Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature 1

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1 Plant Physiology, February 1999, Vol. 119, pp , American Society of Plant Physiologists Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature 1 Toshio Sakamoto and Donald A. Bryant* Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20 C and 38 C, the growth rate decreased with decreasing temperature, and growth ceased at 15 C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38 C remained at 15 C. Oxygen evolution was rapidly inactivated at high light intensity (3 me m 2 s 1 ) at 15 C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 E m 2 s 1 ) for growth at 15 C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15 C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/ nitrite transporter limits growth at low temperature. Ambient temperature is a fundamental physical parameter that can fluctuate substantially in nature, and thus cyanobacteria are expected to exhibit complex adaptive or acclimative responses to changes in temperature. Certain cyanobacteria, such as species of Anabaena, Microcystis, Trichodesmium, and Synechococcus, are capable of forming massive blooms (Waterbury et al., 1986). In the spring and summer when nutrients are abundant, cyanobacterial blooms occur as the water temperature rises, and thus temperature may be an important environmental factor that limits cyanobacterial growth in nature. In spite of the probable importance of temperature as a parameter affecting growth, studies on its effects on cyanobacterial growth and physiology are uncommon. Moreover, identification of the biochemical process responsible for the establishment of the limiting lower temperature for cell growth has not yet been reported, to our knowledge, for any cyanobacterial strain even under laboratory conditions. Until recently, many studies on the low-temperature physiology of cyanobacteria relied on assays of the damage 1 This work was supported by a U.S. Public Health Service grant (no. GM-31625) to D.A.B. * Corresponding author; dab14@psu.edu; fax to photosynthetic activity induced by low-temperature treatments under dark conditions (Murata et al., 1979; Murata and Nishida, 1987; Murata, 1989). However, more recent studies, including those involving genetic manipulation of acyl-lipid desaturation, have focused on the effects of excess illumination in combination with lowtemperature stress in causing damage to PSII (for recent reviews, see Murata and Wada, 1995; Nishida and Murata, 1996). As a working hypothesis to explain chilling injury to cyanobacterial cells, Murata and coworkers initially proposed that a phase separation in the plasma membrane is directly related to the irreversible damage of cells at low temperature that eventually causes cell death (for reviews, see Murata and Nishida, 1987; Murata, 1989). The phase separation of the thylakoid membrane occurs at a higher temperature than the phase separation of the plasma membrane; however, phase separation of the thylakoid membrane causes only reversible loss of photosynthetic activity and does not result in cell death (Murata and Nishida, 1987). The critical temperature at which this irreversible damage to photosynthetic activity occurs can be shifted to lower temperatures as the growth temperature is also decreased (Murata, 1989). Thus, alteration of membrane lipid unsaturation induced at low temperature (Sato and Murata, 1981, 1982) is likely to alter the susceptibility of cells to low temperature in the temperature range from 0 C to 15 C (Ono and Murata, 1982; Murata et al., 1984). Studies performed by gain-of-function analyses with the desa ( 12 acyl-lipid desaturase) gene demonstrated that unsaturation at the 12 position of membrane lipids enhanced the lowtemperature tolerance of oxygen-evolution activity in the cyanobacterium Synechococcus sp. PCC 7942 (the same strain as Anacystis nidulans R2-Spc) (Wada et al., 1990, 1994). The assay used in these studies examined the stability of PSII activity after a low-temperature treatment in the range of 0 C to 15 C without illumination (Wada et al., 1990, 1994). However, no differences in photosynthetic activity were observed between the wild-type strain and the strain transformed with the desa gene in the temperature range of 22 C to 34 C (Wada et al., 1994). Abbreviations: Chl, chlorophyll; PCC, Pasteur Culture Collection; Q 10, the ratio of the rate constants for a reaction at two temperatures 10 C apart. 785

2 786 Sakamoto and Bryant Plant Physiol. Vol. 119, 1999 Low-temperature stress has a synergistic effect with excess-illumination stress in photoinhibition (Powles, 1984). It is believed that an imbalance between energy supply and energy consumption may lead to photoinhibition of PSII, and thus it has been proposed that photosynthetic acclimation to low temperature may overlap with acclimation to excess irradiation (Huner et al., 1996). Recent studies using genetic manipulation of acyl-lipid desaturase genes have suggested that unsaturation of membrane lipids is essential for the establishment of tolerance to low temperature plus excessive light in cyanobacteria (Gombos et al., 1992, 1994; Wada et al., 1992; Tasaka et al., 1996). The recovery of photosynthetic activity at 10 C or 20 C after photoinhibitory damage by exposure of cells to high light intensity was reduced when the desaturation level of membrane lipids decreased (Gombos et al., 1994). Thus, it was proposed that membrane lipid unsaturation may facilitate the repair of the PSII protein complex after photoinhibition at low temperature (Gombos et al., 1992, 1994; Tasaka et al., 1996; Kanervo et al., 1997). Loss-of-function analyses of the desa gene in Synechocystis sp. PCC 6803 (Wada et al., 1992; Tasaka et al., 1996) and in Synechococcus sp. PCC 7002 (Sakamoto et al., 1998) have clearly demonstrated that polyunsaturated fatty acids are necessary for cell growth at temperatures at or below 22 C. However, the inability of mutants lacking polyunsaturated fatty acids to grow at low temperature cannot be explained by the decrease of photosynthetic activity at low temperature, because photosynthetic activity declines in good agreement with Q 10 2 in the temperature range of 15 C to 38 C in these desaturase mutants, although the growth of the mutant cells declines substantially below 22 C (Tasaka et al., 1996; Sakamoto et al., 1998). We have previously demonstrated that growth at low temperature causes the symptoms of nitrogen starvation in the unicellular marine cyanobacterium Synechococcus sp. PCC 7002 and also in the freshwater cyanobacteria Synechococcus sp. PCC 6301 and Synechocystis sp. PCC 6803 when cells are grown with nitrate (Sakamoto and Bryant, 1998). Cells of Synechococcus sp. PCC 7002 became chlorotic and grew arithmetically at 15 C in a medium containing nitrate as the sole nitrogen source. However, when cells were grown at 15 C on urea as the nitrogen source, cells grew exponentially and the symptoms of chlorosis were not observed (Sakamoto and Bryant, 1998). These studies strongly suggested that impairment of nitrate assimilation limits cell growth at low temperature in cyanobacteria. Nitrogen and carbon metabolism have been well characterized in the unicellular cyanobacterium Synechococcus sp. PCC 6301 and the closely related, transformable strain Synechococcus sp. PCC 7942 (Flores and Herrero, 1994). Wild-type cells of these two strains contain only saturated and monounsaturated fatty acids (Murata et al., 1992), and thus these cyanobacteria are known to be rather sensitive to low temperature (Wada et al., 1990). In this study we examined the temperature dependence of cell growth, photosynthesis, photoinhibition, and nitrate assimilation in Synechococcus sp. PCC 6301 to determine which factor limits the growth of this organism at low temperature. MATERIALS AND METHODS Organism and Culture Conditions A laboratory wild-type strain of the freshwater cyanobacterium Synechococcus sp. PCC 6301 was grown photoautotrophically under constant illumination of 50 to 250 E m 2 s 1 from cool-white fluorescent lamps with aeration by 1% (v/v) CO 2 in air in B-Hepes medium (Dubbs and Bryant, 1991). The growth temperature was maintained within 1 C by a water bath. Cell growth was monitored by the increase of light scattering of liquid cultures by measuring the optical density at 550 nm, which was determined with a spectrophotometer (Spectronic 20, Milton Roy, Rochester, NY). The specific growth rate ( ) was calculated by ln 2/(doubling time). A cell suspension from an exponential-phase culture grown at 38 C with an optical density at 550 nm of 1.0 contained g Chl ml 1 (n 5) and cells ml 1 ( 5%; n 5) as determined by a direct microscopic count. Photosynthetic Oxygen-Evolution Rate Cells were collected by centrifugation and resuspended in 25 mm Hepes-NaOH buffer, ph 7.0. The Chl concentration was adjusted to 5 g ml 1, and photosynthetic oxygen evolution (H 2 OtoCO 2 ) from whole cells was measured with a Clark-type oxygen electrode using a saturating concentration of inorganic carbon (10 mm NaHCO 3 ) as the final electron acceptor. Saturating actinic light (3 me m 2 s 1 ) was provided from a tungstenhalogen lamp after passage through a 500-nm cutoff filter and a 3-cm layer of water as a heat filter. The dependence of the rate of oxygen evolution on light intensity was determined by varying the light intensity with neutraldensity filters (Melles Griot, Irvine, CA). The assay temperature was maintained at the specified temperature by circulating water through the jacket of the cuvette. The concentration of oxygen in air-saturated distilled water at a specified temperature was calculated from the equation of Truesdale and Downing (1954). Photoinhibition at 15 C Cells grown at 38 C were suspended in B-Hepes medium supplemented with 10 mm NaHCO 3 to give a Chl concentration of 5 g ml 1. The samples (3 ml) in mm glass culture tubes were maintained at a given temperature by a circulating-water bath. After the illumination treatment at a light intensity of 250 E m 2 s 1 or3mem 2 s 1 provided by a halogen floodlight for the specified period, 60 L of0.5m NaHCO 3 was added to the 3-mL sample to give a final concentration of NaHCO 3 in the assay medium of more than 10 mm, and photosynthetic oxygen evolution (H 2 OtoCO 2 ) from whole cells was measured with a Clarktype oxygen electrode at 38 C. Nitrate Consumption by Whole Cells Exponentially growing cells at 38 C (optical density at 550 nm approximately 0.5) were collected by centrifuga-

3 Low-Temperature Sensitivity of Nitrate Assimilation 787 tion and resuspended at a Chl concentration of 5 g ml 1 in 25 mm Hepes-NaOH buffer, ph 7.0, containing 10 mm NaHCO 3 and 0.5 mm l-met sulfoximine (M-5379, Sigma). The temperature of the sample (25 ml) in a 50-mL jacketed beaker (Kontes Glass, Vineland, NJ) was maintained at the specified temperature ( 0.5 C) by circulating water through the jacket and was monitored by a temperature probe (model T201, Radiometer, Westlake, OH) in the sample chamber. Illumination (250 E m 2 s 1 ) was provided from a halogen floodlight after passage through a 5-cm layer of water as a heat filter. The light intensity was varied between 50 and 1000 E m 2 s 1 by adjusting the voltage supplied to the lamp, and the light intensity was measured directly in the cell suspension using a light meter (model QSL-100, Biospherical Instruments, San Diego, CA). The assay was started by the addition of NaNO 3 to give an initial concentration of 100 m in the assay medium. The disappearance of nitrate from the medium was directly monitored in real time by an ion meter (model PHM240, Radiometer) equipped with a combination nitrate electrode (model 9746 BN, Orion, Beverly, MA). Changes in the electrode potential, and thus the nitrate concentration, were monitored with a chart recorder. Preincubation of cells for about 15 min at 38 C in the presence of l-met sulfoximine was required to obtain the maximum steadystate rate of nitrate consumption. All nitrate consumption rates were calculated from the initial linear phase of nitrate consumption to give the maximal rate at a given temperature. Nitrate Reductase and Nitrite Reductase Activities in Toluene-Permeabilized Cells Nitrate reductase activity was measured essentially as described previously with cells permeabilized with toluene using an artificial electron donor, sodium hydrosulfitereduced methyl viologen (Herrero et al., 1981). Cells were collected by centrifugation and washed in 25 mm Hepes- NaOH buffer, ph 7.0. Toluene (80 L) was added to the concentrated cell suspension (2 ml at approximately 250 g Chl ml 1 )in25mm Hepes-NaOH buffer, ph 7.0, and the cell suspension was vigorously agitated with a Vortex mixer. The toluene-treated cells were kept on ice, and a portion was added to a reaction mixture to start the assay. The reaction mixture (5.0 ml) contained 0.1 m Na 2 CO 3 - NaHCO 3 (ph 10.5), 1 mm KCl, 5 mm NaNO 3,4mm methyl viologen (M-2254, Sigma), 10 mm sodium hydrosulfite, and toluene-treated cells (2.5 g Chl ml 1 for 38 C and 30 C assays, and 5 g Chl ml 1 for 22 C and 15 C assays). Sodium hydrosulfite (50 L of a 1.0 m stock) in N 2 -sparged 0.3 m NaHCO 3 was added to the reaction mixture just before the start of the reaction to reduce the methyl viologen. Aliquots of 0.7 ml were taken from the reaction mixture at 1-min intervals for the 38 C, 30 C, and 22 C assays and at 2-min intervals for the 15 C assay, the reaction was terminated by vigorous agitation to oxidize the sodium hydrosulfite, and the formation of nitrite was then determined. Nitrite reductase activity was measured with toluenepermeabilized cells using sodium hydrosulfite-reduced methyl viologen, essentially as described previously (Herrero and Guerrero, 1986). The reaction mixture (5 ml) contained 25 mm Hepes-NaOH, ph 7.0, 1 mm KCl, 100 m NaNO 2,5mm methyl viologen, 20 mm sodium hydrosulfite, and 5 g Chl ml 1 toluene-treated cells. Sodium hydrosulfite (100 L ofa1.0mstock) in N 2 -sparged 0.3 m NaHCO 3 was added to the reaction mixture just before the start of the reaction to reduce the methyl viologen. Aliquots of 0.7 ml were taken from the reaction mixture at 5-min intervals for the 38 C assay and at 30-min intervals for the 15 C assay, and the reaction was terminated by vigorous agitation. The disappearance of nitrite from the medium was then determined. Nitrite Consumption by Whole Cells Exponentially growing cells at 38 C were collected by centrifugation and resuspended in 25 mm Hepes-NaOH buffer (ph 7.0) or 3-[cyclohexylamino]-1-propanesulfonic acid-naoh buffer (ph 10.0) containing 10 mm NaHCO 3 and 0.5 mm l-met sulfoximine. The Chl concentration was adjusted to 5 g ml 1. The sample (5.0 ml) was maintained at a specified temperature ( 0.5 C) by a circulating water bath. After a 15-min preillumination period (250 E m 2 s 1 ), the reaction was started by the addition of NaNO 2 to an initial concentration of 100 m in the assay medium. Aliquots of 0.7 ml were periodically taken from the reaction mixture, and the disappearance of nitrite from the medium was determined chemically. Determination of Nitrite Nitrite concentrations were determined by the diazo coupling method (Nicholas and Nason, 1957). After cells were removed by centrifugation, 0.5 ml of the supernatant was transferred to a test tube, and 0.5 ml of 1% (w/v) sulfanilamide (S-9251, Sigma) in 3 m HCl, 0.5 ml of 0.02% (w/v) N-(1-napthyl)ethylenediamine (N-5889, Sigma), and 0.8 ml of distilled water was added to the sample. The A 540 was measured with a spectrophotometer (model 14R, Cary, San Fernando, CA) modified for computerized data acquisition by On-Line Instrument Systems (Bogart, GA). The nitrite concentration was determined from a standard curve constructed with known NaNO 2 concentrations (1 100 m). RESULTS Cell Growth at Low Temperature When grown at 38 C under 250 E m 2 s 1 in B-Hepes medium with aeration with 1% (v/v) CO 2 in air, exponentially growing wild-type cells of Synechococcus sp. PCC 6301 have a doubling time of 4.5 to 5 h. When cells growing exponentially at 38 C were transferred to lower temperatures, cell growth slowed after the temperature shift, and no cell growth occurred after a temperature shift to 15 C (Figs. 1 and 2A). When cells grown at 38 C were transferred to 15 C under reduced light intensity (50 E m 2 s 1 ), cell growth was minimal and the cell density did not double even after 7dofincubation (Fig. 2B). However,

4 788 Sakamoto and Bryant Plant Physiol. Vol. 119, 1999 indicate that a decrease in the photosynthetic capacity of the cells was not responsible for the limitation in cell growth at 15 C, and that the photochemical reactions leading to oxygen evolution, ATP and NADPH production, and assimilation of inorganic carbon are still functional at 15 C. Damage of Photosynthesis Activity at 15 C Figure 1. Growth curves for wild-type cells of Synechococcus sp. PCC 6301 after temperature shifts to various temperatures. Cells grown at 38 C were inoculated into fresh B-Hepes medium containing 12 mm NaNO 3 as a nitrogen source and grown at 38 C under 250 E m 2 s 1 (E). After 6hat38 C, the exponentially growing cells at 38 C were transferred to 24 C (F), 20 C ( ), or 15 C (f) under otherwise identical growth conditions. Cell growth was monitored by the optical density at 550 nm. The data shown are from a single experiment, which was repeated four times at 38 C, four times at 24 C, twice at 20 C, and three times at 15 C, and the results were consistent and reproducible. To evaluate damage to the photosynthetic machinery at 15 C under high-light-intensity conditions, the residual oxygen-evolution activity was determined after an exposure of cells to high light intensity at 15 C for varying periods of time. A 1-h treatment in the dark at 15 C had no effect on oxygen-evolution activity (Fig. 5). When cells were treated at 15 C for 1 h under a light intensity of 250 E m 2 s 1 (i.e. at the normal light intensity for cell growth), almost all of the initial oxygen-evolving activity remained (Fig. 5). However, all oxygen-evolution activity was lost when cells were exposed at 15 C for 1htohigh light intensity (3 me m 2 s 1 ); this light intensity is 12-fold higher than that used with the normal cell-growth conditions (Fig. 5). When the light intensity was increased further, oxygen-evolution activity was more rapidly damaged. No oxygen-evolution activity remained after only a 20-min treatment at a very high light intensity (7 me m 2 s 1 ) at 15 C (data not shown). These data demonstrate that photodamage to the photosynthetic machinery does not when the 15 C-treated cells were returned to 38 C, cell growth resumed, with a doubling time of approximately 5 h after a short lag period (Fig. 2). Because the cells resumed growth after the temperature shift to 38 C, the inhibition of cell growth at 15 C is reversible; cells were not killed even after7dofincubation at 15 C under continuous illumination at 250 E m 2 s 1. Figure 3 shows the temperature dependence of the steady-state growth rate. The growth rate decreased linearly as a function of decreasing growth temperature over the temperature range of 15 C to 38 C under constant light intensity (250 E m 2 s 1 ) and CO 2 supply (bubbling with 1% [v/v] CO 2 in air). These results establish that the limiting lower temperature for cell growth of Synechococcus sp. PCC 6301 is 15 C under laboratory growth conditions that provide sufficient light and excess nutrients. Effect of Temperature on Photosynthetic Oxygen-Evolution Rate Figure 4 shows the assay temperature dependence of the rate of photosynthetic oxygen evolution under saturating actinic light and with a saturating supply of NaHCO 3 for Synechococcus sp. PCC 6301 cells grown at 38 C. These data show that the oxygen-evolution rate decreases approximately 4-fold over a 23 C change in temperature (from 38 C to 15 C), and that more than 20% of the oxygenevolution capacity of cells assayed at 38 C still remains at 15 C. The decrease in activity is simply that predicted by the Q 10 2 rule for biochemical reactions. These results Figure 2. Growth curves for Synechococcus sp. PCC 6301 at 15 C. Cells grown at 38 C under 250 E m 2 s 1 were inoculated into fresh B-Hepes medium containing 12 mm NaNO 3 as a nitrogen source and grown at 15 C under 250 E m 2 s 1 (A) and 50 E m 2 s 1 (B). After 168 h at 15 C, the cultures were transferred to 38 C at a light intensity of 250 Em 2 s 1. Cell growth was monitored by the optical density at 550 nm. The data shown are from a single experiment, which was repeated three times, and the results were consistent and reproducible.

5 Low-Temperature Sensitivity of Nitrate Assimilation 789 Figure 3. Temperature dependence of growth rate in Synechococcus sp. PCC Cells were continuously grown at a given temperature under 250 E m 2 s 1 in B-Hepes medium, and the growth rates during the exponential phase were obtained. The growth rate ( ) was calculated (see text). The data shown are the average of seven experiments at 38 C, six experiments at 30 C, three experiments at 24 C, four experiments at 20 C, and six experiments at 15 C. The variance was so small that no error bars are shown for the data points at 15 C, 20 C, and 24 C. inhibit cell growth at 15 C at the normal growth light intensity (250 E m 2 s 1 ), although very strong illumination (3 7 me m 2 s 1 ) at 15 C caused a very rapid loss of photosynthetic oxygen-evolution activity, as reported previously in Synechocystis sp. PCC 6803 (Gombos et al., 1992) and Synechococcus sp. PCC 7942 (Gombos et al., 1997). Effect of Temperature on the Rate of Nitrate Assimilation Figure 6 shows the temperature dependence of the rate of nitrate consumption by cells of Synechococcus sp. PCC Figure 4. Temperature dependence of photosynthetic oxygen evolution in Synechococcus sp. PCC Cells were grown at 38 C under 250 E m 2 s 1, and photosynthetic oxygen evolution from whole cells was measured using 10 mm NaHCO 3 as a final electron acceptor under saturating actinic light (3 me m 2 s 1 ) at various assay temperatures. The data shown are the average of seven experiments at 38 C, five experiments at 30 C, four experiments at 22 C, and six experiments at 15 C. Figure 5. Photodamage of photosynthetic oxygen evolution activity at 15 C in Synechococcus sp. PCC Cells were grown at 38 C under 250 E m 2 s 1, and cells in growth medium supplemented with 10 mm NaHCO 3 were treated at 15 C under a high light intensity of 3 me m 2 s 1 (E), dark (F), and a normal growth light of 250 E m 2 s 1 ( ). Photosynthetic oxygen evolution from whole cells was measured at 38 C. The data shown are the average of two independent experiments for dark incubation conditions and 250 E m 2 s 1, and of three experiments for 3 me m 2 s Cells had the highest nitrate-consumption rate when assayed at 38 C; the nitrate (100 m) in the medium was completely consumed by cells equivalent to 5 g Chl ml 1 in approximately 20 min under these assay conditions (nitrate consumption rate 60 mol nitrate mg 1 Chl h 1 ). Thus, the maximal cellular consumption rate for nitrate was equivalent to approximately 0.66 fmol nitrate cell 1 h 1. Nitrate consumption was light dependent in Synechococcus sp. PCC 6301, and at a light intensity of 125 E m 2 s 1 the rate of nitrate consumption was one-half of the maximal value (data not shown). The nitrate consumption rate was saturated at the light intensity (250 E m 2 s 1 ) normally used for cell growth, and no further increase in the rate of nitrate consumption was observed when the light intensity was increased up to 1 me m 2 s 1. The nitrate-consumption rate decreased as the assay temperature decreased, and no nitrate consumption occurred at 15 C (Fig. 6). Even when the time of the assay at 15 C was extended for up to 45 min, little change in the nitrate concentration in the medium was detected, and the calculated rate of nitrate consumption was less than the detection limit of 0.5 mol nitrate mg 1 Chl h 1 at 15 C (data not shown). These data indicate that Synechococcus sp. PCC 6301 cells are unable to take up nitrate from the medium at a temperature of 15 C or less. To determine whether the cessation of nitrate consumption at 15 C was reversible or irreversible, nitrate consumption was measured by shifting the assay temperature between 38 C and 15 C (Fig. 7). When nitrate consumption was measured at 38 C, cells rapidly consumed nitrate from the medium at the calculated rate of 60 mol nitrate mg 1 Chl h 1. When 100 m sodium nitrate was added back to the medium containing these same cells and the assay

6 790 Sakamoto and Bryant Plant Physiol. Vol. 119, 1999 Nitrate is taken up from the medium by the nitrate transporter and is first reduced to nitrite inside of cells by nitrate reductase; nitrite is subsequently reduced to ammonia through the action of nitrite reductase (Flores and Herrero, 1994). To identify which step in nitrate assimilation is rate limiting at 15 C, nitrate reductase activity was assayed by determining the rate of appearance of nitrite using toluene-permeabilized cells at various temperatures with an artificial electron donor, sodium hydrosulfitereduced methyl viologen (Fig. 8). The nitrate reductase activity decreased as the assay temperature decreased, and about 11% of the nitrate reductase activity at 38 C remained when the enzyme was assayed at 15 C. Nitrite reductase activity was similarly assayed by following the disappearance of nitrite. Although it is technically difficult to assay this enzyme in the presence of saturating substrate to obtain maximal enzymatic activities (the K m value for nitrite is 40 to 230 m; Flores and Herrero, 1994), it is clear that nitrite reductase retains activity at 15 C. When the enzyme was assayed in the presence of 100 m nitrite at 38 C, nitrite was rapidly reduced at a rate of approximately 43 mol nitrite mg 1 Chl h 1. When the enzyme was assayed at 15 C, nitrite reductase activity was still detected with a rate of 7.5 mol nitrite mg 1 Chl h 1 (17% of the activity at 38 C). Because the overall rate of in vivo nitrate consumption decreases at 15 C to less than 1% of the rate at 38 C, these results indicate that the rate-limiting step in nitrate consumption at 15 C is nitrate transport rather than the activities of either nitrate reductase or nitrite reductase. Figure 6. Temperature dependence of nitrate-consumption rate in Synechococcus sp. PCC Disappearance of nitrate from the assay medium (25 mm Hepes-NaOH, ph 7.0, 10 mm NaHCO 3, 0.5 mm L-Met sulfoximine, 100 M NaNO 3, and cells equivalent to 5 g Chl ml 1 ) was directly monitored with a nitrate-specific electrode under continuous illumination at a light intensity of 250 E m 2 s 1. The nitrate-consumption rates were calculated from the slopes of the initial disappearance of nitrate from the medium. The samples were preilluminated until the maximum rate of nitrate consumption was obtained at 38 C. The data shown are the average of five experiments at 38 C, four experiments at 30 C, five experiments at 25 C, three experiments at 20 C, and three experiments at 15 C. The variance was so small that no error bars are shown for the data points at 15 C and 20 C. Effect of Temperature on the Rate of Nitrite Assimilation Nitrate and nitrite are actively taken up from the medium by cells via the nitrate/nitrite transporter, an ATP binding cassette transporter with a periplasmic nitrate- and nitrite-binding protein (Omata et al., 1993; Luque et al., 1994; Maeda and Omata, 1997). Nitrite can also be taken up by cells by passive diffusion of nitrous acid at neutral ph (Flores et al., 1987; Flores and Herrero, 1994; Luque et al., 1994). To examine the effects of low temperature on nitrite uptake, in vivo nitrite consumption from the medium by cells was measured at 38 C and 15 C at ph 7.0 and 10.0 temperature was decreased to 15 C, virtually no nitrate consumption occurred. When the assay temperature was simply increased to 38 C, these same cells resumed nitrate consumption at the original rate (Fig. 7). Thus, the same cells could consume nitrate at 38 C but not at 15 C during these temperature cycles. It should be noted that a slight decrease in the rate of nitrate consumption did occur after the third cycle of uptake at 38 C (Fig. 7). These results indicate that the cessation of nitrate consumption at 15 C is reversible and show that nitrate consumption can quickly resume (within a few minutes) when cells are returned to 38 C. Effect of Temperature on Nitrate Reductase and Nitrite Reductase Activities in Vitro Figure 7. Effect of alternating temperature treatments on nitrate consumption in Synechococcus sp. PCC The disappearance of nitrate from the assay medium (25 mm Hepes-NaOH, ph 7.0, 10 mm NaHCO 3, 0.5 mm L-Met sulfoximine, 100 M NaNO 3, and cells equivalent to 5 g Chl ml 1 ) was directly monitored by a nitratespecific electrode. The temperature was maintained by a circulatingwater bath, and the sample temperature was monitored using a temperature probe in the reaction vessel. Approximately 5 min was required for the temperature changes indicated using this system, and the samples were shaded during the temperature change. The data shown are from a single experiment, which was repeated twice and identical results were obtained.

7 Low-Temperature Sensitivity of Nitrate Assimilation 791 Figure 8. Effect of assay temperature on nitrate reductase. Nitrate reductase activity was measured in toluene-permeabilized cells using an artificial electron donor, sodium hydrosulfite-reduced methyl viologen. The data shown are the average of five experiments at 38 C, four experiments at 30 C, four experiments at 22 C, and five experiments at 15 C. The variance was so small that no error bars are shown for the data points at 15 C and 22 C. (Fig. 9). Cells rapidly consumed nitrite at a rate of approximately 87 mol mg 1 Chl h 1 at 38 C at ph 7.0. Because no nitrite was detected in the medium after only a 15-min incubation at 38 C at ph 7.0, nitrite apparently was taken up by a high-affinity transporter under these conditions. Although the rate of nitrite consumption was low, cells could still slowly consume nitrite from the medium at 15 C at ph 7.0 at a rate of about 8 mol mg 1 Chl h 1. This observation indicates that nitrite reductase is functional in living cells at 15 C; moreover, the observed rate of nitrite consumption was similar to that for the in vitro activity of nitrite reductase measured in toluene-treated cells at 15 C. When nitrite consumption was assayed at ph 10.0, cells could consume nitrite at a rate of 48 mol mg 1 Chl h 1 at 38 C, but little or no nitrite consumption occurred at 15 C at ph 10.0; the calculated rate of nitrite consumption was less than 0.7 mol mg 1 Chl h 1. These results are consistent with the notion that nitrous acid enters cells by passive diffusion at 15 C and neutral ph, but that active transport of nitrite, and thus presumably nitrate as well, ceases at 15 C. At 38 C and neutral ph, nitrite is apparently taken up both by active transport of nitrite and by passive diffusion of nitrous acid. DISCUSSION The Limiting Factor for Cell Growth at 15 C Synechococcus sp. PCC 6301 (formerly called Anacystis nidulans, a strain very closely related to the transformable organism Synechococcus sp. PCC 7942; Golden et al., 1989) is a mesophilic cyanobacterium that is typically cultured at 20 C to 30 C under laboratory growth conditions. Stanier et al. (1971) reported that 43 C was the maximal temperature for growth under laboratory conditions for this organism; however, to our knowledge, no one has reported the lowest temperature for growth of Synechococcus sp. PCC 6301, although this strain is considered to be sensitive to low temperature (Wada et al., 1990). In the present study we demonstrated that cells of Synechococcus sp. PCC 6301 could not grow at 15 C (Figs. 1 and 2) and that the nitrate transporter likewise does not function at 15 C (Figs. 6 and 7). The cessation of nitrate assimilation at 15 C was reversible, because nitrate consumption quickly resumed when cells were returned to 38 C (Fig. 7), and this result is consistent with the rapid recovery of growth after a temperature shift to 38 C (Fig. 2). The decrease in photosynthetic oxygen evolution activity at low temperature was much smaller than the decrease in the growth rate, and substantial photosynthetic activity remained at 15 C (Fig. 4). Little or no loss of oxygen-evolution activity was observed under illumination with 250 E m 2 s 1 (the normal light intensity for cell growth) at 15 C (Fig. 5), demonstrating that cell growth at low temperature is not limited by the decrease in the rate of photosynthesis or by photodamage to the photosynthetic apparatus in this cyanobacterium at 15 C. The data presented here are consistent with our recent report that growth at low temperature causes nitrogen limitation in the cyanobacterium Synechococcus sp. PCC 7002 and other cyanobacteria (Sakamoto and Bryant, 1998), and strongly suggest that nitrate uptake is the limiting step for cell growth at low temperature. Figure 9. Nitrite consumption at 15 C by whole cells of Synechococcus sp. PCC Disappearance of nitrite from the assay medium was measured at 38 C at ph 7.0 (E), 38 C at ph 10.0 ( ), 15 C at ph 7.0 (F), and 15 C at ph 10.0 (f). The samples were preilluminated for 15 min under 250 E m 2 s 1, and 100 M NaNO 2 was added at time 0 to initiate the reaction. The assay medium contained 25 mm Hepes-NaOH (ph 7.0), or 25 mm 3-[cyclohexylamino]-1- propanesulfonic acid-naoh (ph 10.0), 10 mm NaHCO 3, 0.5 mm L-Met sulfoximine, 100 M NaNO 2, and cells equivalent to 5 g Chl ml 1. The nitrite remaining in the medium was determined by the diazo coupling method (see Materials and Methods ). The data shown are the average of four experiments at 38 C at ph 7.0, two experiments at 38 C at ph 10.0, three experiments at 15 C at ph 7.0, and two experiments at 15 C at ph 10.0.

8 792 Sakamoto and Bryant Plant Physiol. Vol. 119, 1999 However, two important questions remain to be answered in future studies: (a) what is the molecular mechanism that causes inactivation of the nitrate transporter at low temperature? and (b) are any other nutrient transporters of the plasma membrane inactivated at low temperature? Regulation of Nitrogen and Carbon Metabolism In this study we developed a system to monitor directly the real-time consumption rate of nitrate by whole cells using a nitrate-specific electrode rather than relying on sampling and assaying to determine the remaining concentration of nitrate in the medium. This system is essential for determining the maximal rate of nitrate consumption at a given temperature. This system also makes it possible to perform continuous measurements with the same sample; by taking advantage of this, it was possible to demonstrate directly that the cessation of nitrate consumption at 15 C is reversible (Fig. 7). Nitrate uptake is generally believed to be the ratelimiting step in nitrate assimilation, and the overall rate of nitrate assimilation is tightly regulated at the nitrate uptake step by the metabolic carbon/nitrogen status of cells (Flores and Herrero, 1994). During the assays of nitrate consumption by whole cells in these studies, no nitrite was detected in the assay medium, but the concentration of ammonium ions increased during the course of assay (data not shown). Thus, the assay described here reflects the overall rate of nitrate consumption; this includes nitrate uptake, the reduction of nitrate to nitrite, and the reduction of nitrite to ammonia inside cells. Because of the presence of l-met sulfoximine, ammonium will not be further assimilated by Gln synthetase, and some ammonia diffuses out of the cells into the medium. Using the nitrate electrode system, we reexamined in Synechococcus sp. PCC 6301 the experiments demonstrating repression of nitrate consumption by ammonia that were reported 22 years ago in Anabaena cylindrica (Ohmori et al., 1977). When l-met sulfoximine, a potent inhibitor of Gln synthetase, was not added to the assay medium, cells of Synechococcus sp. PCC 6301 consumed nitrate from the medium, but the rate of the nitrate consumption was slower than in the presence of l-met sulfoximine (data not shown). When 50 m to1mmammonium chloride was added to the assay medium (without l-met sulfoximine) as cells were consuming nitrate, nitrate consumption stopped very quickly (in as little as 1 min; data not shown). Addition of l-met sulfoximine (0.5 1 mm) to the assay medium containing cells whose nitrate consumption had been arrested by the addition of ammonium chloride caused a very rapid resumption of nitrate consumption (data not shown). These preliminary experiments have shown that the nitrate electrode system has time resolution sufficient to study the kinetics of ammonia repression of nitrate consumption. These studies also showed that the addition of l-met sulfoximine is essential to obtain the maximal rate of nitrate consumption, which reflects the total capacity for nitrate assimilation by whole cells at a given temperature, and clearly showed that the cessation of nitrate assimilation at 15 C does not arise from the regulation of the nitrate transporter through signal transduction mediated by the P II protein (Lee at al., 1998). Nitrate consumption was light dependent in Synechococcus sp. PCC 6301, and the light intensity (250 E m 2 s 1 ) normally used for cell growth was saturating for this process. At this light intensity the rate of photosynthetic oxygen evolution was about mol O 2 mg 1 Chl h 1 at 38 C; this rate is approximately 40% of the lightsaturated rate of approximately 390 mol O 2 mg 1 Chl h 1 at3mem 2 s 1 at 38 C. Thus, as was shown by Hattori (1962), Synechococcus sp. PCC 6301 cells require more light to saturate, and have much greater inherent capacities for, photosynthetic oxygen evolution and carbon assimilation than for nitrate assimilation. This difference can probably be explained by the fact that the number of photosynthetic reaction centers on the thylakoid membranes and the enzymes for carbon fixation are present at much higher levels than the enzymes for the uptake and reduction of nitrate to ammonia. These data also suggest that under optimal growth conditions at 38 C at a light intensity of 250 Em 2 s 1, the ratio of the maximal rates of nitrate assimilation and carbon assimilation is roughly 2.5 (60 mol nitrate mg 1 Chl h 1 versus 150 mol O 2 mg 1 Chl h 1 ). Finally, these data suggest that a metabolic imbalance between nitrogen assimilation and carbon assimilation will inevitably take place when cells are provided with excess light (see below). Photoinhibition at Low Temperature Low-temperature stress has a synergistic effect with irradiation stress in photoinhibition (Powles, 1984). When cells were treated at 38 C for 1 h at a light intensity of 3 me m 2 s 1, approximately 80% of the initial oxygen-evolving activity remained (data not shown), although all oxygenevolving activity was lost when cells were exposed at 15 C for 1 h under the same light intensity (Fig. 5). These results are consistent with previous results that have shown that net damage to the photosynthetic apparatus by excess illumination is much more severe at low temperature. It is believed that PSII is a primary site of damage in photoinhibition (Powles, 1984; Aro et al., 1993), and as a hypothesis for the molecular mechanism of photoinhibition, photoinhibition is postulated to arise from an imbalance between light-induced damage to the D1 protein and the repair of damaged PSII by newly synthesized D1 protein, a protein well known to exhibit a high turnover rate in vivo (Aro et al., 1993; Vasilikiotis and Melis, 1994). The data reported here indicate that the decrease in the maximum capacity for nitrate assimilation is much greater than the decrease in photosynthetic electron transport and carbon assimilation as a function of lowered temperature (compare Figs. 4 and 6). Because no nitrate assimilation occurs at 15 C (Fig. 6), protein synthesis will quickly decrease to the minimal rates allowed by protein turnover in Synechococcus sp. PCC 6301 cells at 15 C. Therefore, D1 protein synthesis will be severely limited, but photochemical reaction rates at 15 C will initially continue at a relatively high

9 Low-Temperature Sensitivity of Nitrate Assimilation 793 level (Fig. 4) under excess illumination until severe photoinhibition occurs (Fig. 5). Membrane Lipid Unsaturation and the Recovery from Photoinhibition It has been proposed that membrane lipid unsaturation may facilitate the recovery from photoinhibition at low temperature (Gombos et al., 1994, 1997; Kanervo et al., 1997) and may be involved in the processing of the D1 protein in PSII (Kanervo et al., 1997). This conclusion was based on loss-of-function studies with the acyl-lipid desaturases in Synechocystis sp. PCC 6803 (Gombos et al., 1994; Tasaka et al., 1996; Kanervo et al., 1997) and gain-offunction studies with the 12 acyl-lipid desaturase for Synechococcus sp. PCC 7942 (Gombos et al., 1997), although membrane lipid unsaturation has no effect on the photosynthetic activity in the physiological temperature range of 15 C to 38 C in cyanobacterial strains of Synechococcus sp. PCC 7942 (Wada et al., 1994), Synechocystis sp. PCC 6803 (Tasaka et al., 1996), and Synechococcus sp. PCC 7002 (Sakamoto et al., 1998). However, other studies demonstrate that nitrogen limitation, and thus limitations on protein synthesis, limits growth of cyanobacteria and acyl-lipid desaturase mutant strains at low temperature (Sakamoto and Bryant, 1998; Sakamoto et al., 1998). An important control experiment was missing in the study by Kanervo et al. (1997): the measurement of the rate of protein synthesis in the desa desd mutant at low temperature. Because a much higher amount of [ 35 S]Met was apparently incorporated into wild-type cells during in vivo pulse-labeling experiments at 18 C (Kanervo et al., 1997), the rate of protein synthesis may have limited the recovery from photoinhibition of the mutant at low temperature. It has been shown that the desa desd mutant cells have a substantially slower growth rate than wild-type cells at 25 C and do not grow at or below 20 C (Wada et al., 1992; Tasaka et al., 1996). These observations, as well as studies indicating that nitrogen limitation probably occurs in Synechocystis sp. PCC 6803 cells grown at 15 C (Sakamoto and Bryant, 1998), suggest that the rate of protein synthesis could be quite slow in the desa desd mutant at 18 C. As shown in the results presented here, growth clearly ceases under conditions that cause nitrogen limitation but not photoinhibition. Changes of the physical phases of the membrane lipids were studied as a function of temperature in A. nidulans (the same or a very closely related strain to strain Synechococcus sp. PCC 6301 used in these studies) by various independent techniques (Ono and Murata, 1982; Murata, 1989). In A. nidulans cells grown at 38 C, the phase separation of the thylakoid membrane takes place at 23 C to 26 C (Ono and Murata, 1982) and the phase separation of the plasma membrane occurs at 13 C to 16 C (Murata, 1989). These observations suggest that a phase separation of the plasma membrane at 15 C might impair the function of the nitrate transporter in the plasma membrane, although this cannot explain why photosynthetic electron transport activity shows greater tolerance to low temperature than nitrate transport. NrtA, the substrate-binding protein of the nitrate transporter, is a lipoprotein that is anchored to the plasma membrane by a fatty acyl moiety (Maeda and Omata, 1997); thus, the function of NrtA might be impaired by a decrease of membrane lipid fluidity or by the phase separation at low temperature. If this suggestion is correct, it would be anticipated that other major nutrient transporters (e.g. those for phosphate and sulfate) might also be functionally impaired at low temperature. However, because nitrogen must be supplied in the greatest amount for growth among elements other than carbon, it is obvious why nitrate transport can still be growth limiting at low temperature (Sakamoto and Bryant, 1998). Two additional issues will be addressed in future studies. Homeoviscous adaptation of the membrane lipids in A. nidulans cells has been reported (Murata, 1989). In cells grown at 28 C the phase-separation temperature of the thylakoid membrane was lowered from 13 C to 16 C, and the phase-separation temperature of the plasma membrane was lowered to 5 C (Murata, 1989). Whether such changes in the physical characteristics of the membrane lipids allow for acclimation of the functionality of the nitrate transporter at low temperature has not yet been investigated. Synechococcus sp. PCC 7002 is a strain that can synthesize polyunsaturated fatty acids for its membrane lipids (Murata et al., 1992; Sakamoto et al., 1997) and has a lower growth-limiting temperature of 12 C (T. Sakamoto and D.A. Bryant, unpublished data). It is interesting that the temperature at which nitrate consumption ceases was found to be 12 C for this organism (T. Sakamoto and D.A Bryant., unpublished data). Whether this 3 C difference in the lower limiting temperature for growth of Synechococcus sp. PCC 7002 is attributable to membrane lipid unsaturation or to a difference in the nitrate transporters of these two organisms is unknown at present. In future investigations, we plan genetic reconstruction studies of membrane lipid unsaturation and nitrate transport through manipulation of the genes encoding the nitrate transporters and acyl-lipid desaturases of the two strains of Synechococcus sp. Such studies should help to identify additional important parameters of both acclimative and adaptive changes to low temperature in cyanobacteria. Low-Temperature Adaptation and Membrane Lipid Unsaturation ACKNOWLEDGMENT We thank Dr. Masayuki Ohmori (Tokyo University, Japan) for his helpful suggestions. Received July 10, 1998; accepted November 18, LITERATURE CITED Aro E-M, Virgin I, Andersson B (1993) Photoinhibition of photosystem II: inactivation, protein damage and turnover. Biochim Biophys Acta 1143: Dubbs JM, Bryant DA (1991) Molecular cloning and transcriptional analysis of the cpeba operon of the cyanobacterium Pseudanabaena sp. PCC Mol Microbiol 5: Flores E, Herrero A (1994) Assimilatory nitrogen metabolism and its regulation. In DA Bryant, ed, The Molecular Biology of

10 794 Sakamoto and Bryant Plant Physiol. Vol. 119, 1999 Cyanobacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp Flores E, Hererro A, Guerrero MG (1987) Nitrite uptake and its regulation in the cyanobacterium Anacystis nidulans. Biochim Biophys Acta 896: Golden SS, Nalty MS, Cho DS (1989) Genetic relationship of two highly studied Synechococcus strains designated Anacystis nidulans. J Bacteriol 171: Gombos Z, Kanervo E, Tsvetkova N, Sakamoto T, Aro E-M, Murata N (1997) Genetic enhancement of the ability to tolerate photoinhibition by introduction of unsaturated bonds into membrane glycerolipids. Plant Physiol 115: Gombos Z, Wada H, Murata N (1992) Unsaturation of fatty acids in membrane lipids enhances tolerance of the cyanobacterium Synechocystis PCC 6803 to low-temperature photoinhibition. Proc Natl Acad Sci USA 89: Gombos Z, Wada H, Murata N (1994) The recovery of photosynthesis from low-temperature photoinhibition is accelerated by the unsaturation of membrane lipids: a mechanism of chilling tolerance. Proc Natl Acad Sci USA 91: Hattori A (1962) Light-induced reduction of nitrate, nitrite and hydroxylamine in a blue-green alga, Anabaena cylindrica. Plant Cell Physiol 3: Herrero A, Flores E, Guerrero MG (1981) Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain J Bacteriol 145: Herrero A, Guerrero MG (1986) Regulation of nitrite reductase in the cyanobacterium Anacystis nidulans. J Gen Microbiol 132: Huner NPA, Maxwell DP, Gray GR, Savitch LV, Krol M, Ivanov AG, Falk S (1996) Sensing environmental temperature change through imbalances between energy supply and energy consumption: redox state of photosystem II. Physiol Plant 98: Kanervo E, Tasaka Y, Murata N, Aro E-M (1997) Membrane lipid unsaturation modulates processing of the photosystem II reaction-center protein D1 at low temperature. Plant Physiol 114: Lee H-M, Flores E, Herrero A, Houmard J, Tandeau de Marsac N (1998) A role of the signal transduction protein P II in the control of nitrate/nitrite uptake in a cyanobacterium. FEBS Lett 427: Luque I, Flores E, Herrero A (1994) Nitrate and nitrite transport in the cyanobacterium Synechococcus sp. PCC 7942 are mediated by the same permease. Biochim Biophys Acta 1184: Maeda S-I, Omata T (1997) Substrate-binding lipoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942 involved in the transport of nitrate and nitrite. J Biol Chem 272: Murata N (1989) Low-temperature effects on cyanobacterial membranes. J Bioenerg Biomembr 21: Murata N, Nishida I (1987) Lipids of blue-green algae (cyanobacteria). In PK Stumpf, ed, The Biochemistry of Plants, Vol 9. Academic Press, San Diego, CA, pp Murata N, Ono T-A, Sato N (1979) Lipid phase of membrane and chilling injury in the blue-green alga Anacystis nidulans. In JM Lyons, D Graham, JK Radison, eds, Low Temperature Stress in Crop Plants: The Role of the Membrane. Academic Press, New York, pp Murata N, Wada H (1995) Acyl-lipid desaturases and their importance in the tolerance and acclimation to cold of cyanobacteria. Biochem J 308: 1 8 Murata N, Wada H, Gombos Z (1992) Modes of fatty-acid desaturation in cyanobacteria. Plant Cell Physiol 33: Murata N, Wada H, Hirasawa R (1984) Reversible and irreversible inactivation of photosynthesis in relation to the lipid phases of membranes in the blue-green algae (cyanobacteria) Anacystis nidulans and Anabaena variabilis. Plant Cell Physiol 25: Nicholas DJ, Nason A (1957) Determination of nitrate and nitrite. Methods Enzymol 3: Nishida I, Murata N (1996) Chilling sensitivity in plants and cyanobacteria: the crucial contribution of membrane lipids. Annu Rev Plant Physiol Plant Mol Biol 47: Ohmori M, Ohmori K, Strotmann H (1977) Inhibition of nitrate uptake by ammonia in a blue-green alga, Anabaena cylindrica. Arch Microbiol 114: Omata T, Andriesse X, Hirano A (1993) Identification and characterization of a gene cluster involved in nitrate transport of the cyanobacterium Synechococcus sp. PCC Mol Gen Genet 236: Ono T-A, Murata N (1982) Chilling susceptibility of the blue-green alga Anacystis nidulans. III. Lipid phase of cytoplasmic membrane. Plant Physiol 69: Powles SB (1984) Photoinhibition of photosynthesis induced by visible light. Annu Rev Plant Physiol 35: Sakamoto T, Bryant DA (1998) Growth at low temperature causes nitrogen limitation in the cyanobacterium Synechococcus sp. PCC Arch Microbiol 169: Sakamoto T, Higashi S, Wada H, Murata N, Bryant DA (1997) Low-temperature induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC FEMS Microbiol Lett 152: Sakamoto T, Shen G, Higashi S, Murata N, Bryant DA (1998) Alternation of low-temperature susceptibility of the cyanobacterium Synechococcus sp. PCC 7002 by genetic manipulation of membrane lipid unsaturation. Arch Microbiol 169: Sato N, Murata N (1981) Studies on the temperature shift induced desaturation of fatty acids in monogalactosyl diacylglycerol in the blue-green alga (cyanobacterium), Anabaena variabilis. Plant Cell Physiol 22: Sato N, Murata N (1982) Lipid biosynthesis in the blue-green alga, Anabaena variabilis. II. Fatty acids and lipid molecular species. Biochim Biophys Acta 710: Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35: Tasaka Y, Gombos Z, Nishiyama Y, Mohanty P, Ohba T, Ohki K, Murata N (1996) Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis. EMBO J 15: Truesdale GA, Downing AL (1954) Solubility of oxygen in water. Nature 173: 1236 Vasilikiotis C, Melis A (1994) Photosystem II reaction center damage and repair cycles: chloroplast acclimation strategy to irradiance stress. Proc Natl Acad Sci USA 91: Wada H, Gombos Z, Murata N (1990) Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation. Nature 347: Wada H, Gombos Z, Murata N (1994) Contribution of membrane lipids to the ability of the photosynthetic machinery to tolerate temperature stress. Proc Natl Acad Sci USA 91: Wada H, Gombos Z, Sakamoto T, Murata N (1992) Genetic manipulation of the extent of desaturation of fatty acids in membrane lipids in the cyanobacterium Synechocystis PCC6803. Plant Cell Physiol 33: Waterbury JB, Watson SW, Valois FW, Franks DG (1986) Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. Can Bull Fish Aquat Sci 214: