Real-time monitoring of chloroplast gene expression by a luciferase reporter: evidence for nuclear regulation of chloroplast circadian period

Chloroplast-encoded genes, like nucleus-encoded genes, exhibit circadian expression. How the circadian clock exerts its control over chloroplast gene expression, however, is poorly understood. To facilitate the study of chloroplast circadian gene expression, we developed a codon-optimized firefly luciferase gene for the chloroplast of Chlamydomonas reinhardtii as a real-time bioluminescence reporter and introduced it into the chloroplast genome. The bioluminescence of the reporter strain correlated well with the circadian expression pattern of the introduced gene and satisfied all three criteria for circadian rhythms. Moreover, the period of the rhythm was lengthened in per mutants, which are phototactic rhythm mutants carrying a long-period gene in their nuclear genome. These results demonstrate that chloroplast gene expression rhythm is a bona fide circadian rhythm and that the nucleus-encoded circadian oscillator determines the period length of the chloroplast rhythm. Our reporter strains can serve as a powerful tool not only for analysis of the circadian regulation mechanisms of chloroplast gene expression but also for a genetic approach to the molecular oscillator of the algal circadian clock.     

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.     

A novel pineal-specific product of the oligopeptide transporter PepT1 gene: circadian expression mediated by cAMP activation of an intronic promoter

The oligopeptide transporter 1, PepT1, is a member of the Slc15 family of 12 membrane-spanning domain transporters; PepT1 has proton/peptide cotransport activity and is selectively expressed in intestinal epithelial cells, where it is responsible for the nutritional absorption of di- and tri-peptides. Here, a novel PepT1 gene product has been identified in the rat pineal gland, termed pgPepT1. It encodes a 150-amino acid protein encompassing the C-terminal 3 membrane-spanning domains of intestinal PepT1 protein, with 3 additional N-terminal residues. Expression of pgPepT1 appears to be restricted to the pineal gland and follows a marked circadian pattern with >100-fold higher levels of mRNA occurring at night; this is accompanied by an accumulation of membrane-associated pgPepT1 protein ( approximately 16 kDa). The daily rhythm in pgPepT1 mRNA is regulated by the well described neural pathway that controls pineal melatonin production. This includes the retina, the circadian clock in the suprachiasmatic nucleus, central structures, and projections from the superior cervical ganglia; activation of this pathway results in the release of norepinephrine. Here it was found that pgPepT1 expression is mediated by a norepinephrine-->cyclic AMP mechanism that activates an alternative promoter located in intron 20 of the gene. pgPepT1 protein was found to have transporter-modulator activity; it could contribute to circadian changes in pineal function through this mechanism.     

Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis

We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function.     

A novel circadianly expressed Drosophila melanogaster gene dependent on the period gene for its rhythmic expression.

The Drosophila melanogaster period (per) gene is required for expression of endogenous circadian rhythms of locomotion and eclosion. per mRNA is expressed with a circadian rhythm that is dependent on Per protein; this feedback loop has been proposed to be essential to the central circadian pacemaker. This model would suggest the Per protein also controls the circadian expression of other genetic loci to generate circadian behavior and physiology. In this paper we describe Dreg-5, a gene whose mRNA is expressed in fly heads with a circadian rhythm nearly identical to that of the per gene. Dreg-5 mRNA continues to cycle in phase with that of per mRNA in conditions of total darkness and also when the daily feeding time is altered. Like per mRNA, Dreg-5 mRNA is not expressed rhythmically in per null mutant flies. Dreg-5 encodes a novel 298 residue protein and Dreg-5 protein isoforms also oscillate in abundance with a circadian rhythm. The phase of Dreg-5 protein oscillation, however, is different from that of Per protein expression, suggesting that Dreg-5 and per have common translational but different post-translational control mechanisms. These results demonstrate that the per gene is capable of modulating the rhythmic expression of other genes; this activity may form the basis of the output of circadian rhythmicity in Drosophila.     

Chloroplast unusual positioning1 is essential for proper chloroplast positioning

The intracellular distribution of organelles is a crucial aspect of effective cell function. Chloroplasts change their intracellular positions to optimize photosynthetic activity in response to ambient light conditions. Through screening of mutants of Arabidopsis defective in chloroplast photorelocation movement, we isolated six mutant clones in which chloroplasts gathered at the bottom of the cells and did not distribute throughout cells. These mutants, termed chloroplast unusual positioning (chup), were shown to belong to a single genetic locus by complementation tests. Observation of the positioning of other organelles, such as mitochondria, peroxisomes, and nuclei, revealed that chloroplast positioning and movement are impaired specifically in this mutant, although peroxisomes are distributed along with chloroplasts. The CHUP1 gene encodes a novel protein containing multiple domains, including a coiled-coil domain, an actin binding domain, a Pro-rich region, and two Leu zipper domains. The N-terminal hydrophobic segment of CHUP1 was expressed transiently in leaf cells of Arabidopsis as a fusion protein with the green fluorescent protein. The fusion protein was targeted to envelope membranes of chloroplasts in mesophyll cells, suggesting that CHUP1 may localize in chloroplasts. A glutathione S-transferase fusion protein containing the actin binding domain of CHUP1 was found to bind F-actin in vitro. CHUP1 is a unique gene identified that encodes a protein required for organellar positioning and movement in plant cells.     

A novel mutation in kaiC affects resetting of the cyanobacterial circadian clock

Light is the most important factor controlling circadian systems in response to day-night cycles. In order to better understand the regulation of circadian rhythms by light in Synechococcus elongatus PCC 7942, we screened for mutants with defective phase shifting in response to dark pulses. Using a 5-h dark-pulse protocol, we identified a mutation in kaiC that we termed pr1, for phase response 1. In the pr1 mutant, a 5-h dark pulse failed to shift the phase of the circadian rhythm, while the same pulse caused a 10-h phase shift in wild-type cells. The rhythm in accumulation of KaiC was abolished in the pr1 mutant, and the rhythmicity of KaiC phosphorylation was reduced. Additionally, the pr1 mutant was defective in mediating the feedback inhibition of kaiBC. Finally, overexpression of mutant KaiC led to a reduced phase shift compared to that for wild-type KaiC. Thus, KaiC appears to play a role in resetting the cellular clock in addition to its documented role in the feedback regulation of circadian rhythms.     

Circadian rhythm-dependent alterations of gene expression in Drosophila brain lacking fragile X mental retardation protein

Fragile X syndrome is caused by the loss of the FMR1 gene product, fragile X mental retardation protein (FMRP). The loss of FMRP leads to altered circadian rhythm behaviors in both mouse and Drosophila; however, the molecular mechanism behind this phenomenon remains elusive. Here we performed a series of gene expression analyses, including of both mRNAs and microRNAs (miRNAs), and identified a number of mRNAs and miRNAs (miRNA-1 and miRNA-281) with circadian rhythm-dependent altered expression in dfmr1 mutant flies. Identification of these RNAs lays the foundation for future investigations of the molecular pathway(s) underlying the altered circadian rhythms associated with loss of dFmr1.     

PAA1, a P-type ATPase of Arabidopsis,functions in copper transport in chloroplasts

Copper (Cu) is an essential trace element with important roles as a cofactor in many plant functions, including photosynthesis. However, free Cu ions can cause toxicity, necessitating precise Cu delivery systems. Relatively little is known about Cu transport in plant cells, and no components of the Cu transport machinery in chloroplasts have been identified previously. Cu transport into chloroplasts provides the cofactor for the stromal enzyme copper/zinc superoxide dismutase (Cu/ZnSOD) and for the thylakoid lumen protein plastocyanin, which functions in photosynthetic electron transport from the cytochrome b(6)f complex to photosystem I. Here, we characterized six Arabidopsis mutants that are defective in the PAA1 gene, which encodes a member of the metal-transporting P-type ATPase family with a functional N-terminal chloroplast transit peptide. paa1 mutants exhibited a high-chlorophyll-fluorescence phenotype as a result of an impairment of photosynthetic electron transport that could be ascribed to decreased levels of holoplastocyanin. The paa1-1 mutant had a lower chloroplast Cu content, despite having wild-type levels in leaves. The electron transport defect of paa1 mutants was evident on medium containing <1 micro M Cu, but it was suppressed by the addition of 10 micro M Cu. Chloroplastic Cu/ZnSOD activity also was reduced in paa1 mutants, suggesting that PAA1 mediates Cu transfer across the plastid envelope. Thus, PAA1 is a critical component of a Cu transport system in chloroplasts responsible for cofactor delivery to plastocyanin and Cu/ZnSOD.     

Sorting signals, N-terminal modifications and abundance of the chloroplast proteome

Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models.     

Independence of genetic variation between circadian rhythm and development time in the seed beetle, Callosobruchus chinensis

A positive genetic correlation between periods of circadian rhythm and developmental time supports the hypothesis that circadian clocks are implicated in the timing of development. Empirical evidence for this genetic correlation in insects has been documented in two fly species. In contrast, here we show that there is no evidence of genetic correlation between circadian rhythm and development time in the adzuki bean beetle, Callosobruchus chinensis. This species has variation that is explained by a major gene in the expression and period length of circadian rhythm between strains. In this study, we found genetic variation in development time between the strains. The development time was not covaried with either the incidence or the period length of circadian rhythm among the strains. Crosses between strains suggest that development time is controlled by a polygene. In the F₂ individuals from the crosses, the circadian rhythm is attributable to allelic variation in the major gene. Across the F₂ individuals, development time was not correlated with either the expression or the period length of circadian rhythm. Thus, we found no effects of major genes responsible for variation in the circadian rhythm on development time in C. chinensis. Our findings collectively give no support to the hypothesis that the circadian clock is involved in the regulation of development time in this species.     

Identification of CDK- and cyclin-like proteins in the eye of Bulla gouldiana

The ocular circadian rhythm in the eye of Bulla gouldiana is generated by a rhythm in membrane potential of retinal neurons that is driven by alterations in potassium conductance. Since potassium conductance may be modulated by the phosphorylation of potassium channels, the circadian rhythm may reflect rhythmic changes in protein kinase activity. Furthermore, the circadian rhythm recorded from the Bulla eye can be phase shifted by agents that affect protein synthesis and protein phosphorylation on tyrosine residues. Interestingly, the eukaryotic cell division residues. Interestingly, the eukaryotic cell division cycle is generated by similar processes. Rhythmic cell division is regulated by periodic synthesis and degradation of a protein, cyclin, and periodic tyrosine phosphorylation of a cyclin-dependent kinase (cdk), p34^cdc2. The interaction between these two proteins results in rhythmic kinase activity of p34^cdc2. Both cyclin and p34^cdc2 are pat of two diverse gene families, some of whose members have been localized to postmitotic cell types with no function yet determined. In the current work, we identify proteins similar to the cdks and cyclin in the eye of Bulla. Neither of these ocular proteins are found in mitotic cells in Bulla, and the cdk-like protein (p40) is specific to the eye. Furthermore, the concentration of the cyclin-like protein (p66) is affected by treatments that phase shift the circadain rhythm. The identification of cdk and cyclin-like proteins in the Bulla eye is consistent with the hypothesis that the biochemical mechanism responsible for generating the ocular circadian rhythm in Bulla is related to the biochemical mechnism that regulates the eukaryotic cell division cycle. 1994 John Wiley & Sons, Inc.