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Summary: R‐loops, three‐stranded nucleic acid structures consisting of a DNA: RNA hybrid and displaced single‐stranded DNA, play critical roles in gene expression and genome stability. How R‐loop homeostasis is integrated into chloroplast gene expression remains largely unknown.We found an unexpected function of FtsHi1, an inner envelope membrane‐bound AAA‐ATPase in chloroplast R‐loop homeostasis of Arabidopsis thaliana.Previously, this protein was shown to function as a component of the import motor complex for nuclear‐encoded chloroplast proteins. However, this study provides evidence that FtsHi1 is an ATP‐dependent helicase that efficiently unwinds both DNA–DNA and DNA–RNA duplexes, thereby preventing R‐loop accumulation. Over‐accumulation of R‐loops could impair chloroplast transcription but not necessarily genome integrity. The dual function of FtsHi1 in both protein import and chloroplast gene expression may be important to coordinate the biogenesis of nuclear‐ and chloroplast‐encoded subunits of multi‐protein photosynthetic complexes.This study suggests a mechanical link between protein import and R‐loop homeostasis in chloroplasts of higher plants. [ABSTRACT FROM AUTHOR]

Most chloroplast proteins are nucleus‐encoded, translated on cytoplasmic ribosomes as precursor proteins, and imported into chloroplasts through TOC and TIC, the translocons of the outer and inner chloroplast envelope membranes. While the composition of the TOC complex is well established, there is still some controversy about the importance of a recently identified TIC complex consisting of Tic20, Tic214, Tic100, and Tic56. TOC and TIC form a supercomplex with a protein channel at the junction of the outer and inner envelope membranes through which preproteins are pulled into the stroma by the ATP‐powered Ycf2 complex consisting of several FtsH‐like ATPases and/or by chloroplast Hsp proteins. Several components of the TOC/TIC system are moonlighting proteins with additional roles in chloroplast gene expression and metabolism. Chaperones and co‐chaperones, associated with TOC and TIC on the cytoplasmic and stromal side of the chloroplast envelope, participate in the unfolding and folding of the precursor proteins and act together with the ubiquitin–proteasome system in protein quality control. Chloroplast protein import is also intimately linked with retrograde signaling, revealing altogether an unsuspected complexity in the regulation of this process. [ABSTRACT FROM AUTHOR]

SUMMARY: The leaf veins of higher plants contain a highly specialized vascular system comprised of xylem and phloem cells that transport water, organic compounds and mineral nutrients. The development of the vascular system is controlled by phytohormones that interact with complex transcriptional regulatory networks. Before the emergence of true leaves, the cotyledons of young seedlings perform photosynthesis that provides energy for the sustainable growth and survival of seedlings. However, the mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood, in part due to the complex cellular composition of this tissue. To better understand the development of leaf veins, we analyzed 14 117 single cells from 3‐day‐old cotyledons using single‐cell RNA sequencing. Based on gene expression patterns, we identified 10 clusters of cells and traced their developmental trajectories. We discovered multiple new marker genes and developmental features of leaf veins. The transcription factor networks of some cell types indicated potential roles of CYCLING DOF FACTOR 5 (CDF5) and REPRESSOR OF GA (RGA) in the early development and function of the leaf veins in cotyledons. These new findings lay a foundation for understanding the early developmental dynamics of cotyledon veins. The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3‐day‐old cotyledons based on single‐cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins and characterized the novel regulators of leaf vein. Significance Statement: The mechanisms underlying the early development of leaf veins in cotyledons are still not fully understood. In this study, we comprehensively characterized the early differentiation and development of leaf veins in 3‐day‐old cotyledons based on single‐cell transcriptome analysis. We identified the cell types and novel marker genes of leaf veins, and characterized the novel regulators of leaf vein. [ABSTRACT FROM AUTHOR]

Fe–S clusters are ancient, ubiquitous and highly essential prosthetic groups for numerous fundamental processes of life. The biogenesis of Fe–S clusters is a multistep process including iron acquisition, sulfur mobilization, and cluster formation. Extensive studies have provided deep insights into the mechanism of the latter two assembly steps. However, the mechanism of iron utilization during chloroplast Fe–S cluster biogenesis is still unknown. Here we identified two Arabidopsis DnaJ proteins, DJA6 and DJA5, that can bind iron through their conserved cysteine residues and facilitate iron incorporation into Fe–S clusters by interactions with the SUF (sulfur utilization factor) apparatus through their J domain. Loss of these two proteins causes severe defects in the accumulation of chloroplast Fe–S proteins, a dysfunction of photosynthesis, and a significant intracellular iron overload. Evolutionary analyses revealed that DJA6 and DJA5 are highly conserved in photosynthetic organisms ranging from cyanobacteria to higher plants and share a strong evolutionary relationship with SUFE1, SUFC, and SUFD throughout the green lineage. Thus, our work uncovers a conserved mechanism of iron utilization for chloroplast Fe–S cluster biogenesis. Synopsis: The identity of the iron donor during chloroplast iron‐sulfur cluster biogenesis has remained unknown. Here, two Arabidopsis DnaJ‐family chaperone proteins are found to promote iron incorporation into chloroplast Fe–S cluster proteins. Arabidopsis DJA6 and DJA5 are essential for photosynthesis, chloroplast development, and plant viability.DJA6 and DJA5 bind iron through their conserved cysteine residuesDJA6 and DJA5 facilitate iron delivery to the SUFBC2D complex for Fe–S cluster assembly.DJA6 and DJA5 are highly conserved in photosynthetic organisms.Expression of cyanobacterial DJA6 rescues dja6 dja5 mutant phenotype in Arabidopsis. [ABSTRACT FROM AUTHOR]

Abstract: Singlet oxygen (1O2) is a prime cause of photo‐damage of the photosynthetic apparatus. The chlorophyll molecules in the photosystem II reaction center and in the light‐harvesting antenna complex are major sources of 1O2 generation. It has been thought that the generation of 1O2 mainly takes place in the appressed regions of the thylakoid membranes, namely, the grana core, where most of the active photosystem II complexes are localized. Apart from being a toxic molecule, new evidence suggests that 1O2 significantly contributes to chloroplast‐to‐nucleus retrograde signalling that primes acclimation and cell death responses. Interestingly, recent studies reveal that chloroplasts operate two distinct 1O2‐triggered retrograde signalling pathways in which β‐carotene and a nuclear‐encoded chloroplast protein EXECUTER1 play essential roles as signalling mediators. The coexistence of these mediators raises several questions: their crosstalk, source(s) of 1O2, downstream signalling components, and the perception and reaction mechanism of these mediators towards 1O2. In this review, we mainly discuss the molecular genetic basis of the mode of action of these two putative 1O2 sensors and their corresponding retrograde signalling pathways. In addition, we also propose the possible existence of an alternative source of 1O2, which is spatially and functionally separated from the grana core. [ABSTRACT FROM AUTHOR]

We have identified hpm91, a Chlamydomonas mutant lacking Proton Gradient Regulation5 (PGR5) capable of producing hydrogen (H2) for 25 days with more than 30-fold yield increase compared to wild type. Thus, hpm91 displays a higher capacity of H2 production than a previously characterized pgr5 mutant. Physiological and biochemical characterization of hpm91 reveal that the prolonged H2 production is due to enhanced stability of PSII, which correlates with increased reactive oxygen species (ROS) scavenging capacity during sulfur deprivation. This anti-ROS response appears to protect the photosynthetic electron transport chain from photo-oxidative damage and thereby ensures electron supply to the hydrogenase. [ABSTRACT FROM AUTHOR]

Genetic analysis of mutants deficient in the biosynthesis of the photosystem I complex has revealed several nucleus-encoded factors that act at different post-transcriptional steps of chloroplast gene expression. Here we have identified and characterized the gene affected in the tab 1-F15 mutant, which is specifically deficient in the translation of the photosystem I reaction center protein PsaB as the result of a single nucleotide deletion. This gene encodes Tab 1, a 1287 amino acid protein that contains 10 tandem 38-40 amino acid degenerate repeats of the PPPEW/OPR (octatricopeptide repeat) family, first described for the chloroplast translation factor Tbc2. These repeats are involved in the binding of Tab 1 to the 5′-untranslated region of the psaB mRNA based on gel mobility shift assays. Tab 1 is part of a large family of proteins in Chlamydomonas that are also found in several bacteria and protozoans, but are rare in land plants. [ABSTRACT FROM AUTHOR]

Chloroplast transformation in microalgae offers great promise for the production of proteins of pharmaceutical interest or for the development of novel biofuels. For many applications, high level expression of transgenes is desirable. We have transformed the chloroplast of Chlamydomonas reinhardtii with two genes, acrV and vapA, which encode antigens from the fish pathogen Aeromonas salmonicida. The promoters and 5′ untranslated regions of four chloroplast genes were compared for their ability to drive expression of the bacterial genes. The highest levels of expression were obtained when they were placed under the control of the cis-acting elements from the psaA-exon1 gene. The expression of these chimeric genes was further increased when a nuclear mutation that affects a factor involved in psaA splicing was introduced in the genetic background of the chloroplast transformants. Accumulation of both the chimeric mRNAs and the recombinant proteins was dramatically increased, indicating that negative feedback loops limit the expression of chloroplast transgenes. Our results demonstrate the potential of manipulating anterograde signalling to alter negative regulatory feedback loops in the chloroplast and improve transgene expression. [ABSTRACT FROM AUTHOR]

We have designed a screen for mutants affected in 3′ maturation of the chloroplast rbcL mRNA in Chlamydomonas reinhardtii. We inserted a spectinomycin resistance cassette, 5′ atpA::aadA::3′ rbcL, in a peripheral domain of tscA, the gene for a small non-coding RNA involved in trans-splicing of psaA. Depending on the orientation of the cassette, a polar effect was observed which was due to processing at the 3′ rbcL element: the chimeric tscA RNA was truncated and splicing of psaA was blocked. We selected phenotypic revertants of this insertion mutant that restored psaA splicing, which correlated with the presence of chimeric transcripts that regained the 3′ part of tscA. We analyzed two nuclear and six chloroplast suppressors. Five chloroplast mutations altered a short element in the center of the second inverted repeat in the 3′ rbcL (IR2), and one deleted a larger region including this element. These mutations revealed a cis-acting element in IR2 which is required for 3′ processing. When the same mutations were inserted in the 3′ untranslated region (UTR) of the native rbcL gene, the rbcL mRNA accumulated to normal levels, but in strong alleles its 3′ end was located upstream, near the end of the first inverted repeat (IR1). Deletion of either IR1 or IR2 allowed stable accumulation of rbcL mRNA, but deletion of both resulted in its complete absence. This indicated that the two inverted repeats function as redundant mRNA stability determinants in the 3′ UTR of rbcL. [ABSTRACT FROM AUTHOR]

Abstract: Photosynthetic organisms are able to adjust to changes in light quality through state transition, a process which leads to a balancing of the light excitation energy between the antennae systems of photosystem II and photosystem I. A genetic approach has been used in Chlamydomonas with the aim of elucidating the signaling chain involved in state transitions. This has led to the identification of a small family of Ser–Thr protein kinases associated with the thylakoid membrane and conserved in algae and land plants. These kinases appear to be involved both in short and long term adaptations to changes in the light environment. [Copyright &y& Elsevier]

We have compared the seed production of Arabidopsis wild-type and mutant plants impaired in the regulation of the photosynthetic light reactions grown under natural conditions in the field. Mutant plants (npq4) lacking feedback de-excitation were, as previously demonstrated, severely affected in seed production. Seed sets of plants deficient in state transitions (stn7) were 19% smaller than those of wild-type plants, whereas plants missing the STN8 kinase required for the phosphorylation of the core photosystem II reaction centre polypeptides (stn8) had a normal seed production. Plants lacking both STN7 and STN8 kinases were strongly affected, indicating that these mutations act synergistically. In contrast, npq4×stn7 double mutants had the same seed set as npq4 mutants. [ABSTRACT FROM AUTHOR]

Plastid translational control depends to a large extent on the light conditions, and is presumably mediated by nucleus-encoded proteins acting on organelle gene expression. However, the molecular mechanisms of light signalling involved in translation are still poorly understood. We investigated the role of the Arabidopsis ortholog of Tab2, a nuclear gene specifically required for translation of the PsaB photosystem I subunit in the unicellular alga Chlamydomonas. Inactivation of ATAB2 strongly affects Arabidopsis development and thylakoid membrane biogenesis and leads to an albino phenotype. Moreover the rate of synthesis of the photosystem reaction center subunits is decreased and the association of their mRNAs with polysomes is affected. ATAB2 is a chloroplast A/U-rich RNA-binding protein that presumably functions as an activator of translation with at least two targets, one for each photosystem. During early seedling development, ATAB2 blue-light induction is lowered in photoreceptor mutants, notably in those lacking cryptochromes. Considering its role in protein synthesis and its photoreceptor-mediated expression, ATAB2 represents a novel factor in the signalling pathway of light-controlled translation of photosystem proteins during early plant development. [ABSTRACT FROM AUTHOR]

The green unicellular alga Chlamydomonas reinhardtii has emerged as a powerful model system for studying the biosynthesis of the photosynthetic apparatus and the acclimation of this system to changes in light conditions. The assembly of the photosynthetic complexes involves the coordinate interaction between the nuclear and chloroplast genetic systems. Many factors involved in specific chloroplast post-transcriptional steps have been identified and characterized. Chlamydomonas is able to adapt to changes in light quality and in cellular ATP content by performing state transition, a process that leads to a redistribution of light excitation energy between photosystem II and photosystem I and that involves the redox state of the plastoquinone pool, the cytochrome b6f complex and one or several kinases specific for the light-harvesting system. Genetic approaches have provided new insights into this process. [Copyright &y& Elsevier]

The energetic metabolism of photosynthetic organisms is profoundly influenced by state transitions and cyclic electron flow around photosystem I. The former involve a reversible redistribution of the light-harvesting antenna between photosystem I and photosystem II and optimize light energy utilization in photosynthesis whereas the latter process modulates the photosynthetic yield. We have used the wild-type and three mutant strains of the green alga Chlamydomonas reinhardtii-locked in state I (stt7), lacking the photosystem II outer antennae (bf4) or accumulating low amounts of cytochrome b6f complex (A-AUU)-and measured electron flow though the cytochrome b6f complex, oxygen evolution rates and fluorescence emission during state transitions. The results demonstrate that the transition from state 1 to state 2 induces a switch from linear to cyclic electron flow in this alga and reveal a strict cause effect relationship between the redistribution of antenna complexes during state transitions and the onset of cyclic electron flow. [ABSTRACT FROM AUTHOR]

In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving trans‐splicing of separate transcripts, encoded at three separate loci of the chloroplast genome. At least 14 nuclear loci and one chloroplast gene, tscA, are needed for this process. We have cloned Raa3, the first nuclear gene implicated in the splicing of intron 1. The predicted sequence of Raa3 consists of 1783 amino acids and shares a small region of homology with pyridoxamine 5′‐phosphate oxidases. Raa3 is present in the soluble fraction of the chloroplast and is part of a large 1700 kDa complex, which also contains tscA RNA and the first psaA exon transcript. These partners, in association with other factors, form a chloroplast RNP particle that is required for the splicing of the first intron of psaA and which may be the counterpart of eukaryotic snRNPs involved in nuclear splicing. [ABSTRACT FROM AUTHOR]

The psbD mRNA, which encodes the D2 reaction center polypeptide of photasystem II, is one of the most abundant chloroplast mRNAs. We have used genomic complementation to isolate the nuclear Nac2 gene, which is required for the stable accumulation of the psbD mRNA in Chlamydomonas reinhardtii. Nac2 encodes a hydrophilic polypeptide of 1385 amino acids with nine tetratricopeptide­like repeats (TPRs) in its C-terminal half. Cell fractionation studies indicate that the Nac2 protein is localized in the stromal compartment of the chloroplast. It is part of a high molecular weight complex that is associated with nonpolysomal RNA. Change of a conserved alanine residue of the fourth TPR motif by site-directed mutagenesis leads to aggregation of Nac2 protein and completely abrogates its function, indicating that this TPR is important for proper folding of the protein and for psbD mRNA stability, processing and/or translation. [ABSTRACT FROM AUTHOR]

Reports on the chloroplast gene psbB that encodes the chlorophyll-a binding protein P5, one of the core subunits of photosystem II. Failure of the psbB mRNA and the downstream psbT and psbH transcripts to accumulate in the Chlamydomonas reinhardtii nuclear mutant 222E affected in the Mbb1 gene; Introduction of chimeric genes to map the Mbb1 target site.

Reports on the Eighth International Conference on the Cell and Molecular Biology of Chlamydomonas held from June 2 to 7, 1998 in Lake Tahoe. Speakers of the event; Topics discussed; Highlights of the event.

Explores the mechanism, implications and connections of post-transcriptional processes in plants, viruses and transgenes. Presence of extraordinary diversity in the post-transcriptional mechanism that promote developmental plasticity and adaptation; Involvement of transcriptional and post-transcriptional mechanism in the silencing of heterologous genes; Exploration of the mechanisms and implications of post-transcriptional processes in plants; Presentation of an overview of post-transcriptional processes in mammals and yeast.

In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving two steps of trans-splicing that remove two group II introns and give rise to the mature mRNA. The products of at least 14 nuclear genes and one chloroplast gene (tscA) are necessary for this process. We have cloned Maea2, one of the nuclear genes involved in trans-splicing of the second intron. Maa2 encodes a protein with similarity to conserved domains of pseudouridine synthases, but mutagenesis of putative catalytic residues showed that this activity may not be required for trans-splicing of psaA RNA. Although it is not clear whether the pseudouridine synthase activity has been maintained in Maa2, it is possible that this enzyme was recruited during evolution as an RNA chaperone for folding or stabilizing the psaA intron. The Maa2 protein appears to be associated through ionic interactions with a low density membrane system in the chloroplast that also contains RNA-binding proteins involved in translation. [ABSTRACT FROM AUTHOR]

Analyzes the photoreduction of flavodoxin by photosystem I (PSI) from the green algae, Chlamydomonas reinhardtii. Absorption changes of the four different types of PSI complexes in the presence of flavodoxin semiquinone; Half life of flavodoxin semiquinone; Molecular mass of cross-linking products.

Using a biolistic device built here and based on the principle of the device described by Klein et al. (1987), we have reproducibly obtained nuclear transformants of Chlamydomonas reinhardtii. The reproducibility of the method has allowed us to examine the maintenance and expression of cloned DNA fragments introduced into C. reinhardtii. [ABSTRACT FROM AUTHOR]

PsaC is the stromal subunit of photosystem I (PSI) which binds the two terminal electron acceptors FA and FB. This subunit resembles 2[4Fe-4S] bacterial ferredoxins but contains two additional sequences: an internal loop and a C-terminal extension. To gain new insights into the function of the internal loop, we used an in vivo degenerate oligonucleotide-directed mutagenesis approach for analysing this region in the green alga Chlamydomonas reinhardtii. Analysis of several psaC mutants affected in PSI function or assembly revealed that K35 is a main interaction site between PsaC and ferredoxin (Fd) and that it plays a key role in the electrostatic interaction between Fd and PSI. This is based upon the observation that the mutations K35T, K35D and K35E drastically affect electron transfer from PSI to Fd, as measured by flash-absorption spectroscopy, whereas the K35R change has no effect on Fd reduction. Chemical cross-linking experiments show that Fd interacts not only with PsaD and PsaE, but also with the PsaC subunit of PSI. Replacement of K35 by T, D, E or R abolishes Fd cross-linking to PsaC, and cross-linking to PsaD and PsaE is reduced in the K35T, K35D and K35E mutants. In contrast, replacement of any other lysine of PsaC does not alter the cross-linking pattern, thus indicating that K35 is an interaction site between PsaC and its redox partner Fd. [ABSTRACT FROM AUTHOR]

The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site-directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA-His676 and PsaB-His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls. [ABSTRACT FROM AUTHOR]

The product of the chloroplast ycf10 gene has been localized in the inner chloroplast envelope membrane (Sasaki et al., 1993) and found to display sequence homology with the cyanobacterial CotA product which is altered in mutants defective in CO2 transport and proton extrusion (Katoh et al., 1996a,b). In Chlamydomonas reinhardtii, ycf10, located between the psbI and atpH genes, encodes a putative hydrophobic protein of 500 residues, which is considerably larger than its higher plant homologue because of a long insertion that separates the conserved N and C termini. Using biolistic transformation, we have disrupted ycf10 with the chloroplast aadA expression cassette and examined the phenotype of the homoplasmic transformants. These were found to grow both photoheterotrophically and photoautotrophically under low light, thereby revealing that the Ycf10 product is not essential for the photosynthetic reactions. However, under high light these transformants did not grow photoautotrophically and barely photoheterotrophically. The increased light sensitivity of the transformants appears to result from a limitation in photochemical energy utilization and/or dissipation which correlates with a greatly diminished photosynthetic response to exogenous (CO2 +HCO3-), especially under conditions where the chloroplast inorganic carbon transport system is not induced. Mass spectrometric measurements with either whole cells or isolated chloroplasts from the transformants revealed that the CO2 and HCO3- uptake systems have a reduced affinity for their substrates. The results suggest the existence of a ycf10-dependent system within the plastid envelope which promotes efficient inorganic carbon (Ci) uptake into chloroplasts. [ABSTRACT FROM AUTHOR]

The chloroplast genes ycf3 and ycf4 from the green alga Chlamydomonas reinhardtii have been characterized. The deduced amino acid sequences of Ycf4 (197 residues) and Ycf3 (172 residues) display 41–52% and 64–78% sequence identity, respectively, with their homologues from algae, land plants and cyanobacteria. In C.reinhardtii, ycf4 and ycf3 are co-transcribed as members of the rps9­ycf4­ycf3­rps18 polycistronic transcriptional unit into RNAs of 8.0 kb and 3.0 kb corresponding to the entire unit and to rps9­ycf4­ycf3, respectively. Using biolistic transformation, ycf4 and ycf3 were disrupted with a chloroplast selectable marker cassette. Transformants lacking ycf4 or ycf3 were unable to grow photoautotrophically and were deficient in photosystem I activity. Western blot analysis showed that the photosystem I (PSI) complex does not accumulate stably in thylakoid membranes of these transformants. Ycf4 and Ycf3 were localized on thylakoid membranes but not stably associated with the PSI complex and accumulated to wild-type levels in mutants lacking PSI. RNA blot hybridizations showed that transcripts of psaA, psaB and psaC accumulate normally in these mutants and use of chimeric reporter genes revealed that Ycf3 is not required for initiation of translation of psaA and psaB mRNA. Our results indicate that Ycf3 and Ycf4 are required for stable accumulation of the PSI complex. [ABSTRACT FROM AUTHOR]

The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site-directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA-His676 and PsaB-His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls. [ABSTRACT FROM AUTHOR]