Cold-responsive Genes Research Articles

ZjMAPKK4 Interacted With ZjNAC78 Regulates Cold Tolerance Response in Jujube.

Jujube (Ziziphus ujuba Mill.) holds great importance as a fruit tree in China, with strong tolerance to drought and saline stress, but its growth is limited by vulnerability to cold stress. Consequently, the role of MAPK cascades in mediating jujube cold stress response remains unclear, with the specific function of ZjMAPKK4 in this context yet to be fully elucidated. Thus, in the current study, it was found that ZjMAPKK4 was significantly upregulated compared with other ZjMAPK cascade genes after cold treatment. Heterologous transformation of ZjMAPKK4 in Arabidopsis, VIGS-induced ZjMAPKK4 transiently silencing and overexpression of ZjMAPKK4 in jujube callus assays demonstrated that ZjMAPKK4 positively regulated the cold resistance of jujube. Furthermore, to elucidate the molecular regulation mechanism behind ZjMAPKK4 under cold stress, 25 key DEGs were screened out by transcriptome analysis. Yeast screening cDNA library, yeast two-hybrid, LCA and Co-IP analysis showed ZjMAPKK4 interacted with ZjNAC78 and VIGS-induced ZjNAC78 silenced sour jujube plants showed cold sensitivity and the expression level of cold response genes were downregulated after cold stress. All the results demonstrated that ZjMAPKK4 could interact with ZjNAC78 to regulate the downstream ZjICE-ZjCBF genes to regulate the cold tolerance of jujube.

Chromatin Accessibility Mediated by CHROMATIN REMODELING 11 Promotes Chilling Tolerance in Rice.

Chromatin remodeling plays a crucial role in controlling gene transcription by modifying chromatin structure. However, the involvement of chromatin remodeling in plant stress responses, especially cold tolerance, through chromatin accessibility remains largely unexplored. Here, we report that rice (Oryza sativa L.)CHROMATIN REMODELING 11 (OsCHR11) positively regulates chilling tolerance by enhancing chromatin accessibility and facilitating changes in gene expression. Loss-of-function mutants of OsCHR11 exhibited increased susceptibility to chilling stress compared to wild-type rice plants. Transcriptome analysis revealed that the chr11 mutant displays diminished transcriptomic responses to chilling. Additionally, assay for transposase-accessible chromatin (ATAC) indicated that chilling treatment increases chromatin accessibility in the promoter regions, and this process depended on OsCHR11 function. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that OsCHR11 is physically associated with the promoters of cold-responsive genes. Integrated multi-omics analysis further demonstrated a correlation between OsCHR11 enrichment and chromatin accessibility, as well as a correlation between chromatin accessibility and gene expression. Furthermore, OsCHR11 is required for the full expression of key cold-response genes, including those involved in trehalose biosynthesis. The exogenous application of trehalose partially rescued the chilling-susceptible phenotype of the chr11 mutant, suggesting that trehalose biosynthesis contributes to the chilling tolerance promoted by OsCHR11. Collectively, these findings indicate that OsCHR11 enhances cold tolerance in plants, likely by increasing chromatin accessibility and elevating the expression levels of cold-response genes in response to chilling.

PiERF1 regulates cold tolerance in Plumbago indica L. through ethylene signalling

Ethylene is a signalling factor that plays a key role in the response of plants to abiotic stresses, such as cold stress. Recent studies have shown that the exogenous application of 1-aminocyclopropane-1-carboxylate (ACC), an ethylene promoter, affects plant cold tolerance. The cold-responsive specific gene DREB plays a crucial role in enhancing cold tolerance in plants by activating several cold-responsive (COR) genes. However, how the ethylene biosynthesis pathway regulates this gene in the cold response of thermophilic plants has yet to be fully elucidated. In this study, the thermophilic plant Plumbago indica L. was used as an example. Physiological experiments and transcriptomic analyses revealed that cold stress treatment induced the synthesis of endogenous ACC and regulated the ethylene signalling activator PiERF1, and cold signalling also activated PiDREB1A. Spray experiments also revealed that ACC-induced upregulation of the PiERF1 gene reduced the cold tolerance of P. indica and decreased the expression level of the PiDREB1A gene. These results indicate that ethylene signalling directly regulates the downstream gene PiERF1 and initiates the DREB‒COR cold-responsive signalling pathway to regulate cold tolerance, resulting in the negative regulation of cold tolerance in thermophilic plants.

The Identification and Characterization of WOX Family Genes in Coffea arabica Reveals Their Potential Roles in Somatic Embryogenesis and the Cold-Stress Response.

WUSCHEL-related homeobox (WOX) genes play significant roles in plant development and stress responses. Difficulties in somatic embryogenesis are a significant constraint on the uniform seedling production and genetic modification of Coffea arabica, hindering efforts to improve coffee production in Yunnan, China. This study comprehensively analyzed WOX genes in three Coffea species. A total of 23 CaWOXs, 12 CcWOXs, and 10 CeWOXs were identified. Transcriptomic profile analysis indicated that about half of the CaWOX genes were actively expressed during somatic embryogenesis. The most represented CaWOXs were CaWOX2a, CaWOX2b, CaWOX8a, and CaWOX8b, which are suggested to promote the induction and development of the embryogenic callus, whereas CaWOX13a and CaWOX13b are suggested to negatively impact these processes. Co-expression analysis revealed that somatic embryogenesis-related CaWOXs were co-expressed with genes involved in embryo development, post-embryonic development, DNA repair, DNA metabolism, phenylpropanoid metabolism, secondary metabolite biosynthesis, and several epigenetic pathways. In addition, qRT-PCR showed that four WOX genes responded to cold stress. Overall, this study offers valuable insights into the functions of CaWOX genes during somatic embryogenesis and under cold stress. The results suggest that certain WOX genes play distinct regulatory roles during somatic embryogenesis, meriting further functional investigation. Moreover, the cold-responsive genes identified here are promising candidates for further molecular analysis to assess their potential to enhance cold tolerance.

TaTCP21-A negatively regulates wheat cold tolerance via repressing expression of TaDREB1C

Cold stress is one of the important harmful factors that seriously affect wheat (Triticum aestivum) yield and quality. TCP transcription factor plays important roles in the process of plant cell proliferation and growth. In this study, we identified 60 TaTCP genes expressed in strong cold resistant winter wheat variety Dongnongdongmai1 (Dn1) under cold stress by previous transcriptome data, of which 13 TaTCPs showed significant differences in expression. The evolution of TaTCPs was analyzed, and the results showed that there were 2 homologous pairs in TaTCPs with AtTCPs and 90 homologous pairs in TaTCPs with OsTCPs. Expression patterns of 20 TaTCPs under cold stress were analyzed by qRT-PCR, and TCP21-A with significant expression differences was screened. We obtained tcp21-A mutant from the EMS mutant library of winter wheat Kenong9204. We observed that the mutation of TaTCP21-A significantly improved its cold resistance. Subsequently, transcriptome analysis revealed that TCP21-A inhibited expression of cold responsive gene TaDREB1C. Finally, subcellular localization and yeast one hybrid were used to verify that TCP21-A can act as a transcription factor to bind to the GGTCCC promoter element. Luciferase reporter gene experiment showed that TCP21-A inhibits the transcriptional activity of the TaDREB1C promoter. In summary, we systematically analyzed the expression patterns of TaTCP family members in Dn1 under cold stress and demonstrated that TaTCP21-A negatively regulated wheat cold tolerance by inhibiting expression of TaDREB1C. These results provide new insights into the functional mechanism of TaTCP transcription factors in response to cold stress.

Genome-wide analysis of fatty acid desaturase genes in moso bamboo (Phyllostachys edulis) reveal their important roles in abiotic stresses responses

BackgroundBamboo is an important nontimber forestry product worldwide, while growth, development and geographic distribution of bamboo are often affected by abiotic stresses. Fatty acid desaturases have important roles in regulating plant abiotic stress tolerance, especially low-temperature. However, there is no report on genome-wide of FAD genes in bamboo under abiotic stresses.ResultsA toltal of 43 PeFAD genes were identified in moso bamboo genome, which were unevenly located in 17 scaffolds. Phylogenetic analysis indicated that PeFAD genes were divided into 6 groups and ADS/FAD5 group was absence in momo bamboo, and gene structure and histidine-motifs remained highly conserved in each group. The expansion of PeFAD genes was mainly caused by tandem and segmental duplications of SAD/FAB2 group. We also identified 59 types of miRNAs targeting PeFAD genes. RNA-seq data indicated that PeFAD genes were transcribed in various organs/tissues with different degrees, and responded to abiotic stresses and hormone treatments, including cold, salt, drought, SA, ABA, BR, NAA and GA. Co-expression analysis under cold stress showed that PeCBF3 might directly bind the promoter of top cold-responsive PeSLD1 gene that contained LTR cis-element and DRE core element. The qRT-PCR assay also validated the expression pattern of PeSLD1 and its upstream regulatory gene PeCBF3.ConclusionIn this study, we performed comprehensive genome-wide survey of PeFAD genes in moso bamboo and analyzed their expression patterns in various tissues and organs, and under abiotic stresses and phytohormones treatment. The qRT-PCR assay validated the cold inducibility of PeSLD1 and PeCBF3. This work showed critical roles of PeFAD genes in abiotic stresses responses. This is the first report on genome-wide analysis of PeFAD genes in moso bamboo, which will provide critical gene resources for molecular breeding of stress-toleranct moso bamboo.

Ectopic expression of HvbHLH132 from hulless barley reduces cold tolerance in transgenic Arabidopsis thaliana.

Overexpression of HvbHLH132 from hulless barley impairs in chilling and freezing tolerance at the seedlings stage in Arabidopsis thaliana The basic helix-loop-helix (bHLH) transcription factors (TF) are ubiquitously existed in eukaryote and play crucial roles in numerous biological processes. However, the characterization of their members and functions in hulless barley remains limited. Here, we conducted a genome-wide identification of the HvbHLH gene family and assessed the role of HvbHLH132 in cold stress tolerance. We identified 141 HvbHLH genes, which were categorized into twelve subfamilies. Subcellular localization predictions indicated that the majority of HvbHLH proteins were localized in the nucleus. cis-Acting element analysis revealed that the promoter regions of the HvbHLH family contain diverse elements associated with various biological processes. Expression profiling of the 141 HvbHLH genes in two extreme varieties revealed that HvbHLH132 was significantly induced and exhibited substantial differential expression under cold stress. Analyses of subcellular localization and transactivation activity confirmed that HvbHLH132 specifically localized in the nucleus and contributed to transcriptional activation. Furthermore, overexpression of HvbHLH132 in Arabidopsis resulted in impaired chilling and freezing tolerance at the seedling stage, leading to biochemical changes unfavorable for freezing stress. Additionally, the expression of some cold-responsive genes (COR) genes was significantly less induced compared to wild type under freezing stress. This study provides comprehensive insight into the HvbHLH gene family and reveals a critical role of HvbHLH132 in regulating cold tolerance in plants.

Full-length single-molecule sequencing uncovers novel insight into the global landscape of the cold stress response in trifoliate orange (Citrus trifoliata).

Trifoliate orange (Citrus trifoliata (L.) Raf.) is a cold-hardy citrus species that contributes to citrus production by frequently serving as a rootstock. Nevertheless, the molecular mechanisms underlying cold tolerance in citrus, particularly post-transcriptional regulation, remain largely unidentified. In this study, we constructed a transcriptome map of trifoliate orange subjected to cold stress by integrating full-length single-molecule sequencing and Illumina short-read sequencing. The hybrid sequencing approach yielded a more comprehensive set of full-length transcripts than was previously available from the reference genome. In particular, the high-quality transcripts enabled the detection of extensive alternative splicing (AS), with intron retention (IR) identified as the predominant AS event in trifoliate orange. Transcriptome analysis revealed that genes associated with starch and sucrose metabolism were significantly enriched among the cold-responsive genes. Consistent with these data, soluble sugar content was elevated by the cold treatments. Additionally, the expression of multiple genes encoding enzymes with antioxidant activity, including PODs and SODs, was induced, which plays a pivotal role in the mitigation of continuous ROS production. Furthermore, we observed that AS and transcriptional regulation modulate distinct pathways. We also found that the expression of genes encoding key transcription factors (TFs) was highly induced by cold stress and that some of the mRNAs encoding these key TFs were differentially spliced. This dataset provides comprehensive transcriptional and post-transcriptional profiles of the response to cold stress in trifoliate orange that may help to identify genes that contribute to cold tolerance in citrus.

The H3K27me3 histone mark correlates with repression of colour and aroma development post-harvest in strawberry fruit.

Strawberry ripening is non-climacteric, and post-harvest fruit enter senescence and deteriorate rapidly. Chilled storage induces transcriptome wide changes in gene expression, including the down-regulation of aroma related genes. Histone marks are associated with transcriptional activation or repression; the H3K27me3 mark is mainly associated with repression of gene expression. Here genes associated with H3K27me3 were identified through ChIP-seq in ripe red strawberry fruit at harvest and after 5 days of chilled storage in the dark. The number of ChIP peaks increased with storage time, indicating an increased role for this mark in regulation of gene expression following chilled dark storage. Comparing ChIP-seq data to RNA-seq data from the same material identified 440 genes whose expression correlates with H3K27me3 repression. Abiotic stress genes, especially cold stress response genes, were down-regulated during storage. Increased association with the H3K27me3 mark indicates that they may be repressed via this epigenetic mark. Other functional groups included cell wall and carbohydrate metabolism. The association with the H3K27me3 mark of two transcription factors (FaHY5 and FaTRAB1) and FaADH, involved in ester biosynthesis, was validated by ChIP-PCR. These three genes are all down-regulated during storage and indicate a network of H3K27me3 gene repression affecting both anthocyanin and ester biosynthesis.

Hydrogen peroxide mediates melatonin-induced chilling tolerance in cucumber seedlings.

MT mitigates chilling damage by enhancing antioxidant system and photosystem activities, and cold-responsive genes expression in cucumbers. H2O2 may act as a downstream signaling molecule in the MT-induced chilling tolerance. Melatonin (MT) and hydrogen peroxide (H2O2) are important endogenous signaling molecules that play multifaceted roles in plant responses to abiotic stress. However, the interactive mechanism by which MT and H2O2 regulate chilling tolerance remains unclear. Here we found that MT exhibited a positive regulatory effect on the chilling tolerance of cucumbers, with an optimum concentration of 100µM. MT markedly enhanced RBOH1 mRNA expression, activity and endogenous H2O2 accumulation in cucumber seedlings. However, 1.0mM H2O2 had no significant effect on mRNA levels of TDC and ASMT, the key genes for MT synthesis, and endogenous MT content. Both MT and H2O2 significantly decreased malondialdehyde (MDA), electrolyte leakage (EL) and chilling injury index (CI) by activating the antioxidant system, thereby alleviating chilling damage in cucumber seedlings. MT and H2O2 improved photosynthetic carbon assimilation, which was primarily attributed to an increase in activity, mRNA expression, and protein levels of RuBPCase and RCA. Meanwhile, MT and H2O2 induced the photoprotection for both PSII and PSI by enhancing the QA's electron transport capacity and elevating protein levels of the photosystems. Moreover, MT and H2O2 significantly upregulated the expression of cold response genes. MT-induced chilling tolerance was attenuated by N', N'-dimethylthiourea (DMTU), a H2O2 specific scavenger. Whereas, the MT synthesis inhibitor (p-chlorophenylalanine, p-CPA) did not influence H2O2-induced chilling tolerance. The positive regulation of MT on the antioxidant system, photosynthesis and cold response gene levels were significantly attenuated in RBOH1-RNAi plants compared with WT plants. These findings suggest that H2O2 may functions as a downstream signaling molecule in MT-induced chilling tolerance in cucumber plants.

Multifunctional Transcription Factor YABBY6 Regulates Morphogenesis, Drought and Cold Stress Responses in Rice

The roles of plant-specific transcription factor family YABBY may vary among different members. OsYABBY6 is a rice YABBY gene, whose function is not well elucidated so far. In this paper, we show that OsYABBY6 is a nucleus-localized protein with transcriptional activation activity. OsYABBY6 is predominantly expressed in the palea and lemma, as well as in the sheath, culm and node. OsYABBY6 RNA interference (RNAi) plants exhibited altered plant height and larger grain size. Under cold treatment, OsYABBY6 overexpression (OE) plants had up-regulated expression of cold responsive genes, and accumulated less reactive oxygen species but more proline compared to wild type, resulting in improved cold tolerance. On the other hand, RNAi plants showed enhanced drought tolerance compared to the wild type by slower water loss, less reactive oxygen species but more proline and soluble sugar accumulation. In addition, endogenous abscisic acid (ABA) level was reduced in OsYABBY6 RNAi plants, and RNAi and OE plants were more and less sensitive to ABA treatment, respectively. Accordingly, we deduce that OsYABBY6 positively regulates cold response but negatively regulates drought response through different pathways. Our study reveals the crucial roles of OsYABBY6 in plant architecture and grain development, as well as in abiotic stress response, providing new insights into the functions of YABBYs in rice.

Enhancing cold tolerance in tobacco through endophytic symbiosis with Piriformospora indica.

Tobacco, a warm-season crop originating from the Americas, is highly susceptible to cold stress. The utilization of symbiotic fungi as a means to bolster crops' resilience against abiotic stresses has been proven to be a potent strategy. In this study, we investigated the effect of endophytic fungus Piriformospora indica on the cold resistance of tobacco. When exposed to cold stress, the colonization of P.indica in tobacco roots effectively stimulates the activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX). This, in turn, reduces the accumulation of reactive oxygen species (ROS), thereby mitigating oxidative damage. Additionally, P. indica elevates the levels of osmolytes, such as soluble sugars, proline, and soluble proteins, thus facilitating the restoration of osmotic balance. Under cold stress conditions, P. indica also induces the expression of cold-responsive genes. Furthermore, this fungus not only enhances photosynthesis in tobacco by stimulating the synthesis of photosynthetic pigments, strengthening Rubisco activity, and elevating PSII efficiency, but also fortifies tobacco's nitrogen assimilation by inducing the expression of nitrate transporter gene and activating enzymes related to nitrogen assimilation. Consequently, this synergistic optimization of nitrogen and carbon assimilation provides a solid material and energetic foundation for tobacco plants to withstand cold stress. Our study demonstrates that a mycorrhizal association between P. indica and tobacco seedlings provides multifaceted protection to tobacco plants against low-temperature stress and offers a valuable insight into how P. indica enhances the cold tolerance of tobacco.

Genome-Wide Identification of the Cyclic Nucleotide-Gated Ion Channel Gene Family and Expression Profiles Under Low-Temperature Stress in Luffa cylindrica L.

Cyclic nucleotide-gated ion channels (CNGCs) are cell membrane channel proteins for calcium ions. They have been reported to play important roles in survival and in the responses to environmental factors in various plants. However, little is known about the CNGC family and its functions in luffa (Luffa cylindrica L.). In this study, a bioinformatics-based method was used to identify members of the CNGC gene family in L. cylindrica. In total, 20 LcCNGCs were detected, and they were grouped into five subfamilies (I, II, Ⅲ, IV-a, and IV-b) in a phylogenetic analysis with CNGCs from Arabidopsis thaliana (20 AtCNGCs) and Momordica charantia (17 McCNGCs). The 20 LcCNGC genes were unevenly distributed on 11 of the 13 chromosomes in luffa, with none on Chromosomes 1 and 5. The members of each subfamily encoded proteins with highly conserved functional domains. An evolutionary analysis of CNGCs in luffa revealed three gene losses and a motif deletion. An examination of gene replication events during evolution indicated that two tandemly duplicated gene pairs were the primary driving force behind the evolution of the LcCNGC gene family. PlantCARE analyses of the LcCNGC promoter regions revealed various cis-regulatory elements, including those responsive to plant hormones (abscisic acid, methyl jasmonate, and salicylic acid) and abiotic stresses (light, drought, and low temperature). The presence of these cis-acting elements suggested that the encoded CNGC proteins may be involved in stress responses, as well as growth and development. Transcriptome sequencing (RNA-seq) analyses revealed tissue-specific expression patterns of LcCNGCs in various plant parts (roots, stems, leaves, flowers, and fruit) and the upregulation of some LcCNGCs under low-temperature stress. To confirm the accuracy of the RNA-seq data, 10 cold-responsive LcCNGC genes were selected for verification by quantitative real-time polymerase chain reaction (RT-qPCR) analysis. Under cold conditions, LcCNGC4 was highly upregulated (>50-fold increase in its transcript levels), and LcCNGC3, LcCNGC6, and LcCNGC13 were upregulated approximately 10-fold. Our findings provide new information about the evolution of the CNGC family in L. cylindrica and provide insights into the functions of the encoded CNGC proteins.

Genome-wide identification of Aux/IAA gene family members in grape and functional analysis of VaIAA3 in response to cold stress.

Twenty-five VvIAA genes and eighteen VaIAA genes were identified from Pinot Noir and Shanputao, respectively. The overexpression of VaIAA3 in transgenic Arabidopsis increased cold tolerance by regulating auxin, ABA and ethylene signaling. Aux/IAA genes are key genes involved in regulating auxin signal transduction in plants. Although IAA genes have been characterized in various plant species, the role of IAA genes in grape cold resistance is unclear. To further explore the members of the Aux/IAA gene family in grape and their functions, in this study, using genomic data for Pinot Noir (Vitis vinifera cv. 'Pinot Noir') and Shanputao (Vitis amurensis), 25 VvIAA genes and 18 VaIAA genes were identified. The VaIAA genes presented different expression patterns at five different temperatures (28 ± 1 °C, 5 ± 1 °C, 0 ± 1 °C, -5 ± 1 °C, and -10 ± 1 °C) according to qRT‑PCR results. VaIAA3 was selected as a candidate gene for further functional analysis because of its high expression level under low-temperature stress. Subcellular localization experiments revealed that VaIAA3 was localized in the nucleus. Additionally, under 4 °C treatment for 24 h, relative expression level of VaIAA3, antioxidant enzyme activity, survival rate, and cold-responsive gene expression in three transgenic lines (OE-1, OE-2, OE-3) were greater, whereas relative electrolytic conductivity (REC), malondialdehyde (MDA) content and hydrogen peroxide (H2O2) content were lower than those of the wild type (WT). Transcriptome sequencing analysis revealed that VaIAA3 regulated cold stress resistance in Arabidopsis thaliana (Arabidopsis) through pathways involving auxin, ABA, JA, or ethylene. Importantly, heterologous overexpression of VaIAA3 increased the resistance of Arabidopsis to cold stress, which provides a theoretical basis for the further use of VaIAA3 to improve cold resistance in grape.

Transcriptome sequencing analysis of overexpressed SikCDPK1 in tobacco reveals mechanisms of cold stress response

BackgroundCold is a significant limiting factor in productivity, particularly in northwestern and eastern China. Calcium-Dependent Protein Kinases (CDPKs), a primary calcium signaling sensor in plants, play an important role in their response to cold. Snow lotus (Sasussured involucrata Kar L) is a plant that thrives in harsh climates and grows in northwest China. However, there were no reports on the transcriptome of OE-SikCDPK1 transgenic tobacco in response to cold.ResultsWhen exposed to cold stress, OE-SikCDPK1 plants displayed a cold-tolerant phenotype compared to non-transgenic tobacco. Under cold conditions, relative water content reduced, relative conductivity increased, malondialdehyde levels rose, and cold-responsive gene expression increased. The OE-SikCDPK1 gene and non-transgenic tobacco were employed for research purposes. The transcriptome of leaves was sequenced using the HISAT2 sequencing platform, and the data were used to examine gene function annotation and differentially expressed genes (DEGs). 53,022 DEGs in tobacco leaves under cold treatment were obtained. The GO enrichment results revealed that it was enriched for biological-process, defense response and other processes under cold stress. The KEGG pathway enrichment analysis revealed that the metabolic pathways of significant enrichment of DEGs under cold stress mainly involved MAPK signaling pathway transduction. The transcription factor identification results showed that the transcription factors with the largest number of differential expressions under cold treatment were mainly from WRKY, AP2, MYB, bHLH, NAC and other transcription factor families.ConclusionThe cold tolerance mechanism of snow lotus SikCDPK1 was comprehensively analyzed at the transcriptional level for the first time using RNA-seq technology. This study demonstrates that SikCDPK1 can respond to cold by participating in the MAPK signaling pathway and regulating the expression levels of transcription factors, including WRKY, AP2, MYB, bHLH, and NAC. These results offer valuable insights for further exploration of the cold tolerance mechanism associated with SikCDPK1.

Environmental salinity differentiates responses to acute hypothermal stress in milkfish

Global warming has led to an increase in the frequency of cold extremes, causing significant economic losses in aquaculture, particularly in subtropical regions. Milkfish (Chanos chanos) holds significant importance in aquaculture within subtropical Asian regions. Despite milkfish's ability to tolerate varying salinity levels, frequent cold snaps associated with extreme weather events have caused substantial mortality. Understanding the molecular and cellular mechanisms underlying cold stress-induced cell death is crucial for developing effective strategies to mitigate such losses. Given the pivotal role of the liver in fish physiology, we established a primary milkfish hepatocyte culture demonstrating robust proliferation and expressing a unique marker leptin A. The molecular characterization of cold-treated hepatocytes at 18 °C showed that the mRNA levels of superoxide dismutase (sod1) and catalase (cat), responsible for neutralizing reactive oxygen species (ROS), were downregulated in freshwater (FW) conditions, while cat expression was upregulated in seawater (SW) conditions. This differential modulation of ROS signaling implies distinct responses in FW and SW, leading to higher ROS levels and increased cell death in FW condition compared to those in SW. Transcriptomic analysis of liver tissues from milkfish reared in FW or SW, with or without cold stress, revealed distinct gene expression patterns. Although cold stress affected a common set of genes in both FW and SW conditions, SW-specific cold responsive genes are associated with metabolic pathways while FW-specific genes were linked to cell proliferation pathways. Notably, gene set enrichment analysis highlighted the downregulation of cytochrome-related genes, a major source of ROS production, in response to cold stress in SW. In summary, our study unveils an insightful mechanism whereby the salinity of SW counteracts cold stress-induced ROS signaling, emphasizing the feasibility and practicality of preconditioning fish in SW as a preventive measure against cold stress-induced mortality.

Predicting Cold-Stress Responsive Genes in Cotton with Machine Learning Models

Machine Learning (ML) serves as a potent tool for data mining and predictive analytics in genomic research. However, its application in identifying stress-responsive genes remains underexplored. This study identified distinct variations in the expression patterns of one-to-one homologous genes responding to cold stress in three cotton species: Gossypium hirsutum, Gossypium barbadense, and Gossypium arboreum. To better understand cold-responsive genes, we developed ML predictive models (LightGBM, XGBoost, and Random Forest) utilizing 121 biochemical features. The incorporating of these features significantly enhanced model accuracy. Moreover, incorporating evolutionary information further refined the models, achieving an impressive 80.80% accuracy in predicting cold-stress responsive genes. Notably, models trained on sequence features from G. hirsutum showed transferability to the closely related species of G. barbadense, with accuracies ranging from 78.65% to 83.04%. This research presents a promising workflow for identifying candidate genes for experimental exploration of cold stress responses and establishes a systematic framework for predicting cold-stress related genes using ML methodologies.

Molecular Mechanisms Underlying Freezing Tolerance in Plants: Implications for Cryopreservation.

Cryopreservation is a crucial technique for the long-term ex situ conservation of plant genetic resources, particularly in the context of global biodiversity decline. This process entails freezing biological material at ultra-low temperatures using liquid nitrogen, which effectively halts metabolic activities and preserves plant tissues over extended periods. Over the past seven decades, a plethora of techniques for cryopreserving plant materials have been developed. These include slow freezing, vitrification, encapsulation dehydration, encapsulation-vitrification, droplet vitrification, cryo-plates, and cryo-mesh techniques. A key challenge in the advancement of cryopreservation lies in our ability to understand the molecular processes underlying plant freezing tolerance. These mechanisms include cold acclimatization, the activation of cold-responsive genes through pathways such as the ICE-CBF-COR cascade, and the protective roles of transcription factors, non-coding RNAs, and epigenetic modifications. Furthermore, specialized proteins, such as antifreeze proteins (AFPs) and late embryogenesis abundant (LEA) proteins, play crucial roles in protecting plant cells during freezing and thawing. Despite its potential, cryopreservation faces significant challenges, particularly in standardizing protocols for a wide range of plant species, especially those from tropical and subtropical regions. This review highlights the importance of ongoing research and the integration of omics technologies to improve cryopreservation techniques, ensuring their effectiveness across diverse plant species and contributing to global efforts regarding biodiversity conservation.

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.

TOR balances plant growth and cold tolerance by orchestrating amino acid-derived metabolism in tomato.

The target of rapamycin (TOR) kinase is a central signaling hub that plays a crucial role in precisely orchestrating plant growth, development, and stress responses. This suggests that TOR is intricately involved in maintaining the balance between plant growth and stress responses. Nevertheless, despite the observed effects, the specific mechanisms through which TOR operates in these processes remain obscure. In this study, we investigated how the tomato (Solanum lycopersicum) TOR (SlTOR) affects plant growth and cold responses. We demonstrated that SlTOR inhibition transcriptionally primes cold stress responses, consequently enhancing tomato cold tolerance. A widely targeted metabolomics analysis revealed the disruption of amino acid metabolism homeostasis under cold stress upon SlTOR inhibition, which led to the accumulation of two important cryoprotective metabolites: salicylic acid (SA) and putrescine (Put). Next, we discovered SlPGH1 (2-PHOSPHO-D-GLYCERATE HYDRO-LYASE 1) as a direct substrate of SlTOR. Inhibiting SlTOR led to increased SlCBF1 (C-REPEAT-BINDING FACTOR 1) expression via SlPGH1, potentially triggering the activation of cold-responsive genes and subsequent metabolic alterations. Our study provides a mechanistic framework that elucidates how SlTOR modulates amino acid-related metabolism to enhance tomato cold tolerance, which sheds light on the complex interplay between growth and stress responses orchestrated by TOR.