I am currently interested in History of Photosynthesis Research and Researchers in it Phone: 1-217-333-1794 Address: Plant Biology, University of Illinois at Urbana-Champaign, 269 Morrill Hall, 505 S. Goodwin Avenue, Urbana,IL 61801, USA
This is a story of a controversy between the 1931 Nobel laureate Otto Warburg and the Midwest Gan... more This is a story of a controversy between the 1931 Nobel laureate Otto Warburg and the Midwest Gang. (The "Midwest Gang" was used by Warburg when he was describing, to Andy Benson, his quarrel with Robert Emerson of UIUC, Urbana, Illinois.) To Warburg, this Midwest Gang also included the 1926 Nobel laureate James Franck, Eugene Rabinowitch, the author of the "Bible" of Photosynthesis (1945, 1951 and 1956), and Hans Gaffron. It turns out that students of Emerson : Govindjee and Rajni Govindjee have played a major role in supporting Emerson even after the death of both the actors: Emerson and Warburg, and that too under Warburg's conditions!
Albert W. Frenkel, a pioneer in photosynthesis
research, and discoverer of photophosphorylation i... more Albert W. Frenkel, a pioneer in photosynthesis research, and discoverer of photophosphorylation in photosynthetic bacteria, is remembered here by two of us: Govindjee (historical corner editor of photosynthesis research) and Susanna Frenkel (SF; Albert Frenkel’s daughter, who provided most of the family information).
t On January 16, 2015, Professor Andrew Alm
Benson, one of the leading plant biochemists of the
t... more t On January 16, 2015, Professor Andrew Alm Benson, one of the leading plant biochemists of the twentieth century, died in La Jolla, California, at the age of 97; he was born on September 24, 1917. Benson was known especially for his pioneering studies on photosynthesis (CO2 assimilation, carbon reduction cycle) and plant lipids (phospholipid phosphatidyl glycerol; and the sulfolipid, sulfoquinovosyl diglyceride).
William Arnold—Bill, to all who knew him—was a plant physiologist, physicist, and biologist all i... more William Arnold—Bill, to all who knew him—was a plant physiologist, physicist, and biologist all in one and a researcher of the highest order in each field. His achievements read like a march of scientific progress. He discovered what came to be called the photosynthetic unit, with Robert Emerson, in 1932. He made the first reliable measurements, in his 1935 Harvard Ph.D. dissertation, of the minimum quantum requirement for the evolution of an oxygen molecule. In 1951, together with J. Robert Oppenheimer, he made critical suggestions on the mechanism of excitation energy transfer in photosynthesis. With Bernard Strehler in that same year he discovered delayed light emission (DLE) in photosyntheticorganisms. He made the first measurements of excitation energy migration by depolarization of fluorescence, with Eleanor Meek in 1956. He discovered thermoluminescence in chloroplasts, with Helen Sherwood in 1957. He elucidated the primary photochemical reactions at the photosynthetic reaction center down to 1K with Rod Clayton in 1960. Bill discovered the presence of electroluminescence, also in chloroplasts, with Jim Azzi in 1971. As this list suggests, Bill Arnold was one of the founding fathers of our understanding of the physical basis for photosynthesis.
Chlorophyll b is synthesized by the oxidation of a methyl group on the B ring of a tetrapyrrole m... more Chlorophyll b is synthesized by the oxidation of a methyl group on the B ring of a tetrapyrrole molecule to a formyl group by chlorophyllide a oxygenase (CAO). The full-length CAO from Arabidopsis (Arabidopsis thaliana) was overexpressed in tobacco (Nicotiana tabacum) that grows well at light intensities much higher than those tolerated by Arabidopsis. This resulted in an increased synthesis of glutamate semialdehyde, 5-aminolevulinic acid, magnesium-porphyrins, and chlorophylls. Overexpression of CAO resulted in increased chlorophyll b synthesis and a decreased chlorophyll a/b ratio in low light grown as well as high light-grown tobacco plants; this effect, however, was more pronounced in high light. The increased potential of the protochlorophyllide oxidoreductase activity and chlorophyll biosynthesis compensated for the usual loss of chlorophylls in high light. Increased chlorophyll b synthesis in CAO-overexpressed plants was accompanied not only by an increased abundance of light-harvesting chlorophyll proteins but also of other proteins of the electron transport chain, which led to an increase in the capture of light as well as enhanced (40%–80%) electron transport rates of photosystems I and II at both limiting and saturating light intensities. Although the quantum yield of carbon dioxide fixation remained unchanged, the lightsaturated photosynthetic carbon assimilation, starch content, and dry matter accumulation increased in CAO-overexpressed plants grown in both low- and high-light regimes. These results demonstrate that controlled up-regulation of chlorophyll b biosynthesis comodulates the expression of several thylakoid membrane proteins that increase both the antenna size and the electron transport rates and enhance carbon dioxide assimilation, starch content, and dry matter accumulation.
As photosynthesis on Earth produces the primary signatures of life that can be detected astronomi... more As photosynthesis on Earth produces the primary signatures of life that can be detected astronomically at the global scale, a strong focus of the search for extrasolar life will be photosynthesis, particularly photosynthesis that has evolved with a different parent star. We take previously simulated planetary atmospheric compositions for Earth-like planets around observed F2V and K2V, modeled M1V and M5V stars, and around the active M4.5V star AD Leo; our scenarios use Earth’s atmospheric composition as well as very low O2 content in case anoxygenic photosynthesis dominates. With a line-by-line radiative transfer model, we calculate the incident spectral photon flux densities at the surface of the planet and under water. We identify bands of available photosynthetically relevant radiation and find that photosynthetic pigments on planets around F2V stars may peak in absorbance in the blue, K2V in the red-orange, and M stars in the near-infrared, in bands at 0.93–1.1 m, 1.1–1.4 m, 1.5–1.8 m, and 1.8–2.5 m. However, underwater organisms will be restricted to wavelengths shorter than 1.4 m and more likely below 1.1 m. M star planets without oxygenic photosynthesis will have photon fluxes above 1.6 m curtailed by methane. Longer-wavelength, multi-photosystem series would reduce the quantum yield but could allow for oxygenic photosystems at longer wavelengths. A wavelength of 1.1 m is a possible upper cutoff for electronic transitions
versus only vibrational energy; however, this cutoff is not strict, since such energetics depend on molecular configuration. M star planets could be a half to a tenth as productive
as Earth in the visible, but exceed Earth if useful photons extend to 1.1 m for anoxygenic photosynthesis. Under water, organisms would still be able to survive ultraviolet flares from
young M stars and acquire adequate light for growth.
Acidocalcisomes are acidic calcium storage compartments
described initially in trypanosomatid and... more Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH4Cl released 45Ca2 from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H- ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H-PPase and the V-H-ATPase activities in addition to high concentrations of PPi and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electrondense vacuoles, PPi-driven proton translocation was stimulated by potassium ions and inhibited by the PPianalog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PPi hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4,6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.
The xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the m... more The xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the most important regulatory and photoprotective mechanisms in photosynthesis. Using parallel time resolved and pulse-amplitude modulation fluorometry, we studied the influence of the intrathylakoid pH and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the ~0.5 ns Chl a fluorescence lifetime (tau) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous effects of the chloroplast trans-thylakoid membrane pH gradient and xanthophyll cycle pigments on the PSII Chl a fluorescence ô distributions and intensity. The model assumes that (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this binding site is activated by a low intrathylakoid pH(pK ~4.5) that increases the affinity for Z (or A), (3) about one Z or A molecule binds to the activated site, and (4) this binding effectively “switches” the fluorescence tau distribution of the PSII unit to a state with a decreased fluorescence tau and emission intensity (a “dimmer switch” concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissipation. Statistical analysis of the data yields an equilibrium association constant, Ka, that ranges from 0.7 to 3.4 per PSII for the protonated/activated binding site for Z (or A). The model explains (1) the relative fraction of the ~0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) the dependence of the ratio of F¢m/Fm on the fraction of the 0.5 ns fluorescence ô component (where F'm and Fm are maximal fluorescence intensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F'm/Fm on the concentration of Z and A and the intrathylakoid pH.
The sequence connecting alpha-helices D and E of the D1 protein in photosystem II (PSII) is longe... more The sequence connecting alpha-helices D and E of the D1 protein in photosystem II (PSII) is longer than that found in
the corresponding loop of the L subunit in the rhodobacterial reaction centre. This sequence was mutated in order
to determine its role in oxygenic photosynthesis. Site-specific mutants, including point mutations and deletions
of different size, of the PEST-like region and the putative cleavage area in the D-E loop of the D1 protein were
constructed in Synechocystis sp. PCC 6803. The effects of mutations on the functional and structural properties
of PSII and turnover of the D1 protein were examined. Our results demonstrate that deletion of either the PESTlike
sequence (DeltaR225-F239) or the putative cleavage region (Delta G240-V249, Delta R225-V249) of the D1 protein
resulted in severe perturbations on the function of the QB electron acceptor of PSII. However, PSII centres of the
mutant with deleted PEST region remained functional enough to support autotrophic growth whereas deletions
of the putative cleavage region prevented autotrophic growth. Although enhanced degradation rates of the mutant
D1 proteins under low-light growth conditions demonstrate that neither the PEST-like sequence nor the putative
cleavage region are required for D1 proteolysis, it became clear that the extension in the D-E loop of the D1
protein is essential for proper PSII assembly and photoautotrophic growth. Moreover, modifications of the D-E
loop resulted in transcriptional activation of the psbA gene, indicating that neither light intensity, as such, nor the
activity of the electron transfer chain are the only determinants in regulation of psbA gene transcription.
Transient absorption measurements of the pheophytin a anion band and Qx band bleach region using ... more Transient absorption measurements of the pheophytin a anion band and Qx band bleach region using preferential excitation of P680 are performed on isolated photosystem II reaction centers to determine the effective rate constant for charge separation. A novel analysis of the Qx band bleach region explicitly takes the changing background into account in order to directly measure the rate of growth of the bleach. Both spectral regions reveal biphasic kinetics, with a ca. (8 ps)-1 rate constant for the faster component, and a ca. (50 ps)-1 rate
constant for the slower component. We propose that the faster component corresponds to the effective rate constant for charge separation from within the equilibrated reaction center core and provides a lower limit for the intrinsic rate constant for charge separation. The slower component corresponds to charge separation that is limited by slow energy transfer from a long-wavelength accessory chlorophyll a.
Photosystem II electron transfer, charge stabilization, and photoinhibition were studied in three... more Photosystem II electron transfer, charge stabilization, and photoinhibition were studied in three site-specific mutants of the D1 polypeptide of Synechocystis PCC 6803: E243K, E229D, and CA1 (deletion of three glutamates 242-244 and a substitution, glutamine- 241 to histidine). The phenotypes of the E229D and E243K mutants were similar to that of the control strain (AR) in all of the studied aspects. The characteristics of CA1 were very different. Formate, which inhibits the QA- to QB- reaction, was several fold less effective in CA1 than in AR. The S2QA- and S2QB- states were stabilized in CA1. It was previously shown that the electron transfer between QA- and QB was modified in CA1 (P Maenpaa, T. Kallio, P. Mulo, C. Salih, E.-M. Aro, E. Tyystjarvi, C. Jansson 11 9931 Plant Mo1 Biol 22: 1-12). A change in the redox potential of the QA/QA-couple, which renders the reoxidation of QA- by back or forward reactions more difficult, could explain the phenotype of CA,. Although the rates of photoinhibition measured as inhibition of oxygen evolution, Chl fluorescence quenching, and decrease of thermoluminescence B and Q bands were similar in AR and CA1, the CA1 strain more quickly reached a state from which the cells were unable to recover their activity. The results described in this paper suggest that a modification in the structure of the D-de loop of D, could influence the properties of the couple QA/QA- in D1, and the mechanism of recovery from photoinhibition.
Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura inno... more Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO2 (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO2 levels (5%). Photoautotrophy was indicated by the requirement for CO2 and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C4 Amaranthus species. The cells showed high dark respiration rates and had lower net CO2 fixation under high 02 conditions. Dark CO2 fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO2 conditions showed the unique properties of a high RuBPcase activation level in ambient CO2 and a stable ability to show net CO2 fixation in 21% 02 conditions.
Disulfiram (tetraethylthiuram disulfide), a metal chelator, inhibits photosynthetic electron tran... more Disulfiram (tetraethylthiuram disulfide), a metal chelator, inhibits photosynthetic electron transport in broken chloroplasts. A major site of inhibition is detected on the electron-acceptor side of photosystem II between QA, the first plastoquinone electron-acceptor, and the second plastoquinone electron-acceptor, QB. This site of inhibition is shown by a several fold increase in the half-time of QA oxidation, as monitored by the decay of the variable chlorophyll a flourescence after an actinic flash. Another site of inhibition is detected in the functioning of the reaction center of photosystem II disulfiram is observed to quench the room temperature variable chlorophyll a fluorescence, as well as the intensity of the 695 nm peak, relative to the 685 nm peak, in the chlorophyll a fluorescence spectrum at 77 K. Electron transport from water to the photosystem II electron-acceptor silicomolybdate is also inhibited. Disulfiram does not inhibit electron flow before the site(s) of donation by exogenous electron donors to photosystem II, and no inhibition is detected in the partial reactions associated with photosystem I.
The effects of cations and abscisic acid on chloroplast activity in guard celis of Viia faba were... more The effects of cations and abscisic acid on chloroplast activity in guard celis of Viia faba were investigated by analysis of the transient of chlorophyli a fluorescence. When epidermal strips containing guard cells as the only living cells were incubated in water and illuminated with strong light, chlorophyll a fluorescence rose rapidly to a high intensity and then declined slowly to a stationary level. The rate of this decline was enhanced by K+ or Na+, and the effect of these cations was greater when added with phosphate than with chloride as the anion. Ca'+ suppressed the enhancement by Na+ and, to a lesser extent, that by K+. Abscisic acid also suppressed the enhancement by K+ and Na+. Since the fluorescence decline reflects the increase of intrathylakod H+ concentration necessary for photophosphorylation, the acceleration of the decline by K+ (or Na+ in the absence of Ca2+ ) implicates chloroplast activity in ion accumulation by guard celis in the light The differential effects of phosphate and chloride suggest that chloroplast actiivty may be Involved In malate formation in guard cells in the light.
Bicarbonate ion, not dissolved CO2 gas, is shown to increase 4-
to 5-fold the rate of dichlorophe... more Bicarbonate ion, not dissolved CO2 gas, is shown to increase 4- to 5-fold the rate of dichlorophenol indophenol reduction by isolated maize (Zea mays) chloroplasts. Glutaraldehyde fixed chloroplasts continue to exhibit bicarbonate-dependent 2,6- dichlorophenol indophenol reduction. Bicarbonate is shown to act close to the oxygen-evolving site, i.e. prior to the electron donation site of diphenyl carbazide to photosystem II. Dark incubation and light pretreatment of chloroplasts in various concentrations of bicarbonate, just prior to assay, indicate that bicarbonate binds to chloroplasts in the dark and is released again as the Hill reaction proceeds in the light. It is suggested that bicarbonate ions may play a critical role in the oxygen-evolving process in photosynthesis.
The Emerson enhancement effect has been shown
to occur in the photoreduction of nicotinamide aden... more The Emerson enhancement effect has been shown to occur in the photoreduction of nicotinamide adenine dinucleotidephosphate by spinach chloroplasts. Greater than additive rates were obtained where far-red light beams were mixed (combined) with supplementary white (fluorescent) or monochromatic 650 nm light in nicotinamide adenine dinucleotidephosphate reduction, and oxygen evolution. In magnitude, wavelength, and intensity dependence of the enhancement effect, as measured in the Hill reaction, appears analogous to that occurring in complete photosynthesis.
Nonsulfur photosynthetic bacteria (Athiorhodaceae) exhibit a time-variable fluorescence in additi... more Nonsulfur photosynthetic bacteria (Athiorhodaceae) exhibit a time-variable fluorescence in addition to a constant fluorescence. All species examined show upon aging a remarkable gain in the variable component at the expense of the constant component while the total fluorescence remains essentially invariant. This result can be rationalized by supposing a change in distribution of bacteriochlorophyll in photosynthetic units as cells age. Alternatively, one may assume operation of two photochemical systems-one cyclic and predominant in young cells, the other noncyclic and predominant in old cells. It is also noted that a hitherto unreported minor fluorescence with maximum emission at - 860 nm exists in addition to the well-known main fluorescence band at -890 nm. The rise in variable fluorescence is associated with the main band, a result in accord with the notion that the bacteriochloro- phyll component responsible and absorbing at 870 nm is directly in contact with the energy trap.
[Basically, these data provided the first clear demonstration of a "dimmer switch" in photosynthe... more [Basically, these data provided the first clear demonstration of a "dimmer switch" in photosynthesis during photo-protection against excess light.]
Excess light triggers protective non-radiative dissipation of excitation energy in photosystem II through the formation of
a trans-thylakoid pH gradient that in turn stimulates formation
of zeaxanthin and antheraxanthin. These xanthophylls when
combined with protonation of antenna pigment-protein complexes may increase nonradiative dissipation and, thus,
quench chlorophyll a fluorescence. Here we measured, in parallel, the chlorophyll a fluorescence lifetime and intensity
to understand the- mechanism of this process. Increasing
the xanthophyll concentration in the presence of a pH gradient
(quenched conditions) decreases the fractional intensity of a
fluorescence lifetime component centered at-2 ns and increases
a component at -0.4 ns. Uncoupling the pH gradient (unquenched conditions) eliminates the 0.4-ns component. Changes in the xanthophyll concentration do not significantly
affect the fluorescence lifetimes in either the quenched or
unquenched sample conditions. However, there are differences
in fluorescence life- times between the quenched and unquenched states that are due to pH-related, but non xanthophyll-related, processes. Quenching of the maximal fluorescence intensity correlates with both the xanthophyll
concentration and the fractional intensity of the 0.4-ns component. The unchanged fluorescence lifetimes and the
proportional quenching of the maximal and dark-level fluorescence intensities indicate that the xanthophylls act on
antenna, not reaction center processes. Further, the fluorescence quenching is interpreted as the combined effect
of the pH gradient and xanthophyll concentration, resulting
in the formation of a quenching complex with a short (~0.4 ns)
fluorescence lifetime.
Photosynthetic conversion of light energy into chemical potential begins in reaction center prote... more Photosynthetic conversion of light energy into chemical potential begins in reaction center protein complexes, where rapid charge separation occurs with nearly unit quantum efficiency. Primary charge separation was studied in isolated photosystem II reaction centers from spinach containing
6 chlorophyll a, 2 pheophytin a (Pheo), 1 cytochrome b559,
and 2 beta -carotene molecules. Time-resolved pump-probe kinetic spectroscopy was carried out with 105-fs time resolution
and with the pump laser polarized parallel, perpendicular, and
at the magic angle (54.7 degrees) relative to the polarized probe beam. The time evolution of the transient absorption changes due to the formation of the oxidized primary electron donor P680+ and the reduced primary electron acceptor Pheo- were measured at 820 nm and 545 nm, respectively. In addition, kinetics were obtained at 680 nm, the wavelength ascribed to the Q transition of the primary electron donor P680 in the reaction center. At each measured probe wavelength the kinetics of the transient absorption changes can be fit to two major kinetic components. The relative amplitudes of these components are strongly dependent on the polarization of the pump beam relative to that of the probe. At the magic angle, where no photoselection occurs, the amplitude of the 3-ps component, which is indicative of the charge separation, dominates. When the primary electron acceptor Pheo is reduced prior to P680 excitation, the 3-ps component is eliminated.
This is a story of a controversy between the 1931 Nobel laureate Otto Warburg and the Midwest Gan... more This is a story of a controversy between the 1931 Nobel laureate Otto Warburg and the Midwest Gang. (The "Midwest Gang" was used by Warburg when he was describing, to Andy Benson, his quarrel with Robert Emerson of UIUC, Urbana, Illinois.) To Warburg, this Midwest Gang also included the 1926 Nobel laureate James Franck, Eugene Rabinowitch, the author of the "Bible" of Photosynthesis (1945, 1951 and 1956), and Hans Gaffron. It turns out that students of Emerson : Govindjee and Rajni Govindjee have played a major role in supporting Emerson even after the death of both the actors: Emerson and Warburg, and that too under Warburg's conditions!
Albert W. Frenkel, a pioneer in photosynthesis
research, and discoverer of photophosphorylation i... more Albert W. Frenkel, a pioneer in photosynthesis research, and discoverer of photophosphorylation in photosynthetic bacteria, is remembered here by two of us: Govindjee (historical corner editor of photosynthesis research) and Susanna Frenkel (SF; Albert Frenkel’s daughter, who provided most of the family information).
t On January 16, 2015, Professor Andrew Alm
Benson, one of the leading plant biochemists of the
t... more t On January 16, 2015, Professor Andrew Alm Benson, one of the leading plant biochemists of the twentieth century, died in La Jolla, California, at the age of 97; he was born on September 24, 1917. Benson was known especially for his pioneering studies on photosynthesis (CO2 assimilation, carbon reduction cycle) and plant lipids (phospholipid phosphatidyl glycerol; and the sulfolipid, sulfoquinovosyl diglyceride).
William Arnold—Bill, to all who knew him—was a plant physiologist, physicist, and biologist all i... more William Arnold—Bill, to all who knew him—was a plant physiologist, physicist, and biologist all in one and a researcher of the highest order in each field. His achievements read like a march of scientific progress. He discovered what came to be called the photosynthetic unit, with Robert Emerson, in 1932. He made the first reliable measurements, in his 1935 Harvard Ph.D. dissertation, of the minimum quantum requirement for the evolution of an oxygen molecule. In 1951, together with J. Robert Oppenheimer, he made critical suggestions on the mechanism of excitation energy transfer in photosynthesis. With Bernard Strehler in that same year he discovered delayed light emission (DLE) in photosyntheticorganisms. He made the first measurements of excitation energy migration by depolarization of fluorescence, with Eleanor Meek in 1956. He discovered thermoluminescence in chloroplasts, with Helen Sherwood in 1957. He elucidated the primary photochemical reactions at the photosynthetic reaction center down to 1K with Rod Clayton in 1960. Bill discovered the presence of electroluminescence, also in chloroplasts, with Jim Azzi in 1971. As this list suggests, Bill Arnold was one of the founding fathers of our understanding of the physical basis for photosynthesis.
Chlorophyll b is synthesized by the oxidation of a methyl group on the B ring of a tetrapyrrole m... more Chlorophyll b is synthesized by the oxidation of a methyl group on the B ring of a tetrapyrrole molecule to a formyl group by chlorophyllide a oxygenase (CAO). The full-length CAO from Arabidopsis (Arabidopsis thaliana) was overexpressed in tobacco (Nicotiana tabacum) that grows well at light intensities much higher than those tolerated by Arabidopsis. This resulted in an increased synthesis of glutamate semialdehyde, 5-aminolevulinic acid, magnesium-porphyrins, and chlorophylls. Overexpression of CAO resulted in increased chlorophyll b synthesis and a decreased chlorophyll a/b ratio in low light grown as well as high light-grown tobacco plants; this effect, however, was more pronounced in high light. The increased potential of the protochlorophyllide oxidoreductase activity and chlorophyll biosynthesis compensated for the usual loss of chlorophylls in high light. Increased chlorophyll b synthesis in CAO-overexpressed plants was accompanied not only by an increased abundance of light-harvesting chlorophyll proteins but also of other proteins of the electron transport chain, which led to an increase in the capture of light as well as enhanced (40%–80%) electron transport rates of photosystems I and II at both limiting and saturating light intensities. Although the quantum yield of carbon dioxide fixation remained unchanged, the lightsaturated photosynthetic carbon assimilation, starch content, and dry matter accumulation increased in CAO-overexpressed plants grown in both low- and high-light regimes. These results demonstrate that controlled up-regulation of chlorophyll b biosynthesis comodulates the expression of several thylakoid membrane proteins that increase both the antenna size and the electron transport rates and enhance carbon dioxide assimilation, starch content, and dry matter accumulation.
As photosynthesis on Earth produces the primary signatures of life that can be detected astronomi... more As photosynthesis on Earth produces the primary signatures of life that can be detected astronomically at the global scale, a strong focus of the search for extrasolar life will be photosynthesis, particularly photosynthesis that has evolved with a different parent star. We take previously simulated planetary atmospheric compositions for Earth-like planets around observed F2V and K2V, modeled M1V and M5V stars, and around the active M4.5V star AD Leo; our scenarios use Earth’s atmospheric composition as well as very low O2 content in case anoxygenic photosynthesis dominates. With a line-by-line radiative transfer model, we calculate the incident spectral photon flux densities at the surface of the planet and under water. We identify bands of available photosynthetically relevant radiation and find that photosynthetic pigments on planets around F2V stars may peak in absorbance in the blue, K2V in the red-orange, and M stars in the near-infrared, in bands at 0.93–1.1 m, 1.1–1.4 m, 1.5–1.8 m, and 1.8–2.5 m. However, underwater organisms will be restricted to wavelengths shorter than 1.4 m and more likely below 1.1 m. M star planets without oxygenic photosynthesis will have photon fluxes above 1.6 m curtailed by methane. Longer-wavelength, multi-photosystem series would reduce the quantum yield but could allow for oxygenic photosystems at longer wavelengths. A wavelength of 1.1 m is a possible upper cutoff for electronic transitions
versus only vibrational energy; however, this cutoff is not strict, since such energetics depend on molecular configuration. M star planets could be a half to a tenth as productive
as Earth in the visible, but exceed Earth if useful photons extend to 1.1 m for anoxygenic photosynthesis. Under water, organisms would still be able to survive ultraviolet flares from
young M stars and acquire adequate light for growth.
Acidocalcisomes are acidic calcium storage compartments
described initially in trypanosomatid and... more Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH4Cl released 45Ca2 from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H- ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H-PPase and the V-H-ATPase activities in addition to high concentrations of PPi and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electrondense vacuoles, PPi-driven proton translocation was stimulated by potassium ions and inhibited by the PPianalog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PPi hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4,6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.
The xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the m... more The xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the most important regulatory and photoprotective mechanisms in photosynthesis. Using parallel time resolved and pulse-amplitude modulation fluorometry, we studied the influence of the intrathylakoid pH and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the ~0.5 ns Chl a fluorescence lifetime (tau) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous effects of the chloroplast trans-thylakoid membrane pH gradient and xanthophyll cycle pigments on the PSII Chl a fluorescence ô distributions and intensity. The model assumes that (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this binding site is activated by a low intrathylakoid pH(pK ~4.5) that increases the affinity for Z (or A), (3) about one Z or A molecule binds to the activated site, and (4) this binding effectively “switches” the fluorescence tau distribution of the PSII unit to a state with a decreased fluorescence tau and emission intensity (a “dimmer switch” concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissipation. Statistical analysis of the data yields an equilibrium association constant, Ka, that ranges from 0.7 to 3.4 per PSII for the protonated/activated binding site for Z (or A). The model explains (1) the relative fraction of the ~0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) the dependence of the ratio of F¢m/Fm on the fraction of the 0.5 ns fluorescence ô component (where F'm and Fm are maximal fluorescence intensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F'm/Fm on the concentration of Z and A and the intrathylakoid pH.
The sequence connecting alpha-helices D and E of the D1 protein in photosystem II (PSII) is longe... more The sequence connecting alpha-helices D and E of the D1 protein in photosystem II (PSII) is longer than that found in
the corresponding loop of the L subunit in the rhodobacterial reaction centre. This sequence was mutated in order
to determine its role in oxygenic photosynthesis. Site-specific mutants, including point mutations and deletions
of different size, of the PEST-like region and the putative cleavage area in the D-E loop of the D1 protein were
constructed in Synechocystis sp. PCC 6803. The effects of mutations on the functional and structural properties
of PSII and turnover of the D1 protein were examined. Our results demonstrate that deletion of either the PESTlike
sequence (DeltaR225-F239) or the putative cleavage region (Delta G240-V249, Delta R225-V249) of the D1 protein
resulted in severe perturbations on the function of the QB electron acceptor of PSII. However, PSII centres of the
mutant with deleted PEST region remained functional enough to support autotrophic growth whereas deletions
of the putative cleavage region prevented autotrophic growth. Although enhanced degradation rates of the mutant
D1 proteins under low-light growth conditions demonstrate that neither the PEST-like sequence nor the putative
cleavage region are required for D1 proteolysis, it became clear that the extension in the D-E loop of the D1
protein is essential for proper PSII assembly and photoautotrophic growth. Moreover, modifications of the D-E
loop resulted in transcriptional activation of the psbA gene, indicating that neither light intensity, as such, nor the
activity of the electron transfer chain are the only determinants in regulation of psbA gene transcription.
Transient absorption measurements of the pheophytin a anion band and Qx band bleach region using ... more Transient absorption measurements of the pheophytin a anion band and Qx band bleach region using preferential excitation of P680 are performed on isolated photosystem II reaction centers to determine the effective rate constant for charge separation. A novel analysis of the Qx band bleach region explicitly takes the changing background into account in order to directly measure the rate of growth of the bleach. Both spectral regions reveal biphasic kinetics, with a ca. (8 ps)-1 rate constant for the faster component, and a ca. (50 ps)-1 rate
constant for the slower component. We propose that the faster component corresponds to the effective rate constant for charge separation from within the equilibrated reaction center core and provides a lower limit for the intrinsic rate constant for charge separation. The slower component corresponds to charge separation that is limited by slow energy transfer from a long-wavelength accessory chlorophyll a.
Photosystem II electron transfer, charge stabilization, and photoinhibition were studied in three... more Photosystem II electron transfer, charge stabilization, and photoinhibition were studied in three site-specific mutants of the D1 polypeptide of Synechocystis PCC 6803: E243K, E229D, and CA1 (deletion of three glutamates 242-244 and a substitution, glutamine- 241 to histidine). The phenotypes of the E229D and E243K mutants were similar to that of the control strain (AR) in all of the studied aspects. The characteristics of CA1 were very different. Formate, which inhibits the QA- to QB- reaction, was several fold less effective in CA1 than in AR. The S2QA- and S2QB- states were stabilized in CA1. It was previously shown that the electron transfer between QA- and QB was modified in CA1 (P Maenpaa, T. Kallio, P. Mulo, C. Salih, E.-M. Aro, E. Tyystjarvi, C. Jansson 11 9931 Plant Mo1 Biol 22: 1-12). A change in the redox potential of the QA/QA-couple, which renders the reoxidation of QA- by back or forward reactions more difficult, could explain the phenotype of CA,. Although the rates of photoinhibition measured as inhibition of oxygen evolution, Chl fluorescence quenching, and decrease of thermoluminescence B and Q bands were similar in AR and CA1, the CA1 strain more quickly reached a state from which the cells were unable to recover their activity. The results described in this paper suggest that a modification in the structure of the D-de loop of D, could influence the properties of the couple QA/QA- in D1, and the mechanism of recovery from photoinhibition.
Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura inno... more Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO2 (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO2 levels (5%). Photoautotrophy was indicated by the requirement for CO2 and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C4 Amaranthus species. The cells showed high dark respiration rates and had lower net CO2 fixation under high 02 conditions. Dark CO2 fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO2 conditions showed the unique properties of a high RuBPcase activation level in ambient CO2 and a stable ability to show net CO2 fixation in 21% 02 conditions.
Disulfiram (tetraethylthiuram disulfide), a metal chelator, inhibits photosynthetic electron tran... more Disulfiram (tetraethylthiuram disulfide), a metal chelator, inhibits photosynthetic electron transport in broken chloroplasts. A major site of inhibition is detected on the electron-acceptor side of photosystem II between QA, the first plastoquinone electron-acceptor, and the second plastoquinone electron-acceptor, QB. This site of inhibition is shown by a several fold increase in the half-time of QA oxidation, as monitored by the decay of the variable chlorophyll a flourescence after an actinic flash. Another site of inhibition is detected in the functioning of the reaction center of photosystem II disulfiram is observed to quench the room temperature variable chlorophyll a fluorescence, as well as the intensity of the 695 nm peak, relative to the 685 nm peak, in the chlorophyll a fluorescence spectrum at 77 K. Electron transport from water to the photosystem II electron-acceptor silicomolybdate is also inhibited. Disulfiram does not inhibit electron flow before the site(s) of donation by exogenous electron donors to photosystem II, and no inhibition is detected in the partial reactions associated with photosystem I.
The effects of cations and abscisic acid on chloroplast activity in guard celis of Viia faba were... more The effects of cations and abscisic acid on chloroplast activity in guard celis of Viia faba were investigated by analysis of the transient of chlorophyli a fluorescence. When epidermal strips containing guard cells as the only living cells were incubated in water and illuminated with strong light, chlorophyll a fluorescence rose rapidly to a high intensity and then declined slowly to a stationary level. The rate of this decline was enhanced by K+ or Na+, and the effect of these cations was greater when added with phosphate than with chloride as the anion. Ca'+ suppressed the enhancement by Na+ and, to a lesser extent, that by K+. Abscisic acid also suppressed the enhancement by K+ and Na+. Since the fluorescence decline reflects the increase of intrathylakod H+ concentration necessary for photophosphorylation, the acceleration of the decline by K+ (or Na+ in the absence of Ca2+ ) implicates chloroplast activity in ion accumulation by guard celis in the light The differential effects of phosphate and chloride suggest that chloroplast actiivty may be Involved In malate formation in guard cells in the light.
Bicarbonate ion, not dissolved CO2 gas, is shown to increase 4-
to 5-fold the rate of dichlorophe... more Bicarbonate ion, not dissolved CO2 gas, is shown to increase 4- to 5-fold the rate of dichlorophenol indophenol reduction by isolated maize (Zea mays) chloroplasts. Glutaraldehyde fixed chloroplasts continue to exhibit bicarbonate-dependent 2,6- dichlorophenol indophenol reduction. Bicarbonate is shown to act close to the oxygen-evolving site, i.e. prior to the electron donation site of diphenyl carbazide to photosystem II. Dark incubation and light pretreatment of chloroplasts in various concentrations of bicarbonate, just prior to assay, indicate that bicarbonate binds to chloroplasts in the dark and is released again as the Hill reaction proceeds in the light. It is suggested that bicarbonate ions may play a critical role in the oxygen-evolving process in photosynthesis.
The Emerson enhancement effect has been shown
to occur in the photoreduction of nicotinamide aden... more The Emerson enhancement effect has been shown to occur in the photoreduction of nicotinamide adenine dinucleotidephosphate by spinach chloroplasts. Greater than additive rates were obtained where far-red light beams were mixed (combined) with supplementary white (fluorescent) or monochromatic 650 nm light in nicotinamide adenine dinucleotidephosphate reduction, and oxygen evolution. In magnitude, wavelength, and intensity dependence of the enhancement effect, as measured in the Hill reaction, appears analogous to that occurring in complete photosynthesis.
Nonsulfur photosynthetic bacteria (Athiorhodaceae) exhibit a time-variable fluorescence in additi... more Nonsulfur photosynthetic bacteria (Athiorhodaceae) exhibit a time-variable fluorescence in addition to a constant fluorescence. All species examined show upon aging a remarkable gain in the variable component at the expense of the constant component while the total fluorescence remains essentially invariant. This result can be rationalized by supposing a change in distribution of bacteriochlorophyll in photosynthetic units as cells age. Alternatively, one may assume operation of two photochemical systems-one cyclic and predominant in young cells, the other noncyclic and predominant in old cells. It is also noted that a hitherto unreported minor fluorescence with maximum emission at - 860 nm exists in addition to the well-known main fluorescence band at -890 nm. The rise in variable fluorescence is associated with the main band, a result in accord with the notion that the bacteriochloro- phyll component responsible and absorbing at 870 nm is directly in contact with the energy trap.
[Basically, these data provided the first clear demonstration of a "dimmer switch" in photosynthe... more [Basically, these data provided the first clear demonstration of a "dimmer switch" in photosynthesis during photo-protection against excess light.]
Excess light triggers protective non-radiative dissipation of excitation energy in photosystem II through the formation of
a trans-thylakoid pH gradient that in turn stimulates formation
of zeaxanthin and antheraxanthin. These xanthophylls when
combined with protonation of antenna pigment-protein complexes may increase nonradiative dissipation and, thus,
quench chlorophyll a fluorescence. Here we measured, in parallel, the chlorophyll a fluorescence lifetime and intensity
to understand the- mechanism of this process. Increasing
the xanthophyll concentration in the presence of a pH gradient
(quenched conditions) decreases the fractional intensity of a
fluorescence lifetime component centered at-2 ns and increases
a component at -0.4 ns. Uncoupling the pH gradient (unquenched conditions) eliminates the 0.4-ns component. Changes in the xanthophyll concentration do not significantly
affect the fluorescence lifetimes in either the quenched or
unquenched sample conditions. However, there are differences
in fluorescence life- times between the quenched and unquenched states that are due to pH-related, but non xanthophyll-related, processes. Quenching of the maximal fluorescence intensity correlates with both the xanthophyll
concentration and the fractional intensity of the 0.4-ns component. The unchanged fluorescence lifetimes and the
proportional quenching of the maximal and dark-level fluorescence intensities indicate that the xanthophylls act on
antenna, not reaction center processes. Further, the fluorescence quenching is interpreted as the combined effect
of the pH gradient and xanthophyll concentration, resulting
in the formation of a quenching complex with a short (~0.4 ns)
fluorescence lifetime.
Photosynthetic conversion of light energy into chemical potential begins in reaction center prote... more Photosynthetic conversion of light energy into chemical potential begins in reaction center protein complexes, where rapid charge separation occurs with nearly unit quantum efficiency. Primary charge separation was studied in isolated photosystem II reaction centers from spinach containing
6 chlorophyll a, 2 pheophytin a (Pheo), 1 cytochrome b559,
and 2 beta -carotene molecules. Time-resolved pump-probe kinetic spectroscopy was carried out with 105-fs time resolution
and with the pump laser polarized parallel, perpendicular, and
at the magic angle (54.7 degrees) relative to the polarized probe beam. The time evolution of the transient absorption changes due to the formation of the oxidized primary electron donor P680+ and the reduced primary electron acceptor Pheo- were measured at 820 nm and 545 nm, respectively. In addition, kinetics were obtained at 680 nm, the wavelength ascribed to the Q transition of the primary electron donor P680 in the reaction center. At each measured probe wavelength the kinetics of the transient absorption changes can be fit to two major kinetic components. The relative amplitudes of these components are strongly dependent on the polarization of the pump beam relative to that of the probe. At the magic angle, where no photoselection occurs, the amplitude of the 3-ps component, which is indicative of the charge separation, dominates. When the primary electron acceptor Pheo is reduced prior to P680 excitation, the 3-ps component is eliminated.
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Papers by FNU Govindjee
research, and discoverer of photophosphorylation in photosynthetic bacteria, is remembered here by two of us:
Govindjee (historical corner editor of photosynthesis research)
and Susanna Frenkel (SF; Albert Frenkel’s daughter, who provided most of the family information).
Benson, one of the leading plant biochemists of the
twentieth century, died in La Jolla, California, at the age of
97; he was born on September 24, 1917. Benson was
known especially for his pioneering studies on photosynthesis
(CO2 assimilation, carbon reduction cycle) and plant
lipids (phospholipid phosphatidyl glycerol; and the sulfolipid,
sulfoquinovosyl diglyceride).
made the first reliable measurements, in his 1935 Harvard Ph.D. dissertation, of the minimum quantum requirement for the evolution of an oxygen molecule. In 1951, together with J. Robert Oppenheimer, he made critical suggestions on the
mechanism of excitation energy transfer in photosynthesis. With Bernard Strehler in that same year he discovered delayed light emission (DLE) in photosyntheticorganisms. He made the first measurements of excitation energy migration by
depolarization of fluorescence, with Eleanor Meek in 1956. He discovered thermoluminescence in chloroplasts, with Helen Sherwood in 1957. He elucidated the primary photochemical reactions at the photosynthetic reaction center down
to 1K with Rod Clayton in 1960. Bill discovered the presence of electroluminescence, also in chloroplasts, with Jim Azzi in 1971. As this list suggests, Bill Arnold was one of the founding fathers of our understanding of the physical basis for photosynthesis.
chlorophyllide a oxygenase (CAO). The full-length CAO from Arabidopsis (Arabidopsis thaliana) was overexpressed in tobacco (Nicotiana tabacum) that grows well at light intensities much higher than those tolerated by Arabidopsis. This resulted in an increased synthesis of glutamate semialdehyde, 5-aminolevulinic acid, magnesium-porphyrins, and chlorophylls.
Overexpression of CAO resulted in increased chlorophyll b synthesis and a decreased chlorophyll a/b ratio in low light grown as well as high light-grown tobacco plants; this effect, however, was more pronounced in high light. The increased
potential of the protochlorophyllide oxidoreductase activity and chlorophyll biosynthesis compensated for the usual loss of
chlorophylls in high light. Increased chlorophyll b synthesis in CAO-overexpressed plants was accompanied not only by an
increased abundance of light-harvesting chlorophyll proteins but also of other proteins of the electron transport chain, which led to an increase in the capture of light as well as enhanced (40%–80%) electron transport rates of photosystems I and II at both limiting and saturating light intensities. Although the quantum yield of carbon dioxide fixation remained unchanged, the lightsaturated photosynthetic carbon assimilation, starch content, and dry matter accumulation increased in CAO-overexpressed plants grown in both low- and high-light regimes. These results demonstrate that controlled up-regulation of chlorophyll b biosynthesis comodulates the expression of several thylakoid membrane proteins that increase both the antenna size and the electron transport rates and enhance carbon dioxide assimilation, starch content, and dry matter accumulation.
versus only vibrational energy; however, this cutoff is not strict, since such energetics depend on molecular configuration. M star planets could be a half to a tenth as productive
as Earth in the visible, but exceed Earth if useful photons extend to 1.1 m for anoxygenic photosynthesis. Under water, organisms would still be able to survive ultraviolet flares from
young M stars and acquire adequate light for growth.
described initially in trypanosomatid and apicomplexan
parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH4Cl released 45Ca2 from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or
polyphosphate bodies of C. reinhardtii showed large
amounts of phosphorus, magnesium, calcium, and zinc.
Immunofluorescence microscopy, using antisera raised
against a peptide sequence of the vacuolar type proton
pyrophosphatase (H-PPase) of Arabidopsis thaliana
which is conserved in the C. reinhardtii enzyme, indicated
localization in the plasma membrane, in intracellular
vacuoles, and the contractile vacuole where it colocalized
with the vacuolar proton ATPase (V-H- ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H-PPase and the V-H-ATPase activities in addition to high concentrations of PPi and short and long chain polyphosphate, but lack of markers
for mitochondria and chloroplasts. In isolated electrondense
vacuoles, PPi-driven proton translocation was stimulated by potassium ions and inhibited by the PPianalog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N-dicyclohexylcarbodiimide,
and N-ethylmaleimide also inhibited PPi hydrolysis
in the isolated organelles in a dose-dependent manner.
These results indicate that the electron-dense vacuoles
of C. reinhardtii are very similar to acidocalcisomes
with regard to their chemical composition and the presence
of proton pumps. Polyphosphate was also localized
to the contractile vacuole by 4,6-diamidino-2-phenylindole
staining, suggesting, with the immunochemical
data, a link between these organelles and the
acidocalcisomes.
and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the ~0.5 ns Chl a fluorescence
lifetime (tau) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous effects of the chloroplast trans-thylakoid membrane pH gradient and xanthophyll cycle pigments on the PSII Chl a fluorescence ô distributions and intensity. The model assumes that (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this binding site is activated by a low intrathylakoid pH(pK ~4.5) that increases the affinity for Z (or A), (3) about one Z or A molecule binds to the activated site, and (4) this binding effectively “switches” the fluorescence tau distribution of the PSII unit to a state with a decreased fluorescence tau and emission intensity (a “dimmer switch” concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissipation. Statistical analysis of the data yields an equilibrium association constant, Ka, that ranges from 0.7 to 3.4 per PSII for the protonated/activated binding site for Z (or A). The model explains (1) the relative fraction of the ~0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) the dependence of the ratio of F¢m/Fm on the fraction of the 0.5 ns fluorescence ô component
(where F'm and Fm are maximal fluorescence intensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F'm/Fm on the concentration of Z and A and the intrathylakoid pH.
the corresponding loop of the L subunit in the rhodobacterial reaction centre. This sequence was mutated in order
to determine its role in oxygenic photosynthesis. Site-specific mutants, including point mutations and deletions
of different size, of the PEST-like region and the putative cleavage area in the D-E loop of the D1 protein were
constructed in Synechocystis sp. PCC 6803. The effects of mutations on the functional and structural properties
of PSII and turnover of the D1 protein were examined. Our results demonstrate that deletion of either the PESTlike
sequence (DeltaR225-F239) or the putative cleavage region (Delta G240-V249, Delta R225-V249) of the D1 protein
resulted in severe perturbations on the function of the QB electron acceptor of PSII. However, PSII centres of the
mutant with deleted PEST region remained functional enough to support autotrophic growth whereas deletions
of the putative cleavage region prevented autotrophic growth. Although enhanced degradation rates of the mutant
D1 proteins under low-light growth conditions demonstrate that neither the PEST-like sequence nor the putative
cleavage region are required for D1 proteolysis, it became clear that the extension in the D-E loop of the D1
protein is essential for proper PSII assembly and photoautotrophic growth. Moreover, modifications of the D-E
loop resulted in transcriptional activation of the psbA gene, indicating that neither light intensity, as such, nor the
activity of the electron transfer chain are the only determinants in regulation of psbA gene transcription.
constant for the slower component. We propose that the faster component corresponds to the effective rate constant for charge separation from within the equilibrated reaction center core and provides a lower limit for the intrinsic rate constant for charge separation. The slower component corresponds to charge separation that is limited by slow energy transfer from a long-wavelength accessory chlorophyll a.
less effective in CA1 than in AR. The S2QA- and S2QB- states were stabilized in CA1. It was previously shown that the electron transfer between QA- and QB was modified in CA1 (P Maenpaa, T. Kallio, P. Mulo, C. Salih, E.-M. Aro, E. Tyystjarvi, C. Jansson 11 9931 Plant Mo1 Biol 22: 1-12). A change in the redox potential of the QA/QA-couple, which renders the reoxidation of QA- by back or forward reactions more difficult, could explain the phenotype of CA,. Although the rates of photoinhibition measured as inhibition of oxygen evolution, Chl fluorescence quenching, and decrease of thermoluminescence
B and Q bands were similar in AR and CA1, the CA1 strain more quickly reached a state from which the cells were
unable to recover their activity. The results described in this paper suggest that a modification in the structure of the D-de loop of D, could influence the properties of the couple QA/QA- in D1, and the mechanism of recovery from photoinhibition.
requirement for CO2 and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate
carboxylase (RuBPcase) activity levels were well below those of
mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C4 Amaranthus species. The cells showed high dark respiration rates and had lower net CO2 fixation under high 02 conditions. Dark CO2 fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna
pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO2 conditions showed the unique properties of a high RuBPcase activation level in ambient CO2 and a stable ability to show net CO2 fixation in 21% 02 conditions.
temperature variable chlorophyll a fluorescence, as well as the intensity of the 695 nm peak, relative to the 685 nm peak, in the chlorophyll a fluorescence spectrum at 77 K. Electron transport from water to the photosystem II electron-acceptor silicomolybdate is also inhibited. Disulfiram does not inhibit electron flow before the site(s) of donation by exogenous electron donors to photosystem II, and no inhibition is detected
in the partial reactions associated with photosystem I.
declined slowly to a stationary level. The rate of this decline was enhanced by K+ or Na+, and the effect of these cations was greater when added with phosphate than with chloride as the anion. Ca'+ suppressed the enhancement by Na+ and, to a lesser extent, that by K+. Abscisic acid also suppressed the enhancement by K+ and Na+. Since the fluorescence
decline reflects the increase of intrathylakod H+ concentration necessary for photophosphorylation, the acceleration of the decline by K+ (or Na+ in the absence of Ca2+ ) implicates chloroplast activity in ion accumulation by guard celis in the light The differential effects of phosphate and chloride
suggest that chloroplast actiivty may be Involved In malate formation in guard cells in the light.
to 5-fold the rate of dichlorophenol indophenol reduction by
isolated maize (Zea mays) chloroplasts. Glutaraldehyde fixed
chloroplasts continue to exhibit bicarbonate-dependent 2,6-
dichlorophenol indophenol reduction. Bicarbonate is shown to
act close to the oxygen-evolving site, i.e. prior to the electron
donation site of diphenyl carbazide to photosystem II. Dark incubation and light pretreatment of chloroplasts in various concentrations of bicarbonate, just prior to assay, indicate that bicarbonate binds to chloroplasts in the dark and is released again as the Hill reaction proceeds in the light. It is suggested that bicarbonate ions may play a critical role in the oxygen-evolving process in photosynthesis.
to occur in the photoreduction of nicotinamide adenine
dinucleotidephosphate by spinach chloroplasts.
Greater than additive rates were obtained where far-red
light beams were mixed (combined) with supplementary
white (fluorescent) or monochromatic 650 nm light
in nicotinamide adenine dinucleotidephosphate reduction,
and oxygen evolution. In magnitude, wavelength,
and intensity dependence of the enhancement effect,
as measured in the Hill reaction, appears analogous
to that occurring in complete photosynthesis.
Excess light triggers protective non-radiative dissipation of excitation energy in photosystem II through the formation of
a trans-thylakoid pH gradient that in turn stimulates formation
of zeaxanthin and antheraxanthin. These xanthophylls when
combined with protonation of antenna pigment-protein complexes may increase nonradiative dissipation and, thus,
quench chlorophyll a fluorescence. Here we measured, in parallel, the chlorophyll a fluorescence lifetime and intensity
to understand the- mechanism of this process. Increasing
the xanthophyll concentration in the presence of a pH gradient
(quenched conditions) decreases the fractional intensity of a
fluorescence lifetime component centered at-2 ns and increases
a component at -0.4 ns. Uncoupling the pH gradient (unquenched conditions) eliminates the 0.4-ns component. Changes in the xanthophyll concentration do not significantly
affect the fluorescence lifetimes in either the quenched or
unquenched sample conditions. However, there are differences
in fluorescence life- times between the quenched and unquenched states that are due to pH-related, but non xanthophyll-related, processes. Quenching of the maximal fluorescence intensity correlates with both the xanthophyll
concentration and the fractional intensity of the 0.4-ns component. The unchanged fluorescence lifetimes and the
proportional quenching of the maximal and dark-level fluorescence intensities indicate that the xanthophylls act on
antenna, not reaction center processes. Further, the fluorescence quenching is interpreted as the combined effect
of the pH gradient and xanthophyll concentration, resulting
in the formation of a quenching complex with a short (~0.4 ns)
fluorescence lifetime.
6 chlorophyll a, 2 pheophytin a (Pheo), 1 cytochrome b559,
and 2 beta -carotene molecules. Time-resolved pump-probe kinetic spectroscopy was carried out with 105-fs time resolution
and with the pump laser polarized parallel, perpendicular, and
at the magic angle (54.7 degrees) relative to the polarized probe beam. The time evolution of the transient absorption changes due to the formation of the oxidized primary electron donor P680+ and the reduced primary electron acceptor Pheo- were measured at 820 nm and 545 nm, respectively. In addition, kinetics were obtained at 680 nm, the wavelength ascribed to the Q transition of the primary electron donor P680 in the reaction center. At each measured probe wavelength the kinetics of the transient absorption changes can be fit to two major kinetic components. The relative amplitudes of these components are strongly dependent on the polarization of the pump beam relative to that of the probe. At the magic angle, where no photoselection occurs, the amplitude of the 3-ps component, which is indicative of the charge separation, dominates. When the primary electron acceptor Pheo is reduced prior to P680 excitation, the 3-ps component is eliminated.
research, and discoverer of photophosphorylation in photosynthetic bacteria, is remembered here by two of us:
Govindjee (historical corner editor of photosynthesis research)
and Susanna Frenkel (SF; Albert Frenkel’s daughter, who provided most of the family information).
Benson, one of the leading plant biochemists of the
twentieth century, died in La Jolla, California, at the age of
97; he was born on September 24, 1917. Benson was
known especially for his pioneering studies on photosynthesis
(CO2 assimilation, carbon reduction cycle) and plant
lipids (phospholipid phosphatidyl glycerol; and the sulfolipid,
sulfoquinovosyl diglyceride).
made the first reliable measurements, in his 1935 Harvard Ph.D. dissertation, of the minimum quantum requirement for the evolution of an oxygen molecule. In 1951, together with J. Robert Oppenheimer, he made critical suggestions on the
mechanism of excitation energy transfer in photosynthesis. With Bernard Strehler in that same year he discovered delayed light emission (DLE) in photosyntheticorganisms. He made the first measurements of excitation energy migration by
depolarization of fluorescence, with Eleanor Meek in 1956. He discovered thermoluminescence in chloroplasts, with Helen Sherwood in 1957. He elucidated the primary photochemical reactions at the photosynthetic reaction center down
to 1K with Rod Clayton in 1960. Bill discovered the presence of electroluminescence, also in chloroplasts, with Jim Azzi in 1971. As this list suggests, Bill Arnold was one of the founding fathers of our understanding of the physical basis for photosynthesis.
chlorophyllide a oxygenase (CAO). The full-length CAO from Arabidopsis (Arabidopsis thaliana) was overexpressed in tobacco (Nicotiana tabacum) that grows well at light intensities much higher than those tolerated by Arabidopsis. This resulted in an increased synthesis of glutamate semialdehyde, 5-aminolevulinic acid, magnesium-porphyrins, and chlorophylls.
Overexpression of CAO resulted in increased chlorophyll b synthesis and a decreased chlorophyll a/b ratio in low light grown as well as high light-grown tobacco plants; this effect, however, was more pronounced in high light. The increased
potential of the protochlorophyllide oxidoreductase activity and chlorophyll biosynthesis compensated for the usual loss of
chlorophylls in high light. Increased chlorophyll b synthesis in CAO-overexpressed plants was accompanied not only by an
increased abundance of light-harvesting chlorophyll proteins but also of other proteins of the electron transport chain, which led to an increase in the capture of light as well as enhanced (40%–80%) electron transport rates of photosystems I and II at both limiting and saturating light intensities. Although the quantum yield of carbon dioxide fixation remained unchanged, the lightsaturated photosynthetic carbon assimilation, starch content, and dry matter accumulation increased in CAO-overexpressed plants grown in both low- and high-light regimes. These results demonstrate that controlled up-regulation of chlorophyll b biosynthesis comodulates the expression of several thylakoid membrane proteins that increase both the antenna size and the electron transport rates and enhance carbon dioxide assimilation, starch content, and dry matter accumulation.
versus only vibrational energy; however, this cutoff is not strict, since such energetics depend on molecular configuration. M star planets could be a half to a tenth as productive
as Earth in the visible, but exceed Earth if useful photons extend to 1.1 m for anoxygenic photosynthesis. Under water, organisms would still be able to survive ultraviolet flares from
young M stars and acquire adequate light for growth.
described initially in trypanosomatid and apicomplexan
parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH4Cl released 45Ca2 from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or
polyphosphate bodies of C. reinhardtii showed large
amounts of phosphorus, magnesium, calcium, and zinc.
Immunofluorescence microscopy, using antisera raised
against a peptide sequence of the vacuolar type proton
pyrophosphatase (H-PPase) of Arabidopsis thaliana
which is conserved in the C. reinhardtii enzyme, indicated
localization in the plasma membrane, in intracellular
vacuoles, and the contractile vacuole where it colocalized
with the vacuolar proton ATPase (V-H- ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H-PPase and the V-H-ATPase activities in addition to high concentrations of PPi and short and long chain polyphosphate, but lack of markers
for mitochondria and chloroplasts. In isolated electrondense
vacuoles, PPi-driven proton translocation was stimulated by potassium ions and inhibited by the PPianalog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N-dicyclohexylcarbodiimide,
and N-ethylmaleimide also inhibited PPi hydrolysis
in the isolated organelles in a dose-dependent manner.
These results indicate that the electron-dense vacuoles
of C. reinhardtii are very similar to acidocalcisomes
with regard to their chemical composition and the presence
of proton pumps. Polyphosphate was also localized
to the contractile vacuole by 4,6-diamidino-2-phenylindole
staining, suggesting, with the immunochemical
data, a link between these organelles and the
acidocalcisomes.
and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the ~0.5 ns Chl a fluorescence
lifetime (tau) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous effects of the chloroplast trans-thylakoid membrane pH gradient and xanthophyll cycle pigments on the PSII Chl a fluorescence ô distributions and intensity. The model assumes that (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this binding site is activated by a low intrathylakoid pH(pK ~4.5) that increases the affinity for Z (or A), (3) about one Z or A molecule binds to the activated site, and (4) this binding effectively “switches” the fluorescence tau distribution of the PSII unit to a state with a decreased fluorescence tau and emission intensity (a “dimmer switch” concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissipation. Statistical analysis of the data yields an equilibrium association constant, Ka, that ranges from 0.7 to 3.4 per PSII for the protonated/activated binding site for Z (or A). The model explains (1) the relative fraction of the ~0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) the dependence of the ratio of F¢m/Fm on the fraction of the 0.5 ns fluorescence ô component
(where F'm and Fm are maximal fluorescence intensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F'm/Fm on the concentration of Z and A and the intrathylakoid pH.
the corresponding loop of the L subunit in the rhodobacterial reaction centre. This sequence was mutated in order
to determine its role in oxygenic photosynthesis. Site-specific mutants, including point mutations and deletions
of different size, of the PEST-like region and the putative cleavage area in the D-E loop of the D1 protein were
constructed in Synechocystis sp. PCC 6803. The effects of mutations on the functional and structural properties
of PSII and turnover of the D1 protein were examined. Our results demonstrate that deletion of either the PESTlike
sequence (DeltaR225-F239) or the putative cleavage region (Delta G240-V249, Delta R225-V249) of the D1 protein
resulted in severe perturbations on the function of the QB electron acceptor of PSII. However, PSII centres of the
mutant with deleted PEST region remained functional enough to support autotrophic growth whereas deletions
of the putative cleavage region prevented autotrophic growth. Although enhanced degradation rates of the mutant
D1 proteins under low-light growth conditions demonstrate that neither the PEST-like sequence nor the putative
cleavage region are required for D1 proteolysis, it became clear that the extension in the D-E loop of the D1
protein is essential for proper PSII assembly and photoautotrophic growth. Moreover, modifications of the D-E
loop resulted in transcriptional activation of the psbA gene, indicating that neither light intensity, as such, nor the
activity of the electron transfer chain are the only determinants in regulation of psbA gene transcription.
constant for the slower component. We propose that the faster component corresponds to the effective rate constant for charge separation from within the equilibrated reaction center core and provides a lower limit for the intrinsic rate constant for charge separation. The slower component corresponds to charge separation that is limited by slow energy transfer from a long-wavelength accessory chlorophyll a.
less effective in CA1 than in AR. The S2QA- and S2QB- states were stabilized in CA1. It was previously shown that the electron transfer between QA- and QB was modified in CA1 (P Maenpaa, T. Kallio, P. Mulo, C. Salih, E.-M. Aro, E. Tyystjarvi, C. Jansson 11 9931 Plant Mo1 Biol 22: 1-12). A change in the redox potential of the QA/QA-couple, which renders the reoxidation of QA- by back or forward reactions more difficult, could explain the phenotype of CA,. Although the rates of photoinhibition measured as inhibition of oxygen evolution, Chl fluorescence quenching, and decrease of thermoluminescence
B and Q bands were similar in AR and CA1, the CA1 strain more quickly reached a state from which the cells were
unable to recover their activity. The results described in this paper suggest that a modification in the structure of the D-de loop of D, could influence the properties of the couple QA/QA- in D1, and the mechanism of recovery from photoinhibition.
requirement for CO2 and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate
carboxylase (RuBPcase) activity levels were well below those of
mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C4 Amaranthus species. The cells showed high dark respiration rates and had lower net CO2 fixation under high 02 conditions. Dark CO2 fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna
pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO2 conditions showed the unique properties of a high RuBPcase activation level in ambient CO2 and a stable ability to show net CO2 fixation in 21% 02 conditions.
temperature variable chlorophyll a fluorescence, as well as the intensity of the 695 nm peak, relative to the 685 nm peak, in the chlorophyll a fluorescence spectrum at 77 K. Electron transport from water to the photosystem II electron-acceptor silicomolybdate is also inhibited. Disulfiram does not inhibit electron flow before the site(s) of donation by exogenous electron donors to photosystem II, and no inhibition is detected
in the partial reactions associated with photosystem I.
declined slowly to a stationary level. The rate of this decline was enhanced by K+ or Na+, and the effect of these cations was greater when added with phosphate than with chloride as the anion. Ca'+ suppressed the enhancement by Na+ and, to a lesser extent, that by K+. Abscisic acid also suppressed the enhancement by K+ and Na+. Since the fluorescence
decline reflects the increase of intrathylakod H+ concentration necessary for photophosphorylation, the acceleration of the decline by K+ (or Na+ in the absence of Ca2+ ) implicates chloroplast activity in ion accumulation by guard celis in the light The differential effects of phosphate and chloride
suggest that chloroplast actiivty may be Involved In malate formation in guard cells in the light.
to 5-fold the rate of dichlorophenol indophenol reduction by
isolated maize (Zea mays) chloroplasts. Glutaraldehyde fixed
chloroplasts continue to exhibit bicarbonate-dependent 2,6-
dichlorophenol indophenol reduction. Bicarbonate is shown to
act close to the oxygen-evolving site, i.e. prior to the electron
donation site of diphenyl carbazide to photosystem II. Dark incubation and light pretreatment of chloroplasts in various concentrations of bicarbonate, just prior to assay, indicate that bicarbonate binds to chloroplasts in the dark and is released again as the Hill reaction proceeds in the light. It is suggested that bicarbonate ions may play a critical role in the oxygen-evolving process in photosynthesis.
to occur in the photoreduction of nicotinamide adenine
dinucleotidephosphate by spinach chloroplasts.
Greater than additive rates were obtained where far-red
light beams were mixed (combined) with supplementary
white (fluorescent) or monochromatic 650 nm light
in nicotinamide adenine dinucleotidephosphate reduction,
and oxygen evolution. In magnitude, wavelength,
and intensity dependence of the enhancement effect,
as measured in the Hill reaction, appears analogous
to that occurring in complete photosynthesis.
Excess light triggers protective non-radiative dissipation of excitation energy in photosystem II through the formation of
a trans-thylakoid pH gradient that in turn stimulates formation
of zeaxanthin and antheraxanthin. These xanthophylls when
combined with protonation of antenna pigment-protein complexes may increase nonradiative dissipation and, thus,
quench chlorophyll a fluorescence. Here we measured, in parallel, the chlorophyll a fluorescence lifetime and intensity
to understand the- mechanism of this process. Increasing
the xanthophyll concentration in the presence of a pH gradient
(quenched conditions) decreases the fractional intensity of a
fluorescence lifetime component centered at-2 ns and increases
a component at -0.4 ns. Uncoupling the pH gradient (unquenched conditions) eliminates the 0.4-ns component. Changes in the xanthophyll concentration do not significantly
affect the fluorescence lifetimes in either the quenched or
unquenched sample conditions. However, there are differences
in fluorescence life- times between the quenched and unquenched states that are due to pH-related, but non xanthophyll-related, processes. Quenching of the maximal fluorescence intensity correlates with both the xanthophyll
concentration and the fractional intensity of the 0.4-ns component. The unchanged fluorescence lifetimes and the
proportional quenching of the maximal and dark-level fluorescence intensities indicate that the xanthophylls act on
antenna, not reaction center processes. Further, the fluorescence quenching is interpreted as the combined effect
of the pH gradient and xanthophyll concentration, resulting
in the formation of a quenching complex with a short (~0.4 ns)
fluorescence lifetime.
6 chlorophyll a, 2 pheophytin a (Pheo), 1 cytochrome b559,
and 2 beta -carotene molecules. Time-resolved pump-probe kinetic spectroscopy was carried out with 105-fs time resolution
and with the pump laser polarized parallel, perpendicular, and
at the magic angle (54.7 degrees) relative to the polarized probe beam. The time evolution of the transient absorption changes due to the formation of the oxidized primary electron donor P680+ and the reduced primary electron acceptor Pheo- were measured at 820 nm and 545 nm, respectively. In addition, kinetics were obtained at 680 nm, the wavelength ascribed to the Q transition of the primary electron donor P680 in the reaction center. At each measured probe wavelength the kinetics of the transient absorption changes can be fit to two major kinetic components. The relative amplitudes of these components are strongly dependent on the polarization of the pump beam relative to that of the probe. At the magic angle, where no photoselection occurs, the amplitude of the 3-ps component, which is indicative of the charge separation, dominates. When the primary electron acceptor Pheo is reduced prior to P680 excitation, the 3-ps component is eliminated.