Cold Tolerance In Transgenic Arabidopsis Research Articles

Brassica rapa BrICE1 and BrICE2 Positively Regulate the Cold Tolerance via CBF and ROS Pathways, Balancing Growth and Defense in Transgenic Arabidopsis.

Winter rapeseed (Brassica rapa) has a good chilling and freezing tolerance. inducer of CBF expression 1 (ICE1) plays a crucial role in cold signaling in plants; however, its role in Brassica rapa remains unclear. In this study, we identified 41 ICE1 homologous genes from six widely cultivated Brassica species. These genes exhibited high conservation, with evolutionary complexity between diploid and allotetraploid species. Cold stress induced ICE1 homolog expression, with differences between strongly and weakly cold-tolerant varieties. Two novel ICE1 paralogs, BrICE1 and BrICE2, were cloned from Brassica rapa Longyou 6. Subcellular localization assays showed that they localized to the nucleus, and low temperature did not affect their nuclear localization. The overexpression of BrICE1 and BrICE2 increased cold tolerance in transgenic Arabidopsis and enhanced reactive oxygen species' (ROS) scavenging ability. Furthermore, our data demonstrate that overexpression of BrICE1 and BrICE2 inhibited root growth in Arabidopsis, and low temperatures could induce the degradation of BrICE1 and BrICE2 via the 26S-proteasome pathway. In summary, ICE1 homologous genes exhibit complex evolutionary relationships in Brassica species and are involved in the C-repeat/DREB binding factor (CBF) pathway and ROS scavenging mechanism in response to cold stress; these regulating mechanisms might also be responsible for balancing the development and cold defense of Brassica rapa.

VvERF117 positively regulates grape cold tolerance through direct regulation of the antioxidative gene BAS1

Cold stress significantly threatens grape quality, yield, and geographical distribution. Although ethylene-responsive factors (ERFs) are recognized for their pivotal roles in cold stress, the regulatory mechanisms of many ERFs contributing to tolerance remain unclear. In this study, we identified the cold-responsive gene VvERF117 and elucidated its positive regulatory function in cold tolerance. VvERF117 exhibits transcriptional activity and localizes to the nucleus. VvERF117 overexpression improved cold tolerance in transgenic Arabidopsis, grape calli, and grape leaves, whereas VvERF117 silencing increased cold sensitivity in grape calli and leaves. Furthermore, VvERF117 overexpression remarkably upregulated the expression of several stress-related genes. Importantly, BAS1, encoding a 2-Cys peroxidase (POD), was confirmed as a direct target gene of VvERF117. Meanwhile, compared to the wild-type, POD activity and H2O2 content were remarkably increased and decreased in VvERF117-overexpressing grape calli and leaves, respectively. Conversely, VvERF117 silencing displayed the opposite trend in grape calli and leaves under cold stress. These findings indicate that VvERF117 plays a positive role in cold resistance by, at least in part, enhancing antioxidant capacity through regulating the POD-encoding gene VvBAS1, leading to effective mitigation of reactive oxygen species.

An AP2/ERF transcription factor VvERF63 positively regulates cold tolerance in Arabidopsis and grape leaves

Cold stress adversely impacts grape growth, development, and yield. Thus, enhancing the cold tolerance of grape is a priority for grape breeding. Several APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors (TFs) play critical roles in plant responses to various abiotic stresses. However, only a few ERF TFs in grape have been identified under various stress. Here, we cloned a novel ERF gene, VvERF63 from ‘Cabernet Sauvignon’ ( Vitis vinifera L . ), which is strongly induced by cold treatment and exogenous abscisic acid (ABA) treatment, and has transcriptional activation activity in yeast. Overexpression of VvERF63 in Arabidopsis improved cold resistance at the seedling and mature stages. Moreover, VvERF63 -overexpressing and silencing grape leaves displayed improved and decreased cold tolerance, respectively. These results indicated that VvERF63 plays a positive role in cold tolerance by enhancing the photosynthetic capacity, and ameliorating cellular damage, which was evidenced by improved maximum photosynthetic efficiency of photosystem II (Fv/Fm), chlorophyll content, lower electrolyte leakage and malondialdehyde, as well as by increased antioxidant enzymes activities in the transgenic Arabidopsis . Additionally, the expression levels of stress- and antioxidant- related genes were upregulated in VvERF63 -overexpressing plants. Our results suggested that VvERF63 positively participated in the cold resistance response and has excellent potential as a candidate gene for improving the cold tolerance of plant. • VvERF63 was an ethylene response factor, the expression of which was induced by cold and ABA treatment. • VvERF63 could increase photosynthetic capacity and enhance membrane stabilization in VvERF63 -overexpressing grape leaves and Arabidopsis under cold stress. • VvERF63 plays a role in cold tolerance by increasing activities of antioxidant enzymes and expression levels of stress-responsive genes. • VvERF63 possibly mediates CBF-COR signal pathway to improve plant cold tolerance in transgenic Arabidopsis and transient transgenic grape leaves.

Characterization of the SWEET Gene Family in Longan (Dimocarpus longan) and the Role of DlSWEET1 in Cold Tolerance

Sugars will eventually be exported transporters (SWEET), a group of relatively novel sugar transporters, that play important roles in phloem loading, seed and fruit development, pollen development, and stress response in plants. Longan (Dimocarpus longan), a subtropic fruit tree with high economic value, is sensitive to cold. However, whether the SWEET gene family plays a role in conferring cold tolerance upon longan remains unknown. Here, a total of 20 longan SWEET (DlSWEET) genes were identified, and their phylogenetic relationships, gene structures, cis-acting elements, and tissue-specific expression patterns were systematically analyzed. This family is divided into four clades. Gene structures and motifs analyses indicated that the majority of DlSWEETs in each clade shared similar exon–intron organization and conserved motifs. Tissue-specific gene expression suggested diverse possible functions for DlSWEET genes. Cis-elements analysis and quantitative real-time PCR (qRT-PCR) analysis revealed that DlSWEET1 responded to cold stress. Notably, the overexpression of DlSWEET1 improved cold tolerance in transgenic Arabidopsis, suggesting that DlSWEET1 might play a positive role in D. longan’s responses to cold stress. Together, these results contribute to a better understanding of SWEET genes, which could serve as a foundation for the further functional identification of these genes.

VaMYB44 transcription factor from Chinese wild Vitis amurensis negatively regulates cold tolerance in transgenic Arabidopsis thaliana and V. vinifera.

Heterologous expression of VaMYB44 gene in Arabidopsis and V. vinifera cv. 'Thompson Seedless' increases cold sensitivity, which is mediated by the interaction of VaMYC2 and VaTIFY5A with VaMYB44 MYB transcription factors play critical roles in plant stress response. However, the function of MYB44 under low temperature stress is largely unknown in grapes. Here, we isolated a VaMYB44 gene from Chinese wild Vitis amurensis acc. 'Shuangyou' (cold-resistant). The VaMYB44 is expressed in various organs and has lower expression levels in stems and young leaves. Exposure of the cold-sensitive V. vinifera cv. 'Thompson Seedless' and cold-resistant 'Shuangyou' grapevines to cold stress (-1°C) resulted in differential expression of MYB44 in leaves with the former reaching 14 folds of the latter after 3h of cold stress. Moreover, the expression of VaMYB44 was induced by exogenous ethylene, abscisic acid, and methyl jasmonate in the leaves of 'Shuangyou'. Notably, the subcellular localization assay identified VaMYB44 in the nucleus. Interestingly, heterologous expression of VaMYB44 in Arabidopsis and 'Thompson Seedless' grape increased freezing-induced damage compared to their wild-type counterparts. Accordingly, the transgenic lines had higher malondialdehyde content and electrolyte permeability, and lower activities of superoxide dismutase, peroxidase, and catalase. Moreover, the expression levels of some cold resistance-related genes decreased in transgenic lines. Protein interaction assays identified VaMYC2 and VaTIFY5A as VaMYB44 interacting proteins, and VaMYC2 could bind to the VaMYB44 promoter and promote its transcription. In conclusion, the study reveals VaMYB44 as the negative regulator of cold tolerance in transgenic Arabidopsis and transgenic grapes, and VaMYC2 and VaTIFY5A are involved in the cold sensitivity of plants by interacting with VaMYB44.

The Kandelia obovata transcription factor KoWRKY40 enhances cold tolerance in transgenic Arabidopsis

BackgroundWRKY transcription factors play key roles in plant development processes and stress response. Kandelia obovata is the most cold-resistant species of mangrove plants, which are the important contributors to coastal marine environment. However, there is little known about the WRKY genes in K. obovata.ResultsIn this study, a WRKY transcription factor gene, named KoWRKY40, was identified from mangrove plant K. obovata. The full-length cDNA of KoWRKY40 gene was 1420 nucleotide bases, which encoded 318 amino acids. The KoWRKY40 protein contained a typical WRKY domain and a C2H2 zinc-finger motif, which were common signatures to group II of WRKY family. The three-dimensional (3D) model of KoWRKY40 was formed by one α-helix and five β-strands. Evolutionary analysis revealed that KoWRKY40 has the closest homology with a WRKY protein from another mangrove plant Bruguiera gymnorhiza. The KoWRKY40 protein was verified to be exclusively located in nucleus of tobacco epidermis cells. Gene expression analysis demonstrated that KoWRKY40 was induced highly in the roots and leaves, but lowly in stems in K. obovata under cold stress. Overexpression of KoWRKY40 in Arabidopsis significantly enhanced the fresh weight, root length, and lateral root number of the transgenic lines under cold stress. KoWRKY40 transgenic Arabidopsis exhibited higher proline content, SOD, POD, and CAT activities, and lower MDA content, and H2O2 content than wild-type Arabidopsis under cold stress condition. Cold stress affected the expression of genes related to proline biosynthesis, antioxidant system, and the ICE-CBF-COR signaling pathway, including AtP5CS1, AtPRODH1, AtMnSOD, AtPOD, AtCAT1, AtCBF1, AtCBF2, AtICE1, AtCOR47 in KoWRKY40 transgenic Arabidopsis plants.ConclusionThese results demonstrated that KoWRKY40 conferred cold tolerance in transgenic Arabidopsis by regulating plant growth, osmotic balance, the antioxidant system, and ICE-CBF-COR signaling pathway. The study indicates that KoWRKY40 is an important regulator involved in the cold stress response in plants.

CdWRKY2-mediated sucrose biosynthesis and CBF-signalling pathways coordinately contribute to cold tolerance in bermudagrass.

SummaryBermudagrass (Cynodon dactylon) is one of the most widely cultivated warm‐season turfgrass species around the world. Cold stress has been a key environmental factor that adversely affects the growth, development, and geographical distribution of bermudagrass; however, the underlying mechanism of bermudagrass responsive to cold stress remains largely unexplored. Here, we identified a cold‐induced WRKY transcription factor CdWRKY2 from bermudagrass and demonstrated its function in cold stress response. Overexpression of CdWRKY2 enhanced cold tolerance in transgenic Arabidopsis and bermudagrass hairy roots, while knocking down CdWRKY2 expression via virus‐induced gene silencing increased cold susceptibility. RNA sequencing showed that overexpression of CdWRKY2 in Arabidopsis activated the expression of genes involved in sucrose synthesis and metabolism, including sucrose synthase 1 (AtSUS1) and sucrose phosphate synthase 2F (AtSPS2F). CdSPS1, the homology gene of AtSPS2F in bermudagrass, was subsequently proven to be the direct target of CdWRKY2 by yeast one‐hybrid, electrophoretic mobility shift assay, and transient expression analysis. As expected, overexpression of CdSPS1 conferred cold tolerance in transgenic Arabidopsis plants, whereas silencing CdSPS1 expression enhanced cold sensitivity in bermudagrass. Besides, CdCBF1 whose expression was dramatically up‐regulated in CdWRKY2‐overexpressing bermudagrass hairy roots but down‐regulated in CdWRKY2‐silencing bermudagrass both under normal and cold stress conditions was confirmed as another target of CdWRKY2. Collectively, this study reveals that CdWRKY2 is a positive regulator in cold stress by targeting CdSPS1 and CdCBF1 promoters and activating their expression to coordinately mediate sucrose biosynthesis and CBF‐signalling pathway, which provides valuable information for breeding cold‐resistant bermudagrass through gene manipulation.

DlICE1, a stress-responsive gene from Dimocarpus longan, enhances cold tolerance in transgenic Arabidopsis

ICE1 (inducer of CBF expression 1) encodes a typical MYC-like basic helix-loop- helix (bHLH) transcription factor that acts as a pivotal component in the cold signalling pathway. In this study, DlICE1, a novel ICE1-like gene, was isolated from the southern subtropical fruit tree longan (Dimocarpus longan Lour.). DlICE1 encodes a nuclear protein with a highly conserved bHLH domain. DlICE1 expression was slightly upregulated under cold stress. Overexpression of DlICE1 in Arabidopsis conferred enhanced cold tolerance via increased proline content, decreased ion leakage, and reduced malondialdehyde (MDA) and reactive oxygen species (ROS) accumulation. Expression of the ICE1-CBF cold signalling pathway genes, including AtCBF1/2/3 and cold-responsive genes (AtRD29A, AtCOR15A, AtCOR47 and AtKIN1), was also significantly higher in DlICE1-overexpressing lines than in wild-type (WT) plants under cold stress. In conclusion, these findings indicate that DlICE1 is a member of the bHLH gene family and positively regulates cold tolerance in D. longan.

The ICE-like transcription factor HbICE2 is involved in jasmonate-regulated cold tolerance in the rubber tree (Hevea brasiliensis).

An ICE-like transcription factor mediates jasmonate-regulated cold tolerance in the rubber tree (Hevea brasiliensis), and confers cold tolerance in transgenic Arabidopsis. The rubber tree (Hevea brasiliensis) is susceptible to low temperatures, and understanding the mechanisms regulating cold stress is of great potential value for enhancing tolerance to this environmental variable. In this study, we find that treatment with exogenous methyl jasmonate (MeJA) could significantly enhance Hevea brasiliensis cold tolerance. In addition, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments show that JASMONATE ZIM-DOMAIN(JAZ) proteins, HbJAZ1 and HbJAZ12, key repressors of JA signaling pathway, interact with HbICE2, a novel ICE (Inducer of CBF Expression)-like protein. HbICE2 was nuclear-localised and bound to the MYC recognition (MYCR) sequence. The transcriptional activation activity of HbICE2 in yeast cells was dependent on the N-terminus, and overexpression of HbICE2 in Arabidopsis resulted in elevated tolerance to chilling stress. Furthermore, dual-luciferase transient assay reveals that HbJAZ1 and HbJAZ12 proteins inhibit the transcriptional function of HbICE2. The expression of C-repeat-binding factor (CBF) signalling pathway genes including HbCBF1, HbCBF2 and HbCOR47 were up-regulated by MeJA. Taken together, our data suggest that the new ICE-like transcription factor HbICE2 is involved in jasmonate-regulated cold tolerance in Hevea brasiliensis.

CsINV5, a tea vacuolar invertase gene enhances cold tolerance in transgenic Arabidopsis

BackgroundVacuolar invertases (VINs) have been reported to regulate plant growth and development and respond to abiotic stresses such as drought and cold. With our best knowledge, the functions of VIN genes little have been reported in tea plant (Camellia sinensis L.). Therefore, it is necessary to develop research in this field.ResultsHere, we identified a VIN gene, CsINV5, which was induced by cold acclimation and sugar treatments in the tea plant. Histochemical assays results showed that the 1154 bp 5′-flanking sequence of CsINV5 drove β-glucuronidase (GUS) gene expression in roots, stems, leaves, flowers and siliques of transgenic Arabidopsis during different developmental stages. Moreover, promoter deletion analysis results revealed that an LTRE-related motif (CCGAAA) and a WBOXHVISO1 motif (TGACT) within the promoter region of CsINV5 were the core cis-elements in response to low temperature and sugar signaling, respectively. In addition, overexpression of CsINV5 in Arabidopsis promoted taproot and lateral root elongation through glucose-mediated effects on auxin signaling. Based on physiological and RNA-seq analysis, we found that overexpression of CsINV5 improved cold tolerance in transgenic Arabidopsis mainly by increasing the contents of glucose and fructose, the corresponding ratio of hexose to sucrose, and the transcription of osmotic-stress-related genes (P5CS1, P5CS2, AtLEA3, COR413-PM1 and COR15B) to adjust its osmotic potential.ConclusionsComprehensive experimental results suggest that overexpression of CsINV5 may enhance the cold tolerance of plant through the modification of cellular sugar compounds contents and osmotic regulation related pathways.

The rice transcription factors OsICE confer enhanced cold tolerance in transgenic Arabidopsis

ABSTRACTCold stress is one of the major constraints for crop yield. Plants have in turn evolved highly sophisticated mechanisms involving altered physiologic and biochemical processes to cope with the cold stress. Previous studies have revealed that the INDUCER OF CBF EXPRESSION 1 (ICE1), a basic helix-loop-helix (bHLH) transcription factor, directly binds and activates the expression of C-Repeat Binding Factor/Dehydration-Responsive-Element-Binding protein (CBF/DREB1) to regulate the cold-response pathway in Arabidopsis thaliana. However, the function of AtICE1 orthologues in rice is largely unknown. Here we identified that OsICE1 and OsICE2 in rice shared highly conserved amino acid sequence with AtICE1 in Arabidopsis. Overexpression of OsICE1 and OsICE2 in Arabidopsis significantly enhanced the cold tolerance of Arabidopsis seedlings and improved the expression of cold-response genes. Furthermore, we showed that both OsICE1 and OsICE2 physically interact with OsMYBS3, a single DNA-binding repeat MYB transcription factor that is essential for cold adaptation in rice, suggesting that OsICE1/OsICE2 and OsMYBS3 probably act through specific signal transduction mechanisms to coordinate cold tolerance in rice. These results demonstrated that the 2 OsICEs are orthologues of AtICE1 and play positive regulators in activation of cold-response genes to regulate the cold tolerance.

Functional Characterization of Cotton GaMYB62L, a Novel R2R3 TF in Transgenic Arabidopsis.

Drought stress can trigger the production of ABA in plants, in response to adverse conditions, which induces the transcript of stress-related marker genes. The R2R3 MYB TFs are implicated in regulation of various plants developmental, metabolic and multiple environmental stress responses. Here, a R2R3-MYB cloned gene, GaMYB62L, was transformed in Arabidopsis and was functionally characterized. The GaMYB62L protein contains two SANT domains with a conserved R2R3 imperfect repeats. The GaMYB62L cDNA is 1,017 bp with a CDS of 879, encodes a 292-residue polypeptide with MW of 38.78 kD and a pI value of 8.91. Overexpressed GaMYB62L transgenic Arabidopsis have increased proline and chlorophyll content, superior seed germination rate under salt and osmotic stress, less water loss rate with reduced stomatal apertures, high drought avoidance as compared to WT on water deprivation and also significant plant survival rates at low temperature. In addition, overexpressed GaMYB62L lines were more sensitive to ABA mediated germination and root elongation assay. Moreover, ABA induced GaMYB62L overexpression, enhanced the expression of ABA stress related marker genes like RD22, COR15A, ADH1, and RD29A. Together, overexpression of GaMYB62L suggested having developed better drought, salt and cold tolerance in transgenic Arabidopsis and thus presented it as a prospective candidate gene to achieve better abiotic stress tolerance in cotton crop.

Cloning of Zoysiagrass CBF Gene and Validation of Cold Tolerance in Transgenic Arabidopsis

Cloning of Zoysiagrass <i>CBF</i> Gene and Validation of Cold Tolerance in Transgenic <i>Arabidopsis</i>

Overexpression of a western white pine PR10 protein enhances cold tolerance in transgenic Arabidopsis

The PmPR10-1.10 protein from western white pine is known to be associated with frost hardiness, and up-regulated by seasonal cold acclimation and biotic and abiotic stresses. To gain insight into the molecular basis of cold hardiness, we investigated the potential physiological role of PmPR10-1.10 by gene overexpression in transgenic Arabidopsis plants. A binary vector was constructed for PmPR10-1.10 synthesis in higher plants and transgenic Arabidopsis lines were generated by Agrobacterium-mediated transformation. Following Western protein blot analysis confirming target protein production, transgenic Arabidopsis lines were tested for cold tolerance by electrolyte leakage analysis post treatment of different freezing temperatures. Our results demonstrate that accumulation of PmPR10-1.10 protein resulted in significantly greater freezing tolerance in transgenic plants than in wild type plants. This indicates that the transfer and selection of cold acclimation proteins like PmPR10-1.10 may be a breeding strategy for the development of freezing tolerance in conifers.

Expression and Function of a Modified AP2/ERF Transcription Factor from Brassica napus Enhances Cold Tolerance in Transgenic Arabidopsis

One of the most rapid and effective defensive mechanisms plants have for protecting themselves, from a variety of biotic and abiotic stresses, is the regulation of plant signal transcription factors. AP2/ERF factors play an important role in plant development as well as in hormonal regulation and cold response. Directed evolution is a powerful tool to modify proteins, improving their properties, and for studying their structure-function relations. Here, the transgenic Arabidopsis plants over-expressed a mutant gene, BnaERF-B3-hy15-mu3, which encoded for a factor that exhibited more binding activity with the GCC box element than the wild-type gene BnaERF-B3-hy15 encode factor, and exhibited more freezing tolerance than transgenic plants containing the original BnaERF-B3-hy15 gene. Real-time PCR analyses also revealed that the expression levels of several stress-regulated genes were altered in the over-expressed BnaERF-B3-hy15-mu3 transgenic lines. The BnaERF-B3-hy15 responded to exogenous ABA. Using RT-PCR analysis, the expression of BnaERF-B3-hy15 at different stages and stress treatments were also analyzed.

The cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1encodes an ICE-like protein in apple

BackgroundPlant growth is greatly affected by low temperatures, and the expression of a number of genes is induced by cold stress. Although many genes in the cold signaling pathway have been identified in Arabidopsis, little is known about the transcription factors involved in the cold stress response in apple.ResultsHere, we show that the apple bHLH (basic helix-loop-helix) gene MdCIbHLH1 (Cold-Induced bHLH1), which encodes an ICE-like protein, was noticeably induced in response to cold stress. The MdCIbHLH1 protein specifically bound to the MYC recognition sequences in the AtCBF3 promoter, and MdCIbHLH1 overexpression enhanced cold tolerance in transgenic Arabidopsis. In addition, the MdCIbHLH1 protein bound to the promoters of MdCBF2 and favorably contributed to cold tolerance in transgenic apple plants by upregulating the expression of MdCBF2 through the CBF (C-repeat-binding factor) pathway. Our findings indicate that MdCIbHLH1 functions in stress tolerance in different species. For example, ectopic MdCIbHLH1 expression conferred enhanced chilling tolerance in transgenic tobacco. Finally, we observed that cold induces the degradation of the MdCIbHLH1 protein in apple and that this degradation was potentially mediated by ubiquitination and sumoylation.ConclusionsBased on these findings, MdCIbHLH1 encodes a transcription factor that is important for the cold tolerance response in apple.

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.