SIG Genes Research Articles

Sigma regulatory network in Rhodococcus erythropolis CCM2595.

The aim of this investigation was to discover the promoters that drive expression of the sig genes encoding sigma factors of RNA polymerase in Rhodococcus erythropolis CCM2595 and classify these promoters according to the sigma factors which control their activity. To analyze the regulation of major sigma factors, which control large regulons that also contain genes expressed under exponential growth and non-stressed conditions, we used the R. erythropolis CCM2595 culture, which grew rapidly in minimal medium. The transcriptional start sites (TSSs) of the genes sigA, sigB, sigD, sigE, sigG, sigH, sigJ, and sigK were detected by primary 5'-end-specific RNA sequencing. The promoters localized upstream of the detected TSSs were defined by their -35 and -10 elements, which were identical or closely similar to these sequences in the related species Corynebacterium glutamicum and Mycobacterium tuberculosis. Regulation of the promoter activities by different sigma factors was demonstrated by two independent techniques (in vivo and in vitro). All analyzed sig genes encoding the sigma factors with extracytoplasmic function (ECF) were found to be also driven from additional housekeeping promoters. Based on the classification of the sig gene promoters, a model of the basic sigma transcriptional regulatory network in R. erythropolis was designed.

Insights into the origin and evolution of plant sigma factors

AbstractPlant sigma factors (SIGs) are key regulators of chloroplast gene expression and chloroplast differentiation. Despite their functional importance, the evolutionary history of these factors remains unclear. Using newly available genomic and transcriptomic data, we undertook a detailed and comprehensive phylogenetic analysis of SIG homologues from land plants and algae. Our results reveal that plants have acquired sigma factors from ancestor cyanobacteria through endosymbiotic gene transfers, forming four major clades, namely, super‐SIG2 (SIG2/3/4/6/SIG2‐like), SIG1, SIG5, and SIGX. The super‐SIG2 clade was confirmed to have evolved from cyanobacterial SIGA factors, and a novel clade (SIGX) specific to non‐angiosperms was revealed here. Gene duplications (mainly whole genome duplications) within lineages and species have contributed to the expansion of sigma factors in plants, especially flowering plants. We hypothesize that plant sigma factors originated from different endosymbiotic ancestors and evolved diverse functions. This not only sheds new light on the evolution of plant SIG genes but also paves the way for understanding the functional diversification of these genes.

OsSIG2A is required for chloroplast development in rice (Oryza sativa L.) at low temperature by regulating plastid genes expression.

The chloroplast is an essential photosynthetic apparatus that is more sensitive to low temperatures than other organelles. Sigma factors were revealed regulating specific gene expression for maintaining photosynthetic efficiency and adapting to physiological and environmental conditions. However, the regulatory mechanisms of SIG genes supporting chloroplast development under low temperature in rice have not yet been reported. Here, we uncovered the essential role of OsSIG2A in rice chloroplast development at low temperatures by a newly reported thermo-sensitive chlorophyll deficient 12 (tcd12) mutant, which exhibited albino leaves with decreased chlorophyll content and malformed chloroplasts at seedling stage under low temperature. OsSIG2A is a typical chloroplast-localised RNA polymerase sigma factor, and constitutively expresses in different rice tissues, especially for young leaves and stems. Moreover, the transcription level of both PEP- and NEP- dependent genes, which are necessary for chloroplast development at early leaf development stage, was greatly affected in the tcd12 mutant under low temperature. Taken together, our findings indicate that OsSIG2A is required for early chloroplast differentiation under low temperatures by regulating plastid genes expression.

Analysis of the evolution of the family of the Sig genes encoding plant sigma factors

Within the latter decade, sigma subunits of bacterial-type RNA polymerases were found in eukaryote cells. In the higher plants and algae, these subunits determine the promoter specificity of the chloroplast multisubunit RNA polymerase. In the higher plants, sigma subunits are encoded by the family of nuclear Sig genes comprising 5–6 genes. Several conclusions as to the evolution of this gene family were deduced from the comparison of the deduced amino acid sequences and the sites of intron location.

Microarray Profiling of Plastid Gene Expression in a Unicellular Red Alga, Cyanidioschyzon merolae

Plastid genomes of red algae contain more genes than those of green plant lineages, and it is of special interest that four transcription factors derived from ancestral cyanobacteria are encoded therein. However, little is known about transcriptional regulation of the red algal plastid genome. In this study, we constructed a red algal plastid DNA microarray of Cyanidioschyzon merolae covering almost all protein coding genes, and found that plastid genes are differentially activated by illumination. Run-on transcription assays using isolated plastids confirmed that activation takes place at the transcriptional level. In bacteria and plants, sigma factors determine the genes that are to be transcribed, and four plastid sigma factors (Cm_SIG1-4) encoded in the nuclear genome of C. merolae may be responsible for differential gene expression of the plastid genome. We found that transcripts for all Cm_SIG genes accumulated transiently after a shift from dark to light, whereas only the Cm_SIG2 transcript was increased after a shift from low to high light, suggesting that Cm_SIG2 is a sigma factor that responds to high light. Phylogenetic analysis of plastid sigma factors suggested that sigma factors of red and green algal plastids and the group 1 sigma factors of cyanobacteria form a monophyletic group.

Differential expression of three plastidial sigma factors, OsSIG1, OsSIG2A, and OsSIG2B, during leaf development in rice.

We isolated and characterized two rice nuclear genes, OsSIG2A and OsSIG2B, encoding the putative sigma-factor of the plastid RNA polymerase. Deduced protein sequences predicted a plastid-localizing signal in the N-terminus and subsequent polypeptides similar to known SIG2 proteins. Gene expression analysis revealed that the OsSIG2A transcript is more abundant than the OsSIG2B transcript in all tissues tested and that both rice SIG2s are expressed from earlier stages of leaf development than that in the case of OsSIG1. These results indicate differential expression of SIG genes in leaf morphogenesis, suggesting the existence of tissue- and stage-specific functions of SIG proteins for transcriptional regulation of chloroplast genes in plant development.

Blue light specific and differential expression of a plastid σ factor, Sig5 in Arabidopsis thaliana

The transcription of plastid gene psbD is under the control of the BLRP ( b lue- l ight- r esponsive p romoter) recognized by plastid-encoded RNA polymerase, in which nuclear-encoded σ factors play a crucial role in the promoter recognition. We examined the effects of light on mRNA levels of six different SIG genes in Arabidopsis and found that blue light extensively induced the accumulation of SIG5 transcripts, but red light did not. The blue light specificity was not observed in the accumulations of remaining five SIG genes. The blue light dependency of the SIG5 expression well explains the light-dependent behavior of the psbD BLRP.

An Arabidopsis sigma factor (SIG2)-dependent expression of plastid-encoded tRNAs in chloroplasts.

A eubacteria-type RNA polymerase (PEP) plays crucial roles for chloroplast development in higher plants. The core subunits are encoded on plastid DNA (rpo genes) while the regulatory sigma factors are encoded on the nuclear DNA (SIG genes). However, the definite gene specificity of each sigma factor is unknown. We recently identified an Arabidopsis recessive pale-green mutant abc1 in which T-DNA is inserted in SIG2 (sigB). In this mutant, almost normal etioplasts were developed under dark conditions while the small chloroplasts with poor thylakoid membranes and stacked lamellar were developed under light conditions. The sig2-1 mutant was deficient in accumulating enough photosynthetic and photosynthesis-related proteins as well as chlorophyll. However, mRNAs of their structural genes were not significantly reduced. Further analyses revealed that several plastid-encoded tRNAs including trnE-UUC that has dual function for protein and ALA biosyntheses were drastically reduced in the sig2-1 mutant. In contrast, nucleus-encoded T7 phage-type RNA polymerase (NEP)-dependent gene transcripts were steadily accumulated in the mutant. These results indicate that progress of chloroplast development requires SIG2-dependent expression of plastid genes, particularly some of the tRNA genes.

Analysis of promoter selectivity of plastid sigma factors in Arabidopsis thaliana

In higher plants, chloroplasts have at least two types of RNA polymerases(RNAPs), the bacterial-type RNAP (PEP; plastid-encoded RNAP) and the T7 bacteriophage type RNAP (NEP; nuclear-encoded RNAP). PEP transcribes preferentially the genes encoding the proteins implicated in the characteristic functions of chloroplasts and is composed of plastid-encoded core enzyme subunits and a nuclear-encoded sigma factor, which presumably confer promoter-specific transcription initiation on RNAP. In higher plant genome analysis, a number of putative sigma factors have been identified. These multiple sigma factors are considered to recognize different sets of promoter sequences and regulate the activity of PEP in response to the developmental stage and various environmental clues. To analyze the promoter selectivity of sigma factors in Arabidopsis thaliana, each of six sig genes was expressed transiently in protoplasts and the transcription of 12 plastid genes in each transformant was measured by means of the run-on transcription assay. In this assay, three sigma factors were found to elevate the PEP activity for different plastid genes, indicating that each sigma factor has different promoter recognition property. The regulation mechanism of transcription of plastid genes through sigma factors will be discussed.

EXPRESSION PATTERNS OF THREE PUTATIVE SEXUAL‐SPECIFIC GENES IN THE CENTRIC DIATOM THALASSIOSIRA WEISSFLOGII

An intriguing feature of the diatom life cycle is that sexual reproduction not only generates genetic diversity but also controls cell size. Very little is known, however, about either the frequency or the potential ramifications of sexual events in field populations, due in large part to the difficulties associated with recognizing sexual stages. Our approach has been to identify genes involved in the diatom sexual cycle, with the goal of developing molecular probes to determine when and where sexual reproduction occurs. We are investigating the expression pattern of three genes identified in a sexual reproduction screen. The Sig1, Sig2 and Sig3 genes comprise a gene family, and encode proteins with strong sequence homology to extracellular matrix proteins. We have hypothesized that the Sig proteins are necessary for sperm/egg recognition. Using RT‐PCR, we have shown that transcription of the Sig genes is highly upregulated during the early stages of the sexual cycle, before any morphological changes are apparent. The three genes show significant differences in the exact timing and levels of expression, suggesting different roles for each member of the gene family. We are now employing quantitative RT‐PCR to study their relative transcription levels in more detail. We are also using antibodies made against a recombinant version of the Sig2 protein to look at protein expression levels before and after sexual induction, and to determine where the Sig2 protein localizes in the cell.

Three new nuclear genes, sigD, sigE and sigF, encoding putative plastid RNA polymerase σ factors in Arabidopsis thaliana

Three new nuclear genes ( sigD, sigE and sigF) of Arabidopsis thaliana, encoding putative plastid RNA polymerase σ factors, were identified and analyzed. Phylogenetic analysis revealed that higher plant σ factors fell into at least four distinct subgroups within a diverse protein family. In addition, Arabidopsis sig genes contained conserved chromosomal intron sites, indicating that these genes arose by DNA duplication events during plant evolution. Transcript analyses revealed two alternatively spliced transcripts generated from the sigD region, one of which is predicted to encode a σ protein lacking the carboxy-terminal regions 3 and 4. Finally, the amino-terminal sequence of the sigF gene product was shown to function as a plastid-targeting signal using green fluorescent protein fusions.

Plastidic RNA polymerase sigma factors in Arabidopsis.

In plant cells, plastid DNA is transcribed by at least two types of RNA polymerase, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). PEP is homologous to eubacterial transcription machinery, but its regulatory subunit, sigma (sigma) factor, is not encoded on the plastid DNA. We previously cloned the three nuclear-encoded sigma factor genes from Arabidopsis thaliana and designated them as sigA, sigB, and sigC. By means of RFLP mapping, sigA and sigB were mapped on chromosome I and sigC on the chromosome III. Based on comparison of the genomic structure of the three sig genes, intron sites in the 3' half of the genes were shown to be identical between sigB and sigC but divergent in sigA, consistent with the phylogenetic relevance of the three gene products. A transient expression assay of GFP fusions in Arabidopsis protoplasts showed that the N-termini of all three sig gene products functioned as chloroplast-targeting signals. We also constructed transgenic Arabidopsis lines harboring the sigA-promoter or the sigB-promoter uidA fusion. Both the sigA- and sigB-promoters were similarly activated at cotyledons, hypocotyls, rosette leaves, cauline leaves, sepals, and siliques but not at roots, seeds, or other flower organs. In addition, the two promoters were repeatedly activated in young seedlings under continuous light, possibly in an oscillated fashion.