Role of subunits in eukaryotic Photosystem I.

Photosystem I (PSI) of eukaryotes has a number of features that distinguishes it from PSI of cyanobacteria. In plants, the PSI core has three subunits that are not found in cyanobacterial PSI. The remaining 11 subunits of the core are conserved but several of the subunits have a different role in eukaryotic PSI. A distinguishing feature of eukaryotic PSI is the membrane-imbedded peripheral antenna. Light-harvesting complex I is composed of four different subunits and is specific for PSI. Light-harvesting complex II can be associated with both PSI and PSII. Several of the core subunits interact with the peripheral antenna proteins and are important for proper function of the peripheral antenna. The review describes the role of the different subunits in eukaryotic PSI. The emphasis is on features that are different from cyanobacterial PSI.

The PSI-H subunit of photosystem I is essential for state transitions in plant photosynthesis.

Photosynthesis in plants involves two photosystems responsible for converting light energy into redox processes. The photosystems, PSI and PSII, operate largely in series, and therefore their excitation must be balanced in order to optimize photosynthetic performance. When plants are exposed to illumination favouring either PSII or PSI they can redistribute excitation towards the light-limited photosystem. Long-term changes in illumination lead to changes in photosystem stoichiometry. In contrast, state transition is a dynamic mechanism that enables plants to respond rapidly to changes in illumination. When PSII is favoured (state 2), the redox conditions in the thylakoids change and result in activation of a protein kinase. The kinase phosphorylates the main light-harvesting complex (LHCII) and the mobile antenna complex is detached from PSII. It has not been clear if attachment of LHCII to PSI in state 2 is important in state transitions. Here we show that in the absence of a specific PSI subunit, PSI-H, LHCII cannot transfer energy to PSI, and state transitions are impaired.

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.

Chlorina and viridis mutants of barley (Hordeum vulgare L.) allow assignment of long-wavelength chlorophyll forms to individual Lhca proteins of photosystem I in vivo.

The isolated subcomplex LHCI-730 of plant photosystem I (PSI) chlorophyll (Chl) alb binding antenna is a heterodimer of Lhca1 and Lhca4 and has a 77 K fluorescence emission peak at 730 nm (F730). Recently, three Chl spectral forms with 77 K fluorescence emission peaks at 720 nm, 730 nm and 742 nm were identified in native PSI. In an attempt to assign the two longest wavelength emission maxima to peripheral PSI antenna proteins, we performed 77 K fluorescence emission spectroscopy on intact leaves of chlorina and viridis mutants from barley which lack individual LHCI-730 proteins. This approach indicates that F732 is found only in Lhca1 and F742 only in Lhca4, when these proteins are associated with the PSI reaction centre.

Import of the barley PSI-F subunit into the thylakoid lumen of isolated chloroplasts.

A full-length cDNA clone encoding the PSI-F subunit of barley photosystem I has been isolated and sequenced. The open reading frame encodes a precursor polypeptide with a deduced molecular mass of 24837 Da. The barley PSI-F precursor contains a bipartite presequence with characteristics similar to the presequences of proteins destined to the thylakoid lumen. In vitro import studies demonstrate that an in vitro synthesized precursor is transported across the chloroplast envelope and directed to the thylakoid membrane, where it accumulates in a protease-resistant form. Incubation of the precursor with a chloroplast stromal extract results in processing to a form intermediate in size between the precursor and mature forms. Hydrophobicity analysis of the barley PSI-F protein reveals a hydrophobic region predicted to be a membrane spanning alpha-helix. The hydrophobic nature of PSI-F combined with a bipartite presequence is unusual. We postulate that the second domain in the bipartite presequence of the PSI-F precursor proteins is required to ensure the proper orientation of PSI-F in the thylakoid membrane. The expression of the PsaF gene is light-induced similar to other barley photosystem I genes.

Import of barley photosystem I subunit N into the thylakoid lumen is mediated by a bipartite presequence lacking an intermediate processing site. Role of the delta pH in translocation across the thylakoid membrane.

Translocation across the thylakoid membrane of the recently identified photosystem I polypeptide, PSI-N, has been analyzed in pea (Pisum sativum) and barley (Hordeum vulgare). PSI-N from barley is synthesized in the cytosol with a bipartite presequence similar in structural terms to those of other cytosolically synthesized proteins routed to the thylakoid lumen. In vitro reconstitution assays demonstrate that translocation into thylakoids is absolutely dependent on the transthylakoidal delta pH, but that nucleotide triphosphates are not required; the translocation mechanism is thus similar in these respects to those utilized by the 23- and 16-kDa proteins of the oxygen-evolving complex. The translocation of PSI-N is unique in that the presequence of PSI-N does not contain an intermediate cleavage site for the stromal processing peptidase; important experiments using intact chloroplasts depleted of a delta pH by nigericin treatment demonstrate the accumulation of the full precursor protein in the stroma. Translocation across the thylakoid membrane can take place in the absence of stromal factors, although the presence of stromal extracts leads to a consistent but slight enhancement of translocation efficiency. We also show that efficient translocation of the 33-kDa protein of the oxygen-evolving complex can take place in the complete absence of a delta pH, in apparent contradiction with earlier findings; the translocation of this protein is thus similar in several respects to that of plastocyanin. The data indicate the operation of two very different types of translocation mechanism, with PSI-N exhibiting additional separate characteristics.

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 alpha-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.

Identification of the photosystem I antenna polypeptides in barley. Isolation of three pigment-binding antenna complexes.

The light-harvesting antenna of barley photosystem I (LHCI) was isolated from native photosystem I (PSI) complexes and fractionated into three pigment-protein subcomplexes using two consecutive rounds of green gel electrophoresis. Each complex showed a characteristic polypeptide composition and low-temperature fluorescence emission spectrum; they were designated as LHCI-730, LHCI-680A and LHCI-680B. Their four apoproteins of 21, 22, 23 and 25 kDa were purified and NH2-terminal sequences were determined; in the case of the NH2-terminally blocked 25-kDa protein, an internal sequence was obtained after cleavage with endoproteinase Lys-C. This made possible an assignment of the four proteins to the four types (I-IV) of genes coding for chlorophyll a/b proteins of PSI (cab or lha genes). The LHCI-730 complex was isolated as a heterodimer composed of the 21-kDa (LHCI type IV) and the 22-kDa (LHCI type I) polypeptides. Each LHCI-680 complex had a single apoprotein. LHCI-680A consisted of the 25-kDa (LHCI type III) and LHCI-680B of the 23-kDa (LHCI type II) polypeptides. LHCI-680B was associated with the non-pigmented PSI-E subunit, indicating that this protein may function in the binding of this antenna to the reaction centre.

Expression and organisation of antenna proteins in the light-and temperature-sensitive barley mutant chlorina-(104.).

The nuclear gene mutant chlorina-(104) of barley (Hordeum vulgare L.) is chlorophyll-deficient when grown under high irradiance, particularly at low temperatures. Chlorina- (104) chloroplasts had fewer thylakoids than the wild type, and fewer appressed lamellae relative to non-appressed lamellae. The freeze-fracture ultrastructure showed a loss of particles from the protoplasmic fracture face of the stacked thylakoid region (PFs), consistent with the loss of most of the light-harvesting complex (LHC) II, and a loss of some of the large particles from the same face of the unstacked thylakoid region (PFu), indicating a loss of photosystem-I particles. The mutant is remarkable for the high density of particles on the exoplasmic fracture face of the unstacked thylakoid region (EFu), levels of which fell to normal after transfer to low light. The chlorophyll deficiency was shown to be primarily caused by the loss of LHCII and LHCI-680, with the consequent loss of much of the chlorophyll (Chl) b and the xanthophylls neoxanthin and lutein. The use of a monoclonal antibody which recognises the 23-kDa polypeptide of LHCI-680, confirmed that it was severely depleted in chloroplasts from chlorina-(104) grown under restrictive conditions. The 77 K fluorescence emission spectrum was characterised by a pronounced shoulder at 720 nm, arising from the photosystem-I reaction centre (CPI). Since fluorescence from CPI is normally quenched by LHCI-730, this indicates that LHCI-680 mediates excitation energy transfer between LHCI-730 and the reaction centre. After moving seedlings to permissive conditions, LHCII and LHCI-680 began to accumulate in the chlorotic leaves and the fluorescence emission spectrum resembled that of wild-type leaves. Measurement of the steady-state mRNA levels with specific Cab probes, showed no difference between wild type and mutant, indicating that control of LHCII and LHCI-680 accumulation was at a post-transcriptional level.