Role In Cold Response Research Articles

A cold-inducible MYB like transcription factor, CsHHO2, positively regulates chilling tolerance of cucumber fruit by enhancing CsGR-RBP3 expression

Plant HRS1 (hypersensitive to low phosphate (Pi)-elicited primary root shortening 1) and its homologs have been primarily characterized in Pi and nitrogen responses. However, their regulatory roles in cold responses remain largely unexplored. Here, we characterized the functional roles of HRS1 homolog 2 (CsHHO2) in regulating cold resistance in harvested cucumber fruit and examined the dependency of the role on glycine-rich RNA-binding protein 3 (CsGR-RBP3) gene expression. Cold treatments strongly induced CsHHO2 expression in cucumber fruit. GUS staining and expression analysis revealed that cold treatment stimulated CsHHO2 expression, at least partially, by enhancing the promoter activity. Subcellular localization results indicated that the green fluorescent protein (GFP)-tagged CsHHO2 proteins were localized to both the nucleus and the cytoplasm (potentially the plasma membrane), with confirmed nuclear localization when co-overexpressed with mCherry. Reducing CsHHO2 expression in cucumber fruit through VIGS significantly compromised the chilling tolerance, while its overexpression in cucumber fruit and Arabidopsis plants enhanced cold tolerance. The results of electrophoretic mobility shift assay (EMSA), yeast one-hybrid (Y1H) assay and dual-luciferase reporter (DLR) assay indicated that CsHHO2 specifically bound to the promoter of CsGR-RBP3, and enhanced its transcription. Transcriptomic analysis indicated that most differentially expressed genes (DEGs) modulated by CsHHO2 and CsGR-RBP3 proteins were identical, and the common DEGs were mainly enriched in several stress defense pathways, such as plant-pathogen interaction, phenylpropanoid biosynthesis, and plant hormone signal transduction. Functional complementation analysis of CsHHO2 and CsGR-RBP3 in cucumber fruit revealed that CsGR-RBP3 expression were indispensable for CsHHO2-modulated cold resistance. These results collectively suggest that CsHHO2, activated by cold signals, positively regulates the chilling tolerance of cucumber fruit by enhancing CsGR-RBP3 expression. Overall, this work suggests CsHHO2 as a key regulator of cold tolerance in cucumber fruit, provides key genetic resources for breeding cold-resistant cucumbers, and sheds novel insight into the roles of HRS1 family members in regulating plant development under environmental stresses.

The pepper MYB transcription factor CaMYB306 accelerates fruit coloration and negatively regulates cold resistance

The extensively studied MYB transcription factor (TF) family has significant functions in plant development and stress responses. However, little is known about the role of MYB TFs in fruit coloration and cold resistance in pepper (Capsicum annuum L.). In this study, we identified the novel MYB gene CaMYB306 in pepper. CaMYB306 expression was higher in red fruit. Knockdown of CaMYB306 inhibited carotenoid biosynthesis and decreased expression of carotenoid-related genes in pepper fruit. CaMYB306-overexpressing (CaMYB306-OE) tomato plants showed earlier coloration with increased carotenoid accumulation and decreased chlorophyll and anthocyanin contents. The expression levels of key carotenoid biosynthesis genes (SlPSY and SlPDS) and key chlorophyll catabolic enzymes genes (SlSGR and SlPAO) were increased in CaMYB306-OE fruit, whereas key anthocyanin biosynthesis genes (SlF3H and SlCHS) were down-regulated. Additionally, CaMYB306 expression was up-regulated under cold treatment. CaMYB306 played a negative role in cold response via the regulation of the reactive oxygen species system and the expression of cold-related genes. CaMYB306 suppressed the transcriptional activity of Calcineurin B-Like-Interacting Protein Kinase gene 13 (CaCIPK13), which was a positive regulator of cold resistance. These findings clarify the role of MYB TFs in fruit coloration and cold resistance in pepper.

Elevation of GhDREB1B transcription by a copy number variant significantly improves chilling tolerance in cotton.

The elevation of transcript levels of GhDREB1B causes the accumulation of osmoregulants and mitigation of reactive oxygen species, which contributes to the enhanced resistance to chilling stress in AiSheng98 cotton. Low temperature is one of the key environmental stresses that impairs cotton growth and restricts fiber productivity. Dehydration responsive element binding (DREB) transcription factors play an important role in cold response in plants by modulating the transcription level of cold-responsive genes to protect the plants from low-temperature stress. Here, we showed that GhDREB1B, a copy number variant in the AiSheng98 (AS98) cotton mutant, significantly improved chilling tolerance in cotton seedlings, while silencing of GhDREB1B made transgenic cotton sensitive to chilling stress in AS98 cotton compared with control plants. Elevated GhDREB1B transcript level activated the expression of major cold-responsive genes. Genome-wide expression profiling by RNA sequencing revealed the upregulation of genes related to fatty acids, lipid proteins, osmoprotection, and anti-oxidative enzymes in AiSheng98. Excessive accumulation of malondialdehyde (MDA) and higher ion leakage rates occurred in wild-type LFH10 plants when compared to those of Aisheng98 during chilling stress, signifying lower chilling tolerance in the wild-type than in Aisheng98. Furthermore, the Aisheng98 mutant under chilling stress accumulated higher levels of free proline and soluble sugar than LFH10 accumulated. These results suggest that GhDREB1B is a positive regulator and its variant can alter the expression patterns of major low-temperature stress-related genes and enhance chilling tolerance in cotton.

Comparative physiology and transcriptome response patterns in cold-tolerant and cold-sensitive varieties of Zanthoxylum bungeanum Maxim

Zanthoxylum bungeanum is an economically important tree species of edible, medicinal, and ornamental value. The trees grow in varied climates from tropical to subtropical regions owing to their high resistance to adverse environment. The unstable environmental temperature is one of the important factors affecting the yield and quality of Z. bungeanum. To investigate complex responses to cold stress in Z. bungeanum, we compared cold-tolerant “Fuguhuajiao” (FG) and cold-sensitive “Fengxiandahongpao” (FX) varieties. Physiological measurements and transcriptome sequencing were performed using plant leaves collected at six different timepoints (0, 1, 3, 6, 12, and 24 h) of 4 °C treatment. Chemometrics analysis showed that the physiological cold response in FX at late stage was similar to that in FG at early stage. Based on orthogonal partial least squares discriminant analysis (OPLS-DA), the variable important in projection (VIP) values of peroxidase (POD) and soluble protein were 1.11 and 1.41, which were identified as important physiological indices in the two varieties. Under low temperature stress, there were 3513 differentially expressed genes in FG and 25,157 in FX. Weighted gene co-expression network analysis (WGCNA) revealed that many cold-response genes and pathways were enriched in the MEfirebrick4 module which had a high correlation with POD. Hub genes identified in this module indicated that Z. bungeanum can respond and adapt to cold stress by altering signal transduction, plant hormones, transcription factors, protein modification, functional proteins, metabolism, cell structure, light response, and the circadian clock. Endocytosis-related gene ERD7, abscisic acid pathway-related gene PP2C, late embryogenesis abundant gene LEA D-29, and two plasma membrane/lipid raft-associated remorin genes ZB01477 and pp34 are located at the core of the network, and all play pivotal roles in cold responses. These results of this study highlight cold response mechanisms at the physiological and molecular levels in Z. bungeanum and will contribute to a better understanding of low temperature resistance in related plants.

An eggplant SmICE1a gene encoding MYC-type ICE1-like transcription factor enhances freezing tolerance in transgenic Arabidopsis thaliana.

Low temperature is a crucial environmental factor affecting the quality and production of eggplant. Therefore, it is necessary to explore the molecular mechanisms of low temperature response. We isolated an ICE (inducer of CBF expression) gene from Solanum melongena, named SmICE1a. We then analysed structure, transcriptional activity and expression patterns of SmICE1a. Moreover, we also expressed SmICE1a in Arabidopsis thaliana. Bioinformatics and expression analysis showed that SmICE1a has a typical S-rich motif, ZIP region, bHLH and ACT-like domain. The gene SmICE1a had transcriptional activity in yeast and was localized to the nucleus following transient expression in tobacco leaves, which suggests that SmICE1a is a transcription factor. A dual-LUC assay revealed that SmICE1a can enhance expression of SmCBF. Overexpression of SmICE1a in Arabidopsis increased freezing tolerance and caused multiple biochemical changes: transgenic lines have higher proline content and lower electrolyte leakage and malondialdehyde than the wild type in cold conditions. The expression of AtCBF and their target genes, AtCOR15A, AtCOR47, AtKIN1 and AtRD29A, were up-regulated in SmICE1a-overexpressing plants under low temperatures. Based on these results, we suggest that SmICE1a plays an important role in cold response, which may help to understand the cold response mechanism in eggplant and could be used to enhance cold tolerance of eggplant in future.

The DEAD-box RNA helicase SHI2 functions in repression of salt-inducible genes and regulation of cold-inducible gene splicing.

Gene regulation is central for growth, development, and adaptation to environmental changes in all living organisms. Many genes are induced by environmental cues, and the expression of these inducible genes is often repressed under normal conditions. Here, we show that the SHINY2 (SHI2) gene is important for repressing salt-inducible genes and also plays a role in cold response. The shi2 mutant displayed hypersensitivity to cold, abscisic acid (ABA), and LiCl. Map-based cloning demonstrates that SHI2 encodes a DEAD- (Asp-Glu-Ala-Asp) box RNA helicase with similarity to a yeast splicing factor. Transcriptomic analysis of the shi2 mutant in response to cold revealed that the shi2 mutation decreased the number of cold-responsive genes and the magnitude of their response, and resulted in the mis-splicing of some cold-responsive genes. Under salt stress, however, the shi2 mutation increased the number of salt-responsive genes but had a negligible effect on mRNA splicing. Our results suggest that SHI2 is a component in a ready-for-transcription repressor complex important for gene repression under normal conditions, and for gene activation and transcription under stress conditions. In addition, SHI2 also serves as a splicing factor required for proper splicing of cold-responsive genes and affects 5' capping and polyadenylation site selection.

The involvements of calcium-dependent protein kinases and catechins in tea plant [Camellia sinensis (L.) O. Kuntze] cold responses

Temperature is one of the most important environmental factors limiting tea plant growth and tea production. Previously we reported that both Ca2+ and ROS signals play important roles in tea plant cold acclimation. Here, we identified 26 CsCPK transcripts, analyzed their phylogenetic and sequence characters, and detected their transcriptions to monitor Ca2+ signaling status. Tissue-specific expression profiles indicated that most CsCPK genes were constitutively expressed in tested tissues, suggesting their possible roles in development. Cold along with calcium inhibitor assays suggested that CsCPKs are important cold regulators and CsCPK30/5/4/9 maybe the key members. Moreover, LaCl3 or EGTA pre-treatment could result in impaired Ca2+ signaling and compromised cold-responding network, but higher catechins accumulation revealed their potential positive roles in cold responses. Those findings indicated that catechins and other secondary metabolites in tea plant may form an alternative cold-responding network that closely correlated with Ca2+ signaling status.

Ethylene biosynthesis is involved in regulating chilling tolerance and SlCBF1 gene expression in tomato fruit

Ethylene plays positive role in cold response of tomato fruit. CBFs (C-repeat/dehydration-responsive element binding factors) have been proved important in regulating plant chilling tolerance. Till now, the role that ethylene biosynthesis played in chilling response of tomato fruit is still unclear. In this study, we used antisense SlACS2 and wild-type Lichun tomato fruit as inhibited- and normal- ethylene biosynthesis models, in order to investigate whether inhibiting ethylene biosynthesis could affect chilling tolerance and SlCBF1 relative expression in tomato fruit. Results showed that antisense SlACS2 tomato fruit suffered more chilling injury, accompanied by higher malondialdehyde content and ion leakage, less proline and soluble protein content, as well as lower antioxidant enzymes activities, suggesting that inhibiting ethylene biosynthesis reduced tomato fruit chilling tolerance. Furthermore, SlCBF1 expression in transgenic fruit was lower than in WT, the second peak of SlCBF1 expression was coincidence with the peaks of both endogenous ethylene production and ACS activity, and there was significant correlation between ACC content and SlCBF1 relative expression, which implied that endogenous ethylene biosynthesis could contribute to upregulate SlCBF1 expression under cold stress. These results revealed that ethylene biosynthesis played important roles in regulating tomato fruit chilling tolerance and SlCBF1 relative expression in tomato fruit.

Overexpression of the Medicago falcata NAC transcription factor MfNAC3 enhances cold tolerance in Medicago truncatula

Cold stress is the main factor underlying the reduction in productivity of Medicago. Medicago falcata and Medicago truncatula are two subspecies of Medicago, whose geographic adaption is limited by water, salinity and temperature. However, the regulatory signaling pathway under cold stress in Medicago is unclear. In this study, we identified a gene, MfNAC3, induced under salt, drought and cold stress. By generating the overexpression lines of MfNAC3, we observed a typical cold-resistant phenotype under both cold-acclimated and non-acclimated conditions, featured by an increased survival rate and significantly higher expression levels of the cold-responsive genes MtCBFs and MtCASs. Further investigations revealed that this gene encodes a NAC-type transcriptional factor that is localized in the nucleus and exhibits transcription activity. By performing an electrophoretic mobility shift assay, we found that MfNAC3 could bind to the CATGTG and CACG motifs in the promoter region of MtCBF4. Taken together, our results demonstrate that MfNAC3 exerts a positive role in cold response and provide evidence that MfNAC3 is a positive regulator of MtCBF4.

Arabidopsis thaliana ICE2 gene: Phylogeny, structural evolution and functional diversification from ICE1

The ability to tolerate environmental stresses is crucial for all living organisms, and gene duplication is one of the sources for evolutionary novelties. Arabidopsis thaliana INDUCER OF CBF EXPRESSION1 and 2 (ICE1 and ICE2) encode MYC-type bHLH (basic helix–loop–helix) transcription factors. They confer cold stress tolerance by induction of the CBF/DREB1 regulon and regulate stomata formation. Although ICE2 is closely related to ICE1, its origin and role in cold response remains uncertain. Here, we used a bioinformatics/phylogenetic approach to uncover the ICE2 evolutionary history, structural evolution and functional divergence from the putative ancestral gene. Sequence diversification from ICE1 included the gain of cis-acting elements in ICE2 promoter sequence that may provide meristem-specific and defense-related gene expression. By analyzing transgenic Arabidopsis lines with ICE2 over-expression we showed that it contributes to stomata formation, flowering time regulation and cold response. Constitutive ICE2 expression led to induced meristem freezing tolerance, resulting from activation of CBF1 and CBF3 genes and ABA biosynthesis by NCED3 induction. We presume that ICE2 gene has originated from a duplication event about 17.9MYA followed by sub- and neofunctionalization of the ancestral ICE1 gene. Moreover, we predict its role in pathogen resistance and flowering time regulation.

Expressing a Citrus ortholog of Arabidopsis ERF1 enhanced cold-tolerance in tobacco

The ERF1 gene was well characterized in Arabidopsis to be responsive to salt, drought, heat and necrotrophic fungi. In this paper, an ortholog of ERF1, designated as CsERF that was isolated originally as an ethylene induced gene from Citrus sinensis (L.) Osbeck, was characterized. Sequence analysis revealed that CsERF and ERF1 belonged to the same AP2/ERF superfamily, or more specifically, the same IXc subgroup of the ERF subfamily. Transient expression experiment revealed that CsERF protein was localized in nuclei. Quantitative real-time PCR results showed that the expression of CsERF was induced not only by various stresses including salt, dehydration and cold, but also by stress-related hormones, such as ethylene, jasmonic acid and abscissic acid, with ethylene and cold as the two most potent inducers. Its ethylene induction could be blocked by 1-MCP, indicating ethylene signaling pathway was required for its activation. Overexpressing CsERF conferred tobacco plants with much stronger tolerance to chilling stress, and activated constitutively four indicator genes: two cold responsive transcription factor genes, NtCBF1 and NtCBF3, and two cold-induced genes, NtERD10B and NtERD10C. Under cold treatment, proline content and superoxide dismutase activity increased more rapidly and were much higher in overexpression lines than in wild type. These data suggest that CsERF plays a role in cold response in addition to the roles shared with its ortholog ERF1.

Ectopic Overexpression of SsCBF1, a CRT/DRE-Binding Factor from the Nightshade Plant Solanum lycopersicoides, Confers Freezing and Salt Tolerance in Transgenic Arabidopsis

The C-repeat (CRT)/dehydration-responsive element (DRE) binding factor (CBF/DREB1) transcription factors play a key role in cold response. However, the detailed roles of many plant CBFs are far from fully understood. A CBF gene (SsCBF1) was isolated from the cold-hardy plant Solanum lycopersicoides. A subcellular localization study using GFP fusion protein indicated that SsCBF1 is localized in the nucleus. We delimited the SsCBF1 transcriptional activation domain to the C-terminal segment comprising amino acid residues 193–228 (SsCBF1193–228). The expression of SsCBF1 could be dramatically induced by cold, drought and high salinity. Transactivation assays in tobacco leaves revealed that SsCBF1 could specifically bind to the CRT cis-elements in vivo to activate the expression of downstream reporter genes. The ectopic overexpression of SsCBF1 conferred increased freezing and high-salinity tolerance and late flowering phenotype to transgenic Arabidopsis. RNA-sequencing data exhibited that a set of cold and salt stress responsive genes were up-regulated in transgenic Arabidopsis. Our results suggest that SsCBF1 behaves as a typical CBF to contribute to plant freezing tolerance. Increased resistance to high-salinity and late flowering phenotype derived from SsCBF1 OE lines lend more credence to the hypothesis that plant CBFs participate in diverse physiological and biochemical processes related to adverse conditions.

CsICE1 and CsCBF1: two transcription factors involved in cold responses in Camellia sinensis

C-repeat/dehydration-responsive element binding factors (CBFs) can induce the expression of a suite of cold-responsive genes to increase plant cold tolerance, and inducer of CBF expression 1 (ICE1) is a major activator for CBF. In the present study, we isolated the full-length cDNAs of ICE1 and CBF from Camellia sinensis, designated as CsICE1 and CsCBF1, respectively. The deduced protein CsICE1 contains a highly conserved basic helix-loop-helix (bHLH) domain and C-terminal region of ICE1-like proteins. CsCBF1 contains all conserved domains of CBFs in other plant species and can specifically bind to the C-repeat/dehydration-responsive element (CRT/DRE) as confirmed by electrophoretic mobility shift assay. The transcription of CsICE1 had no apparent alteration after chilling treatment (4°C). CsCBF1 expression was not detected in normal temperature (20°C) but was induced immediately and significantly by low temperature (4°C). Our results suggest that ICE1-CBF cold-response pathway is conserved in tea plants. CsICE1 and CsCBF1, two components of this pathway, play roles in cold responses in tea plants.

CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana

The family of calcineurin B-like (CBL) proteins is a unique group of Ca 2+ sensors in plants. CBLs relay the calcium signal by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). Extensive studies have demonstrated that the CBL–CIPK complexes mediate plant responses to a variety of external stresses. However, there are few reports on the CBL–CIPK involved in cold stress responses. In this study, we analyzed expression of CIPK7 and CBL1 in Arabidopsis during cold treatments. Expression of CIPK7 was induced by cold, and CIPK7 interacted with CBL1 in vitro. Moreover, affinity chromatography purification of CIPK7 from Arabidopsis plants using CBL1 suggested that CIPK7 may associate with CBL1 in vivo. Expression of CBL1 was cold inducible, and CBL1 had a role in regulating cold response. By comparing expression patterns of CIPK7 between wild-type and cbl1 mutant plants, we found the induction of CIPK7 by cold stress was influenced by CBL1. This is the first report to demonstrate that CIPK7 may play a role in cold response via its interaction with CBL1.

Increased Transcript Level of RBM3, a Member of the Glycine-Rich RNA-Binding Protein Family, in Human Cells in Response to Cold Stress

Although the cold-shock responses of microorganisms have been extensively investigated, those of mammalian cells are just beginning to be understood. Recently, CIRP, a member of the glycine-rich RNA-binding protein (GRP) family, has been identified as the first cold-shock protein in mammalian cells. Here, we report that RBM3, another member of the GRP family, is induced in human cells in response to cold stress (32°C).RBM3transcripts were constitutively expressed in all cell lines examined including K562, HepG2, NC65, HeLa, and T24 cells. In all of them, the transcript levels ofRBM3were increased at 24 h after the 37 to 32°C temperature down-shift. In NC65 cells, the kinetics ofRBM3induction was different from that ofCIRP.Protein synthesis inhibitors cycloheximide and puromycin inducedRBM3transcripts, but cadmium chloride, H2O2, ethanol, and osmotic shock had no effect. Combined with the different tissue distribution of expression, these results suggest that RBM3 and CIRP play distinct roles in cold responses of human cells.