Detection of 5′ capping of the transcripts of <i>LUC</i>, <i>AtSOT12</i>, <i>At5g25280</i>, <i>COR15A</i>, and <i>COR47</i> in wild type, <i>shi1</i> and <i>shi4</i> mutants.

<p>(A) Relative capping ratios of the five selected genes transcripts determined by using the RLM-qRT-PCR method. Values are shown as mean ± SD (n = 3). (B) Percentages of the capped mRNA of three selected genes determined by using the 5′ RACE based method.</p

Map-based cloning of the <i>SHI1</i> locus.

<p>(A) Identification of the <i>shi1</i> mutation. Recombination events from 1858 chromosomes analyzed for each marker are shown. The locations of the <i>shi1</i> mutation of G1494A and two T-DNA insertions are shown. (B) Complementation tests for the <i>shi1</i> mutation. Left panel, genetic complementation between the <i>shi1</i> mutation and the T-DNA knockouts; Right panel, molecular complementation of the <i>shi1</i> mutation by the wild type <i>SHI1</i> gene.</p

Detection of polyadenylation site selection of the transcripts of <i>LUC</i>, <i>AtSOT12</i>, <i>At5g25280</i>, <i>COR15A</i>, and <i>COR47</i> wild type, <i>shi1</i> and <i>shi4</i> mutants.

<p>(A) DNA sequences at the 3′ end of the five selected genes. Triangles indicate the major polyadenylation sites in the transcripts. Arrow shows the polyadenylation sites downstream of the indicated nucleotide in the <i>AtSOT12</i> transcripts. The stop codon and the putative polyadenylation signal sequence are highlighted in red. The last nucleotides at the polyadenylation sites are in uppercase and bolded. (B) Percentages of polyadenylation at the major polyadenylation sites in wild type, <i>shi1</i> and <i>shi4</i> mutants.</p

Gene expression and subcellular localization of the SHI1.

<p>(A) Promoter-GUS analysis showing GUS expression at different developmental stages and different organs. (B) Northern blot showing the transcript levels of <i>SHI1</i> gene in different organs. (C) The subcellular localization of the SHI-GFP fusion protein. The left panel shows the nuclear localization of the fusion protein and the right panel shows fluorescent staining of the nucleus by Hoechst 33342. (D) Northern blot showing the <i>SHI1</i> gene expression in response to different abiotic stress treatments. Control, control without treatment; NaCl, 100 mM NaCl for 12 h; NaNO₃, 100 mM NaNO₃ for 12 h; KCl, 100 mM KCl for 12 h; LiCl, 100 mM LiCl for 12 h; Sorbitol, 200 mM sorbitol for 12 h; Cold, 4°C for 24 h; ABA, 100 µM ABA for 3 h; Desicc, desiccation for 15 min; pH 3.0, pH 3.0 for 12 h; pH 8.5, pH 8.5 for 12 h. Tubulin is shown as a loading control.</p

Map-based cloning and characterization of the <i>SHI4</i> locus.

<p>(A) Identification of the <i>shi4</i> mutation. (B) Hypersensitive phenotype of the <i>shi4</i> mutant to low temperature. (C) Subcellular localization of SHI4-GFP fusion protein. Showing is GFP fluorescence and merged image of GFP fluorescence in the root.</p

The Arabidopsis RNA Binding Protein with K Homology Motifs, SHINY1, Interacts with the C-terminal Domain Phosphatase-like 1 (CPL1) to Repress Stress-Inducible Gene Expression

<div><p>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 <i>shi1</i> 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 <i>shi1</i> mutant is more sensitive to cold treatment during vegetative growth and insensitive to abscisic acid in seed germination, resembling the phenotypes of <i>shi4</i> that is allelic to the <i>cpl1</i> mutant. Both SHI1 and SHI4/CPL1 are nuclear-localized proteins. SHI1 interacts with SHI4/CPL1 <i>in vitro</i> and <i>in vivo</i>. Loss-of-function mutations in <i>shi1</i> and <i>shi4</i> resulted in similar changes in the expression of some stress-inducible genes. Moreover, both <i>shi1</i> and <i>shi4</i> 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.</p></div

Stress response phenotype of the <i>shi1</i> mutant.

<p>(A) Seed germination with or without 1.0 µM ABA. (B) Seed germination at different concentrations of ABA. Values are means ± SD (n = 3). (C) Plant growth at room temperature and low temperature for indicated days.</p

Detection of stress-inducible gene expression in wild type, <i>shi1</i> and <i>shi4</i> mutants.

<p>(A) Northern blot showing the transcript levels of stress-inducible genes in wild type, <i>shi1</i> and <i>shi4</i> mutants. Tubulin is shown as a loading control. (B) Quantitative measurements of the relative signal strengths compared with the signal strength in the wild type control. The signal strengths were measured by Imaging J. Each signal strength was normalized with the signal strength of the corresponding tubulin.</p

The hypersensitivity of <i>med18</i> in response to ABA and salt stress.

<p>(A) The root growth of Col-0 wild type and <i>med18</i> mutants on control ½ MS and ABA or NaCl-containing MS medium at 7days after transfer. (B) Quantification of the primary root length of Col-0 wild type and <i>med18</i> mutants at 7 days after transfer to control and ABA or NaCl- containing MS medium. Three-day-old seedlings were transferred to the ½ MS or ABA/NaCl containing media and primary root length was measured after 7 days. Values indicate means ± SD (n = 20). Asterisks indicate significant differences compared to WT Col under the same treatments. Significance between the mean values were analyzed with Student’s <i>t</i> test (* P< 0.05).</p

An <i>Arabidopsis</i> Nucleoporin <i>NUP85</i> modulates plant responses to ABA and salt stress

<div><p>Several nucleoporins in the nuclear pore complex (NPC) have been reported to be involved in abiotic stress responses in plants. However, the molecular mechanism of how NPC regulates abiotic stress responses, especially the expression of stress responsive genes remains poorly understood. From a forward genetics screen using an abiotic stress-responsive luciferase reporter (<i>RD29A-LUC</i>) in the <i>sickle-1</i> (<i>sic-1</i>) mutant background, we identified a suppressor caused by a mutation in <i>NUCLEOPORIN 85</i> (<i>NUP85</i>), which exhibited reduced expression of <i>RD29A-LUC</i> in response to ABA and salt stress. Consistently, the ABA and salinity induced expression of several stress responsive genes such as <i>RD29A</i>, <i>COR15A</i> and <i>COR47</i> was significantly compromised in <i>nup85</i> mutants and other nucleoporin mutants such as <i>nup160</i> and <i>hos1</i>. Subsequently, Immunoprecipitation and mass spectrometry analysis revealed that NUP85 is potentially associated with HOS1 and other nucleoporins within the nup107-160 complex, along with several mediator subunits. We further showed that there is a direct physical interaction between MED18 and NUP85. Similar to <i>NUP85</i> mutations, <i>MED18</i> mutation was also found to attenuate expression of stress responsive genes. Taken together, we not only revealed the involvement of <i>NUP85</i> and other nucleoporins in regulating ABA and salt stress responses, but also uncovered a potential relation between NPC and mediator complex in modulating the gene expression in plants.</p></div