Cuscuta europaea plastid apparatus in various developmental stages: localization of THF1 protein.

It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder. Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1) is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein in "get together" tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C. europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions of this protein in the complex relationship between C. europaea and its host.

Introduction

Parasitic plants have a specialized absorptive organ, called haustorium, which is used to penetrate through the host tissues and connect to xylem or phloem. Cuscuta (dodder) is the only parasitic genus found in the morning-glory“ family Convolvulaceae. The genus Cuscuta has been considered to be a holoparasitic angiosperm, obtaining both water and organic nutrients from its host plants. Some species like C. subinclusa D., C. gronovii Willd., C. campestris Yunck. possess thylakoids and contain both chlorophyll a and b especially in the growth tips of the seedlings.- Plastids of C. grandiflora R. et al. and C. odorata H.B.K. do not contain chlorophyll, the enzyme Rubisco and developed thylakoids. Using ultrastructural studies, Sherman et al. showed no differences between the structure of C. pentagona thylakoids in seedlings germinated and grown in the dark and seedlings grown in the light. Plastids had poorly differentiated endomembrane system with prolamellar bodies, which are prominent structures of etioplasts. It has been shown that C. reflexa Roxb. possesses a number of photosynthesis-related genes with significant homology to those found in higher plants., Immunoblots revealed chloroplast proteins associated with PSI and PSII, as well as cytochrome f and plastocyanin. Up to date literature provides information that C. europaea is more specialized for the parasitic lifestyle than other Cuscuta species, with no detectable chlorophylls, no capacity for CO2 assimilation and no thylakoids in plastids. Large deletions of photosynthesis-related genes has been documented as well.,

Several nucleus- and plastid-encoded Arabidopsis genes that can ultimately affecting thylakoid formation or chloroplast development have been identified. One of many, the thf1 gene, was found in various plant species.- Based on sequence similarity and different functions that were initially attributed to the protein, various names of the same protein do exist—THF1,, ToxA-binding protein or Psb29. Previously, it was suggested that THF1 protein is involved in thylakoid membrane differentiation, in responses to fungal and bacterial attacks and as a G-protein (GPA1) interacting partner in sugar (D-glucose) signaling pathway. Huang et al. provided evidence that the C-terminal 162 residues of THF1 interact with the constitutively active form of GPA1 (GTP-bound form of GPA1). Zhang et al. proposed a model for G protein-mediated chloroplast development via FtsH proteases, which play a critical role during chloroplast development in Arabidopsis., Immunofluorescence microscopy revealed that THF1 localizes to the stroma (soluble THF1) and to the membranes of plastids in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. Zhang et al. have postulated that the THF1-mediated regulation of chloroplast development may be achieved via G protein-dependent and/or -independent pathways. In the G protein-independent pathway, stroma- and/or thylakoid-localized THF1 controls chloroplast development through affecting the stability of FtsH protease. In the G protein-dependent pathway, the THF1 is at least in part, localized to the outer membrane of plastid and to stromules,, which appear to associate with the plasma membrane. After the binding to GPA1, this activated form can transmit signals from the plasma membrane to nuclei via second messengers and ultimately control the expression of the FtsH genes needed for chloroplast development.

Our results proved that photosynthesis is not a main process in the plastids in C. europaea haustorium. Localization of THF1 protein in plasma membrane and plasmodesmata leading us to pronounce assumption, that this protein has probably other functions, for example in sugar sensing within the dodder haustorial cells. Furthermore, result from western blot showing high levels of THF1 in doder and tobbaco tissues which are actively involved in the development of parasitic connection. To clarify the precise role of THF1 in host-parasitic interactions could be argued and needs further investigations.

Results

Plant material cultivation

Seeds of Cuscuta started to germinate approximately 10 d after inoculation to the soil around the base of 8-week-old tobacco plants, but germination was very asynchronous. After 20 d of cultivation, dodder vines attacked their host plant. Cuscuta set up flowers approximately 90 d after the host plant-parasite connection establishment.

Pigment analysis

Early developmental stages and flowers of C. europaea accumulated chlorophylls. Seven-day-old seedlings show the highest level of these photosynthetic pigments (Fig. 1A, column 2). We detected only trace amounts of the Chls (a+b) in 30-d-old dodder stems compared with young seedlings and flowers. The decrease was statistically significant (Fig. 1A). Dodder flowers and 30-d-old vines accumulate high amounts of carotenoids (Fig. 1B, columns 1, 3). The lowest level of carotenoids was identified in young dodder seedlings (Fig. 1B, column 2). The high levels of Chls (a+b) and carotenoids were observed in tobacco leaves (Fig. 1A and B, column 4). Significantly lower content of Chls (a+b) and carotenoids were observed in tobacco stems (Fig. 1A and B, column 5).

Figure 1. Concentration of Chl a, Chl b (A) and carotenoids (B) in C. europaea and N. benthamiana. (1) Cuscuta flowers, (2) 7-d-old Cuscuta seedlings, (3) 30-d-old Cuscuta stems, (4) Nicotiana leaves, (5) Nicotiana stems. FM, fresh mass. Presented data are means (± standard errors) of three independent experiments. The small letters inside the graphs (a, b, c, d) denote significant difference at p < 0.05 (Student’s t-test).

Chlorophyll a fluorescence

Maximum quantum yield of PSII (Fv/Fm), which is proportional to the quantum yield of O2 evolution, was relatively high and did not differ significantly between Cuscuta stem and flower. Effective photochemical quantum yield of PSII (ΦPSII), which measures the proportion of the light absorbed by chlorophylls associated with PSII, did not shown any significant changes between Cuscuta stem and flower. However, the values of Fv/Fm and ΦPSII were lower in comparison to the host plant, as documented by the 2-D chlorophyll fluorescence imaging system (Fig. 2).

Figure 2. Chlorophyll a fluorescence in dodder (C. europaea, f-flowers, c-stems) attached to the host (N. benthamiana, n-stems). Fv/Fm, maximum quantum yield of PSII; ΦPSII, effective photochemical quantum yield of PSII.

Plastid ultrastructure

Detailed ultrastructural study revealed differences in the plastid apparatus between young and older developmental stages of the dodder stem. In growing tips of the young dodder seedlings, chloroplasts with poorly differentiated endomembrane system are shown (Fig. 3A). Chloro-amyloplasts in the cortical cells of young dodder stems contained both thylakoids and large starch grains (Fig. 3B). Thirty-day-old stems represented advanced ontogenesis stage and their cells contained only amyloplasts with enlarged and sometimes numerous starch grains (Fig. 3C and D).

Figure 3. Ultrastructure of plastids in dodder stems. (A) Chloroplasts in growth tips, (B) chloro-amyloplasts in cortex of 7-d-old seedlings (C and D) amyloplasts in cortical cells of 30-d-old stem. Arrows denote thylakoids, stars denote starch grains. Electron microscopy. Bars: 200 nm, (A); 500 nm, (B–D).

Western blot analysis

The highest levels of THF1 protein were detected in Cuscuta stems attached to the host, Cuscuta haustorium and tobacco stems attacked by the parasite (Fig. 4A, columns 2, 4 and 5). In dodder seedlings and tobacco stem unattacked by Cuscuta we observed notable decrease in THF1 protein accumulation (Fig. 4A, columns 1and 3). Presence of RbcL and D1 proteins were proved in dodder seedlings, tobacco leaves, tobacco stems and stems attacked by dodder, although the levels of these proteins were lower in dodder seedlings (Fig. 4A, columns 1, 3, 5 and 6). LHC complex polypeptides were observed in all examined samples, the highest accumulation was in tobacco tissues. FLP protein was detected only in tobacco. We found the accumulation of the GluTR in dodder young seedlings and stems attached to the host (Fig. 4A, columns 1and 2), but it was not detected in haustorium (Fig. 4A, column 4).

Figure 4. (A) Western blot analysis of the THF1, GluTR, FLP, D1, LHCI complex and RbcL in C. europaea: (1) 7-d-old seedlings, (2) 30-d-old Cuscuta stems attached to the host, (4) haustorium and in N. benthamiana, (3) stems, (5) stems attacked by Cuscuta, and (6) leaves. Twenty-five µg proteins have been loaded. (B) Western blot analysis of the THF1 protein in C. europaea, (1) plasma membrane fraction isolated from dodder haustoria. Purity of fraction was tested using anti-LHCI antibody, (2) plastid membrane fraction isolated from chloroplasts of 7-d-old dodder seedlings, (3) plastid soluble (stromal) fraction isolated from chloroplasts of 7-d-old dodder seedlings. Purity of fractions were tested using anti-D1 and anti-RbcL antibodies. Fifteen microgram proteins have been loaded.

Using western blot analysis we detected the THF1 protein accumulation in plastid membrane and stromal fractions isolated from chloroplasts of 7-d-old seedlings (Fig. 4B, columns 2 and 3). In plasma membrane fractions originated from dodder haustorium, we confirmed lower levels of THF1 protein (Fig. 4B, column 1).

THF1 protein in situ immulocalization

Microscopical analysis and THF1 protein immulocalization were performed on C. europaea stems attached to the host plant (N. benthamiana) at the stage of dodder haustorium penetration into the phloem area of the host plant (Fig. 5). Based on immunofluorescence microscopy results we found high accumulation of THF1 protein in endophytic tissue of the haustorium and in cortical cells of the parasite. Tissues of the host plant accumulated low levels of THF1 protein (Fig. 5B and C).

Figure 5. Light microscopy and Immunolocalization of THF1 protein in C. europaea and N. benthamiana. Stem segments of C. europaea and their host were fixed and embedded in Steedman’s wax, 10 µm-thick cross sections were used for anti-THF1:IgG-FITC immunostaining. (A) C. europaea (c) and their host N. benthamiana (n) interconnected by dodder haustorium (h). (B) Overall immunofluorescence localization of the THF1 protein in stems of dodder and tobacco closely associated together. (C) Detailed view to vascular connection of the host and the parasite. Arrows denote host plant tissue, stars denote parasite cortical cells. Bars: 200 µm, (A and B); 100 µm, (C).

Subcellular localization by confocal microscopy revealed plastid-related but also plastid-unrelated (extra-plastidial) distribution of the THF1 protein. In host tobacco plants the protein was found mainly in the stroma of plastids in cells of the stem, however in parasite cells, in addition of plastid-localized protein, the plasma membrane expressed strong labeling (Fig. 6). In general, a higher accumulation of this protein was detected in dodder tissues. Chloro-amyloplasts with one starch grain in cortical cells of the host stem, as well as amyloplasts with 2 to 3 small starch grains in parasite cortex and haustorial cells were evenly distributed at the cell periphery (Fig. 6). Amyloplasts in digitate haustorial cells comprised 4 to 5 large starch grains which depressed THF1-positive stroma to margins and to the center of plastids (Fig. 6G–I). In cortex and haustorial cells of the C. europaea stems the THF1 protein was distributed in stroma and outer membrane of plastids and in the plasma membrane (Fig. 6G–I). Especially in digitate haustorial cells of the dodder, the protein was detected also in plasmodesmata (Fig. 6J-L).

Figure 6. THF1 protein immunofluorescence localization by confocal microscopy in N. benthamiana (A–C) and C. europaea (D–L) cells. (A–C) Cortical cells of N. benthamiana stem. (D–F) Cortical cells of dodder at the zone of haustorium penetration into the host plant. (G–L) Digitate cells of the endophyte primordium. Arrows denote chloro-amyloplasts in cortex cells of N. benthamiana (A–C) and plasmodesmata in digitate haustorial cells of the endophyte primordium (J–L). Stars denote starch grains in amyloplasts in digitate haustorial cells of the endophyte primordium (G–I). Bars: 100 µm, (A–Cand G–L); 200 µm, (D–F).

Discussion

The current literature provides some information about in vitro and in vivo cultivation of parasitic plants.,- It is generally known, that before inoculation of dodder seeds on the cultivation medium or in the soil, it is necessary to treat dodder seeds with concentrated H2SO4. After this step, the germination is running faster and more effectively. Lee scarified seeds of C. japonica Choisy with concentrated H2SO4 for 45 min and after washing with distilled water, seeds were placed on moist paper. Seeds of C. europaea were not able to germinate after this procedure and thus, we shortened the time period for H2SO4 treatment to 15 min, which yielded effective germination.

The genus Cuscuta is considered to be a holoparasitic angiosperm, obtaining both water and organic nutrients from its host plants. Previously, it has been shown that pre-parasitic seedlings of C. campestris, C. subinclusa and C. gronovii contain chlorophylls, enzyme Rubisco and thylakoids, especially in their growth tips., According to Haberhausen and Zetsche, C. reflexa possesses a number of photosynthesis-related genes with significant homology to those found in higher plants. Machado and Zetsche reported a very low chlorophyll content for C. reflexa {112 ± 24 [µg (g FW)−1]}. C. odorata and C. grandiflora had an amoeboid-like shape of proplastid and did not contain visible thylakoid structures. Western blotting revealed that LSU, the product of rbcL gene, was not detected in protein extracts of C. odorata and C. grandiflora. It has been stated that C. europaea appears to be the best adapted to a parasitic lifestyle with a number of deletions in photosynthesis-related genes, without development of thylakoid membranes and with no detectable levels of chlorophylls.-, However, we found out that young developmental stages of C. europaea accumulate relatively high levels of chlorophyll a and b. Significantly lower content of chlorophylls and increase in carotenoids concentration were observed only in 30-d-old dodder stems. These facts are supported by electron microscopy observations. Ultrastructural analysis has shown presence of chloroplasts in growth tips and chloro-amyloplasts in cortical cells of young dodder stems, while no chloroplasts were present in cells of older dodder stems. At points of contact with the host, the coiled dodder stems produces haustoria that penetrate host tissues and form vascular connections. Haustorial cells contained only amyloplasts with 4 to 5 starch grains. Our results shown low photochemical activity of PSII (Fig. 2) as well as low content of examined photosynthesis-related proteins (Fig. 4A, column 4). GluTR, the key regulatory enzyme in biosynthesis of 5-aminolevulinic acid, universal precursor of tetrapyrroles, is tightly controlled at transcriptional and posttranslational levels. In young dodder seedlings (7-d-old) we observed relatively high GluTR accumulation. These results are in correlation with measuring of Chls (a+b) concentrations. FLP protein, which is thought to bind to GluTR and represses its activity to prevent overproduction of Pchlide, was not detected in dodder seedlings, stems, haustoria. Photosystem I (PSI) is a membrane-bound multisubunit protein complex located in the chloroplast thylakoids. It utilizes light energy to oxidize plastocyanin or cytochrome c and to reduce ferredoxin or flavodoxin. In higher plants, green and red algae, the outer light-harvesting system associated with PSI is made up of LHCa proteins that are encoded by the cab genes and known collectively as LHCI (light-harvesting complex I). Some studies have explored the structural basis by which LHCI act as the outer light-harvesting system of PSI. LHCI polypeptide complex was observed in all examined Cuscuta and tobbaco samples, but the highest accumulation was detected in tobacco. Accumulation of D1 protein, which represents a reaction center protein of photosystem II (PSII), was observed in 7-d-old dodder seedlings and in tobacco (Fig. 4A) and never in older dodder stems and in haustoria. Results of Zhang et al. strongly suggest that THF1 and G proteins are the new regulators for FtsH protease and THF1 probably functions as a regulator for FtsH expression. The FtsH, a well-characterized family of membrane bound proteases, are required for damaged D1 protein degradation, which is a core protein of the PSII reaction center. The low activity of FtsH proteases leads to the accumulation of damaged D1 protein, and to the inhibition of photosynthesis. We can conclude that 7-d-old seedlings of C. europaea have some characteristics of photosynthetic plants, while older plants have lost this ability and are not photosynthetic.

Development of thylakoid membranes depends upon the transport of membrane vesicles from the chloroplast inner envelope and subsequent fusion of vesicles within the interior of the plastid. Several nuclear- and plastid-encoded Arabidopsis genes have been identified as they can ultimately affect thylakoid formation or chloroplast development. One of them, the thf1 gene, has been previously identified in various plant species. The Arabidopsis thf1 gene product controls specifically an important step required for leaf development, the normal organization of vesicles into mature thylakoid stacks. Disruption of the thf1 gene via T-DNA insertion results in variegated leaf patterns. Non-green sectors of variegated leaves lacking thf1 expression contain plastids that accumulate membrane vesicles on the interior, but lack organized thylakoid structures. It has been reported, that THF1 protein localizes to the outer plastid membrane, stroma, thylakoids and stromules.-,, Our results have shown presence of THF1 protein in both, parasite and host plant tissues (Fig. 4A). Confocal microscopy revealed localization of THF1 protein to outer plastid membrane and stroma of haustorial cells (Fig. 6G–L), what agree with previous observations of other authors,, but in addition to this, we observed localization of this protein also in plasma membrane and plasmodesmata (Fig. 6J–L). The western blot of haustorial plasma membrane fractions verified the presence of THF1 protein in the plasma membrane (Fig. 4B, column 1). Huang et al. predicted that THF1 protein plays a role as a G-protein (GPA1) interacting partner in sugar (D-glucose) signaling pathway. This interaction requires proximity of the plastid or its stromule with the plasma membrane. Stromules may also play a role in stress response. Wangdi et al. considered that THF1 have a role in COR signaling pathway in bacterial speck disease development.

In conclusion, localization of THF1 protein in plasma membrane and plasmodesmata leading us to pronounce assumption, that this protein has probably other functions, for example in sugar sensing within the dodder haustorial cells. Furthermore, result from western blot showing high levels of THF1 in doder and tobacco tissues, which are actively involved in the development of parasitic connection. To clarify the precise role of THF1 in host-parasitic interactions could be argued and needs further investigations.

Materials and Methods

Seeds sterilization and growth conditions

In vivo cultivated stems of parasitic dodder (Cuscuta europaea) and tobacco (Nicotiana benthamiana) as a host plant were used in our study. Seeds of C. europaea originate from the locality Ivanka pri Dunaji (2009, Slovak Republic), the N. benthamiana seeds were obtained from Gene Bank in Gatersleben, Germany. Dodder seeds were scarified by soaking in concentrated H2SO4 for 15 min, washed with distilled water and planted in soil around the base of 8-week-old tobacco plants. The plant material in soil (tobacco plants with dodder) was cultivated in the growth chamber (16/8 h photoperiod) at 23 ± 2°C, 40 µmol m−2 s−1 PAR.

Protein isolation and western blot analysis

Samples (100 mg) were ground in liquid nitrogen and suspended in protein extraction buffer [28 mM dithiotreitol, 175 mM sucrose, 28 mM Na2CO3, 10 mM EDTA, 5% (w/v) SDS, chemicals from Sigma-Aldrich] with antiprotease pill (Roche). After 30 min incubation at 70°C and 15 min centrifugation (12,100 × g), supernatant was used for determination of protein concentration using Bichinonic Acid Kit for Protein Determination (Sigma-Aldrich). Protein samples (25 μg) were separated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Millipore) using Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). For protein immunodetection, specific primary antibodies were used. Antibodies against THF1, GluTR, FLP, D1, LHCI and RbcL were purchased from Agrisera. Secondary antibody Goat Anti-Rabbit IgG (H+L)-HRP Conjugate (Bio-Rad) was used. Signal was revealed using chemiluminiscent kit Immobilon Western (Millipore).

Preparation of plasma membrane, plastid membrane and stromal fractions

The plasma membrane fraction from 6 g of dodder haustorium (4 mo-long picking) was enriched by partitioning microsomes in an aqueous dextran/polyethylene glycol two-phase system. The purified plasma membrane fraction was treated with 40 mM CHAPS {3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate} for 1 h at 4°C and centrifuged at 50,000 g for 30 min at 4°C. Purity of plasma membrane fraction was tested using anti-LHC antibody (Agrisera).

Plastids were isolated and fractionated as described previously by Inaba et al. Essentially, chloroplasts were isolated from 10 g of 7-d-old seedlings (2 mo-long picking). Isolated intact chloroplasts were separated into membrane and soluble fractions by lysis in 0.1 M Na2CO3, pH 11.5, followed by centrifugation at 200,000 g for 20 min. The pellets (membrane fraction) were directly dissolved in SDS-PAGE sample buffer. The soluble proteins were recovered by precipitation with trichloroacetic acid and dissolved into SDS-PAGE sample buffer. Purity of membrane fraction was tested using anti-RbcL antibody and purity of soluble (stroma) fraction was tested using anti-D1 antibody (Agrisera). Samples (15 μg) were separated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane using Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). THF1 protein was detected by western blot analysis using rabbit polyclonal anti-THF1 antibody (Agrisera). Secondary antibody Goat Anti-Rabbit IgG (H+L)-HRP Conjugate (Bio-Rad) was used. Signal was revealed using chemiluminiscent kit Immobilon Western (Millipore). Protein concentrations were determined according to Bichinonic Acid Kit for Protein Determination (Sigma-Aldrich).

Chlorophyll (a+b) and carotenoids were extracted with 80% (v/v) chilled acetone and spectrophotometrically (Jenway 6400) quantified: Chl a at 663.2 nm, Chl b at 646.8 nm, carotenoids at 470 nm. Concentration was calculated according to Lichtenthaler.

Measurements of chlorophyll a fluorescence

Prior to measurements, the parasitic plants with their hosts were dark-adapted for 30 min. Chlorophyll a fluorescence was measured by fluorcam FC1000-LC (Photon Systems Instruments). Minimal fluorescence (F0, < 0.1 μmol m−2 s−1 PAR) and maximal fluorescence (Fm) were measured using a saturation pulse (4,000 μmol m−2 s−1 PAR, 800-ms duration, λ = 620 nm) and maximal quantum yield of PSII (Fv/Fm) was calculated as (Fm − F0)/Fm., Then an actinic light of 100 μmol m−2 s−1 PAR (λ = 620 nm) was switched on for induction of photosynthesis and steady-state fluorescence was measured (Ft). Saturation pulses were applied in 60 sec intervals, for estimation of maximum chlorophyll fluorescence in the light-adapted state (F’m). Effective photochemical quantum yields of photosystem II (ΦPSII) were calculated according to Roháček and to Maxwell and Johnson as (F’m – Ft)/F’m.

THF1 protein in situ immunolocalization

Steedman’s wax embedding for THF1 visualization in dodder plants attached to host was performed according to Vitha et al. with some modifications: sections (10 μm-thick) were incubated for 1 h at room temperature in the primary antibody (1:200 dilution in PBS buffer: 0.14 M NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.3, chemicals from Sigma-Aldrich), washed 2 × 10 min in stabilizing buffer (SB buffer: 50 mM PIPES, 5 mM MgSO4, 5 mM EGTA, pH 6.9, chemicals from Sigma-Aldrich) and incubated overnight at RT, in the dark in secondary antibody (1:100 dilution in PBS). Antibody against THF1 protein was purchased from Agrisera. Secondary antibody Goat Anti-Rabbit IgG (whole molecule) F(ab´)fragment-FITC was purchased from Sigma-Aldrich. Sections were examined under the fluorescence microscope (Axioskop 2 plus, Zeiss), equipped with 485/20 nm exciter filter, 510 nm beamsplitter and 515 nm LP barrier filter (filter Zeiss set 16, 25) and using confocal microscope (CLSM Fluoview FV1000, Olympus) with the excitation laser line 488 nm and BA505-550 barrier emission filter.

Electron microscopy

Samples were fixed in 5% (v/v) glutaraldehyde in 0.06% (w/v) cacodylate buffer (pH 6.8) for 4 h, washed with 0.06% (w/v) cacodylate buffer (pH 6.8) 6 × 10 min and postfixed in 1% (v/v) osmium tetroxide in the same buffer overnight. Fixed specimens were dehydrated in ethanol and propyleneoxide series and embedded in Spurr medium (BIOTECH). Ultrathin sections were cut on ultramicrotome Reichert-Jung Ultracut E and observed with JEOL 2000 FX electron microscope (Jeol Ltd.).