Mutations in CHLOROPLAST RNA BINDING provide evidence for the involvement of the chloroplast in the regulation of the circadian clock in Arabidopsis

The Arabidopsis circadian system regulates the expression of up to 36% of the nuclear genome, including many genes that encode photosynthetic proteins. The expression of nuclear-encoded photosynthesis genes is also regulated by signals from the chloroplasts, a process known as retrograde signaling. We have identified CHLOROPLAST RNA BINDING (CRB), a putative RNA-binding protein, and have shown that it is important for the proper functioning of the chloroplast. crb plants are smaller and paler than wild-type plants, and have altered chloroplast morphology and photosynthetic performance. Surprisingly, mutations in CRB also affect the circadian system, altering the expression of both oscillator and output genes. In order to determine whether the changes in circadian gene expression are specific to mutations in the CRB gene, or are more generally caused by the malfunctioning of the chloroplast, we also examined the circadian system in mutations affecting STN7, GUN1, and GUN5, unrelated nuclear-encoded chloroplast proteins known to be involved in retrograde signaling. Our results provide evidence that the functional state of the chloroplast may be an important factor that affects the circadian system.     

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

Summary We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.     

Sequencing and phylogenetic analysis of the chloroplast genome of Pseudosasa japonica f. Akebonosuji

Pseudosasa japonica f. Akebonosuji H. Okamura is a bamboo species with variable leaf colors, including albino, green, and green-white stripes. To determine whether variation in leaf color is due to mutations in the chloroplast genome, we sequenced the chloroplast genomes of green and albino leaves of P. japonica f. Akebonosuji. The results indicated that the chloroplast genome included 86 protein-coding genes, seven ribosomal RNA genes, and 31 tRNA genes. The similarity of chloroplast genomes for the two leaf types was 99.98%, with variation between genes encoding for trnfM and trnT. We observed that the relative expression patterns of trnfM and trnT were reversed in green and albino leaves. Whether the differential expression of trnfM and trnT is involved in leaf color variation among P. japonica f. Akebonosuji remains unclear.With many bamboo chloroplast genomes available, we aligned the chloroplast genomes of 28 bamboo species, including P. japonica f. Akebonosuji, to analyze polymorphisms. This comparison revealed that noncoding regions possessed more nucleotide polymorphisms than coding regions. Chloroplast genomes and the nuclear gene “granule-bound starch synthase I” (GBSSI) of 28 bamboo species were used to construct evolutionary trees. Both evolutionary trees indicated that P. japonica f. Akebonosuji was clustered into Subtrib. Arundinariinae.     

A Possible Role for Short Introns in the Acquisition of Stroma-Targeting Peptides in the Flagellate Euglena gracilis

The chloroplasts of Euglena gracilis bounded by three membranes arose via secondary endosymbiosis of a green alga in a heterotrophic euglenozoan host. Many genes were transferred from symbiont to the host nucleus. A subset of Euglena nuclear genes of predominately symbiont, but also host, or other origin have obtained complex presequences required for chloroplast targeting. This study has revealed the presence of short introns (41–93 bp) either in the second half of presequence-encoding regions or shortly downstream of them in nine nucleus-encoded E. gracilis genes for chloroplast proteins (Eno29, GapA, PetA, PetF, PetJ, PsaF, PsbM, PsbO, and PsbW). In addition, the E. gracilis Pbgd gene contains two introns in the second half of presequence-encoding region and one at the border of presequence-mature peptide-encoding region. Ten of 12 introns present within presequence-encoding regions or shortly downstream of them identified in this study have typical eukaryotic GT/AG borders, are T-rich, 45–50 bp long, and pairwise sequence identities range from 27 to 61%. Thus single recombination events might have been mediated via these cis-spliced introns. A double crossing over between these cis-spliced introns and trans-spliced introns present in 5′-UTRs of Euglena nuclear genes is also likely to have occurred. Thus introns and exon-shuffling could have had an important role in the acquisition of chloroplast targeting signals in E. gracilis. The results are consistent with a late origin of photosynthetic euglenids.     

Nuclear Suppressors of the Photosensitivity Associated with Defective Photosynthesis in Chlamydomonas reinhardii

Several nuclear mutations were recovered that suppress the photosensitivity associated with the Chamydomonas reinhardii chloroplast mutant rcl-u-1-10-6C, which is defective in ribulose-1,5-bisphosphate carboxylase/oxygenase. Two of the suppressor mutations affect other components of photosynthesis. These results show that suppressors of photosensitivity are sufficiently common to permit the recovery of photosensitive, photosynthesis-deficient mutants in bright light, and indicate that photosynthesis-deficient mutants selected and maintained in the light may accumulate suppressors which can confuse the biochemical analysis of lesions in photosynthesis. One of the suppressor mutations inhibits photosystem II activity, indicating that photosensitivity can be mediated by partial reactions of the photosynthetic electron transport chain.     

Nuclear mutations specifically affect the synthesis and/or degradation of the chloroplast-encoded D2 polypeptide of photosystem II in Chlamydomonas reinhardtii

To study the interaction of the nuclear and chloroplast genomes in the biogenesis of the photosynthetic apparatus, nuclear mutants of Chlamydomonas reinhardtii deficient in photosystem II (PSII) activity were analyzed. Two independently-isolated, allelic nuclear mutants show a pleiotropic reduction in a set of functionally related PSII polypeptides. Immunoblot analysis reveals that the two mutants, nac-1-18 and nac-1-11, accumulate reduced amounts of the chloroplast-encoded polypeptides P5 and P6 and are completely deficient in polypeptides D1 and D2. Polypeptides of the oxygen-evolving and light-harvesting complexes associated with PSII, however, are present at wild-type levels. Analysis of mRNAs encoding PSII polypeptides from these mutants indicates that all messages are present, although some species, including the D2 message, are significantly elevated. When mutant cells are pulse-labeled for 10 min with [¹⁴C]acetate, a greatly reduced amount of labeled D2 protein is observed, while all other PSII polypeptides are synthesized normally. These data indicate that the mutations present in nac-1-18 and nac-1-11 affect a nuclear gene whose product specifically controls the translation and/or degradation of the chloroplast-encoded D2 polypeptide.     

Evidence that the plastid signal and light operate via the samecis-acting elements in the promoters of nuclear genes for plastid proteins

Nuclear-encoded genes for proteins of the photosynthetic maschinery represent a particular subset of genes. Their expression is cooperatively stimulated by discrete factors including the developmental stage of plastids and light. We have analyzed in transgenic tobacco the plastid- and light-dependent expression of a series of 5′ promoter deletions of various nuclear genes from spinach, of fusions of defined promoter segments with the 90-bp 35S RNA CaMV minimal promoter, as well as with mutations in sequences with homologies to characterizedcis-elements, to address the question of whether the plastid signal and light operate via the same or differentcis-acting elements. In none of the 160 different transgenic lines (representing 32 promoter constructs from seven genes) analyzed, could significant differences be identified in the responses to the two regulatory pathways. The data are compatible with the idea that both signals control the expression of nuclear genes for plastid proteins via the samecis-acting elements.     

Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis

Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these dual functional proteins. We previously described the RNA binding activity of a 63 kDa chloroplast protein from Chlamydomonas reinhardtii, which has been implicated in expression of the psbA mRNA, encoding the D1 protein of photosystem II. Here, we identify this factor as dihydrolipoamide acetyltransferase (DLA2), a subunit of the chloroplast pyruvate dehydrogenase complex (cpPDC), which is known to provide acetyl-CoA for fatty acid synthesis. Analyses of RNAi lines revealed that DLA2 is involved in the synthesis of both D1 and acetyl-CoA. Gel filtration analyses demonstrated an RNP complex containing DLA2 and the chloroplast psbA mRNA specifically in cells metabolizing acetate. An intrinsic RNA binding activity of DLA2 was confirmed by in vitro RNA binding assays. Results of fluorescence microscopy and subcellular fractionation experiments support a role of DLA2 in acetate-dependent localization of the psbA mRNA to a translation zone within the chloroplast. Reciprocally, the activity of the cpPDC was specifically affected by binding of psbA mRNA. Beyond that, in silico analysis and in vitro RNA binding studies using recombinant proteins support the possibility that RNA binding is an ancient feature of dihydrolipoamide acetyltransferases. Our results suggest a regulatory function of DLA2 in response to growth on reduced carbon energy sources. This raises the intriguing possibility that this regulation functions to coordinate the synthesis of lipids and proteins for the biogenesis of photosynthetic membranes.     

A method for complementation analysis of nuclear and chloroplast mutants of photosynthesis in chlamydomonas

The photosynthetic properties of young zygotes of Chlamydomonas reinhardi were analyzed. In heterozygotes for two nuclear or two chloroplast mutations affecting photosynthesis, recovery of photosynthetic activity was observed that is most likely the result of intergenic complementation.——We observed that chloramphenicol inhibited the recovery of activity in double heterozygotes for mutants lacking at least one thylakoid polypeptide of chloroplast origin, while it had not effect on wild-type homozygotes. This indicates that the recovery of activity in double heterozygotes could result from the repair of existing thylakoid membranes by de novo synthesis of the missing polypeptides.