Interaction of the soluble recombinant PsaD subunit of spinach photosystem I with ferredoxin I.

Photosystem I of higher plants functions in photosynthesis as a light-driven oxidoreductase producing reduced ferredoxin. Its peripheral subunit PsaD has been identified as the docking site for ferredoxin I. With the aim of elucidating the structure-function relationship and the role of this subunit, a recombinant form of the spinach protein was produced by heterologous expression in Escherichia coli. The PsaD protein was synthesized in soluble form and purified to homogeneity. The interaction of the PsaD subunit with ferredoxin I was investigated using three different approaches: chemical cross-linking between the two purified proteins in solution, affinity chromatography of the PsaD subunit on a ferredoxin-coupled resin, and titration with ferredoxin of the protein fluorescence of the subunit. All these studies indicated that the isolated PsaD in solution has a definite conformation and maintains the ability to bind ferredoxin I with high affinity and specificity. The Kd value of the complex of PsaD and ferredoxin is in the nanomolar range, which is consistent with reported Km values for ferredoxin I photoreduction by thylakoid membranes. The ionic strength dependence of the K(d) suggests that the protein-protein interaction is at least partially electrostatic in nature. Nevertheless, none of the glutamate residues of the acidic cluster of residues 92-94 of ferredoxin I, which have been reported to be involved in the interaction with the subunit, seems to be essential for PsaD binding, as borne out by experiments using ferredoxin I mutants in positions 92-94.     

Structural features and assembly of the soluble overexpressed PsaD subunit of photosystem I

PsaD is a peripheral protein on the reducing side of photosystem I (PS I). We expressed the psaD gene from the thermophilic cyanobacterium Mastigocladus laminosus in Escherichia coli and obtained a soluble protein with a polyhistidine tag at the carboxyl terminus. The soluble PsaD protein was purified by Ni-affinity chromatography and had a mass of 16716 Da by MALDI-TOF. The N-terminal amino acid sequence of the overexpressed PsaD matched the N-terminal sequence of the native PsaD from M. laminosus. The soluble PsaD could assemble into the PsaD-less PS I. As determined by isothermal titration calorimetry, PsaD bound to PS I with 1.0 binding site per PS I, the binding constant of 7.7x10(6) M-1, and the enthalpy change of -93.6 kJ mol-1. This is the first time that the binding constant and binding heat have been determined in the assembly of any photosynthetic membrane protein. To identify the surface-exposed domains, purified PS I complexes and overexpressed PsaD were treated with N-hydroxysuccinimidobiotin (NHS-biotin) and biotin-maleimide, and the biotinylated residues were mapped. The Cys66, Lys21, Arg118 and Arg119 residues were exposed on the surface of soluble PsaD whereas the Lys129 and Lys131 residues were not exposed on the surface. Consistent with the X-ray crystallographic studies on PS I, circular dichroism spectroscopy revealed that PsaD contains a small proportion of alpha-helical conformation.     

The assembly of the PsaD subunit into the membranal photosystem I complex occurs via an exchange mechanism.

PsaD is a peripheral stromal-facing subunit of photosystem I (PSI), a multisubunit complex of the thylakoid membranes. PsaD plays a major role in both the function and assembly of PSI. Past studies with radiolabeled PsaD indicated that PsaD is able to assemble in vitro specifically into the PSI complex. To unravel the mechanism by which this assembly takes place, the following steps were taken. (i) Mature PsaD of spinach and PsaD of the prokaryotic caynobacterium Mastigocladus laminosus, both bearing a six-histidine tag at their C-termini, were overexpressed in Escherichia coli and purified to homogeneity. (ii) The purified recombinant protein was introduced into the isolated PSI complex. (iii) Following incubation, the PsaD that assembled into PSI was separated from the nonassembled PsaD by a sucrose gradient. Differential Western blot analysis was used to determine whether the native and the recombinant PsaD were present as free or assembled proteins of the PSI complex. Antibodies that can recognize only the recombinant PsaD (anti-his) or both the native and recombinant PsaD (anti-PsaD) were used. The findings indicated that an exchange mechanism enables the assembly of a newly introduced PsaD into PSI. The latter replaces the PsaD subunit that is present in situ within the complex. In vivo studies that followed the assembly of PsaD in Chlamydomonas reinhardtii cells supported this in vitro-characterized exchange mechanism. In C. reinhardtii, in the absence of synthesis and assembly of new PSI complexes, newly synthesized PsaD assembled into pre-existing PSI complexes.     

Multiple functions for the C terminus of the PsaD subunit in the cyanobacterial photosystem I complex

PsaD subunit of Synechocystis sp PCC 6803 photosystem I (PSI) plays a critical role in the stability of the complex and is part of the docking site for ferredoxin (Fd). In the present study we describe major physiological and biochemical effects resulting from mutations in the accessible C-terminal end of the protein. Four basic residues were mutated: R111, K117, K131, and K135, and a large 36-amino acid deletion was generated at the C terminus. PSI from R111C mutant has a 5-fold decreased affinity for Fd, comparable with the effect of the C terminus deletion, and NADP+ is photoreduced with a 2-fold decreased rate, without consequence on cell growth. The K117A mutation has no effect on the affinity for Fd, but decreases the stability of PsaE subunit, a loss of stability also observed in R111C and the deletion mutants. The double mutation K131A/K135A does not change Fd binding and reduction, but decreases the overall stability of PSI and impairs the cell growth at temperatures above 30 degrees C. Three mutants, R111C, K117A, and the C-terminal deleted exhibit a higher content of the trimeric form of PSI, in apparent relation to the removal of solvent accessible positive charges. Various regions in the C terminus of cyanobacterial PsaD thus are involved in Fd strong binding, PSI stability, and accumulation of trimeric PSI.     

Role of acidic amino acid residues of PsaD subunit on limiting the affinity of photosystem I for ferredoxin

The PsaD subunit of photosystem I is one of the central polypeptides for the interaction with ferredoxin, its acidic electron acceptor. In the cyanobacterium Synechocystis 6803, this role is partly performed by a sequence extending approximately from histidine 97 to arginine 119, close to the C-terminus. In the present work, acidic amino acids D100, E105, and E109 are shown to moderate the affinity of Photosystem I for ferredoxin. Most single replacements of these residues by neutral amino acids increased the affinity for ferredoxin, resulting in a dissociation constant as low as 0.015 microM for the E105Q mutant (wild-type K(D) = 0.4 microM). This is the first report on the limitation of photosystem I affinity for ferredoxin due to acidic amino acids from PsaD subunit. It highlights the occurrence of a negative control on the binding during the formation of transient complexes between electron carriers.     

The plastocyanin binding domain of photosystem I.

The molecular recognition between plastocyanin and photosystem I was studied. Photosystem I and plastocyanin can be cross-linked to an active electron transfer complex. Immunoblots and mass spectrometric analysis of proteolytic peptides indicate that the two negative patches conserved in plant plastocyanins are cross-linked with lysine residues of a domain near the N-terminus of the PsaF subunit of photosystem I. Conversion of these negative to uncharged patches of plastocyanin by site-directed mutation D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N respectively, reveals the first patch to be essential for the electrostatic interaction in the electron transfer complex with photosystem I and the second one to lower the redox potential. The domain in PsaF, not found in cyanobacteria, is predicted to fold into two amphipathic alpha-helices. The interacting N-terminal helix lines up six lysines on one side which may guide a fast one-dimensional diffusion of plastocyanin and provide the electrostatic attraction at the attachment site, in addition to the hydrophobic interaction in the area where the electron is transferred to P700 in the reaction center of photosystem I. This two-step interaction is likely to increase the electron transfer rate by more than two orders of magnitude in plants as compared with cyanobacteria. Our data resolve the controversy about the function of PsaF.     

Absence of PsaC subunit allows assembly of photosystem I core but prevents the binding of PsaD and PsaE in Synechocystis sp. PCC6803

In photosystem I (PSI) of oxygenic photosynthetic organisms the psaC polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] clusters, F_A and F_B. Unlike other cyanobacteria, two different psaC genes have been reported in the cyanobacterium Synechocystis 6803, one (copy 1) with a deduced amino acid sequence identical to that of tobacco and another (copy 2) with a deduced amino acid sequence similar to those reported for other cyanobacteria. Insertion of a gene encoding kanamycin resistance into copy 2 resulted in a photosynthesis-deficient strain, CDK25, lacking the PsaC, PsaD and PsaE polypeptides in isolated thylakoid membranes, while the PsaA/PsaB and PsaF subunits were found. Growth of the mutant cells was indistinguishable from that of wild-type cells under light-activated heterotrophic growth (LAHG). A reversible P700⁺ signal was detected by EPR spectroscopy in the isolated thylakoids during illumination at low temperature. Under these conditions, the EPR signals attributed to F_A and F_B were absent in the mutant strain, but a reversible F_x signal was present with broad resonances at g=2.079, 1.903, and 1.784. Addition of PsaC and PsaD proteins to the thylakoids gave rise to resonances at g=2.046, 1.936, 1.922, and 1.880; these values are characteristic of an interaction-type spectrum of F_A ^- and F_B ^-. In room-temperature optical spectroscopic analysis, addition of PsaC and PsaD to the thylakoids also restored a 30 ms kinetic transient which is characteristic of the P700⁺ [F_A/F_B]^- backreaction. Expression of copy 1 was not detected in cells grown under LAHG and under mixotrophic conditions. These results demonstrate that copy 2 encodes the PsaC polypeptide in PSI in Synechocystis 6803, while copy 1 is not involved in PSI; that the PsaC polypeptide is necessary for stable assembly of PsaD and PsaE into PSI complex in vivo; and that PsaC, PsaD and PsaE are not needed for assembly of PsaA-PsaB dimer and electron transport from P700 to F_x.     

Organization of photosystem I polypeptides. Identification of PsaB domains that may interact with PsaD.

PsaA and PsaB are homologous integral membrane-proteins that form the heterodimeric core of photosystem i (PSI). We used subunit-deficient PSI complexes from the mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 to examine interactions between PsaB and other PSI subunits. Incubation of the wild-type PSI with thermolysin yielded 22-kD C-terminal fragments of PsaB that were resistant to further proteolysis. Modification of the wild-type PSI with N-hydroxysuccinimidobiotin and subsequent cleavage by thermolysin showed that the lysyl residues in the 22-kD C-terminal domain were inaccessible to modification by N-hydroxysuccinimidobiotin. The absence of PsaE, PsaF, PsaI, PsaJ, or PsaL facilitated accumulation of 22-kD C-terminal fragments of PsaB but did not alter their resistance to further proteolysis. When the PsaD-less PSI was treated with thermolysin, the 22-kD C-terminal fragments of PsaB were rapidly cleaved, with concomitant accumulation of a 16-kD fragment and then a 3.4-kD one. We mapped the N termini of these fragments by N-terminal amino acid sequencing and the C termini from their positive reaction with an antibody against the C-terminal peptide of PsaB. The cleavage sites were proposed to be in the extramembrane loops on the cytoplasmic side. Western blot analyses showed resistance of PsaC and PsaI to proteolysis prior to cleavage of the 22-kD fragments. Therefore, we propose that PsaD shields two extramembrane loops of PsaB and protects the C-terminal domain of PsaB from in vitro proteolysis.     

Mutational analysis of domain I of Pseudomonas exotoxin. Mutations in domain I of Pseudomonas exotoxin which reduce cell binding and animal toxicity

Pseudomonas exotoxin (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three structural domains. Domain I is responsible for cell recognition, II for translocation of PE across membranes and III for ADP ribosylation of elongation factor 2. Treatment of PE with reagents that react with lysine residues has been shown to lead to a reduction in cytotoxic activity apparently due to a modification of domain I (Pirker, R., FitzGerald, D. J. P., Hamilton, T. C., Ozols, R. F., Willingham, M. C., and Pastan, S. (1985) Cancer Res. 45, 751-757). To determine which lysine residues are important in cell recognition, all 12 lysines in domain I were converted to glutamates by site-directed mutagenesis. Also, two deletion mutants encompassing almost all of domain I (amino acids 4-252) or most of domain I (amino acids 4-224) were studied. The mutant proteins were produced in Escherichia coli, purified, and tested for their cytotoxic activity against Swiss 3T3 cells and in mice. The data indicate that conversion of lysine 57 to glutamate reduces cytotoxic activity towards 3T3 cells 50-100-fold and in mice about 5-fold. Deletion of amino acids 4-224 causes a similar reduction in toxicity towards cells and mice. Deletion of most of the rest of domain I (amino acids 4-252) causes a further reduction in toxicity toward cells and mice indicating this second region between amino acids 225 and 252 of domain I is also important in the toxicity of PE. Competition assays indicated that the ability of PEGlu57 to bind to 3T3 cells was greatly diminished, accounting for its diminished cytotoxic activity.     

Cosuppression of photosystem I subunit PSI-H in Arabidopsis thaliana. Efficient electron transfer and stability of photosystem I is dependent upon the PSI-H subunit

PSI-H is an intrinsic membrane protein of 10 kDa that is a subunit of photosystem I (PSI). PSI-H is one of the three PSI subunits found only in eukaryotes. The function of PSI-H was characterized in Arabidopsis plants transformed with a psaH cDNA in sense orientation. Cosuppressed plants containing less than 3% PSI-H are smaller than wild type when grown on sterile media but are similar to wild type under optimal conditions. PSI complexes lacking PSI-H contain 50% PSI-L, whereas other PSI subunits accumulate in wild type amounts. PSI devoid of PSI-H has only 61% NADP+ photoreduction activity compared with wild type and is highly unstable in the presence of urea as determined from flash-induced absorbance changes at 834 nm. Our data show that PSI-H is required for stable accumulation of PSI and efficient electron transfer in the complex. The plants lacking PSI-H compensate for the less efficient PSI with a 15% increase in the P700/chlorophyll ratio, and this compensation is sufficient to prevent overreduction of the plastoquinone pool as evidenced by normal photochemical quenching of fluorescence. Nonphotochemical quenching is approximately 60% of the wild type value, suggesting that the proton gradient across the thylakoid membrane is decreased in the absence of PSI-H.     

The PSI-K subunit of photosystem I is involved in the interaction between light-harvesting complex I and the photosystem I reaction center core

PSI-K is a subunit of photosystem I. The function of PSI-K was characterized in Arabidopsis plants transformed with a psaK cDNA in antisense orientation, and several lines without detectable PSI-K protein were identified. Plants without PSI-K have a 19% higher chlorophyll a/b ratio and 19% more P700 than wild-type plants. Thus, plants without PSI-K compensate by making more photosystem I. The photosystem I electron transport in vitro is unaffected in the absence of PSI-K. Light response curves for oxygen evolution indicated that the photosynthetic machinery of PSI-K-deficient plants have less capacity to utilize light energy. Plants without PSI-K have less state 1-state 2 transition. Thus, the redistribution of absorbed excitation energy between the two photosystems is reduced. Low temperature fluorescence emission spectra revealed a 2-nm blue shift in the long wavelength emission in plants lacking PSI-K. Furthermore, thylakoids and isolated PSI without PSI-K had 20-30% less Lhca2 and 30-40% less Lhca3, whereas Lhca1 and Lhca4 were unaffected. During electrophoresis under mildly denaturing conditions, all four Lhca subunits were partially dissociated from photosystem I lacking PSI-K. The observed effects demonstrate that PSI-K has a role in organizing the peripheral light-harvesting complexes on the core antenna of photosystem I.     

The interaction between plastocyanin and photosystem I is inefficient in transgenic Arabidopsis plants lacking the PSI-N subunit of photosystem I

The PSI-N subunit of photosystem I (PSI) is restricted to higher plants and is the only subunit located entirely in the thylakoid lumen. The role of the PSI-N subunit in the PSI complex was investigated in transgenic Arabidopsis plants which were generated using antisense and co-suppression strategies. Several lines without detectable levels of PSI-N were identified. The plants lacking PSI-N assembled a functional PSI complex and were capable of photoautotrophic growth. When grown on agar media for several weeks the plants became chlorotic and developed significantly more slowly. However, under optimal growth conditions, the plants without PSI-N were visually indistinguishable from the wild-type although several photosynthetic parameters were affected. In the transformants, the second-order rate constant for electron transfer from plastocyanin to P700+, the oxidized reaction centre of PSI, was only 55% of the wild-type value, and steady-state NADP+ reduction was decreased to a similar extent. Quantum yield of oxygen evolution and PSII photochemistry were about 10% lower than in the wild-type at leaf level. Photochemical fluorescence quenching was lowered to a similar extent. Thus, the 40-50% lower activity of PSI at the molecular level was much less significant at the whole-plant level. This was partly explained by a 17% increase in PSI content in the plants lacking PSI-N.     

Identification of the plastocyanin binding subunit of photosystem I

Plastocyanin is specifically cross-linked by incubation with N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to a subunit of photosystem I in stroma lamellae and in isolated photosystem I complex. SDS-PAGE shows the disappearance of a 18.5 kDa subunit and the appearance of a new 31.5 kDa protein which was recognized by anti-plastocyanin antibodies. The isolated subunit was identified by its N-terminal amino acid sequence as the mature peptide coded by the nuclear gene psaF [Steppuhn et al. (1988) FEBS Lett. 237, 218–224]. P700⁺ was reduced by cross-linked plastocyanin with the same halftime of 13 μs as found in the native complex. This is evidence that cross-linking conserved the orientation of the complex and that the 18.5 kDa subunit provides the conformation of photosystem I necessary for the extremely rapid electron transfer from plastocyanin to P700⁺.     

PsaD is required for the stable binding of PsaC to the photosystem I core protein of Synechococcus sp. PCC 6301.

The psaC gene product from Synechococcus sp. PCC 7002 and the psaD gene product from Nostoc sp. PCC 8009 were synthesized in Escherichia coli and purified to homogeneity. Incubation of the PsaC apoprotein with the Synechoccus sp. PCC 6301 photosystem I core protein in the presence of FeCl3, Na2S, and beta-mercaptoethanol resulted in a time-dependent transition in the flash-induced absorption change from a 1.2-ms, P700+ FX- back-reaction to a long-lived, P700+ [FA/FB]- back-reaction. ESR studies showed that FB and FA were photoreduced about equally at 19 K, and while the resonances were shifted upfield, they remained as broad as in the free PsaC holoprotein. When the reconstituted complex was purified in a sucrose gradient containing 0.1% Triton X-100, most of the optical absorption transient reverted to that characteristic of the P700+ FX- back-reaction. Addition of purified PsaD to the incubation mixture led to a greater extent of recovery of electron flow to FA/FB for any given concentration of PsaC. ESR studies showed that FA, rather than FB, became the preferred electron acceptor at 19 K; moreover, the resonances moved upfield and sharpened to become nearly identical with those of a control photosystem I complex. When the sample was purified in a sucrose gradient containing 0.1% Triton X-100, the long-lived P700+ [FA/FB]- optical transient remained stable. Analysis by denaturing polyacrylamide gel electrophoresis showed that the PsaC and PsaD proteins had rebound to the photosystem I core. The data indicate that although PsaC can bind loosely, the presence of PsaD leads to a stable, isolatable photosystem I complex which is spectroscopically indistinguishable from the native complex. Since a PsaC1 fusion protein which contains an amino-terminal extension of five amino acids (MEHSM...) does not bind in the absence of PsaD [Zhao, J., et al. (1990) FEBS Lett. 276, 175-180], the N-terminus of the PsaC protein could provide a site of interaction with the photosystem I core. We propose that the binding of PsaC to the PsaA/PsaB heterodimer is potentiated by insertion of the FA/FB clusters into PsaC, and stabilized by the presence of PsaD.     

Mutations in the D1 subunit of photosystem II distinguish between quinone and herbicide binding sites.

The structure-activity relationships of the plastoquinone QB binding domain in the D1 subunit of photosystem II (PSII) were investigated by characterization of mutations introduced in the D1 protein. Eight novel point mutations in the gene psbA, which encodes D1, were generated in the cyanobacterium Synechocystis PCC6803 by site-specific mutagenesis in vitro. The effects of the resulting modifications in D1 on electron transfer in PSII and on herbicide binding were analyzed. The results extend the structural analogies between the secondary quinone binding site in D1 and in subunit L of the photosynthetic reaction center in purple bacteria. The involvement of Phe255, Ser264, and Leu271 of D1 in plastoquinone binding and electron transfer in PSII was established. An indirect effect of Tyr254 on the binding of QB was demonstrated. Changes in binding of herbicides and QB to D1 as a result of the mutations revealed specific interactions between amino acid residues in D1 and the plastoquinone and distinguished between the binding sites of QB and herbicides.     

Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum

The plastid-encoded psaJ gene encodes a hydrophobic low-molecular-mass subunit of photosystem I (PSI) containing one transmembrane helix. Homoplastomic transformants with an inactivated psaJ gene were devoid of PSI-J protein. The mutant plants were slightly smaller and paler than wild-type because of a 13% reduction in chlorophyll content per leaf area caused by an approximately 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI-J is not involved in binding of light-harvesting complex I. The specific PSI activity, measured as NADP(+) photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second-order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI-J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis revealed a secondary loss of the luminal PSI-N subunit in PSI particles devoid of PSI-J. Presumably PSI-J affects the conformation of PSI-F, which in turn affects the binding of PSI-N. This together renders a fraction of the PSI particles inactive. Thus, PSI-J is an important subunit that, together with PSI-F and PSI-N, is required for formation of the plastocyanin-binding domain of PSI. PSI-J is furthermore important for stability or assembly of the PSI complex.     

Evidence for the involvement of PSI-E subunit in the reduction of ferredoxin by photosystem I.

Of the stroma-accessible proteins of photosystem I (PSI) from Synechocystis sp. PCC 6803, the PSI-C, PSI-D and PSI-E subunits have already been characterized, and the corresponding genes isolated. PCR amplification and cassette mutagenesis were used in this work to delete the psaE gene. PSI particles were isolated from this mutant, which lacks subunit PSI-E, and the direct photoreduction of ferredoxin was investigated by flash absorption spectroscopy. The second order rate constant for reduction of ferredoxin by wild type PSI was estimated to be approximately 10(9) M-1s-1. Relative to the wild type, PSI lacking PSI-E exhibited a rate of ferredoxin reduction decreased by a factor of at least 25. After reassociation of the purified PSI-E polypeptide, the original rate of electron transfer was recovered. When a similar reconstitution was performed with a PSI-E polypeptide from spinach, an intermediate rate of reduction was observed. Membrane labeling of the native PSI with fluorescein isothiocyanate allowed the isolation of a fluorescent PSI-E subunit. Peptide analysis showed that some residues following the N-terminal sequence were labeled and thus probably accessible to the stroma, whereas both N- and C-terminal ends were probably buried in the photosystem I complex. Site-directed mutagenesis based on these observations confirmed that important changes in either of the two terminal sequences of the polypeptide impaired its correct integration in PSI, leading to phenotypes identical to the deleted mutant. Less drastic modifications in the predicted stroma exposed sequences did not impair PSI-E integration, and the ferredoxin photoreduction was not significantly affected. All these results lead us to propose a structural role for PSI-E in the correct organization of the site involved in ferredoxin photoreduction.     

Directed mutagenesis of the transmembrane domain of the PsbL subunit of photosystem II in Synechocystis sp. PCC 6803

The PsbL protein is one of three low-molecular-weight subunits identified at the monomer-monomer interface of photosystem II (PSII) [Ferreira et al. (2004) Science 303:1831-1838; Loll et al. (2005) Nature 438:1040-1044]. We have employed site-directed mutagenesis to investigate the role of PsbL in Synechocystis sp. PCC 6803 cells. Truncation of the C-terminus by deleting the last four residues (Tyr-Phe-Phe-Asn) prevented association of PsbL with the CP43-less monomeric sub-complex and therefore blocked PSII assembly resulting in an obligate photoheterotrophic strain. Replacement of these residues with Ala created four photoautotrophic mutants. Compared to wild type, the F37A, F38A, and N39A strains had reduced levels of assembled PSII centers and F37A and F38A cells were readily photodamaged. In contrast, Y36A and Y36F mutants were similar to wild type. However, each of these strains had elevated levels of the CP43-less inactive monomeric complex. Mutations targeting a putative hydrogen bond between Arg-16 and sulfoquinovosyldiacylglycerol resulted in mutants that were also highly susceptible to photodamage. Similarly mutations targeting a conserved Tyr residue (Tyr-20) also destabilized PSII under high light and suggest that Tyr-20-lipid interactions or interactions of Tyr-20 with PsbT influence the ability of PSII to recover from photodamage.     

Polymorphism of a photosystem I subunit caused by alloploidy in Nicotiana

The photosystem I complex from Nicotiana tabacum, which has an alloploid genome, contains subunits of 17.5 and 18.5 kilodaltons whose N-terminal amino acid sequences are highly homologous. Comparative analysis of photosystem I subunits among N. tabacum and its ancestral plants, N. tomentosiformis and N. sylvestris, revealed that the 17.5 kilodalton subunit of N. tabacum derives from N. sylvestris, and the 18.5 kilodalton subunit from N. tomentosiformis.