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(+).     

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.     

Comparison of the subunit compositions of the PSI-LHCI supercomplex and the LHCI in the green alga Chlamydomonas reinhardtii

Although the light-harvesting chlorophyll protein complex I (LHCI) of photosystem I (PSI) is intimately associated with the PSI core complex and forms the PSI-LHCI supercomplex, the LHCI is normally synthesized in PSI-deficient mutants. In this paper, we compared the subunit compositions of the PSI-LHCI supercomplex and the LHCI by immunoblot analysis and two-dimensional gel electrophoresis combined with mass spectrometry. The PSI-LHCI supercomplex and the LHCI were purified by sucrose density gradient centrifugation and (diethylamino)ethyl column chromatography from n-dodecyl-beta-D-maltoside-solubilized thylakoids of the wild-type and DeltapsaB mutant of the green alga Chlamydomonas reinhardtii. The PSI-LHCI supercomplex contained all of the nine Lhca polypeptides (Lhca1-9) that are detected in wild-type thylakoids. In contrast, the LHCI retained only six Lhca polypeptides, whereas Lhca3 and two minor polypeptides, Lhca2 and Lhca9, were lost during the purification procedure. Sucrose density gradient centrifugation showed that the purified LHCI retains an oligomeric structure with an apparent molecular mass of 300-400 kDa. We therefore concluded that Lhca2, Lhca3, and Lhca9 are not required for the stable oligomeric structure of the LHCI and that the association of these polypeptides in the LHCI is stabilized by the presence of the PSI core complex. Finally, we discuss the possible localization and function of Lhca polypeptides in the LHCI.     

Photosystem I lacking the PSI-G subunit has a higher affinity for plastocyanin and is sensitive to photodamage

PSI-G is an 11 kDa subunit of PSI in photosynthetic eukaryotes. Arabidopsis thaliana plants devoid of PSI-G have a decreased PSI content and an increased activity of NADP(+) photoreduction in vitro but otherwise no obvious phenotype. To investigate the biochemical basis for the increased activity, the kinetic parameters of the reaction between PSI and plastocyanin were determined. PSI-G clearly plays a role in the affinity for plastocyanin since the dissociation constant (K(D)) is only 12 muM in the absence of PSI-G compared to 32 muM for the wild type. On the physiological level, plants devoid of PSI-G have a more reduced Q(A). This indicates that the decreased PSI content is due to unstable PSI rather than an adaptation to the increased activity. In agreement with this indication of decreased stability, plants devoid of PSI-G were found to be more photo-inhibited both at low temperature and after high light treatment. The decreased PSI stability was confirmed in vitro by measuring PSI activity after illumination of a thylakoid suspension which clearly showed a faster decrease in PSI activity in the thylakoids lacking PSI-G. Light response of the P700 redox state in vivo showed that in the absence of PSI-G, P700 is more reduced at low light intensities. We conclude that PSI-G is involved in the binding dynamics of plastocyanin to PSI and that PSI-G is important for the stability of the PSI complex.     

Three-dimensional reconstruction of a light-harvesting complex I-photosystem I (LHCI-PSI) supercomplex from the green alga Chlamydomonas reinhardtii. Insights into light harvesting for PSI

A supercomplex containing the photosystem I (PSI) and chlorophyll a/b light-harvesting complex I (LHCI) has been isolated using a His-tagged mutant of Chlamydomonas reinhardtii. This LHCI-PSI supercomplex contained approximately 215 chlorophyll molecules of which 175 were estimated to be chlorophyll a and 40 to be chlorophyll b, based on P700 oxidation and chlorophyll a/b ratio measurements. Its room temperature long wavelength absorption peak was at 680 nm, and it emitted chlorophyll fluorescence maximally at 715 nm (77 K). The LHCI was composed of four or more different types of Lhca polypeptides including Lhca3. No LHCII proteins or other phosphoproteins were detected in the LHCI-PSI supercomplexes suggesting that the cells from which they were isolated were in State 1. Electron microscopy of negatively stained samples followed by image analysis revealed the LHCI-PSI supercomplex to have maximal dimensions of 220 A by 180 A and to be approximately 105 A thick. An averaged top view was used to model in x-ray and electron crystallographic data for PSI and Lhca proteins respectively. We conclude that the supercomplex consists of a PSI reaction center monomer with 11 Lhca proteins arranged along the side where the PSI proteins, PsaK, PsaJ, PsaF, and PsaG are located. The estimated molecular mass for the complex is 700 kDa including the bound chlorophyll molecules. The assignment of 11 Lhca proteins is consistent with a total chlorophyll level of 215 assuming that the PSI reaction center core binds approximately 100 chlorophylls and that each Lhca subunit binds 10 chlorophylls. There was no evidence for oligomerization of Chlamydomonas PSI in contrast to the trimerization of PSI in cyanobacteria.     

Specific substitutions of light‐harvesting complex I proteins associated with photosystem I are required for supercomplex formation with chloroplast NADH dehydrogenase‐like complex

In Arabidopsis, the chloroplast NADH‐dehydrogenase‐like (NDH) complex is sandwiched between two copies of photosystem I (PSI) supercomplex, consisting of a PSI core and four light‐harvesting complex I (LHCI) proteins (PSI‐LHCI) to form the NDH–PSI supercomplex. Two minor LHCI proteins, Lhca5 and Lhca6, contribute to the interaction of each PSI–LHCI copy with the NDH complex. Here, large‐pore blue‐native gel electrophoresis revealed that, in addition to this complex, there were at least two types of higher‐order association of more LHCI copies with the NDH complex. In single‐particle images, this higher‐order association of PSI–LHCI preferentially occurs at the left side of the NDH complex when viewed from the stromal side, placing subcomplex A at the top (Yadav et al., Biochim. Biophys. Acta ‐ Bioenerg., 1858, 2017, 12). The association was impaired in the lhca6 mutant but not in the lhca5 mutant, suggesting that the left copy of PSI–LHCI was linked to the NDH complex via Lhca6. From an analysis of subunit compositions of the NDH–PSI supercomplex in lhca5 and lhca6 mutants, we propose that Lhca6 substitutes for Lhca2 in the left copy of PSI–LHCI, whereas Lhca5 substitutes for Lhca4 in the right copy. In the lhca2 mutant, Lhca3 was specifically stabilized in the NDH–PSI supercomplex through heterodimer formation with Lhca6. In the left copy of PSI–LHCI, subcomplex B, Lhca6 and NdhD likely formed the core of the supercomplex interaction. In contrast, a larger protein complex, including at least subcomplexes B and L and NdhB, was needed to form the contact site with Lhca5 in the right copy of PSI–LHCI.     

Photosystem I trimers from Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits bind an IsiA ring of 17 units

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of Photosystem I (PSI) and the chlorophyll-binding protein IsiA from a mutant of the cyanobacterium Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits. The circular complex consists of a central PSI trimer surrounded by a ring of 17 IsiA units, one less than in the wild-type supercomplex. We conclude that PsaF and PsaJ are not obligatory for the binding of the IsiA ring, and that the size of the PSI complex determines the number of IsiA units in the ring. The resulting number of 17 copies implies that each PSI monomer has a different association to the IsiA ring.     

Photosystem I lacking the PSI-G subunit has a higher affinity for plastocyanin and is sensitive to photodamage

PSI-G is an 11 kDa subunit of PSI in photosynthetic eukaryotes. Arabidopsis thaliana plants devoid of PSI-G have a decreased PSI content and an increased activity of NADP⁺ photoreduction in vitro but otherwise no obvious phenotype [P.E. Jensen, L. Rosgaard, J. Knoetzel, H.V. Scheller, Photosystem I activity is increased in the absence of the PSI-G subunit. J. Biol. Chem. 277, (2002) 2798-2803.]. To investigate the biochemical basis for the increased activity, the kinetic parameters of the reaction between PSI and plastocyanin were determined. PSI-G clearly plays a role in the affinity for plastocyanin since the dissociation constant (K_D) is only 12 μM in the absence of PSI-G compared to 32 μM for the wild type. On the physiological level, plants devoid of PSI-G have a more reduced Q_A. This indicates that the decreased PSI content is due to unstable PSI rather than an adaptation to the increased activity. In agreement with this indication of decreased stability, plants devoid of PSI-G were found to be more photoinhibited both at low temperature and after high light treatment. The decreased PSI stability was confirmed in vitro by measuring PSI activity after illumination of a thylakoid suspension which clearly showed a faster decrease in PSI activity in the thylakoids lacking PSI-G. Light response of the P700 redox state in vivo showed that in the absence of PSI-G, P700 is more reduced at low light intensities. We conclude that PSI-G is involved in the binding dynamics of plastocyanin to PSI and that PSI-G is important for the stability of the PSI complex.     

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.     

N-terminal processing of Lhca3 Is a key step in remodeling of the photosystem I-light-harvesting complex under iron deficiency in Chlamydomonas reinhardtii

Iron deficiency induces a remodeling of the photosynthetic apparatus in Chlamydomonas reinhardtii. In this study we showed that a key mechanistic event in the remodeling process of photosystem I (PSI) and its associated light-harvesting proteins (LHCI) is the N-terminal processing of Lhca3. N-terminal processing of Lhca3 is documented independently by two-dimensional gel electrophoresis and tandem mass spectrometric (MS/MS) analysis as well as by quantitative comparative MS/MS peptide profiling using isotopic labeling of proteins. Dynamic remodeling of the LHCI complex under iron deficiency is further exemplified by depletion of Lhca5 and up-regulation of Lhca4 and Lhca9 polypeptides in respect to photosystem I. Most importantly, the induction of N-terminal processing of Lhca3 by progression of iron deficiency correlates with the functional drop in excitation energy transfer efficiency between LHCI and PSI as assessed by low temperature fluorescence emission spectroscopy. Using an RNA interference (RNAi) strategy, we showed that the truncated form of Lhca3 is essential for the structural stability of LHCI. Depletion of Lhca3 by RNAi strongly impacted the efficiency of excitation energy transfer between PSI and LHCI, as is the case for iron deficiency. However, in contrast to iron deficiency, comparative MS/MS peptide profiling using isotopic labeling of proteins demonstrated that RNAi depletion of Lhca3 caused strong reduction of almost all Lhca proteins in isolated PSI particles.     

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.     

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.     

Lhca5 interaction with plant photosystem I

In the outer antenna (LHCI) of higher plant photosystem I (PSI) four abundantly expressed light-harvesting protein of photosystem I (Lhca)-type proteins are organized in two heterodimeric domains (Lhca1/Lhca4 and Lhca2/Lhca3). Our cross-linking studies on PSI-LHCI preparations from wildtype Arabidopsis and pea plants indicate an exclusive interaction of the rarely expressed Lhca5 light-harvesting protein with LHCI in the Lhca2/Lhca3-site. In PSI particles with an altered LHCI composition Lhca5 assembles in the Lhca1/Lhca4 site, partly as a homodimer. This flexibility indicates a binding-competitive model for the LHCI assembly in plants regulated by molecular interactions of the Lhca proteins with the PSI core.     

Photosystem I trimers from Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits bind an IsiA ring of 17 units

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of Photosystem I (PSI) and the chlorophyll-binding protein IsiA from a mutant of the cyanobacterium Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits. The circular complex consists of a central PSI trimer surrounded by a ring of 17 IsiA units, one less than in the wild-type supercomplex. We conclude that PsaF and PsaJ are not obligatory for the binding of the IsiA ring, and that the size of the PSI complex determines the number of IsiA units in the ring. The resulting number of 17 copies implies that each PSI monomer has a different association to the IsiA ring.     

Isolation and characterization of a large photosystem I–light‐harvesting complex II supercomplex with an additional Lhca1–a4 dimer in Arabidopsis

The biological conversion of light energy into chemical energy is performed by a flexible photosynthetic machinery located in the thylakoid membranes. Photosystems I and II (PSI and PSII) are the two complexes able to harvest light. PSI is the last complex of the electron transport chain and is composed of multiple subunits: the proteins building the catalytic core complex that are well conserved between oxygenic photosynthetic organisms, and, in green organisms, the membrane light‐harvesting complexes (Lhc) necessary to increase light absorption. In plants, four Lhca proteins (Lhca1–4) make up the antenna system of PSI, which can be further extended to optimize photosynthesis by reversible binding of LHCII, the main antenna complex of photosystem II. Here, we used biochemistry and electron microscopy in Arabidopsis to reveal a previously unknown supercomplex of PSI with LHCII that contains an additional Lhca1–a4 dimer bound on the PsaB–PsaI–PsaH side of the complex. This finding contradicts recent structural studies suggesting that the presence of an Lhca dimer at this position is an exclusive feature of algal PSI. We discuss the features of the additional Lhca dimer in the large plant PSI–LHCII supercomplex and the differences with the algal PSI. Our work provides further insights into the intricate structural plasticity of photosystems.     

The properties of the chlorophyll a/b-binding proteins Lhca2 and Lhca3 studied in vivo using antisense inhibition

The specific functions of the light-harvesting proteins Lhca2 and Lhca3 were studied in Arabidopsis ecotype Colombia antisense plants in which the proteins were individually repressed. The antisense effect was specific in each plant, but levels of Lhca proteins other than the targeted products were also affected. The contents of Lhca1 and Lhca4 were unaffected, but Lhca3 (in Lhca2-repressed plants) was almost completely depleted, and Lhca2 decreased to about 30% of wild-type levels in Lhca3-repressed plants. This suggests that the Lhca2 and Lhca3 proteins are in physical contact with each other and that they require each other for stability. Photosystem I fluorescence at 730 nm is thought to emanate from pigments bound to Lhca1 and Lhca4. However, fluorescence emission and excitation spectra suggest that Lhca2 and Lhca3, which fluoresce in vitro at 680 nm, also could contribute to far-red fluorescence in vivo. Spectral forms with absorption maxima at 695 and 715 nm, apparently with emission maxima at 702 and 735 nm, respectively, might be associated with Lhca2 and Lhca3.     

Isolation of a psaF-deficient mutant of Chlamydomonas reinhardtii: efficient interaction of plastocyanin with the photosystem I reaction center is mediated by the PsaF subunit.

The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membrane and its precise role is not yet fully understood. Here we describe the isolation of a psaF-deficient mutant of the green alga Chlamydomonas reinhardtii generated by co-transforming the nuclear genome of the cw15-arg7A strain with two plasmids: one harboring a mutated version of the psaF gene and the other containing the argininosuccinate lyase gene conferring arginine prototrophy. This psaF mutant still assembles a functional PSI complex and is capable of photoautotrophic growth. However, electron transfer from plastocyanin to P700+, the oxidized reaction center chlorophyll dimer, is dramatically reduced in the mutant, indicating that the PsaF subunit plays an important role in docking plastocyanin to the PSI complex. These results contrast with those obtained previously with a cyanobacterial psaF-, psaJ- double mutant where no phenotype was apparent.     

Lhca5 – an LHC-Type Protein Associated with Photosystem I

The light-harvesting antenna of higher plant photosystem (PS) I is known to be composed of four different types of light-harvesting complex (LHC) proteins (Lhca1-4). However, the genomic sequence of Arabidopsis thaliana contains open reading frames coding for two additional LHC type proteins (Lhca5-6) that are presumably associated with PSI. While Lhca6 might not be expressed at all, ESTs have been detected for the Lhca5 gene in Arabidopsis and a number of other plant species. Here we demonstrate the presence of the Lhca5 gene product in the thylakoid membrane of Arabidopsis as an additional type of Lhca-protein associated with PSI. Lhca5 seems to be regulated differently from the other LHC proteins since Lhca5 mRNA levels increase under high light conditions. Analyses reported here of Lhca5 in plants lacking individual Lhca1-4 proteins show that it is more abundant in plants lacking Lhca1/4, and suggest that it interacts in a direct physical fashion with Lhca2 or Lhca3. We propose that Lhca5 binds chlorophylls in a similar fashion to the other Lhca proteins and is associated with PSI only in sub-stoichiometric amounts.     

Excitation-energy transfer dynamics of higher plant photosystem I light-harvesting complexes

Photosystem I (PSI) plays a major role in the light reactions of photosynthesis. In higher plants, PSI is composed of a core complex and four outer antennas that are assembled as two dimers, Lhca1/4 and Lhca2/3. Time-resolved fluorescence measurements on the isolated dimers show very similar kinetics. The intermonomer transfer processes are resolved using target analysis. They occur at rates similar to those observed in transfer to the PSI core, suggesting competition between the two transfer pathways. It appears that each dimer is adopting various conformations that correspond to different lifetimes and emission spectra. A special feature of the Lhca complexes is the presence of an absorption band at low energy, originating from an excitonic state of a chlorophyll dimer, mixed with a charge-transfer state. These low-energy bands have high oscillator strengths and they are superradiant in both Lhca1/4 and Lhca2/3. This challenges the view that the low-energy charge-transfer state always functions as a quencher in plant Lhc's and it also challenges previous interpretations of PSI kinetics. The very similar properties of the low-energy states of both dimers indicate that the organization of the involved chlorophylls should also be similar, in disagreement with the available structural data.