JXB Advance Access published online on June 11, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm085
RESEARCH PAPER |
High salt stress induces swollen prothylakoids in dark-grown wheat and alters both prolamellar body transformation and reformation after irradiation
1Department of Plant and Environmental Sciences, Göteborg University, Box 461, SE-405 30 Göteborg, Sweden
2Department of Plant Anatomy, Eötvös University, Pázmány P.s. 1/c, Budapest, H-1117 Hungary
* To whom correspondence should be addressed. E-mail: Christer.Sundqvist{at}dpes.gu.se
Received 19 January 2007; Revised 21 March 2007 Accepted 26 March 2007
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High salinity causes ion imbalance and osmotic stress in plants. Leaf sections from 8-d-old dark-grown wheat (Triticum aestivum cv. Giza 168) were exposed to high salt stress (600 mM) and the native arrangements of plastid pigments together with the ultrastructure of the plastids were studied using low-temperature fluorescence spectroscopy and transmission electron microscopy. Although plastids from salt-treated leaves had highly swollen prothylakoids (PTs) the prolamellar bodies (PLBs) were regular. Accordingly, a slight intensity decrease of the short-wavelength protochlorophyllide (Pchlide) form was observed, but no change was found in the long-wavelength Pchlide form emitting at 656 nm. After irradiation, newly formed swollen thylakoids showed traversing stromal strands. The PLB dispersal was partly inhibited and remnants of the PLBs formed an electron-dense structure, which remained after prolonged (8 h) irradiation. The difference in fluorescence emission maximum of the main chlorophyll form in salt-stressed leaves (681 nm) and in control leaves (683 nm) indicated a restrained formation of the photosynthetic apparatus. Overall chlorophyll accumulation during prolonged irradiation was inhibited. Salt-stressed leaves returned to darkness after 3 h of irradiation had, compared with the control, a reduced amount of Pchlide and reduced re-formation of regular net-like PLBs. Instead, the size of the electron-dense structures increased. This study reports, for the first time, the salt-induced swelling of PTs and reveals traversing stromal strands in newly formed thylakoids. Although the PLBs were intact and the Pchlide fluorescence emission spectra appeared normal after salt stress in darkness, plastid development to chloroplasts was highly restricted during irradiation.
Key words: Chloroplasts, electron microscopy, etioplasts, fluorescence spectrum, morphometric analysis, prolamellar bodies, prothylakoids, protochlorophyllide, salt stress
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High salinity is a crucial problem affecting plant cultivation in many parts of the world. High salt concentrations cause ion imbalance and hyperosmotic stress in plants. As a consequence, secondary stress reactions such as oxidative damage often occur (Zhu, 1997). Ionic imbalance occurs due to excessive accumulation of Na+ and Cl–, which reduces the uptake of other mineral nutrients, such as K+, Ca2+, and Mn2+ (Lutts et al., 1999). One important aspect of salt tolerance is the accumulation of compatible solutes in the cytoplasm to balance the ions accumulated in the vacuole osmotically (Inan et al., 2004). Non-tolerant plants usually do not have this capacity and the metabolic processes are disturbed by the ion imbalance and water stress reactions (Sairam and Tyagi, 2004). Taking into account the chlorotic symptoms of plants grown at high salinity conditions (Fedina et al., 2003; Meloni et al., 2003), the chlorophyll accumulation occurring in irradiated dark-grown leaves is a process suitable for the study of metabolic effects of salinity stress. In addition, the metabolic disorder allows a disclosure of mechanisms regulating chloroplast differentiation.
Dark-grown angiosperms lack chlorophyll. Instead they contain protochlorophyllide (Pchlide) which is transformed to chlorophyllide (Chlide) by the light-driven enzyme NADPH:Pchlide oxidoreductase (POR, EC. 1.3.1.33 [EC] ). The Chlide is then esterified to form chlorophyll and in continuous light the amount of chlorophyll is increased about a hundred times. Pchl(ide) is present in dark-grown plants as short-wavelength and long-wavelength spectral forms due to various molecular interactions. Thus Pchlide esters (Pchl) and Pchlide can appear as different spectral forms. The short-wavelength forms have fluorescence emission peaks between 631–643 nm (Kis-Petik et al., 1999) and are phototransformed by continuous irradiation on the time scale of minutes and hours (reviewed by Schoefs and Franck, 2003). The main long-wavelength Pchlide form has a fluorescence emission band located around 656 nm and is phototransformed even with ms light flashes.
The long-wavelength Pchlide form represents a large aggregate of the ternary complex of NADPH, Pchlide, and POR located in prolamellar bodies (PLBs, Ryberg and Dehesh, 1986; Böddi et al., 1989). By contrast, the short-wavelength Pchlide forms are suggested to be located in the prothylakoids (PTs, Böddi et al., 1989), but may also be found in the chloroplast envelope (Barthélemy et al., 2000).
After irradiation, the newly formed Chlide undergoes a spectral shift (the Shibata shift) seen as a change in the fluorescence emission maximum from 694 nm to 680 nm. The rate of the Shibata shift depends on, for example, leaf age and temperature (Henningsen and Boynton, 1969; Henningsen, 1970; Smeller et al., 2003). In parallel with the shift, the newly formed Chlide is esterified to form chlorophyll a and this process is shown to be biphasic (Domanski and Rüdiger, 2001). During continuous irradiation, a part of the newly formed Chl(ide) a is converted to chlorophyll b (Rüdiger, 2002).
The PLB forms a more or less spherical structure with a diameter of approximately 1 µm. The structural unit of the PLB is a tetrahedral 4-armed unit with six units forming a hexagon with symmetry similar to that of the carbon atoms in a diamond crystal (Gunning and Steer, 1975; Murakami et al., 1985; Williams et al., 1998). The bilayer forming the tetrahedral units is continuous throughout the PLB, separating the stroma of the etioplast from the lumen of the PLB membranes (Selstam and Widell-Wigge, 1993). At the periphery of the regular net-like PLB structure, PTs are found as flat perforated membranes. The stroma and the space between the tubules of the PLBs make up one compartment within the etioplast, while the lumen of the PTs and the lumen of the PLB tubules form another (Selstam and Widell-Wigge, 1993). Isolated PTs as well as thylakoids behave as ordinary membranes and can be more or less swollen depending on the osmotic potential of the isolation medium, whereas the PLBs do not react by swelling (Ryberg and Sundqvist, 1982; Opanasenko et al., 1999).
During illumination of dark-grown leaves, the PLBs in the plastids are rapidly dispersed and disappear within a few hours (Virgin et al., 1963; Henningsen, 1970; Minkov et al., 1988). At the same time the PTs increase in length. Some components of the PLBs are transported into the PTs, which are gradually transformed into photosynthetically active appressed and non-appressed thylakoid membranes (Virgin and Egneus, 1983). However, PLBs are re-formed whenever illuminated plants with immature chloroplasts are kept in darkness or in low light intensity, indicating the universal applicability of PLBs for chloroplast differentiation (Solymosi et al., 2004). The composition of the re-formed PLBs is, in many aspects, similar to PLBs of etiolated leaves (Minkov et al., 1988).
In a previous publication (Abdelakader et al., 2007), dark-grown leaves exposed to salt stress were shown to have a reduced chlorophyll accumulation, a delayed Shibata-shift and a retarded Pchlide resynthesis. In this work, the impact of salt stress on pigment formation was complemented with studies on the ultrastructure of the etioplasts in dark-grown plants, plastids developing during irradiation, as well as the formation of etio/chloroplasts when plants, that had been irradiated for a short time, were returned to darkness. In corresponding samples, various pigment–protein complexes from etioplasts and differentiating etio/chloroplasts were also characterized by fluorescence emission.
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Plant material and growth conditions
Wheat grains (Triticum aestivum cv. Giza 168, National Research Institute, Cairo, Egypt) were soaked in tap water for 12 h at room temperature in darkness. The grains were sown in soil and the seedlings were grown for 8 d at room temperature in darkness. In some experiments dark-grown plants were irradiated with continuous white light (Osram, L20 W/30S warm white, light intensity 50 µmol m–2 s–1) for various periods of time as indicated in the Table and Figure legends.
The primary leaves were used for measurements. The 1 cm tip section of each leaf was discarded and the next 2 cm section was used for measurements. The leaf sections were floated in Petri dishes on Hoagland's solution (Hoagland and Arnon, 1950) with or without 600 mM of salt as indicated in the Table and Figure legends. The salt was a mixture of NaCl:KCl (1:1, v:v) on a molar basis. The osmolarity values (measured with a Osmometer automatic type 13, Roebling, Berlin, Germany) were 16 mOsm for the nutrient solution and 1050 mOsm for the 600 mM salt solution. Before irradiation, the dark-grown leaves were pretreated on Hoagland's solution or on salt solutions in the dark for 1.5 h and then irradiated for the time period given in the Table and Figure legends. The handling of the dark-grown seedlings was done under dim green light.
Experimental series
Three different experimental series were performed as follows: (i) Leaf sections from 8-d-old wheat seedlings were floated in Petri-dishes on either salt-free Hoagland's solution or on Hoagland's solution with 600 mM salt in darkness for 4.5 h prior to sampling for transmission electron microscopy and low temperature (77 K) fluorescence emission measurements. (ii) Leaf sections incubated in the dark on either salt-free Hoagland's solution or on Hoagland's solution with 600 mM salt for 1.5 h were exposed to continuous white light for 3 h or 8 h and then sampled for electron microscopy and low temperature (77 K) fluorescence emission measurements. (iii) Leaf sections incubated in the dark either on salt-free Hoagland's solution or on Hoagland's solution with 600 mM salt for 1.5 h were exposed to continuous white light for 3 h and then returned to darkness for 12 h. In this experiment, some leaf sections were allowed to recover by transferring them from salt-containing solutions to salt-free Hoagland's solution at the time they were returned to darkness. After a 12 h dark period the leaf sections were sampled for electron microscopy and low temperature (77 K) fluorescence emission measurements.
Transmission electron microscopy
Leaf pieces (1x1 mm) were fixed in 2.5% glutaraldehyde at room temperature for 3 h or at 4 °C overnight. The samples were washed three times for 15 min with 70 mM Na-K phosphate buffer (pH 7.2). Samples were post-fixed for 2 h in 1% osmium tetroxide dissolved in 70 mM Na–K phosphate buffer (pH 7.2). The fixed samples were rinsed three times for 15 min with the same phosphate buffer. Samples were dehydrated in ethanol series, put in propylene oxide, and then embedded in Durcupan ACM resin (Fluka Chemie AG, Buchs, Switzerland). By means of a Reichert Jung Ultracut-E microtome (Reichert Jung AG, Vienna, Austria) ultrathin sections (70 nm) were cut with a diamond knife and mounted on grids. Uranyl acetate and Reynold's lead citrate were used for staining. The samples were examined with a Hitachi 7100 transmission electron microscope (Hitachi, Tokyo, Japan) at 75 kV accelerating voltage. More than 30 pictures were taken per treatment condition.
A morphometric analysis was performed according to the method described by Steer (1981). A 1 cm based grid on a transparent overlay was used at a magnification of about x35 000 to determine the fractional ratio of different structures within the etioplast (Mostowska, 1986). The results are mean values from about 40 etioplasts given with the standard deviation.
Fluorescence spectroscopy
Low temperature (77 K) in vivo fluorescence emission spectra were recorded using a Fluorolog 3 spectrofluorimeter (Spex Instruments S.A. Inc. New Jersey, USA). Data from the fluorimeter was retrieved over a PC interface using the GRAM/32 program (Galactic Industries Corporation, Salem, USA). The emission was measured as photon emission per unit interval of wavelength. Intact samples were placed into small cylindrical glass cuvettes and kept immersed in liquid nitrogen. The geometry of the sample holder and the cuvettes minimized the self-absorbance. The results represent the average of five repetitions of each experiment.
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Effect of salt stress on the ultrastructure of plastids in dark-grown leaves
Leaves floated for 4.5 h in darkness on nutrients (Hoagland's solution) or nutrients with 600 mM salt were sampled for electron microscopy. The electron micrographs were analysed with the morphometric method described by Steer (1981). The percentage fraction of the plastid occupied with PLBs were similar for the two samples, i.e. 20.7% and 21.9% for leaves floating on nutrients and salt, respectively (Table 1). However, the etioplast ultrastructure of the samples analysed had clear differences (Fig. 1A, B). In the salt-treated samples (Fig. 1B) the lumen of the PTs had expanded leaving a clear space between the two PT membranes. In this way the PTs appeared to be swollen. The fraction of the plastid occupied by the swollen PTs was 10% (Table 1). The corresponding PTs in nutrient-treated samples had a shape typical for etioplasts (Fig. 1A).
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The PLBs maintained intact and similar ultrastructure in the nutrient- and salt-treated samples, i.e. no swelling occurred in any of the compartments formed by the regular network of the PLB membranes. Interestingly, when the PTs between two PLBs were swollen none of the two PLBs were affected (Fig. 2). The swollen area could extend up to the PLB structures without affecting the tetrahedral units of the PLBs (Fig. 2B, C, D). In addition, the etioplast envelope remained intact without any tendency to become swollen (Figs 1, 2A).
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The fluorescence emission spectra of the salt-treated leaves had a lower intensity at 633 nm (when normalized at 656 nm), corresponding to the non-photoactive Pchlide 633 form, compared with the spectra of leaves floating on nutrients (Fig. 3). Neither the position nor the half-bandwidth values were different.
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Ultrastructural alterations in salt-stressed plastids during irradiation
After 3 h irradiation the plastids from leaves floated on nutrient solution showed a complete dispersal of the PLBs (Fig. 1C; Table 1). A large number of non-stacked thylakoid membranes were observed in the chloroplast. By contrast, plastids from leaves floated on salt solution had incomplete dispersal of PLBs and an absence of a normal formation of thylakoids (Fig. 1D; Table 1). The PLBs still covered 9.2% of the plastid and contained condensed dark electron-dense areas. These dark areas occupied 3.2% of the plastids. Many etioplasts had a less electron-dense area, in the centre of the partially transformed PLBs, surrounded by a more dense structure (Fig. 4). The central area contained vesicle-like structures indicating that the tube transformation had occurred. The dark electron-dense area had a disordered structure where, occasionally, PLB-tubuli could be recognized (Fig. 4B). The PTs/thylakoids were often swollen and, in a few cases, stromal strands could be found traversing the lumen of the swollen thylakoids (Fig. 4A). The plastids in the salt-stressed leaves had a round shape with an axial ratio, length to width (l/w), of 1.2 (Table 1). The plastids from the non-stressed control leaves, which were typically lens-shaped had an axial ratio of 1.7. The fluorescence emission spectra from the salt-treated leaves had a dominating band at 681 nm while the fluorescence emission peak from the leaves floating on nutrient solution had the band at 683 nm (Fig. 5). The fluorescing pigment was identified in both cases by HPLC as chlorophyll a and chlorophyll b (results not shown).
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Samples were also collected after 8 h of irradiation to find out if the above results represented the final stage of plastid development in the salt-stressed leaves (Fig. 6). The plastids from leaves floated on nutrients contained well-developed thylakoids with incipient grana formation (Fig. 6A; Table 1). In a number of samples starch-grains were also found (data not shown). Plastids from salt-stressed leaves had remnants of PLBs, even after 8 h irradiation (Table 1), in the form of electron-dense structures (Fig. 6B). In contrast to the salt-stressed samples irradiated for 3 h, no electron transparent central areas were found in plastids from salt-stressed leaves irradiated for 8 h. In some sections PLBs in the process of vesicle dispersal could be found (Fig. 6C) and in other sections the PLB remnants appeared as an electron-dense structure of regular narrow-spaced configuration (Fig. 6D). In a number of sections, large translucent areas were observed in close proximity to the PLB remnants (Fig. 6D). The plastids contained more thylakoids than after 3 h irradiation (Table 1) and many of them were highly swollen. The distance between the thylakoid membranes became wider after prolonged irradiation under salt stress. A number of osmiophilic globules were found, but neither grana formation nor starch grains were found. It should also be noted that the difference in the shape of the plastids between the salt-stressed and the non-stressed leaves had increased compared to 3 h of irradiation. The plastids from the salt-stressed leaves were almost circular in shape (l/w=1.2) in contrast to the lens-shaped (l/w=2.4) plastids from unstressed leaves (Table 1).
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The fluorescence emission spectra of chloroplasts of leaves incubated in nutrient solution had emission bands at 687 nm and 742 nm after 8 h irradiation (Fig. 7). The spectra of salt-treated leaves had a single band at 681 nm similar to that of leaves irradiated for 3 h.
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Re-formation of PLBs in darkness in plastids recovering from salt stress
To find out whether the salt treatment also influenced the regeneration of PLBs and different Pchlide forms, salt-stressed and nutrient treated leaves were irradiated for 3 h then incubated for 12 h in darkness. The leaves were floated on nutrients or salt-containing solutions during the whole experiment (Figs 8, 9A, B). In the spectrum from leaves floated on nutrients, both the 633 and 656 nm bands were recognized. However, in the spectrum of the salt-stressed leaves, only the 656 nm band appeared (Fig. 8). To demonstrate clearly the connection between salt stress and the lack of the 633 nm Pchlide form, previously salt-treated leaves (1.5 h preincubation in darkness and 3 h irradiation on a salt-containing solution) were transferred to nutrient solution for 12 h dark incubation. The spectrum of the leaves treated this way had both the 633 nm and 656 nm Pchlide maxima (Fig. 8) and the plastids lacked the large swollen PTs (Fig. 9C).
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Plastids, from the re-etiolated leaves floated on nutrient solution, had PLBs with a regular ultrastructure (Fig. 9A). When counting, etioplasts from dark-grown samples had, as a mean, 1.3 PLBs per plastid section. In the previously irradiated samples, the re-formation of PLBs led to an increased number, i.e. 2.8 PLBs per plastid. In the salt-treated leaves there was no such increase in the number of PLBs. The plastids from salt-stressed leaves had re-formed PLBs appearing as electron-dense structures with a regular narrow-spaced configuration (Fig. 9B, E). A large translucent area was often found in close proximity to the reformed PLBs. In addition, in a number of plastids from the leaves returned to darkness for 12 h, there were large, dark, dense formations lacking internal structure (Fig. 9D). The plastids of leaves transferred to the nutrient solution for a 12 h dark incubation were almost fully recovered. They had a delimited PLB area and lacked the dark electron-dense structures. In addition to ordinary thylakoids there was also a number of vesicles (Fig. 9C; Table 1).
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Discussion |
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The chlorophyll content is often decreased in plants grown either under conditions of increased salinity (Le Dily et al., 1993; Kahn, 2003) or treated with salt solutions (Siew and Klein, 1968; Abdelkader et al., 2007). It has recently been shown that dark-grown leaves exposed to high concentrations of salt have a reduced chlorophyll accumulation, a delayed Shibata-shift, and a retarded Pchlide resynthesis (Abdelkader et al., 2007). A co-ordinated effect of salt stress on the chlorophyll accumulation and the ultrastructural changes during the transformation of etioplasts to chloroplasts has not earlier been described.
Spectral properties of pigment–protein complexes
Pchl(ide) present in dark-grown leaves has fluorescence emission peaks at 633 nm and 656 nm (Fig. 3). The phototransformable Pchlide with fluorescence emission at 656 nm is known to be sensitive to different stress conditions leading to a decreased fluorescence ratio at 656/633 nm after prolonged stress (Bengtson et al., 1978; Le Lay et al., 2001). However, no reduction in the fluorescence at 656 nm occurred in leaves floated on salt, instead a relative decrease took place at 633 nm (Fig. 3).
The oligomers of the ternary POR complexes, fluorescing at 656 nm, are located in PLBs (Ryberg and Dehesh, 1986; Böddi et al., 1989) and this structure does not seem to be influenced by the salt treatment (Fig. 1). The short-wavelength Pchlide forms are suggested to be located in the PTs (Böddi et al., 1989) which are swollen due to the salt treatment (Figs 1, 2). If the swelling leads to a destruction of the pigment fluorescing at 633 nm, or to an increased energy transfer to the 656 nm fluorescing form, has not been deduced. Actually a conversion of the 636 nm form to the 656 nm form seems most probable and is favoured by the fact that the Pchlide fluorescing at 656 nm was the form regenerated in darkness after irradiation of salt-treated leaves (Fig. 8; Abdelkader et al., 2007). Pchlide fluorescing at 633 nm was formed first after recovery from the salt stress.
The ultrastructure of PLBs and PTs
The PLB is regarded as being a cubic phase membrane structure in direct continuation with the planar PT membrane (Selstam and Widell-Wigge, 1993). The results found here raise the question about the connection between the compartments present in the PLBs and PTs and their contact with the stroma. The lipid bilayer forming the tetrahedral basic units of the PLBs is continuous throughout the PLB, separating two systems of water-containing compartments, i.e. the wider stroma conduit and the inner lumen of the PLB membranes (Gunning and Steer, 1975; Murakami et al., 1985; Selstam and Widell-Wigge, 1993). The stroma in the wider PLB compartment is continuous with the rest of the etioplast stroma. This renders the diffusion of ions possible into the PLBs. The area inside the PLB membranes, the PLB lumen, corresponding to the lumen of PTs and thylakoids, form an inner space. The possible connection between the PLB lumen and the lumen of the PTs is discussed below.
Electron micrographs from salt-treated leaves showed etioplasts with normal PLBs but a considerable swelling of the PTs (Figs 1, 2). A similar resistance of the PLBs to salt-induced swelling was also found by Mitsuya et al. (2000). This implies that the lumen of the PLBs and the lumen of the PTs are separate compartments in as much as ions can not move freely between the two compartments. It could be reasoned that the three-dimensional structure of the PLB could hinder the swelling of the PLB-tubules. However, in such a case, swelling would be possible in the peripheral part of the PLBs but such tendency was not observed (Fig. 2). If one presumes that the PLB cubic membranes allow free diffusion of ions and that a free connection between the PT and PLB lumen exists then the substance causing the swelling of the PTs should have leaked out this way and the swelling of the PTs would have been suppressed. Furthermore, the perforations found in young and newly formed PTs (Henningsen and Boynton, 1969, 1970; Klein and Schiff, 1972) are obviously not open perforations into the PT lumen in 7-d-old etioplast as substances otherwise should have leaked back into the stroma. The perforations seem to facilitate a stroma connection through the PTs/young thylakoids. When the thylakoids became swollen the stroma could be stretched and form strands traversing the thylakoids over a considerable distance (Fig. 4). The strands are possibly kept together by surface proteins since there was no indication of membranes around the strands (Fig 4C). The perforations and the stroma strands going through will increase the contact area between the PT lumen and the stroma, which might be important for the exchange of proteins. The salt stress could cause a delay in the closure of the perforations possibly coupled to the delayed dispersal of the PLBs.
However, the existence of perforations in the PTs and newly formed thylakoids raise the question whether this can be considered an additional route for protein import to the lumen. Nucleus-encoded luminal proteins contain a specific bipartite sequence that targets them first into the plastid and thereafter to the lumen (Jarvis and Robinson, 2004). Possibly the luminal import step is facilitated by perforations in PTs/thylakoids and not restricted by the presence of the specific luminal target sequence. Also plastid-encoded luminal proteins may get transported into the lumen of PTs/thylakoids more easily by the occurrence of perforations. This kind of facilitated import might occur as long as the perforations are open, i.e. in young etioplasts and during early greening. In addition, in contrast to the normal thylakoid import of non-folded proteins, the observed thylakoid import of folded protein domains (Clark and Theg, 1997) may be assisted by this route.
The formation of swollen PTs
Are the translucent areas formed in connection with the PTs really swollen PTs? Some facts point in this direction. First, a single membrane borders the translucent area (Fig. 2). Second, PTs swollen to different degrees could be found in the same plastid (Figs 1, 2). Thus, the translucent area forms through an increase of the lumen of the PTs. This is similar to the swelling of thylakoids in salt-treated green plants (Salama et al., 1994; Lechno et al., 1997; Mitsuya et al., 2000; Sam et al., 2003). Such swelling can be found to influence various parts of the thylakoid system, for example, stroma thylakoids (Lechno et al., 1997) or the grana region (Salama et al., 1994; Hasan et al., 2005). Often the most typical feature is a disorientation of thylakoids (Salama et al., 1994).
The process behind the formation of swollen PTs is not clear. As the sodium, potassium, and chloride ion concentrations are strongly increased in the solution surrounding the leaves, it is tempting to speculate that these ions are taken up by the leaf cells, imported into the plastids, accumulating inside the PTs and attracting water. As there is no swelling of the envelope membrane (Figs 1, 6) diffusion and uptake through ion channels in the plastid envelope increase the stromal content of ions (Heiber et al., 1995). Several types of ion channels have been found in the thylakoids (Tester and Blatt, 1989; Pottosin and Schönknecht, 1995, 1996). Ion channels have not been identified in the PTs, but they can be supposed to work in a similar manner as in the thylakoids. Isolated PTs behave as regular membranes and can be swollen depending on the osmotic potential of the isolation medium, whereas isolated PLBs do not react osmotically (Ryberg and Sundqvist, 1982).
Ultrastructural changes after irradiation
Irradiation of dark-grown leaves leads to the phototransformation of Pchlide to Chlide and induces a morphological change of the plastid ultrastructure. The PLBs lose their regular appearance and tubuli are transformed and dispersed (Fig. 1C, D; Henningsen and Boynton, 1969). However, in salt-treated leaves the dispersal after irradiation is inhibited. The areas corresponding to PLBs are still dense and heavily stained. In a similar manner, during reaccumulation of Pchlide in darkness after irradiation, the reforming PLBs are dense and heavily stained indicating a strong influence of the salt treatment on the tube-transformation process, the dispersal and the re-formation of PLBs. It was found earlier that high concentrations of ions could have a condensing effect on the PLBs in darkness (Grevby et al., 1989; Widell-Wigge and Selstam, 1990), similar to that obtained here after irradiation.
The tube transformation and the dispersal of the PLB membranes are multistep processes partly reflected in the spectral changes that Pchlide and Chlide undergo when forming chlorophyll. A lipid phase transition occurs (Klement et al., 2000) together with a conformational change (Akoyunoglou and Michalopoulos, 1971; Wiktorsson et al., 1993) and a disaggregation (Böddi et al., 1990) of POR-aggregates. Fluorescence spectroscopy at high pressure showed that the blue shift (Shibata shift) can proceed without protein disaggregation, but that the lipid phase transition is important (Smeller et al., 2003). The Shibata shift can occur in the salt-treated leaves with a reduced rate (Abdelkader et al., 2007). At the same time the tube transformation and the dispersal of the PLBs are restrained, which also points to the importance of the lipid phase transition for the ultrastructural transformation.
The irradiated salt-treated leaves contained PLBs with electron-dense membrane residues, or in some plastids, centrally a less electron-dense area (Fig. 4). This lighter area is probably the consequence of a reduced rate in transformation and dispersal of the PLBs. This is also in accordance with a delayed Shibata shift after irradiation (Abdelkader et al., 2007). Furthermore, this light area was never found after 8 h irradiation, when much of the electron-dense structure was still present. The highly electron-dense areas with or without structure (Fig. 9D, E) had some resemblance to ferritin. However, an EELS test for Fe3+ carried out as in Solymosi et al. (2004) gave negative results and thus ferritin can be ruled out (data not shown).
PLB re-formation in leaves returned to darkness
Returning leaves floated on nutrients to darkness for 12 h resulted in a reformation of PLBs (Fig. 9). However, the plastids from salt-stressed leaves showed the presence of electron-dense structures with a regular narrow-spaced configuration (Fig. 9B, E). The fluorescence spectra showed the presence of reformed Pchlide fluorescing at 656 nm in spite of the fact that no normal PLBs had formed. This indicates that other factors than the ternary complex of NADPH, Pchlide, and POR is necessary to form normal PLBs. Overall, it seems as if the lipid metabolism is hampered by the salt treatment since the membranes, not properly dispersed, could not re-form. The electron-dense structures were not present in the dark-grown material but were formed in connection with irradiation. It is well-known that salt treatment, together with light, creates oxidative stress in plants (Menezes-Benavente et al., 2004; Katsuhara et al., 2005). The oxidative stress can be induced in the chloroplasts and leads to a disorganization of the thylakoids and the increased formation of plastoglobuli (Hernández et al., 1995). An increased lipid peroxidation (Elkahoui et al., 2005; Katsuhara et al., 2005) and thus a decreased reuse of the PLB lipids for formation of thylakoids can also be the explanation for the formation of the electron-dense areas obtained here (Figs 6, 9).
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A relative decrease of the PT-localized short-wavelength Pchlide form is correlated with swelling of PT during salt stress. Newly formed swollen thylakoids have traversing stromal strands suggested to participate in an enhanced protein import into the thylakoid lumen necessary for young developing plastids. The PLBs are unaltered by the salt stress and have an intact fully phototransformable ternary NADPH–Pchlide–POR complex fluorescing at 656 nm. The fact that PTs but not PLBs are influenced by the salt treatment in darkness emphasizes the difference between the PLB and PT membrane structures. The hampered dispersal of the PLBs and reduced formation of thylakoids lead to an inability of the plastids to accumulate chlorophyll during irradiation. This is coupled with the formation of dark osmiophilic structures replacing the PLBs during irradiation, indicating an obstructed lipid rearrangement in the salt-stressed leaves. An inhibiting effect of salt stress on chlorophyll formation in greening dark-grown leaves is consistent with the reduced chlorophyll content found in salt-stressed leaves under otherwise natural growing conditions.
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Acknowledgements |
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The authors thank Zoltán Kristóf for running the EELS test and Csilla Jónás for her assistance with the electron microscopic sample preparation. Support from the Hungarian Scientific Research Fund (KS, BB, grant OTKA T038003) and from the Swedish Scientific Research Council (CS) and the Swedish Research Council FORMAS (HA) is greatly appreciated.
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Abbreviations |
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Chlide, chlorophyllide; Chl(ide), chlorophyll and chlorophyllide; Pchlide, protochlorophyllide; Pchl(ide), protochlorophyll and protochlorophyllide; PLB, prolamellar body; POR, NADPH:protochlorophyllide oxidoreductase; PT, prothylakoid.
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