Effects of Salt Stress on PSII Function and Photoinhibition in the Cyanobacterium Spirulina platensis

11 .OUR.AL OF •

j. Plant Physiol. Vol. 155. pp. 740-745 (1999)

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http://www.urbanfischer.de/journals/j pp

© 1999 URBAN & FISCHER

Effects of Salt Stress on PSI! Function and Photoinhibition in the Cyanobacterium Spirulina platensis CONG-MING Lu

and JIAN-HuA

ZHANG*

Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong Received December 7, 1998· Accepted May 4, 1999

Summary

The changes in PSII function and susceptibility to photo inhibition in the cyanobacterium Spirulina platensis adapted to 0.75 mollL NaCl were evaluated by use of chlorophyll fluorescence. In salt-stressed cells, photochemical quenching (qp) decreased while the proportion of Qs-non-reducing PSII reaction centres increased. However, salt stress had small effect on the maximal efficiency of PSII photochemistry (F)Fm) and the efficiency of excitation energy capture by open PSII reaction centres (F/IF m'). These results suggest that the decreased quantum yield of PSII electron transport (
Key words: Chlorophyll fluorescence, photosystem I!, photoinhibition, salt stress, Spirulina platensis (cyanobacterium). Abbreviations: Fj = intermediate level phase in a rapid fluorescence induction kinetics curve; Fm(dark) = maximal fluorescence in dark-adapted state; Fm = maximal fluorescence with all quenching mechanisms at a minimum, measured in the presence of DCMU; F m' = maximal fluorescence in light-adapted state; F0 = minimal fluorescence in dark-adapted state; Fo' = minimal fluorescence in light-adapted state; Fp = maximal level phase in a rapid fluorescence induction kinetics curve; Fy = maximal variable fluorescence in dark-adapted state; Fv' = maximal variable fluorescence in light-adapted state; Fs = steady-state fluorescence yield at qp>O; F)F m= maximal quantum efficiency ofPSII photochemistry;
Salt stress causes a decrease in photosynthetic activity in algal cells. It has been suggested that a decrease in photosystem II * Correspondence.

(PSII) activity is one of the major factors responsible for such a decrease (Kirst, 1990). However, the mechanisms for the decreased PSII activity are still not clear. It was reported that in the green alga Dunaliella tertiolecta, the decrease in PSII activity was associated with state-2 transition after the cells had been exposed to high salinity for 5 min in the dark (Gil0176-1617/991155/740 $ 12.0010

PSII Photochemistry, Photo inhibition and Salt Stress

mour et aI., 1984, 1985). Endo et aI. (1995) also showed that the inhibition of the quantum yield of PSII in Chlamydomonas reinhardtii by salt stress was attributed to the state-2 transition. In cells of red alga Porphyra perforata stressed by high salinity for 5 min in the dark, the decrease in the excitation energy reaching PSII reaction centres and the inhibition of the oxidising side of PSII were demonstrated to result in a decrease in PSII activity (Satoh et aI., 1983). In cyanobacteria, it has been shown that salt stress leads to a decrease in the PSII activity (Schubert and Hagemann, 1990) and in the excitation energy transfer from phycobilisomes to PSII in Synechocystis sp. (Schubert et al., 1993), but Jeanjean et aI. (1993) reported that salt stress induced no change in the PSII activity in the same species. Considerable interest has been invested in outdoor cultivation of Spirulina platensis, a filamentous cyanobacterium, for commercial biomass production because of its high content of proteins (Vonshak, 1990). In cultures grown outdoors in open ponds under arid and semiarid conditions, daily evaporation amounts to 1- 2 cm of water level, leading to a progressive increase in the salt concentration in the culture (Vonshak, 1987). Besides salt stress, these cultures also suffer from photoinhibition (Vonshak and Guy, 1992). It has been shown that Spirulina platensis is capable of adapting to high salinity up to 1.0 mollL NaCl by the buildup of organic osmotica and the extrusion of sodium from the cell (Vonshak et aI., 1988). After fully adapting to high salinity, a partial or nearly full recovery of the photosynthetic activity, depending on the strain and the level of salinity, has been observed (Vonshak et aI., 1995). Although Spirulina platensis has some capability of adapting to high salinity, it has been observed that salt stress increases susceptibility to photoinhibition (Vonshak et aI., 1996). A better understanding of the interaction of light and salt stress on photosynthesis may help to optimise the productivity of the algal cultures grown outdoors. In this study, we first characterized PSII photochemistry in Spirulina platensis when fully adapted to high salinity (0.75 mollL NaCl). We then investigated the relationship between these characteristics of PS II photochemistry and susceptibility ofpSII to photoinhibition in salt-adapted cells.

Materials and Methods Cell culture Spirulina platensis M2 was grown in Zarouk's medium, containing 200 mmollL sodium bicarbonate (Vonshak et ai., 1982) at an irradiance of 50 Ilmol m -2 s-I provided by fluorescent lamps at 25 0c.

741

cell suspensions were then placed in a temperature-controlled cell at 25 °C and exposed to an irradiance of 600 Ilmol m -2 s-I provided by a halogen lamp (Flecta, Germany) for different periods (0-30 min).

Fluorescence quenching analysis Chlorophyll fluorescence quenching analysis was carried out at room temperature with a portable fluorometer (PAM-2000, Walz, Effeltrich, Germany). The fluorometer was connected to a computer with data acquisition software (DA-2000, Walz). The procedure follows in general that of Campbell et ai. (1996). The minimal fluorescence level in the dark-adapted state (Fa) was determined by measuring the modulated light, which was sufficiently low «0.1 Ilmolm-2 s-I) as not to induce any significant variable fluorescence. A 0.8 s flash of saturating white light (8000llmol m- 2 s-I) was then given to determine the maximal fluorescence in the dark-adapted state, Fm(dark)' The actinic light (50 Ilmol m- 2 S-I) was thereafter turned on. The steady-state fluorescence, Fs' was reached within 2.5 min and thereafter a saturating light flash was given again to determine the maximal fluorescence in the lightadapted state, Fm'. Mter Fs was established again, the minimal fluorescence level in the light-adapted state, Fa', was measured by briefly interrupting the actinic light and illuminating the cells with far-red light for 3s (7Ilmolm-2s-I). Thereafter, the actinic light was turned on again. Mter the steady-state fluorescence was achieved, the true maximum fluorescence, Fm was determined by adding 3-(3,4-dichlorophenyl)-l,l-dimethyl urea (DCMU) (10llmollL final) to the cuvette. Using both light and dark fluorescence parameters, we calculated: (1) the maximum efficiency of PSII photochemistry in the darkadapted state (F)F m ), (2) the photochemical quenching coefficient, qP = (Fm' -Fs)/(Fm' -Fa'), which measures the proportion of open PSII reaction centres (van Kooten and Sne!, 1990), (3) the nonphotochemical quenching coefficient, qN = l-(Fm' -Fa')/(Fm-Fa), (4) the quantum yield ofPSII electron transport,
Fast fluorescence induction kinetics The fast fluorescence induction kinetics was measured by PAM2000 in the dark-adapted samples suddenly illuminated with moderate white light (40 Ilmol m- 2 s-I) at a sampling rate of 1 ms point -I. In order to avoid an incomplete reoxidation of the plastoquinone pool in the dark, which could result in an increase in fluorescence level at phase I, the dark-adapted samples were illuminated with 3 s far-red light prior to the measurements of the fluorescence induction kinetics. All samples were dark-adapted for 10 min before chlorophyll fluorescence was determined.

Results Salt stress treatment To obtain a salt-adapted Spirulina platensis culture, exponentially growing cells were diluted and grown for at least 14 days under 0.75 mollL NaCI in Zarouk's medium.

Photo inhibition treatment Spirulina cells in the log phase were harvested and resuspended in a fresh medium to a chlorophyll concentration of 5 Ilg mL -I. The

Chlorophyll fluorescence characteristics in dark-adapted state Figure 1 shows the changes in the maximal efficiency ofPSII photochemistry (FJFm), the minimal fluorescence yield (Fa) and the maximal fluorescence yield (Fm) in control and saltstressed cells in the dark-adapted state. FJF m showed a small but significant decrease in salt-stressed cells. Such a decrease in FJF m was the result of an increase in Fa because of no significant changes in Fm.

742

CONG-MING Lv andJIAN-HvA ZHANG

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PS II electron transport (PSII), the efficiency of excitation energy capture by open PSII reaction centres (F/IF m') and photochemical quenching (qp) in response to different PFOs in control and salt-stressed cells. PSII decreased significantly with the increase of PFO and was approximately 25 % lower in salt-stressed cells than in control cells (Fig. 3A). F/IF m' decreased slightly with the increase of PFO in both control and salt-stressed cells and there was a small decrease (about 6 %) in F/IF m' in salt-stressed cells relative to control cells (Fig. 3 B). qP decreased with the increase of PFO, and an average decrease of 21 % in qP was observed in salt-stressed cells at all PFOs (Fig. 3 C).

Effects ofsalt stress on susceptibility ofPSII to photoinhibition Fir. Fig. 1: Changes in the maximal efficiency of PSI! photochemistry (FvIFm), the minimal fluorescence (Fo) and the maximal fluorescence (F m) in dark-adapted control cells and salt-stressed cells. Values are means ± SE of four measurements. Although FjFm is most frequently used as an indicator of the maximal efficiency of PSII photochemistry (Krause and Weis, 1991), it gave no direct information on the heterogeneity ofPSII reaction centres. To evaluate the effects of salt stress on the heterogeneity of PSII reaction centres, the fast fluorescence induction kinetics was investigated in salt-adapted cells. When a dark-adapted cell suspension was illuminated with a white light of moderate intensity, the fluorescence induction kinetics curve in Spirulina platensis displayed a rapid rise of chlorophyll fluorescence from the minimal level (0) to an intermediate level (1) followed by a very fast rise to the maximum level (P) (the control in Fig. 2). This induction kinetics was similar to that of higher plants, such as wheat leaves (data not shown). The 0-1 phase has been attributed to OA reduction in the OB-non-reducing PSII reaction centres, in which the electron transfer from OA - to OB is inhibited and phase 1- P reflects the accumulation of OA - in the active PSII reaction centres with efficient electron transfer to the plastoquinone pool (Chylla and Whitmarsh, 1989; Cao and Govindjee, 1990); the ratio (Fj-Fo)/(Fy-F o) can thus be considered a measure of the percentage ot those OB-non-reducing PSII reaction centres. Figure 2 shows that salt stress resulted in an increase in phase I. The (Fj-Fo)/(F -Fo) ratio increased from 0.22 in control cells to 0.32 in sJ't-stressed cells, suggesting an increase in the proportion of OB-non-reducing PSII reaction centres.

Susceptibility of PSII in salt-stressed cells to photoinhibition was examined. Cell suspensions were ex~osed to a photoinhibitory illumination of 600 Ilmol m -2 s- . The ratio Fj Fm was then determined. Figure 4 displays the decline in the ratio Fy/Fm when control and salt-stressed cells were photoinhibited. A greater decrease in F jF mwas observed in salt-stressed cells, indicating higher susceptibility of PSII to photoinhibition for the cells grown under salt stress. The changes in fluorescence parameters in the light-adapted state, F/ IF m', PSII, qp, and qN during photoinhibition in control and salt-stressed cells were further examined. As demonstrated in Fig. 5, F/IF m', PSII and qP decreased during photo inhibition and a greater decrease was also observed in salt-stressed cells than that in control cells (Fig. 5A, 5 B, 5 C). qN showed no big difference between control and salt-stressed cells although it increased during photoinhibition (Fig. 5 D).

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Fig. 2: Changes in the rapid fluorescence induction kinetics curves in control and the salt-stressed cells. The curves were recorded by PAM-2000 in the dark-adapted samples suddenly illuminated with moderate white light (40 Ilmol m- 2 s-I) and started from the same point.

PSII Photochemistry, Photoinhibition and Salt Stress

Discussion

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Our results show that the quantum yield of PSII electron transport (PSII) decreased considerably by 25 % after Spirutina cells became adapted to 0.75 mollL NaCl (Fig. 3 A). A small decrease in the maximal efficiency of PSI! photochemistry (FvlFm) and the efficiency of excitation energy capture by open PSII reaction centres (Fy'/Fm') in salt-stressed cells indicate that the decrease in PSII was mainly due to the decrease in the photochemical quenching (qp) (PSII = qp x Fv' / Fm', Genty et al., 1989, Figs. 1, 3). The decreased qP in saltstressed cells may indicate an increase in the proportion of the reduced state of OA in salt-stressed cells (Bradbury and Baker, 1981; Genty et al., 1989; van Kooten and Snel, 1990). What caused an increase in the proportion of the reduced state of OA, i.e. a decrease in qp? As shown in this study, an increase in the proportion of the OB-non-reducing PSI! reaction centres was observed in salt-adapted cells. Since the key feature of these PSII reaction centres is the inhibition of electron transport from the OA - to OB pool (Chylla and Whitmarsh, 1989; Cao and Govindjee, 1990), it is evident that an increase in the proportion of the OB-non-reducing PSII reaction centres would inevitably lead to an accumulation of a reduced state of OA, which brought about an increase in the fraction of the reduction state of OA as indicated by the decreased qp. In this study, a greater decrease in both FJF m and PSII in salt-stressed cells than that in control cells when exposed to high irradiance suggests that salt stress induced increased susceptibility of PSII to photo inhibition (Figs. 4, 5). It seems

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To evaluate whether higher susceptibility of the salt-stressed cells to photoinhibition resulted from the higher proportion of inactivated PS II reaction centres, the changes in the proportion of the OB-non-reducing PSII reaction centres were investigated. Figure 6 shows that the (Fi-Fo)/(Fp-Fo) ratio increased during photoinhibition and a greater increase of this ratio occurred in salt-stressed cells, suggesting a larger increase in the proportion of the OB-non-reducing PSII reaction centres in salt-stressed cells than in control cells.

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CONG-MING Lu and JIAN-HuA ZHANG

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salt stress was a consequence of an increase in the proportion of the reduction state of OA, which was due to an increase in the proportion of the OB-non-reducing PSII centres in saltstressed cells.

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Acknowledgements

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We are grateful to Hong Kong Baptist University (FRG grant), the Groucher Foundation, for their financial support to this research.

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References

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Time (min) Fig. 6: Changes in the ratio (F;-Fo)/(Fp-Fo) determined from the rapid fluorescence induction kinetics curves during photoinhibition in Spirulina platensis cells grown under culture medium (e) or culture medium plus 0.75 mol/L NaCl (_), respectively. Values are means ± SE of 4 replications.

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