Serum response factor (SRF) plays a central role during myogenesis, being required for the expression of striated alpha-actin genes. As shown here, the small GTPase RhoA-dependent activation of SRF results in the expression of muscle-specific genes, thereby promoting myogenic differentiation in myoblast cell lines. Co-expression of activated V14-RhoA and SRF results in an approximately 10-fold activation of the skeletal alpha-actin promoter in replicating myoblasts, while SRFpm1, a dominant negative SRF mutant, blocks RhoA dependent skeletal alpha-actin promoter activity. Serum withdrawal further potentiates RhoA- and SRF-mediated activation of alpha-actin promoter to about 30-fold in differentiated myotubes. In addition, the proximal SRE1 in the skeletal alpha-actin promoter is sufficient to mediate RhoA signaling via SRF. Furthermore, SRFpm1 and to a lesser extent dominant negative N19-RhoA inhibit myoblast fusion, postreplicative myogenic differentiation, and expression of direct SRF targets such as skeletal alpha-actin and indirect targets such as myogenin and alpha-myosin heavy chain. Moreover, RhoA also stimulates the autoregulatable murine SRF gene promoter in myoblasts, and the expression level of SRF is reduced in myoblasts overexpressing N19-RhoA. Our study supports the concept that RhoA signaling via SRF serves as an obligatory muscle differentiation regulatory pathway.

Arsenic (As) bioavailability to rice plants is elevated in flooded paddy soils due to reductive mobilization of arsenite [As(III)]. However, some microorganisms are able to mediate anaerobic As(III) oxidation by coupling to nitrate reduction, thus attenuating As mobility. In this study, we investigated the impact of nitrate additions on As species dynamics in the porewater of four As-contaminated paddy soils. The effects of nitrate on microbial community structure and the abundance and diversity of the As(III) oxidase (aioA) genes were quantified using 16S rRNA sequencing, quantitative PCR, and aioA gene clone libraries. Nitrate additions greatly stimulated anaerobic oxidation of As(III) to As(V) and decreased total soluble As in the porewater in flooded paddy soils. Nitrate additions significantly enhanced the abundance of aioA genes and changed the microbial community structure by increasing the relative abundance of the operational taxonomic units (OTUs) from the genera Acidovorax and Azoarcus. The aioA gene sequences from the Acidovorax related OTU were also stimulated by nitrate. A bacterial strain (ST3) belonging to Acidovorax was isolated from nitrate-amended paddy soil. The strain was able to oxidize As(III) and Fe(II) under anoxic conditions using nitrate as the electron acceptor. Abiotic experiments showed that Fe(II), but not As(III), could be oxidized by nitrite. These results show that nitrate additions can stimulate As(III) oxidation in flooded paddy soils by enhancing the population of anaerobic As(III) oxidizers, offering a potential strategy to decrease As mobility in As-contaminated paddy soils.

No abstract

Primary transcripts encoding the MADS box superfamily of proteins, such as MEF2 in animals and ZEMa in plants, are alternatively spliced, producing several isoformic species. We show here that murine serum response factor (SRF) primary RNA transcripts are alternatively spliced at the fifth exon, deleting approximately one-third of the C-terminal activation domain. Among the different muscle types examined, visceral smooth muscles have a very low ratio of SRF⌬5 to SRF. Increased levels of SRF⌬5 correlates well with reduced smooth muscle contractile gene activity within the elastic aortic arch, suggesting important biological roles for differential expression of SRF⌬5 variant relative to wild-type SRF. SRF⌬5 forms DNA binding-competent homodimers and heterodimers. SRF⌬5 acts as a naturally occurring dominant negative regulatory mutant that blocks SRF-dependent skeletal ␣-actin, cardiac ␣-actin, smooth ␣-actin, SM22␣, and SRF promoter-luciferase reporter activities. Expression of SRF⌬5 interferes with differentiation of myogenic C2C12 cells and the appearance of skeletal ␣-actin and myogenin mRNAs. SRF⌬5 repressed the serum-induced activity of the c-fos serum response element. SRF⌬5 fused to the yeast Gal4 DNA binding domain displayed low transcriptional activity, which was complemented by overexpression of the coactivator ATF6. These results indicate that the absence of exon 5 might be bypassed through recruitment of transcription factors that interact with extra-exon 5 regions in the transcriptional activating domain. The novel alternatively spliced isoform of SRF, SRF⌬5, may play an important regulatory role in modulating SRF-dependent gene expression.Alternative splicing is a commonly used molecular strategy for creating diverse gene products from a single genetic locus in most eucaryotic cells. The modular organization of transcription factor genes, through exon-encoded structural domains, may be conducive for forming a variety of alternatively spliced isoforms that affect DNA binding avidity and specificity, transactivation, subcellular localization, responsiveness to signaling pathways, and developmental regulation (reviewed in reference 31). For example, alternative splicing within the DNA binding domain of Pax-6 (9) and Wilm's tumor-associated protein 1 (5, 21) alters their DNA binding specificities. Alternative splicing of exons encoding the transactivating domains in the paired family proteins Pax-3 (59), , and Pax-9 (39), the POU homeodomain family proteins Pit-1 (37), Oct-1 (8), Oct-2 (1), and Brn-3a (38), and the zinc finger transcription factor GATA-5 (32) results in isoforms possessing activation domains with different potencies. Splicing out of select exons from the activation domain as in AML1a (56), Oct-2 (29), and CREB family proteins CREB (reviewed in reference 11), Drosophila CREB/CREM (61), and CREM (12) produces isoforms with dominant negative activity. Similarly, several splice variants containing only the DNA binding domain act as dominant negative isoforms presumably by heterodimerizing w...

Jasmonic acid (JA) is an important plant hormone involved in regulation of many aspects of plant growth and development including secondary metabolism and JASMONATE ZIM-DOMAIN (JAZ) proteins are key components in JA signal processes. In this study, two new JAZ genes named SmJAZ3 and SmJAZ9 were cloned from S. miltiorrhiza hairy roots and characterized. Expression profiles under methyl jasmonate (MJ) treatment revealed that SmJAZ3 and SmJAZ9 were both MJ-responsive. Subcellular localization assay showed that SmJAZ3 was located in nucleus while SmJAZ9 was preferentially in nucleus. Expression of SmJAZ3 and SmJAZ9 in S. miltiorrhiza hairy roots differently affected the production of tanshinone. Over-expression of SmJAZ3 or SmJAZ9 in hairy roots produced lower level of tanshinone compared with the control, tanshinone production was as low as 0.077 mg/g DW in line SmJAZ3-3 and 0.266 mg/g DW in line SmJAZ9-22. Whereas, down-regulation of SmJAZs enhanced tanshione production, the content of tanshinone increased to 2.48 fold in anti-SmJAZ3-3 line, and 1.35-fold in anti-SmJAZ9-23 line. Our work indicated that SmJAZ3 and SmJAZ9 are involved in regulation of tanshinone biosynthesis and act as repressive transcriptional regulators in the JA signaling pathway, which paves the way to further dissect molecular mechanism in details in the future.