Through sos3 (salt overly sensitive 3) suppressor screening, two allelic suppressor mutants that are weak alleles of the strong sos3 suppressor sos3hkt1-1 were recovered. Molecular characterization identified T-DNA insertions in the distal promoter region of the Arabidopsis thaliana HKT1 (AtHKT1, At4g10310) in these two weak sos3 suppressors, which results in physical separation of a tandem repeat from the proximal region of the AtHKT1 promoter. The tandem repeat is approximately 3.9 kb upstream of the ATG start codon and functions as an enhancer element to promote reporter gene expression. A putative small RNA target region about 2.6 kb upstream of the ATG start codon is heavily methylated. CHG and CHH methylation but not CG methylation is significantly reduced in the small RNA biogenesis mutant rdr2, indicating that non-CG methylation in this region is mediated by small RNAs. Analysis of AtHKT1 expression in rdr2 suggests that non-CG methylation in the putative small RNA target region represses AtHKT1 expression in shoots. The DNA methylation-deficient mutant met1-3 has nearly complete loss of total cytosine methylation in the putative small RNA target region and is hypersensitive to salt stress. The putative small RNA target region and the tandem repeat are essential for maintaining AtHKT1 expression patterns crucial for salt tolerance.

The phosphorylation state of the C-terminal domain (CTD) of the RNA polymerase II plays crucial roles in transcription and mRNA processing. Previous studies showed that the plant CTD phosphatase-like 1 (CPL1) dephosphorylates Ser-5-specific CTD and regulates abiotic stress response in Arabidopsis. Here, we report the identification of a K-homology domain-containing protein named SHINY1 (SHI1) that interacts with CPL1 to modulate gene expression. The shi1 mutant was isolated from a forward genetic screening for mutants showing elevated expression of the luciferase reporter gene driven by a salt-inducible promoter. The shi1 mutant is more sensitive to cold treatment during vegetative growth and insensitive to abscisic acid in seed germination, resembling the phenotypes of shi4 that is allelic to the cpl1 mutant. Both SHI1 and SHI4/CPL1 are nuclear-localized proteins. SHI1 interacts with SHI4/CPL1 in vitro and in vivo. Loss-of-function mutations in shi1 and shi4 resulted in similar changes in the expression of some stress-inducible genes. Moreover, both shi1 and shi4 mutants display higher mRNA capping efficiency and altered polyadenylation site selection for some of the stress-inducible genes, when compared with wild type. We propose that the SHI1-SHI4/CPL1 complex inhibits transcription by preventing mRNA capping and transition from transcription initiation to elongation.

Inducible gene expression is a gene regulatory mechanism central to plant response to environmental cues. The inducible genes are often repressed under normal growth conditions while their expression levels are significantly elevated by conditions such as abiotic stresses. Induction of gene expression requires both cis-acting DNA elements and trans-acting proteins that are modulated through signal transduction pathways. Here we report several molecular events that affect salt induced expression of the Arabidopsis AtSOT12 gene. Promoter deletion analysis revealed that DNA elements residing in the 5′ UTR are required for the salt induced expression of AtSOT12. Cytosine methylation in the promoter was low and salt stress slightly increased the DNA methylation level, suggesting that DNA methylation may not contribute to AtSOT12 gene repression. Co-transcriptional processing of AtSOT12 mRNA including capping and polyadenylation site selection was also affected by salt stress. The percentage of capped mRNA increased by salt treatment, and the polyadenylation sites were significantly different before and after exposure to salt stress. The expression level of AtSOT12 under normal growth conditions was markedly higher in the oxi1 mutant defective of reactive oxygen species (ROS) signaling than in the wild type. Moreover, AtSOT12 transcript level was elevated by treatments with DPI and DMTU, two chemicals preventing ROS accumulation. These results suggest that repression of AtSOT12 expression may require physiological level of ROS and ROS signaling.

Gene regulation is central for growth, development, and adaptation to environmental changes in all living organisms. Many genes are induced by environmental cues, and the expression of these inducible genes is often repressed under normal conditions. Here, we show that the SHINY2 (SHI2) gene is important for repressing salt-inducible genes and also plays a role in cold response. The shi2 mutant displayed hypersensitivity to cold, abscisic acid (ABA), and LiCl. Map-based cloning demonstrates that SHI2 encodes a DEAD- (Asp-Glu-Ala-Asp) box RNA helicase with similarity to a yeast splicing factor. Transcriptomic analysis of the shi2 mutant in response to cold revealed that the shi2 mutation decreased the number of cold-responsive genes and the magnitude of their response, and resulted in the mis-splicing of some cold-responsive genes. Under salt stress, however, the shi2 mutation increased the number of salt-responsive genes but had a negligible effect on mRNA splicing. Our results suggest that SHI2 is a component in a ready-for-transcription repressor complex important for gene repression under normal conditions, and for gene activation and transcription under stress conditions. In addition, SHI2 also serves as a splicing factor required for proper splicing of cold-responsive genes and affects 5' capping and polyadenylation site selection.