Regulates Cold Tolerance Research Articles

Dissecting the effect of ethylene in the transcriptional regulation of chilling treatment in grapevine leaves

Ethylene (ETH) plays important roles in various development programs and stress responses in plants. In grapevines, ETH increased dramatically under chilling stress and is known to positively regulate cold tolerance. However, the role of ETH in transcriptional regulation during chilling stress of grapevine leaves is still not clear. To address this gap, targeted hormone profiling and transcriptomic analysis were performed on leaves of Vitis amurensis under chilling stress with and without aminoethoxyvinylglycine (AVG, a inhibitor of ETH synthesis) treatment. APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) and WRKY transcription factors (TF) were only the two highly enriched TF families that were consistently up-regulated during chilling stress but inhibited by AVG. The comparison of leaf transcriptomes between chilling treatment and chilling with AVG allowed the identification of potential ETH-regulated genes. Potential genes that are positively regulated by ETH are enriched in solute transport, protein biosynthesis, phytohormone action, antioxidant and carbohydrate metabolism. Conversely, genes related to the synthesis and signaling of ETH, indole-3-acetic acid (IAA), abscisic acid (ABA) were up-regulated by chilling treatment but inhibited by AVG. The contents of ETH, ABA and IAA also paralleled with the transcriptome data, which suggests that the response of ABA and IAA during chilling stress may regulate by ETH signaling, and together may belong to an integrated network of hormonal signaling pathways underpinning chilling stress response in grapevine leaves. Together, these findings provide new clues for further studying the complex regulatory mechanism of ETH under low-temperature stress in plants more generally and new opportunities for breeding cold-resilient grapevines.

Proteome and phosphoproteome analysis of 2,4-epibrassinolide-mediated cold stress response in cucumber seedlings.

The 2, 4-epibrassinolide (EBR) significantly increased plants cold tolerance. However, mechanisms of EBR in regulating cold tolerance in phosphoproteome and proteome levels have not been reported. The mechanism of EBR regulating cold response in cucumber was studied by multiple omics analysis. In this study, phosphoproteome analysis showed that cucumber responded to cold stress through multi-site serine phosphorylation, while EBR further upregulated single-site phosphorylation for most of cold-responsive phosphoproteins. Association analysis of the proteome and phosphoproteome revealed that EBR reprogrammed proteins in response to cold stress by negatively regulating protein phosphorylation and protein content, and phosphorylation negatively regulated protein content in cucumber. Further functional enrichment analysis of proteome and phosphoproteome showed that cucumber mainly upregulated phosphoproteins related to spliceosome, nucleotide binding and photosynthetic pathways in response to cold stress. However, different from the EBR regulation in omics level, hypergeometric analysis showed that EBR further upregulated 16 cold-up-responsive phosphoproteins participated photosynthetic and nucleotide binding pathways in response to cold stress, suggested their important function in cold tolerance. Analysis of cold-responsive transcription factors (TFs) by correlation between proteome and phosphoproteome showed that cucumber regulated eight class TFs may through protein phosphorylation under cold stress. Further combined with cold-related transcriptome found that cucumber phosphorylated eight class TFs, and mainly through targeting major hormone signal genes by bZIP TFs in response to cold stress, while EBR further increased these bZIP TFs (CsABI5.2 and CsABI5.5) phosphorylation level. In conclusion, the EBR mediated schematic of molecule response mechanisms in cucumber under cold stress was proposed.

Mdm-miR160-MdARF17-MdWRKY33 module mediates freezing tolerance in apple.

Apple (Malus domestica) trees are vulnerable to freezing temperatures. Cold resistance in woody perennial plants can be improved through biotechnological approaches. However, genetic engineering requires a thorough understanding of the molecular mechanisms of the tree's response to cold. In this study, we demonstrated that the Mdm-miR160-MdARF17-MdWRKY33 module is crucial for apple freezing tolerance. Mdm-miR160 plays a negative role in apple freezing tolerance, whereas MdARF17, one of the targets of Mdm-miR160, is a positive regulator of apple freezing tolerance. RNA sequencing analysis revealed that in apple, MdARF17 mediates the cold response by influencing the expression of cold-responsive genes. EMSA and ChIP-qPCR assays demonstrated that MdARF17 can bind to the promoter of MdWRKY33 and promotes its expression. Overexpression of MdWRKY33 enhanced the cold tolerance of the apple calli. In addition, we found that the Mdm-miR160-MdARF17-MdWRKY33 module regulates cold tolerance in apple by regulating reactive oxygen species (ROS) scavenging, as revealed by (i) increased H2 O2 levels and decreased peroxidase (POD) and catalase (CAT) activities in Mdm-miR160e OE plants and MdARF17 RNAi plants and (ii) decreased H2 O2 levels and increased POD and CAT activities in MdmARF17 OE plants and MdWRKY33 OE calli. Taken together, our study uncovered the molecular roles of the Mdm-miR160-MdARF17-MdWRKY33 module in freezing tolerance in apple, thus providing support for breeding of cold-tolerant apple cultivars.

Elongases of Long-Chain Fatty Acids ELO2 and ELO9 Are Involved in Cuticle Formation and Function in Fecundity in the Yellow Fever Mosquito, Aedes aegypti

Long-chain fatty acid elongases (ELOs) play important roles in the metabolism of fatty acids in insects. In this study, the genes for two elongases from Aedes aegypti were identified, AeELO2 and AeELO9. Quantitative real time PCR showed that AeELO2 and AeELO9 are expressed at all developmental stages and some body parts, but with different expression patterns. RNAi-mediated knockdown of AeELO2 and AeELO9 was performed to investigate their roles in the development, growth, osmotic balance, and cold tolerance of Ae. aegypti. Knockdown of AeELO2 slowed larval growth and development by causing molting abnormalities. Additionally, 33% ± 3.3% of adults died during oviposition, accompanied by an abnormal extension of cuticles in AeELO2-dsRNA knockdown mosquitos. Knockdown of AeEL09 resulted in abnormal balance of cuticular osmotic pressure and a reduction in egg production. The maximal mRNAs of AeELO2 and AeELO9 were detected in eggs at 72 h after oviposition. Moreover, AeELO2 knockdown reduced the egg hatching rates and AeELO9 knockdown larvae did not develop well. In summary, AeELO2 is involved in larval molting and growth, and its knockdown affects the flexibility and elasticity of adult mosquito cuticles. AeELO9 regulates cold tolerance, osmotic balance, and egg development in Ae. aegypti.

A natural promoter variation of SlBBX31 confers enhanced cold tolerance during tomato domestication.

Cold stress affects crop growth and productivity worldwide. Understanding the genetic basis of cold tolerance in germplasms is critical for crop improvement. Plants can coordinate environmental stimuli of light and temperature to regulate cold tolerance. However, it remains unknown which gene in germplasms could have such function. Here, we utilized genome-wide association study (GWAS) to investigate the cold tolerance of wild and cultivated tomato accessions and discovered that increased cold tolerance is accompanied with tomato domestication. We further identified a 27-bp InDel in the promoter of the CONSTANS-like transcription factor (TF) SlBBX31 is significantly linked with cold tolerance. Coincidentally, a key regulator of light signalling, SlHY5, can directly bind to the SlBBX31 promoter to activate SlBBX31 transcription while the 27-bp InDel can prevent S1HY5 from transactivating SlBBX31. Parallel to these findings, we observed that the loss of function of SlBBX31 results in impaired tomato cold tolerance. SlBBX31 can also modulate the cold-induced expression of several ERF TFs including CBF2 and DREBs. Therefore, our study has uncovered that SlBBX31 is possibly selected during tomato domestication for cold tolerance regulation, providing valuable insights for the development of hardy tomato varieties.

QTL Mapping and a Transcriptome Integrative Analysis Uncover the Candidate Genes That Control the Cold Tolerance of Maize Introgression Lines at the Seedling Stage.

Chilling injury owing to low temperatures severely affects the growth and development of maize (Zea mays.L) seedlings during the early and late spring seasons. The existing maize germplasm is deficient in the resources required to improve maize's ability to tolerate cold injury. Therefore, it is crucial to introduce and identify excellent gene/QTLs that confer cold tolerance to maize for sustainable crop production. Wild relatives of maize, such as Z. perennis and Tripsacum dactyloides, are strongly tolerant to cold and can be used to improve the cold tolerance of maize. In a previous study, a genetic bridge among maize that utilized Z. perennis and T. dactyloides was created and used to obtain a highly cold-tolerant maize introgression line (MIL)-IB030 by backcross breeding. In this study, two candidate genes that control relative electrical conductivity were located on MIL-IB030 by forward genetics combined with a weighted gene co-expression network analysis. The results of the phenotypic, genotypic, gene expression, and functional verification suggest that two candidate genes positively regulate cold tolerance in MIL-IB030 and could be used to improve the cold tolerance of cultivated maize. This study provides a workable route to introduce and mine excellent genes/QTLs to improve the cold tolerance of maize and also lays a theoretical and practical foundation to improve cultivated maize against low-temperature stress.

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.

COG2 negatively regulates chilling tolerance through cell wall components altered in rice.

Chilling-tolerant QTL gene COG2 encoded an extensin and repressed chilling tolerance by affecting the compositions of cell wall. Rice as a major crop is susceptible to chilling stress. Chilling tolerance is a complex trait controlled by multiple quantitative trait loci (QTLs). Here, we identify a QTL gene, COG2, that negatively regulates cold tolerance at seedling stage in rice. COG2 overexpression transgenic plants are sensitive to cold, whereas knockout transgenic lines enhance chilling tolerance. Natural variation analysis shows that Hap1 is a specific haplotype in japonica/Geng rice and correlates with chilling tolerance. The SNP1 in COG2 promoter is a specific divergency and leads to the difference in the expression level of COG2 between japonica/Geng and indica/Xian cultivars. COG2 encodes a cell wall-localized extensin and affects the compositions of cell wall, including pectin and cellulose, to defense the chilling stress. The results extend the understanding of the adaptation to the environment and provide an editing target for molecular design breeding of cold tolerance in rice.

A WRKY Transcription Factor PmWRKY57 from Prunus mume Improves Cold Tolerance in Arabidopsis thaliana.

Prunus mume, a woody perennial tree, is valued for its ornamental traits and has been cultivated for a long history. Low temperature is the main environmental factor restricting the distribution and affecting the growth of P. mume. In plants, some WRKY transcription factors have been reported to participate in regulating cold tolerance. However, there were few researches about functional characterization of WRKYs involving in P. mume cold response. Here, a cold-induced WRKY gene named as PmWRKY57 was cloned from a P. mume cultivar 'Guhong Zhusha.' PmWRKY57 protein harboring a WRKY domain and a C2H2 zinc finger motif belongs to Group IIc of WRKY family. The PmWRKY57 protein was located to the nucleus and has transcriptional activation activity. PmWRKY57-overexpresing Arabidopsis thaliana lines showed improved cold tolerance, compared to wild-type plants. Under cold treatment, the leaves of transgenic lines contained significantly lower malondialdehyde content, and higher levels of superoxide dismutase activity, peroxidase activity, and proline content than wild-type plants. Furthermore, the expression levels of cold-response genes such as AtCOR6.6, AtCOR47, AtKIN1, and AtRCI2A were up-regulated in leaves of transgenic A. thaliana compared to those in wild-type plants. This study characterized the function of PmWRKY57 in improving cold tolerance of plants.

Oligomeric Proanthocyanidins Confer Cold Tolerance in Rice through Maintaining Energy Homeostasis.

Oligomeric proanthocyanidins (OPCs) are abundant polyphenols found in foods and botanicals that benefit human health, but our understanding of the functions of OPCs in rice plants is limited, particularly under cold stress. Two rice genotypes, named Zhongzao39 (ZZ39) and its recombinant inbred line RIL82, were subjected to cold stress. More damage was caused to RIL82 by cold stress than to ZZ39 plants. Transcriptome analysis suggested that OPCs were involved in regulating cold tolerance in the two genotypes. A greater increase in OPCs content was detected in ZZ39 than in RIL82 plants under cold stress compared to their respective controls. Exogenous OPCs alleviated cold damage of rice plants by increasing antioxidant capacity. ATPase activity was higher and poly (ADP-ribose) polymerase (PARP) activity was lower under cold stress in ZZ39 than in RIL82 plants. Importantly, improvements in cold tolerance were observed in plants treated with the OPCs and 3-aminobenzamide (PARP inhibitor, 3ab) combination compared to the seedling plants treated with H2O, OPCs, or 3ab alone. Therefore, OPCs increased ATPase activity and inhibited PARP activity to provide sufficient energy for rice seedling plants to develop antioxidant capacity against cold stress.

Jasmonate Positively Regulates Cold Tolerance by Promoting ABA Biosynthesis in Tomato.

As a cold-sensitive species, tomato is frequently challenged by cold stress during vegetative and reproductive growth. Understanding how tomato responds to cold stress is of critical importance for sustainable tomato production. In this work, we demonstrate that jasmonate (JA) plays a crucial role in tomato response to cold stress by promoting abscisic acid (ABA) biosynthesis. It was observed that both JA and ABA levels were substantially increased under cold conditions, whereas the suppression of JA biosynthesis abated ABA accumulation. The ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE2 (NCED2) was subsequently found to be associated with JA-mediated ABA biosynthesis in tomato plants in response to cold stress. NCED2 was rapidly induced by exogenous MeJA and cold treatment. Silencing NCED2 led to a decrease in ABA accumulation that was concurrent with increased cold sensitivity. Moreover, blocking ABA biosynthesis using a chemical inhibitor impaired JA-induced cold tolerance in tomato. Furthermore, MYC2, a core component of the JA signaling pathway, promoted the transcription of NCED2, ABA accumulation and cold tolerance in tomato. Collectively, our results support that JA signaling promotes ABA biosynthesis to confer cold tolerance in tomato.

Transcriptome and Expression Analysis of Genes Related to Regulatory Mechanisms in Holly (Ilex dabieshanensis) under Cold Stress

Ilex dabieshanensis (K. Yao and M. B. Deng) is not only an important economic tree species, but also has the characteristics of evergreens in all seasons, as well as strong cold resistance. In order to understand the molecular mechanism of holly’s response to cold stress, we used transcriptome analysis to identify the main signaling pathways and key genes involved in cold stress. The result showed that 5750 differentially expressed genes (DEGs) were identified under different cold treatment times compared with the control (cold—0 h). The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs showed that seven phytohormone signal transduction were the most highly enriched, including abscisic acid (ABA), ethylene (ET), cytokinin (CK), auxin (IAA), gibberellin (GA), jasmonate (JA), and brassinosteroids (BR). In addition, proline metabolism, arginine metabolism, flavonoid biosynthesis, and anthocyanin biosynthesis were also implicated in response to cold stress. The weighted gene co-expression network analysis (WGCNA) showed that the genes in two modules were significantly up-regulated after 12 h and 24 h treatments, suggesting these two module genes may participate in the cold stress. The gene ontology (GO) results of the two module genes showed that calcium, scavenging reactive oxygen species, and nitric oxide might act as signaling molecules to regulate cold tolerance in holly. By calculating the connectivity and function prediction of genes in the two modules, five genes (evm.TU.CHR2.244, evm.TU.CHR1.1507, evm.TU.CHR1.1821, evm.TU.CHR2.89, and evm.TU.CHR2.210) were identified as the key hub genes of I. dabieshanensis response to cold stress. These results provided candidate genes and clues for further studies on the molecular genetic mechanism of cold stress in holly.

Membrane lipid metabolism is involved in regulating γ‐aminobutyric acid‐mediated cold tolerance in peach fruit

AbstractThe cold‐sensitive peach fruits are prone to chilling injury (CI). γ‐aminobutyric acid (GABA) has been confirmed to effectively improve peaches’ cold tolerance. This research aimed to probe the role of dynamic changes in membrane lipid metabolism in GABA‐mediated peaches’ cold tolerance and reveal the underlying mechanisms based on lipid metabolomics. GABA treatment improved peaches’ cold tolerance by reducing phosphatidic acid (PA) accumulation via inhibiting phospholipid degradation, attenuating PA biosynthesis, and maintaining higher phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels. Under chilling conditions, GABA treatment protected peaches against low‐temperature stress and delayed CI by maintaining higher PC and PE levels and enhancing the massive accumulation of diacylglycerol (DAG) and triacylglycerol (TAG). This study provides a novel mechanism regulating cold tolerance of postharvest peach fruits.

SEC1-C3H39 module fine-tunes cold tolerance by mediating its target mRNA degradation in tomato.

Plants adapt to cold stress at the physiological and biochemical levels, thus enabling them to maintain growth and development. However, the molecular mechanism of fine-tuning cold signals remains largely unknown. We addressed the function of SlSEC1-SlC3H39 module in cold tolerance by using SlSEC1 and SlC3H39 knockout and overexpression tomato lines. A tandem CCCH zinc-finger protein SlC3H39 negatively modulates cold tolerance in tomato. SlC3H39 binds to AU-rich elements in the 3'-untranslated region (UTR) to induce mRNA degradation and regulates gene expression post-transcriptionally. We further validate that SlC3H39 participates in post-transcriptional regulation of a variety of cold-responsive genes. An O-linked N-acetylglucosamine transferase SlSEC1 physically interacts with SlC3H39 proteins and negatively regulates cold tolerance in tomato. Further study shows that SlSEC1 is essential for SlC3H39 protein stability and maintains SlC3H39 function in cold tolerance. Genetic analysis shows that SlC3H39 is epistatic to SlSEC1 in cold tolerance. The findings indicate that SlC3H39 negatively modulates plant cold tolerance through post-transcriptional regulation by binding to cold-responding mRNA 3'-UTR and reducing those transcripts. SlSEC1 promotes the O-GlcNAclation status of SlC3H39 and maintains SlC3H39 function in cold tolerance. Taken together, we propose a SlSEC1-SlC3H39 module, which allows plants to balance defense responses and growth processes.

VaBAM1 weakens cold tolerance by interacting with the negative regulator VaSR1 to suppress β-amylase expression

Cold stress is a key climatic factor that limits grape productivity and quality. Although β-amylase (BAM) is known to play an important role as a mediator of starch degradation under conditions of cold stress, the mechanism by which BAM regulates cold tolerance in grape remains unclear. Here, we identified VaBAM1 from Vitis amurensis and characterized its interactive regulating mechanism under cold stress in Arabidopsis thaliana and grape. VaBAM1-overexpressing A. thaliana plants (OEs) exhibited high freezing tolerance. Soluble sugar content and amylase activity were increased in OEs and VaBAM1-overexpressing grape calli (VaBAM1-OEs) under cold stress; however, they were decreased in grape calli in which VaBAM1 was edited using CRISPR/Cas9. The results of yeast two-hybrid, bimolecular fluorescence complementation, and pull-down experiments showed that serine/arginine-rich splicing factor 1 (VaSR1) interacted with VaBAM1. Furthermore, the expression of VaSR1 was opposite that of VaBAM1 in phloem tissue of Vitis amurensis during winter dormancy. In VaSR1-overexpressing grape calli (VaSR1-OEs), BAM activity and the expression levels of C-repeat binding transcription factor and cold response genes were all significantly lower than that in untransformed calli subjected to cold stress. Moreover, VvBAM1 was downregulated in VaSR1-OEs under cold stress. Overall, we identified that VaSR1 interacts with VaBAM1, negatively regulating BAM activity and resulting in decreased plant cold tolerance.

OsMKKK70 Negatively Regulates Cold Tolerance at Booting Stage in Rice.

Cold stress at the booting stage leads to a lower seed setting rate and seriously threatens the production of rice (Oryza sativa L.), which has become a major yield-limiting factor in higher-altitude and -latitude regions. Because cold tolerance at the booting stage (CTB) is a complex trait and is controlled by multiple loci, only a few genes have been reported so far. In this study, a function of OsMKKK70 (Mitogen Activated Protein Kinase Kinase Kinase 70) in response to CTB was characterized. OsMKKK70 expression was rapidly induced by cold stress at the booting stage. OsMKKK70 overexpression (OsMKKK70-OE) plants were more sensitive to cold stress at the booting stage with a lower seed setting and pollen fertility, but there was no significant difference between the osmkkk70 mutant and WT. Considering the effect of functional redundancy, we further tested the CTB response of osmkkk62/70 and osmkkk55/62/70, the double and triple mutants of OsMKKK70 with its closest homologs OsMKKK62 and OsMKKK55, and found that osmkkk62/70 and osmkkk55/62/70 displayed significantly increased CTB with a higher seed setting and pollen fertility, indicating that OsMKKK70 negatively regulates rice CTB. Moreover, under the low-temperature (LT) condition, the osmkkk62/70 mutant had slightly higher Gibberellin (GA) contents, increased expression of GA biosynthesis genes, and lower protein level of OsSLR1 in anthers than those in WT. By contrast, OsMKKK70-OE anther had a lower GA biosynthesis than that of WT. Together, these findings suggest that OsMKKK70 negatively regulates rice CTB by fine-tuning GA levels in anthers.

Overexpression of TaMYC2 confers freeze tolerance by ICE-CBF-COR module in Arabidopsis thaliana.

Dongnongdongmai No.1 (Dn1) is one of the few winter wheat varieties that can successfully overwinter at temperatures as low as -25°C or even lower. To date, few researches were carried to identify the freeze tolerance genes in Dn1 and applied them to improve plant resistance to extreme low temperatures. The basic helix-loop-helix (bHLH) transcription factor MYC2 is a master regulator in JA signaling, which has been reported to involve in responses to mild cold stress (2°C and 7°C). We hypothesized that MYC2 might be part of the regulatory network responsible for the tolerance of Dn1 to extreme freezing temperatures. In this study, we showed that wheat MYC2 (TaMYC2) was induced under both extreme low temperature (-10°C and-25°C) and JA treatments. The ICE-CBF-COR transcriptional cascade, an evolutionary conserved cold resistance pathway downstream of MYC2, was also activated in extreme low temperatures. We further showed that overexpression of any of the MYC2 genes from Dn1 TaMYC2A, B, D in Arabidopsis led to enhanced freeze tolerance. The TaMYC2 overexpression lines had less electrolyte leakage and lower malondialdehyde (MDA) content, and an increase in proline content, an increases antioxidant defences, and the enhanced expression of ICE-CBF-COR module under the freezing temperature. We further verified that TaMYC2 might function through physical interaction with TaICE41 and TaJAZ7, and that TaJAZ7 physically interacts with TaICE41. These results elucidate the molecular mechanism by which TaMYC2 regulates cold tolerance and lay the foundation for future studies to improve cold tolerance in plants.

Grape CIPK18 acts as a positive regulator of CBF cold signaling pathway by modulating ROS homeostasis

Cold temperatures trigger a diverged Ca 2+ decoding paradigm that includes the proteins CBL (Calcineurin B-like proteins) and CIPK (CBL-interacting protein kinases). Here, putative CBL and CIPK genes were surveyed in cold-tolerant wild Amur grape ( Vitis amurensis ), designated as VaCBLs and VaCIPKs , respectively. The transcripts of 8 VaCBLs and 19 VaCIPKs encoded in this genome were confirmed, among which VaCIPK18 physically interacted with all 8 VaCBLs through the C-terminal regulatory domain (NAF), indicating that it may serve as a node regulator. VaCIPK18 was ubiquitously expressed in various tissues and was highly induced by cold, drought, salt, and ABA . The NAF domain of VaCIPK18 was required for functional homodimerization in yeast as well as in planta . Complementation tests confirmed that VaCIPK18 rescued the cold sensitivity of ArabidopsisAtcipk3 mutant, in which the expressions of CBFs were suppressed and is sensitive to cold and freezing stresses. In addition, overexpression of VaCIPK18 enhanced the cold tolerance by upregulating the C-repeat binding transcription factor (CBF) transcriptional pathway under normal or cold conditions, and reducing reactive oxygen species production. Thus, our study demonstrates that VaCIPK18 positively regulates cold signaling at least partly through the direct transcriptional control of cold-regulated CBFs and modulation of ROS homeostasis . Moreover, VaCIPK18 was found to interact with VaMYB4a in vivo and in vitro . Our findings provide evidence that VaCIPK18 functions as a key node in the CBL-CIPK network and may interact with VaMYB4a to contribute to cold response in grape plants. • An Amur grape VaCIPK18 could function as a node regulator of CBL-CIPK network. • VaCIPK18 modulates cold tolerance by directly controlling CBFs and ROS homeostasis. • 'VaCIPK18-VaMYB4a' module may regulate cold tolerance.

Tomato SlMYB15 transcription factor targeted by sly-miR156e-3p positively regulates ABA-mediated cold tolerance.

Cold is a common abiotic stress that seriously affects plant growth and development. MYB transcription factors are regulatory molecules that play important roles in various biological processes. We have previously demonstrated that SlMYB15 positively regulates cold tolerance in tomato. However, the underlying mechanism of SlMYB15-induced cold tolerance remains largely unexplored. Here, cold-induced SlMYB15 was found to be targeted by Solanum lycopersicum (sly)-miR156e-3p, which was decreased by cold stimulus in tomato. Tomato plants overexpressing sly-MIR156e-3p displayed significant enhancement in susceptibility to cold stress, while silencing of sly-miR156e-3p by an artificial microRNA interference strategy caused tomato plants to be more tolerant to cold. Moreover, both overexpression of SlMYB15 and silencing of sly-miR156e-3p increased the accumulation of ABA. SlMYB15 directly binds to the promoter regions of ABA biosynthesis and signalling genes, SlNCED1 and SlABF4, resulting in enhanced cold tolerance. Further experiments showed that SlMYB15 and sly-miR156e-3p also coordinated the cold tolerance of tomato via the reactive oxygen species (ROS) signalling pathway, as reflected by the increased expression of SlRBOH1, enhanced H2O2 and O2•-accumulation, and amplified activity of antioxidant enzymes in SlMYB15-overexpressing and sly-miR156e-3p-silenced plants. Taken together, our results demonstrate that SlMYB15 targeted by sly-miR156e-3p confers higher survivability to cold stress via ABA and ROS signals. This study provides valuable information for breeding improved crop cultivars better equipped with cold tolerance.

A single-nucleotide polymorphism in WRKY33 promoter is associated with the cold sensitivity in cultivated tomato.

Natural variations in cis-regulatory regions often affect crop phenotypes by altering gene expression. However, the mechanism of how promoter mutations affect gene expression and crop stress tolerance is still poorly understood. In this study, by analyzing RNA-sequencing (RNA-Seq) data and reverse transcription quantitative real-time PCR validation in the cultivated tomato and its wild relatives, we reveal that the transcripts of WRKY33 are almost unchanged in cold-sensitive cultivated tomato Solanum lycopersicum L. 'Ailsa Craig' but are significantly induced in cold-tolerant wild tomato relatives Solanum habrochaites LA1777 and Solanum pennellii LA0716 under cold stress. Overexpression of SlWRKY33 or ShWRKY33 positively regulates cold tolerance in tomato. Variant of the critical W-box in SlWRKY33 promoter results in the loss of self-transcription function of SlWRKY33 under cold stress. Analysis integrating RNA-Seq and chromatin immunoprecipitation sequencing data reveals that SlWRKY33 directly targets and induces multiple kinases, transcription factors, and molecular chaperone genes, such as CDPK11, MYBS3, and BAG6, thus enhancing cold tolerance. In addition, heat- and Botrytis-induced WRKY33s expression in both wild and cultivated tomatoes are independent of the critical W-box variation. Our findings suggest nucleotide polymorphism in cis-regulatory regions is crucial for different cold sensitivity between cultivated and wild tomato plants.