool accessible to the enzyme. The inhibition of nitrate reductase activity might be also due to the inhibition of protein synthesis or it might have stemmed out from decreased rate of photosynthate supply in the infected needles. These results allow us to draw the following general conclusion: cypress canker infection causes non-specific, general stress responses: the Chl and photosystem processes of the infected tissues are delayed and the senescence or ageing phenomena are accelerated. Thus, the chlorotic or necrotic symptoms can be the result of Chl and Car inhibition, as well as decomposition of functioning photosynthetic apparatus formed before inhibition. In addition, the cypress canker infection induced changes on the donor side of PSII.

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Acknowledgements This work is part of the project ECOCYPRE (Ecological assessment and sustainable managements of cypress in the landscape of Trentino). It was financially supported by the Provincia Autonoma of Trento with deliberation no. 437 of Dr Nicola la Porta. The authors gratefully acknowledge the three anonymous reviewers for improving the manuscript.

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