The evolutionarily conserved MOS4-associated complex

Recent work in plant immunity has shown that MOS4, a known intermediate in R protein mediated resistance, is a core member of the nuclear MOS4-associated complex (MAC). This complex is highly conserved in eukaryotes, as orthologous complexes known as the CDC5L-SNEV^Prp19-Pso4 complex and the Nineteen complex (NTC) were previously identified in human and yeast, respectively. The involvement of these complexes in pre-mRNA splicing and spliceosome assembly suggests that the MAC probably has a similar function in plants. Double mutants of any two MAC components are lethal, whereas single mutants of the MAC core components mos4, Atcdc5, mac3, and prl1 are all viable and display pleiotropic defects. This suggests that while the MAC is required for some essential biological function such as splicing, individual MAC components are not crucial for complex functionality and likely have regulatory roles in other biological processes such as plant immunity and flowering time control. Future studies on MAC components in Arabidopsis will provide further insight into the regulatory mechanisms of the MAC on specific biological processes.     

Two putative RNA-binding proteins function with unequal genetic redundancy in the MOS4-associated complex

The MOS4-associated complex (MAC) is a highly conserved nuclear protein complex associated with the spliceosome. We recently purified the MAC from Arabidopsis (Arabidopsis thaliana) nuclei, identified its potential components by mass spectrometry, and showed that at least five core proteins in the MAC are required for defense responses in plants. Here, we report the characterization of a putative RNA-binding protein identified in the MAC named MAC5A and its close homolog MAC5B. We confirmed that MAC5A is a component of the MAC through coimmunoprecipitation with the previously described MAC protein CELL DIVISION CYCLE5 from Arabidopsis. In addition, like all other characterized MAC proteins, MAC5A fused to the Green Fluorescent Protein localizes to the nucleus. Double mutant analysis revealed that MAC5A and MAC5B are unequally redundant and that a double mac5a mac5b mutant results in lethality. Probably due to this partial redundancy, mac5a and mac5b single mutants do not exhibit enhanced susceptibility to virulent or avirulent pathogen infection. However, like other MAC mutations, mac5a-1 partially suppresses the autoimmune phenotypes of suppressor of npr1-1, constitutive1 (snc1), a gain-of-function mutant that expresses a deregulated Resistance protein. Our results suggest that MAC5A is a component of the MAC that contributes to snc1- mediated autoimmunity.     

HD2C interacts with HDA6 and is involved in ABA and salt stress response in Arabidopsis

HD2 proteins are plant specific histone deacetylases. Four HD2 proteins, HD2A, HD2B, HD2C, and HD2D, have been identified in Arabidopsis. It was found that the expression of HD2A, HD2B, HD2C, and HD2D was repressed by ABA and NaCl. To investigate the function of HD2 proteins further, two HD2C T-DNA insertion lines of Arabidopsis, hd2c-1 and hd2c-3 were identified. Compared with wild-type plants, hd2c-1 and hd2c-3 plants displayed increased sensitivity to ABA and NaCl during germination and decreased tolerance to salt stress. These observations support a role of HD2C in the ABA and salt-stress response in Arabidopsis. Moreover, it was demonstrated that HD2C interacted physically with a RPD3-type histone deacetylase, HDA6, and bound to histone H3. The expression of ABA-responsive genes, ABI1 and ABI2, was increased in hda6, hd2c, and hda6/hd2c-1 double mutant plants, which was associated with increased histone H3K9K14 acetylation and decreased histone H3K9 dimethylation. Taken together, our results suggested that HD2C functionally associates with HDA6 and regulates gene expression through histone modifications.     

Identification of quantitative trait loci for ABA responsiveness at the seedling stage associated with ABA-regulated gene expression in common wheat

Responsiveness to abscisic acid (ABA) during vegetative growth plays an important role in regulating adaptive responses to various environmental conditions, including activation of a number of ABA-responsive genes. However, the relationship between gene expression and responsiveness to ABA at the seedling stage has not been well studied in wheat. In the present study, quantitative trait locus (QTL) analysis for ABA responsiveness at the seedling stage was performed using recombinant inbred lines derived from a cross between common wheat cultivars showing different ABA responsiveness. Five QTLs were found to be significant, located on chromosomes 1B, 2A, 3A, 6D and 7B. The QTL with the greatest effect was located on chromosome 6D and explained 11.12% of the variance in ABA responsiveness. The other QTLs each accounted for approximately 5–8% of the phenotypic variation. Expression analyses of three ABA-responsive Cor/Lea genes, Wdhn13, Wrab15 and Wrab17, showed that allelic differences in QTLs on chromosomes 2A, 6D and 7B influenced expression of these genes in seedlings treated with ABA. The 3A QTL appeared to be involved in the regulatory system of Wdhn13 and Wrab15, but not Wrab17. The effects of the 2A and 6D QTLs on gene expression were relatively large. The combination of alleles at the QTLs resulted in an additive or synergistic effect on Cor/Lea expression. These results indicate that the QTLs influencing ABA responsiveness are associated with ABA-regulated gene expression and suggest that the QTL on chromosome 6D with the largest effect acts as a key regulator of ABA responses including seedling growth arrest and gene expression during the vegetative stage.     

CYCLIN-DEPENDENT KINASE G1 Is Associated with the Spliceosome to Regulate CALLOSE SYNTHASE5 Splicing and Pollen Wall Formation in Arabidopsis

Arabidopsis thaliana CYCLIN-DEPEDENT KINASE G1 (CDKG1) belongs to the family of cyclin-dependent protein kinases that were originally characterized as cell cycle regulators in eukaryotes. Here, we report that CDKG1 regulates pre-mRNA splicing of CALLOSE SYNTHASE5 (CalS5) and, therefore, pollen wall formation. The knockout mutant cdkg1 exhibits reduced male fertility with impaired callose synthesis and abnormal pollen wall formation. The sixth intron in CalS5 pre-mRNA, a rare type of intron with a GC 5′ splice site, is abnormally spliced in cdkg1. RNA immunoprecipitation analysis suggests that CDKG1 is associated with this intron. CDKG1 contains N-terminal Ser/Arg (RS) motifs and interacts with splicing factor Arginine/Serine-Rich Zinc Knuckle-Containing Protein33 (RSZ33) through its RS region to regulate proper splicing. CDKG1 and RS-containing Zinc Finger Protein22 (SRZ22), a splicing factor interacting with RSZ33 and U1 small nuclear ribonucleoprotein particle (snRNP) component U1-70k, colocalize in nuclear speckles and reside in the same complex. We propose that CDKG1 is recruited to U1 snRNP through RSZ33 to facilitate the splicing of the sixth intron of CalS5.     

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.     

Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3' end formation

tRNA splicing is a fundamental process required for cell growth and division. The first step in tRNA splicing is the removal of introns catalyzed in yeast by the tRNA splicing endonuclease. The enzyme responsible for intron removal in mammalian cells is unknown. We present the identification and characterization of the human tRNA splicing endonuclease. This enzyme consists of HsSen2, HsSen34, HsSen15, and HsSen54, homologs of the yeast tRNA endonuclease subunits. Additionally, we identified an alternatively spliced isoform of SEN2 that is part of a complex with unique RNA endonuclease activity. Surprisingly, both human endonuclease complexes are associated with pre-mRNA 3' end processing factors. Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA. These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.     

Optically pure abscisic Acid analogs-tools for relating germination inhibition and gene expression in wheat embryos

We report an examination of the structural requirements of the abscisic acid (ABA) recognition response in wheat dormant seed embryos using optically pure isomers of ABA analogs. These compounds include permutations to the ABA structure with either an acetylene or a trans bond at C-4 C-5, and either a single or double bond at the C-2′ C-3′ double bond. (R)-ABA and the three isomers with the same configuration at C-1′ as natural ABA were found to be effective germination inhibitors. The biologically active ABA analogs exhibited differential effects on ABA-responsive gene expression. All the ABA analogs that inhibited germination induced two ABA-responsive genes, wheat group 3 lea and dhn (rab). However, (R)-ABA and (S)-dihydroABA were less effective in inducing the ABA-responsive gene Em within the time that embryonic germination was inhibited.     

Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.