PhotosynthesisResearch 42: 173-183, 1994. © 1994KluwerAcademicPublishers. Printedin the Netherlands. Regular paper
Changes in the cyanobacterial photosynthetic apparatus during acclimation to macronutrient deprivation J a c k i e L. C o l l i e r 2, S t e p h e n K. H e r b e r t 3, D a v i d C. F o r k 1 & A r t h u r R. G r o s s m a n 1,*
lDepartment of Plant Biology, Carnegie Institution of Washington, 290 Panama Street, Stanford, CA 94305, USA; 2Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-020, USA; 3Department of Biological Sciences, University of Idaho, Moscow, ID 83843, USA; *Author for correspondence and~or reprints Received9 June 1994;acceptedin revisedform28 September1994
Key words: cyanobacteria, cyclic photophosphorylation, nutrient deprivation, photoacoustic spectroscopy, photoinhibition, Photosystem I
Abstract
When the cyanobacterium Synechococcus sp. Strain PCC 7942 is deprived of an essential macronutrient such as nitrogen, sulfur or phosphorus, cellular phycobiliprotein and chlorophyll contents decline. The level of t-carotene declines proportionately to chlorophyll, but the level of zeaxanthin increases relative to chlorophyll. In nitrogen- or sulfur-deprived cells there is a net degradation of phycobiliproteins. Otherwise, the declines in cellular pigmentation are due largely to the diluting effect of continuedcell division after new pigment synthesis ceases and not to net pigment degradation. There was also a rapid decrease in 02 evolution when Synechococcus sp. Strain PCC 7942 was deprived of macronutrients. The rate of O2 evolution declined by more than 90% in nitrogen- or sulfur-deprived cells, and by approximately 40% in phosphorus-deprived cells. In addition, in all three cases the fluorescence emissions from Photosystem II and its antennae were reduced relative to that of Photosystem I and the remaining phycobilisomes. Furthermore, state transitions were not observed in cells deprived of sulfur or nitrogen and were greatly reduced in cells deprived of phosphorus. Photoacoustic measurements of the energy storage capacity of photosynthesis also showed that Photosystem II activity declined in nutrient-deprived cells. In contrast, energy storage by Photosystem I was unaffected, suggesting that Photosystem I-driven cyclic electron flow persisted in nutrient-deprived cells. These results indicate that in the modified photosynthetic apparatus of nutrient-deprived cells, a much larger fraction of the photosynthetic activity is driven by Photosystem I than in nutrient-replete cells.
Abbreviations: ES - energy storage; N - nitrogen; P - phosphorus; PBS -phycobilisomes; S - sulfur Introduction
It has been frequently observed that cyanobacteria exhibit a marked reduction in pigmentation and thylakoid membrane content, accumulate densely staining inclusion bodies, and stop growing when they are deprived of macronutrients such as nitrogen (N), sulfur (S) or phosphorus (P) (Jensen and Rachlin 1984; Healey 1982; Allen 1984; Bryant 1986; Simon 1987; Reithman et al. 1988; Bryant 1991; Grossman et al. 1993). The most striking effect of N or S deficien-
cy is the active and specific degradation of the lightharvesting phycobilisomes (PBS) (Allen and Smith 1969; Yamanaka and Glazer 1980; Collier and Grossman 1992). In contrast, during P deprivation little PBS degradation occurs, although the cellular PBS level is reduced by the cell divisions that occur after new PBS synthesis ceases (Collier and Grossman 1992). The cellular chlorophyll (Chl) content and quantity of thylakoid membranes also decline following nutrient deprivation, largely as a consequence of cell divisions that occur after new Chl (and presumably thy-
174 lakoid membrane) synthesis ceases; there is little net Chl degradation (Allen and Smith 1969; Collier and Grossman 1992). N-deprived cells exhibit an increase in the total carotenoid content relative to Chl (Allen and Smith 1969). Among the carotenoids, the content of fl-carotene declines in parallel with Chl, while the content of zeaxanthin relative to Chl increases in nutrientdeprived cells (Gombos and Vigh 1986; Fresnedo et al. 1991; Grossman et al. 1992). While pigment levels have been monitored during macronutrient limitation in a number of cyanobacteria, there is little information concerning the activities of the photosystems themselves in macronutrient-deprived cells. In the work presented here we show that there is a decrease in Photosystem II (PS II) activity during nutrient limitation while the energy storage capacity of Photosystem I (PS I) is not dramatically altered.
Materials and methods
Cell growth The cyanobacterium Synechococcus sp. Strain PCC 7942 was grown in batch culture in BG-11 medium (Allen 1969) at 32 °C, bubbled with 3% CO2 in air and illuminated at an intensity of 50/~moles m -2 s- l from incandescent bulbs. Cells that were in the midlogarithmic phase of growth were deprived of either N, S, or P, as previously described (Collier and Grossman 1992).
Pigment measurements PBS and Chl contents were determined from whole cell spectra, as previously described (Collier and Grossman 1992). Carotenoids and Chl were extracted in 80% acetone and the total carotenoid to Chl ratio was estimated from the absorbances of the extract at 460 nm (mainly due to carotenoids) and 663 nm (mainly due to Chl) (Myers and Kratz 1955; Eley 1971). Spectral measurements were made using a Beckman (Fullerton, CA) DU-70 spectrophotometer. The whole cell spectra presented in Fig. 2A were measured through the frosted side of glass cuvettes and corrected for scattering at 800 nm. The spectra of acetone extracted pigments shown in Fig. 2B were normalized to the Chl absorbance peak at 663 nm. The relative ratios of the individual carotenoids to Chl were determined by HPLC separation of the pigments, as previously described (Thayer and BjSrkman 1990; Thayer and Bj0rkman 1992).
Oxygen evolution 02 evolution measurements were performed with a Clark-type 02 electrode (Rank Brothers, Cambridge UK). The samples were maintained at 30 °C in a water jacketed cuvette. A 500 W light source was used to illuminate the sample at a light intensity (1200 #moles m -2 s- l) that saturated photosynthesis.
Fluorescence emission spectra The fluorescence emission spectra of cyanobacterial cultures were determined with a Photon Technologies International (New Brunswick, NJ) single beam fluorometer equipped with both excitation and detection monochromators with bandpasses of 5 nm. Samples were placed in a cuvette that could be immersed in liquid nitrogen, and light was transmitted to and from the cuvette by a fiber optic cable system. Fluorescence emission spectra were measured from 600 to 750 nm, with excitation at either 570 or 440 nm. The background fluorescence measured from sterile medium was subtracted from each spectrum. No corrections were made for the spectral sensitivity of the instrument. To achieve state transitions, samples were placed in the cuvette at room temperature under either 440 nm (to produce state 1) or 570 nm (to produce state 2) excitation light for 5 minutes, and then immersed in liquid nitrogen. For each sample, 77 K fluorescence emission spectra were measured upon excitation with 440 nm or 570 nm light, yielding four emission spectra: state 1, 440 nm and 570 nm excitation; state 2, 440 nm and 570 nm excitation. Fluorescence emission spectra of state 1 and state 2 cells excited with either 440 nm or 570 nm light were normalized to the peak of phycobiliprotein fluorescence (phycobiliprotein fluorescence does not change significantly during state transitions). For the nutrient deprivation time-course experiments presented in Fig. 2, measurements were performed on a single culture. The Chl content of the samples changed little during N deprivation and increased slightly during S deprivation (Collier and Grossman 1992).
Photoacoustic measurements of energy storage Photoacoustic measurements of energy storage were made using a continuously-modulated photoacoustic spectrometer described previously (Herbert et al. 1990; Fork and Herbert 1991). Samples were prepared by filtering cells onto a nitrocellulose disc (0.45 #m, Millipore), which was then loaded into a photoacoustic cell.
175 The measuring light was modulated at 150 Hz. At this frequency, photosynthetic oxygen evolution did not contribute to the photoacoustic signal from the sample (Herbert et al. 1990). The sample was supplied with 5% CO2 in air during energy storage (ES) versus irradiance measurements to avoid CO2 limitation. CO2 was supplied through a gas permeable backing plate, as described elsewhere (Fork and Herbert 1991). ES was calculated as (PAm-PAs) / PAm and expressed as a percentage. PAre was the photoacoustic signal produced by the modulated measuring light during illumination with 1200/~E m -2 s- 1 non-modulated background light, which acted to saturate the photochemistry. PAs was the photoacoustic signal in modulated actinic light alone. ES represents the percentage of absorbed modulated light that is maintained in stable photochemical products. ES values are strongly dependent upon the intensity of the modulated light. A series of ES measurements taken over a range of light intensities may be used to determine two separate ES parameters, the maximum ES of the sample and its ES capacity (Carpentier et al. 1988; Herbert et al. 1990). Correlations with photosynthetic oxygen evolution indicate that ES capacity is proportional to the rate of photosynthetic electron transport (Carpentier et al. 1988). Carpentier et al. (1988, 1989a) elaborated upon the earlier analysis of Malkin et al. (1981) and adapted the Michaelis-Menten model of enzyme kinetics to determine maximum ES and ES capacity. Their method of calculating ES capacity has been further modified in the present study. For given measurement conditions the value of PAm PAs is assumed to be proportional to the 'rate' at which light energy is stored by the sample. The rate of energy storage is likely to represent the extent to which electron carriers at the rate limiting steps of electron transport can become fully oxidized between flashes of the modulated light. Accordingly, a plot of PAm-PAs versus the modulated light intensity yields a curve that is kinetically similar to a photosynthesis versus irradiance curve, exhibiting light saturation at high intensities of modulated light (Fig. 1A). A double reciprocal plot of the same data yields a straight line that may be extrapolated to the y-axis to determine ES capacity (Fig. 1B). Values of ES capacity determined in this way were very similar to values determined from the slope of a plot of 1/ES versus light intensity, which is the method of analysis used by Malkin et al. (1981) and Carpentier et al. (1988, 1989a). The present method has the advantage that the double reciprocal plots do not become non-linear at very low intensities
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Changes in the cyanobacterial photosynthetic apparatus during acclimation to macronutrient deprivation J a c k i e L. C o l l i e r 2, S t e p h e n K. H e r b e r t 3, D a v i d C. F o r k 1 & A r t h u r R. G r o s s m a n 1,*
lDepartment of Plant Biology, Carnegie Institution of Washington, 290 Panama Street, Stanford, CA 94305, USA; 2Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-020, USA; 3Department of Biological Sciences, University of Idaho, Moscow, ID 83843, USA; *Author for correspondence and~or reprints Received9 June 1994;acceptedin revisedform28 September1994
Key words: cyanobacteria, cyclic photophosphorylation, nutrient deprivation, photoacoustic spectroscopy, photoinhibition, Photosystem I
Abstract
When the cyanobacterium Synechococcus sp. Strain PCC 7942 is deprived of an essential macronutrient such as nitrogen, sulfur or phosphorus, cellular phycobiliprotein and chlorophyll contents decline. The level of t-carotene declines proportionately to chlorophyll, but the level of zeaxanthin increases relative to chlorophyll. In nitrogen- or sulfur-deprived cells there is a net degradation of phycobiliproteins. Otherwise, the declines in cellular pigmentation are due largely to the diluting effect of continuedcell division after new pigment synthesis ceases and not to net pigment degradation. There was also a rapid decrease in 02 evolution when Synechococcus sp. Strain PCC 7942 was deprived of macronutrients. The rate of O2 evolution declined by more than 90% in nitrogen- or sulfur-deprived cells, and by approximately 40% in phosphorus-deprived cells. In addition, in all three cases the fluorescence emissions from Photosystem II and its antennae were reduced relative to that of Photosystem I and the remaining phycobilisomes. Furthermore, state transitions were not observed in cells deprived of sulfur or nitrogen and were greatly reduced in cells deprived of phosphorus. Photoacoustic measurements of the energy storage capacity of photosynthesis also showed that Photosystem II activity declined in nutrient-deprived cells. In contrast, energy storage by Photosystem I was unaffected, suggesting that Photosystem I-driven cyclic electron flow persisted in nutrient-deprived cells. These results indicate that in the modified photosynthetic apparatus of nutrient-deprived cells, a much larger fraction of the photosynthetic activity is driven by Photosystem I than in nutrient-replete cells.
Abbreviations: ES - energy storage; N - nitrogen; P - phosphorus; PBS -phycobilisomes; S - sulfur Introduction
It has been frequently observed that cyanobacteria exhibit a marked reduction in pigmentation and thylakoid membrane content, accumulate densely staining inclusion bodies, and stop growing when they are deprived of macronutrients such as nitrogen (N), sulfur (S) or phosphorus (P) (Jensen and Rachlin 1984; Healey 1982; Allen 1984; Bryant 1986; Simon 1987; Reithman et al. 1988; Bryant 1991; Grossman et al. 1993). The most striking effect of N or S deficien-
cy is the active and specific degradation of the lightharvesting phycobilisomes (PBS) (Allen and Smith 1969; Yamanaka and Glazer 1980; Collier and Grossman 1992). In contrast, during P deprivation little PBS degradation occurs, although the cellular PBS level is reduced by the cell divisions that occur after new PBS synthesis ceases (Collier and Grossman 1992). The cellular chlorophyll (Chl) content and quantity of thylakoid membranes also decline following nutrient deprivation, largely as a consequence of cell divisions that occur after new Chl (and presumably thy-
174 lakoid membrane) synthesis ceases; there is little net Chl degradation (Allen and Smith 1969; Collier and Grossman 1992). N-deprived cells exhibit an increase in the total carotenoid content relative to Chl (Allen and Smith 1969). Among the carotenoids, the content of fl-carotene declines in parallel with Chl, while the content of zeaxanthin relative to Chl increases in nutrientdeprived cells (Gombos and Vigh 1986; Fresnedo et al. 1991; Grossman et al. 1992). While pigment levels have been monitored during macronutrient limitation in a number of cyanobacteria, there is little information concerning the activities of the photosystems themselves in macronutrient-deprived cells. In the work presented here we show that there is a decrease in Photosystem II (PS II) activity during nutrient limitation while the energy storage capacity of Photosystem I (PS I) is not dramatically altered.
Materials and methods
Cell growth The cyanobacterium Synechococcus sp. Strain PCC 7942 was grown in batch culture in BG-11 medium (Allen 1969) at 32 °C, bubbled with 3% CO2 in air and illuminated at an intensity of 50/~moles m -2 s- l from incandescent bulbs. Cells that were in the midlogarithmic phase of growth were deprived of either N, S, or P, as previously described (Collier and Grossman 1992).
Pigment measurements PBS and Chl contents were determined from whole cell spectra, as previously described (Collier and Grossman 1992). Carotenoids and Chl were extracted in 80% acetone and the total carotenoid to Chl ratio was estimated from the absorbances of the extract at 460 nm (mainly due to carotenoids) and 663 nm (mainly due to Chl) (Myers and Kratz 1955; Eley 1971). Spectral measurements were made using a Beckman (Fullerton, CA) DU-70 spectrophotometer. The whole cell spectra presented in Fig. 2A were measured through the frosted side of glass cuvettes and corrected for scattering at 800 nm. The spectra of acetone extracted pigments shown in Fig. 2B were normalized to the Chl absorbance peak at 663 nm. The relative ratios of the individual carotenoids to Chl were determined by HPLC separation of the pigments, as previously described (Thayer and BjSrkman 1990; Thayer and Bj0rkman 1992).
Oxygen evolution 02 evolution measurements were performed with a Clark-type 02 electrode (Rank Brothers, Cambridge UK). The samples were maintained at 30 °C in a water jacketed cuvette. A 500 W light source was used to illuminate the sample at a light intensity (1200 #moles m -2 s- l) that saturated photosynthesis.
Fluorescence emission spectra The fluorescence emission spectra of cyanobacterial cultures were determined with a Photon Technologies International (New Brunswick, NJ) single beam fluorometer equipped with both excitation and detection monochromators with bandpasses of 5 nm. Samples were placed in a cuvette that could be immersed in liquid nitrogen, and light was transmitted to and from the cuvette by a fiber optic cable system. Fluorescence emission spectra were measured from 600 to 750 nm, with excitation at either 570 or 440 nm. The background fluorescence measured from sterile medium was subtracted from each spectrum. No corrections were made for the spectral sensitivity of the instrument. To achieve state transitions, samples were placed in the cuvette at room temperature under either 440 nm (to produce state 1) or 570 nm (to produce state 2) excitation light for 5 minutes, and then immersed in liquid nitrogen. For each sample, 77 K fluorescence emission spectra were measured upon excitation with 440 nm or 570 nm light, yielding four emission spectra: state 1, 440 nm and 570 nm excitation; state 2, 440 nm and 570 nm excitation. Fluorescence emission spectra of state 1 and state 2 cells excited with either 440 nm or 570 nm light were normalized to the peak of phycobiliprotein fluorescence (phycobiliprotein fluorescence does not change significantly during state transitions). For the nutrient deprivation time-course experiments presented in Fig. 2, measurements were performed on a single culture. The Chl content of the samples changed little during N deprivation and increased slightly during S deprivation (Collier and Grossman 1992).
Photoacoustic measurements of energy storage Photoacoustic measurements of energy storage were made using a continuously-modulated photoacoustic spectrometer described previously (Herbert et al. 1990; Fork and Herbert 1991). Samples were prepared by filtering cells onto a nitrocellulose disc (0.45 #m, Millipore), which was then loaded into a photoacoustic cell.
175 The measuring light was modulated at 150 Hz. At this frequency, photosynthetic oxygen evolution did not contribute to the photoacoustic signal from the sample (Herbert et al. 1990). The sample was supplied with 5% CO2 in air during energy storage (ES) versus irradiance measurements to avoid CO2 limitation. CO2 was supplied through a gas permeable backing plate, as described elsewhere (Fork and Herbert 1991). ES was calculated as (PAm-PAs) / PAm and expressed as a percentage. PAre was the photoacoustic signal produced by the modulated measuring light during illumination with 1200/~E m -2 s- 1 non-modulated background light, which acted to saturate the photochemistry. PAs was the photoacoustic signal in modulated actinic light alone. ES represents the percentage of absorbed modulated light that is maintained in stable photochemical products. ES values are strongly dependent upon the intensity of the modulated light. A series of ES measurements taken over a range of light intensities may be used to determine two separate ES parameters, the maximum ES of the sample and its ES capacity (Carpentier et al. 1988; Herbert et al. 1990). Correlations with photosynthetic oxygen evolution indicate that ES capacity is proportional to the rate of photosynthetic electron transport (Carpentier et al. 1988). Carpentier et al. (1988, 1989a) elaborated upon the earlier analysis of Malkin et al. (1981) and adapted the Michaelis-Menten model of enzyme kinetics to determine maximum ES and ES capacity. Their method of calculating ES capacity has been further modified in the present study. For given measurement conditions the value of PAm PAs is assumed to be proportional to the 'rate' at which light energy is stored by the sample. The rate of energy storage is likely to represent the extent to which electron carriers at the rate limiting steps of electron transport can become fully oxidized between flashes of the modulated light. Accordingly, a plot of PAm-PAs versus the modulated light intensity yields a curve that is kinetically similar to a photosynthesis versus irradiance curve, exhibiting light saturation at high intensities of modulated light (Fig. 1A). A double reciprocal plot of the same data yields a straight line that may be extrapolated to the y-axis to determine ES capacity (Fig. 1B). Values of ES capacity determined in this way were very similar to values determined from the slope of a plot of 1/ES versus light intensity, which is the method of analysis used by Malkin et al. (1981) and Carpentier et al. (1988, 1989a). The present method has the advantage that the double reciprocal plots do not become non-linear at very low intensities
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