Chlorophyll fluorescence emission spectroscopy of oxygenic organisms at 77 K

Photosynthetic fluorescence emission spectra measurement at the temperature of 77 K (–196°C) is an often-used technique in photosynthesis research. At low temperature, biochemical and physiological processes that modulate fluorescence are mostly abolished, and the fluorescence emission of both PSI and PSII become easily distinguishable. Here we briefly review the history of low-temperature chlorophyll fluorescence methods and the characteristics of the acquired emission spectra in oxygen-producing organisms. We discuss the contribution of different photosynthetic complexes and physiological processes to fluorescence emission at 77 K in cyanobacteria, green algae, heterokont algae, and plants. Furthermore, we describe practical aspects for obtaining and presenting 77 K fluorescence spectra.     

Use of the fluorochrome 4''6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA

Use of the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) makes it possible to examine in situ the structure of chloroplast DNA (chDNA) with the fluorescence microscope. This simplifies the study of genetic and developmental changes in chloroplast DNA. Three examples are presented. (a) Wild-type Euglena gracilis B contains several chloroplast DNA nucleoids per chloroplast. A yellow mutant lacking functional chloroplasts is similar, but such nucleoids are absent in an aplastidic mutant strain known from biochemical studies to have lost its chDNA. (b) In vegetative cells of the giant-celled marine algae Acetabularia and Batophora, only about a quarter of the chloroplasts have even one discernible chloroplast DNA particle, and such particles vary in size, showing a 30-fold variation in the amount of DNA-bound DAPI fluorescence detected per chloroplast. By contrast, 98% of chloroplasts in developing Acetabularia cysts contain chDNA, with as many as nine nucleoids per chloroplast. (c) DAPI-stained chloroplasts of chromophyte algae display the peripheral ring of DNA expected from electron microscope studies. However, these rings are not uniform in thickness, but are necklace-like, with the appearance of beads on a string. Since the multiple nucleoids in plastids of chlorophyte algae also appear to be interconnected throughout the chloroplast, a common structural plan may underlie chDNA morphology in both groups of algae.     

Dependence of fluorescence spectra of chloroplasts from the activity of photosystem 2]

By means of high sensitive spectrofluorometer the fluorescence spectra have been measured of normal chloroplasts and those with blocked photosystem 2 activity due to photoinhibition or treatment with 0.6 M tris-buffer. At room temperature fluorescence spectra of inactivated chloroplasts are similar to the spectrum of normal chloroplasts measured at low light intensity. Under excitation by intense light a decrease of intensity at 685 nm is appeared (about 3-4 times) in the fluorescence spectra of inactivated chloroplasts as compared to the spectrum of normal chloroplasts. The sharp intensity decrease of maxima at 685 and 695 nm (3-4 times) and small decrease at 680 and 730 nm (by 30-50%) are observed in low temperature fluorescence spectra of inactivated chloroplasts. Thus, the damage of photosystem 2 reaction centres is not accompanied by the preferential decrease of the only fluorescence band. The similarity of fluorescence difference spectra of chloroplasts distinguished by the state of photosystem 2 reaction centre, and the complex structure of difference spectra indicate that the variable fluorescence of chloroplasts during the induction is due to the emission of bulk chlorophyll alpha of the photosystem 2.     

Changes in the Cyclic AMP Content during Growth and Development of Acetabularia

The 3',5'-adenosine monophosphate (cyclic-AMP) content of the unicellular alga Acetabularia has been examined at various developmental stages. It has been found that very young algae, less than 10 mm in length, have a high cAMP content [more than 7 pmoles per 100 mg wet weight (WW)], but that with the growth of the algae, the cAMP content decreases rapidly, reaching the low level of 0.5–1.0 pmoles per 100 mg WW. The cAMP content remains at this level until cap differentiation, after which an increase in cAMP content accompanies cap enlargement. It has been shown that these results are unlikely to be affected by changes in the cAMP content induced by variations in circadian rhythm.
Treatment with theophylline (2.10⁻³ M), a phosphodiesterase inhibitor, results in an increase in the cAMP content and delays growth and cap formation. Experiments on the effects of theophylline upon the circadian rhythm of oxygen evolution have shown that the continuous presence of theophylline in the culture medium does not induce a phase shift in the rhythm.
The cAMP content of anucleate Acetabularia shows development stage variations parallel to that of the whole algae.     

Changes in the cyclic AMP content during growth and development of Acetabularia.

The 3',5'-adenosine monophosphate (cyclic-AMP) content of the unicellular alga Acetabularia has been examined at various developmental stages. It has been found that very young algae, less than 10mm in length, have a high cAMP content [more than 7 pmoles per 100 mg wet weight (WW)], but that with the growth of the algae, the cAMP content decreases rapidly, reaching the low level of 0.5--1.0 pmoles per 100mg WW. The cAMP content remains at this level until cap differentiation, after which an increase in cAMP content accompanies cap enlargement. It has been shown that these results are unlikely to be affected by changes in the cAMP content induced by variations in circadian rhythm. Treatment with theophylline (2.10(-3) M), a phosphodieterase inhibitor, results in an increase in the cAMP content and delays growth and cap formation. Experiments on the effects of theophylline upon the circadian rhythm of oxygen evolution have shown that the continuous presence of theophylline in the culture medium does not induce a phase shift in the rhythm. The cAMP content of anucleate Acetabularia shows development stage variations parallel to that of the whole algae.     

Characterization of Photosynthetic Rhythms in Marine Dinoflagellates: II. Photosynthesis-Irradiance Curves and in Vivo Chlorophyll a Fluorescence

Using data from light-dark cultures of Gonyaulax polyedra entrained to a 24-hour cycle, whole cell absorption curves and photosynthesis-irradiance curves were constructed for various circadian times. While whole cell absorbance and half-saturation constants of photosynthesis showed no statistical difference that could be directly related to the photosynthetic rhythm, the initial slope of the photosynthesis-irradiance curve was a time-dependent parameter which altered in direct proportion to the change in photosynthetic capacity. The results indicated a temporal change in the relative quantum yield of photosynthesis, and the circadian rhythmicity of light-limited photosynthesis was established under constant conditions. Circadian rhythmicity was detected in room temperature chlorophyll fluorescence yield. Low temperature fluorescence kinetics also showed fluctuations. The results suggest that regulation of photosynthesis by the biological clock of Gonyaulax may be mediated through the membrane-bound light reactions and a partial explanation of the underlying mechanism is proposed.     

Thermal Acclimation of Respiration and Photosynthesis in the Marine Macroalga Gracilaria lemaneiformis (Gracilariales,Rhodophyta)

The responses of respiration and photosynthesis to temperature fluctuations in marine macroalgae have the potential to significantly affect coastal carbon fluxes and sequestration. In this study, the marine red macroalga Gracilaria lemaneiformis was cultured at three different temperatures (12, 19, and 26°C) and at high‐ and low‐nitrogen (N) availability, to investigate the acclimation potential of respiration and photosynthesis to temperature change. Measurements of respiratory and photosynthetic rates were made at five temperatures (7°C–33°C). An instantaneous change in temperature resulted in a change in the rates of respiration and photosynthesis, and the temperature sensitivities (i.e., the Q₁₀ value) for both the metabolic processes were lower in 26°C‐grown algae than 12°C‐ or 19°C‐grown algae. Both respiration and photosynthesis acclimated to long‐term changes in temperature, irrespective of the N availability under which the algae were grown; respiration displayed strong acclimation, whereas photosynthesis only exhibited a partial acclimation response to changing growth temperatures. The ratio of respiration to gross photosynthesis was higher in 12°C‐grown algae, but displayed little difference between the algae grown at 19°C and 26°C. We propose that it is unlikely that respiration in G. lemaneiformis would increase significantly with global warming, although photosynthesis would increase at moderately elevated temperatures.     

Selective Formation of Photosystem 1 under Illumination at Low Intensity

Analyses of chlorophylls a and b and P700 in the wheat leaves grown for 8 days under illumination with white light at different intensities suggested selective formation of photosystem 1 of the photosynthesis at low light intensities. This was confirmed for the two types of chloroplasts isolated from leaves grown at light intensities of 1.1 and 240 μ W/cm², respectively, by measuring their pigment compositions, activities of photosystems 1 and 2, and absorption and fluorescence spectra. The chloroplasts developed at the low intensity showed properties only of photosystem 1 while those developed at the high intensity showed properties of both photosystems 1 and 2. Only photosystem 1 particles were obtained by fractionation of low intensity chloroplasts by treatment with digitonin followed by centrifugation, while high intensity chloroplasts could be fractionated into photosystem-1 and photosystem-2 particles. When the leaves grown at low light intensity were illuminated with strong light, photosystem 2 was developed. The fluorescence emission spectrum of low intensity chloroplasts at 77°K showed two peaks at 685 and 734 nm, and the spectrum of high intensity chloroplasts showed three peaks at 685, 697 and 740 nm.     

The circadian rhythm in photosynthesis in Acetabularia in the presence of actinomycin D, puromycin, and chloramphenicol

Anucleate Acetabularia crenulata shows a circadian rhythm in photosynthesis. In this study, an oxygen electrode was employed to measure this photosynthetic rhythm in the presence and absence of the inhibitors, actinomycin D, chloramphenicol, and puromycin. High concentrations of the inhibitors were used: actinomycin D, 20-40 micrograms ml-1; puromycin, 30 and 100 micrograms ml-1; and chloramphenicol, 250 micrograms ml-1. The effectiveness of these inhibitors on protein synthesis was also measured under the same conditions used for the determination of rhythmicity. In spite of large effects of all three inhibitors on the incorporation of 14C leucine, no effect on the period or the phase of the photosynthetic rhythm was observed. The higher concentration of puromycin and chloramphenicol produced toxic effects which were expressed as a reduction in the amount of photosynthesis, but rhythmicity was still apparent. After 3 or 4 days' exposure to actinomycin, Acetabularia became resistant to its effect. Recovery was also observed in the ability to incorporate leucine. The implications of these results for theories of the basic oscillator responsible for circadian rhythmicity are discussed.     

Thermal acclimation and photoacclimation of photosynthesis in the brown alga Laminaria saccharina

Thermal acclimation and photoacclimation of photosynthesis were compared in Laminaria saccharina sporophytes grown at temperatures of 5 and 17 °C and irradiances of 15 and 150μmol photons m^?2 s^?1. When measured at a standard temperature (17°C), rates of light-saturated photosynthesis (P_max) were higher in 5 °C-grown algae (c. 3.0 μmol O₂ m^?2 s^?1) than in 17 °C-grown algae (c. 0.9 μmol O₂ m_-2 s_-1). Concentrations of Rubisco were also 3-fold higher (per unit protein) in 5 °C-grown algae than in algae grown at 17 °C. Light-limited photosynthesis responded similarly to high temperature and low light Photon yields (α) were higher in algae grown at high temperature (regardless of light), and at 5 °C in low light, than in algae grown at 5 °C in high light Differences in a were correlated with light absorption; both groups of 17 °C algae and 5 °C low-light algae absorbed c. 75% of incident light, whereas 5 °C high-light algae absorbed c. 55%. Increased absorption was correlated with increases in pigment content PSII reaction centre densities and the fucoxanthin-Chl ale protein complex (FCP). Changes in a were also attributed, in part, to changes in the maximum photon yield of photosynthesis (0_max). PSI reaction centre densities were unaffected by growth temperature, but the areal concentration of PSI in low-light-grown algae was twice that of high-light-grown algae (c. 160.0 versus 80.0 nmol m^?2). We suggest that complex metabolic regulation allows L, saccharina to optimize photosynthesis over the wide range of temperatures and light levels encountered in nature.     

Modification of Photosystem I Light Harvesting of Bundle-Sheath Chloroplasts Occurred during the Evolution of NADP-Malic Enzyme C4 Photosynthesis

Low-temperature emission spectra and excitation spectra for chlorophyll fluorescence were recorded from leaves of species of the genus Flaveria (Asteraceae) with C3, C3-C4-intermediate, C4-like, and C4 photosynthesis. Among the latter two groups, high chlorophyll b absorption was observed in excitation spectra for photosystem I (PSI) fluorescence. By comparing leaf data with those from isolated chloroplast fractions, the high chlorophyll b absorption was attributed to the specific properties of the bundle-sheath chloroplasts in leaves from C4 plants. The deconvolution of the PSI excitation spectra and the use of a model revealed that the contribution of photosystem II absorption to the functional antenna of PSI was markedly increased in leaves from three of the five C4-like and C4 species investigated in detail. The two other species exhibited normal, C3-like light-harvesting properties of PSI. The former species are known for efficient carbon assimilation, the latter for decreased efficiencies of carbon assimilation. It is concluded that photosystem II becomes a substantial part of the functional PSI antenna late in the evolution of C4 photosynthesis, and that the composite antenna optimizes the light-harvesting of PSI in bundle-sheath chloroplasts to meet the energy requirements of C4 photosynthesis.     

DISINTEGRATION OF THE CELLS OF SIPHONOUS GREEN ALGA CODIUM EDULE (BRYOPSIDALES,CHLOROPHYTA) UNDER MILD HEAT STRESS1

The siphonous green alga Codium edule P. C. Silva (Bryopsidales, Chlorophyta) has the highest covering ratio among the macroalgae on the coral reef of Nanwan Bay in southern Taiwan, but its population in the subtidal region drastically decreases from July to September each year. The objective of this study was to determine whether the high temperature of summer could be the basis for this population decrease. Chlorophyll fluorescence measurements revealed that when the algae were incubated at 35°C (a temperature that can be reached in southern Taiwan during the summer), their photosynthetic activities were almost completely inhibited after about 8 h. The circadian rhythm of photosynthesis was disrupted at a temperature as low as 32°C. TEM studies showed that 4 h incubation at 35°C induced a decrease in turgidity accompanied by vacuole shrinkage and plasmolysis. The marked disintegrative changes, including damage to organelles, such as chloroplasts and nuclei, occurred after about 8 h, at which time central vacuoles collapsed and the cell interior was then filled with numerous small vesicles. Our results suggested that the rise in seawater temperature during the summer could be one of the major causes of the massive death of C. edule in the field.     

The coleoptile chloroplast: Distinct distribution of xanthophyll cycle pigments,and enrichment in Photosystem II

Recent studies have shown that coleoptile chloroplasts operate the xanthophyll cycle, and that their zeaxanthin concentration co-varies with their sensitivity to blue light. The present study characterized the distribution of photosynthetic pigments in thylakoid pigment–protein complexes from dark-adapted and light-treated coleoptile and mesophyll chloroplasts, the low temperature fluorescence emission spectra, and the rates of PS I and PS II electron transport in both types of chloroplasts from 5-day-old corn seedlings. Pigments were extracted from isolated PS I holocomplex, LHC IIb trimeric and LHC II monomeric complexes and analyzed by HPLC. Chlorophyll distribution in coleoptile thylakoids showed 31% of the total collected Chl in PS I and 65% in the light harvesting complexes of PS II. In mesophyll thylakoids, the values were 44% and 54%, respectively. Mesophyll and coleoptile PS I holocomplexes differed in their Chl t a/Chl t b ratios (8.1 and 6.1, respectively) and -carotene content. In contrast, mesophyll and coleoptile LHC IIb trimers and LHC II monomers had similar Chl t a/Chl t b ratios and -carotene content. The three analyzed pigment–protein complexes from dark-adapted coleoptile chloroplasts contained zeaxanthin, whereas there was no detectable zeaxanthin in the complexes from dark-adapted mesophyll chloroplasts. In both chloroplast types, zeaxanthin and antheraxanthin increased markedly in the three pigment–protein complexes upon illumination, while violaxanthin decreased. In mesophyll thylakoids, zeaxanthin distribution as a percentage of the xanthophyll cycle pool was: LHC II monomers > LHC IIb trimers > PS I holocomplex, and in coleoptile thylakoids, it was: LHC IIb trimers > LHC II monomers = PS I holocomplex. Low temperature (77 K) fluorescence emission spectra showed that the 686 nm emission of coleoptile chloroplasts was approximately 50% larger than that of mesophyll chloroplasts when normalized at 734 nm. The pigment and fluorescence analysis data suggest that there is relatively more PS II per PS I and more LHC I per CC I in coleoptile chloroplasts than in mesophyll chloroplasts. Measurements of t in vitro uncoupled photosynthetic electron transport showed approximately 60% higher rates of electron flow through PS II in coleoptile chloroplasts than in mesophyll chloroplasts. Electron transport rates through PS I were similar in both chloroplast types. Thus, when compared to mesophyll chloroplasts, coleoptile chloroplasts have a distinct PS I pigment composition, a distinct chlorophyll distribution between PS I and PS II, a distinct zeaxanthin percentage distribution among thylakoid pigment–protein complexes, a higher PS II-related fluorescence emission, and higher PS II electron transport capacity. These characteristics may be associated with a sensory transducing role of coleoptile chloroplasts.     

In vivo fluorometric method for early detection of cyanobacterial waterblooms

A specific method was developed for monitoring the concentration of cyanobacteria (blue-green algae) before waterblooms, based on their characteristics ofin vivo fluorescence. The excitation and emission spectra of cyanobacteria are very different from those of eukaryotic algae, due to the importance of phycocyanin, rather than chlorophylla, in determining the fluorescence characteristics. Our results, based on four cyanobacteria:Microcystis aeruginosa, Anabaena cylindrica, Phormidium tenue andSpirulina platensis, indicate that excitation at 620 nm and its emission at 645 nm is a sensitive and specific method for their detection. Furthermore, the addition of 10 M photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) induced only 3% increase in phycocyanin fluorescence, suggesting that this measurement is almost independent of the ongoing rate of photosynthesis.Author for correspondence     

The circadian rhythms of photosynthesis,ATP content and cell division in Microcystis aeruginosa PCC7820

Microcystis aeruginosa is one of the most common blue-green algae species that forms harmful water bloom, which frequently causes serious ecological pollution and poses a health hazard to animals and humans. To understand the progression of algal blooms and to provide a theoretical basis for predicting and preventing the occurrence of algal blooms and reducing the harm of algal bloom to environment, we investigated the diurnal variation of photosynthesis, ATP content and cell division in M. aeruginosa PCC7820. The results showed that the photosynthesis and ATP content of M. aeruginosa PCC7820 exhibited clear circadian rhythm with a period of approximately 24 h and that the periodic rhythms continued for at least three cycles under continuous light conditions. Furthermore, the period length showed that a temperature compensation effect and changes in light cycle or temperature could reset the phase of circadian rhythm. These results indicate that the circadian rhythms of physiological process in M. aeruginosa PCC7820 are controlled by the endogenous circadian clock. Examinations of the number, size and cytokinin content of cells also reveal that the cell division of M. aeruginosa PCC7820 with the generation time of 38.4 h exhibits robust circadian rhythms with a period close to 24 h. The circadian rhythms of cell division may be generated by a biological clock through regulation of the cell division phase of M. aeruginosa PCC7820 via a gating mechanism. The phases in which cell division slows or stop recur with a circadian periodicity of about 24 h.     

Auslösung der circadianen Photosynthese-Rhythmik beiAcetabularia durch Blaulicht

Summary Additional irradiation with blue light of low intensity ofAcetabularia mediterranea cells, pretreated by prolonged irradiation with red, induces an increase of photosynthetic activity. This induction is accompanied by the appearance of rhythmic changes of the O₂ production. Maxima are found about 6, 30, 54, 78, ... hours after the onset of blue light irradiation. Thus blue light not only induces an increase of the rate of photosynthesis but also acts as a Zeitgeber for the circadian rhythm of photosynthesis inAcetabularia.

     

Effect of Light Quality on the Circadian Expression of Nitrate Reductase in the Red Macroalga Gracilaria tenuistipitata

A circadian rhythm in the activity of nitrate reductase (NR: EC 1.6.6.1) isolated from the marine red algae Gracilaria tenuistipitata is shown to be attributable to the daily oscillation of protein levels. The experiments reported here indicate that light quality has differential effects over NR expression. In extracts of algae grown under white light : dark, red light : dark and blue light : dark cycle, the activity of NR peaks during photophase, as does photosynthesis. Staining with a monoclonal antibody (NR10), raised against NR purified from Porphyra yezoensis, shows that the amount of protein changes by a factor of about 20, with a maximum occurring during photophase when algae are submitted to white and blue light. Red light changes the circadian rhythm of NR protein levels and also inhibits its night degradation. Illumination with blue light is able to restore the NR activity as well as its protein levels only when the light irradiance was the same of the white light. Surprisingly, the red light promoted 40% induction on NR activity under the same conditions.     

Influence of carbon supply on the circadian rhythmicity of photosynthesis and its stimulation by blue light in Ectocarpus siliculosus: clues to the mechanism of inorganic carbon acquisition in lower brown algae

Stimulation or light-saturated rates of photosynthesis in Ectocarpus siliculosus (Dillwyn) Lyngb. by blue light was eliminated by increasing dissolved inorganic carbon (DIC) or by lowering pH in natural seawater. The amplitude of the circadian rhythm of photosynthesis was also diminished under these conditions, and the pH compensation points in a closed system were higher in the presence of blue light and during the circadian day. These observations suggest that blue light and the circadian clock regulate the activity of a carbon acquisition system in these plants. The inhibitor of external carbonic anhydrase, acetazolamide, reduced overall rates of photosynthesis by only about 30%, but ethoxyzolamide suppressed the circadian rhythm of photosynthesis almost completely and markedly reduced the duration of responses to blue light pulses. Similar patterns were obtained when photosynthesis was measured in strongly limiting DIC concentrations (0–0.5 mol m^?3). Since blue light stimulated photosynthesis under these conditions of strong carbon limitation, we suggest that blue light activates the release of CO₂ from an internal CO₂ store. We propose a metabolic pathway with similarities to that of CAM plants. Non-photosynthetic fixation leads to the accumulation of a storage metabolite. The circadian clock and blue light control the mobilization of CO₂ at the site of decarboxylation of this metabolite. In the presence of continuous blue light the pathway is proposed to cycle and act as a pump for CO₂ into the chloroplasts. This hypothesis helps to explain a number of previously reported peculiarities of brown algal photosynthesis.     

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20°C (nonhardened rye, RNH) or 5°C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.

Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20°C indicated that RNH and RH thylakoids contained the same proportions of fast (α) and slow (β) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for α components increased and the rate constant for β components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5°C.