CBF Genes Research Articles

Increased Drought and Salinity Tolerance in Citrus aurantifolia (Mexican Lemon) Plants Overexpressing Arabidopsis CBF3 Gene

Citrus are a globally important fruit crop. Abiotic stressors such as drought and salinity adversely affect physiological citrus performance and survival. With the aim of improving drought tolerance in citrus plants, we constructed transgenic lines of Citrus lemon overexpressing the Arabidopsis transcription factor CBF3. Molecular, physiological, and quantitative real-time analyses showed high expression of AtCBF3 in three selected transgenic lines. During a 15-day treatment of water deficit by cessation of irrigation, the transgenic lines LM2 and LM14 showed lower stomatal conductance and transpiration paired with lower photosynthesis, whereas transgenic line LM7 maintained its photosynthesis, declining stomatal conductance, and transpiration compared to WT plants, which is manifested into more efficient water use. The genes CsRafS1 and CsGolS1 showed similar or greater expression in one of the transgenic lines with respect to control plants. Moreover, transgenic lines were more tolerant to saline stress and presented a greener phenotype with increased chlorophyll content in leaf discs compared to WT plants. In addition, a lower electrical conductivity in solution was observed in transgenic lines. Furthermore, all transgenic lines exhibited significantly less accumulation of reactive oxygen species than WT plants. Together, these results suggest the potential for heterologous expression of the AtCBF3 gene to mediate tolerance to hydric and saline stress in citrus plants.

Studies on expression of CBF1 and CBF2 genes and anti-oxidant enzyme activities in papaya genotypes exposed to low temperature stress

Papaya, a crop of tropical and subtropical regions, is a herbaceous plant. Frost damage is a serious problem of papaya cultivation in sub-tropical areas. The C-repeat binding factor (CBF) genes are transcriptional activators that bind to the promoter regions of cold tolerance genes. The present experiment was conducted under completely controlled conditions of National Phytotron Facility, ICAR-IARI, New Delhi to understand the effect of temperature stress and its interaction in five papaya genotypes namely, Pusa Nanha, Red Lady, P-7-2, P-7-9, P-7-14 and one cold tolerant distant relative genus, i.e., Vasconcellea cundinamarcensis. RNA was isolated from all the above genotypes under control and low temperature stress conditions of 12° and 6 °C for 2 h and the expression analysis was done using quantitative Real-Time PCR (qRT-PCR). The expression of CBF2 was found to be the lowest in V. cundinamarcensis followed by P-7-9, while the highest in Red Lady. However the expression of CBF1 was observed to increase under the low temperature regimes and was maximum in V. cundinamarcensis and P-7-9, while minimum in Red Lady. This clearly explains the molecular basis of cold tolerance in V. cundinamarcensis and P-7-9 and cold susceptibility of Red Lady. The maximum activity of superoxide dismutase (SOD) was recorded in the V. cundinamarcensis (9.36 unit mg–1 TSP min–1), which was statistically different from all other genotypes. The highest mean activity of ascorbate peroxidase (APX) was observed in V. cundinamarcensis (5.79 unit mg–1 TSP) followed by P-7-9 (5.47 unit mg–1 TSP).

Overexpression of Two Upstream Phospholipid Signaling Genes Improves Cold Stress Response and Hypoxia Tolerance, but Leads to Developmental Abnormalities in Barley

Phosphatidylinositol transfer protein (PITP) and phosphatidylinositol 4-kinase (PI4K) are very upstream regulatory elements of the phospholipid signaling pathway in the signal transduction network. Unlike in animal systems, their role in stress signaling is poorly understood in plants. To study this area, PITP- and PI4K-overexpressing transgenic barley lines were developed. Morphological and developmental abnormalities were surveyed and characterized. It was revealed that the overexpression of the upstream signaling genes led to more phenotypic abnormalities than in other transgenic studies working with effector genes or even transcription factors. We hypothesize that this high level of abnormalities is the consequence of the modulation of the very upstream signal transduction pathway elements. On the other hand, we also revealed that overexpression of the PITP and PI4K genes increased stress tolerance during hypoxic cold stress, but not during salinity stress. Differences were also found in the level of frost tolerance between the transgenic overexpression plants and the recipient Golden Promise line. Molecular analysis showed that this improvement was not related to the most important cold responsive transcription factors, the CBF genes. We conclude that the transgenic method may be useful to prove the role of an upstream signaling element; however, due to the many developmental consequences that occur as side effects, it is a less advisable approach to achieve improved stress tolerance.

Differential gene expression in non-transgenic and transgenic \u201cM.26\u201d apple overexpressing a peach CBF gene during the transition from eco-dormancy to bud break

The CBF signal pathway is responsible for a significant portion of plant responses to low temperature and freezing. Overexpression of CBF genes in model organisms such as Arabidopsis thaliana enhances abiotic stress tolerance but also reduces growth. In addition to these effects, overexpression of the peach (Prunus persica [L.] Batsch) CBF1 gene in transgenic apple (Malus x domestica Borkh.) line T166 also results in early entry into and late exit from dormancy. Although the regulation of dormancy-induction and dormancy-release occur while the CBF regulon is operative in perennial, woody plants, how overexpression of CBF1 affects these dormancy-related changes in gene expression is incompletely understood. The objective of the present study was to characterize global changes in gene expression in peach CBF1-overexpressing and non-transformed apple bark tissues at different states of dormancy via RNA-seq. RNA-seq bioinformatics data was confirmed by RT-qPCR on a number of genes. Results indicate that the greatest number of significantly differentially expressed genes (DEGs) occurred in April when dormancy release and bud break normally occur but are delayed in Line T166. Genes involved in storage and inactivation of auxin, GA, and cytokinin were generally upregulated in T166 in April, while those for biosynthesis, uptake or signal transduction were generally downregulated in T166. Genes for cell division and cambial growth were also downregulated in T166 relative to the non-transformed line. These data suggest that overexpression of the peach CBF1 gene impacts growth hormone homeostasis and as a result the activation of growth in the spring, and most likely growth cessation in the fall as well.

A Role for PICKLE in the Regulation of Cold and Salt Stress Tolerance in Arabidopsis.

Arabidopsis PICKLE (PKL) is a putative CHD3-type chromatin remodeling factor with important roles in regulating plant growth and development as well as RNA-directed DNA methylation (RdDM). The role of PKL protein in plant abiotic stress response is still poorly understood. Here, we report that PKL is important for cold stress response in Arabidopsis. Loss-of-function mutations in the PKL gene lead to a chlorotic phenotype in seedlings under cold stress, which is caused by the alterations in the transcript levels of some chlorophyll metabolism-related genes. The pkl mutant also exhibits increased electrolyte leakage after freezing treatment. These results suggest that PKL is required for proper chilling and freezing tolerance in plants. Gene expression analysis shows that CBF3, encoding a key transcription factor involved in the regulation of cold-responsive genes, exhibits an altered transcript level in the pkl mutant under cold stress. Transcriptome data also show that PKL regulates the expression of a number of cold-responsive genes, including RD29A, COR15A, and COR15B, possibly through its effect on the expression of CBF3 gene. Mutation in PKL gene also results in decreased cotyledon greening rate and reduced primary root elongation under high salinity. Together, our results suggest that PKL regulates plant responses to cold and salt stress.

Sequencing of a Wild Apple (Malus baccata) Genome Unravels the Differences Between Cultivated and Wild Apple Species Regarding Disease Resistance and Cold Tolerance.

Malus baccata is one of four wild apple species that can hybridize with the cultivated apple species (Malus domestica). It is widely used in high-latitude apple-producing areas as a rootstock and breeding resource because of its disease resistance, and cold tolerance. A lack of a reference genome has limited the application of M. baccata for apple breeding. We present a draft reference genome for M. baccata. The assembled sequence consisting of 665 Mb, with a scaffold N50 value of 452 kb, included transposable elements (413 Mb) and 46,114 high-quality protein-coding genes. According to a genetic map derived from 390 sibling lines, 72% of the assembly and 85% of the putative genes were anchored to 17 linkage groups. Many of the M. baccata genes under positive selection pressure were associated with plant–pathogen interaction pathways. We identified 2,345 Transcription factor-encoding genes in 58 families in the M. baccata genome. Genes related to disease defense and cold tolerance were also identified. A total of 462 putative nucleotide-binding site (NBS)-leucine-rich-repeat (LRR) genes, 177 Receptor-like kinase (RLK) and 51 receptor-like proteins (RLP) genes were identified in this genome assembly. The M. baccata genome contained 3978 cold-regulated genes, and 50% of these gene promoter containing DREB motif which can be induced by CBF gene. We herein present the first M. baccata genome assembly, which may be useful for exploring genetic variations in diverse apple germplasm, and for facilitating marker-assisted breeding of new apple cultivars exhibiting resistance to disease and cold stress.

ZmMYB31, a R2R3-MYB transcription factor in maize, positively regulates the expression of CBF genes and enhances resistance to chilling and oxidative stress.

Maize (Zea mays L.) is an important model plant with an important role in agriculture and national economies all over the world. The optimum growth temperature of maize is between 25 and 28°C. At temperatures below 12°C, maize is vulnerable to damage by chilling stress. MYB transcription factors play important roles in plants' response to low temperature stress. Maize ZmMYB31 encodes a R2R3-MYB transcription factor, ZmMYB31, which localized in the nucleus. ZmMYB31 expression was induced by chilling stress and the highest expression level was detected with the 24h chilling treatment. ZmMYB31 expression also increased in overexpressing Arabidopsis lines. The minimal fluorescence (Fo) with all photosystem II reaction centers open increased in wild type (WT) and transgenic plants under chilling stress, with the highest increase in WT. The maximal photochemical efficiency of photosystem II (Fv/Fm) decreased more in WT than in transgenic plants during chilling stress. Furthermore, the ZmMYB31-overexpressing lines showed higher superoxide dismutase and ascorbate peroxidase activity and lower reactive oxygen species (ROS) content than the WT. The expression of genes related to chilling stress was higher in transgenic plants than in WT. These results suggest that ZmMYB31 plays a positive regulatory role in chilling and peroxide stress by regulating the expression of chilling stress-related genes to reduce ion extravasation, ROS content, and low-temperature photoinhibition, thereby improving low temperature resistance.

An update to database TraVA: organ-specific cold stress response in Arabidopsis thaliana

BackgroundTranscriptome map is a powerful tool for a variety of biological studies; transcriptome maps that include different organs, tissues, cells and stages of development are currently available for at least 30 plants. Some of them include samples treated by environmental or biotic stresses. However, most studies explore only limited set of organs and developmental stages (leaves or seedlings). In order to provide broader view of organ-specific strategies of cold stress response we studied expression changes that follow exposure to cold (+ 4 °C) in different aerial parts of plant: cotyledons, hypocotyl, leaves, young flowers, mature flowers and seeds using RNA-seq.ResultsThe results on differential expression in leaves are congruent with current knowledge on stress response pathways, in particular, the role of CBF genes. In other organs, both essence and dynamics of gene expression changes are different. We show the involvement of genes that are confined to narrow expression patterns in non-stress conditions into stress response. In particular, the genes that control cell wall modification in pollen, are activated in leaves. In seeds, predominant pattern is the change of lipid metabolism.ConclusionsStress response is highly organ-specific; different pathways are involved in this process in each type of organs. The results were integrated with previously published transcriptome map of Arabidopsis thaliana and used for an update of a public database TraVa: http://travadb.org/browse/Species=AthStress.

CBF-dependent and CBF-independent regulatory pathways contribute to the differences in freezing tolerance and cold-regulated gene expression of two Arabidopsis ecotypes locally adapted to sites in Sweden and Italy.

Arabidopsis thaliana (Arabidopsis) increases in freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation. The CBF regulatory pathway, which contributes to cold acclimation, includes three genes—CBF1, CBF2 and CBF3—encoding closely-related transcription factors that regulate the expression of more than 100 genes—the CBF regulon—that impart freezing tolerance. Here we compare the CBF pathways of two Arabidopsis ecotypes collected from sites in Sweden (SW) and Italy (IT). Previous studies showed that the SW ecotype was more freezing tolerant than the IT ecotype and that the IT ecotype had a nonfunctional CBF2 gene. Here we present results establishing that the difference in CBF2 alleles contributes to the difference in freezing tolerance between the two ecotypes. However, other differences in the CBF pathway as well as CBF-independent pathways contribute the large majority of the difference in freezing tolerance between the two ecotypes. The results also provided evidence that most cold-induced CBF regulon genes in both the SW and IT ecotypes are coregulated by CBF-independent pathways. Additional analysis comparing our results with those published by others examining the Col-0 accession resulted in the identification of 44 CBF regulon genes that were conserved among the three accessions suggesting that they likely have important functions in life at low temperature. The comparison further supported the conclusion that the CBF pathway can account for a large portion of the increase in freezing tolerance that occurs with cold acclimation in a given accession, but that CBF-independent pathways can also make a major contribution.

The arginine decarboxylase gene ADC1, associated to the putrescine pathway, plays an important role in potato cold-acclimated freezing tolerance as revealed by transcriptome and metabolome analyses.

Low temperature severely influences potato production as the cultivated potato (Solanum tuberosum) is frost sensitive, however the mechanism underlying the freezing tolerance of the potato is largely unknown. In the present research, we studied the transcriptome and metabolome of the freezing-tolerant wild species Solanum acaule (Aca) and freezing-sensitive cultivated S.tuberosum (Tub) to identify the main pathways and important factors related to freezing tolerance. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation indicated that polyamine and amino acid metabolic pathways were specifically upregulated in Aca under cold treatment. The transcriptome changes detected in Aca were accompanied by the specific accumulation of putrescine, saccharides, amino acids and other metabolites. The combination of transcriptome and metabolome analyses revealed that putrescine exhibited an accumulative pattern in accordance with the expression of the arginine decarboxylase gene ADC1. The primary role of putrescine was further confirmed by analyzing all three polyamines (putrescine, spermidine, and spermine) and the genes encoding the corresponding enzymes in two sets of potato genotypes with distinct freezing tolerance, implying that only putrescine and ADC1 were uniquely enhanced by cold in the freezing-tolerant genotypes. The function of putrescine was further analyzed by its exogenous application and the overexpression of SaADC1 in S.tuberosum cv. E3, indicating its important role(s) in cold-acclimated freezing tolerance, which was accompanied with the activation of C-repeat binding factor genes (CBFs). The present research has identified that the ADC1-associated putrescine pathway plays an important role in cold-acclimated freezing tolerance of potato, probably by enhancing the expression of CBF genes.

Overexpression of Brassica campestris BcICE1 gene increases abiotic stress tolerance in tobacco

In this study, a cDNA of ICE1 (inducer of CBF expression 1) gene, named BcICE1, was isolated from Brassica campestris ‘Longyou 6’. The deduced protein has 499 amino acids containing a typical bHLH domain and is highly identical with AtICE1 (85.9%) from Arabidopsis thaliana. BcICE1 is located in the nucleus. The activities of SOD, CAT, POD, and APX and the transcriptional levels of SOD, CAT, and POD genes were higher in BcICE1-transgenic tobacco than in wild-type (WT) tobacco under cold stress. Compared with WT tobacco, proline, soluble sugar, and chlorophyll were enhanced, whereas malondialdehyde and relative conductivity were decreased in BcICE1-transgenic tobacco. The overexpression of BcICE1 in tobacco increased the expression of CBF1, CBF2, and other stress-related genes. Moreover, under salt and PEG (25%) stress, the activities of APX and GPX and content of soluble sugar and chlorophyll in BcICE1-transgenic tobacco were higher than those in WT tobacco. Our results suggest that BcICE1 plays an important role in the response to abiotic stress.

Employing exogenous melatonin applying confers chilling tolerance in tomato fruits by upregulating ZAT2/6/12 giving rise to promoting endogenous polyamines, proline, and nitric oxide accumulation by triggering arginine pathway activity

In the present study, the mechanisms employed by exogenous melatonin applying for conferring chilling tolerance in tomato fruits during storage at 4 °C for 28 days were investigated. Conferring chilling tolerance in tomato fruits in response to exogenous melatonin applying at 100 µM may arise from upregulating SlZAT2/6/12 giving rise to triggering CBF1 gene expression. Employing higher arginine pathway activity in tomato fruits by exogenous melatonin applying demonstrating by higher endogenous polyamines accumulation arising from higher ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) genes expression and enzymes activity, higher endogenous proline accumulation arising from higher pyroline 5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) genes expression and enzymes activity accompanying by lower proline dehydrogenase (PDH) gene expression and enzyme activity and higher endogenous nitric oxide (NO) accumulation arising from higher nitric oxide synthase (NOS) gene expression and enzyme activity may be responsible for keeping safe membrane integrity.

Freezing Tolerance of Lactuca sativa and Induction of CBF and GolS Genes during Cold Treatment

Freezing Tolerance of Lactuca sativa and Induction of CBF and GolS Genes during Cold Treatment

Anti-freezing-protein type III strongly influences the expression of relevant genes in cryopreserved potato shoot tips.

AFP improved cryopreservation efficiency of potato (Solanum tuberosum cv. Superior) by regulating transcript levels of CBF1 and DHN1. However, the optimal AFP concentration required for strong induction of the genes was dependent on the type of vitrification solution to which the AFP was added: This finding suggests that AFP increased cryopreservation efficiency by transcriptional regulation of these genes, which might protect plant cell membranes from cold stress during cryopreservation. Despite the availability of many studies reporting the benefits of anti-freeze protein III (AFP III) as a cryoprotectant, the role of AFP III in this process has not been well demonstrated using molecular analysis. In addition, AFP III has not been exploited in the cryopreservation of potato thus far. Therefore, we studied the effects of AFP III on the cryopreservation of potato (Solanum tuberosum cv. Superior). We found that CBF1 and DHN1 genes are low temperature-inducible in potato leaves (S. tuberosum cv. Superior). Transcript levels of these genes expressed in shoot tips cryopreserved with AFP III were higher than those of the controls. However, the optimal AFP III concentration required for strong induction of the genes was dependent on the type of cryoprotection solution to which the AFP III was added: 500ng/mL worked best for PVS2, while 1500ng/mL was optimal for LS. Interestingly, the involvement of AFP III in the cryoprotection solutions improved cryopreservation efficiency as compared to the control, and expression levels of the detected genes were associated with cryopreservation efficiency. This finding suggests that AFP III increased cryopreservation efficiency by transcriptional regulation of these genes, which might protect plant cell membranes from cold stress during cryopreservation. Therefore, we expect that our findings will lead to the successful application of AFP III as a potent cryoprotectant in the cryopreservation of rare and commercially important plant germplasms.

CBF/DREB transcription factor genes play role in cadmium tolerance and phytoaccumulation in Ricinus communis under molybdenum treatments

The C-repeat binding factor/dehydration responsive element binding proteins (CBF/DREB) constitute a large group of transcriptional factors. Their role in abiotic stresses such as drought, salinity and low temperature tolerance in plants have been well established, while little information about their role in metals stress tolerance is available. Transcriptomic analyses of four genes (DREB-1A, DREB-1B, DREB-1F and CBF) were carried out in industrially important plant Ricinus communis under cadmium (Cd) and molybdenum (Mo) treatments. Cadmium (in soil) and Mo (as foliar spray) were used separate as well as in combinations. All the genes (except DREB 1A) expressed under Cd stress, while Mo further enhanced their expression. The proline (55.68 ± 5.51 ppm) and phenolic (120.00 ± 14.40 ppm) contents were significant increase in combination treatments of Cd and Mo. Positive and significant correlations of DREB 1B, DREB 1F and CBF genes expressions with free proline (0.92, 0.93 and 0.88 respectively), phenolic (075, 0.77 and 0.62 respectively) contents and Cd accumulation were demonstrated. Nucleotide sequence of R. comunis DREB1F and CBF genes showed more than 80% homology with related genes of other flowering plants. Predicted amino acids sequence of R. communis DREB 1F and CBF protein fragment demonstrated more than 75% homology with related proteins from other flowering plants.

Isolation and functional characterization of the SpCBF1 gene from Solanum pinnatisectum.

Low temperature causes a negative impact on plant growth and development, but plants evolve a series of mechanisms to respond to chilling stress, and one of them is CBF [C-repeat (CRT)/dehydration-responsive element (DRE) binding factor] gene family which has been well studied in different crops. In this paper, a new CBF1 gene, named as SpCBF1, was isolated from frost-tolerant Solanum pinnatisectum by PCR and analyzed for its function in cold-tolerance by over-expression technique. The ORF of SpCBF1 was 666bp long and encoded a protein of 221 amino acids with a predicted molecular mass 24.5821kDa and theoretically isoelectric point 5.0. SpCBF1 protein contained a highly conserved specific AP2/ERF domain. SpCBF1 was expressed in all tested tissues with the highest level in tuber and the lowest in root, and induced by chilling stress (0°C). Under natural low temperature condition (1-10°C), plants over-expressing SpCBF1 (OE) exhibited slighter necrotic lesion and lower necrotic injury, compared with untransformed Solanum tuberosum cv. Désirée (WT) and antisense-StCBF1 control lines. Over-expression of CBF1 increased the level of COR (cold-regulated) gene transcripts in OE lines, and the physiological indexes related to cold tolerance like the contents of SOD, soluble protein, MDA, proline and soluble sugar were higher in OE lines than in WT except RWC which was lower. All these results indicated that SpCBF1 gene plays a promoting role in potato responding to cold stress.

Heterotrimeric G-Protein γ Subunit CsGG3.2 Positively Regulates the Expression of CBF Genes and Chilling Tolerance in Cucumber.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of alpha (Gα), beta (Gβ), and gamma (Gγ) subunits are central signal transducers mediating the cellular response to multiple stimuli, such as cold, in eukaryotes. Plant Gγ subunits, divided into A, B, and C three structurally distinct types, provide proper cellular localization and functional specificity to the heterotrimer complex. Here, we demonstrate that a type C Gγ subunit CsGG3.2 is involved in the regulation of the CBF regulon and plant tolerance to cold stresses in cucumber (Cucumis sativus L.). We showed that CsGG3.2 transcript abundance was positively induced by cold treatments. Transgenic cucumber plants (T1) constitutively over-expressing CsGG3.2 exhibits tolerance to chilling conditions and increased expression of CBF genes and their regulon. Antioxidative enzymes, i.e., superoxide dismutase, catalase, peroxidase, and glutathione reductase activities increased in cold-stressed transgenic plants. The reactive oxygen species, oxygen free radical and H2O2, production, as well as membrane lipid peroxidation (MDA) production decreased in transgenic plants, suggesting a better antioxidant system to cope the oxidative-damages caused by cold stress. These findings provide evidence for a critical role of CsGG3.2 in mediating cold signal transduction in plant cells.

Arabidopsis PCaP2 Plays an Important Role in Chilling Tolerance and ABA Response by Activating CBF- and SnRK2-Mediated Transcriptional Regulatory Network.

Chilling stress affects plant growth and productivity. However, the multi-underlying mechanisms of chilling tolerance are not well understood. Arabidopsis PCaP2 is involved in regulating the dynamic of microtubules (MTs) and F-actin and Ca2+-binding ability. Here, the results showed that the PCaP2 expression was highly induced in roots, cotyledons, true leaves, lateral roots and flowers under cold stress. Compared with the wild type, PCaP2-overexpressing plants displayed the enhanced tolerance, whereas its RNAi and mutant were more sensitive in seed germination, seedling and reproductive growth under chilling stress in Arabidopsis. In addition, PCaP2 was also a positive regulator of ABA signaling pathway by analyzing the expression of PCaP2 and the phenotypes of PCaP2-overexpressing, mutant and RNAi plants under ABA treatment. Interestingly, disruption of PCaP2 inhibited the expression of CBF1, -3 and CBF-target COR genes, while increased the CBF2 expression in response to cold or ABA. Moreover, we found that SnRK2s were involved in cold stress and PCaP2 mutants down-regulated the transcription level of SnRK2.2, -2.3 and SnRK2-mediated downstream genes including ABF2, RD29A, KIN1, KIN2, but up-regulated SnRK2.6, ABF1, -3, -4 in ABA and cold treatments. It is well-accepted that PCaP2 as a Ca2+-binding protein triggers the gene expression to enhance plant chilling tolerance. Our further studies showed that MT destabilizing activity of PCaP2, but not F-actin-severing function, may be involved in chilling stress. Taken together, our results highlight that PCaP2 plays an important role in chilling tolerance and ABA response by triggering the CBF- and SnRK2-meditated transcriptional regulatory pathways, providing novel evidences of underlying mechanisms of multi-pathways in chilling stress.

Accession-Dependent CBF Gene Deletion by CRISPR/Cas System in Arabidopsis.

The CRISPR/Cas system became a powerful genome editing tool for basic plant research and crop improvement. Thus far, CRISPR/Cas has been applied to many plants, including Arabidopsis, rice and other crop plants. It has been reported that CRISPR/Cas efficiency is generally high in many plants. In this study, we compared the genome editing efficiency of CRISPR/Cas in three different Arabidopsis accessions [Col-0, Ler, and C24RDLUC (C24 accession harboring the stress-responsive RD29A promoter-driven luciferase reporter)]. For the comparison, we chose to target the cold-responsive C-repeat/DRE-Binding Factor (CBF) genes. CBF1, CBF2, and CBF3 genes are tandemly located on Arabidopsis chromosome 4 with redundant functions as the key transcription factors functioning in cold stress signaling and tolerance. Due to the close proximity of these CBFs on the chromosome, it is impossible to generate cbf1, cbf2, cbf3 triple mutants (cbf123) by traditional genetic crosses. Therefore, using the CRISPR/Cas tool, we aimed to generate cbf123 mutants and compared the genome editing efficiency in different Arabidopsis accessions. Among the accessions, Ler was the most resilient to the CRISPR/Cas deletion with the lowest gene deletion ratio in both T1 and T2 generations. Interestingly, while C24RDLUC showed a high CBF123 deletion frequency in T2 only when the gene deletion was observed in T1 generation, Col-0 displayed high ratios of the CBF123 deletions in T2 regardless of the presence or absence of the CBF123 deletion in T1. Isolated cbf123 mutants in C24RDLUC background showed no expression of CBF1, CBF2, and CBF3 genes and proteins with reduction in the CBF target gene expression under cold stress.

MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability

Mitogen-activated protein kinase cascades are important signaling modules that convert environmental stimuli into cellular responses. We show that MPK3, MPK4, and MPK6 are rapidly activated after cold treatment. The mpk3 and mpk6 mutants display increased expression of CBF genes and enhanced freezing tolerance, whereas constitutive activation of the MKK4/5-MPK3/6 cascade in plants causes reduced expression of CBF genes and hypersensitivity to freezing, suggesting that the MKK4/5-MPK3/6 cascade negatively regulates the cold response. MPK3 and MPK6 can phosphorylate ICE1, a basic-helix-loop-helix transcription factor that regulates the expression of CBF genes, and the phosphorylation promotes the degradation of ICE1. Interestingly, the MEKK1-MKK2-MPK4 pathway constitutively suppresses MPK3 and MPK6 activities and has a positive role in the cold response. Furthermore, the MAPKKK YDA and two calcium/calmodulin-regulated receptor-like kinases, CRLK1 and CRLK2, negatively modulate the cold activation of MPK3/6. Our results uncover important roles of MAPK cascades in the regulation of plant cold response.