Expression Of Cold-responsive Genes Research Articles

A nuclear-localized protein, KOLD SENSITIV-1, affects the expression of cold-responsive genes during prolonged chilling in Arabidopsis

Plants respond to cold by transcriptional and metabolic responses which underlie tolerance and acclimation mechanisms, but details at the molecular level are incomplete. Here we describe KOLD SENSITIV-1 ( KOS1), a new gene required for responses to cold. KOS1 protein is predicted to have coiled-coil, Structural Maintenance of Chromosomes and nuclear-targeting domains. GFP-labeled KOS1 localizes to the nucleus. Null mutants could not be isolated but two independent knockdown T-DNA mutants were obtained. Growth and development of kos1 knockdown mutant plants was comparable to wild type when grown at 21 °C. However, when grown at 4 °C these mutants exhibited accelerated leaf yellowing and smaller rosette size than wild type. Quantitative RT-PCR revealed that in the cold kos1 mutants had reduced expression of cold-responsive transcripts COR15A, COR15B, BAM3 and AMY3. Metabolite profiling revealed that ascorbate levels were lower in the mutants in the cold relative to wild type. KOS1 therefore represents a new gene that influences the regulation of transcript and metabolite levels in response to prolonged chilling temperatures.

Overexpression of a homopeptide repeat-containing bHLH protein gene (OrbHLH001) from Dongxiang Wild Rice confers freezing and salt tolerance in transgenic Arabidopsis

Dongxiang Wild Rice (Oryza rufipogon) is the northernmost wild rice in the world known to date and has extremely high cold tolerance and many other adversity-resistant properties. To identify the genes responsible for the high stress tolerance, we isolated and characterized a basic helix-loop-helix (bHLH) protein gene OrbHLH001 from Dongxiang Wild Rice. The gene encodes an ICE1-like protein containing multiple homopeptide repeats. Expression of OrbHLH001 is induced by salt stress and is predominant in the shoots of wild rice seedlings. Overexpression of OrbHLH001 enhanced the tolerance to freezing and salt stresses in transgenic Arabidopsis. Examination of the expression of cold-responsive genes in transgenic Arabidopsis showed that the function of OrbHLH001 differs from that of ICE1 and is independent of a CBF/DREB1 cold-response pathway.

Overexpression of SlCZFP1, a Novel TFIIIA-type Zinc Finger Protein from Tomato, Confers Enhanced Cold Tolerance in Transgenic Arabidopsis and Rice

The C2H2-type (also called TFIIIA-type) zinc finger protein family is a major family of eukaryotic transcription factors. C2H2-type zinc finger proteins in plants play important roles in regulating various developmental processes or responses to abiotic stresses. We isolated a complementary deoxyribonucleic acid (cDNA) clone, designated Solanumlycopersicumcold zinc finger protein 1 (SlCZFP1), from tomato by analyzing the previous microarray data of the low-temperature transcriptome of tomato. SlCZFP1 encodes a novel TFIIIA-type zinc finger protein, which contains two typical zinc finger motifs, CX2–4 CX3FX3QALGGHX3–5H, and a potential nuclear localization signal (NLS). The SlCZFP1–GFP fusion protein was localized to the nucleus in a transient expression assay. Expression of SlCZFP1 was strongly induced by cold stress, dehydration, and salt treatment, but not by abscisic acid (ABA). Overexpression of SlCZFP1 in transgenic Arabidopsis and rice induced constitutive expression of cold-regulated (COR) or cold-responsive genes and conferred enhanced tolerance to freezing or cold treatments for non-acclimate transgenic plants, compared with wild-type plants. However, there was no obvious enhancement observed in drought and salt tolerance. Our data suggest that SlCZFP1 plays an important role in plant responses to cold stress by regulating cold-responsive gene expression and that SlCZEP1 might be a useful gene for improving cold tolerance in crop plants.

Proteolytic processing of anArabidopsismembrane-bound NAC transcription factor is triggered by cold-induced changes in membrane fluidity

Changes in membrane fluidity are the earliest cellular events that occur in plant cells upon exposure to cold. This subsequently triggers physiological processes, such as calcium influx and reorganization of actin cytoskeletons, and induces expression of cold-responsive genes. The plasma-membrane-anchored NAC (NAM/ATAF/CUC) transcription factor NTL6 is of particular interest. Cold triggers proteolytic activation of the dormant NTL6 protein, which in turn elicits pathogen-resistance responses by inducing a small group of cold-inducible PR (pathogenesis-related) genes in Arabidopsis. In the present study, we show that proteolytic processing of NTL6 is regulated by cold-induced remodelling of membrane fluidity. NTL6 processing was stimulated rapidly by cold. The protein stability of NTL6 was also enhanced by cold. The effects of cold on NTL6 processing and protein stability were significantly reduced in cold-acclimatized plants, supporting the regulation of NTL6 processing by membrane fluidity. Consistent with this, although NTL6 processing was stimulated by pharmacological agents that reduce membrane fluidity and thus mimic cold, it was inhibited when plants were treated with a 18:3 unsaturated fatty acid, linolenic acid. In addition, the pattern of NTL6 processing was changed in Arabidopsis mutants with altered membrane lipid compositions. Assays employing chemicals that inhibit activities of the proteasome and proteases showed that NTL6 processing occurs via the regulated intramembrane proteolysis mechanism. Interestingly, a metalloprotease inhibitor blocked the NTL6 processing. These observations indicate that a metalloprotease activity is responsible for NTL6 processing in response to cold-induced changes in membrane fluidity.

Cucumber ( Cucumis sativus L.) over-expressing cold-induced transcriptome regulator ICE1 exhibits changed morphological characters and enhances chilling tolerance

Cold is an environmental factor that limits the growing season of cucumber and adversely affects fruit quality and productivity. ICE1 is a positive regulator of CBF3 and has a critical role in cold stress ( Chinnusamy et al., 2003). To test the function and potential use of cold-induced transcriptome regulator ICE1 (inducer of CBF expression) in improving the chill tolerance of cucumbers, the Arabidopsis ICE1 gene driven by super promoter was introduced into cucumbers by Agrobacterium-mediated transformation. Transgenic plants were obtained, and were confirmed by PCR and southern blot, then the expression of ICE1 was tested by RT-PCR. Transgenic plants exhibited dwarf phenotypes, and shorter internodes, with higher nodes in the first female flowers and a lower female flower rate of every node than wild type plants. Under cold stress, the chilling index of four transgenic lines (IF3, IF6, IF7, and IF10) was decreased significantly, indicating their improved chilling tolerance. Over-expression of ICE1 in cucumbers could induce the expression of cold-responsive genes, further activating the accumulation of soluble sugars and free proline, and inhibiting malondialdehyde (MDA) accumulation. This leads to electrolyte leakage and regulation of osmotic potential to osmotic stress under cold stress.

Cold resistance in plants: A mystery unresolved

Herbaceous temperate plants are capable of developing freezing tolerance when they are exposed to low nonfreezing temperatures. Acquired freezing tolerance involves extensive reprogramming of gene expression and metabolism. Recent full-genome transcript profiling studies, in combination with mutational and transgenic plant analyses, have provided a snapshot of the complex transcriptional network that operates under cold stress. The changes in expression of hundreds of genes in response to cold temperatures are followed by increases in the levels of hundreds of metabolites, some of which are known to have protective effects against the damaging effects of cold stress. Genetic analysis has revealed important roles for cellular metabolic signals, and for RNA splicing, export and secondary structure unwinding, in regulating cold-responsive gene expression and chilling and freezing tolerance. These results along with many of the others summarized here further our understanding of the basic mechanisms that plants have evolved to survive freezing temperatures. In addition, the findings have potential practical applications, as freezing temperatures are a major factor limiting the geographical locations suitable for growing crop and horticultural plants and periodically account for significant losses in plant productivity. Although, great progress has been made in the field but lacunae still remain since it appears that the cold resistance is more complex than perceived and involves more than one pathway.

Effects of molybdenum on expression of cold-responsive genes in abscisic acid (ABA)-dependent and ABA-independent pathways in winter wheat under low-temperature stress

Molybdenum (Mo) is an essential trace element for higher plants. It has been shown that application of Mo enhances the cold resistance of winter wheat. In order to improve our understanding of the molecular mechanisms of cold resistance arising from application of Mo in winter wheat, investigations were made regarding the transcription of cold-responsive (COR) genes in abscisic acid (ABA)-dependent and ABA-independent pathways in winter wheat regulated by Mo application under low-temperature stress. Two cultivars of winter wheat (Triticum aestivum), Mo-efficient cultivar '97003' and Mo-inefficient cultivar '97014', were grown in control (-Mo) and Mo fertilizer (+Mo) treatments for 40 d at 15/12 degrees C (day/night), and the temperature was then reduced to 5/2 degrees C (day/night) to create low-temperature stress. Aldehyde oxidase (AO) activities, ABA contents, the transcripts of basic leucine zipper (bZIP)-type transcription factor (TF) genes, ABA-dependent COR genes, CBF/DREB transcription factor genes and ABA-independent COR genes were investigated at 0, 3, 6 and 48 h post cold stress. Mo application significantly increased AO activity, ABA levels, and expression of bZIP-type TF genes (Wlip19 and Wabi5) and ABA-dependent COR genes (Wrab15, Wrab17, Wrab18 and Wrab19). Mo application increased expression levels of CBF/DREB transcription factor genes (TaCBF and Wcbf2-1) and ABA-independent COR genes (Wcs120, Wcs19, Wcor14 and Wcor15) after 3 and 6 h exposure to low temperature. Mo might regulate the expression of ABA-dependent COR genes through the pathway: Mo --> AO --> ABA --> bZIP --> ABA-dependent COR genes in winter wheat. The response of the ABA-dependent pathway to Mo was prior to that of the ABA-independent pathway. Similarities and differences between the Mo-efficient and Mo-inefficient wheat cultivars in response to Mo under cold stress are discussed.

Overexpression of the Arabidopsis 10-Kilodalton Acyl-Coenzyme A-Binding Protein ACBP6 Enhances Freezing Tolerance

Small 10-kD acyl-coenzyme A-binding proteins (ACBPs) are highly conserved proteins that are prevalent in eukaryotes. In Arabidopsis (Arabidopsis thaliana), other than the 10-kD ACBP homolog (designated Arabidopsis ACBP6), there are five larger forms of ACBPs ranging from 37.5 to 73.1 kD. In this study, the cytosolic subcellular localization of Arabidopsis ACBP6 was confirmed by analyses of transgenic Arabidopsis expressing autofluorescence-tagged ACBP6 and western-blot analysis of subcellular fractions using ACBP6-specific antibodies. The expression of Arabidopsis ACBP6 was noticeably induced at 48 h after 4 degrees C treatment by northern-blot analysis and western-blot analysis. Furthermore, an acbp6 T-DNA insertional mutant that lacked ACBP6 mRNA and protein displayed increased sensitivity to freezing temperature (-8 degrees C), while ACBP6-overexpressing transgenic Arabidopsis plants were conferred enhanced freezing tolerance. Northern-blot analysis indicated that ACBP6-associated freezing tolerance was not dependent on the induction of cold-regulated COLD-RESPONSIVE gene expression. Instead, ACBP6 overexpressors showed increased expression of mRNA encoding phospholipase Ddelta. Lipid profiling analyses of rosettes from cold-acclimated, freezing-treated (-8 degrees C) transgenic Arabidopsis plants overexpressing ACBP6 showed a decline in phosphatidylcholine (-36% and -46%) and an elevation of phosphatidic acid (73% and 67%) in comparison with wild-type plants. From our comparison, the gain in freezing tolerance in ACBP6 overexpressors that was accompanied by decreases in phosphatidylcholine and an accumulation of phosphatidic acid is consistent with previous findings on phospholipase Ddelta-overexpressing transgenic Arabidopsis. In vitro filter-binding assays indicating that histidine-tagged ACBP6 binds phosphatidylcholine, but not phosphatidic acid or lysophosphatidylcholine, further imply a role for ACBP6 in phospholipid metabolism in Arabidopsis, including the possibility of ACBP6 in the cytosolic trafficking of phosphatidylcholine.

Interplay between cold-responsive gene regulation, metabolism and RNA processing during plant cold acclimation

Temperate plants are capable of developing freezing tolerance when they are exposed to low nonfreezing temperatures. Acquired freezing tolerance involves extensive reprogramming of gene expression and metabolism. Recent full-genome transcript profiling studies, in combination with mutational and transgenic plant analyses, have provided a snapshot of the complex transcriptional network that operates under cold stress. Ubiquitination-mediated proteosomal protein degradation has a crucial role in regulating one of the upstream transcription factors, INDUCER OF CBF EXPRESSION 1 (ICE1), and thus in controlling the cold-responsive transcriptome. The changes in expression of hundreds of genes in response to cold temperatures are followed by increases in the levels of hundreds of metabolites, some of which are known to have protective effects against the damaging effects of cold stress. Genetic analysis has revealed important roles for cellular metabolic signals, and for RNA splicing, export and secondary structure unwinding, in regulating cold-responsive gene expression and chilling and freezing tolerance.

Perception, transduction, and networks in cold signaling

Cold temperature is one of the most critical environmental conditions that limit the geographic distribution of plants and account for significant reductions in the yields of agriculturally important crops. As part of their stress-adaptation, plants respond to cold by changing various aspects of their physiological processes, including gene expression. Numerous genes are induced by cold at the transcriptional level, encoding proteins that protect against freezing injury. While the expression of stress-inducible genes is mainly mediated by ABA-dependent and ABA-independent pathways, the C-repeat/dehydration-responsive element (CRT/DRE), containing a core sequence of-CCGAC-in the ABA-independent pathway, is essential for transcriptional activation in response to abiotic stresses, including cold. The CRT/DRE-binding factors (CBFs) and DRE-binding proteins (DREBs) can confer freezing and drought tolerances in transgenic plants by up-regulating the genes involved in enhancing stress tolerance when overexpressed. However, microarray analysis and characterization of variousArabidopsis mutants with altered response to cold and freezing temperatures suggest the existence of other cold-signaling pathways that do not involve CBF/DREB transcription factors. This review describes potential mechanisms for cold-perception, the functions of genes induced by cold temperatures in freezing tolerance, and the regulation of cold-responsive gene expression. Genetic approaches, including classical methods and reporter-gene-based screening, have revealed many signaling components in this stress response, suggesting that complicated, multiple pathways and cross-talk are involved. Recent advances in our understanding of the roles for these components as well as calcium-signaling networks are also discussed here.

A Putative Arabidopsis Nucleoporin, AtNUP160, Is Critical for RNA Export and Required for Plant Tolerance to Cold Stress

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.

The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1

Plant responses to cold stress are mediated by a transcriptional cascade, in which the transcription factor ICE1 and possibly related proteins activate the expression of C-repeat (CRT)-binding factors (CBFs), leading to the transcription of downstream effector genes. The variant RING finger protein high expression of osmotically responsive gene (HOS)1 was identified genetically as a negative regulator of cold responses. We present evidence here that HOS1 is an E3 ligase required for the ubiquitination of ICE1. HOS1 physically interacts with ICE1 and mediates the ubiquitination of ICE1 both in vitro and in vivo. We found that cold induces the degradation of ICE1 in plants, and this degradation requires HOS1. Consistent with enhanced cold-responsive gene expression in loss-of-function hos1 mutant plants, overexpression of HOS1 represses the expression of CBFs and their downstream genes and confers increased sensitivity to freezing stress. Our results indicate that cold stress responses in Arabidopsis are attenuated by a ubiquitination/proteasome pathway in which HOS1 mediates the degradation of the ICE1 protein.

Functional Analysis of Rice DREB1/CBF-type Transcription Factors Involved in Cold-responsive Gene Expression in Transgenic Rice

The transcription factors dehydration-responsive element-binding protein 1s (DREB1s)/C-repeat-binding factors (CBFs) specifically interact with the DRE/CRT cis-acting element and control the expression of many stress-inducible genes in Arabidopsis. The genes for DREB1 orthologs, OsDREB1A and OsDREB1B from rice, are induced by cold stress, and overexpression of DREB1 or OsDREB1 induced strong expression of stress-responsive genes in transgenic Arabidopsis plants, resulting in increased tolerance to high-salt and freezing stresses. In this study, we generated transgenic rice plants overexpressing the OsDREB1 or DREB1 genes. These transgenic rice plants showed not only growth retardation under normal growth conditions but also improved tolerance to drought, high-salt and low-temperature stresses like the transgenic Arabidopsis plants overexpressing OsDREB1 or DREB1. We also detected elevated contents of osmoprotectants such as free proline and various soluble sugars in the transgenic rice as in the transgenic Arabidopsis plants. We identified target stress-inducible genes of OsDREB1A in the transgenic rice using microarray and RNA gel blot analyses. These genes encode proteins that are thought to function in stress tolerance in the plants. These results indicate that the DREB1/CBF cold-responsive pathway is conserved in rice and the DREB1-type genes are quite useful for improvement of stress tolerance to environmental stresses in various kinds of transgenic plants including rice.

Two AP2 domain containing genes isolated from the cold-hardy Citrus relative Poncirus trifoliata are induced in response to cold.

Poncirus trifoliata (L.) Raf. is a cold-hardy, interfertile Citrus relative able to tolerate temperatures as low as -26°C when cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus varieties. A cold-induced cDNA library was constructed by subtractive hybridisation of non-acclimated and 2-d cold-acclimated P. trifoliata seedlings and many genes induced in response to cold were identified. Two of these cDNAs, PI-B05 and PI-C10, were selected from this library for further characterisation. Full-length cDNA sequences of these genes were obtained by 5' and 3' rapid amplification of cDNA ends (RACE). Sequence analysis revealed that PI-B05 contained an apetala2 / ethylene response factor (AP2 / ERF) domain and showed homology with ERF proteins from other plants, some of which are involved in environmental stress-induced gene expression. PI-C10 contained both AP2 / ERF and B3 DNA binding domains and showed homology with other plant proteins in the RAV subfamily of the AP2 / ERF transcription factors, some of which are induced in response to cold and other environmental stresses. Expression patterns of these genes in cold-tolerant P. trifoliata and cold-sensitive pummelo [Citrus grandis (L.) Osb.] in response to cold and drought at different time points were analysed by northern blots. Expression analysis showed that both genes were induced in response to cold, but not under drought conditions in cold-hardy P. trifoliata. However, little or no expression of these genes was detected by northern analysis in cold-sensitive pummelo under cold or drought conditions. The sequence analysis and expression data indicated that these genes may play a role in cold-responsive gene expression in cold-hardy P. trifoliata and could possibly be used for improving cold tolerance in cold-sensitive citrus cultivars.

A genetic link between cold responses and flowering time through FVE in Arabidopsis thaliana.

Cold induces expression of a number of genes that encode proteins that enhance tolerance to freezing temperatures in plants. A cis-acting element responsive to cold and drought, the C-repeat/dehydration-responsive element (C/DRE), was identified in the Arabidopsis thaliana stress-inducible genes RD29A and COR15a and found in other cold-inducible genes in various plants. C/DRE-binding factor/DRE-binding protein (CBF/DREB) is an essential component of the cold-acclimation response, but the signaling pathways and networks are mostly unknown. Here we used targeted genetic approach to isolate A. thaliana mutants with altered cold-responsive gene expression (acg) and identify ACG1 as a negative regulator of the CBF/DREB pathway. acg1 flowered late and had elevated expression of FLOWERING LOCUS C (FLC), a repressor of flowering encoding a MADS-box protein. We showed that acg1 is a null allele of the autonomous pathway gene FVE. FVE encodes a homolog of the mammalian retinoblastoma-associated protein, a component of a histone deacetylase (HDAC) complex involved in transcriptional repression. We also showed that plants sense intermittent cold stress through FVE and delay flowering with increasing expression of FLC. Dual roles of FVE in regulating the flowering time and the cold response may have an evolutionary advantage for plants by increasing their survival rates.

Cold induction of Arabidopsis CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature.

The Arabidopsis CBF1, 2, and 3 genes (also known as DREB1b, c, and a, respectively) encode transcriptional activators that have a central role in cold tolerance. CBF1-3 are rapidly induced upon exposing plants to low temperature, followed by expression of CBF-targeted genes, the CBF regulon, resulting in an increase in plant freezing tolerance. At present, little is known about the cold-sensing mechanism that controls CBF expression. Results presented here indicate that this mechanism does not require a cold shock to bring about the accumulation of CBF transcripts, but instead, absolute temperature is monitored with a greater degree of input, i.e. lower temperature, resulting in a greater output, i.e. higher levels of CBF transcripts. Temperature-shift experiments also indicate that the cold-sensing mechanism becomes desensitized to a given low temperature, such as 4 degrees C, and that resensitization to that temperature requires between 8 and 24 h at warm temperature. Gene fusion experiments identified a 125-bp section of the CBF2 promoter that is sufficient to impart cold-responsive gene expression. Mutational analysis of this cold-responsive region identified two promoter segments that work in concert to impart robust cold-regulated gene expression. These sequences, designated ICEr1 and ICEr2 (induction of CBF expression region 1 or 2), were also shown to stimulate transcription in response to mechanical agitation and the protein synthesis inhibitor, cycloheximide.

OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression.

The transcription factors DREBs/CBFs specifically interact with the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element (core motif: G/ACCGAC) and control the expression of many stress-inducible genes in Arabidopsis. In rice, we isolated five cDNAs for DREB homologs: OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, and OsDREB2A. Expression of OsDREB1A and OsDREB1B was induced by cold, whereas expression of OsDREB2A was induced by dehydration and high-salt stresses. The OsDREB1A and OsDREB2A proteins specifically bound to DRE and activated the transcription of the GUS reporter gene driven by DRE in rice protoplasts. Over-expression of OsDREB1A in transgenic Arabidopsis induced over-expression of target stress-inducible genes of Arabidopsis DREB1A resulting in plants with higher tolerance to drought, high-salt, and freezing stresses. This indicated that OsDREB1A has functional similarity to DREB1A. However, in microarray and RNA blot analyses, some stress-inducible target genes of the DREB1A proteins that have only ACCGAC as DRE were not over-expressed in the OsDREB1A transgenic Arabidopsis. The OsDREB1A protein bound to GCCGAC more preferentially than to ACCGAC whereas the DREB1A proteins bound to both GCCGAC and ACCGAC efficiently. The structures of DREB1-type ERF/AP2 domains in monocots are closely related to each other as compared with that in the dicots. OsDREB1A is potentially useful for producing transgenic monocots that are tolerant to drought, high-salt, and/or cold stresses.

Molecular genetic analysis of cold-regulated gene transcription.

Chilling and freezing temperatures adversely affect the productivity and quality of crops. Hence improving the cold hardiness of crop plants is an important goal in agriculture, which demands a clear understanding of cold stress signal perception and transduction. Pharmacological and biochemical evidence shows that membrane rigidification followed by cytoskeleton rearrangement, Ca(2+) influx and Ca(2+)-dependent phosphorylation are involved in cold stress signal transduction. Cold-responsive genes are regulated through C-repeat/dehydration-responsive elements (CRT/DRE) and abscisic acid (ABA)-responsive element cis elements by transacting factors C-repeat binding factors/dehydration-responsive element binding proteins (CBFs/DREBs) and basic leucine zippers (bZIPs) (SGBF1), respectively. We have carried out a forward genetic analysis using chemically mutagenized Arabidopsis plants expressing cold-responsive RD29A promoter-driven luciferase to dissect cold signal transduction. We have isolated the fiery1 (fry1) mutant and cloned the FRY1 gene, which encodes an inositol polyphosphate 1-phosphatase. The fry1 plants showed enhanced induction of stress genes in response to cold, ABA, salt and dehydration due to higher accumulation of the second messenger, inositol (1,4,5)- triphosphate (IP(3)). Thus our study provides genetic evidence suggesting that cold signal is transduced through changes in IP(3) levels. We have also identified the hos1 mutation, which showed super induction of cold-responsive genes and their transcriptional activators. Molecular cloning and characterization revealed that HOS1 encodes a ring finger protein, which has been implicated as an E3 ubiquitin conjugating enzyme. HOS1 is present in the cytoplasm at normal growth temperatures but accumulates in the nucleus upon cold stress. HOS1 appears to regulate temperature sensing by the cell as cold-responsive gene expression occurs in the hos1 mutant at relatively warm temperatures. Thus HOS1 is a negative regulator, which may be functionally linked to cellular thermosensors to modulate cold-responsive gene transcription.

An AP2 domain transcription factor HvCBF1 activates expression of cold-responsive genes in barley through interaction with a (G/a)(C/t)CGAC motif

A HvCBF1 cDNA encoding an AP2 domain-containing protein was isolated from barley leaves. Expression of HvCBF1 was induced in barley leaves by exposure to a low temperature (2 °C), but not by drought or abscisic acid (ABA) treatment. Functional analysis showed that HvCBF1 was a transcriptional activator, capable of activating expression of reporter genes driven by a cold- and drought-responsive HVA1s promoter in barley leaves. Transactivation analysis of HvCBF1 on a number of synthetic oligonucleotides containing potential C-repeat (CRT)/dehydration-responsive element (DRE) derived from cold-responsive barley genes revealed that HvCBF1 interacted much more efficiently with a GCCGAC motif than a previously identified barley low-temperature-responsive element (LTRE), CCGAAA. A detailed base-substitution mutagenesis study revealed that only the CGAC sequence of the GCCGAC motif was highly conserved for interacting with HvCBF1. The promoter activity of the mutant motifs, ACCGAC and GTCGAC, was 36% and 75% of that of the GCCGAC motif, respectively. The base composition surrounding the GCCGAC motif also had a significant effect on the efficiency of the motif. These data suggest that barley HvCBF1 protein interacts with the (G/a)(C/t)CGAC motif and is involved in regulation of cold-responsive genes in barley via an ABA-independent signal transduction pathway.

HOS1, a genetic locus involved in cold-responsive gene expression in arabidopsis.

Low-temperature stress induces the expression of a variety of genes in plants. However, the signal transduction pathway(s) that activates gene expression under cold stress is poorly understood. Mutants defective in cold signaling should facilitate molecular analysis of plant responses to low temperature and eventually lead to the identification and cloning of a cold stress receptor(s) and intracellular signaling components. In this study, we characterize a plant mutant affected in its response to low temperatures. The Arabidopsis hos1-1 mutation identified by luciferase imaging causes superinduction of cold-responsive genes, such as RD29A, COR47, COR15A, KIN1, and ADH. Although these genes are also induced by abscisic acid, high salt, or polyethylene glycol in addition to cold, the hos1-1 mutation only enhances their expression under cold stress. Genetic analysis revealed that hos1-1 is a single recessive mutation in a nuclear gene. Our studies using the firefly luciferase reporter gene under the control of the cold-responsive RD29A promoter have indicated that cold-responsive genes can be induced by temperatures as high as 19 degrees C in hos1-1 plants. In contrast, wild-type plants do not express the luciferase reporter at 10 degrees C or higher. Compared with the wild type, hos1-1 plants are l ess cold hardy. Nonetheless, after 2 days of cold acclimation, hos1-1 plants acquired the same degree of freezing tolerance as did the wild type. The hos1-1 plants flowered earlier than did the wild-type plants and appeared constitutively vernalized. Taken together, our findings show that the HOS1 locus is an important negative regulator of cold signal transduction in plant cells and that it plays critical roles in controlling gene expression under cold stress, freezing tolerance, and flowering time.