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.     

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.     

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.     

Pigment organization and energy transfer dynamics in isolated photosystem I (PSI) complexes from Arabidopsis thaliana depleted of the PSI-G, PSI-K, PSI-L, or PSI-N subunit

Green plant photosystem I (PSI) consists of at least 18 different protein subunits. The roles of some of these protein subunits are not well known, in particular those that do not occur in the well characterized PSI complexes from cyanobacteria. We investigated the spectroscopic properties and excited-state dynamics of isolated PSI-200 particles from wild-type and mutant Arabidopsis thaliana plants devoid of the PSI-G, PSI-K, PSI-L, or PSI-N subunit. Pigment analysis and a comparison of the 5 K absorption spectra of the various particles suggests that the PSI-L and PSI-H subunits together bind approximately five chlorophyll a molecules with absorption maxima near 688 and 667 nm, that the PSI-G subunit binds approximately two red-shifted beta-carotene molecules, that PSI-200 particles without PSI-K lack a part of the peripheral antenna, and that the PSI-N subunit does not bind pigments. Measurements of fluorescence decay kinetics at room temperature with picosecond time resolution revealed lifetimes of ~0.6, 5, 15, 50, 120, and 5000 ps in all particles. The 5- and 15-ps phases could, at least in part, be attributed to the excitation equilibration between bulk and red chlorophyll forms, though the 15-ps phase also contains a contribution from trapping by charge separation. The 50- and 120-ps phases predominantly reflect trapping by charge separation. We suggest that contributions from the core antenna dominate the 15-ps trapping phase, that those from the peripheral antenna proteins Lhca2 and Lhca3 dominate the 50-ps phase, and that those from Lhca1 and Lhca4 dominate the 120-ps phase. In the PSI-200 particles without PSI-K or PSI-G protein, more excitations are trapped in the 15-ps phase and less in 50- and 120-ps phases, which is in agreement with the notion that these subunits are involved in the interaction between the core and peripheral antenna proteins.     

Insertion of the plant photosystem I subunit G into the thylakoid membrane

Subunit G of photosystem I is a nuclear-encoded protein, predicted to form two transmembrane alpha-helices separated by a loop region. We use in vitro import assays to show that the positively charged loop domain faces the stroma, whilst the N- and C-termini most likely face the lumen. PSI-G constructs in which a His- or Strep-tag is placed at the C-terminus or in the loop region insert with the same topology as wild-type photosystem I subunit G (PSI-G). However, the presence of the tags in the loop make the membrane-inserted protein significantly more sensitive to trypsin, apparently by disrupting the interaction between the loop and the PSI core. Knock-out plants lacking PSI-G were transformed with constructs encoding the C-terminal and loop-tagged PSI-G proteins. Experiments on thylakoids from the transgenic lines show that the C-terminally tagged versions of PSI-G adopt the same topology as wild-type PSI-G, whereas the loop-tagged versions affect the sensitivity of the loop region to trypsin, thus confirming the in vitro observations. Furthermore, purification of PSI complexes from transgenic plants revealed that all the tagged versions of PSI-G are incorporated and retained in the PSI complex, although the C-terminally tagged variants of PSI-G were preferentially retained. This suggests that the loop region of PSI-G is important for proper integration into the PSI core. Our experiments demonstrate that it is possible to produce His- and Strep-tagged PSI in plants, and provide further evidence that the topology of membrane proteins is dictated by the distribution of positive charges, which resist translocation across membranes.     

Arabidopsis thaliana plants lacking the PSI-D subunit of photosystem I suffer severe photoinhibition,have unstable photosystem I complexes,and altered redox homeostasis in the chloroplast stroma

The PSI-D subunit of photosystem I is a hydrophilic subunit of about 18 kDa, which is exposed to the stroma and has an important function in the docking of ferredoxin to photosystem I. We have used an antisense approach to obtain Arabidopsis thaliana plants with only 5-60% of PSI-D. No plants were recovered completely lacking PSI-D, suggesting that PSI-D is essential for a functional PSI in plants. Plants with reduced amounts of PSI-D showed a similar decrease in all other subunits of PSI including the light harvesting complex, suggesting that in the absence of PSI-D, PSI cannot be properly assembled and becomes degraded. Plants with reduced amounts of PSI-D became light-stressed even in low light although they exhibited high non-photochemical quenching (NPQ). The high NPQ was generated by upregulating the level of violaxanthin de-epoxidase and PsbS, which are both essential components of NPQ. Interestingly, the lack of PSI-D affected the redox state of thioredoxin. During the normal light cycle thioredoxin became increasingly oxidized, which was observed as decreasing malate dehydrogenase activity over a 4-h light period. This result shows that photosynthesis was close to normal the first 15 min, but after 2-4 h photoinhibition dominated as the stroma progressively became less reduced. The change in the thiol disulfide redox state might be fatal for the PSI-D-less plants, because reduction of thioredoxin is one of the main switches for the initiation of CO2 assimilation and photoprotection upon light exposure.     

Down-regulation of the PSI-F subunit of photosystem I (PSI) in Arabidopsis thaliana. The PSI-F subunit is essential for photoautotrophic growth and contributes to antenna function

The PSI-F subunit of photosystem I is a transmembrane protein with a large lumenal domain. The role of PSI-F was investigated in Arabidopsis plants transformed with an antisense construct of the psaF cDNA. Several plant lines with reduced amounts of the PSI-F subunit were generated. Many of the transgenic plants died, apparently because they were unable to survive without the PSI-F subunit. Plants with 5% of PSI-F were capable of photoautotrophic growth but were much smaller than wild-type plants. The plants suffered severely under normal growth conditions but recovered somewhat in the dark indicating chronic photoinhibition. Photosystem I lacking PSI-F was less stable, and the stromal subunits PSI-C, PSI-D, and PSI-E were present in lower amounts than in wild type. The lack of PSI-F resulted in an inability of light-harvesting complex I-730 to transfer energy to the P700 reaction center. In thylakoids deficient in PSI-F, the steady state NADP(+) reduction rate was only 10% of the wild-type levels indicating a lower efficiency in oxidation of plastocyanin. Surprisingly, the lack of PSI-F also gave rise to disorganization of the thylakoids. The strict arrangement in grana and stroma lamellae was lost, and instead a network of elongated and distorted grana was observed.     

Molecular aspects of photosystem I

Photosystem I (PSI) in higher plants consists of 17 polypeptide subunits. Cofactors are chlorophyll a and b , β-carotene, phylloquinone and iron-sulfur clusters. Eight subunits are specific for higher plants while the remaining ones are also present in cyanobacteria. Two 80-kDa subunits (PSI-A and -B) constitute the major part of PSI and bind most of the pigments and electron donors and acceptors. The 9-kDa PSI-C carries the remaining electron acceptors which are [4Fe-4S] iron sulfur clusters. PSI-D, -E and -H have importance for integrity and function at the stromal face of PSI while PSI-F has importance for function at the lumenal face. PSI-N is localized at the lumenal side, but its function is unknown. Four subunits are light-harvesting chlorophyll a/b -binding proteins. The remaining subunits are integral membrane proteins with poorly understood function. Subunit interactions have been studied in reconstitution experiments and by cross-linking studies. Based on these data, it is concluded that iron-sulfur cluster F_B is proximal to F_X and that F_A is the terminal acceptor in PSI. Similarities between PSI and the reaction center from green sulfur bacteria are discussed.     

The chloroplast NAD(P)H dehydrogenase complex interacts with photosystem I in Arabidopsis

The chloroplast NAD(P)H dehydrogenase (NDH) complex is involved in photosystem I (PSI) cyclic and chlororespiratory electron transport in higher plants. Although biochemical and genetic evidence for its subunit composition has accumulated, it is not enough to explain the complexes putative activity of NAD(P)H-dependent plastoquinone reduction. We analyzed the NDH complex by using blue native PAGE and found that it interacts with PSI to form a novel supercomplex. Mutants lacking NdhL and NdhM accumulated a pigment-protein complex with a slightly lower molecular mass than that of the NDH-PSI supercomplex; this may be an intermediate supercomplex including PSI. This intermediate is unstable in mutants lacking NdhB, NdhD, or NdhF, implying that it includes some NDH subunits. Analysis of thylakoid membrane complexes using sucrose density gradient centrifugation supported the presence of the NDH-PSI supercomplex in vivo. Although the NDH complex exists as a monomer in etioplasts, it interacts with PSI to form a supercomplex within 48 h during chloroplast development.     

A large fraction of PsaF is nonfunctional in photosystem I complexes lacking the PsaJ subunit

PsaJ is a small hydrophobic subunit of the photosystem I complex (PSI) whose function is not yet fully understood. Here we describe mutants of the green alga Chlamydomonas reinhardtii, in which the psaJ chloroplast gene has been inactivated either in a wild-type or in a PsaF-deficient nuclear background. Cells lacking one or both subunits grow photoautotrophically and contain normal levels of PSI. Flash-absorption spectroscopy performed with isolated PSI particles isolated from the PsaJ-deficient strain indicates that only 30% of the PSI complexes oxidize plastocyanin (Pc) or cytochrome c6 (Cyt c6) with kinetics identical to wild type, whereas the remaining 70% follow slow kinetics similar to those observed with PsaF-deficient PSI complexes. This feature is not due to partial loss of PsaF, as the PsaJ-less PSI complex contains normal levels of the PsaF subunit. The N-terminal domain of PsaF can be cross-linked to Pc and Cyt c6 indicating that in the absence of PsaJ, this domain is exposed in the lumenal space. Therefore, the decreased amount of functional PsaF revealed by the electron-transfer measurements is best explained by a displacement of the N-terminal domain of PsaF which is known to provide the docking site for Pc and Cyt c6. We propose that one function of PsaJ is to maintain PsaF in a proper orientation which allows fast electron transfer from soluble donor proteins to P700(+).     

Brassica rapa plants adapted to microgravity with reduced photosystem I and its photochemical activity

The photosynthetic apparatus contains several protein complexes, many of which are regulated by environmental conditions. In this study, the influences of microgravity on PSI and PSII in Brassica rapa plants grown aboard the space shuttle were examined. We found that Brassica plants grown in space had a normal level of growth relative to controls under similar conditions on Earth. Upon return to Earth, cotyledons were harvested and thylakoid membranes were isolated. Analysis of chlorophyll contents showed that the Chl a/b ratio (3.5) in flight cotyledons was much higher than a ratio of 2.42 in the ground controls. The flight samples also had a reduction of PSI complexes and a corresponding 30% decrease of PSI photochemical activity. Immunoblotting showed that the reaction centre polypeptides of PSI were more apparently decreased (e.g. by 24-33% for PsaA and PsaB, and 57% for PsaC) than the light-harvesting complexes. In comparison, the accumulation of PSII complex was less affected in microgravity, thus only a slight reduction in D1, D2 and LHCII was observed in protein blots. However, there was a 32% decrease of OEC1 in the flight samples, indicating a defective OEC subcomplex. In addition, an average 54% increase of the 54 kDa CF1-beta isoform was found in the flight samples, suggesting that space-grown plants suffered from certain stresses, consistent with implications of the increased Chl a/b ratio. Taken together, the results demonstrated that Brassica plants can adapt to spaceflight microgravity, but with significant alterations in chloroplast structures and photosynthetic complexes, and especially reduction of PSI and its activity.     

Single and double knockouts of the genes for photosystem I subunits G,K, and H of Arabidopsis. Effects on photosystem I composition,photosynthetic electron flow,and state transitions

Photosystem I (PSI) of higher plants contains 18 subunits. Using Arabidopsis En insertion lines, we have isolated knockout alleles of the genes psaG, psaH2, and psaK, which code for PSI-G, -H, and -K. In the mutants psak-1 and psag-1.4, complete loss of PSI-K and -G, respectively, was confirmed, whereas the residual H level in psah2-1.4 is due to a second gene encoding PSI-H, psaH1. Double mutants, lacking PSI-G, and also -K, or a fraction of -H, together with the three single mutants were characterized for their growth phenotypes and PSI polypeptide composition. In general, the loss of each subunit has secondary, in some cases additive, effects on the abundance of other PSI polypeptides, such as D, E, H, L, N, and the light-harvesting complex I proteins Lhca2 and 3. In the G-less mutant psag-1.4, the variation in PSI composition suggests that PSI-G stabilizes the PSI-core. Levels of light-harvesting complex I proteins in plants, which lack simultaneously PSI-G and -K, indicate that PSI subunits other than G and K can also bind Lhca2 and 3. In the same single and double mutants, psag-1.4, psak-1, psah2-1.4, psag-1.4/psah2-1.4, and psag-1.4/psak-1 photosynthetic electron flow and excitation energy quenching were analyzed to address the roles of the various subunits in P700 reduction (mediated by PSI-F and -N) and oxidation (PSI-E), and state transitions (PSI-H). Based on the results, we also suggest for PSI-K a role in state transitions.     

The primary structure of a cDNA for PsaN,encoding an extrinsic lumenal polypeptide of barley photosystem I

A cDNA clone encoding a 15.501 Da photosystem I (PSI) subunit of barley was isolated using an oligonucleotide based on the NH₂-terminal amino acid sequence of the isolated protein. The polypeptide, which migrates with an apparent molecular mass of 9.5 kDa on denaturing SDS-PAGE, has been designated PSI-N, and the corresponding gene is PsaN. Analysis of the deduced protein sequence indicates a mature protein of 85 amino acid residues and a molecular mass of 9818 Da. PSI-N is a hydrophilic, extrinsic protein with no predicted membrane-spanning regions. The transit peptide of 60 residues (5683 Da) contains a predicted hydrophobic -helix, suggesting that the protein is routed into the thylakoid lumen. Thus, PSI-N is the second known lumenal protein component associated with PSI, together with PSI-F.     

Light-harvesting complex II binds to several small subunits of photosystem I

Mobile light-harvesting complex II (LHCII) is implicated in the regulation of excitation energy distribution between Photosystem I (PSI) and Photosystem II (PSII) during state transitions. To investigate how LHCII interacts with PSI during state transitions, PSI was isolated from Arabidopsis thaliana plants treated with PSII or PSI light. The PSI preparations were made using digitonin. Chemical cross-linking using dithio-bis(succinimidylpropionate) followed by diagonal electrophoresis and immunoblotting showed that the docking site of LHCII (Lhcb1) on PSI is comprised of the PSI-H, -L, and -I subunits. This was confirmed by the lack of energy transfer from LHCII to PSI in the digitonin-PSI isolated from plants lacking PSI-H and -L. Digitonin-PSI was purified further to obtain an LHCII.PSI complex, and two to three times more LHCII was associated with PSI in the wild type in State 2 than in State 1. Lhcb1 was also associated with PSI from plants lacking PSI-K, but PSI from PSI-H, -L, or -O mutants contained only about 30% of Lhcb1 compared with the wild type. Surprisingly, a significant fraction of the LHCII bound to PSI in State 2 was not phosphorylated. Cross-linking prior to sucrose gradient purification resulted in copurification of phosphorylated LHCII in the wild type, but not with PSI from the PSI-H, -L, and -O mutants. The data suggest that migration of LHCII during state transitions cannot be explained sufficiently by different affinity of phosphorylated and unphosphorylated LHCII for PSI but is likely to involve structural changes in thylakoid organization.     

The PSI-O subunit of plant photosystem I is involved in balancing the excitation pressure between the two photosystems

PSI-O is a subunit of photosystem I in eukaryotes. The function of PSI-O was characterized in Arabidopsis plants using RNA interference. Several transformants with the psaO-RNAi construct were generated, and a high proportion of the plants contained only very little or virtually no residual PSI-O. Plants lacking PSI-O have a 50% reduction in state transitions indicating a role for PSI-O in the balancing of excitation energy between the two photosystems. PSI-H and -L have been shown previously to be involved in state transitions, and immunoblot analysis revealed that plants devoid of PSI-L or -H also have 80-90% reduction in the abundance of PSI-O. In contrast, down-regulation of PSI-O has no negative effect on the content of PSI-H and -L. The interaction between PSI-O and the PSI-L was confirmed by chemical cross-linking. A model of PSI is proposed in which PSI-L as the most ancient subunit is closest to the reaction center, and PSI-O is positioned close to PSI-L on the PSI-H/-L/-I side of the PSI complex. PSI-H, -L, -O, and possibly -I are all involved in forming a domain in PSI that is involved in the interaction with light-harvesting complex II.     

Genetic decrease in fatty acid unsaturation of phosphatidylglycerol increased photoinhibition of photosystem I at low temperature in tobacco leaves

Leaves of transgenic tobacco plants with decreased levels of fatty acid unsaturation in phosphatidylglycerol (PG) exhibited a slightly lower level of the steady state oxidation of the photosystem I (PSI) reaction center P700 (P700(+)) than wild-type plants. The PSI photochemistry of wild-type plants was only marginally affected by high light treatments. Surprisingly, all plants of transgenic lines exhibited much higher susceptibility to photoinhibition of PSI than wild-type plants. This was accompanied by a 2.5-fold faster re-reduction rate of P700(+) in the dark, indicating a higher capacity for cyclic electron flow around PSI in high light treated transgenic leaves. This was associated with a much higher intersystem electron pool size suggesting over-reduction of the PQ pool in tobacco transgenic lines with altered PG unsaturation compared to wild-type plants. The physiological role of PG unsaturation in PSI down-regulation and modulation of the capacity of PSI-dependent cyclic electron flows and distribution of excitation light energy in tobacco plants under photoinhibitory conditions at low temperatures is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Supercomplexes of IsiA and photosystem I in a mutant lacking subunit PsaL

The cyanobacterium Synechocystis PCC 6803 grown under short-term iron-deficient conditions assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 IsiA complexes. Furthermore, it has been shown that single or double rings of IsiA with up to 35 copies in total can surround monomeric PSI. Here we present an analysis by electron microscopy and image analysis of the various PSI-IsiA supercomplexes from a Synechocystis PCC 6803 mutant lacking the PsaL subunit after short- and long-term iron-deficient growth. In the absence of PsaL, the tendency to form complexes with IsiA is still strong, but the average number of complete rings is lower than in the wild type. The majority of IsiA copies binds into partial double rings at the side of PsaF/J subunits rather than in complete single or double rings, which also cover the PsaL side of the PSI monomer. This indicates that PsaL facilitates the formation of IsiA rings around PSI monomers but is not an obligatory structural component in the formation of PSI-IsiA complexes.     

Decreased stability of photosystem I in dgd1 mutant of Arabidopsis thaliana

The dgd1 mutant of Arabidopsis thaliana provides us with a powerful tool for revealing the specific role of digalactosyldiacylglycerol (DGDG) in photosynthesis. Blue-native polyacrylamide gel electrophoresis analysis revealed that photosystem I (PSI) subunits are assembled into a PSI complex, and that a PSI subcomplex lacking stroma side subunits was also present. PSI subunits in the dgd1 mutant were decreased to a similar level compared with that in the wild type (WT) Arabidopsis. Further experiments showed that PSI subunits in the stroma side, PsaD and PsaE, in the dgd1 mutant were more susceptible to removal by chaotropic agents than those in the WT plant, indicating that the stability of PsaD and PsaE is impaired in the dgd1 mutant. These results provide evidence that DGDG is important for the stability of the PSI complex.