Cold Tolerance In Arabidopsis Research Articles

Characterization and Cloning of Grape Circular RNAs Identified the Cold Resistance-Related Vv-circATS1.

Circular RNAs (circRNAs) are widely distributed and play essential roles in a series of developmental processes, although none have been identified or characterized in grapevines (Vitis vinifera). In this study, we characterized the function of grape circRNA and uncovered thousands of putative back-splicing sites by global transcriptome analysis. Our results indicated that several reported circRNA prediction algorithms should be used simultaneously to obtain comprehensive and reliable circRNA predictions in plants. Furthermore, the length of introns flanking grape circRNAs was closely related to exon circularization. Although the longer introns flanking grape circRNAs appeared to circularize more efficiently, a 20- to 50-nt region seemed large enough to drive grape circRNA biogenesis. In addition, the endogenous introns flanking circularized exon(s) in conjunction with reverse complementary sequences could support the accurate and efficient circularization of various exons in grape, which constitutes a new tool for exploring the functional consequences caused by circRNA expression. Finally, we identified 475 differentially expressed circRNAs in grape leaves under cold stress. Overexpression of Vv-circATS1, a circRNA derived from glycerol-3-P acyltransferase, improved cold tolerance in Arabidopsis (Arabidopsis thaliana), while the linear RNA derived from the same sequence cannot. These results indicate the functional difference between circRNA and linear RNA, and provide new insight into plant abiotic stress resistance.

The glutamate receptors AtGLR1.2 and AtGLR1.3 increase cold tolerance by regulating jasmonate signaling in Arabidopsis thaliana

Plant glutamate-like receptors (GLRs), which are homologs of mammalian ionotropic glutamate receptors (iGluRs), are thought to be involved in plant growth, development, and environmental stress responses. In this study, we demonstrated that two members of Arabidopsis glutamate-like receptors, AtGLR1.2 and AtGLR1.3, play positive roles in the plant response to cold stress. Genetic and biochemical experiments revealed that exogenous jasmonate could attenuate the cold sensitivity of glr1.2 and glr1.3 mutants, and the overexpression of GLR1.2 or GLR1.3 enhanced cold tolerance by increasing endogenous jasmonate levels under cold stress. In addition, the expression of genes in the CBF/DREB1 signaling pathway was decreased in the glr1.2 and glr1.3 mutants, but was promoted in GLR1.2-OE and GLR1.3-OE transgenic plants compared with the wild-type during cold treatment. Further investigation revealed that AtGLR1.2 and AtGLR1.3 independently drove similar functions without directly interacting. Together, our findings suggest that AtGLR1.2 and 1.3 positively enhance cold tolerance in Arabidopsis by activating endogenous jasmonate accumulation and subsequently promoting the downstream CBF/DREB1 cold response pathway during cold stress.

Overexpression of ethylene response factors VaERF080 and VaERF087 from Vitis amurensis enhances cold tolerance in Arabidopsis

Low temperature is a vital environmental factor that affects plant growth, development and crop yields. Ethylene response factor (ERF) is a plant specific transcription factor with important roles in stress adaptation. However, the regulatory mechanism on how grapevine responds to cold stress is still unclear. In this study, we isolated and characterized two ERF genes from Vitis amurensis designated as VaERF080 and VaERF087. The function of these two genes was investigated in transgenic Arabidopsis. We found that both VaERF080 and VaERF087 were induced by cold stress and ethylene. The overexpression of these genes in Arabidopsis resulted in enhanced cold tolerance. The VaERF080- and VaERF087-overexpressing (OE) Arabidopsis lines showed lower levels of malondialdehyde (MDA) content and higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress compared to wild-type (WT) Arabidopsis. Nine representative cold-responsive genes, CBF1, CBF2, ICE1, ZAT12, KIN1, SIZ1, RD29A, COR15A, and COR47, were significantly upregulated in OE Arabidopsis. These results revealed that both VaERF080 and VaERF087 may improve the cold tolerance of transgenic Arabidopsis through increasing antioxidant enzyme activities and regulating the expression of cold-related genes.

MYC-type transcription factors, MYC67 and MYC70, interact with ICE1 and negatively regulate cold tolerance in Arabidopsis

The expression of hundreds of genes is induced by low temperatures via a cold signaling pathway. ICE1, a MYC-type transcription factor, plays an important role in the induction of CBF3/DREB1A to control cold-responsive genes and cold tolerance. To elucidate other molecular factors, a yeast 2-hybrid screening was performed. Two MYC-type transcription factors, MYC67 and MYC70, were identified as ICE1-interacting proteins. The myc mutants were more tolerant to freezing temperatures than wild type. CBF3/DREB1A and other cold-responsive genes were up-regulated in the myc mutants. Overexpression of the MYC genes increased the cold sensitivity and down-regulated the expression of cold-responsive genes. The MYC proteins interacted with the cis-elements in the CBF3/DREB1A promoter, probably to interfere interaction between ICE1 and the cis-elements. Taken together, these results demonstrate that MYC67 and MYC70, ICE1 interactors, negatively regulate cold-responsive genes and cold tolerance.

Identification of cold-related miRNAs in sugarcane by small RNA sequencing and functional analysis of a cold inducible ScmiR393 to cold stress

Sugarcane, the main sugar crop harvested around the world, is sensitive to cold stress. miRNA plays an important role in plant response to abiotic stress. In order to discover the miRNAs that play a continuing role in sugarcane response to cold stress in a later period (2 d), four sugarcane small RNA libraries were constructed. In this study, a total of 144 miRNAs were identified. Among them, 32 miRNAs showed differential expression in two relatively cold tolerant and sensitive sugarcane cultivars (FN39 and ROC22). The expression tendency for ten randomly selected differentially expressed miRNAs and their predicted targets by qRT-PCR were consistent with those from sequencing data. To further investigate the miRNA role, ScmiR393 in sugarcane was selected and overexpressed in Arabidopsis. After a relatively long cold treatment, the over-expressed ScmiR393 Arabidopsis showed better growth than the wild type, and this phenomenon was supported by the higher anthocyanin and proline contents, lower MDA and H2O2 contents, and higher SOD, POD and CAT activities, as well as the induction of some cold resistant genes including CBFs. In addition, transcriptome analysis revealed that the overexpression of ScmiR393 inhibited the auxin signaling pathway by targeting TIR1, and this may also lead to ethylene signaling inhibition. Inhibited ethylene signaling may induce the cold-resistant genes and promote anthocyanin accumulation and bolting, eventually, the cold tolerance of Arabidopsis may be improved. This study contributes to the present sugarcane miRNA resource and shows an important role of ScmiR393 in plant response to cold stress.

Epigenetic switch from repressive to permissive chromatin in response to cold stress

Switching from repressed to active status in chromatin regulation is part of the critical responses that plants deploy to survive in an ever-changing environment. We previously reported that HOS15, a WD40-repeat protein, is involved in histone deacetylation and cold tolerance in Arabidopsis However, it remained unknown how HOS15 regulates cold responsive genes to affect cold tolerance. Here, we show that HOS15 interacts with histone deacetylase 2C (HD2C) and both proteins together associate with the promoters of cold-responsive COR genes, COR15A and COR47 Cold induced HD2C degradation is mediated by the CULLIN4 (CUL4)-based E3 ubiquitin ligase complex in which HOS15 acts as a substrate receptor. Interference with the association of HD2C and the COR gene promoters by HOS15 correlates with increased acetylation levels of histone H3. HOS15 also interacts with CBF transcription factors to modulate cold-induced binding to the COR gene promoters. Our results here demonstrate that cold induces HOS15-mediated chromatin modifications by degrading HD2C. This switches the chromatin structure status and facilitates recruitment of CBFs to the COR gene promoters. This is an apparent requirement to acquire cold tolerance.

Tea plant SWEET transporters: expression profiling, sugar transport, and the involvement of CsSWEET16 in modifying cold tolerance in Arabidopsis.

Thirteen SWEET transporters were identified in Camellia sinensis and the cold-suppression gene CsSWEET16 contributed to sugar compartmentation across the vacuole and function in modifying cold tolerance in Arabidopsis. The sugars will eventually be exported transporters (SWEET) family of sugar transporters in plants is a recently identified protein family of sugar uniporters that contain seven transmembrane helices harbouring two MtN3 motifs. SWEETs play important roles in various biological processes, including plant responses to environmental stimuli. In this study, 13 SWEET transporters were identified in Camellia sinensis and were divided into four clades. Transcript abundances of CsSWEET genes were detected in various tissues. CsSWEET1a/1b/2a/2b/2c/3/9b/16/17 were expressed in all of the selected tissues, whereas the expression of CsSWEET5/7/9a/15 was not detected in some tissues, including those of mature leaves. Expression analysis of nine CsSWEET genes in leaves in response to abiotic stresses, natural cold acclimation and Colletotrichum camelliae infection revealed that eight CsSWEET genes responded to abiotic stress, while CsSWEET3 responded to C. camelliae infection. Functional analysis of 13 CsSWEET activities in yeast revealed that CsSWEET1a/1b/7/17 exhibit transport activity for glucose analogues and other types of hexose molecules. Further characterization of the cold-suppression gene CsSWEET16 revealed that this gene is localized in the vacuolar membrane. CsSWEET16 contributed to sugar compartmentation across the vacuole and function in modifying cold tolerance in Arabidopsis. Together, these findings demonstrate that CsSWEET genes play important roles in the response to abiotic and biotic stresses in tea plants and provide insights into the characteristics of SWEET genes in tea plants, which could serve as the basis for further functional identification of such genes.

Ca2+-permeable mechanosensitive channels MCA1 and MCA2 mediate cold-induced cytosolic Ca2+ increase and cold tolerance in Arabidopsis

Cold shock triggers an immediate rise in the cytosolic free calcium concentration ([Ca2+]cyt) in Arabidopsis thaliana and this cold-induced elevation of [Ca2+]cyt is inhibited by lanthanum or EGTA. It is suggested that intracellular calcium mainly contributes to the cold-induced [Ca2+]cyt response by entering into the cytosol. Two calcium-permeable mechanosensitive channels, MCA1 and MCA2 (mid1-complementing activity), have been identified in Arabidopsis. Here, we demonstrate that MCA1 and MCA2 are involved in a cold-induced increase in [Ca2+]cyt. The cold-induced [Ca2+]cyt increase in mca1 and mca2 mutants was markedly lower than that in wild types. The mca1 mca2 double mutant exhibited chilling and freezing sensitivity, compared to wild-type plants. Expression of At5g61820, At3g51660, and At4g15490, which are not regulated by the CBF/DREB1s transcription factor, was down-regulated in mca1 mca2. These results suggest that MCA1 and MCA2 are involved in the cold-induced elevation of [Ca2+]cyt, cold tolerance, and CBF/DREB1-independent cold signaling.

The inhibition of protein translation mediated by AtGCN1 is essential for cold tolerance in Arabidopsis thaliana.

In yeast, the interaction of General Control Non-derepressible 1 (GCN1) with GCN2 enables GCN2 to phosphorylate eIF2α (the alpha subunit of eukaryotic translation initiation factor 2) under a variety of stresses. Here, we cloned AtGCN1, an Arabidopsis homologue of GCN1. We show that AtGCN1 directly interacts with GCN2 and is essential for the phosphorylation of eIF2α under salicylic acid (SA), ultraviolet (UV), cold stress and amino acid deprivation conditions. Two mutant alleles, atgcn1-1 and atgcn1-2, which are defective in the phosphorylation of eIF2α, showed increased sensitivity to cold stress, compared with the wild type. Ribosome-bound RNA profiles showed that the translational state of mRNA was higher in atgcn1-1 than in the wild type. Our result also showed that cold treatment reduced the tendency of the tor mutant seedlings to produce purple hypocotyls. In addition, the kinase activity of TOR was transiently inhibited when plants were exposed to cold stress, suggesting that the inhibition of TOR is another pathway important for plants to respond to cold stress. In conclusion, our results indicate that the AtGCN1-mediated phosphorylation of eIF2α, which is required for inhibiting the initiation of protein translation, is essential for cold tolerance in Arabidopsis.

De novo Transcriptome Sequencing of Cold-treated Kentucky Bluegrass (Poa pratensis) and Analysis of the Genes Involved in Cold Tolerance

Kentucky bluegrass (Poa pratensis) is strongly resistant to cold stress. Although the molecular mechanism of the plant response to cold stress has been widely documented in model plants, little is known about the cold tolerance of Kentucky bluegrass at the genomic level. Here, we compared the transcriptomes of Kentucky bluegrass under cold treatment (-5°C) and a control treatment (at 20°C) by RNA-seq and de novo assembly. Totally 75,934 unigenes were generated, among which 53,762 were successfully annotated in public databases. Upon comparing the transcriptomes of the control and cold-treated plants, 3,896 unigenes were identified as differentially expressed. Among these genes, 2,410 were down-regulated and 1,486 were up-regulated in the cold-treated plants. A few previously reported cold-induced proteins, antioxidant enzymes, and osmoregulation proteins were identified, and their expression levels were estimated. Moreover, ten differentially expressed genes were selected for qRT-PCR verification. Their expression patterns were consistent with the results of the RNA-seq. Additionally, the transcription factor families, i.e., ethylene response factors, heat stress transcription factors, NAC proteins, WRKY domaincontaining proteins, and auxin response factors, were identified as differentially expressed genes. The identification of these involved genes will facilitate studies on the transcriptional regulation of cold tolerance in plants. Keywords: Kentucky bluegrass; Cold tolerance; Transcriptome; Differentially expressed genes; Transcriptional regulation Introduction Cold stress, such as chilling and freezing temperatures, is a major environmental factor that influences plant distribution, growth and development [1]. A few plants cope with cold stress by acquiring cold tolerance, a process termed cold acclimation. During this process, various biochemical and physiological changes occur and make the plant more tolerant under chilling or freezing conditions [2]. Cold acclimation involves the signal transduction of various genes responding to cold stress, transcriptional regulation and posttranslational regulation of transcription factors [1]. The induction of cold responsive (COR) genes have been shown to result in cold acclimation for alfalfa [3]. Many transcription factors involved in cold tolerance have been identified [4]. C-repeat binding factors (CBFs), also known as dehydration-responsive element-binding (DREB) proteins, are a group of transcription factors that are well-studied in plant cold stress. CBFs can bind to the promoters of COR genes and activate their expression [1]. The ectopic expression of CBF1 activates the expression of COR and induces cold tolerance in Arabidopsis [5]. Regulators upstream of CBF have also been identified, including ICE1, MYB15, CAMTA and HOS1 [1,6-8]. Although considerable studies have documented the molecular mechanism of plant cold tolerance, deep studies are necessary to highlight the transcriptional regulation of cold tolerance. Kentucky bluegrass (Poa pratensis) is a perennial, cool-season species of grass native to Europe and is widely used for making lawns in parks, gardens, golf courses and sports fields [9]. This plant is strongly resistant to cold stress and can survive at temperatures as low as -38°C. This adaptability suggests that Kentucky bluegrass

The DEAD-Box RNA Helicase AtRH7/PRH75 Participates in Pre-rRNA Processing, Plant Development and Cold Tolerance in Arabidopsis.

DEAD-box RNA helicases belong to an RNA helicase family that plays specific roles in various RNA metabolism processes, including ribosome biogenesis, mRNA splicing, RNA export, mRNA translation and RNA decay. This study investigated a DEAD-box RNA helicase, AtRH7/PRH75, in Arabidopsis. Expression of AtRH7/PRH75 was ubiquitous; however, the levels of mRNA accumulation were increased in cell division regions and were induced by cold stress. The phenotypes of two allelic AtRH7/PRH75-knockout mutants, atrh7-2 and atrh7-3, resembled auxin-related developmental defects that were exhibited in several ribosomal protein mutants, and were more severe under cold stress. Northern blot and circular reverse transcription-PCR (RT-PCR) analyses indicated that unprocessed 18S pre-rRNAs accumulated in the atrh7 mutants. The atrh7 mutants were hyposensitive to the antibiotic streptomycin, which targets ribosomal small subunits, suggesting that AtRH7 was also involved in ribosome assembly. In addition, the atrh7-2 and atrh7-3 mutants displayed cold hypersensitivity and decreased expression of CBF1, CBF2 and CBF3, which might be responsible for the cold intolerance. The present study indicated that AtRH7 participates in rRNA biogenesis and is also involved in plant development and cold tolerance in Arabidopsis.

Overexpression of pigeonpea stress-induced cold and drought regulatory gene (CcCDR) confers drought, salt, and cold tolerance in Arabidopsis.

A potent cold and drought regulatory protein-encoding gene (CcCDR) was isolated from the subtractive cDNA library of pigeonpea plants subjected to drought stress. CcCDR was induced by different abiotic stress conditions in pigeonpea. Overexpression of CcCDR in Arabidopsis thaliana imparted enhanced tolerance against major abiotic stresses, namely drought, salinity, and low temperature, as evidenced by increased biomass, root length, and chlorophyll content. Transgenic plants also showed increased levels of antioxidant enzymes, proline, and reducing sugars under stress conditions. Furthermore, CcCDR-transgenic plants showed enhanced relative water content, osmotic potential, and cell membrane stability, as well as hypersensitivity to abscisic acid (ABA) as compared with control plants. Localization studies confirmed that CcCDR could enter the nucleus, as revealed by intense fluorescence, indicating its possible interaction with various nuclear proteins. Microarray analysis revealed that 1780 genes were up-regulated in CcCDR-transgenics compared with wild-type plants. Real-time PCR analysis on selected stress-responsive genes, involved in ABA-dependent and -independent signalling networks, revealed higher expression levels in transgenic plants, suggesting that CcCDR acts upstream of these genes. The overall results demonstrate the explicit role of CcCDR in conferring multiple abiotic stress tolerance at the whole-plant level. The multifunctional CcCDR seems promising as a prime candidate gene for enhancing abiotic stress tolerance in diverse plants.

Over-expression of BcFLC1 from non-heading Chinese cabbage enhances cold tolerance in Arabidopsis

A gene (named BcFLC1) homologous to the AtFLC gene, which encodes a floral repressor, was isolated from the nonheading Chinese cabbage (Brassica campestris L. ssp. chinensis) cv. NJ074. The gene showed high similarity to AtFLC. For studying the gene function, we designed to introduce the BcFLC1 gene into Arabidopsis thaliana. The results showed that BcFLC1 had effects on flowering time similar to AtFLC. We also found that Arabidopsis cold-tolerance was enhanced by BcFLC1 overexpression. Under low temperature stress, the BcFLC1 transgenic plants exhibited stronger growth than wild-type plants. The elevated cold tolerance of the BcFLC1 over-expressing plants was also confirmed by the changes of electrolyte leakage and malonyldialdehyde and proline content.

AtMYB14 Regulates Cold Tolerance in Arabidopsis

Low temperature affects plant growth and crop productivity. The CBF genes are a class of transcription factors that play important roles in cold response. Here we report that AtMYB14 participates in freezing tolerance in Arabidopsis by affecting expression of CBF genes. The AtMYB14 gene was down-regulated by cold treatment. AtMYB14 encodes a nuclear protein that functions as an R2R3-MYB transcription activator. Knock-down of AtMYB14 by artificial microRNA increased the tolerance to freezing stress. Both the CBF genes and the downstream genes were induced to a much higher level in AtMYB14 knock-down plants than in wild type under cold treatment. Our results suggest that AtMYB14 plays an important role in the plant response to cold stress.

Cold-inducible expression of AZI1 and its function in improvement of freezing tolerance of Arabidopsis thaliana and Saccharomyces cerevisiae

AZI1 ( AZELAIC ACID INDUCED 1) of Arabidopsis thaliana could be induced by azelaic acid and was involved in priming of systemic plant immunity. In the present work, expression of AZI1 in response to low temperature was investigated via RNA gel blot analysis. AZI1 could be induced slowly by cold stress and more than 6 h treatment at 4 °C was required to detect an increase in mRNA abundance. However, the high expression state could not be maintained stably and would decline to basal level when the plants were transferred to room temperature. In order to clarify the function of AZI1 in resistance to abiotic stresses, overexpressing, RNA interference and T-DNA knockout lines of this gene were used in electrolyte leakage assays. Overexpression of AZI1 resulted in reduced electrolyte leakage during freezing damage. In contrast, AZI1 knockdown and knockout lines showed increased tendencies in cellular damage after freezing treatment. To further validate the potential resistance of AZI1 to low-temperature stress, Saccharomyces cerevisiae cells were transformed with pESC- AZI1 in which AZI1 was under the control of GAL1 promoter. Compared to yeast cells containing empty pESC-URA, the survival rate of yeast cells harboring AZI1 increased obviously after freezing treatment. All these results suggested that AZI1 might be multifunctional and associated with cold tolerance of Arabidopsis.

Comparative analysis of Arabidopsis zinc finger-containing glycine-rich RNA-binding proteins during cold adaptation

Among the three zinc finger-containing glycine-rich RNA-binding proteins, named AtRZ-1a, AtRZ-1b, and AtRZ-1c, in the Arabidopsis thaliana genome, AtRZ-1a has previously been shown to enhance cold and freezing tolerance in Arabidopsis. Here, we determined and compared the functional roles of AtRZ-1b and AtRZ-1c in Arabidopsis and Escherichia coli under cold stress conditions. AtRZ-1b, but not AtRZ-1c, successfully complemented the cold sensitivity of E. coli BX04 mutant cells lacking four cold shock proteins. Domain deletion and site-directed mutagenesis showed that the zinc finger motif of AtRZ-1b is important for its complementation ability, and that the truncated N- and C-terminal domains of AtRZ-1b and AtRZ-1c harbor the complementation ability. Despite an increase in transcript levels of AtRZ-1b and AtRZ-1c under cold stress, overexpression or loss-of-function mutations did not affect seed germination or seedling growth of Arabidopsis under cold stress conditions. AtRZ-1b and AtRZ-1c proteins, being localized to the nucleus, have been shown to bind non-specifically to RNA sequences in vitro, in comparison to AtRZ-1a that is localized to both the nucleus and the cytoplasm and binds preferentially to G- or U-rich RNA sequences. Taken together, these results demonstrate that the three AtRZ-1 family members showing different cellular localization and characteristic nucleic acid-binding property have a potential to contribute differently to the enhancement of cold tolerance in Arabidopsis and E. coli.

Short term temperature drops do not enhance cold tolerance

To successfully transplant agricultural species in the spring, prior hardening is of great significance. Low, non-freezing temperature increases cold tolerance in many species. Also, diurnal temperature drops have been suggested to improve cold tolerance, as assessed by ultrastructural studies after short term freezing of leaf discs. Pre-treatment with lower day than night temperature prior to hardening has also been reported to enhance cold resistance in winter rape. This study investigated the effect of temperature drops on cold resistance of different species. In contrast to a period of continuous low temperature, short diurnal temperature drops did not enhance cold tolerance in Arabidopsis, swede, white cabbage or pea, compared to control plants. Exposure to low temperature of 6°C for 6 days increased cold tolerance by 2–5°C compared to plants exposed to diurnal temperature drops or control plants. Pre-treatment with diurnal temperature drops in the entire growth period prior to hardening with constant low temperature did not give any additional hardening in swede and pea. In conclusion, by freeze testing of whole plants under controlled conditions we have found no evidence supporting the hypothesis that diurnal temperature drops improve cold tolerance. However, temperature drops reduce plants size like shown earlier for a number of other species, and thus is a tool to produce compact, robust plants.

The Role of a Zinc Finger-containing Glycine-rich RNA-binding Protein During the Cold Adaptation Process in Arabidopsis thaliana

The mechanistic role of a glycine-rich RNA-binding protein designated atRZ-1a that contributes to enhance cold tolerance in Arabidopsis was investigated. Overexpression of atRZ-1a did not affect the expression of various cold-responsive genes such as COR6.6, COR15a, COR47, RD29A, RD29B and LTI29. Proteome analyses revealed that overexpression of atRZ-1a modulated the expression of several stress-responsive genes, and the transcript levels and RNA stability of these target genes were not affected by atRZ-1a. atRZ-1a successfully complements the cold sensitivity of Escherichia coli lacking four cold shock proteins. These results strongly suggest that atRZ-1a plays a role as an RNA chaperone during the cold adaptation process.