Identification of cis-acting RNA leader elements required for chloroplast psbD gene expression in Chlamydomonas.

The psbD mRNA of Chlamydomonas reinhardtii is one of the most abundant chloroplast transcripts and encodes the photosystem II reaction center polypeptide D2. This RNA exists in two forms with 5' untranslated regions of 74 and 47 nucleotides. The shorter form, which is associated with polysomes, is likely to result from processing of the larger RNA. Using site-directed mutagenesis and biolistic transformation, we have identified two major RNA stability determinants within the first 12 nucleotides at the 5' end and near position -30 relative to the AUG initiation codon of psbD. Insertion of a polyguanosine tract at position -60 did not appreciably interfere with translation of psbD mRNA. The same poly(G) insertion in the nac2-26 mutant, which is known to be deficient in psbD mRNA accumulation, stabilized the psbD RNA. However, the shorter psbD RNA did not accumulate, and the other psbD RNAs were not translated. Two other elements were found to affect translation but not RNA stability. The first comprises a highly U-rich sequence (positions -20 to -15), and the second, called PRB1 (positions -14 to -11), is complementary to the 3' end of the 16S rRNA. Changing the PRB1 sequence from GGAG to AAAG had no detectable effect on psbD mRNA translation. However, changing this sequence to CCUC led to a fourfold diminished rate of D2 synthesis and accumulation. When the psbD initiation codon was changed to AUA or AUU, D2 synthesis was no longer detected, and psbD RNA accumulated to wild-type levels. The singular organization of the psbD 5' untranslated region could play an important role in the control of initiation of psbD mRNA translation.

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.

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.

Molecular and biophysical analysis of herbicide-resistant mutants of Chlamydomonas reinhardtii: structure-function relationship of the photosystem II D1 polypeptide.

Plants and green algae can develop resistance to herbicides that block photosynthesis by competing with quinones in binding to the chloroplast photosystem II (PSII) D1 polypeptide. Because numerous herbicide-resistant mutants of Chlamydomonas reinhardtii with different patterns of resistance to such herbicides are readily isolated, this system provides a powerful tool for examining the interactions of herbicides and endogenous quinones with the photosynthetic membrane, and for studying the structure-function relationship of the D1 protein with respect to PSII electron transfer. Here we report the results of DNA sequence analysis of the D1 gene from four mutants not previously characterized at the molecular level, the correlation of changes in specific amino acid residues of the D1 protein with levels of resistance to the herbicides atrizine, diuron, and bromacil, and the kinetics of fluorescence decay for each mutant, which show that changes at two different amino acid residues dramatically slow PSII electron transfer. Our analyses, which identify a region of 57 amino acids of the D1 polypeptide involved in herbicide binding and which define a D1 binding niche for the second quinone acceptor, QB of PSII, provide a strong basis of support for structural and functional models of the PSII reaction center.

Analysis of the genes of the OEE1 and OEE3 proteins of the photosystem II complex from Chlamydomonas reinhardtii.

The sequences of the nuclear genes of the 33 kDa (OEE1) and the 16 kDa (OEE3) polypeptides of the oxygen evolving complex of Chlamydomonas reinhardtii have been established. Comparison between the OEE1 protein sequences of C. reinhardtii and higher plants and cyanobacteria reveals 67 and 47% homology. In contrast, C. reinhardtii and higher plants have only 28% overall homology for OEE3 which is mostly limited to the central portion of the protein. The transit peptides of the C. reinhardtii proteins consist of 52 (OEE1) and, most likely, 51 (OEE1) amino acids. They have a basic amino terminal region and, at least in the case of OEE1, a hydrophobic segment at their carboxy terminal end typical of thylakoid lumen proteins. Comparison of the genomic and cDNA clones indicates that the OEE1 and OEE3 genes contain five and four introns, respectively, some of which are located within the coding sequences of the transit peptides.

Expression of the nuclear gene encoding oxygen-evolving enhancer protein 2 is required for high levels of photosynthetic oxygen evolution in Chlamydomonas reinhardtii.

We have cloned a cDNA encoding a 20-kDa polypeptide, oxygen-evolving enhancer protein 2 (OEE2), in Chlamydomonas reinhardtii. This polypeptide has been implicated in photosynthetic oxygen evolution, and it is associated with the photosystem II complex, the site of oxygen evolution in all higher plants and algae. The sequence of OEE2 cDNA, the deduced amino acid sequence of the preprotein, the N-terminal protein sequence of mature OEE2 protein, and the coding regions of the single OEE2 gene are presented. The protein is synthesized with a 57-amino acid N-terminal transit peptide that directs the transport of this polypeptide across three cellular membranes. A nuclear mutant of C. reinhardtii, deficient in oxygen-evolving activity, is shown to be specifically missing OEE2 polypeptides. This mutation, which results in the complete absence of all OEE2 mRNA and protein, does not affect the accumulation of other photosystem II polypeptides or their mRNAs.

Sequence, evolution and differential expression of the two genes encoding variant small subunits of ribulose bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii.

We have sequenced the two genes for the small subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) in Chlamydomonas reinhardtii and analyzed their expression. The two genes encode variant small subunits that differ by four amino acid residues. Both genes are expressed and each is transcribed into an RNA of distinct size. The accumulation of the two RNAs changes depending on the growth conditions, so the small subunit composition of Rubisco may be expected to differ in response to the environment. The C. reinhardtii small subunit sequence is homologous to those of vascular plants or cyanobacteria, but is longer at the amino terminus and in internal positions. The number and location of the intervening sequences in the genes from C. reinhardtii and from other plants differ. In several cases, internal length differences in the polypeptide coincide with the positions of introns in the coding sequence. Thus, changes in the exon structure of the genes during evolution may have been accompanied by substantial changes in the encoded protein. The translation and splicing signals in C. reinhardtii are similar to those of other eukaryotes, but the transcription signals are less conserved and the highly biased codon usage is very unusual.

The chloroplast ribosomal intron of Chlamydomonas reinhardii codes for a polypeptide related to mitochondrial maturases.

The sequences of the 888bp chloroplast ribosomal intron and of the flanking 23S rRNA gene regions of Chlamydomonasreinhardii have been established. The intron can be folded with a secondary structure which is typical of group I introns of fungal mitochondrial genes. It contains a 489bp open reading frame encoding a potential polypeptide that is related to mitochondrial maturases.

Sequence homology between the 32K dalton and the D2 chloroplast membrane polypeptides of Chlamydomonas reinhardii.

The region of the chloroplast genome of Chlamydomonas reinhardii containing the gene of the thylakoid polypeptide D2 (psbD) has been sequenced. A unique open reading frame of 350 codons exists in this region. Because the first ATG is followed 11 codons downstream by a second one, the D2 polypeptide consists of either 339 or 350 amino acids. Comparison of the sequences of D2 and the 32K dalton polypeptides, both of which are associated with photosystem II, reveals partial homology. Although, the overall homology of these two polypeptides is only 27%, they contain several related regions and their hydropathic profiles are strikingly similar. These data suggest that the two polypeptides may have related functions and/or that their genes may have originated from a common ancestor. Alternatively, convergent evolution of these polypeptides may be due to structural constraints in the thylakoid membrane. Limited sequence homology is also observed between the D2 polypeptide and some of the subunits of the reaction centers of photosynthetic bacteria.