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Phycology |
Laboratory of Plant Cytophysiology, Department of Biology and Evolution, University of Ferrara, Corso Ercole I d'Este, 32 – 44100 Ferrara, Italy; Department of Earth Sciences, University of Ferrara, Via Saragat, 1 – 44100 Ferrara, Italy
Received for publication March 6, 2007. Accepted for publication September 21, 2007.
ABSTRACT
Plants differ in their ability to tolerate salt stress. In aquatic ecosystems, it is important to know the responses of microalgae to increased salinity levels, especially considering that global warming will increase salinity levels in some regions of the Earth, e.g., Ethiopia. A green microalga, Kirchneriella sp. (Selenastraceae, Chlorophyta), isolated from freshwater Lake Awasa in the Rift Valley, Ethiopia, was cultured in media amended with 0, 0.4, 1.9, 5.9, and 19.4 g NaCl·L–1 adjusted with NaCl to five salinity levels adjusted with NaCl. Growth was monitored for 3 mo, then samples were collected for photosynthetic pigment determinations, microspectrofluorimetric analyses, and micro- and submicroscopic examinations. The best growth was found at 1.9 g NaCl·L–1. In the chloroplast, excess NaCl affected the coupling of light harvesting complex II and photosystem II (LHCII-PSII), but changes in thylakoid architecture and in the PSII assembly state allowed sufficient integrity of the photosynthetic membrane. The mucilaginous capsule around the cell probably provided partial protection against NaCl excess. On the whole, the microalga is able to acclimate to a range of NaCl concentrations, and this plasticity indicates that Kirchneriella sp. may survive future changes in water quality.
Key Words: chloroplast • Ethiopian Rift Valley • Kirchneriella • Lake Awasa • microspectrofluorimetry • photosystem II • salinity • ultrastructure
In some regions of the Earth, aquatic ecosystems may undergo increased salt concentration because of global warming. Both algae and land plants differ greatly in the ability to tolerate NaCl in the environment. In particular, shifts in salt content may change the phytoplankton communities according to the tolerance capability of the microalgae. Tolerance depends on specific metabolic adjustments, which allow the maintenance of photosynthetic performance under unfavorable conditions.
Salt stress affects basic processes of photosynthesis. Several studies have shown that photosystem II (PSII) is a major target of increased Na+, mainly due to the degradation of the D1 protein of the reaction center (RCII) and also due to alterations of the water oxidation complex (reviewed by Sudhir and Murthy, 2004
). Sudhir and coworkers (2005)
have recently shown that NaCl also induces degradation of the CP47 inner antenna of PSII, leading to impaired energy transfer to the PSII reaction center. NaCl-dependent changes in the light harvesting complex II and photosystem II (LHCII-PSII) coupling occur in the halotolerant green alga Dunaliella salina (Liu and Shen, 2006
). In plants adapted to high salt conditions (halophytes), PSII presents interesting, unique features such as high tolerance to photoinhibition (Qiu et al., 2003
) and increased thermostability (Lu et al., 2003
; Wen et al., 2005
). The photosynthetic membranes of nonhalophytes also can acclimate following chronic exposures to NaCl. Acclimation of the thylakoid system to high salinity may include changes in membrane lipid composition (Müller and Santarius, 1978
), activation of salt-tolerant photosynthesis (Locy et al., 1996
), and beneficial effects of compatible solutes against PSII photodamage (Ohnishi and Murata, 2006
). The thylakoid architecture itself is strongly influenced by the ionic environment of the membranes (reviewed by Chow et al., 2005
). In studies on microalgae of the genus Dunaliella, acclimation to high salinity includes compression of a major portion of the thylakoid membrane surface with an altered density of particles (Pfeifhofer and Belton, 1975
). However, this different organization, highlighted by freeze–fracture of the thylakoid membranes, does not modify the ultrastructure of the chloroplast in ultrathin sections (Pfeifhofer and Belton, 1975
; Bérubé et al., 1999
). Conversely, changes in thylakoid ultrastructure have been observed in potato (Fidalgo et al., 2004
) or maize (Hasan et al., 2005
, 2006
).
The water in the lakes of the Ethiopian Rift Valley is subjected to overall long-term changes, due to a combination of hydrogeological, climatic, and human factors (Zinabu et al., 2002
; Zinabu and Pearce, 2003
). Considering that Ethiopia has undergone repeated droughts in the past three decades, an increase in salinity is expected as a common feature among these tropical lakes with closed drainage (Wood and Talling, 1988
; Zinabu et al., 2002
). However, in the freshwater Lake Awasa the trend is opposite because of the seepage of more dilute underground water into the lake, documented since 1964–1966 (Zinabu et al., 2002
). Most floristic surveys of the algal flora in the Ethiopian lakes were derived from expeditions or short visits; identification of algae was affected by taxonomic uncertainties, synonymy, and nomenclatural changes (Wood and Talling, 1988
). The instability of the lacustrine system in the Ethiopian Rift causes us to hypothesize that the algae could have evolved some plasticity in surviving past fluctuations in salinity. In the only study on the subject, a wide tolerance to salinity (total salts, 13–88 g·L–1) was documented in the cyanobacterium Spirulina platensis from the soda crater Lake Chitu (Kebede, 1997
).
This work focuses on a green microalga, a Kirchneriella species (Selenastraceae), which we have isolated from water of the Lake Awasa (Rift Valley, Ethiopia). Selenastraceae are common green microalgae and were even dominant in Lake Awasa in the past (Cannicci and Almagià, 1947
). The presence of a Kirchneriella species (K. aperta) in Lake Awasa has been more recently reported by Kebede and Belay (1994)
. Cells live solitary or joined in colonies by a mucilaginous capsule and have a characteristic curved or twisted shape (Krienitz et al., 2001
). The microalga reproduces by forming autospores (Pickett-Heaps, 1970
). The majority of the cell volume is occupied by the chloroplast, which contains a large pyrenoid (Krienitz et al., 2001
).
We tested the hypothesis of the acclimative plasticity against increased NaCl concentrations in Kirchneriella sp. The microalga was cultured at five NaCl levels for 3 mo. The lowest salinity condition (total salt concentration, 0.6 g·L–1, no NaCl added) approximately corresponds to recent surveys in Lake Awasa (total salts, 0.7 g·L–1; main ions, g·L–1: Na+ 0.16, K+ 0.03, Ca2+ 0.01, (bi)carbonate 0.47, Cl– 0.03, SO42– 0.01; Zinabu et al., 2002
). The NaCl concentrations added to the culture medium (0.4, 1.9, 5.9, and 19.4 g·L–1) were chosen to give final salinities comparable to current conditions of other lakes in the Ethiopian Rift (Kebede et al., 1994
; Zinabu et al., 2002
). The cell content in photosynthetic pigments and the microspectrofluorimetric responses at room temperature were correlated with the changes in the thylakoid organization (Pancaldi et al., 2002
). The general morphology of the cells was also investigated. This study on Kirchneriella sp. provides an instructive example of the acclimative ability of green microalgae living in an instable environment.
MATERIALS AND METHODS
Isolation and culture conditions of the organism
Samples of water were collected from Lake Awasa (Rift Valley, Ethiopia, 7°05'N, 38°50'E) during the summer of 2002 and transported to the Laboratory of Plant Cytophysiology (Department of Biology and Evolution, University of Ferrara, Italy). Standard techniques were employed to isolate microalgae on the following medium: 2 mM NaNO3, 0.1 mM NaH2PO4, 1 µM ZnCl2, 1 µM MnCl2, 1 µM Na2MoO4, 0.1 µM CoCl3, 0.1 µM CuSO4, 20 µM Fe(III) citrate, 26.4 µM Na2EDTA, 35 µg·L–1 thiamine, 50 µg·L–1 biotin, and 15 g·L–1 agar agar (Fabregás et al., 1984
). The pH was adjusted to 8.8 with NaOH, corresponding to the value measured in the water of Lake Awasa (Kebede et al., 1994
). Colonies were kept in a growth chamber at 24° ± 1°C and 20 µmol·m–2·s–1 of photosynthetically active radiation (PAR); an axenic culture of Kirchneriella sp. strain AW15 (Selenastraceae, Chlorophyceae, Chlorophyta; Krienitz et al., 2001
) was obtained by subculturing. The microalgae were then added to Erlenmeyer flasks containing liquid Bristol GR+ growth medium (pH 8.8, total salt content 0.6 g·L–1) (Bold, 1949
). Cells were grown in static cultures without air bubbling at 24° ± 1°C and 20 µmol·m–2·s–1 PAR in a growth chamber; cultures were stirred once each day. Subsequently, cells were inoculated into the same culture medium without any addition of NaCl or with the addition of 0.4, 1.9, 5.9, or 19.4 g NaCl·L–1. The density at inoculation was approximately 3 x 106 cells·mL–1. Cells were acclimated to the different NaCl concentrations for at least 3 mo in triplicate cultures under a low irradiance regime (20 µmol·m–2·s–1 PAR) to avoid concomitant photoinhibitory effects. Growth was monitored weekly. After 3 mo of cultivation, cells were sampled for analyses.
Growth kinetics
Cells were counted with a Thoma's hemacytometer (HBG, Giessen, Germany). The growth rate of Kirchneriella sp. was evaluated by the following equation:
|
| (1) |
). The equation was used with data recorded in the exponential phase of growth.
Photosynthetic pigment analyses
Photosynthetic pigments were extracted following the procedure used by Issa et al. (1995)
for Kirchneriella lunaris with absolute methanol for 5 min at 70°–80°C. For determining chlorophyll a (Chl a), chlorophyll b (Chl b), and total carotenoids, the extracts were measured at 665, 645, and 470 nm, respectively, with a Pharmacia Ultrospec 2000 UV-Vis spectrophotometer (1 nm bandwidth) (Amersham Biosciences, Piscataway, New Jersey, USA). Concentrations were calculated using the simultaneous equations proposed by Wellburn (1994)
.
Room temperature microspectrofluorimetry
Room temperature microspectrofluorimetric analyses were performed to study the assembly state of the Chl–protein complexes of PSII in the alga grown at different salinities (Pancaldi et al., 2002
). Data acquisition was as described in Pancaldi et al. (2002)
. Fluorescence emission spectra were recorded at room temperature (25°C) using a microspectrofluorimeter (RCS, Firenze, Italy), combined with a Zeiss model Axiophot photomicroscope (Carl Zeiss, Oberkochen, Germany). Cells were mounted on poly-lysine microslides (Menzel-Gläser, Braunschweig, Germany), and all spectra were recorded on single living cells viewed under fluorescent light. The excitation light (436 nm) was focused on a single cell at a time. Autolab software (RCS) set the emission recording range (620–750 nm) and optimized the photomultiplier response. At least three spectra were recorded for each replicated culture.
Spectra were processed with Origin 6.0 software (OriginLab, Northampton, Massachusetts, USA) as previously reported (Pancaldi et al., 2002
; Baldisserotto et al., 2004
; Ferroni et al., 2007
). In particular, average spectra were smoothed with the Fast Fourier Transform filtering function and deconvoluted into Gaussian components after fourth derivative analysis.
Fluorescence emission bands were interpreted according to previous work (Pancaldi et al., 2002
; Baldisserotto et al., 2004
, 2005a
, b
, 2007
; Ferroni et al., 2004
, 2007
). The attribution of bands is reported in Table 1. The contributions of the emission components of interest (PSII reaction center, RCII; inner antennae of PSII, CP43–47; light harvesting complex II, LHCII) were evaluated as the areas subtended under the corresponding Gaussian curves.
|
). Semithin sections (approximately 5 µm thick) of fixed samples embedded in Lowicryl K4M resin (see Transmission electron microscopy) were stained with 0.02% (m/v) ruthenium red (RuR; Merck) in distilled water for 10 min at room temperature (Engels and Jung, 1998
).
Confocal laser scanning microscopy
The topographical distribution of Chl fluorescence was obtained with a Zeiss LSM410 confocal laser scanning microscope (CLSM). Cells were collected by centrifugation and placed onto poly-lysine-coated microslides (Menzel-Gläser). Images were acquired and processed as previously described (Ferroni et al., 2004
).
Transmission electron microscopy
Cells, harvested by centrifugation, were fixed with 3% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h at 4°C and postfixed overnight with 1% (m/v) OsO4 in the same buffer. Embedding and staining procedures for transmission electron microscopy (TEM) were routinely performed as described in previous reports (Pancaldi et al., 2002
; Ferroni et al., 2004
).
For RuR cytochemical analyses, the cells were fixed for 2 h at 4°C in 0.1 M phosphate buffer (pH 7.4) containing 2% (m/v) paraformaldehyde and 1% (v/v) glutaraldehyde. After rinsing, samples were maintained overnight in buffer and subsequently treated with 0.5 M NH4Cl and gradually embedded in Lowicryl K4M as previously described (Pancaldi et al., 2002
). Thin sections were mounted on nickel grids, and acidic polysaccharides were stained by floating the grids on a 2% (m/v) aqueous solution of RuR for 1 h at room temperature (Merck).
All samples were observed with a Hitachi H800 electron microscope (Hitachi, Tokyo, Japan) (Electron Microscopy Center, Ferrara University, Italy). Morphometric parameters were evaluated in longitudinal radial sections of cells in TEM micrographs. For observations of RuR-stained sections, the smallest diaphragm of the electron microscope was used.
Statistical analyses
Where appropriate, data were compared using analysis of variance (ANOVA). When differences were statistically significant, the data were further analyzed with a Student's t test. Statistical analyses were performed with Origin 6.0 software.
RESULTS
The cultures used for the experiments were static, with no air bubbling. Because most cells tended to sediment in all NaCl concentrations, especially in the lowest ones, the cultures were gently stirred once each day. Static culture conditions were chosen because in Lake Awasa the phytoplankton develops mainly with the onset of the thermal stratification following mixing in December (stratification lasts from January to May) (Kebede and Belay, 1994
).
Growth kinetics
Different NaCl concentrations had strong effects on the growth kinetics of the cultures of Kirchneriella sp. (Fig. 1A). The cells in the medium without the addition of NaCl grew exponentially without any lag until the end of the experiment, with a doubling time of 
30 d (Fig. 1B). Total salt concentration of this medium (0.6 g·L–1) approximates current values in Lake Awasa (0.7 g·L–1; Zinabu et al., 2002
). The addition of 0.4 and 1.9 g NaCl·L–1 to the medium caused a 4-d lag, followed by a strong growth activation (doubling time, 10 d) during the 4–14-d interval (Fig. 1A). Subsequently, exponential growth was maintained to the end of the experiment, with doubling times of 28 and 22 d, respectively (Fig. 1B). Addition of 5.9 g NaCl·L–1 caused a 4-d lag, followed by growth recovery (doubling time, 34 d) (Fig 1A, B). Eighty days after inoculation, these cultures entered a stationary phase of growth. Growth was not completely inhibited even with 19.4 g NaCl·L–1; indeed, after a lag of 7 d, the cells started growing exponentially with a doubling time of 40 d. These cultures were also stationary 80 d after inoculation (Fig. 1A, B).
|
2.2–2.3 independently of the salinity (Fig. 2B). Conversely, the Chl to carotenoid molar ratio was markedly salinity dependent and tended to decrease, from 2.6 (no NaCl added) to 1.8 (19.4 g NaCl·L–1), indicating that NaCl affected the Chl content more than the carotenoids (Fig. 2B).
|
; Baldisserotto et al., 2005b
; Ferroni et al., 2007
). In our experiment, the reference condition is represented by the cells cultured in a medium without addition of NaCl. In the fluorescence spectra obtained from these cells, the main deconvoluted emission sources were easily recognized as the inner antennae CP43 (687 nm) and CP47 (696 nm) of PSII (Fig. 3A). Emissions from CP43 and CP47, indicated jointly as the inner antenna CP43–47, are indeed prominent at room temperature, when proper energy transfer processes occur (Pancaldi et al., 2002
). They, in fact, accounted for 50% of the total fluorescence emission. According to the band attribution reported in Table 1, the emission peak at 678–680 nm was attributed to the RCII (i.e., the pheophytin/Chl-containing complex) at which the photochemical reactions occur (Omata et al., 1984
). The Gaussian bands with a maximum within 699 to 715 nm were attributed to LHCII. Multiple bands in the LHCII emission range most probably depended on the heterogeneity in the peripheral antenna complexes. Fluorescence emissions at wavelengths longer than 715 nm were attributed to PSI. The fluorescence yield of PSI is normally very low because in the reaction center the P700 is an efficient fluorescence quencher at room temperature. Photosystem I accounted for less than 10% of the total emission. Finally, the short-wavelength emissions (675 nm) were attributed to loosely bound, uncoupled Chl.
|
). In a steady-state system, the relative intensity of the emission components is linked to the efficiency in the energy transfer processes among the complexes, which is closely related to their assembly state (Pancaldi et al., 2002
). Events preventing energy transfer to RCII increase the relative emission from RCII and/or LHCII (Vassiliev et al., 1995
; Pancaldi et al., 2002
). Two fluorescence emission ratios were used to obtain information on the yield of the energy transfer. Because in a properly assembled PSII core, most of the emission derives from CP43–47, the RCII/CP43–47 provides an indication of the energy transfer efficiency within the PSII core. In Kirchneriella sp. cultured with no addition of NaCl, the RCII/CP43–47 ratio was 0.34 (Fig. 4). The coupling of LHCII with PSII causes the transfer of energy to PSII, resulting in low emission from LHCII (Vassiliev et al., 1995
). Therefore, the energy transfer from the peripheral antennae to the PSII core is reflected in the LHCII/PSII fluorescence emission ratio, which was 0.12 in Kirchneriella sp. cultured with no additional NaCl (Fig. 4).
|
An increase in the LHCII/PSII emission ratio can be accompanied by more intense emission from PSI-LHCI, because the PSII-LHCII uncoupling can be followed by the redistribution of the excitation energy from PSII to PSI (Ferroni et al., 2004
). In Kirchneriella sp. acclimated to different NaCl concentrations, the contribution of the PSI components to total emission was essentially unaffected, and no correlation was found with the changes in the LHCII/PSII ratio. PSI always yielded a very low emission (9%) that was independent of salinity (Fig. 3).
Cell morphology
As seen with light microscopy, as NaCl concentrations increased, the cells formed fewer, smaller colonies and cell size increased (Fig. 5A, D, F). Morphometric analyses using TEM sections showed that the cell length and width increased exponentially as a function of salinity (Fig. 6A). Up to 5.9 g NaCl·L–1, cells tended to maintain a width-to-length ratio of 0.4; conversely, with 19.4 g NaCl·L–1, the ratio increased to 0.6, leading to a quite squat appearance of the cells.
|
|
) (Fig. 5A). The cells were joined in colonies by extracellular mucilaginous polysaccharides. Cells observed with a fluorescence microscope and with CLSM were characterized by a uniform distribution of Chl fluorescence, only interrupted by a pyrenoid, suggesting the absence of grana/intergrana differentiations (Gunning and Schwartz, 1999
) (Fig. 5B, C). The TEM observations confirmed that in the chloroplast the thylakoids were appressed in groups of about six for their entire length (Fig. 7A, B). The thylakoid groups, which ran more or less straight, were also connected to a simple homogeneous pyrenoid (Fig. 7A, B). The organelle also contained several ovoid starch granules, which hid the thylakoid system in some cell sections (Fig. 7C). Outside of the chloroplast, typical eukaryotic organelles were observed: the nucleus containing chromatin clumps, the endoplasmic reticulum, the Golgi apparatus, and one or two mitochondria (Fig. 7A, D). Moreover, large dark inclusions, most probably polyphosphate granules, were always visible, as is quite common in microalgae (Dodge, 1973
) (Fig. 7A, C). In addition, at 1.9 g NaCl·L–1, the cells contained some pale lipid globules (Fig. 7E). The cell covering (100–110 nm thick) consisted of the cell wall with a homogeneous texture and, outside the cell wall, a mucilaginous capsule as a thin layer of loose, amorphous material (Fig. 7F). Faint curls with the same appearance were visible near the cells (Fig. 7C, F).
|
). Thus, in these cells, fluorescence images suggested the presence of a grana-like organization of the thylakoids, which was further analyzed with TEM (Fig. 7G). In the chloroplast, the mean number of appressed thylakoids per group was increased by 50%, from ca. six to ca. nine (P < 0.05) (Figs. 6B and 7H). The increase in the number of appressed thylakoids per group also involved intermediate stages, with variable degrees of thylakoid appression, as if the inner membrane system was reorganizing (Fig. 7I). Although in these intermediate stages the thylakoids formed an interconnected system, the resulting thylakoid groups essentially appeared isolated in sections (Fig. 7G, I). However, it is very likely that the thylakoid groups were actually interconnected; their isolated appearance in sections could depend on the interpolation of abundant starch granules (Fig. 7G, J). The cells grown at 5.9 g NaCl·L–1 usually contained lipid globules in the cytoplasm (Fig. 7J). The cell covering included the electron-dense, uniformly thin cell wall (30 nm); a layer of amorphous pale material; and a less electron-dense, thin layer (12–15 nm) that formed a wavy and loose outline (Fig. 7K). Mucilaginous material was present outside of the cell covering.
The marked increase in dimension of the cells cultured at 19.4 g NaCl·L–1 was accompanied by strong alterations of the cell ultrastructure (Figs. 5F, 8A). In the chloroplast, the mean number of thylakoids per group rose to 12, i.e., doubled with respect to the low salinity conditions (Figs. 6B, 8A, B). This was in accordance with the spot distribution of Chl fluorescence inside the chloroplast (Fig. 5G, H). Starch granules and pyrenoid were still present (Fig. 8A). The cytoplasm was filled with lipid globules, and polyphosphate granules were also present but were smaller in diameter (0.44 vs. 0.56 µm with no NaCl added, P < 0.01) (Fig. 8A). The cell covering was strongly, but asymmetrically, thickened (Fig. 8A). Its stratification was similar to that found in the cells grown in 5.9 g NaCl·L–1. The cell wall proper (30 nm thick) was still visible (Fig. 8C). The outermost layer, dense and devoid of any stratification, increased in thickness from 30 nm at the thin side to 3–400 nm at the thick side (Fig. 8C, D). Although the external outline was substantially linear, the materials seemed to be irregularly deposited in this layer. A pale granular matrix was interposed between the cell wall and the external layer of the cell covering (Fig. 8C, D).
|
). The reaction with Alcian blue in vivo (Fig. 5I–K) and with the RuR on embedded material (not shown) showed that the acidic polysaccharides of the extracellular matrix were mainly localized near the cell at 19.4 g NaCl·L–1 and, to a lesser extent, at 5.9 g NaCl·L–1. The TEM observations of ultrathin sections stained with RuR indicated that most of the extruded acidic polysaccharides accumulated in the outermost layer of the cell covering (Fig. 8E, F). The cell wall was not reactive (Fig. 8F). Flaky materials were dispersed in the space between the cell wall and the compact extracellular matrix (Fig. 8F). DISCUSSION
Increased salt concentration in water usually causes changes in phytoplankton composition, depending on the acclimative ability of the microalgae. These changes have been documented in some Ethiopian lakes, like Lake Abijata, with strong effects on the food chain and, consequently, on the local economy (Zinabu et al., 2002
). Other lakes, which are not currently subjected to salinization, may have similar problems in the future. In our work, we have used a strain of a green microalgal species of Kirchneriella isolated from freshwater Lake Awasa (Ethiopia) to test whether this organism was able to grow in culture media with increased NaCl concentrations and whether its growth capability was linked to special acclimation features of the chloroplast. The results presented in this paper were obtained using cells cultured at low irradiance so that effects of NaCl at the chloroplast level were not confounded by photoinhibition and photodamage. However, parallel experiments performed in higher light (200 µmol·m–2·s–1 PAR) gave an essentially comparable outcome, except for the lower pigment content due to photo-acclimation (data not shown).
Kirchneriella sp. has two alternative thylakoid architectures depending on the NaCl concentration: thylakoid appressed in groups of 5–6 at low NaCl and thylakoids appressed in larger groups at high NaCl. The thylakoid system of the green lineage has a complex organization, in which different domains can be distinguished. The appressed domains are enriched in PSII, while the domains exposed to the stroma are enriched in PSI (Albertsson, 2001
). The formation and the extent of the thylakoid stacking depend on many interacting factors, including electrostatic repulsion, van der Waals attraction, and steric hindrance (reviewed by Chow et al., 2005
). Because of its abundance, LHCII is generally considered the main protein responsible for the attractive forces sustaining the membrane appression (Anderson, 1986
). In Kirchneriella sp., the Chl a/b molar ratio, which is a good indicator for antenna size (Anderson, 1986
; Yamazaki et al., 2006
), is unaffected by salinity, thus excluding major alterations in the stoichiometry of PSII and LHCII. Conversely, the fluorescence ratios suggest changes in the assembly state of LHCII with PSII (Pancaldi et al., 2002
). In fact, at high NaCl concentrations, the increased LHCII/PSII fluorescence emission ratio indicates defective energy transfer from LHCII to PSII in comparison to the transfer at low salinities. In the alga Dunaliella salina, the salt-induced LHCII-PSII uncoupling represents an acclimation mechanism (Liu and Shen, 2006
). The salt shock activates LHCII phosphorylation and state II transition, which favors cyclic electron flow around PSI and ATP synthesis, thus contributing to glycerol synthesis and ion extrusion (Liu and Shen, 2004
, 2006
). Similarly, in the marine green macroalgae Bryopsis maxima and Ulva pertusa, which are clearly adapted to high NaCl, a substantial part of LHCII serves as an antenna of PSI, responding to high ATP demand through cyclic photophosphorylation (Yamazaki et al., 2005
, 2006
). Although PSII-LHCII uncoupling in salt-acclimated Kirchneriella sp. could conceivably have a similar meaning, the ultrastructural observations do not support this interpretation. The PSII-LHCII uncoupling is expected to promote less pronounced thylakoid appression, due to a weaker association between LHCII and PSII (Dekker and Boekema, 2005
). Surprisingly, the degree of thylakoid appression in Kirchneriella sp. greatly increases at high NaCl. As a consequence, the relative reduction in the stroma-exposed (i.e., PSI-enriched) thylakoid regions is not evidence to support that PSII-LHCII uncoupling preferentially drives the transfer of energy to PSI. Moreover, the redistribution of the excitation energy to PSI would have increased the emission from PSI-LHCI (Ferroni et al., 2004
), which is not observed in Kirchneriella sp. Therefore, one can reasonably reject the inference that PSII-LHCII uncoupling in Kirchneriella sp. represents an acclimative strategy; instead, the uncoupling appears to be a toxic effect due to the chronic exposure to high NaCl. In these conditions, the rate of energy transfer among the various peripheral and core antenna proteins to the RCII is probably the most limiting step for the photosynthetic performance of PSII (Dekker and Boekema, 2005
). The structural rearrangement of the thylakoid membranes can ameliorate the energy transfer. Thylakoid stacking enhances light capture via the intricate macro-organization of LHCII-PSII supercomplexes, which results in a large functional antenna size (reviewed by Chow et al., 2005
; Dekker and Boekema, 2005
). In NaCl-acclimated Kirchneriella sp., increased thylakoid appression may promote light harvesting and compensate for the LHCII-PSII uncoupling. Furthermore, considering that salt-stressed organisms normally undergo greater PSII photoinhibition (Misra et al., 1999
; Sudhir and Murthy, 2004
), appressed regions could serve as reservoir of inactive PSII (Chow et al., 2005
). In this way, the PSII repair system is not overloaded, and the energy flow is driven until it is trapped in a functional PSII (Anderson and Aro, 1994
). Accumulation of nonfunctional PSII centers in appressed domains may prevent PSII cores from disassembly by virtue of being inaccessible to D1 protein degradation (Baena-González and Aro, 2002
). This easily explains the low RCII/CP43–47 ratio, which excludes the disassembly of PSII units (Pancaldi et al., 2002
).
In contrast to high concentrations of NaCl, 1.9 g NaCl·L–1 is more congenial to the alga in view of its growth kinetics, but concentrations lower than 1.9 g·L–1, which approximate the current value recorded in Lake Awasa (Zinabu et al., 2002
), appear to favor the higher pigment content and the lower LHCII/PSII emission ratio, which is linked with a tight LHCII-PSII association (Baldisserotto et al., 2005a
; Ferroni et al., 2007
). But at the lowest salinities, in a PSII subpopulation an increased RCII/CP43–47 ratio indicates that the energy transfer from the inner antennae to the RCII is impaired (Pancaldi et al., 2002
). The differences among the three low NaCl conditions do not have any evident impact on the thylakoid architecture.
No obvious effect of NaCl stress on photosynthetic pigments has been reported (reviewed by Sudhir and Murthy, 2004
). The trend toward a decrease in pigments observed in Kirchneriella sp. with rising NaCl (the increase observed at 19.4 g NaCl·L–1 is due to increased cell size) is a frequent response in salt-susceptible plants. More interesting is the salinity-dependent decrease in the Chl to carotenoids ratio. The cells are presumably more liable to PSII photoinhibition at high NaCl than at low NaCl (Misra et al., 1999
), and the change in the Chl/carotenoids stoichiometry can reduce the energy pressure to PSII by virtue of the well-known photoprotective role of carotenoids (reviewed by Demmig-Adams et al., 1996
). Microalgal cells normally are subject to increased irradiance when they colonize the surface layers of the water column. In this regard, the accumulation of lipid globules in the cytoplasm is interesting because they decrease the density of the cell and promote buoyancy (Dodge, 1973
).
The ultrastructure of the cell covering of Kirchneriella sp. was affected by the level of NaCl in the culture medium. In Kirchneriella, the cell wall is surrounded by a mucilaginous capsule formed by polysaccharides made up of mannose, rhamnose, and uronic acids (Lombardi and Vieira, 1999
). The capsule plays an ecological role in Kirchneriella; it provides some passive resistance to heavy metals because of its abundance of negative charges (Issa et al., 1995
; Lombardi and Vieira, 1999
; Lombardi et al., 2002
). Our work further supports the important role of the capsule in creating a favorable ionic environment around the cells. The relevance of Na+ for the cell physiology has been shown in alkaliphilic cyanobacteria, in which the acidic intracellular pH is ensured by an Na+/H+ antiporter activity (Krulwich, 1995
; Pogoryelov et al., 2005
). In Lake Awasa, Kirchneriella lives at pH values near 9 (Zinabu et al., 2002
); therefore, a similar mechanism could operate in the microalga as well. At high Na+ concentrations, the negative charges of the uronic acids of the extruded polysaccharides could benefit the cells by forming a protective layer against excessive Na+ uptake.
Conclusions
From our research on Kirchneriella sp., we conclude that (1) the alga acclimates to a variety of NaCl concentrations; (2) at the chloroplast level, NaCl affects the balance between energy transfer and trapping through alterations of the LHCII-PSII coupling; and (3) at high NaCl concentrations, changes in the thylakoid architecture and in the PSII assembly state allow sufficient integrity of the photosynthetic membrane. Although the mucilaginous capsule probably plays an important role in regulating the ionic balance, the cell is not completely protected against chronic NaCl excess. Because of its ability to acclimate to unfavorable salinity, Kirchneriella sp. has the potential to survive future changes in water chemistry.
FOOTNOTES
1 The authors thank C. Andreoli (University of Padova, Italy) for help in organism identification and E. Ferroni for help in language editing. Funding was provided by the Fondo per gli Investimenti della Ricerca di Base (FIRB2001) of the Italian MIUR (Ministero per l'Istruzione, l'Università e la Ricerca) and by the University of Ferrara. 
4 Author for correspondence (e-mail: fsm{at}unife.it
)
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