Regulates Cold Tolerance Research Articles

COLD6-OSM1 module senses chilling for cold tolerance via 2′,3′-cAMP signaling in rice

While it is known that temperature sensors trigger calcium (Ca2+) signaling to confer cold tolerance in cells, less is known about sensors that couple with other secondary messengers. Here, we identify a cold sensor complex of CHILLING-TOLERANCE DIVERGENCE 6 (COLD6) and osmotin-like 1 (OSM1), which triggers 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) production to enhance cold tolerance in rice. COLD6, which is encoded by a major quantitative trait locus (QTL) gene, interacts with the rice G protein α subunit (RGA1) at the plasma membrane under normal conditions. Upon exposure to chilling, cold-induced OSM1 binds to COLD6, kicking out RGA1 from interaction. This triggers an elevation of 2',3'-cAMP levels for enhancing chilling tolerance. Genetic data show that COLD6 negatively regulates cold tolerance and functionally depends on OSM1 in chilling stress. COLD6 alleles were selected during rice domestication. Knockout and natural variation of COLD6 in hybrid rice enhanced chilling tolerance, hinting design potential for breeding. This highlighted a module triggering 2',3'-cAMP to improve chilling tolerance in crops.

OsWRKY70 Plays Opposite Roles in Blast Resistance and Cold Stress Tolerance in Rice

The transcription factor WRKYs play pivotal roles in the adapting to adverse environments in plants. Prior research has demonstrated the involvement of OsWRKY70 in resistance against herbivores and its response to abiotic stress. Here, we reported the functional analysis of OsWRKY70 in immunity against fungal diseases and cold tolerance. The results revealed that OsWRKY70 was induced by various Magnaporthe oryzae strains. Knock out mutants of OsWRKY70, which were generated by the CRISPR/Cas9 system, exhibited enhanced resistance to M. oryzae. This was consistent with fortifying the reactive oxygen species (ROS) burst after inoculation in the mutants, elevated transcript levels of defense-responsive genes (OsPR1b, OsPBZ1, OsPOX8.1 and OsPOX22.3) and the observation of the sluggish growth of invasive hyphae under fluorescence microscope. RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) validations demonstrated that differentially expressed genes were related to plant-pathogen interactions, hormone transduction and MAPK cascades. Notably, OsbHLH6, a key component of the JA signaling pathway, was down-regulated in the mutants compared to wild type plants. Further investigation confirmed that OsWRKY70 bound to the promoter of OsbHLH6 by semi-in vivo chromatin immunoprecipitation (ChIP). Additionally, the loss-function of OsWRKY70 impaired cold tolerance in rice. The enhanced susceptibility in the mutants characterized by excessive ROS production, elevated ion leakage rate and increased malondialdehyde content, as well as decreased activity of catalase (CAT) and peroxidase (POD) under low temperature stress was, which might be attributed to down-regulation of cold-responsive genes (OsLti6b and OsICE1). In conclusion, our findings indicate that OsWRKY70 negatively contributes to blast resistance but positively regulates cold tolerance in rice, providing a strategy for crop breeding with tolerance to stress.

SlHB8 Is a Novel Factor in Enhancing Cold Resistance in Tomato Anthers by Modulating Tapetal Cell Death.

Tomato plants favor warmth, making them particularly susceptible to cold conditions, especially their reproductive development. Therefore, understanding how pollen reacts to cold stress is vital for selecting and improving cold-resistant tomato varieties. The programmed cell death (PCD) in the tapetum is particularly susceptible to cold temperatures which could hinder the degradation of the tapetal layer in the anthers, thus affecting pollen development. However, it is not clear yet how genes integral to tapetal degradation respond to cold stress. Here, we report that SlHB8, working upstream of the conserved genetic module DYT1-TDF1-AMS-MYB80, is crucial for regulating cold tolerance in tomato anthers. SlHB8 expression increases in the tapetum when exposed to low temperatures. CRISPR/Cas9-generated SlHB8-knockout mutants exhibit improved pollen cold tolerance due to the reduced temperature sensitivity of the tapetum. SlHB8 directly upregulates SlDYT1 and SlMYB80 by binding to their promoters. In normal anthers, cold treatment boosts SlHB8 levels, which then elevates the expression of genes like SlDYT1, SlTDF1, SlAMS, and SlMYB80; however, slhb8 mutants do not show this gene activation during cold stress, leading to a complete blockage of delayed tapetal programmed cell death (PCD). Furthermore, we found that SlHB8 can interact with both SlTDF1 and SlMYB80, suggesting the possibility that SlHB8 might regulate tapetal PCD at the protein level. This study sheds light on molecular mechanisms of anther adaptation to temperature fluctuations.

Overexpression of VaSS4 negatively regulates cold tolerance by disturbing ROS balance and decreasing soluble sugar content

Cold stress is one of the major limiting factors for grapevine growth and yield. Sucrose synthetase (SS) is a glycosyl transferase enzyme involved in plant sugar metabolism, which is essential for plant growth and development and response to abiotic stress. However, the function of SS genes in grape cold resistance has not been systematically understood. In the present study, a SS gene from Vitis amurensis named VaSS4 was identified, which was significantly suppressed in response to cold stress. The physicochemical properties, evolutionary relationships, synteny, and selection pressure of VaSS4 were analyzed. Furthermore, VaSS4-overexpressed Arabidopsis and grape calli were obtained to characterize their tolerance to cold stress. Amino acid sequence analysis demonstrated that VaSS4 protein encoded 806 amino acids and contained conserved sucrose synthase domain and glycosyl transferase domain. Overexpression of VaSS4 decreased SS decomposition activity and soluble sugar accumulation under cold stress. Meanwhile, VaSS4 overexpression decreased the reactive oxygen scavenging ability and cold tolerance of Arabidopsis and grape calli. Importantly, overexpression of VaSS4 negatively regulated the expression of cold-related genes in the CBF-dependent pathway compared with WT plants. Overall, these findings not only provide extensive systematic information for Vitis amurensis VaSS4 gene research, but also provide valuable germplasm resources for grape cold resistance breeding.

MiR1432 negatively regulates cold tolerance by targeting OsACAs.

miRNAs function as negative regulators that significantly influence plant growth and stress responses. Within rice and other monocotyledonous plants, miR1432 plays a conserved role in seed development and disease resistance. However, its involvement in the response to abiotic stresses remains unclear. Our study aimed to elucidate this mechanism by predicting the targeting of the rice P-type IIB Ca2+ ATPase gene OsACAs by miR1432 and identifying its cleavage sites via 5'RACE. We observed induced expression of miR1432 and its target gene, OsACA6, under abiotic stresses. Overexpression (OX) of miR1432 and suppression of OsACA6 resulted in reduced cold, salt, and drought tolerance, while OsACA6 suppression/knockout and OX had opposite effects on cold tolerance. Additionally, miR1432 may target other OsACA6 homologs. RNA-sequencing data highlighted the differential expression of stress-related genes in miR1432-overexpressing rice. Furthermore, miR1432-overexpressing rice exhibited weakened vigor, dwarfism, yellowing leaves and reduced fertility. Collectively, our results strongly suggest that miR1432 not only negatively modulates abiotic stress tolerance by suppressing Ca2+ ATPase gene(s) but also influences plant growth and development.

Genetic variation in a heat shock transcription factor modulates cold tolerance in maize

Understanding how maize (Zea mays) responds to cold stress is crucial for facilitating breeding programs of cold-tolerant varieties. Despite extensive utilization of the genome-wide association study (GWAS) approach for exploring favorable natural alleles associated with maize cold tolerance, few studies have successfully identified candidate genes that contribute to maize cold tolerance. In this study, we used a diverse panel of inbred maize lines collected from different germplasm sources to perform a GWAS on variations in the relative injured area of maize true leaves during cold stress—a trait very closely correlated with maize cold tolerance. We identified HSF21, which encodes a B-class heat shock transcription factor (HSF) that positively regulates cold tolerance at both the seedling and germination stages. Natural variations in the promoter of the cold-tolerant HSF21Hap1 allele led to increased HSF21 expression under cold stress by inhibiting binding of the basic leucine zipper bZIP68 transcription factor, a negative regulator of cold tolerance. By integrating transcriptome deep sequencing, DNA affinity purification sequencing, and targeted lipidomic analysis, we revealed the function of HSF21 in regulating lipid metabolism homeostasis to modulate cold tolerance in maize. In addition, we found that HSF21 confers maize cold tolerance without incurring yield penalties. Collectively, this study establishes HSF21 as a key regulator that enhances cold tolerance in maize, providing valuable genetic resources for breeding of cold-tolerant maize varieties.

PHD17 acts as a target of miR1320 to negatively control cold tolerance via JA-activated signaling in rice

Plant Homeo Domain (PHD) proteins are involved in diverse biological processes during plant growth. However, the regulation of PHD genes on rice cold stress response remains largely unknown. Here, we reported that PHD17 negatively regulated cold tolerance in rice seedlings as a cleavage target of miR1320. PHD17 expression was greatly induced by cold stress, and was down-regulated by miR1320 overexpression and up-regulated by miR1320 knockdown. Through 5′RACE and dual luciferase assays, we found that miR1320 targeted and cleaved the 3′UTR region of PHD17. PHD17 was a nuclear-localized protein and acted as a transcriptional activator in yeast. PHD17 overexpression reduced cold tolerance of rice seedlings, while knockout of PHD17 increased cold tolerance, partially via the CBF cold signaling. By combining transcriptomic and physiological analyses, we demonstrated that PHD17 modulated ROS homeostasis and flavonoid accumulation under cold stress. K-means clustering analysis revealed that differentially expressed genes in PHD17 transgenic lines were significantly enriched in the jasmonic acid (JA) biosynthesis pathway, and expression of JA biosynthesis and signaling genes was verified to be affected by PHD17. Cold stress tests applied with MeJA or IBU (JA synthesis inhibitor) further suggested the involvement of PHD17 in JA-mediated cold signaling. Taken together, our results suggest that PHD17 acts downstream of miR1320 and negatively regulates cold tolerance of rice seedlings through JA-mediated signaling pathway.

FveDREB1B improves cold tolerance of woodland strawberry by positively regulating FveSCL23 and FveCHS.

Cold stress has seriously inhibited the growth and development of strawberry during production. CBF/DREB1 is a key central transcription factor regulating plant cold tolerance, but its regulatory mechanisms are varied in different plants. Especially in strawberry, the molecular mechanism of CBF/DREB1 regulating cold tolerance is still unclear. In this study, we found that FveDREB1B was most significantly induced by cold stress in CBF/DREB1 family of diploid woodland strawberry. FveDREB1B was localized to the nucleus, and DREB1B sequences were highly conserved in diploid and octoploid strawberry, and even similar in Rosaceae. And FveDREB1B overexpressed strawberry plants showed delayed flowering and increased cold tolerance, while FveDREB1B silenced plants showed early flowering and decreased cold tolerance. Under cold stress, FveDREB1B activated FveSCL23 expression by directly binding to its promoter. Meanwhile, FveDREB1B and FveSCL23 interacted with FveDELLA, respectively. In addition, we also found that FveDREB1B promoted anthocyanin accumulation in strawberry leaves by directly activating FveCHS expression after cold treatment and recovery to 25°C. DREB1B genes were also detected to be highly expressed in cold-tolerant strawberry resources 'Fragaria mandschurica' and 'Fragaria nipponica'. In conclusion, our study reveals the molecular mechanism of FveDREB1B-FveSCL23-FveDELLA module and FveDREB1B-FveCHS module to enhance the cold tolerance of woodland strawberry. It provides a new idea for improving the cold tolerance of cultivated strawberry and evaluating the cold tolerance of strawberry germplasm resources.

The gene CmPYL6 strongly contributes to cold tolerance in oriental melon.

The current simple and crude facilities make melon production more susceptible to cold stress during off-season cultivation in China. The ABA signalling pathway is an important target for breeding cold-tolerant melon. Cold-tolerant No. 330 and cold-sensitive No. 410 oriental melon genotypes were used to analyse the relationship between ABA and cold tolerance. 12 CmPYLs, ABA receptors, were identified from the melon genome database according to sequence alignment and phylogenetic analysis. Gene function of CmPYL6 in cold tolerance was analysed using VIGS in No. 330 and overexpression in Arabidopsis WT. A total of 12 CmPYL members contain the representative domain and conserved sites. Under cold treatment, No.330 seedlings had lower electrolyte leakage and MDA content, higher ABA content and CmPYL6 expression than seedlings of No. 410. Exogenous application of ABA upregulated expression of CmPYL6 and enhanced cold tolerance of both genotypes, while inhibiting ABA accumulation reduced expression of CmPYL6 and cold tolerance of both genotypes. CmPYL6-silenced No. 330 seedlings had reduced cold tolerance, increased electrolyte leakage and MDA content as well as limited proline and soluble sugar content, while CmPYL6 overexpressed transgenic Arabidopsis plants had enhanced cold tolerance, with limited electrolyte leakage and MDA content, as well as increased proline and soluble sugar content. The CmPYL6 gene is probably an important ABA receptor in regulating cold tolerance of oriental melon. Our study provides a direction for improving breeding of cold tolerance of oriental melon.

Identification of candidate genes controlling cold tolerance at the early seedling stage from Dongxiang wild rice by QTL mapping, BSA-Seq and RNA-Seq

BackgroundThe cold tolerance of rice is closely related to its production and geographic distribution. The identification of cold tolerance-related genes is of important significance for developing cold-tolerant rice. Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) is well-adapted to the cold climate of northernmost-latitude habitats ever found in the world, and is one of the most valuable rice germplasms for cold tolerance improvement.ResultsTranscriptome analysis revealed genes differentially expressed between Xieqingzao B (XB; a cold sensitive variety) and 19H19 (derived from an interspecific cross between DXWR and XB) in the room temperature (RT), low temperature (LT), and recovery treatments. The results demonstrated that chloroplast genes might be involved in the regulation of cold tolerance in rice. A high-resolution SNP genetic map was constructed using 120 BC5F2 lines derived from a cross between 19H19 and XB based on the genotyping-by-sequencing (GBS) technique. Two quantitative trait loci (QTLs) for cold tolerance at the early seedling stage (CTS), qCTS12 and qCTS8, were detected. Moreover, a total of 112 candidate genes associated with cold tolerance were identified based on bulked segregant analysis sequencing (BSA-seq). These candidate genes were divided into eight functional categories, and the expression trend of candidate genes related to ‘oxidation-reduction process’ and ‘response to stress’ differed between XB and 19H19 in the RT, LT and recovery treatments. Among these candidate genes, the expression level of LOC_Os12g18729 in 19H19 (related to ‘response to stress’) decreased in the LT treatment but restored and enhanced during the recovery treatment whereas the expression level of LOC_Os12g18729 in XB declined during recovery treatment. Additionally, XB contained a 42-bp deletion in the third exon of LOC_Os12g18729, and the genotype of BC5F2 individuals with a survival percentage (SP) lower than 15% was consistent with that of XB. Weighted gene coexpression network analysis (WGCNA) and modular regulatory network learning with per gene information (MERLIN) algorithm revealed a gene interaction/coexpression network regulating cold tolerance in rice. In the network, differentially expressed genes (DEGs) related to ‘oxidation-reduction process’, ‘response to stress’ and ‘protein phosphorylation’ interacted with LOC_Os12g18729. Moreover, the knockout mutant of LOC_Os12g18729 decreased cold tolerance in early rice seedling stage signifcantly compared with that of wild type.ConclusionsIn general, study of the genetic basis of cold tolerance of rice is important for the development of cold-tolerant rice varieties. In the present study, QTL mapping, BSA-seq and RNA-seq were integrated to identify two CTS QTLs qCTS8 and qCTS12. Furthermore, qRT-PCR, genotype sequencing and knockout analysis indicated that LOC_Os12g18729 could be the candidate gene of qCTS12. These results are expected to further exploration of the genetic mechanism of CTS in rice and improve cold tolerance of cultivated rice by introducing the cold tolerant genes from DXWR through marker-assisted selection.

OsJRL negatively regulates rice cold tolerance via interfering phenylalanine metabolism and flavonoid biosynthesis.

The identification of new genes involved in regulating cold tolerance in rice is urgent because low temperatures repress plant growth and reduce yields. Cold tolerance is controlled by multiple loci and involves a complex regulatory network. Here, we show that rice jacalin-related lectin (OsJRL) modulates cold tolerance in rice. The loss of OsJRL gene functions increased phenylalanine metabolism and flavonoid biosynthesis under cold stress. The OsJRL knock-out (KO) lines had higher phenylalanine ammonia-lyase (PAL) activity and greater flavonoid accumulation than the wild-type rice, Nipponbare (NIP), under cold stress. The leaves had lower levels of reactive oxygen species (ROS) and showed significantly enhanced cold tolerance compared to NIP. In contrast, the OsJRL overexpression (OE) lines had higher levels of ROS accumulation and showed lower cold tolerance than NIP. Additionally, the OsJRL KO lines accumulated more abscisic acid (ABA) and jasmonic acid (JA) under cold stress than NIP. The OsJRL OE lines showed increased sensitivity to ABA compared to NIP. We conclude that OsJRL negatively regulates the cold tolerance of rice via modulation of phenylalanine metabolism and flavonoid biosynthesis.

LUX ARRHYTHMO links CBF pathway and jasmonic acid metabolism to regulate cold tolerance of tea plants.

Cold stress declines the quality and yield of tea, yet the molecular basis underlying cold tolerance of tea plants (Camellia sinensis) remains largely unknown. Here, we identified a circadian rhythm component LUX ARRHYTHMO (LUX) that potentially regulates cold tolerance of tea plants through a genome-wide association study and transcriptomic analysis. The expression of CsLUX phased with sunrise and sunset and was strongly induced by cold stress. Genetic assays indicated that CsLUX is a positive regulator of freezing tolerance in tea plants. CsLUX was directly activated by CsCBF1 and repressed the expression level of CsLOX2, which regulates the cold tolerance of tea plants through dynamically modulating jasmonic acid content. Furthermore, we showed that the CsLUX-CsJAZ1 complex attenuated the physical interaction of CsJAZ1 with CsICE1, liberating CsICE1 with transcriptional activities to withstand cold stress. Notably, a single-nucleotide variation of C-to-A in the coding region of CsLUX was functionally validated as the potential elite haplotype for cold response, which provided valuable molecular markers for future cold resistance breeding in tea plants.

MaHsf24, a novel negative modulator, regulates cold tolerance in banana fruits by repressing the expression of HSPs and antioxidant enzyme genes.

Transcriptional regulation mechanisms underlying chilling injury (CI) development have been widely investigated in model plants and cold-sensitive fruits, such as banana (Musa acuminata). However, unlike the well-known NAC and WRKY transcription factors (TFs), the function and deciphering mechanism of heat shock factors (HSFs) involving in cold response are still fragmented. Here, we showed that hot water treatment (HWT) alleviated CI in harvested banana fruits accomplishing with reduced reactive oxygen species (ROS) accumulation and increased antioxidant enzyme activities. A cold-inducible but HWT-inhibited HSF, MaHsf24, was identified. Using DNA affinity purification sequencing (DAP-seq) combined with RNA-seq analyses, we found three heat shock protein (HSP) genes (MaHSP23.6, MaHSP70-1.1 and MaHSP70-1.2) and three antioxidant enzyme genes (MaAPX1, MaMDAR4 and MaGSTZ1) were the potential targets of MaHsf24. Subsequent electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and dual-luciferase reporter (DLR) analyses demonstrated that MaHsf24 repressed the transcription of these six targets via directly binding to their promoters. Moreover, stably overexpressing MaHsf24 in tomatoes increased cold sensitivity by suppressing the expressions of HSPs and antioxidant enzyme genes, while HWT could recover cold tolerance, maintaining higher levels of HSPs and antioxidant enzyme genes, and activities of antioxidant enzymes. In contrast, transiently silencing MaHsf24 by virus-induced gene silencing (VIGS) in banana peels conferred cold resistance with the upregulation of MaHSPs and antioxidant enzyme genes. Collectively, our findings support the negative role of MaHsf24 in cold tolerance, and unravel a novel regulatory network controlling bananas CI occurrence, concerning MaHsf24-exerted inhibition of MaHSPs and antioxidant enzyme genes.

Genome-wide identification of the plant homeodomain-finger family in rye and ScPHD5 functions in cold tolerance and flowering time.

111 PHD genes were newly identified in rye genome and ScPHD5's role in regulating cold tolerance and flowering time was suggested. Plant homeodomain (PHD)-finger proteins regulate the physical properties of chromatin and control plant development and stress tolerance. Although rye (Secale cereale L.) is a major winter crop, PHD-finger proteins in rye have not been studied. Here, we identified 111 PHD genes in the rye genome that exhibited diverse gene and protein sequence structures. Phylogenetic tree analysis revealed that PHDs were genetically close in monocots and diverged from those in dicots. Duplication and synteny analyses demonstrated that ScPHDs have undergone several duplications during evolution and that high synteny is conserved among the Triticeae species. Tissue-specific and abiotic stress-responsive gene expression analyses indicated that ScPHDs were highly expressed in spikelets and developing seeds and were responsive to cold and drought stress. One of these genes, ScPHD5, was selected for further functional characterization. ScPHD5 was highly expressed in the spike tissues and was localized in the nuclei of rye protoplasts and tobacco leaves. ScPHD5-overexpressing Brachypodium was more tolerant to freezing stress than wild-type (WT), with increased CBF and COR gene expression. Additionally, these transgenic plants displayed an extremely early flowering phenotype that flowered more than two weeks earlier than the WT, and vernalization genes, rather than photoperiod genes, were increased in the WT. RNA-seq analysis revealed that diverse stress response genes, including HSPs, HSFs, LEAs, and MADS-box genes, were also upregulated in transgenic plants. Our study will help elucidate the roles of PHD genes in plant development and abiotic stress tolerance in rye.

VvJAZ13 Positively Regulates Cold Tolerance in Arabidopsis and Grape.

Cold stress adversely impacts grape growth, development, and yield. Therefore, improving the cold tolerance of grape is an urgent task of grape breeding. The Jasmonic acid (JA) pathway responsive gene JAZ plays a key role in plant response to cold stress. However, the role of JAZ in response to low temperatures in grape is unclear. In this study, VvJAZ13 was cloned from the 'Pinot Noir' (Vitis vinefera cv. 'Pinot Noir') grape, and the potential interacting protein of VvJAZ13 was screened by yeast two-hybrid (Y2H). The function of VvJAZ13 under low temperature stress was verified by genetic transformation. Subcellular localization showed that the gene was mainly expressed in cytoplasm and the nucleus. Y2H indicated that VvF-box, VvTIFY5A, VvTIFY9, Vvbch1, and VvAGD13 may be potential interacting proteins of VvJAZ13. The results of transient transformation of grape leaves showed that VvJAZ13 improved photosynthetic capacity and reduced cell damage by increasing maximum photosynthetic efficiency of photosystem II (Fv/Fm), reducing relative electrolyte leakage (REL) and malondialdehyde (MDA), and increasing proline content in overexpressed lines (OEs), which played an active role in cold resistance. Through the overexpression of VvJAZ13 in Arabidopsis thaliana and grape calli, the results showed that compared with wild type (WT), transgenic lines had higher antioxidant enzyme activity and proline content, lower REL, MDA, and hydrogen peroxide (H2O2) content, and an improved ability of scavenging reactive oxygen species. In addition, the expression levels of CBF1-2 and ICE1 genes related to cold response were up-regulated in transgenic lines. To sum up, VvJAZ13 is actively involved in the cold tolerance of Arabidopsis and grape, and has the potential to be a candidate gene for improving plant cold tolerance.

SlGAD2 is the target of SlTHM27, positively regulates cold tolerance by mediating anthocyanin biosynthesis in tomato.

Cold stress significantly limits the yield and quality of tomato. Deciphering the key genes related to cold tolerance is important for selecting and breeding superior cold-tolerant varieties. γ-aminobutyric acid (GABA) responds to various types of stress by rapidly accumulating in plant. In this study, glutamic acid decarboxylase (GAD2) was a positive regulator to enhance cold stress tolerance of tomato. Overexpression of SlGAD2 decreased the extent of cytoplasmic membrane damage and increased the endogenous GABA content, antioxidant enzyme activities, and reactive oxygen species (ROS) scavenging capacity in response to cold stress, whereas Slgad2 mutant plants showed the opposite trend. In addition, SlGAD2 induced anthocyanin biosynthesis in response to cold stress by increasing the content of endogenous GABA. Further study revealed that SlGAD2 expression was negatively regulated by the transcription factor SlTHM27. However, the transcript levels of SlTHM27 were repressed under cold stress. Antioxidant enzyme activities, SlGAD2 transcript levels, GABA and anthocyanin contents were significantly increased in Slthm27 mutant plants. Further, our study demonstrated that SlTHM27 decreases SlGAD2-promoted cold resistance in tomato by repressing SlGAD2 transcription. Overall, our results showed that the SlTHM27-SlGAD2 model regulates the cold tolerance in tomato by regulating GABA and anthocyanin.

Transcriptome and Metabolome Analyses Reveal Ascorbic Acid Ameliorates Cold Tolerance in Rice Seedling Plants

Ascorbic acid (AsA) plays a crucial role in the physiological processes of rice plants when they encounter various biotic and abiotic stresses. However, the specific mechanism by which AsA affects cold tolerance in rice seedlings remains unclear. Two rice genotypes, Zhongzao39 (ZZ39) and its recombinant inbred line RIL82, were exposed to cold stress, resulting in more damage observed in RIL82 compared to ZZ39. This damage included higher levels of relative electrolytic leakage (REC), malondialdehyde (MDA), H2O2, a lower Fv/Fm, and a lower survival rate. A comprehensive analysis of transcriptome and metabolome data indicated that AsA was involved in regulating cold tolerance in ZZ39 and RIL82 seedling plants. AsA content increased in ZZ39 while it decreased in RIL82 under cold stress. Additionally, analysis of carbohydrate contents highlighted their important role in the responses to cold stress of these two genotypes. Importantly, exogenous AsA and sucrose, either alone or in combination, enhanced the values of maximum fluorescence quantum yield (Fv/Fm) and effective quantum yield (YII) as well as decreased H2O2 and MDA levels to improve cold tolerance in both genotypes compared with plants treated with H2O. These findings highlight the potential significance of AsA in mitigating the effects of cold stress on rice seedling plants.

Whole-transcriptome profiling and identification of cold tolerance-related ceRNA networks in japonica rice varieties.

Low-temperature stress negatively impacts rice yield, posing a significant risk to food security. While previous studies have explored the physiological and linear gene expression alterations in rice under low-temperature conditions, the changes in competing endogenous RNA (ceRNA) networks remain largely unexamined. We conducted RNA sequencing on two japonica rice varieties with differing cold-tolerance capabilities to establish ceRNA networks. This enabled us to investigate the transcriptional regulatory network and molecular mechanisms that rice employs in response to low-temperature stress. We identified 364 differentially expressed circular RNAs (circRNAs), 224 differentially expressed microRNAs (miRNAs), and 12,183 differentially expressed messenger RNAs (mRNAs). WRKY family was the most prominent transcription factor family involved in cold tolerance. Based on the expression patterns and targeted relationships of these differentially expressed RNAs, we discerned five potential ceRNA networks related to low-temperature stress in rice: osa-miR166j-5p from the miR166 family was associated with cold tolerance; osa-miR528-3p and osa-miR156j-3p were linked to stress response; and osa-miR156j-3p was involved in the antioxidant system. In addition, Os03g0152000 in the antioxidant system, as well as Os12g0491800 and Os05g0381400, correlated with the corresponding stress response and circRNAs in the network. A gene sequence difference analysis and phenotypic validation of Os11g0685700 (OsWRKY61) within the WRKY family suggested its potential role in regulating cold tolerance in rice. We identified Os11g0685700 (OsWRKY61) as a promising candidate gene for enhancing cold tolerance in japonica rice. The candidate miRNAs, mRNAs, and circRNAs uncovered in this study are valuable targets for researchers and breeders. Our findings will facilitate the development of cold-tolerant rice varieties from multiple angles and provide critical directions for future research into the functions of cold-tolerance-related miRNAs, mRNAs, and circRNAs in rice.

Screening for cold tolerance genes in C. elegans, whose expressions are affected by anticancer drugs camptothecin and leptomycin B

Temperature is a vital environmental factor affecting organisms’ survival as they determine the mechanisms to tolerate rapid temperature changes. We demonstrate an experimental system for screening chemicals that affect cold tolerance in Caenorhabditis elegans. The anticancer drugs leptomycin B and camptothecin were among the 4000 chemicals that were screened as those affecting cold tolerance. Genes whose expression was affected by leptomycin B or camptothecin under cold stimuli were investigated by transcriptome analysis. Abnormal cold tolerance was detected in several mutants possessing genes that were rendered defective and whose expression altered after exposure to either leptomycin B or camptothecin. The genetic epistasis analysis revealed that leptomycin B or camptothecin may increase cold tolerance by affecting a pathway upstream of the insulin receptor DAF-2 that regulates cold tolerance in the intestine. Our experimental system combining drug and cold tolerance could be used for a comprehensive screening of genes that control cold tolerance at a low cost and in a short time period.

The cold-responsive C-repeat binding factors in Betula platyphylla Suk. positively regulate cold tolerance

Cold stress is one of the most destructive abiotic stresses limiting plant growth and development. CBF (C-repeat binding factor) transcription factors and their roles in cold response have been identified in Arabidopsis as well as several other plant species. However, the biological functions and related molecular mechanisms of CBFs in birch (Betula platyphylla Suk.) remain undetermined. In this study, five cold-responsive BpCBF genes, BpCBF1, BpCBF2, BpCBF7, BpCBF10 and BpCBF12 were cloned. Via protoplast transformation, BpCBF7 was found to be localized in nucleus. The result of yeast one hybrid assay validated the binding of BpCBF7 to the CRT/DRE (C-repeat/dehydration responsive element) elements in the promoter of BpERF1.1 gene. By overexpressing and repressing BpCBFs in birch plants, it was proven that BpCBFs play positive roles in the cold tolerance. At the metabolic level, BpCBFs OE lines had lower ROS accumulation, as well as higher activities of antioxidant enzymes (SOD, POD and CAT) and higher accumulation of protective substances (soluble sugar, soluble protein and proline). Via yeast one hybrid and co-transformation of effector and reporter vectors assay, it was proven that BpCBF7 can regulate the expression of BpERF5 and BpZAT10 genes by directly binding to their promoters. An interacting protein of BpCBF7, BpWRKY17, was identified by yeast two hybrid library sequencing and the interaction was validated with in vivo methods. These results indicates that BpCBFs can increase the cold tolerance of birch plants, partly by gene regulation and protein interaction. This study provides a reference for the research on CBF transcription factors and genetic improvement of forest trees upon abiotic stresses.