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Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 degrees C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing "activity gels". Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being "self-digested", also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 degrees C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg(2+), and inhibited by Zn(2+), Cd(2+), EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing "activity gels" or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.

The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochromeb 6 /f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the ΔpH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH2-terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of ΔpH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways.

The interaction between plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant plastocyanin to elucidate the site of electron transfer and the docking regions of the molecule. Mutation of Tyr-83 to Arg or Leu provides no evidence for a second electron transfer path via Tyr-83 of plastocyanin, which has been proposed to be the site of electron transfer from cytochrome f. The data found with mutations of acidic residues indicate that both conserved negative patches are essential for the binding of plastocyanin to the intact cytochrome bf complex. Replacing Ala-90 and Gly-10 at the flat hydrophobic surface of plastocyanin by larger residues slowed down and accelerated, respectively, the rate of electron transfer as compared with wild-type plastocyanin. These opposing effects reveal that the hydrophobic region around the electron transfer site at His-87 is divided up into two regions, of which only that with Ala-90 contributes to the attachment to the cytochrome bf complex. These binding sites of plastocyanin are substantially different from those interacting with photosystem I. It appears that each of the two binding regions of plastocyanin is split into halves, which are used in different combinations in the molecular recognition at the two membrane complexes.

The observation that chloroplasts and mitochondria have retained relics of eubacterial genomes and a protein-synthesizing machinery has long puzzled biologists. If most genes have been transferred from organelles to the nucleus during evolution, why not all? What selective pressure maintains genomes in organelles? Electron transport through the photosynthetic and respiratory membranes is a powerful – but dangerous – source of energy. Recent evidence suggests that organelle genomes have persisted because structural proteins that maintain redox balance within bioenergetic membranes must be synthesized when and where they are needed, to counteract the potentially deadly side effects of ATP-generating electron transport.

The isolated OEC23 (oxygen evolving complex) protein of Photosystem II can be affinity-labeled by ATP and GTP in the presence of UV light. Labeling is competed by ATP, AMP, and NAD as well as by the ATP and ATP-γ-S and 5′-p-Fluorosulfonylbenzoyl-adenosine (FSBA) and is inhibited by Dithiothreitol (DTT). The physiological significance of this finding and the possible implication of ATP and GTP binding in thylakoid membrane dynamics remains to be further investigated.

The D1 reaction center protein of the Photosystem II complex in green plants is synthesized with a short carboxyl-terminal extension. Proteolytic cleavage and removal of this extension peptide in the thylakoid lumen are necessary for the assembly of a manganese cluster that is essential for the oxygen evolution activity of Photosystem II. We have isolated cDNAs encoding CtpA, the carboxyl-terminal processing protease for the D1 protein, from two higher plants, spinach and barley. In each of these organisms, CtpA is encoded by a single copy nuclear gene, and its steady-state mRNA levels are light-regulated. The CtpA protein is detectable in etiolated material, and its level increases approximately 5-fold upon illumination. Moreover, the CtpA gene is expressed in shoot tissues and not in roots. In its precursor form, the CtpA protein harbors a bipartite transit sequence characteristic for thylakoid lumenal proteins. Cell fractionation studies demonstrated that CtpA is associated with thylakoid membranes and is resistant to treatments with thermolysin, consistent with its localization in the lumen of thylakoids. Comparisons of the sequence of the higher plant CtpA enzyme with those of other related carboxyl-terminal processing proteases suggest that these proteins constitute a new family of proteases.

The nuclear-encoded proteins of the oxygen-evolving complex (OEC) of photosystem II are bound on the lumenal side of the thylakoid membrane and stabilize the manganese ion cluster forming the photosystem II electron donor side. The OEC proteins are released from their binding site(s) following light-induced degradation of reaction center II (RCII)-D1 protein in Chlamydomonas reinhardtii. The kinetics of OEC proteins release correlates with that of RCII-D1 protein degradation. Only a limited amount of RCII-D2 protein is degraded during the process, and no loss of the core proteins CP43 and CP47 is detected. The release of the OEC proteins is prevented when the photoinactivated RCII-D1 protein degradation is retarded by addition of 3-(3,5-dichlorophenyl)-1,1-dimethylurea or by a high PQH2/PQ ratio prevailing in membranes of the plastocyanin-deficient mutant Ac208. The released proteins are not degraded but persist in the thylakoid lumen for up to 8 h and reassociate with photosystem II when new D1 protein is synthesized in cells exposed to low light, thus allowing recovery of photosystem II function. Reassociation also occurs following D1 protein synthesis in darkness when RCII activity is only partially recovered. These results indicate that (i) the D1 protein participates in the formation of the lumenal OEC proteins binding site(s) and (ii) the photoinactivation of RCII-D1 protein does not alter the conformation of the donor side of photosystem II required for the binding of the OEC proteins.

A Sec-type system is responsible for the translocation of a subset of proteins across the thylakoid membrane in higher plant chloroplasts. Previous studies have suggested that the thylakoidal ΔpH plays a minor role in this translocation mechanism, but we show here that it can be essential for the translocation process, depending on the identity of the passenger protein and the concentration of ATP. Studies using chimeric proteins show that, whereas the presequence dictates the translocation pathway, the ΔpH requirement is dictated exclusively by the passenger protein; some passenger proteins are virtually ΔpH-independent whereas others are absolutely dependent. ΔpH requirement is not related to charge characteristics of the passenger proteins, ruling out an electrophoretic effect. Analysis of the 33-kDa photosystem II protein reveals an inverse relationship between ΔpH requirement and ATP concentration; import into isolated thylakoids is inhibited 14-fold by nigericin at moderate ATP concentrations, and totally inhibited when the ATP concentration is reduced to 2 μM. The results indicate that the roles of the ΔpH and ATP overlap and suggest that the ΔpH may be obligatory when the passenger protein is abnormally difficult to translocate, possibly due to the folding of the polypeptide chain. We compare the energetics of this system with those of prokaryotic systems from which the chloroplast system is believed to have evolved.

Thylakoid membranes of chloroplasts in higher plants harbor different pathways for the translocation of proteins. One of these routes is related to the prokaryotic Sec pathway, which mediates the secretion of particular proteins into the periplasmic space and involves the SecA protein as an essential component. We have isolated a full size cDNA of 3739 nucleotides encoding the SecA homologue from spinach. It contains an open reading frame of 1036 codons corresponding to a polypeptide with a calculated mass of 117 kDa. The deduced amino acid sequence shows between 43 and 49% identity to SecA proteins from bacteria and lower algae and 62% identity to SecA of the cyanobacterium Synechococcus sp. PCC7942. Compared with the Escherichia coli protein, spinach SecA carries an amino-terminal extension of approximately 80 residues. In organello experiments performed with the protein made in vitro by transcription of the cDNA and cell-free translation of the resulting RNA showed that this extension comprises a transit peptide that mediates the import of the protein into the chloroplast. The processed product of approximately 107 kDa accumulates predominantly in the stroma and to a lower extent associates with the thylakoid membrane. Comparably to E. coli, in which SecA activity can be inhibited by sodium azide, thylakoid translocation of a subset of lumenal proteins is sensitive to sodium azide in pea but not in spinach chloroplasts, suggesting that the latter contain an azide-resistant SecA variant.

Subunit 3 of photosystem I (PSI-3), the product of the nuclear psaF gene, is the docking protein for plastocyanin during photosynthetic electron transport in thylakoid membranes and is synthesized in the cytosol with a transit peptide that resembles structurally the bipartite targeting signals of hydrophilic, lumenal components such as plastocyanin. In organello import experiments performed with the authentic PSI-3 precursor and chimeric polypeptides consisting of residue-correct fusions of transit peptides and mature proteins derived from different plastid proteins demonstrate that the PSI-3 transit peptide is indeed capable of translocating proteins into the thylakoid lumen and that, conversely, mature PSI-3 depends on a bipartite transit peptide for its thylakoid transfer. Of the three recently described translocation/integration pathways for nucleus-encoded proteins carrying bipartite transit peptides that are distinct in their physiological requirements and strictly protein-specific, PSI-3, like plastocyanin and the 33-kDa protein of the oxygen-evolving complex, is translocated by a pathway that involves stromal factors but no proton gradient across the membrane. It is not affected by saturating amounts of the precursor for the 23-kDa protein of the oxygen-evolving complex that follows the latter route. Thylakoid translocation of PSI-3 is, however, impaired in the presence of sodium azide, which indicates that a homolog to the bacterial SecA protein might be involved in this process suggesting, thus, a prokaryote-like translocation pathway. The azide-sensitive factor appears to interact predominantly with the transit peptide of a precursor protein, since chimeras consisting of a presequence from an azide-resistant precursor and a mature part of an azide-sensitive polypeptide are still translocated in the presence of the inhibitor.

Plastocyanin and the 16-, 23-, and 33-kDa polypeptides of the oxygen-evolving complex associated with photosystem II are hydrophilic, nuclear encoded components of the photosynthetic machinery that are all located in the lumen of thylakoid membranes. All four proteins are therefore imported into chloroplasts and, in addition, translocated across the thylakoid membrane. They share functionally equivalent, bipartite transit peptides, which are removed in two steps during or after import into the organelle and translocation across the thylakoid membrane, respectively. The transit peptides lack any homology at the sequence level but possess remarkably similar predicted secondary structures. We have studied the targeting potential of the authentic precursor molecules and all possible chimeric combinations generated by a specific, commonly applicable cassette system, which facilitates codon-correct reciprocal exchanges of transit peptides and mature parts. An unexpected specificity of import and sorting processes was found. All constructs can be imported into the organelle, though with greatly differing efficiency. On the other hand, the lumen-targeting parts are essential but not in all cases sufficient for correct intraorganellar routing. This implies that translocation across the thylakoid membrane appears not to depend merely on simple interactions of charged or hydrophobic regions between protein and membrane but requires an additional quality of information that includes the functional co-evolution of a transit peptide with its mature protein. Signaling and sorting appear to be essential at almost every step of the entire process for proper traffic regulation since distinct steps can be impaired (rate-limiting or arrested) in the individual combinations: the transfer across the envelope membranes (e.g. 16/PC) and the interaction with (e.g. 16/33) or the translocation across the thylakoid membrane (e.g. 33/23).

We have isolated the genes encoding 11 photosystem I (PSI) subunits from Synechococcus sp., from which this reaction center has been crystallized. The recombinant DNAs, including psaA, psaB, psaC, psaD, psaE, psaF, psal, psaJ, psaK and psaL , were obtained by heterologous hybridization with probes from appropriate cDNAs or genes from spinach and Synechocystis sp. PCC 6803, or with synthetic oligodeoxyribonucleotides. Genes psaA/psaB, psaF/psaJ and psaL/psal are each closely linked. The open reading frames predict polypeptides of 83 kDa (subunits la and Ib, encoded by genes psaA and psaB , respectively), 15.4 kDa (II, psaD ), 17.7 kDa (III, psaF ), 8.4 kDa (IV, psaE ), 8.8 kDa (VII, psaC ), 4.6 kDa (VIII, psal ), 4.8 kDa (IX, psaJ ), 8.5 kDa (X, psaK ) and 15.5 kDa (XI, psaL ). A novel subunit (XII, psaM ) was also identified. Subunits II, III, IV and VII seem to be peripheral, while the others seem to be intrinsic components of the reaction center. These data imply a striking similarity of cyanobacterial and eukaryotic PSI. All subunits studied are encoded by single-copy genes which seem to be transcribed into monocistronic ( psaC, psaD, psaC, psaK ) or dicistronic ( psaA/psaB, psaF/psaJ, psaL/psal ) RNA species. Subunit III is translated as a 17.7-kDa precursor, including a transit peptide of 23 amino acid residues. This is consistent with its location in the thylakoid lumen.

The targeting of cytosolically synthesized proteins into the thylakoid lumen is mediated by an aminoterminal pre-sequence consisting of an "envelope transit" and a "thylakoid transfer" signal in tandem. We have investigated the structural characteristics of several thylakoid transfer signals by determining the intermediate sites at which the stromal processing peptidase cleaves to remove the transit sequences. Using this approach we have found that the thylakoid transfer signals of Silene pratensis plastocyanin, 23-kDa oxygen-evolving complex protein from wheat, and 33-kDa oxygen-evolving complex protein from wheat, are 25, 39, and 48 residues in length, respectively. All of the transfer signals contain hydrophobic core sequences and a "-3,-1" motif reminiscent of those found in signal sequences, but the amino-terminal regions of the transfer signals of the 23- and 33-kDa proteins are both longer and more highly charged. The net charge of each amino-terminal region of the transfer sequences is +1, including the amino-terminal amino group. In each case, the stromal processing peptidase cleaves immediately after a positively charged residue, but otherwise the cleavage sites exhibit no common elements of either primary or secondary structure.

We have isolated recombinant lambda gt11 phages which carry cDNA clones for the major light-harvesting chlorophyll a/b -binding proteins of photosystem I (LHCPI) and II (LHCPII), subunit II of photosystem I, a chlorophyll a/b -binding protein of photosystem II (CP24), the Rieske iron—sulphur protein of the cytochrome b 6 / f complex, and the 33, 23 and 16 kDa proteins of the water-oxidizing complex of photosystem II from Nicotiana tabacum . The nucleotide sequences of cDNA clones encoding the precursors for LHCPI and the FeS protein are presented. If tobacco or tomato seedlings, or seedlings of a phytochrome-deficient aurea mutant of tomato which lacks more than 95% of the phytochrome of the isogenic wild type, are kept in blue light, the transcript level of each of these genes is higher than in seedlings grown in red light suggesting the involvement of a blue—UVA-light photoreceptor. In the case of LHCPI, a 1 min blue-light pulse applied to red-light-grown seedlings is sufficient to increase the transcript levels to those present in blue-light-grown seedlings, whereas almost no increase is observed for transcripts encoding the FeS and 33 kDa proteins. If dark-grown tomato seedlings receive a single far-red-light pulse, significant stimulation is detected for LHCPI transcripts, whereas transcripts encoding the FeS and 33 kDa proteins are not stimulated. It is concluded that the lower light requirement for the increase in the LHCPI transcript level is not specific for one of the light-dependent signal transduction chains.

Nucleotide sequence data for thylakoid membrane proteins, and calculation of the distribution of hydrophobic residues from these sequences, allows prediction of hydrophobic membrane-spanning helices and the position of attached redox groups. This provides a new perspective for considering the structure and function of the proteins. This review discusses the conclusions reached so far for several plant photosynthetic electron transport proteins. In cytochrome b 6 the arrangement of two hemes across the hydrophobic core of the membrane may illustrate a general structural principle for a transmembrane redox reaction.

By screening lambda gt11 cDNA expression libraries prepared from poly(A+) RNA of illuminated (4 and 16 h) spinach seedlings (Spinacia oleracea var. Monatol), we have recovered phage containing inserts corresponding to the complete "10-kDa" precursor polypeptide associated with the water oxidizing photosystem II. The amino acid sequence deduced from the nucleotide sequence indicates a polypeptide of 140 amino acid residues (15.0 kDa). The mature protein is predicted to consist of 99 amino acid residues (10.8 kDa). The identity of the recombinant DNAs was established serologically and by determining an N-terminal sequence of 25 amino acid residues from the purified authentic protein. This sequence was found in the cDNA-derived amino acid sequence. In addition, a monospecific antiserum detected the appropriate polypeptides when copy-RNA was translated into protein which was imported into isolated unbroken chloroplasts. The transit sequence (41 amino acid residues; 4.2 kDa) is markedly dissimilar to those of the three extrinsic, luminal photosystem II proteins. Secondary structure prediction suggests a transmembrane arrangement of the 10-kDa polypeptide in the thylakoid membrane, with the N terminus in the thylakoid lumen and a 17-amino acid C-terminal sequence at the outside partitions. This is consistent with biochemical and functional data for the protein. A major transcript of 1.0 kilobases has been observed in polyadenylated RNA.

Spinach chloroplast 4S RNAs has been separated by two-dimensional polyacrylamide gel electrophoresis into about 35 species. After extraction from the gel, 27 of these RNA species were identified by aminoacylation as tRNAs specific for 16 amino acids. Individual tRNAs were labeled in vitro with 125I and hybridized to DNA fragments obtained by digestion of spinach chloroplast DNA with KpnI, PstI, SalI and XmaI restriction endonucleases. A minimum of 21 genes corresponding to tRNAs for 14 different amino acids have been localized on the restriction endonuclease cleavage site map of the DNA molecule. Of these, 15 genes corresponding to tRNAs for 12 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. Each copy of this repeat region contains a set of genes for the ribosomal RNAs and a gene for tRNA2Ile in the "spacer" sequence between the 16S and 23S ribosomal RNAs. The genes for tRNA1Ile, tRNA2Leu and tRNA3Leu also map in the repeat region, but outside the ribosomal DNA unit. At present, two more chloroplast tRNAs (for Pro and Lys) have been identified, but not mapped, while 4 unidentified 4S RNAs have been mapped in the large single-copy region of the DNA molecule. Evidence is presented that isoaccepting tRNA species can be transcripts from different loci.

The Oenothera plastome mutant Iα is deficient in ribulosebisphosphate carboxylase / oxygenase activity. Transcription-translation of the plastid DNA or translation of the plastid RNA of the mutant both give rise to a large subunit of this enzyme which has an M r of about 30 000, whereas that from the wild-type is 52 000. The mutant possesses mRNA of the same size as that of the wild-type, indicating that the lesion is a mutation within the coding region of the gene which causes premature termination of translation. The truncated polypeptide is expressed in vivo. The small subunit of the carboxylase is also expressed in vivo in the mutant but the free pool of processed protein is low. The data presented suggest that control mechanisms after transcription are involved in the biogenesis of ribulosebisphosphate carboxylase / oxygenase.

The 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized as precursors in the cytoplasm and transported into the thylakoid lumen of higher plant chloroplasts. In this report we have analyzed the import and maturation of these precursors, using reconstituted protein import assays and partially purified preparations of the processing peptidases involved. Precursors of the 33- and 23-kDa proteins from Spinacia and Triticum aestivum are processed by a stromal peptidase to intermediate forms; polypeptides of similar size are observed during the transport of these precursors and possibly that of the 16-kDa protein, into isolated chloroplasts. Complete maturation of the 33- and 23-kDa proteins is carried out by a thylakoidal peptidase shown previously to be involved in plastocyanin biogenesis. The data support an import mechanism involving successive cleavages by the stromal and thylakoidal processing peptidases.