P700: the primary electron donor of photosystem I.

The primary electron donor of photosystem I, P700, is a chlorophyll species that in its excited state has a potential of approximately -1.2 V. The precise chemical composition and electronic structure of P700 is still unknown. Recent evidence indicates that P700 is a dimer of one chlorophyll (Chl) a and one Chl a'. The Chl a' and Chl a are axially coordinated by His residues provided by protein subunits PsaA and PsaB, respectively. The Chl a', but not the Chl a, is also H-bonded to the protein. The H-bonding is likely responsible for selective insertion of Chl a' into the reaction center. EPR studies of P700(+*) in frozen solution and single crystals indicate a large asymmetry in the electron spin and charge distribution towards one Chl of the dimer. Molecular orbital calculations indicate that H-bonding will specifically stabilize the Chl a'-side of the dimer, suggesting that the unpaired electron would predominantly reside on the Chl a. This is supported by results of specific mutagenesis of the PsaA and PsaB axial His residues, which show that only mutations of the PsaB subunit significantly alter the hyperfine coupling constants associated with a single Chl molecule. The PsaB mutants also alter the microwave induced triplet-minus-singlet spectrum indicating that the triplet state is localized on the same Chl. Excitonic coupling between the two Chl a of P700 is weak due to the distance and overlap of the porphyrin planes. Evidence of excitonic coupling is found in PsaB mutants which show a new bleaching band at 665 nm that likely represents an increased intensity of the upper exciton band of P700. Additional properties of P700 that may give rise to its unusually low potential are discussed.

Increased Accumulation of Carbohydrates and Decreased Photosynthetic Gene Transcript Levels in Wheat Grown at an Elevated CO2 Concentration in the Field.

Repression of photosynthetic genes by increased soluble carbohydrate concentrations may explain acclimation of photosynthesis to elevated CO2 concentration. This hypothesis was examined in a field crop of spring wheat (Triticum aestivum L.) grown at both ambient (approximately 360 [mu]mol mol-1) and elevated (550 [mu]mol mol-1) atmospheric CO2 concentrations using free-air CO2 enrichment at Maricopa, Arizona. The correspondence of steady-state levels of mRNA transcripts (coding for the 83-kD photosystem I apoprotein, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, phosphoglycerokinase, and the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase) with leaf carbohydrate concentrations (glucose-6-phosphate, glucose, fructose, sucrose, fructans, and starch) was examined at different stages of crop and leaf development and through the diurnal cycle. Overall only a weak correspondence between increased soluble carbohydrate concentrations and decreased levels for nuclear gene transcripts was found. The difference in soluble carbohydrate concentration between leaves grown at elevated and current ambient CO2 concentrations diminished with crop development, whereas the difference in transcript levels increased. In the flag leaf, soluble carbohydrate concentrations declined markedly with the onset of grain filling; yet transcript levels also declined. The results suggest that, whereas the hypothesis may hold well in model laboratory systems, many other factors modified its significance in this field wheat crop.

Photosystem I reaction-centre proteins contain leucine zipper motifs. A proposed role in dimer formation.

The photosystem I (PS I) reaction-centre polypeptides, encoded by the psaA and psaB genes, are shown to contain several highly conserved leucine repeats, consisting of a leucine residue every seventh amino acid, similar to the leucine zipper motifs known to mediate DNA-binding polypeptide dimerisation. In each of the PSI reaction-centre subunits the leucine zipper motif precedes highly conserved cysteine residues which have been proposed to ligate the interpolypeptide [4Fe-4S] centre, Fx. We propose that PS I reaction-centre dimerisation and [4Fe-4S] centre formation are mediated through the leucine zipper.

Transformation of chloroplasts with the psaB gene encoding a polypeptide of the photosystem I reaction center.

A chloroplast photosystem I reaction center mutation, ac-u-g-2.3, of Chlamydomonas reinhardtii has been complemented with a wild type psaB gene to restore photosynthetic competence. The mutation was mapped in the psaB coding sequence by chloroplast transformation using subcloned restriction fragments of psaB. The mutation was found to be a single base pair deletion resulting in a reading frame shift and premature termination of the polypeptide. Transformants were verified by insertion of a site-directed mutation which created a new restriction enzyme site. These transformations demonstrate the feasibility of insertion of site-directed mutations into the psaB gene in order to elucidate amino acid residues involved in photosystem I assembly and function.

A 10 kDa polypeptide associated with the oxygen-evolving complex of photosystem II has a putative C-terminal non-cleavable thylakoid transfer domain.

The N-terminal amino acid sequence of the 10 kDa polypeptide associated with the oxygen-evolving complex of wheat photosystem II has been determined and shown to be homologous to the amino acid sequence of the product of the ST-LS1 gene from potato. The N-terminal sequence of the mature protein indicates that the polypeptide is synthesized with a 39 amino acid N-terminal presequence which is similar to chloroplast import sequences but which lacks a hydrophobic domain for transfer of the protein across the thylakoid membrane. The mature polypeptide has a C-terminal hydrophobic region which shows homology to the hydrophobic thylakoid transfer domain of other lumenal proteins and this hydrophobic region of the 10 kDa polypeptide is suggested to facilitate transfer of the protein across the thylakoid membrane.

Photosynthetic water oxidation. A new chemical model.

A sequential four-step chemical model for the water oxidation process in photosystem II is presented, based on the observation that a peroxide-linked biquinone complex can be chemically formed as a result of hydroxide ion addition to quinone. In our model, the hydroxide ion intermediate is generated in photosystem II as a result of proton abstraction from water. In the model, the first two flashes of light raise the oxidation state of the bimanganese center, while the third and fourth flashes of light sequentially generate the peroxide-linked biquinone which is then directly oxidized by the bimanganese center to produce oxygen and regenerate quinone.

Acclimation response of spring wheat in a free-air CO(2) enrichment (FACE) atmosphere with variable soil nitrogen regimes. 1. Leaf position and phenology determine acclimation response.

We have examined the photosynthetic acclimation of wheat leaves grown at an elevated CO(2) concentration, and ample and limiting N supplies, within a field experiment using free-air CO(2) enrichment (FACE). To understand how leaf age and developmental stage affected any acclimation response, measurements were made on a vertical profile of leaves every week from tillering until maturity. The response of assimilation (A) to internal CO(2) concentration (C(i)) was used to estimate the in vivo carboxylation capacity (Vc(max)) and maximum rate of ribulose-1,5-bisphosphate limited photosynthesis (A (sat)). The total activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and leaf content of Rubisco and the Light Harvesting Chlorophyll a/b protein associated with Photosystem II (LHC II), were determined. Elevated CO(2) did not alter Vc(max) in the flag leaf at either low or high N. In the older shaded leaves lower in the canopy, acclimatory decline in Vc(max) and A (sat) was observed, and was found to correlate with reduced Rubisco activity and content. The dependency of acclimation on N supply was different at each developmental stage. With adequate N supply, acclimation to elevated CO(2) was also accompanied by an increased LHC II/Rubisco ratio. At low N supply, contents of Rubisco and LHC II were reduced in all leaves, although an increased LHC II/Rubisco ratio under elevated CO(2) was still observed. These results underscore the importance of leaf position, leaf age and crop developmental stage in understanding the acclimation of photosynthesis to elevated CO(2) and nutrient stress.

Acclimation response of spring wheat in a free-air CO(2) enrichment (FACE) atmosphere with variable soil nitrogen regimes. 3. Canopy architecture and gas exchange.

The response of whole-canopy net CO(2) exchange rate (CER) and canopy architecture to CO(2) enrichment and N stress during 1996 and 1997 for open-field-grown wheat ecosystem (Triticum aestivum L. cv. Yecora Rojo) are described. Every Control (C) and FACE (F) CO(2) treatment (defined as ambient and ambient +200 mumol mol(-1), respectively) contained a Low- and High-N treatment. Low-N treatments constituted initial soil content amended with supplemental nitrogen applied at a rate of 70 kg N ha(-1) (1996) and 15 kg N ha(-1) (1997), whereas High-N treatments were supplemented with 350 kg N ha(-1) (1996 and 1997). Elevated CO(2) enhanced season-long carbon accumulation by 8% and 16% under Low-N and High-N, respectively. N-stress reduced season-long carbon accumulation 14% under ambient CO(2), but by as much as 22% under CO(2) enrichment. Averaging both years, green plant area index (GPAI) peaked approximately 76 days after planting at 7.13 for FH, 6.00 for CH, 3.89 for FL, and 3.89 for CL treatments. Leaf tip angle distribution (LTA) indicated that Low-N canopies were more erectophile than those of High-N canopies: 48 degrees for FH, 52 degrees for CH, and 58 degrees for both FL and CL treatments. Temporal trends in canopy greenness indicated a decrease in leaf chlorophyll content from the flag to flag-2 leaves of 25% for FH, 28% for CH, 17% for CL, and 33% for FL during 1997. These results indicate that significant modifications of canopy architecture occurs in response to both CO(2) and N-stress. Optimization of canopy architecture may serve as a mechanism to diminish CO(2) and N-stress effects on CER.

Acclimation response of spring wheat in a free-air CO(2) enrichment (FACE) atmosphere with variable soil nitrogen regimes. 2. Net assimilation and stomatal conductance of leaves.

Atmospheric CO(2) concentration continues to rise. It is important, therefore, to determine what acclimatory c hanges will occur within the photosynthetic apparatus of wheat (Triticum aestivum L. cv. Yecora Rojo) grown in a future high-CO(2) world at ample and limited soil N contents. Wheat was grown in an open field exposed to the CO(2) concentration of ambient air [370 mumol (CO(2)) mol(-1); Control] and air enriched to approximately 200 mumol (CO(2)) mol(-1) above ambient using a Free-Air CO(2) Enrichment (FACE) apparatus (main plot). A High (35 g m(-2)) or Low (7 and 1.5 g m(-2) for 1996 and 1997, respectfully) level of N was applied to each half of the main CO(2) treatment plots (split-plot). Under High-N, FACE reduced stomatal conductance (g (s)) by 30% at mid-morning (2 h prior to solar noon), 36% at midday (solar noon) and 27% at mid-afternoon (2.5 h after solar noon), whereas under Low-N, g (s) was reduced by as much as 31% at mid-morning, 44% at midday and 28% at mid-afternoon compared with Control. But, no significant CO(2) x N interaction effects occurred. Across seasons and growth stages, daily accumulation of carbon (A') was 27% greater in FACE than Control. High-N increased A' by 18% compared with Low-N. In contrast to results for g (s), however, significant CO(2) x N interaction effects occurred because FACE increased A' by 30% at High-N, but by only 23% at Low-N. FACE enhanced the seasonal accumulation of carbon (A'') by 29% during 1996 (moderate N-stress), but by only 21% during 1997 (severe N-stress). These results support the premise that in a future high-CO(2) world an acclimatory (down-regulation) response in the photosynthetic apparatus of field-grown wheat is anticipated. They also demonstrate, however, that the stimulatory effect of a rise in atmospheric CO(2) on carbon gain in wheat can be maintained if nutrients such as nitrogen are in ample supply.

Site-directed mutagenesis and analysis of second-site revertants indicates a requirement for C-terminal amino acids of PsaB for stable assembly of the photosystem I reaction center complex in Chlamydomonas reinhardtii.

The PsaA and PsaB polypeptides form the reaction center core heterodimer of photosystem I (PSI). Both PsaA and PsaB are predicted to have 11 hydrophobic domains, although it is unclear how both polypeptides fold within the thylakoid membrane. If all 11 hydrophobic regions form membrane-spanning domains, the N- and C-terminus must be located on opposite sides of the membrane. The C-terminus of PsaB is very conserved in a wide range of organisms and may be important for PSI assembly or function. Using chloroplast transformation in Chlamydomonas reinhardtii we have generated a series of C-terminal extension and deletion mutants of the PsaB polypeptide. Analysis of these mutants and spontaneous revertants indicates that the C-terminus may be extended by at least 14 amino acids without impairing PSI assembly. Deletion of amino acids 732-736 also has no impact on PSI, whereas deletion of amino acids 727-736 results in no accumulation of the complex. The site of truncation in the 727-736 deletion coincides with the end of the hydrophobic domain XI supporting a location of the C-terminus of PsaB on the lumenal side of PSI.

Genetic engineering of thylakoid protein complexes by chloroplast transformation in Chlamydomonas reinhardtii.

Chloroplast transformation of Chlamydomonas reinhardtii has developed into a powerful tool for studying the structure, function and assembly of thylakoid protein complexes in a eukaryotic organism. In this article we review the progress that is being made in the development of procedures for efficient chloroplast transformation. This focuses on the development of selectable markers and the use of Chlamydomonas mutants, individually lacking thylakoid protein complexes, as recipients. Chloroplast transformation has now been used to engineer all four major thylakoid protein complexes, photosystem II, photosystem I, cytochrome b 6/f and ATP synthase. These results are discussed with an emphasis on new insights into assembly and function of these complexes in chloroplasts as compared with their prokaryotic counterparts.

Increased mRNA accumulation in a psaB frame-shift mutant of Chlamydomonas reinhardtii suggests a role for translation in psaB mRNA stability.

Regulation of mRNA stability is an important control in the differential accumulation of chloroplast mRNAs that occurs in response to developmental and environmental signals. The mechanism by which differential mRNA accumulation is achieved is unknown. We have examined mRNA accumulation in a chloroplast mutant of Chlamydomonas reinhardtii previously shown to contain a single AT base-pair deletion in the psaB gene. In this mutant, steady-state levels of mRNA from psaB accumulate to a level more than twice that found in cells that have had the mutation repaired by chloroplast transformation. In vivo pulse labeling of RNA shows that increased mRNA accumulation is due to a more stable transcript. We show that inhibitors of chloroplast protein synthesis also increase mRNA accumulation from the psaB gene. The results are consistent with a link between polysome association, active synthesis and stability of psaB transcripts.

Acclimation of photosynthetic proteins to rising atmospheric CO2.

In this review we discuss how the photosynthetic apparatus, particularly Rubisco, acclimates to rising atmospheric CO2 concentrations (ca). Elevated ca alters the control exerted by different enzymes of the Calvin cycle on the overall rate of photosynthetic CO2 assimilation, so altering the requirement for different functional proteins. A decreased flux of carbon through the photorespiratory pathway will decrease requirements for these enzymes. From modeling of the response of CO2 uptake (A) to intracellular CO2 concentration (ci) it is shown that the requirement for Rubisco is decreased at elevated ca, whilst that for proteins limiting ribulose 1,5 bisphosphate regeneration may be increased. This balance may be altered by other interactions, in particular plasticity of sinks for photoassimilate and nitrogen supply; hypotheses on these interactions are presented. It is speculated that increased accumulation of carbohydrate in leaves developed at elevated ca may signal the 'down regulation' of Rubisco. The molecular basis of this 'down regulation' is discussed in terms of the repression of photosynthetic gene expression by the elevated carbohydrate concentrations. This molecular model is then used to predict patterns of acclimation of perennials to long term growth in elevated ca.

Function of 3' non-coding sequences and stop codon usage in expression of the chloroplast psaB gene in Chlamydomonas reinhardtii.

The rate of mRNA decay is an important step in the control of gene expression in prokaryotes, eukaryotes and cellular organelles. Factors that determine the rate of mRNA decay in chloroplasts are not well understood. Chloroplast mRNAs typically contain an inverted repeat sequence within the 3' untranslated region that can potentially fold into a stem-loop structure. These stem-loop structures have been suggested to stabilize the mRNA by preventing degradation by exonuclease activity, although such a function in vivo has not been clearly established. Secondary structures within the translation reading frame may also determine the inherent stability of an mRNA. To test the function of the inverted repeat structures in chloroplast mRNA stability mutants were constructed in the psaB gene that eliminated the 3' flanking sequences of psaB or extended the open reading frame into the 3' inverted repeat. The mutant psaB genes were introduced into the chloroplast genome of Chlamydomonas reinhardtii. Mutants lacking the 3' stem-loop exhibited a 75% reduction in the level of psaB mRNA. The accumulation of photosystem I complexes was also decreased by a corresponding amount indicating that the mRNA level is limiting to PsaB protein synthesis. Pulse-chase labeling of the mRNA showed that the decay rate of the psaB mRNA was significantly increased demonstrating that the stem-loop structure is required for psaB mRNA stability. When the translation reading frame was extended into the 3' inverted repeat the mRNA level was reduced to only 2% of wild-type indicating that ribosome interaction with stem-loop structures destabilizes chloroplast mRNAs. The non-photosynthetic phenotype of the mutant with an extended reading frame allowed us to test whether infrequently used stop codons (UAG and UGA) can terminate translation in vivo. Both UAG and UGA are able to effectively terminate PsaB synthesis although UGA is never used in any of the Chlamydomonas chloroplast genes that have been sequenced.

Mutants of Sweetclover (Melilotus alba) Lacking Chlorophyll b: Studies on Pigment-Protein Complexes and Thylakoid Protein Phosphorylation.

Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Using thin-layer chromatography and absorption spectroscopy, three of these mutants were found to lack chlorophyll (Chl) b. One of these three mutants, U374, possessed thylakoid membranes lacking the three Chl b-containing pigment-protein complexes (AB-1, AB-2, and AB-3) while still containing A-1 and A-2, Chl a complexes derived from photosystems I and II, respectively. Complete solubilization and denaturation of the thylakoid proteins from this mutant revealed very little apoprotein from the Chl b-containing light-harvesting complexes, the major thylakoid proteins in normal plants. The normal and mutant sweetclover plants had active thylakoid protein kinase activities and numerous polypeptides were labeled following incubation with [gamma-(32)P]ATP. With the U374 mutant, however, there was very little detectable label co-migrating with the light-harvesting complex apoproteins on polyacrylamide gels. The Chl b-deficient chlorina-f2 mutant of barley (Hordeum vulgare) also had an active protein kinase activity capable of phosphorylating numerous polypeptides, including ones migrating with the same mobility as the light-harvesting complex apoproteins. These results indicate that the sweetclover mutants may be useful systems for studies on the function and organization of Chl b in thylakoid membranes of higher plants.

Site-directed mutagenesis of the photosystem I reaction center in chloroplasts. The proline-cysteine motif.

Site-directed mutagenesis has been used to introduce specific amino acid changes into the photosystem I reaction center in the green alga Chlamydomonas reinhardtii. Plasmids containing mutated copies of the chloroplast psaB gene, encoding a polypeptide of the photosystem I reaction center heterodimer, were introduced into the chloroplast genome by particle bombardment. Successful transformants were selected by two procedures. The first involved complementation of a nonphotosynthetic mutant of Chlamydomonas, CC-2341 (ac-u-g-2.3), which has a frameshift mutation in the psaB gene, and selection of photosynthetic transformants on minimal medium. The second procedure utilized a co-transformation procedure with a plasmid containing a rRNA gene that confers spectinomycin resistance. Homologous replacement of the psaB gene was confirmed by screening for a unique restriction enzyme site within the transforming psaB sequences. These procedures have been used to specifically mutate a highly conserved proline-cysteine motif suggested to be important in coordinating the [4Fe-4S] iron-sulfur center Fx. Our results show that the cysteine is essential for assembly of the photosystem I reaction center although the adjacent proline fulfills no identifiable function. The approach described in this paper will be of value to future studies of the structure, function, and assembly of photosystem I.

Evidence that the FX domain in photosystem I interacts with the subunit PsaC: site-directed changes in PsaB destabilize the subunit interaction in Chlamydomonas reinhardtii.

The highly conserved amino acid sequence PCDGPGRGGTC in both photosystem I reaction center core proteins PsaA and PsaB has been predicted to contribute the four cysteine ligands for coordination of the 4Fe-4S iron-sulfur cluster FX, and we have proposed a working model for the binding of PsaC to this domain of the reaction center core heterodimer [Rodday et al. (1993) Photosynth. Res. 36, 1-9]. We have investigated structure-function relationships between this domain and the PsaC subunit by site-directed mutagenesis of the conserved prolines P560 and P564, and the charged residues D562 and R566 in the eucaryotic alga Chlamydomonas reinhardtii. The D562N and R566E mutants did not accumulate the PsaA and PsaB reaction center proteins, indicating that these residues are essential for the stable assembly of photosystem I. The P560A, P560L, and P564L mutants accumulated functional reaction centers but showed an impaired interaction between the reaction center core complex and the PsaC subunit. We observed that the reaction centers of the proline mutants dissociated more readily in urea, and reconstitution of the mutant core preparations using PsaC and Fe-S cluster insertion protocols in vitro were incomplete. We suggest that P560 and D562 contribute to the stability of the FX cluster, most likely by providing essential hydrogen bonding to the C561 ligand. The data obtained from the P564 and R566 replacements provide direct evidence that the intercysteinyl region in PsaB is a domain involved in the interaction between PsaC and the reaction center core.

Site-directed mutagenesis of conserved histidines in the helix VIII domain of PsaB impairs assembly of the photosystem I reaction center without altering spectroscopic characteristics of P700.

The chloroplast psaB gene encodes one of the polypeptides of the photosystem I reaction center heterodimer that coordinates the electron transfer components P700, A0, and A1. Histidine residues in the most highly conserved region of the PsaB protein are predicted to coordinate the P700 reaction center chlorophyll(s) and the initial electron acceptor, A0. Oligonucleotide-mediated site-directed mutagenesis and chloroplast transformation of Chlamydomonas reinhardtii have been used to determine the importance of these conserved histidines in photosystem I reaction center biogenesis and function. It is demonstrated that these histidine residues are essential for stable accumulation of the photosystem I reaction center. Protein pulse-labeling shows that changing the histidine residues impairs a post-translational step in reaction center assembly. Photosystem I complexes from the mutants have been characterized by Electron Nuclear Double Resonance and Electron Spin Echo Envelope Modulation spectroscopy to determine the impact of any mutations on P700+. In all cases we determine that spectroscopic characteristics of P700+ remain unchanged. The implications of these results to current models of the photosystem I reaction center and related bacterial reaction centers are discussed.

Catalytic and EPR studies of the beta E204Q mutant of the chloroplast F1-ATPase from Chlamydomonas reinhardtii.

The mutation E204Q in the beta subunit of the chloroplast F1-ATPase was made by biolistic transformation of Chlamydomonas reinhardtii. The yield of chloroplast F1-ATPase (CF1) purified from thylakoids was unaltered, suggesting that the mutation did not affect protein assembly. However, photoautotrophic growth of Chlamydomonas strains containing beta E204Q was virtually abolished, and the effect of the mutation on the light-driven ATPsynthase activity catalyzed by purified thylakoids was comparable to the change in the photoautotrophic growth rate. The loss of ATPsynthase activity in the mutant was not the result of uncoupling. Addition of wild-type CF1 to mutant thylakoids depleted of CF1 reconstituted ATPsynthase activity indicating that the mutation did not affect assembly of F0. Furthermore, the mutant CF1F0 was capable of catalyzing ATPase-dependent proton pumping as measured by fluorescence quenching of 9-amino acridine. Although the mutation significantly affected the apparent kcat/K(m) of the Mg(2+)-ATPase activity of the purified CF1-ATPase, no significant effect on the apparent kcat was observed with the mutant compared to wild-type. No significant changes in the ability of Mg2+ or Mn2+ to serve either as a cofactor or as an inhibitor of ATPase activity were observed in the mutants relative to the wild-type CF1-ATPase. EPR spectra were also taken of VO2+ bound at catalytic site 3 in its latent form. In a large fraction of the latent enzyme, a carboxyl group has displaced the nucleotide-phosphate coordination to the metal which results in the free-metal inhibited form (M3). No significant effects on the gII and AII 51V hyperfine parameters were observed between wild-type and mutant. However, the mutation increased the abundance of the M3 form relative to the M3-N3 form (metal-nucleotide-coordinated form). On the basis of these results, beta E204 is not the carboxyl group that displaces the nucleotide phosphate as a ligand to form the free-metal inhibited enzyme form which predominates in site 3 in the latent state. Instead, the data are consistent with a role in which beta E204 is essential to protonate an inorganic phosphate-oxygen to make that oxygen a good leaving group to facilitate ATP synthesis and, via this role in H-bonding, increases the abundance of the functional metal-nucleotide complex bound to the catalytic site.

Universality of energy and electron transfer processes in photosystem I.

Femtosecond transient absorption spectroscopy has been used to investigate the photoinduced energy and electron transfer processes in photosystem I (PS I) particles from cyanobacteria, green algae, and higher plants. At room temperature, the kinetics observed in all three species are very similar: Following 590 nm excitation, an equilibration process(es) with a 3.7-7.5 ps lifetime was observed, followed by a 19-24 ps process that is associated with trapping. In all three species long-wavelength pigments (pigments that absorb at longer wavelengths than the primary electron donor) were observed. The difference spectrum associated with reduction of the primary electron acceptor [Ao(-)-Ao) difference spectrum] was obtained for all three species. The (Ao(-)-Ao) difference spectra obtained from measurements using detergent-isolated PS I particles from spinach and Chlamydomonas reinhardtii are similar but clearly membrane fragments. In all three species the reduced primary electron acceptor (Ao(-)) is reoxidized extremely rapidly, in about 20 ps. The difference spectrum associated with Ao reduction appears to contain contributions from more than a single chlorophyll pigment.