Cold temperatures trigger the expression of the CBF family of transcription factors, which in turn activate many downstream genes that confer freezing tolerance to plants. It has been shown previously that the cold regulation of CBF3 involves an upstream bHLH-type transcription factor, ICE1. ICE1 binds to the Myc recognition sequences in the CBF3 promoter. Apart from Myc recognition sequences, CBF promoters also have Myb recognition sequences. We report here that the Arabidopsis MYB15 is involved in cold-regulation of CBF genes and in the development of freezing tolerance. The MYB15 gene transcript is up-regulated by cold stress. The MYB15 protein interacts with ICE1 and binds to Myb recognition sequences in the promoters of CBF genes. Overexpression of MYB15 results in reduced expression of CBF genes whereas its loss-of-function leads to increased expression of CBF genes in the cold. The myb15 mutant plants show increased tolerance to freezing stress whereas its overexpression reduces freezing tolerance. Our results suggest that MYB15 is part of a complex network of transcription factors controlling the expression of CBFs and other genes in response to cold stress.Cold temperatures have a huge impact on the survivability and distribution of living organisms. Plants, being sessile, have evolved efficient mechanisms to sense and adapt to low temperature stress. Plant responses to adverse low temperature are manifested at physiological, molecular and biochemical levels. Many temperate plants have the potential to increase their freezing tolerance after a prior exposure to nonfreezing temperatures, a process known as cold acclimation (1-3). At the molecular level, a specific set of proteins is induced in response to low temperature, which helps plants cope with chilling and freezing stress (4 -8). Proteins induced during cold acclimation include enzymes involved in respiration and metabolism of carbohydrates, lipids, phenylpropanoids, and antioxidants, molecular chaperones, antifreeze proteins, and many others with a presumed function in tolerance to cellular dehydration caused by apoplastic freezing (1, 4, 9).Promoters of many of the cold-responsive genes have the DRE/CRT/LTRE (dehydration responsive element/C-repeat/ low temperature responsive element) sequence, a cis element necessary and sufficient for gene transcription under cold stress (10 -12). The CBF/DREB family of transcription factors binds to this sequence and activates cold-responsive genes (11, 13). The CBF transcription factor genes are also induced by cold, and their induction is regulated by components upstream in the cold response pathways (14 -17). In addition, it has been shown that a loss-of-function mutation in CBF2 results in increased expression of CBF1 and CBF3, implying that CBF2 negatively regulates the expression of CBF1 and CBF3 (18).In addition to the CBF pathway, recent studies have revealed the presence of parallel pathways associated with cold acclimation (19 -21). Some important components mediating cold tolerance through CBF-inde...

Argonautes (AGOs) are conserved proteins that contain an RNA-binding PAZ domain and an RNase H-like PIWI domain. In Arabidopsis, except for AGO1, AGO4 and AGO7, the roles of seven other AGOs in gene silencing are not known. We found that a mutation in AGO6 partially suppresses transcriptional gene silencing in the DNA demethylase mutant ros1-1. In ago6-1ros1-1 plants, RD29A promoter short interfering RNAs (siRNAs) are less abundant, and cytosine methylation at both transgenic and endogenous RD29A promoters is reduced, compared to that in ros1-1. Interestingly, the ago4-1 mutation has a stronger suppression of the transcriptional silencing phenotype of ros1-1 mutant. Analysis of cytosine methylation at the endogenous MEA-ISR, AtREP2 and SIMPLEHAT2 loci revealed that the CpNpG and asymmetric methylation levels are lower in either of the ago6-1 and ago4-1 single mutants than those in the wild type, and the levels are the lowest in the ago6-1ago4-1 double mutant. These results suggest that AGO6 is important for the accumulation of specific heterochromatin-related siRNAs, and for DNA methylation and transcriptional gene silencing, this function is partly redundant with AGO4.

To study the genetic control of plant responses to cold stress, Arabidopsis thaliana mutants were isolated by a screen for mutations that impair cold-induced transcription of the CBF3-LUC reporter gene. We report here the characterization and cloning of a mutated gene, atnup160-1, which causes reduced CBF3-LUC induction under cold stress. atnup160-1 mutant plants display altered cold-responsive gene expression and are sensitive to chilling stress and defective in acquired freezing tolerance. AtNUP160 was isolated through positional cloning and shown to encode a putative homolog of the animal nucleoporin Nup160. In addition to the impaired expression of CBF genes, microarray analysis revealed that a number of other genes important for plant cold tolerance were also affected in the mutants. The atnup160 mutants flower early and show retarded seedling growth, especially at low temperatures. AtNUP160 protein is localized at the nuclear rim, and poly(A)-mRNA in situ hybridization shows that mRNA export is defective in the atnup160-1 mutant plants. Our study suggests that Arabidopsis AtNUP160 is critical for the nucleocytoplasmic transport of mRNAs and that it plays important roles in plant growth and flowering time regulation and is required for cold stress tolerance.In eukaryotic cells, the genome is enclosed within the nucleus. Nucleocytoplasmic transport of macromolecules across the nuclear membrane occurs through channels formed by nuclear pore complexes (NPCs). Embedded in the double-lipid bilayer nuclear envelope, NPCs form a ringlike structure surrounding a central pore that is believed to facilitate the bidirectional transport of RNAs, proteins, and ribonucleoprotein particles and, at the same time, to allow the diffusion of small molecules and ions across the double membrane (reviewed in reference 4). The overall three-dimensional architecture and transport mechanisms seem to be highly conserved from yeasts to mammals. In Saccharomyces cerevisiae, NPCs are constructed from ϳ30 different nucleoporins with a combined mass of ϳ50 MDa (26). The mammalian NPCs are much larger complexes (ϳ120 MDa) composed of ϳ80 different proteins (10). Although the structural organization of NPCs, the transport mechanism across the channels, and the function of individual nucleoporins have been extensively studied in yeast and vertebrates, very little is known about NPCs in plants.Recently, it was reported that the Arabidopsis thaliana proteins MOS3/SAR3 and SAR1 share high sequence similarities with human nucleoporins Nup96 and Nup160, respectively (25, 34). The putative nucleoporin MOS3/SAR3 was localized at the nuclear rim. The studies suggested that nucleocytoplasmic trafficking plays an important role in plant disease resistance, hormone signaling, and development (25,34). In the present report, we provide evidence that Arabidopsis nucleoporin AtNUP160/SAR1 controls nucleocytoplasmic transport of RNAs and plays important roles in seedling growth, flowering time regulation, and cold stress tolerance.Low temperature is one of...

Ten eleven translocation (TET) enzymes (TET1/TET2/TET3) and thymine DNA glycosylase (TDG) play crucial roles in early embryonic and germ cell development by mediating DNA demethylation. However, the molecular mechanisms that regulate TETs/TDG expression and their role in cellular differentiation, including that of the pancreas, are not known. Here, we report that (i) TET1/2/3 and TDG can be direct targets of the microRNA miR-26a, (ii) murine TETs, especially TET2 and TDG, are down-regulated in islets during postnatal differentiation, whereas miR-26a is up-regulated, (iii) changes in 5-hydroxymethylcytosine accompany changes in TET mRNA levels, (iv) these changes in mRNA and 5-hydroxymethylcytosine are also seen in an in vitro differentiation system initiated with FACS-sorted adult ductal progenitor-like cells, and (v) overexpression of miR-26a in mice increases postnatal islet cell number in vivo and endocrine/acinar colonies in vitro. These results establish a previously unknown link between miRNAs and TET expression levels, and suggest a potential role for miR-26a and TET family proteins in pancreatic cell differentiation.T en eleven translocation (TET) enzymes and thymine DNA glycosylase (TDG) are implicated in active DNA demethylation (1-3). The three TET family enzymes oxidize 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), and subsequently to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (1, 2, 4, 5). TDG, a base excision repair glycosylase, replaces 5fC and 5caC with an unmodified cytosine via DNA repair (5, 6). Despite these advances, the molecular mechanisms underlying TETs/ TDG regulation are still not known. In addition, although recent data suggest a role of TET and 5hmC in embryonic stem cells and primordial germ cells (2, 7-12), evidence for enzymatic demethylation by TET enzymes during differentiation of cells of later stages, such as the postnatal and adult stem cells of various organs including pancreas, remains very limited (13-16).MicroRNAs (miRNAs) are an abundant class of small, highly conserved noncoding RNAs that bind the 3′-untranslated regions (UTRs) of protein-coding genes to suppress gene expression. Accumulating data have demonstrated that miRNAs are critical for many developmental and cellular processes, including organogenesis and differentiation (17). However, the role of miRNAs in TET expression and active DNA demethylation remains unclear.Three major cell lineages exist in the adult pancreas-duct, acinar, and endocrine cells. The endocrine pancreas is composed of several hormone-releasing cells, including the insulin-secreting beta cells and glucagon-secreting alpha cells. Many transcription factors are known to control pancreas development (18). For example, the expression of pancreatic and duodenal homeobox 1 (Pdx1) in embryonic foregut region induces pancreas commitment (19,20), and those early progenitor cells have the potential to give rise to all three pancreatic lineages (21,22). Subsequent activation of another transcription factor, neurogenin 3 (N...

DNA methylation is an important epigenetic mark for transcriptional gene silencing (TGS) in diverse organisms [1][2][3][4][5][6] . Recent studies suggest that the methylation status of a number of genes is dynamically regulated by methylation and demethylation [7][8][9][10] . In Arabidopsis, active DNA demethylation is mediated by the ROS1 (repressor of silencing 1) subfamily of 5-methylcytosine DNA glycosylases through a base excision repair pathway 8,[10][11][12][13] . These demethylases play critical roles in erasing DNA methylation and preventing TGS of target genes 7,8,10 . However, it is not known how the demethylases are targeted to specific sequences. We report here the identification of ROS3, an essential regulator of DNA demethylation that contains an RNA recognition motif. Analysis of ros3 mutant and ros1ros3 double mutant suggests that ROS3 acts in the same genetic pathway as ROS1 to prevent DNA hypermethylation and TGS. Gel mobility shift assays and analysis of ROS3 immunoprecipitate from plant extracts showed that ROS3 binds to small RNAs in vitro and in vivo. Immunostaining shows that ROS3 and ROS1 proteins colocalize in discrete foci dispersed throughout the nucleus. These results demonstrate a critical role for ROS3 in preventing DNA hypermethylation and suggest that DNA demethylation by ROS1 may be guided by RNAs bound to ROS3.We developed a sensitive assay system in Arabidopsis to genetically dissect active DNA demethylation 10,14 . The system consists of the RD29A-LUC transgene (firefly luciferase reporter driven by the stress-responsive RD29A promoter) and the non-allelic endogenous RD29A gene. The RD29A promoter is subjected to continuous siRNA-directed DNA methylation such that active DNA demethylation is required to keep the RD29A and RD29A-LUC genes transcriptionally active. In ros1 mutants, the RD29A promoter for both the transgene and endogenous gene becomes hypermethylated and both genes are silenced 10 . In addition, the 35S-NPTII transgene linked to RD29A-LUC is also silenced such that ros1 mutant plants are sensitive to kanamycin. We isolated the ros3 mutant from a T-DNA mutagenized population 15 Fig. 1a and Supplementary Fig. 1a and 1b) as well as sensitivity to kanamycin (Fig. 1b). Genetic analysis indicated that the ros3 mutation is recessive and affects a nuclear gene (data not shown).Northern blot ( Fig. 1c) and nuclear run-on ( Fig. 1d) Fig. 2a and 2b). Treatment with the cytosine methylation inhibitor 5-aza-2'-deoxycytidine increased RD29A-LUC expression in the ros3 mutant to the wild type level ( Supplementary Fig. 3). These results suggest that DNA hypermethylation is responsible for the TGS in ros3 mutant plants.The nrpd1a-1 mutation in the largest subunit of RNA polymerase IVa blocks the accumulation of 24-nt siRNAs corresponding to the RD29A promoter (data not shown). Analysis of nrpd1aros3 double mutant showed that the nrpd1a mutation causes a significant increase in RD29A-LUC expression ( Fig. 2c and 2d) and substantial decrease in CpG, CpNpG and CpNpN methyl...