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Kintake Sonoike, Yukako Hihara, Masahiko Ikeuchi, Physiological Significance of the Regulation of Photosystem Stoichiometry upon High Light Acclimation of Synechocystis sp. PCC 6803, Plant and Cell Physiology, Volume 42, Issue 4, 15 April 2001, Pages 379–384, https://doi.org/10.1093/pcp/pce046
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Abstract
We characterized the photosynthetic properties of the pmgA mutant of Synechocystis PCC 6803, which cannot change its photosystem stoichiometry under a high-light condition (200 µmol m–2 s–1), in order to clarify the physiological significance of the regulation of photosystem stoichiometry. We found that (1) PSII activity was inhibited more in wild-type cells on the first day under the high-light conditions than in mutant cells. (2) The growth of the mutants following the initial imposition of high light was faster than that of wild-type cells. (3) However, growth was severely inhibited in the mutants after the third day of exposure to high light. (4) The growth inhibition in the mutants under the extended high-light conditions was reversed by the addition of sublethal concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which seemed to mimic photoinhibition of PSII. These results suggest that the main role of adjusting the photosystem stoichiometry with respect to light intensity is not to maintain efficient photosynthesis, but to down regulate electron transfer. Failure to down regulate electron flow leads to cell death under prolonged exposure to high light in this cyanobacterium.
(Received August 17, 2000; Accepted January 20, 2001).
Introduction
The photosynthetic machinery of higher plants and cyanobacteria contains two photosystems (PSI and PSII) that absorb light energy and convert it to electrochemical potential. Since PSI and PSII participate in linear electron flow, it is essential to regulate photosystem stoichiometry for efficient photosynthesis in plants and cyanobacteria. In cyanobacteria, the antenna system for PSI is totally different from that for PSII. The light-harvesting antenna for PSI is exclusively comprised of Chl a while that for PSII consists mainly of phycobilisome. Since the antenna systems absorb light of different qualities, regulation of photosystem stoichiometry (PSI/PSII ratio) could allow efficient photosynthetic electron transport when the quality of light in the environment changes. This is supported by the finding that the content of PSI relative to PSII decreased when cells were exposed to light absorbed primarily by PSI and increased when cells were exposed to light primarily absorbed by PSII (Fujita et al. 1985, Manodori and Melis 1986, Melis et al. 1989, Cunningham et al. 1990, for a review, see Fujita et al. 1994). Also, in many photosynthetic organisms the PSI/PSII ratio is decreased in response to high light. Cyanobacterial cells grown under high light have a lower PSI/PSII ratio than that of cells grown under low light (Kawamura et al. 1979, Murakami and Fujita 1991, Hihara et al. 1998). This change tends to be considered as compensation for PSII antenna size, since the size of the phycobilisome is preferentially reduced under a high-light condition. However, it has been difficult to directly examine the importance of these adaptation phenomena to light because of the lack of an experimental technique.
A novel gene, pmgA was first identified as an essential factor for the growth of Synechocystis PCC 6803 under photomixotrophic (light in the presence of glucose) conditions (Hihara and Ikeuchi 1997). Characterization of the pmgA mutant revealed that it showed little change in photosystem stoichiometry upon a shift to high light (Hihara et al. 1998). Since other responses to high light such as a decrease in the PSII antenna size seemed normal, this mutant could be exploited to elucidate the physiological significance of the regulation of photosystem stoichiometry upon transferring Synechocystis to high light. Here, we report that pmgA mutants show less photoinhibition of PSII and faster growth under short-term high light conditions compared to the wild type. However, severe growth inhibition occurred under long-term exposure to high light. We propose that the decrease in the rate of electron transport due to the change of photosystem stoichiometry is indispensable for the growth under continuous high-light conditions.
Materials and Methods
A glucose tolerant wild-type strain of Synechocystis sp. PCC 6803 and mutants (pmgA(L65F) and pmgA::SpR) (Hihara and Ikeuchi 1997) were grown at 32°C in BG-11 medium with 20 mM HEPES-NaOH (pH 7.0) under continuous illumination provided by fluorescent lamps (Hihara et al. 1998). The pmgA(L65F) was a spontaneous mutant isolated from the wild-type, while pmgA::SpR was a directed disruptant of pmgA by insertion of a spectinomycin-resistant cassette (aadA gene). Cell density was measured by A730 with a spectrophotometer (model UV-160A, Shimadzu, Kyoto, Japan). Fluorescence measurements were carried out essentially according to Campbell et al. (1998) using a PAM fluorometer (PAM 101/102/103, Heinz Waltz, Effeltrich, Germany). Cells were dark-adapted for 5 min and then the measuring light (ML) was turned on to obtain the minimal fluorescence level, Fo (Fig. 1). The fluorescence level with fully reduced QA (Fm′) was obtained by applying multiple turnover (MT) flashes (XMT-103, Heinz Waltz, Effeltrich, Germany). The stable level of fluorescence (Fs) was determined during exposure of cells to actinic light (AL) with defined photon flux density (KK 1500, Schott, Wiesbaden, Germany). The far-red light was from a photodiode (FR-102, Heinz Waltz, Effeltrich, Germany) and was applied just after turning off the actinic light to determine Fo′. The maximum fluorescence (Fm) was obtained by adding 10 µM DCMU to the sample. Fv and Fv′ were defined as Fm–Fo and Fm′–Fo′, respectively. Photochemical quenching (qP), non-photochemical quenching (qN) and effective quantum yield of electron transport through PSII (ϕII) were calculated as (Fm′–Fs)/ (Fm′–Fo′), 1–[(Fm′–Fo′)/ (Fm–Fo)] and (Fm′–Fs)/Fm′, respectively.
Results
We first determined whether or not the mutation in pmgA interfered with the mechanism involved in sensing the light environment. The light intensity to which the cells are acclimated could be predicted by the light response curve of the fluorescence parameter qN (Campbell and Öquist 1996). In a wide range of cyanobacteria with different pigment contents, morphologies and light histories, qN was shown to reach a minimum near the photon flux densities (PFD) in which the cells were grown. As expected, in wild-type Synechocystis sp. PCC 6803 the qN reached a minimum near the growth PFD (Fig. 2, closed circle). Both the pmgA point mutant (L65F) and the disrupted mutant obtained with a spectinomycin-resistance cassette (pmgA::SpR) showed the same phenomena (Fig. 2, open circle and open square, respectively). These results suggest that the photosynthetic organization is modified in the pmgA mutants with respect to growth PFD; that is, the mutation does not affect the light sensing mechanism.
We then determined the fluorescence characteristics of the wild-type and pmgA mutants in order to elucidate the effect of inability of the mutants to change their photosystem stoichiometry. One of the fluorescence parameters, qP, is an indicator of the redox state of plastoquinone pool. The light response curves of qP between the wild type and mutants when the cells were grown at the PFD of 20 µmol m–2 s–1 (low light) were identical (Fig. 3, upper panel). However, when the growth PFD was raised to 300 µmol m–2 s–1 (high light), the two mutant strains showed a higher qP (more oxidized plastoquinone pool) near the growth PFD (Fig. 3, lower panel), which may reflect the higher PSI content relative to wild type.
Quantum yield of electron transfer through PSII (ϕII) is also affected by the mutation. ϕII is not different between wild-type and mutant cells grown in low light (Fig. 4, time 0), and steeply decreased just after the shift of the cells to high light. This initial decrease may be ascribed to the temporal photoinhibition in cells acclimated to the low light. By 15 h in high light, ϕII gradually recovered to a level that was higher than the original level and the mutant strains exhibited a higher ϕII than the wild-type strain. The higher ϕII was maintained for at least several more h under high light after the inoculation of the cells to new culture (indicated by an arrow in Fig. 4). Under a comparable condition, PSI/PSII ratio started to decrease after 10 h in high light (Hihara et al. 1998). Within 22 h, the decrease of PSI/PSII ratio from 2.08 to 1.37 was observed. The low PSI/PSII ratio seemed to be maintained under the high-light condition when the cultures were inoculated every 24 h, judging from the Chl content (roughly represents PSI content) on a cell basis (Hihara and Ikeuchi 1998).
ϕII can be expressed as the product of qP by Fv′/Fm′, the effective quantum yield of open PSII centers. The higher ϕII in the mutants grown under a high-light condition is due to both a higher qP and a higher Fv′/Fm′ (Table 1). The mutants grown under high light also showed higher maximum quantum yield of PSII (Fv/Fm). When cells acclimated to high light were illuminated for 5 min at various PFD, the Fv/Fm decreased as a result of photoinhibition of PSII (Fig. 5). Since the Fv/Fm in wild-type cells is more readily decreased than pmgA mutants (Fig. 5), the lower Fv/Fm (Table 1) in wild-type cells can be ascribed to the photoinhibition of PSII under high-light conditions.
We consistently observed 25% higher growth in the mutant cells on the first day after the shift to high light. This seems to be due to the higher quantum yield of photosynthesis in the mutant. However, if the cultures were inoculated every 24 h to minimize the self-shading effect, the difference in the growth was not observed on the second day, and the growth of the mutants was severely suppressed in the third and fourth day (Fig. 6). Thus, the mutant cells grow better than wild-type cells under short-term high-light conditions, but the situation is totally different during long-term, high-light growth. To examine whether the lower quantum yield of photosynthesis in the wild-type strain is related to the continuous growth under long-term high-light condition, the effect of a sublethal concentration (0.03 µM) of DCMU on the growth of the mutants was examined. This concentration of DCMU suppressed the growth of the mutant cells almost to the levels in the wild-type cells, but cells could continue to grow even after 3 d (Fig. 7, double circles and double squares) in contrast to the decreased growth of the mutants without DCMU on the third day (Fig. 7, open circles and open squares). These results suggest that down regulation of the electron transport is necessary for the extended growth under high-light conditions.
Discussion
Although acclimation of plants and cyanobacteria to high light has been extensively characterized, the molecular mechanism for the acclimation is practically unknown (Anderson et al. 1995, Hihara 1999). With the acclimation of cyanobacteria to high light, many changes in the photosynthetic and other components were observed; decrease of phycobilin and Chl, decrease of PSI content relative to PSII content, and induction of several genes which have putatively protective functions against high light. Among these responses, pmgA mutants seemed to specifically lack the modulation of photosystem stoichiometry; the change in phycocyanin content was normal and the initial decrease in the Chl content was also not affected in the mutant (Hihara et al. 1998). The specific defect in photosystem modulation in pmgA mutants was confirmed in this study. Cells grown in high light showed a different light response curve of qN from the cells grown in low light for both wild-type and pmgA mutants (Fig. 2), which is a good indicator of PFD during growth (Campbell and Öquist 1996). This suggests that the general response of pmgA mutants to the light environment is normal, and that the defect is localized in photosystem modulation.
There are many cyanobacterial mutants with a photosystem stoichiometry different from that in wild-type cells. The disruption of genes encoding PSI or PSII subunits may lead to the decrease in PSI or PSII content. Inactivation of the factors that play a role in transcription, translation or assembly of PSI or PSII can also change photosystem stoichiometry. The pmgA mutants, however, are unique since they cannot be distinguished from wild-type cells during growth at low PFD (Hihara and Ikeuchi 1997, and also see Fig. 3, upper panel in this paper), but show a significant difference when grown at high PFD. Only the modulation system for the photosystem stoichiometry in high light is defective in pmgA mutants.
The plastoquinone pool is more oxidized in the mutant than in wild-type cells under high-light conditions (Fig. 3 and Table 1) possibly because the mutant does not decrease PSI content. pmgA mutants have about 36% more PSI than the wild type when grown under high light (Hihara et al. 1998). More oxidized plastoquinone pool, i.e. higher qP, must lead to higher rate of photosynthetic electron transport. In fact, the higher rate of the electron transport from water to bicarbonate was reported for the pmgA mutants grown under high-light conditions (Hihara et al. 1998). The higher ϕII in pmgA mutants observed in Fig. 4 also supports more efficient photosynthesis in the mutants. Thus, the modulation of the photosystem stoichiometry does not work to keep high rates of photosynthesis after transfer to high-light conditions.
This is in contrast with the case of acclimation of cells to light quality. The relative PSI/PSII ratio is high under condition of PSII excitation (PSII light) and low under condition of PSI excitation (PSI light) in higher plant chloroplasts (Chow et al. 1990, Melis and Harvey 1981), in green algae (Melis et al. 1996) and in phycobilisome-containing organisms (Fujita et al. 1985, Manodori and Melis 1986, Melis et al. 1989, Cunningham et al. 1990). A quantum efficiency of photosynthesis under given light quality conditions was shown to be improved by the modulation of the photosystem stoichiometry (Murakami and Fujita 1988, Melis et al. 1989, Chow et al. 1990, Melis et al. 1996).
The higher ϕII in the pmgA mutants is achieved not only by higher qP but also by higher Fv′/Fm′, the parameter representing effective quantum yield of open PSII (Table 1). Since Fv/Fm, the maximum quantum yield of PSII is also high in the pmgA mutants (Table 1) and Fv/Fm is more sensitive to high light in wild type (Fig. 5), the low Fv′/Fm′ in the wild type compared with the pmgA mutants can be attributed to the photoinhibition of PSII under growth light. The extent of the photoinhibition of PSII is well correlated with the redox state of plastoquinone pool (Huner et al. 1996). The high sensitivity of PSII in the wild type to photoinhibition may be another consequence of the more reduced plastoquinone pool due to the decreased PSI content under high-light conditions.
Then why is the PSI content decreased under high light in the wild-type strain? Although the wild-type strain showed slower growth in the first day following the shift to high light, wild-type cells continued growing even under a prolonged high-light condition. In contrast, the pmgA mutants could not survive under such condition. We hypothesize that the higher initial electron transfer rate in the pmgA mutants, which results in better growth, ultimately causes cell death. It was previously shown that PSI electron transfer rate of cells mediated by DAD increased after 48 h following the shift to high light (Hihara and Ikeuchi 1998). Since PSI electron transport is limited by the transfer of DAD into cells, the membrane permeability of the pmgA mutant cells was assumed to be increased after 48 h following the shift to high light. The higher electron transport may lead to higher production of reactive oxygen species, and this may further affect membrane permeability through the peroxidation of membrane lipid. Although we have no evidence of the involvement of the reactive oxygen species at present, the enhanced electron transfer seems to be in fact the cause of the defect in the pmgA mutants under prolonged exposure to high light. This hypothesis is in agreement with the result that the addition of a sublethal concentration of DCMU rescued the phenotype of decreased growth rate after 3 d in high light (Fig. 7). Alternatively, change of the redox state of plastoquinone pool may be another possible cause of the cell death. Since various metabolic processes are under the control of redox state, the altered redox state (more oxidized plastoquinone pool) in the pmgA mutants would cause the cell death though abnormal metabolic processes. In this case, however, the rescuing effect of DCMU, which would make plastoquinone pool more oxidized, seems to be difficult to interpret.
Stress conditions other than high-light stress also lead to a down regulation of PSII. In cyanobacteria the phycobilin content is usually decreased under nutrient stress. A mutant strain of Synechococcus sp. strain PCC 7942, in which a gene encoding a response regulator, NblR, was inactivated, did not degrade phycobilisome during nutrient limitation and died rapidly when deprived of either sulfur or nitrogen, or when exposed to high light (Schwarz and Grossman 1998). Similarly, in Chlamydomonas reinhardtii, the efficiency of PSII electron transfer was reduced during sulfur deprivation, while the SacI mutant did not show such an acclimating process (Wykoff et al. 1998). This mutant became light-sensitive during sulfur deprivation because it could not down regulate photosynthetic electron transport. Thus, the down regulation of PSII is important for survival under nutrient stress as well as high-light stress. However, the mechanism to down regulate PSII activity under high-light stress seems to be different from that under nutrient stress.
In conclusion, down regulation of photosynthetic electron transport was observed in wild-type cells under high-light conditions by the decrease in the PSI content and photoinhibition of PSII. Without the decrease in the PSI/PSII ratio, pmgA mutants showed better growth in a short-term high-light condition, but could not grow under long-term high-light conditions. Thus, physiological role of the modulation of photosystem stoichiometry seems not to maintain efficient photosynthesis but to protect the cells from oxidative damage under prolonged high-light conditions.
Acknowledgements
We thank Prof. Himadri Pakrasi for the critical reading of the manuscript. This research was supported in part by Grant-in-Aid for Scientific Research (B) (11554035 to M.I. and 11440233 to K.S.) from the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Priority Area (C) Genome Biology (12206029 to K.S.) from the Ministry of Education, Science, Sports and Culture, and a grant for Scientific Research from Human Frontier Science Program (to M.I.).
Corresponding author: E-mail, sonoike@k.u-tokyo.ac.jp.
WT | pmgA(L65F) | pmgA::SpR | |
ϕII | 0.243±0.003 | 0.296±0.010 | 0.304±0.020 |
qP | 0.643±0.003 | 0.703±0.018 | 0.702±0.016 |
Fv′/Fm′ | 0.378±0.003 | 0.422±0.007 | 0.431±0.002 |
qN | 0.575±0.012 | 0.557±0.013 | 0.553±0.029 |
Fv/Fm | 0.586±0.006 | 0.625±0.001 | 0.631±0.005 |
WT | pmgA(L65F) | pmgA::SpR | |
ϕII | 0.243±0.003 | 0.296±0.010 | 0.304±0.020 |
qP | 0.643±0.003 | 0.703±0.018 | 0.702±0.016 |
Fv′/Fm′ | 0.378±0.003 | 0.422±0.007 | 0.431±0.002 |
qN | 0.575±0.012 | 0.557±0.013 | 0.553±0.029 |
Fv/Fm | 0.586±0.006 | 0.625±0.001 | 0.631±0.005 |
The values represent mean±standard deviation. Standard deviation was calculated for three independent measurements with one same sample. Please note that the standard deviation among the values obtained with samples from different culture experiments is very large, and only the values obtained with the samples from the same set of experiments can be compared.
WT | pmgA(L65F) | pmgA::SpR | |
ϕII | 0.243±0.003 | 0.296±0.010 | 0.304±0.020 |
qP | 0.643±0.003 | 0.703±0.018 | 0.702±0.016 |
Fv′/Fm′ | 0.378±0.003 | 0.422±0.007 | 0.431±0.002 |
qN | 0.575±0.012 | 0.557±0.013 | 0.553±0.029 |
Fv/Fm | 0.586±0.006 | 0.625±0.001 | 0.631±0.005 |
WT | pmgA(L65F) | pmgA::SpR | |
ϕII | 0.243±0.003 | 0.296±0.010 | 0.304±0.020 |
qP | 0.643±0.003 | 0.703±0.018 | 0.702±0.016 |
Fv′/Fm′ | 0.378±0.003 | 0.422±0.007 | 0.431±0.002 |
qN | 0.575±0.012 | 0.557±0.013 | 0.553±0.029 |
Fv/Fm | 0.586±0.006 | 0.625±0.001 | 0.631±0.005 |
The values represent mean±standard deviation. Standard deviation was calculated for three independent measurements with one same sample. Please note that the standard deviation among the values obtained with samples from different culture experiments is very large, and only the values obtained with the samples from the same set of experiments can be compared.
Abbreviations
- Fo, Fm, Fv
minimal, maximal and variable level of Chl a fluorescence
- Fo′, Fm′, Fv′
minimal, maximal and variable level of Chl a fluorescence under excitation light
- PFD
photon flux density
- qP, qN
photochemical and non-photochemical quenching of Chl a fluorescence
- ϕII
effective quantum yield of electron transfer through PSII.
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