The chloroplast ycf7 (petL) open reading frame of Chlamydomonas reinhardtii encodes a small functionally important subunit of the cytochrome b6f complex.

The small chloroplast open reading frame ORF43 (ycf7) of the green unicellular alga Chlamydomonas reinhardtii is cotranscribed with the psaC gene and ORF58. While ORF58 has been found only in the chloroplast genome of C.reinhardtii, ycf7 has been conserved in land plants and its sequence suggests that its product is a hydrophobic protein with a single transmembrane alpha helix. We have disrupted ORF58 and ycf7 with the aadA expression cassette by particle-gun mediated chloroplast transformation. While the ORF58::aadA transformants are indistinguishable from wild type, photoautotrophic growth of the ycf7::aadA transformants is considerably impaired. In these mutant cells, the amount of cytochrome b6f complex is reduced to 25-50% of wild-type level in mid-exponential phase, and the rate of transmembrane electron transfer per b6f complex measured in vivo under saturating light is three to four times slower than in wild type. Under subsaturating light conditions, the rate of the electron transfer reactions within the b6f complex is reduced more strongly in the mutant than in the wild type by the proton electrochemical gradient. The ycf7 product (Ycf7) is absent in mutants deficient in cytochrome b6f complex and present in highly purified b6f complex from the wild-type strain. Ycf7-less complexes appear more fragile than wild-type complexes and selectively lose the Rieske iron-sulfur protein during purification. These observations indicate that Ycf7 is an authentic subunit of the cytochrome b6f complex, which is required for its stability, accumulation and optimal efficiency. We therefore propose to rename the ycf7 gene petL.     

Disruption of the plastid ycf10 open reading frame affects uptake of inorganic carbon in the chloroplast of Chlamydomonas.

The product of the chloroplast ycf10 gene has been localized in the inner chloroplast envelope membrane (Sasaki et al., 1993) and found to display sequence homology with the cyanobacterial CotA product which is altered in mutants defective in CO2 transport and proton extrusion (Katoh et al., 1996a,b). In Chlamydomonas reinhardtii, ycf10, located between the psbI and atpH genes, encodes a putative hydrophobic protein of 500 residues, which is considerably larger than its higher plant homologue because of a long insertion that separates the conserved N and C termini. Using biolistic transformation, we have disrupted ycf10 with the chloroplast aadA expression cassette and examined the phenotype of the homoplasmic transformants. These were found to grow both photoheterotrophically and photoautotrophically under low light, thereby revealing that the Ycf10 product is not essential for the photosynthetic reactions. However, under high light these transformants did not grow photoautotrophically and barely photoheterotrophically. The increased light sensitivity of the transformants appears to result from a limitation in photochemical energy utilization and/or dissipation which correlates with a greatly diminished photosynthetic response to exogenous (CO2 + HCO3-), especially under conditions where the chloroplast inorganic carbon transport system is not induced. Mass spectrometric measurements with either whole cells or isolated chloroplasts from the transformants revealed that the CO2 and HCO3- uptake systems have a reduced affinity for their substrates. The results suggest the existence of a ycf10-dependent system within the plastid envelope which promotes efficient inorganic carbon (Ci) uptake into chloroplasts.     

Abnormal regulation of photosynthetic electron transport in a chloroplast ycf9 inactivation mutant

The ycf9 (orf62) gene of the plastid genome encodes a 6.6-kDa protein (ORF62) of thylakoid membranes. To elucidate the role of the ORF62 protein, the coding region of the gene was disrupted with an aadA cassette, yielding mutant plants that were nearly (more than 95%) homoplasmic for ycf9 inactivation. The ycf9 mutant had no altered phenotype under standard growth conditions, but its growth rate was severely reduced under suboptimal irradiances. On the other hand, it was less susceptible to photodamage than the wild type. ycf9 inactivation resulted in a clear reduction in protein amounts of CP26, the NAD(P)H dehydrogenase complex, and the plastid terminal oxidase. Furthermore, depletion of ORF62 led to a faster flow of electrons to photosystem I without a change in the maximum electron transfer capacity of photosystem II. Despite the reduction of CP26 in the mutant thylakoids, no differences in PSII oxygen evolution rates were evident even at low light intensities. On the other hand, the ycf9 mutant presented deficiencies in the capacity for PSII-independent electron transport (ferredoxin-dependent cyclic electron transport and NAD(P)H dehydrogenase-mediated plastoquinone reduction). Altogether, it is shown that depletion of ORF62 leads to anomalies in the photosynthetic electron transfer chain and in the regulation of electron partitioning among the different routes of electron transport.     

Loss of Albino3 leads to the specific depletion of the light-harvesting system

The chloroplast Albino3 (Alb3) protein is a chloroplast homolog of the mitochondrial Oxa1p and YidC proteins of Escherichia coli, which are essential components for integrating membrane proteins. In vitro studies in vascular plants have revealed that Alb3 is required for the integration of the light-harvesting complex protein into the thylakoid membrane. Here, we show that the gene affected in the ac29 mutant of Chlamydomonas reinhardtii is Alb3.1. The availability of the ac29 mutant has allowed us to examine the function of Alb3.1 in vivo. The loss of Alb3.1 has two major effects. First, the amount of light-harvesting complex from photosystem II (LHCII) and photosystem I (LHCI) is reduced >10-fold, and total chlorophyll represents only 30% of wild-type levels. Second, the amount of photosystem II is diminished 2-fold in light-grown cells and nearly 10-fold in dark-grown cells. The accumulation of photosystem I, the cytochrome b(6)f complex, and ATP synthase is not affected in the ac29 mutant. Mild solubilization of thylakoid membranes reveals that Alb3 forms two distinct complexes, a lower molecular mass complex of a size similar to LHC and a high molecular mass complex. A homolog of Alb3.1, Alb3.2, is present in Chlamydomonas, with 37% sequence identity and 57% sequence similarity. Based on the phenotype of ac29, these two genes appear to have mostly nonredundant functions.     

Ycf12 is a core subunit in the photosystem II complex

The latest crystallographic model of the cyanobacterial photosystem II (PS II) core complex added one transmembrane low molecular weight (LMW) component to the previous model, suggesting the presence of an unknown transmembrane LMW component in PS II. We have investigated the polypeptide composition in highly purified intact PS II core complexes from Thermosynechococcus elongatus, the species which yielded the PS II crystallographic models described above, to identify the unknown component. Using an electrophoresis system specialized for separation of LMW hydrophobic proteins, a novel protein of approximately 5 kDa was identified as a PS II component. Its N-terminal amino acid sequence was identical to that of Ycf12. The corresponding gene is known as one of the ycf (hypothetical chloroplast reading frame) genes, ycf12, and is widely conserved in chloroplast and cyanobacterial genomes. Nonetheless, the localization and function of the gene product have never been assigned. Our finding shows, for the first time, that ycf12 is actually expressed as a component of the PS II complex in the cell, revealing that a previously unidentified transmembrane protein exists in the PS II core complex.     

The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes

The chloroplast genomes of most higher plants contain two giant open reading frames designated ycf1 and ycf2. In tobacco, ycf1 potentially specifies a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the functions of ycf1 and ycf2, we have constructed several mutant alleles for targeted disruption and/or deletion of these two reading frames. The mutant alleles were introduced into the tobacco plastid genome by biolistic chloroplast transformation to replace the corresponding wild-type alleles by homologous recombination. Chloroplast transformants were obtained for all constructs and tested for their homoplastomic state. We report here that all transformed lines remained heteroplastomic even after repeated cycles of regeneration under high selective pressure. A balanced selection was observed in the presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild-type versus transformed genome copies. Upon removal of the antibiotic and therewith release of the selective pressure, sorting out towards the wild-type plastid genome occurred in all transplastomic lines. These findings suggest that ycf1 and ycf2 are functional genes and encode products that are essential for cell survival. The two reading frames are thus the first higher plant chloroplast genes identified as being indispensable.     

The plastid genome-encoded Ycf4 protein functions as a nonessential assembly factor for photosystem I in higher plants

Photosystem biogenesis in the thylakoid membrane is a highly complicated process that requires the coordinated assembly of nucleus-encoded and chloroplast-encoded protein subunits as well as the insertion of hundreds of cofactors, such as chromophores (chlorophylls, carotenoids) and iron-sulfur clusters. The molecular details of the assembly process and the identity and functions of the auxiliary factors involved in it are only poorly understood. In this work, we have characterized the chloroplast genome-encoded ycf4 (for hypothetical chloroplast reading frame no. 4) gene, previously shown to encode a protein involved in photosystem I (PSI) biogenesis in the unicellular green alga Chlamydomonas reinhardtii. Using stable transformation of the chloroplast genome, we have generated ycf4 knockout plants in the higher plant tobacco (Nicotiana tabacum). Although these mutants are severely affected in their photosynthetic performance, they are capable of photoautotrophic growth, demonstrating that, different from Chlamydomonas, the ycf4 gene product is not essential for photosynthesis. We further show that ycf4 knockout plants are specifically deficient in PSI accumulation. Unaltered expression of plastid-encoded PSI genes and biochemical analyses suggest a posttranslational action of the Ycf4 protein in the PSI assembly process. With increasing leaf age, the contents of Ycf4 and Y3IP1, another auxiliary factor involved in PSI assembly, decrease strongly, whereas PSI contents remain constant, suggesting that PSI is highly stable and that its biogenesis is restricted to young leaves.     

Alternative mRNA processing increases the complexity of microRNA-based gene regulation in Arabidopsis

During trans-splicing of discontinuous organellar introns, independently transcribed coding sequences are joined together to generate a continuous mRNA. The chloroplast psaA gene from Chlamydomonas reinhardtii encoding the P(700) core protein of photosystem I (PSI) is split into three exons and two group IIB introns, which are both spliced in trans. Using forward genetics, we isolated a novel PSI mutant, raa4, with a defect in trans-splicing of the first intron. Complementation analysis identified the affected gene encoding the 112.4 kDa Raa4 protein, which shares no strong sequence identity with other known proteins. The chloroplast localization of the protein was confirmed by confocal fluorescence microscopy, using a GFP-tagged Raa4 fusion protein. RNA-binding studies showed that Raa4 binds specifically to domains D2 and D3, but not to other conserved domains of the tripartite group II intron. Raa4 may play a role in stabilizing folding intermediates or functionally active structures of the split intron RNA.     

Cloning and characterization of the nuclear AC115 gene of Chlamydomonas reinhardtii

The nuclear ac115 mutant of Chlamydomonas reinhardtii is specifically blocked in the synthesis of the chloroplast encoded D2 protein of the photosystem II reaction center at a point after translation initiation. Here, we report the identification of the AC115 gene through complementation rescue of the ac115 mutant strain, using an indexed cosmid library of Chlamydomonas genomic DNA. AC115 is a small, novel, intronless nuclear gene which encodes a protein of 113 amino acids. The amino terminal end of the Ac115 protein is rich in basic amino acids and has features which resemble a chloroplast transit sequence. A hydrophobic stretch of amino acids at the protein's carboxyl terminus is sufficiently large to be a membrane spanning or a protein/protein interaction domain. Various models are discussed to account for the mechanism by which Ac115p works in D2 synthesis. The ac115 mutant allele was sequenced and determined to be an A-to-T transversion at the first position of the fourth codon of the coding sequence. This mutation changes an AAG codon to a TAG nonsense codon and results in a null phenotype.     

A nuclear-encoded function essential for translation of the chloroplast psaB mRNA in chlamydomonas.

We report the analysis of a photosystem I-deficient mutant of Chlamydomonas reinhardtii, F15, that contains a mutation at the TAB1 (for translation of psaB mRNA) nuclear locus. Pulse labeling of chloroplast proteins revealed that the synthesis of the two photosystem I reaction center polypeptides PSAA and PSAB was undetectable in this mutant. The mRNA levels of these proteins were only moderately reduced, suggesting that the primary defect occurs at a step during or after translation. We constructed chimeric genes consisting of the psaA and psaB 5' untranslated region (5' UTR) fused to the aminoglycoside adenyltransferase (aadA) coding sequence, which confers spectinomycin resistance. Insertion of these genes into the chloroplast genome through biolistic transformation and analysis of their expression in the TAB1 mutant nuclear background revealed that the psaB (but not the psaA) 5' UTR is the target of the wild-type TAB1 function. This suggests that TAB1 is required for the initiation of psaB mRNA translation. The dependence of PSAA synthesis or accumulation on PSAB synthesis is strongly suggested by the identification of a suppressor mutation within the psaB 5' UTR. The suppressor specifically restores the synthesis of both proteins in the presence of the tab1-F15 mutation. The location of the suppressor mutation within a putative base-paired region near the psaB initiation codon suggests a role for TAB1 in the activation of translation of the psaB mRNA.     

Xanthophyll cycle mutants from Chlamydomonas reinhardtii indicate a role for zeaxanthin in the D1 protein turnover

Photosynthetic activity, pigment conversion and D1 protein degradation under high light stress has been investigated in a wild type strain and two xanthophyll cycle mutants (npq1 and npq2) of Chlamydomonas reinhardtii. Wild type cells exhibited the well-known inactivation of photosystem II in high light, which was accompanied by the loss of β-carotene and a concomitant increase of zeaxanthin. Complete degradation of D1 protein was found after 2 h of illumination in the presence of chloramphenicol, an inhibitor of chloroplast protein synthesis. The npq1 mutant, which is unable to convert violaxanthin to zeaxanthin, showed a very similar behaviour. For the npq2 mutant, however, which is unable to form violaxanthin from zeaxanthin and thus contains high amounts of zeaxanthin even in low light, photosystem II inactivation was less pronounced. This was paralleled by a much slower D1 protein degradation in chloramphenicol treated cells. Our results support a protective role for zeaxanthin against high light-induced photosystem II inactivation resulting in a slowed-down D1 protein turnover.     

The chloroplast gene ycf9 encodes a photosystem II (PSII) core subunit, PsbZ, that participates in PSII supramolecular architecture

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.     

Nuclear and chloroplast mutations affect the synthesis or stability of the chloroplast psbC gene product in Chlamydomonas reinhardtii.

The psbC gene of Chlamydomonas reinhardtii encodes P6, the 43 kd photosystem II core polypeptide. The sequence of P6 is highly homologous to the corresponding protein in higher plants with the exception of the N-terminal region where the first 12 amino acids are missing. Translation of P6 is initiated at GUG in C. reinhardtii. The chloroplast mutant MA16 produces a highly unstable P6 protein. The mutation in this strain maps near the middle of the psbC gene and consists of a 6 bp duplication that creates a Ser-Leu repeat at the end of one transmembrane domain. Two nuclear mutants, F34 and F64, and one chloroplast mutant, FuD34, are unable to synthesize P6. All of these mutants accumulate wild-type levels of psbC mRNA. The FuD34 mutation has been localized near the middle of the 550 bp 5' untranslated region of psbC where the RNA can be folded into a stem-loop structure. A chloroplast suppressor of F34 has been isolated that partially restores synthesis of the 43 kd protein. The mutation of this suppressor is near that of FuD34, in the same stem-loop region. These chloroplast mutations appear to define the target site of a nuclear factor that is involved in P6 translation.     

The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii

The sites of synthesis of the major thylakoid membrane polypeptides have been studied in the green alga Chlamydomonas reinhardtii by pulse labeling of cells with [14C]acetate in the presence of inhibitors specific for chloroplast and cytoplasmic protein synthesis. The labeled membrane polypeptides were separated by an improved method of sodium dodecyl sulfate (SDS) gradient gel electrophoresis, and autoradiographs were made of the dried gels. The results demonstrate that of the 33 polypeptides resolved in the gels, at least nine are made on chloroplast ribosomes. Two of these (polypeptides 2 and 6) are associated with the reaction centers of photosystems I and II. Another polypeptide (polypeptide 5) appears from genetic data to be coded by chloroplast DNA. Experiments with a mutant whose chloroplast ribosomes are resistant to spectinomycyn (spr-u-1-6-2) show that polypeptides whose synthesis takes place on chloroplast ribosomes are made in the presence of spectinomycin in the mutant although their synthesis is blocked by this antibiotic in wild type cells.     

Ycf12 is a core subunit in the photosystem II complex

The latest crystallographic model of the cyanobacterial photosystem II (PS II) core complex added one transmembrane low molecular weight (LMW) component to the previous model, suggesting the presence of an unknown transmembrane LMW component in PS II. We have investigated the polypeptide composition in highly purified intact PS II core complexes from Thermosynechococcus elongatus, the species which yielded the PS II crystallographic models described above, to identify the unknown component. Using an electrophoresis system specialized for separation of LMW hydrophobic proteins, a novel protein of ∼ 5 kDa was identified as a PS II component. Its N-terminal amino acid sequence was identical to that of Ycf12. The corresponding gene is known as one of the ycf (hypothetical chloroplast reading frame) genes, ycf12, and is widely conserved in chloroplast and cyanobacterial genomes. Nonetheless, the localization and function of the gene product have never been assigned. Our finding shows, for the first time, that ycf12 is actually expressed as a component of the PS II complex in the cell, revealing that a previously unidentified transmembrane protein exists in the PS II core complex.     

A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii.

The light-independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild-type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light-independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.