Cold temperatures trigger the expression of the CBF family of transcription factors, which in turn activate many downstream genes that confer chilling and freezing tolerance to plants. We report here the identification of ICE1 (inducer of CBF expression 1), an upstream transcription factor that regulates the transcription of CBF genes in the cold. An Arabidopsis ice1 mutant was isolated in a screen for mutations that impair cold-induced transcription of a CBF3 promoter-luciferase reporter gene. The ice1 mutation blocks the expression of CBF3 and decreases the expression of many genes downstream of CBFs, which leads to a significant reduction in plant chilling and freezing tolerance. ICE1 encodes a MYC-like bHLH transcriptional activator. ICE1 binds specifically to the MYC recognition sequences in the CBF3 promoter. ICE1 is expressed constitutively, and its overexpression in wild-type plants enhances the expression of the CBF regulon in the cold and improves freezing tolerance of the transgenic plants.

SummaryThe abiotic stresses of drought, salinity and freezing are linked by the fact that they all decrease the availability of water to plant cells. This decreased availability of water is quantified as a decrease in water potential. Plants resist low water potential and related stresses by modifying water uptake and loss to avoid low water potential, accumulating solutes and modifying the properties of cell walls to avoid the dehydration induced by low water potential and using protective proteins and mechanisms to tolerate reduced water content by preventing or repairing cell damage. Salt stress also alters plant ion homeostasis, and under many conditions this may be the predominant factor affecting plant performance. Our emphasis is on experiments that quantify resistance to realistic and reproducible low water potential (drought), salt and freezing stresses while being suitable for genetic studies where a large number of lines must be analyzed. Detailed protocols for the use of polyethylene glycol-infused agar plates to impose low water potential stress, assay of salt tolerance based on root elongation, quantification of freezing tolerance and the use of electrolyte leakage experiments to quantify cellular damage induced by freezing and low water potential are also presented.

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...

cold stress ͉ RING finger protein I n response to low temperatures, numerous genes are induced in plants (1). Three transcription factors known as C-repeat (CRT)-binding factors (CBFs) or dehydration-responsive element (DRE)-binding protein can bind to CRT͞DRE cis elements in the promoters and activate transcription of many of the cold-responsive genes (2-4). The CBF genes are transiently induced by low temperature, and this induction precedes that of the downstream cold-responsive genes (4-6). ICE1 is an upstream constitutively expressed transcription factor that controls the cold induction of CBF3 (7).Previously, a genetic screen using Arabidopsis plants that express the firefly luciferase reporter gene driven by the CRT͞ DRE element-containing RD29A promoter led to the identification of HOS1 (high expression of osmotically responsive gene 1), a negative regulator of the CBF regulon (8). CBFs and their downstream genes show enhanced cold induction in loss-offunction hos1 mutant plants (8). HOS1 was predicted to encode a 915-aa protein containing a short motif near the N terminus that is similar to the RING finger domain found in a group of animal proteins known as inhibitor of apoptosis (9). Recently, several plant proteins containing RING finger domain have been shown to function as ubiquitin E3 ligases in hormonal signaling and development (10).Transfer and covalent ligation of the 76-aa protein ubiquitin to target proteins is a central part of the ubiquitination pathway, involving three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3 is responsible for recruiting specific target proteins for ubiquitination (10). Polyubiquitinated proteins are recognized by the 26S proteasome and degraded. Arabidopsis has two E1 isoforms (11) and at least 37 E2 enzymes (12). In contrast, Ͼ1,300 genes are predicted to encode for E3 ubiquitin ligases (10). The large number and diversity of E3 ligases confer specificity upon the ubiquitination pathway. On the basis of subunit composition and action mode, four E3 types have been described in plants: homology to E6AP C terminus; RING͞U box; a complex of Skp1, CDC53, and F-box protein (SCF); and anaphasepromoting complex. Arabidopsis contains at least 469 predicted RING-domain-containing proteins (13). However, the physiological functions for most of them are not known. The importance of RING proteins in plant responses to cold stress through the ubiquitin͞proteasome pathway has not been investigated. In fact, the involvement of the ubiquitin͞proteasome pathway in cold stress has not been explored in any system.In this study, we have found that cold response in Arabidopsis is attenuated by the proteasome pathway. We demonstrated that the Arabidopsis HOS1 is a functional RING finger protein that has ubiquitin E3 ligase activity. We found that HOS1 physically interacts with ICE1. Both in vitro and in vivo ubiquitination assays showed that HOS1 is required for ICE1 ubiquitination. Furthermore, we discovered that cold induces...

The phytohormone abscisic acid (ABA) modulates the expression of many genes important to plant growth and development and to stress adaptation. In this study, we found that an APETALA2/EREBP-type transcription factor, AtERF7, plays an important role in ABA responses. AtERF7 interacts with the protein kinase PKS3, which has been shown to be a global regulator of ABA responses. AtERF7 binds to the GCC box and acts as a repressor of gene transcription. AtERF7 interacts with the Arabidopsis thaliana homolog of a human global corepressor of transcription, AtSin3, which in turn may interact with HDA19, a histone deacetylase. The transcriptional repression activity of AtERF7 is enhanced by HDA19 and AtSin3. Arabidopsis plants overexpressing AtERF7 show reduced sensitivity of guard cells to ABA and increased transpirational water loss. By contrast, AtERF7 and AtSin3 RNA interference lines show increased sensitivity to ABA during germination. Together, our results suggest that AtERF7 plays an important role in ABA responses and may be part of a transcriptional repressor complex and be regulated by PKS3.

Proteins containing the forkhead-associated domain (FHA) are known to act in biological processes such as DNA damage repair, protein degradation, and signal transduction. Here we report that DAWDLE (DDL), an FHA domain-containing protein in Arabidopsis, acts in the biogenesis of miRNAs and endogenous siRNAs. Unlike mutants of genes known to participate in the processing of miRNA precursors, such as dcl1, hyponastic leaves1, and serrate, ddl mutants show reduced levels of pri-miRNAs as well as mature miRNAs. Promoter activity of MIR genes, however, is not affected by ddl mutations. DDL is an RNA binding protein and is able to interact with DCL1. In addition, we found that SNIP1, the human homolog of DDL, is involved in miRNA biogenesis and interacts with Drosha. Therefore, we uncovered an evolutionarily conserved factor in miRNA biogenesis. We propose that DDL participates in miRNA biogenesis by facilitating DCL1 to access or recognize pri-miRNAs.class of sequence-specific repressors of gene expression in eukaryotes is 20-to 24-nt small RNAs, which include miRNAs and siRNAs. miRNAs are processed from stem-loop precursor RNAs, called pri-miRNAs. In animals, pri-miRNAs are processed in the nucleus by Drosha to form pre-miRNAs, which are exported to the cytoplasm by exportin 5 and further processed by Dicer to produce mature miRNAs (reviewed in ref. 1). In Arabidopsis, mature miRNAs are produced through two processing steps (primiRNAs to pre-miRNAs and pre-miRNAs to miRNAs) in the nucleus by DCL1 with the assistance of HYL1 and SERRATE (reviewed in ref. 2). After processing, miRNAs are 2Ј-O-methylated by HEN1 (3). siRNAs are produced from long, double-stranded RNAs. Plants contain several classes of endogenous siRNAs, such as transacting siRNAs (ta-siRNAs), natural antisense siRNAs (nat-siRNAs), and siRNAs from endogenous repeat sequences and transposons (reviewed in ref. 4).The forkhead-associated (FHA) domain is an 80-to 100-aa module that is thought to recognize phosphothreonine-containing motifs and mediate protein-protein interactions in prokaryotes and eukaryotes (reviewed in ref. 5). DAWDLE (DDL) is a nuclearlocalized FHA domain-containing protein in Arabidopsis (6). DDL appears to act in multiple developmental processes such as growth, fertility, and root, shoot, and floral morphogenesis (6).Smad nuclear interacting protein 1 (SNIP1) is a human FHA domain-containing protein that functions as an inhibitor of TGF-␤ and NF-B signaling pathways by competing with the TGF-␤ signaling protein Smad4 and the NF-B transcription factor p65/ RelA for binding to the transcriptional coactivator p300 (7,8). Recently, Fujii et al. (9) reported that SNIP1 interacts with the transcription factor/oncoprotein c-Myc and enhances its activity by bridging its interaction with p300.Here we report that DDL is required for the accumulation of miRNAs and endogenous siRNAs in Arabidopsis. Its affinity for RNA, its potential association with DCL1, and the reduction in pri-miRNA levels in ddl loss-of-function mutants suggest that DDL is...

Stem cells are crucial in morphogenesis in plants and animals. Much is known about the mechanisms that maintain stem cell fates or trigger their terminal differentiation. However, little is known about how developmental time impacts stem cell fates. Using Arabidopsis floral stem cells as a model, we show that stem cells can undergo precise temporal regulation governed by mechanisms that are distinct from, but integrated with, those that specify cell fates. We show that two microRNAs, miR172 and miR165/166, through targeting APETALA2 and type III homeodomain-leucine zipper (HD-Zip) genes, respectively, regulate the temporal program of floral stem cells. In particular, we reveal a role of the type III HD-Zip genes, previously known to specify lateral organ polarity, in stem cell termination. Both reduction in HD-Zip expression by over-expression of miR165/166 and mis-expression of HD-Zip genes by rendering them resistant to miR165/166 lead to prolonged floral stem cell activity, indicating that the expression of HD-Zip genes needs to be precisely controlled to achieve floral stem cell termination. We also show that both the ubiquitously expressed ARGONAUTE1 (AGO1) gene and its homolog AGO10, which exhibits highly restricted spatial expression patterns, are required to maintain the correct temporal program of floral stem cells. We provide evidence that AGO10, like AGO1, associates with miR172 and miR165/166 in vivo and exhibits “slicer” activity in vitro. Despite the common biological functions and similar biochemical activities, AGO1 and AGO10 exert different effects on miR165/166 in vivo. This work establishes a network of microRNAs and transcription factors governing the temporal program of floral stem cells and sheds light on the relationships among different AGO genes, which tend to exist in gene families in multicellular organisms.

The adverse effects of high salt on plants include Na ؉ toxicity and hyperosmotic and oxidative stresses. The plasma membrane-localized Na ؉ ͞H ؉ antiporter SOS1 functions in the extrusion of toxic Na ؉ from cells and is essential for plant salt tolerance. We report here that, under salt or oxidative stress, SOS1 interacts through its predicted cytoplasmic tail with RCD1, a regulator of oxidativestress responses. Without stress treatment, RCD1 is localized in the nucleus. Under high salt or oxidative stress, RCD1 is found not only in the nucleus but also in the cytoplasm. Like rcd1 mutants, sos1 mutant plants show an altered sensitivity to oxidative stresses. The rcd1mutation causes a decrease in salt tolerance and enhances the salt-stress sensitivity of sos1 mutant plants. Several genes related to oxidative-stress tolerance were found to be regulated by both RCD1 and SOS1. These results reveal a previously uncharacterized function of a plasma membrane Na ؉ ͞H ؉ antiporter in oxidativestress tolerance and shed light on the cross-talk between the ion-homeostasis and oxidative-stress detoxification pathways involved in plant salt tolerance.salt stress ͉ reactive oxygen species ͉ hydrogen peroxide stress