Selective Activation of Chloroplast psbD Light-Responsive Promoter and psaA/B Promoter in Transplastomic Tobacco Plants Overexpressing Arabidopsis Sigma Factor AtSIG5.

BACKGROUND: Plastid-encoded eubacterial-type RNA polymerase (PEP) plays a critical role in the transcription of photosynthesis genes in chloroplasts. Notably, some of the reaction center genes, including psaA, psaB, psbA, and psbD genes, are differentially transcribed by PEP in mature chloroplasts. However, the molecular mechanism of promoter selection in the reaction center gene transcription by PEP is not well understood. OBJECTIVE: Sigma factor proteins direct promoter selection by a core PEP in chloroplasts as well as bacteria. AtSIG5 is a unique chloroplast sigma factor essential for psbD light-responsive promoter (psbD LRP) activity. To analyze the role of AtSIG5 in chloroplast transcription in more detail, we assessed the effect of AtSIG5 hyper-expression on the transcription of plastid-encoded genes in chloroplast transgenic plants. RESULTS: The chloroplast transgenic tobacco (CpOX-AtSIG5) accumulates AtSIG5 protein at extremely high levels in chloroplasts. Due to the extremely high-level expression of recombinant AtSIG5, most PEP holoenzymes are most likely to include the recombinant AtSIG5 in the CpOXAtSIG5 chloroplasts. Thus, we can assess the promoter preference of AtSIG5 in vivo. The overexpression of AtSIG5 significantly increased the expression of psbD LRP transcripts encoding PSII reaction center D2 protein and psaA/B operon transcripts encoding PSI core proteins. Furthermore, run-on transcription analyses revealed that AtSIG5 preferentially recognizes the psaA/B promoter, as well as the psbD LRP. Moreover, we found that psbD LRP is constitutively active in CpOX-AtSIG5 plants irrespective of light and dark. CONCLUSION: AtSIG5 probably plays a significant role in differential transcription of reaction center genes in mature chloroplasts.

Comparative Analysis of Chloroplast psbD Promoters in Terrestrial Plants.

The transcription of photosynthesis genes encoded by the plastid genome is mainly mediated by a prokaryotic-type RNA polymerase called plastid-encoded plastid RNA polymerase (PEP). Standard PEP-dependent promoters resemble bacterial sigma-70-type promoters containing the so-called -10 and -35 elements. On the other hand, an unusual light- and stress-responsive promoter (psbD LRP) that is regulated by a 19-bp AAG-box immediately upstream of the -35 element has been mapped upstream of the psbD-psbC operon in some angiosperms. However, the occurrence of the AAG-box containing psbD LRP in plant evolution remains elusive. We have mapped the psbD promoters in eleven embryophytes at different evolutionary stages from liverworts to angiosperms. The psbD promoters were mostly mapped around 500-900 bp upstream of the psbD translational start sites, indicating that the psbD mRNAs have unusually long 5'-UTR extensions in common. The -10 elements of the psbD promoter are well-conserved in all embryophytes, but not the -35 elements. We found that the AAG-box sequences are highly conserved in angiosperms and gymnosperms except for gnetaceae plants. Furthermore, partial AAG-box-like sequences have been identified in the psbD promoters of some basal embryophytes such as moss, hornwort, and lycophyte, whereas liverwort has the standard PEP promoter without the AAG-box. These results suggest that the AAG-box sequences of the psbD LRP may have evolved from a primitive type of AAG-box of basal embryophytes. On the other hand, monilophytes (ferns) use another type of psbD promoter composed of a distinct cis-element upstream of the potential -35 element. Furthermore, we found that psbD expression is not regulated by light in gymnosperms or basal angiosperms, although they have the well-conserved AAG-box sequences. Thus, it is unlikely that acquisition of the AAG-box containing psbD promoter is directly associated with light-induced transcription of the psbD-psbC operon. Light- and stress-induced transcription may have evolved independently and multiple times during terrestrial plant evolution.

Ferritin 2 domain-containing protein found in lacquer tree (Toxicodendron vernicifluum) sap has negative effects on laccase and peroxidase reactions.

Lacquer tree sap, a raw material of traditional paints in East Asia, is hardened through laccase-catalyzed oxidation and the following polymerization of phenolic compound urushiol. In the sap's water-insoluble fraction, we found two plantacyanins and a ferritin 2 domain-containing protein (TvFe2D, a homolog of Arabidopsis AT1G47980 and AT3G62730). The recombinant TvFe2D protein suppressed the accumulation of laccase-catalyzed oxidation products of a model substrate syringaldazine without decreasing oxygen consumption, the second substrate of laccase. The suppression was also observed when another substrate guaiacol or another oxidizing enzyme peroxidase was used. The functional domain of the suppression was the C-terminal half, downstream of the ferritin 2 domain. The results suggest that this protein may be involved in regulating the sap polymerization/hardening. We also discuss the possibility that homologous proteins of TvFe2D in other plants might be involved in the laccase- or peroxidase-mediated polymerization of phenolic compounds, such as lignin and flavonoids.

Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type plastid RNA polymerase.

Plastid transcription is mediated by two distinct types of RNA polymerases (RNAPs), bacterial-type RNAP (PEP) and phage-type RNAP (NEP). Recent genomic and proteomic studies revealed that higher plants have lost most prokaryotic transcription regulators and have acquired eukaryotic-type proteins during plant evolution. However, in vivo dynamics of chloroplast RNA polymerases and eukaryotic-type plastid nucleoid proteins have not been directly characterized experimentally. Here, we examine the association of the α-subunit of PEP and eukaryotic-type protein, plastid transcriptionally active chromosome 3 (pTAC3) with transcribed regions in vivo by using chloroplast chromatin immunoprecipitation (cpChIP) assays. PEP α-subunit preferentially associates with PEP promoters of photosynthesis and rRNA genes, but not with NEP promoter regions, suggesting selective and accurate recognition of PEP promoters by PEP. The cpChIP assays further demonstrate that the peak of PEP association occurs at the promoter-proximal region and declines gradually along the transcribed region. pTAC3 is a putative DNA-binding protein that is localized to chloroplast nucleoids and is essential for PEP-dependent transcription. Density gradient and immunoprecipitation analyses of PEP revealed that pTAC3 is associated with the PEP complex. Interestingly, pTAC3 associates with the PEP complex not only during transcription initiation, but also during elongation and termination. These results suggest that pTAC3 is an essential component of the chloroplast PEP complex. In addition, we demonstrate that light-dependent chloroplast transcription is mediated by light-induced association of the PEP-pTAC3 complex with promoters. This study illustrates unique dynamics of PEP and its associated protein pTAC3 during light-dependent transcription in chloroplasts.

Role of DNA methylation and histone H3 lysine 27 methylation in tissue-specific imprinting of mouse Grb10.

Mouse Grb10 is a tissue-specific imprinted gene with promoter-specific expression. In most tissues, Grb10 is expressed exclusively from the major-type promoter of the maternal allele, whereas in the brain, it is expressed predominantly from the brain type promoter of the paternal allele. Such reciprocally imprinted expression in the brain and other tissues is thought to be regulated by DNA methylation and the Polycomb group (PcG) protein Eed. To investigate how DNA methylation and chromatin remodeling by PcG proteins coordinate tissue-specific imprinting of Grb10, we analyzed epigenetic modifications associated with Grb10 expression in cultured brain cells. Reverse transcriptase PCR analysis revealed that the imprinted paternal expression of Grb10 in the brain implied neuron-specific and developmental stage-specific expression from the paternal brain type promoter, whereas in glial cells and fibroblasts, Grb10 was reciprocally expressed from the maternal major-type promoter. The cell-specific imprinted expression was not directly related to allele-specific DNA methylation in the promoters because the major-type promoter remained biallelically hypomethylated regardless of its activity, whereas gametic DNA methylation in the brain type promoter was maintained during differentiation. Histone modification analysis showed that allelic methylation of histone H3 lysine 4 and H3 lysine 9 were associated with gametic DNA methylation in the brain type promoter, whereas that of H3 lysine 27 regulated by the Eed PcG complex was detected in the paternal major-type promoter, corresponding to its allele-specific silencing. Here, we propose a molecular model that gametic DNA methylation and chromatin remodeling by PcG proteins during cell differentiation cause tissue-specific imprinting in embryonic tissues.

A nuclear-encoded sigma factor, Arabidopsis SIG6, recognizes sigma-70 type chloroplast promoters and regulates early chloroplast development in cotyledons.

Eubacterial-type multi-subunit plastid RNA polymerase (PEP) is responsible for the principal transcription activity in chloroplasts. PEP is composed of plastid-encoded core subunits and one of multiple nuclear-encoded sigma factors that confer promoter specificity on PEP. Thus, the replacement of sigma factors associated with PEP has been assumed to be a major mechanism for the switching of transcription patterns during chloroplast development. The null mutant (sig6-1) of plastid sigma factor gene AtSIG6 exhibited a cotyledon-specific pale green phenotype. Light-dependent chloroplast development was significantly delayed in the sig6-1 mutant. Genetic complementation of the mutant phenotype by the AtSIG6 cDNA demonstrated that AtSIG6 plays a key role in light-dependent chloroplast development. Northern and array-based global analyses for plastid transcripts revealed that the transcript levels of most PEP-dependent genes were greatly reduced in the sig6-1 mutant, but that the accumulation of nuclear-encoded RNA polymerase (NEP)-dependent transcripts generally increased. As the PEP alpha subunit and PEP-dependent trnV accumulated at normal levels in the sig6-1 mutant, the AtSIG6 knockout mutant probably retained functional PEP, and the transcriptional defects are likely to have been directly caused by AtSIG6 deficiency. Most of the AtSIG6-dependent genes are preceded by sigma70-type promoters comprised of conserved -35/-10 elements. Thus, AtSIG6 may act as a major general sigma factor in chloroplasts during early plant development. On the other hand, the mutant phenotype was restored in older seedlings. Arabidopsis probably contains another late general sigma factor, the promoter specificity of which widely overlaps with that of AtSIG6.

Blue light-induced transcription of plastid-encoded psbD gene is mediated by a nuclear-encoded transcription initiation factor, AtSig5.

Light is one of the most important environmental factors regulating expression of photosynthesis genes. The plastid psbD gene encoding the photosystem II reaction center protein D2 is under the control of a unique blue light responsive promoter (BLRP) that is transcribed by a bacterial-type plastid RNA polymerase (PEP). Promoter recognition of PEP is mediated by one of the six nuclear-encoded sigma factors in Arabidopsis. The replacement of the plastid sigma factor associated with PEP may be the major mechanism for switching of plastid transcription pattern in response to environmental and developmental signals. This study demonstrates that AtSig5 is a unique sigma factor that is essential for psbD BLRP activity. A T-DNA insertional mutant with reduced AtSIG5 expression resulted in loss of primary transcripts from the psbD BLRP. Furthermore, transient overexpression of AtSig5 in dark-adapted protoplasts specifically elevated psbD and psbA transcription activities. On the other hand, overproduction of AtSig2 enhanced the transcription of psbA gene and trnE operon, but not psbD transcription. The AtSIG5 gene is phylogenetically distinct from other plastid sigma factors, and its expression is induced exclusively by blue light. We propose that AtSig5 acts as a mediator of blue light signaling that specifically activates the psbD BLRP in response to blue light in Arabidopsis.

A role of the -35 element in the initiation of transcription at psbA promoter in tobacco plastids.

Most plastid promoters recognized by bacteria-like plastid RNA polymerase (PEP) are similar to E. coli sigma(70)-type promoters comprising "-35" and "-10" elements. Among them, psbA promoter is unique in bearing additional elements between the conserved -35 and -10 elements. The psbA promoter activity is differentially maintained in the mature chloroplasts where the activity of most PEP promoters declines. Previously, we identified two types of PEP activities in wheat seedlings [Satoh et al. (1999) Plant J. 18: 407]; PEP present in the mature chloroplasts of the leaf tip (tip-type PEP) can initiate transcription from the -35-destructed psbA promoter, but the -35 element is essential for transcription by PEP present in immature chloroplasts of the leaf base (base-type PEP). To reveal which type of PEP functions in various types of plastids in tobacco, we analyzed the tobacco psbA promoter by means of a transplastomic approach. The promoter core context (-42 to +9) was sufficient for developmental regulation of the psbA promoter activity. The -35 promoter element was important for transcription initiation at the psbA promoter in all types of plastids, including chloroplasts in mature leaves, leucoplasts in roots, etioplasts in etiolated cotyledons. The conclusion is that the PEP bearing a promoter preference, similar to the wheat base-type PEP, functions dominantly in tobacco chloroplasts.