Promoter recognition in eubacteria is carried out by the initiation factor sigma, which binds RNA polymerase and initiates transcription. Cells have one housekeeping factor and a variable number of alternative sigma factors that possess different promoter-recognition properties. The cell can choose from its repertoire of sigmas to alter its transcriptional program in response to stress. Recent structural information illuminates the process of initiation and also shows that the two key sigma domains are structurally conserved, even among diverse family members. We use the sigma repertoire of Escherichia coli, Bacillus subtilis, Streptomyces coelicolor, and cyanobacteria to illustrate the different strategies utilized to organize transcriptional space using multiple sigma factors.

Multiple sigma subunits and the partitioning of bacterial transcription space.

Following the acquisition of chloroplasts and mitochondria by eukaryotic cells during endosymbiotic evolution, most of the genes in these organelles were either lost or transferred to the nucleus. Encoding organelle-destined proteins in the nucleus allows for host control of the organelle. In return, organelles send signals to the nucleus to coordinate nuclear and organellar activities. In photosynthetic eukaryotes, additional interactions exist between mitochondria and chloroplasts. Here we review recent advances in elucidating the intracellular signalling pathways that coordinate gene expression between organelles and the nucleus, with a focus on photosynthetic plants.

Coordination of gene expression between organellar and nuclear genomes.

Chloroplasts are the ancestral members of the plastid organelle family. Their identity, division and biogenesis require the import of nucleus-encoded proteins and tight coordination between the organellar genetic system and the nucleocytosolic system. The ubiquitin–proteasome system also links plastid homeostasis and biogenesis to organismal development. Chloroplasts are the organelles that define plants, and they are responsible for photosynthesis as well as numerous other functions. They are the ancestral members of a family of organelles known as plastids. Plastids are remarkably dynamic, existing in strikingly different forms that interconvert in response to developmental or environmental cues. The genetic system of this organelle and its coordination with the nucleocytosolic system, the import and routing of nucleus-encoded proteins, as well as organellar division all contribute to the biogenesis and homeostasis of plastids. They are controlled by the ubiquitin–proteasome system, which is part of a network of regulatory mechanisms that integrate plastid development into broader programmes of cellular and organismal development.

Biogenesis and homeostasis of chloroplasts and other plastids

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

/pdf/in-posidonia-oceanica-cadmium-induces-changes-in-dna-3cu7uj83mr.pdf

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

DNA methylation is a major epigenetic modification in the genomes of higher eukaryotes. In vertebrates, DNA methylation occurs predominantly on the CpG dinucleotide, and approximately 60% to 90% of these dinucleotides are modified. Distinct DNA methylation patterns, which can vary between different tissues and developmental stages, exist on specific loci. Sites of DNA methylation are occupied by various proteins, including methyl-CpG binding domain (MBD) proteins which recruit the enzymatic machinery to establish silent chromatin. Mutations in the MBD family member MeCP2 are the cause of Rett syndrome, a severe neurodevelopmental disorder, whereas other MBDs are known to bind sites of hypermethylation in human cancer cell lines. Here, we review the advances in our understanding of the function of DNA methylation, DNA methyltransferases, and methyl-CpG binding proteins in vertebrate embryonic development. MBDs function in transcriptional repression and long-range interactions in chromatin and also appear to play a role in genomic stability, neural signaling, and transcriptional activation. DNA methylation makes an essential and versatile epigenetic contribution to genome integrity and function.

/pdf/dna-methylation-and-methyl-cpg-binding-proteins-515i9rf4s6.pdf

DNA methylation and methyl-CpG binding proteins: developmental requirements and function

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.

/pdf/plastidic-rna-polymerase-s-factors-in-arabidopsis-52c7jec7lo.pdf

Plastidic RNA polymerase σ factors in Arabidopsis

Chloroplast transcription in higher plants is performed by two types of RNA polymerases, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). PEP is a eubacteria-type multisubunit enzyme whose catalytic core subunits are encoded by the chloroplast genome, whereas NEP is the nuclear encoded T7 phage-type single subunit enzyme. PEP is critical for the biogenesis and maintenance of chloroplasts, and is finely tuned by the nuclear encoded sigma subunits. Of the six Arabidopsis sigma subunits, SIG2 is involved in the transcription of several chloroplast tRNA genes, including trnE encoding tRNA-Glu. SIG2 possibly couples translation and pigment synthesis in chloroplasts. On the other hand, SIG5 is induced by various stresses and contributes to repair of damaged photosystem II (PSII) through transcription of the psbD and psbC genes. Thus target genes and the physiological role of each sigma subunit are becoming clearer.

https://www.tandfonline.com/doi/pdf/10.1271/bbb.68.2215

Roles of chloroplast RNA polymerase sigma factors in chloroplast development and stress response in higher plants

Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor Sigma B

Cyclic GMP (cGMP) is an important signaling molecule that controls a range of cellular functions. So far, however, only a few genes have been found to be regulated by cGMP in higher plants. We investigated the cGMP-responsiveness of several genes encoding flavonoid-biosynthetic enzymes in soybean (Glycine max L.) involved in legume-specific isoflavone, phytoalexin and anthocyanin biosynthesis, such as phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, chalcone synthase, chalcone reductase, chalcone isomerase, 2-hydroxyisoflavanone synthase, 2-hydroxyisoflavanone dehydratase, anthocyanidin synthase, UDP-glucose:isoflavone 7-O-glucosyltransferase, and isoflavone reductase, and found that the majority of these genes were induced by cGMP but not by cAMP. All cGMP-induced genes were also stimulated by sodium nitroprusside (SNP), a nitric oxide (NO) donor, and illumination of cultured cells with white light. The NO-dependent induction of these genes was blocked by 6-anilino-5,8-quinolinedione, an inhibitor of guanylyl cyclase. Moreover, cGMP levels in cultured cells were transiently increased by SNP. Consistent with the increases of these transcripts, the accumulation of anthocyanin in response to cGMP, NO, and white light was observed. The treatment of soybean cotyledons with SNP resulted in a high accumulation of isoflavones such as daidzein and genistein. Loss- and gain-of-function experiments with the promoter of chalcone reductase gene indicated the Unit I-independent activation of gene expression by cGMP. Together, these results suggest that cGMP acts as a second messenger to activate the expression of genes for enzymes involved in the flavonoid biosynthetic pathway in soybean.

/pdf/cyclic-gmp-acts-as-a-common-regulator-for-the-52dq3zzlw0.pdf

Cyclic GMP acts as a common regulator for the transcriptional activation of the flavonoid biosynthetic pathway in soybean.

5′-Aminolevulinic acid (ALA) is widely used in the pharmaceutical industry, healthcare, and food production, and is a substrate for the biosynthesis of heme, which is required for respiration and photosynthesis. Enhancement of ALA biosynthesis has never been developed in Saccharomyces cerevisiae, which is a well-known model microorganism used for bioproduction of many value-added compounds. We demonstrated that metabolic engineering significantly improved ALA production in S. cerevisiae. First, we found that overexpression of HEM1, which encodes ALA synthetase, increased ALA production. Furthermore, addition of an optimal amount of glycine, a substrate for ALA biosynthesis, or levulinic acid, an inhibitor of ALA dehydrogenase, effectively increased ALA production. Next, we developed an assay for multiple metabolites including ALA and found that aconitase, encoded by ACO1 and ACO2, is the rate-limiting enzyme of ALA biosynthesis when sufficient glycine is supplied. Overexpression of ACO2 further enhanced ALA production in S. cerevisiae overexpressing HEM1. In this study, ALA production in S. cerevisiae was enhanced by metabolic engineering. This study also shows a strategy to identify the rate-limiting step of a target synthetic pathway by assay for multiple metabolites alongside the target product. This strategy can be applied to improve production of other valuable products in the well-studied and well-industrialized microorganism S. cerevisiae.

/pdf/5-aminolevulinic-acid-fermentation-using-engineered-3zbdwkcvw6.pdf

Kengo Kanamaru

Papers

Plastidic RNA polymerase σ factors in Arabidopsis

Roles of chloroplast RNA polymerase sigma factors in chloroplast development and stress response in higher plants

Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor Sigma B

Cyclic GMP acts as a common regulator for the transcriptional activation of the flavonoid biosynthetic pathway in soybean.

5-Aminolevulinic acid fermentation using engineered Saccharomyces cerevisiae