RESUMEN
Unusually low temperatures caused by global climate change adversely affect rice production. Sensing cold to trigger signal network is a key base for improvement of chilling tolerance trait. Here, we report that Oryza sativa Calreticulin 3 (OsCRT3) localized at the endoplasmic reticulum (ER) exhibits conformational changes under cold stress, thereby enhancing its interaction with CBL-interacting protein kinase 7 (OsCIPK7) to sense cold. Phenotypic analyses of OsCRT3 knock-out mutants and transgenic overexpression lines demonstrate that OsCRT3 is a positive regulator in chilling tolerance. OsCRT3 localizes at the ER and mediates increases in cytosolic calcium levels under cold stress. Notably, cold stress triggers secondary structural changes of OsCRT3 and enhances its binding affinity with OsCIPK7, which finally boosts its kinase activity. Moreover, Calcineurin B-like protein 7 (OsCBL7) and OsCBL8 interact with OsCIPK7 specifically on the plasma membrane. Taken together, our results thus identify a cold-sensing mechanism that simultaneously conveys cold-induced protein conformational change, enhances kinase activity, and Ca2+ signal generation to facilitate chilling tolerance in rice.
Asunto(s)
Calreticulina , Oryza , Calreticulina/metabolismo , Oryza/genética , Oryza/metabolismo , Temperatura , Frío , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Plants respond to cold stress at multiple levels, including increasing cytosolic calcium (Ca2+) influx and triggering the expression of cold-responsive genes. Here we show that the Ca2+-permeable channel CYCLIC NUCLEOTIDE GATED CHANNEL20 (CNGC20) positively regulates freezing tolerance in Arabidopsis (Arabidopsis thaliana) by mediating cold-induced Ca2+ influx. Moreover, we demonstrate that the leucine-rich repeat receptor-like kinase PLANT PEPTIDE CONTAINING SULFATED TYROSINE1 RECEPTOR (PSY1R) is activated by cold, phosphorylating and enhancing the activity of CNGC20. The psy1r mutant exhibited decreased cold-evoked Ca2+ influx and freezing tolerance. Conversely, COLD-RESPONSIVE PROTEIN KINASE1 (CRPK1), a protein kinase that negatively regulates cold signaling, phosphorylates and facilitates the degradation of CNGC20 under prolonged periods of cold treatment, thereby attenuating freezing tolerance. This study thus identifies PSY1R and CRPK1 kinases that regulate CNGC20 activity and stability, respectively, thereby antagonistically modulating freezing tolerance in plants.
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The Cys2/His2 (C2H2)-type zinc finger family has been reported to regulate multiple aspects of plant development and abiotic stress response. However, the role of C2H2-type zinc finger proteins in cold tolerance remains largely unclear. Through RNA-sequence analysis, a cold-responsive zinc finger protein, named as PtrZAT12, was identified and isolated from trifoliate orange (Poncirus trifoliata L. Raf.), a cold-hardy plant closely related to citrus. Furthermore, we found that PtrZAT12 was markedly induced by various abiotic stresses, especially cold stress. PtrZAT12 is a nuclear protein, and physiological analysis suggests that overexpression of PtrZAT12 conferred enhanced cold tolerance in transgenic tobacco (Nicotiana tabacum) plants, while knockdown of PtrZAT12 by virus-induced gene silencing (VIGS) increased the cold sensitivity of trifoliate orange and repressed expression of genes involved in stress tolerance. The promoter of PtrZAT12 harbors a DRE/CRT cis-acting element, which was verified to be specifically bound by PtrCBF1 (Poncirus trifoliata C-repeat BINDING FACTOR1). VIGS-mediated silencing of PtrCBF1 reduced the relative expression levels of PtrZAT12 and decreased the cold resistance of trifoliate orange. Based on these results, we propose that PtrZAT12 is a direct target of CBF1 and plays a positive role in modulation of cold stress tolerance. The knowledge gains new insight into a regulatory module composed of CBF1-ZAT12 in response to cold stress and advances our understanding of cold stress response in plants.
Asunto(s)
Citrus , Poncirus , Poncirus/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Respuesta al Choque por Frío/fisiología , Dedos de Zinc , Citrus/genética , Regulación de la Expresión Génica de las Plantas , Plantas Modificadas Genéticamente/metabolismo , FríoRESUMEN
Chilling stress causes banana fruit softening disorder and severely impairs fruit quality. Various factors, such as transcription factors, regulate fruit softening. Herein, we identified a novel regulator, MaC2H2-IDD, whose expression is closely associated with fruit ripening and softening disorder. MaC2H2-IDD is a transcriptional activator located in the nucleus. The transient and ectopic overexpression of MaC2H2-IDD promoted "Fenjiao" banana and tomato fruit ripening. However, transient silencing of MaC2H2-IDD repressed "Fenjiao" banana fruit ripening. MaC2H2-IDD modulates fruit softening by activating the promoter activity of starch (MaBAM3, MaBAM6, MaBAM8, MaAMY3, and MaISA2) and cell wall (MaEXP-A2, MaEXP-A8, MaSUR14-like, and MaGLU22-like) degradation genes. DLR, Y1H, EMSA, and ChIP-qPCR assays validated the expression regulation. MaC2H2-IDD interacts with MaEBF1, enhancing the regulation of MaC2H2-IDD to MaAMY3, MaEXP-A2, and MaGLU22-like. Overexpressing/silencing MaC2H2-IDD in banana and tomato fruit altered the transcript levels of the cell wall and starch (CWS) degradation genes. Several differentially expressed genes (DEGs) were authenticated between the overexpression and control fruit. The DEGs mainly enriched biosynthesis of secondary metabolism, amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, starch and sucrose metabolism, and plant hormones signal transduction. Overexpressing MaC2H2-IDD also upregulated protein levels of MaEBF1. MaEBF1 does not ubiquitinate or degrade MaC2H2-IDD. These data indicate that MaC2H2-IDD is a new regulator of CWS degradation in "Fenjiao" banana and cooperates with MaEBF1 to modulate fruit softening, which also involves the cold softening disorder.
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Respuesta al Choque por Frío , Frutas , Regulación de la Expresión Génica de las Plantas , Musa , Proteínas de Plantas , Musa/genética , Musa/metabolismo , Musa/fisiología , Frutas/genética , Frutas/metabolismo , Frutas/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Respuesta al Choque por Frío/genética , Solanum lycopersicum/genética , Solanum lycopersicum/fisiología , Solanum lycopersicum/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Plantas Modificadas Genéticamente , Pared Celular/metabolismo , Almidón/metabolismoRESUMEN
Lipocalins constitute a conserved protein family that binds to and transports a variety of lipids while fatty acid desaturases (FADs) are required for maintaining the cell membrane fluidity under cold stress. Nevertheless, it remains unclear whether plant lipocalins promote FADs for the cell membrane integrity under cold stress. Here, we identified the role of OsTIL1 lipocalin in FADs-mediated glycerolipid remodeling under cold stress. Overexpression and CRISPR/Cas9 mediated gene edition experiments demonstrated that OsTIL1 positively regulated cold stress tolerance by protecting the cell membrane integrity from reactive oxygen species damage and enhancing the activities of peroxidase and ascorbate peroxidase, which was confirmed by combined cold stress with a membrane rigidifier dimethyl sulfoxide or a H2 O2 scavenger dimethyl thiourea. OsTIL1 overexpression induced higher 18:3 content, and higher 18:3/18:2 and (18:2 + 18:3)/18:1 ratios than the wild type under cold stress whereas the gene edition mutant showed the opposite. Furthermore, the lipidomic analysis showed that OsTIL1 overexpression led to higher contents of 18:3-mediated glycerolipids, including galactolipids (monoglactosyldiacylglycerol and digalactosyldiacylglycerol) and phospholipids (phosphatidyl glycerol, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and phosphatidyl inositol) under cold stress. RNA-seq and enzyme linked immunosorbent assay analyses indicated that OsTIL1 overexpression enhanced the transcription and enzyme abundance of four ω-3 FADs (OsFAD3-1/3-2, 7, and 8) under cold stress. These results reveal an important role of OsTIL1 in maintaining the cell membrane integrity from oxidative damage under cold stress, providing a good candidate gene for improving cold tolerance in rice.
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Respuesta al Choque por Frío , Oryza , Especies Reactivas de Oxígeno/metabolismo , Oryza/metabolismo , Estrés Oxidativo , Membrana Celular/metabolismo , Frío , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genéticaRESUMEN
ERFs (ethylene-responsive factors) are known to play a key role in orchestrating cold stress signal transduction. However, the regulatory mechanisms and target genes of most ERFs are far from being well deciphered. In this study, we identified a cold-induced ERF, designated as PtrERF110, from trifoliate orange (Poncirus trifoliata L. Raf., also known as Citrus trifoliata L.), an elite cold-hardy plant. PtrERF110 is a nuclear protein with transcriptional activation activity. Overexpression of PtrERF110 remarkably enhanced cold tolerance in lemon (Citrus limon) and tobacco (Nicotiana tabacum), whereas VIGS (virus-induced gene silencing)-mediated knockdown of PtrERF110 drastically impaired the cold tolerance. RNA sequence analysis revealed that PtrERF110 overexpression resulted in global transcriptional reprogramming of a range of stress-responsive genes. Three of the genes, including PtrERD6L16 (early responsive dehydration 6-like transporters), PtrSPS4 (sucrose phosphate synthase 4), and PtrUGT80B1 (UDP-glucose: sterol glycosyltransferases 80B1), were confirmed as direct targets of PtrERF110. Consistently, PtrERF110-overexpressing plants exhibited higher levels of sugars and sterols compared to their wild type counterparts, whereas the VIGS plants had an opposite trend. Exogenous supply of sucrose restored the cold tolerance of PtrERF110-silencing plants. In addition, knockdown of PtrSPS4, PtrERD6L16, and PtrUGT80B1 substantially impaired the cold tolerance of P. trifoliata. Taken together, our findings indicate that PtrERF110 positively modulates cold tolerance by directly regulating sugar and sterol synthesis through transcriptionally activating PtrERD6L16, PtrSPS4, and PtrUGT80B1. The regulatory modules (ERF110-ERD6L16/SPS4/UGT80B1) unraveled in this study advance our understanding of the molecular mechanisms underlying sugar and sterol accumulation in plants subjected to cold stress.
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Epigenetic regulation of gene expression plays a crucial role in plant development and environmental adaptation. The H3K4me3 and H3K27me3 have not only been discovered in the regulation of gene expression in multiple biological processes but also in responses to abiotic stresses in plants. However, evidence for the presence of both H3K4me3 and H3K27me3 on the same nucleosome is sporadic. Cold-induced deposition of bivalent H3K4me3-H3K27me3 modifications and nucleosome depletion over a considerable number of active genes is documented in potato tubers and provides clues on an additional role of the bivalent modifications. Limited by the available information of genes encoding PcG/TrxG proteins as well as their corresponding mutants in potatoes, the molecular mechanism underlying the cold-induced deposition of the bivalent mark remains elusive. In this study, we found a similar deposition of the bivalent H3K4me3-H3K27me3 mark over 2129 active genes in cold-treated Arabidopsis Col-0 seedlings. The expression levels of the bivalent mark-associated genes tend to be independent of bivalent modification levels. However, these genes were associated with greater chromatin accessibility, presumably to provide a distinct chromatin environment for gene expression. In mutants clf28 and lhp1, failure to deposit H3K27me3 in active genes upon cold treatment implies that the CLF is potentially involved in cold-induced deposition of H3K27me3, with assistance from LHP1. Failure to deposit H3K4me3 during cold treatment in atx1-2 suggests a regulatory role of ATX1 in the deposition of H3K4me3. In addition, we observed a cold-induced global reduction in nucleosome occupancy, which is potentially mediated by LHP1 in an H3K27me3-dependent manner.
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Proteínas de Arabidopsis , Arabidopsis , Histonas/genética , Histonas/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Epigénesis Genética , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Regulación de la Expresión Génica de las Plantas/genéticaRESUMEN
Citrus is one of the most important fruit crop genera in the world, but many Citrus species are vulnerable to cold stress. Ichang papeda (Citrus ichangensis), a cold-hardy citrus species, holds great potential for identifying valuable metabolites that are critical for cold tolerance in Citrus. However, the metabolic changes and underlying mechanisms that regulate Ichang papeda cold tolerance remain largely unknown. In this study, we compared the metabolomes and transcriptomes of Ichang papeda and HB pummelo (Citrus grandis 'Hirado Buntan', a cold-sensitive species) to explore the critical metabolites and genes responsible for cold tolerance. Metabolomic analyses led to the identification of common and genotype-specific metabolites, consistent with transcriptomic alterations. Compared to HB pummelo under cold stress, Ichang papeda accumulated more sugars, flavonoids, and unsaturated fatty acids, which are well-characterized metabolites involved in stress responses. Interestingly, sphingosine and chlorogenic acid substantially accumulated only in Ichang papeda. Knockdown of CiSPT (C. ichangensis serine palmitoyltransferase) and CiHCT2 (C. ichangensis hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase2), two genes involved in sphingosine and chlorogenic acid biosynthesis, dramatically decreased endogenous sphingosine and chlorogenic acid levels, respectively. This reduction in sphingosine and chlorogenic acid notably compromised the cold tolerance of Ichang papeda, whereas exogenous application of these metabolites increased plant cold tolerance. Taken together, our findings indicate that greater accumulation of a spectrum of metabolites, particularly sphingosine and chlorogenic acid, promotes cold tolerance in cold-tolerant citrus species. These findings broaden our understanding of plant metabolic alterations in response to cold stress and provide valuable targets that can be manipulated to improve Citrus cold tolerance.
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In plant cells, changes in fluidity of the plasma membrane may serve as the primary sensor of cold stress; however, the precise mechanism and how the cell transduces and fine-tunes cold signals remain elusive. Here we show that the cold-activated plasma membrane protein cold-responsive protein kinase 1 (CRPK1) phosphorylates 14-3-3 proteins. The phosphorylated 14-3-3 proteins shuttle from the cytosol to the nucleus, where they interact with and destabilize the key cold-responsive C-repeat-binding factor (CBF) proteins. Consistent with this, the crpk1 and 14-3-3κλ mutants show enhanced freezing tolerance, and transgenic plants overexpressing 14-3-3λ show reduced freezing tolerance. Further study shows that CRPK1 is essential for the nuclear translocation of 14-3-3 proteins and for 14-3-3 function in freezing tolerance. Thus, our study reveals that the CRPK1-14-3-3 module transduces the cold signal from the plasma membrane to the nucleus to modulate CBF stability, which ensures a faithfully adjusted response to cold stress of plants.
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Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Membrana Celular/enzimología , Núcleo Celular/enzimología , Frío , Respuesta al Choque por Frío , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Sensación Térmica , Factores de Transcripción/metabolismo , Proteínas 14-3-3/genética , Transporte Activo de Núcleo Celular , Adaptación Fisiológica , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Activación Enzimática , Genotipo , Fluidez de la Membrana , Mutación , Fenotipo , Fosforilación , Plantas Modificadas Genéticamente , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Estabilidad Proteica , Proteolisis , Factores de Tiempo , Factores de Transcripción/genéticaRESUMEN
Cassava, a crucial tropical crop, faces challenges from cold stress, necessitating an exploration of its molecular response. Here, we investigated the role of DNA methylation in moderating the response to moderate cold stress (10 °C) in cassava. Using whole-genome bisulfite sequencing, we examined DNA methylation patterns in leaf blades and petioles under control conditions, 5 h, and 48 h of cold stress. Tissue-specific responses were observed, with leaf blades exhibiting subtle changes, while petioles displayed a pronounced decrease in methylation levels under cold stress. We identified cold stress-induced differentially methylated regions (DMRs) that demonstrated both tissue and treatment specificity. Importantly, these DMRs were enriched in genes with altered expression, implying functional relevance. The cold-response transcription factor ERF105 associated with DMRs emerged as a significant and conserved regulator across tissues and treatments. Furthermore, we investigated DNA methylation dynamics in transposable elements, emphasizing the sensitivity of MITEs with bHLH binding motifs to cold stress. These findings provide insights into the epigenetic regulation of response to cold stress in cassava, contributing to an understanding of the molecular mechanisms underlying stress adaptation in this tropical plant.
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Respuesta al Choque por Frío , Metilación de ADN , Regulación de la Expresión Génica de las Plantas , Manihot , Proteínas de Plantas , Manihot/genética , Manihot/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Epigénesis Genética , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Elementos Transponibles de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2 O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response.
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Frío , Solanum tuberosum , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/metabolismo , Metabolismo de los Hidratos de Carbono , Hexosas/metabolismo , Sacarosa/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Tubérculos de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Cold stress poses a significant threat to the quality and productivity of lychee (Litchi chinensis Sonn.). While previous research has extensively explored the genomic and transcriptomic responses to cold stress in lychee, the translatome has not been thoroughly investigated. This study delves into the translatomic landscape of the 'Xiangjinfeng' cultivar under both control and low-temperature conditions using RNA sequencing and ribosome profiling. We uncovered a significant divergence between the transcriptomic and translatomic responses to cold exposure. Additionally, bioinformatics analyses underscored the crucial role of codon occupancy in lychee's cold tolerance mechanisms. Our findings reveal that the modulation of translation via codon occupancy is a vital strategy to abiotic stress. Specifically, the study identifies ribosome stalling, particularly at the E site AAU codon, as a key element of the translation machinery in lychee's response to cold stress. This work enhances our understanding of the molecular dynamics of lychee's reaction to cold stress and emphasizes the essential role of translational regulation in the plant's environmental adaptability.
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Codón , Respuesta al Choque por Frío , Biosíntesis de Proteínas , Respuesta al Choque por Frío/genética , Codón/genética , Ribosomas/metabolismo , Ribosomas/genética , Regulación de la Expresión Génica de las Plantas , TranscriptomaRESUMEN
BACKGROUND: The heat shock transcription factor (Hsf) is a crucial regulator of plant stress resistance, playing a key role in plant stress response, growth, and development regulation. RESULTS: In this study, we utilized bioinformatics tools to screen 25 VbHsf members, which were named VbHsf1-VbHsf25. We used bioinformatics methods to analyze the sequence structure, physicochemical properties, conserved motifs, phylogenetic evolution, chromosome localization, promoter cis-acting elements, collinearity, and gene expression of Hsf heat shock transcription factor family members under low-temperature stress. The results revealed that the majority of the Hsf genes contained motif1, motif2, and motif3, signifying that these three motifs were highly conserved in the Hsf protein sequence of Verbena bonariensis. Although there were some variations in motif deletion among the members, the domain remained highly conserved. The theoretical isoelectric point ranged from 4.17 to 9.71, with 21 members being unstable proteins and the remainder being stable proteins. Subcellular localization predictions indicated that all members were located in the nucleus. Phylogenetic analysis of the Hsf gene family in V. bonariensis and Arabidopsis thaliana revealed that the Hsf gene family of V. bonariensis could be categorized into three groups, with group A comprising 17 members and group C having at least two members. Among the 25 Hsf members, there were 1-3 exons located on seven chromosome fragments, which were unevenly distributed. Collinearity analysis demonstrated the presence of seven pairs of homologous genes in the VbHsf gene family. The Ka/Ks ratios were less than one, indicating that the VbHsf gene underwent purification selection pressure. Additionally, nine genes in V. bonariensis were found to have collinearity with A. thaliana. Promoter analysis revealed that the promoters of all VbHsf genes contained various types of cis-acting elements related to hormones and stress. Based on RNA-seq data, qRT-PCR analysis of six highly expressed genes was performed, and it was found that VbHsf5, VbHsf14, VbHsf17, VbHsf18, VbHsf20 and VbHsf21 genes were highly expressed at 12 h of low-temperature treatment, and the expression decreased after 24 h, among which VbHsf14 was up-regulated at 12 h of low-temperature by 70-fold. CONCLUSIONS: Our study may help reveal the important roles of Hsf in plant development and show insight for the further molecular breeding of V. bonariensis.
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Regulación de la Expresión Génica de las Plantas , Factores de Transcripción del Choque Térmico , Filogenia , Proteínas de Plantas , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Familia de Multigenes , Respuesta al Choque por Frío/genética , Frío , Estrés Fisiológico/genética , Regiones Promotoras Genéticas , Genoma de Planta , Arabidopsis/genética , Perfilación de la Expresión GénicaRESUMEN
BACKGROUND: The core regulation of the abscisic acid (ABA) signalling pathway comprises the multigenic families PYL, PP2C, and SnRK2. In this work, we conducted a genome-wide study of the components of these families in Cucurbita pepo. RESULTS: The bioinformatic analysis of the C. pepo genome resulted in the identification of 19 CpPYL, 102 CpPP2C and 10 CpSnRK2 genes. The investigation of gene structure and protein motifs allowed to define 4 PYL, 13 PP2C and 3 SnRK2 subfamilies. RNA-seq analysis was used to determine the expression of these gene families in different plant organs, as well as to detect their differential gene expression during germination, and in response to ABA and cold stress in leaves. The specific tissue expression of some gene members indicated the relevant role of some ABA signalling genes in plant development. Moreover, their differential expression under ABA treatment or cold stress revealed those ABA signalling genes that responded to ABA, and those that were up- or down-regulated in response to cold stress. A reduced number of genes responded to both treatments. Specific PYL-PP2C-SnRK2 genes that had potential roles in germination were also detected, including those regulated early during the imbibition phase, those regulated later during the embryo extension and radicle emergence phase, and those induced or repressed during the whole germination process. CONCLUSIONS: The outcomes of this research open new research lines for agriculture and for assessing gene function in future studies.
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Proteínas de Arabidopsis , Cucurbita , Ácido Abscísico/farmacología , Ácido Abscísico/metabolismo , Cucurbita/genética , Cucurbita/metabolismo , Estudio de Asociación del Genoma Completo , Plantas/genética , Respuesta al Choque por Frío , Regulación de la Expresión Génica de las Plantas , Proteínas de Arabidopsis/genéticaRESUMEN
Eukaryotic messenger RNAs (mRNAs) are often modified with methyl groups at the N6 position of adenosine (m6A), and these changes are interpreted by YTH domain-containing proteins to regulate the metabolism of m6A-modified mRNAs. Although alfalfa (Medicago sativa) is an established model organism for forage development, the understanding of YTH proteins in alfalfa is still limited. In the present investigation, 53 putative YTH genes, each encoding a YT521 domain-containing protein, were identified within the alfalfa genome. These genes were categorized into two subfamilies: YTHDF (49 members) and YTHDC (four members). Each subfamily demonstrates analogous motif distributions and domain architectures. Specifically, proteins encoded by MsYTHDF genes incorporate a single domain structure, while those corresponding to MsYTH5, 8, 12, 16 who are identified as members of the MsYTHDC subfamily, exhibit CCCH-type zinc finger repeats at their N-termini. It is also observed that the predicted aromatic cage pocket that binds the m6A residue of MsYTHDC consists of a sequence of two tryptophan residues and one tyrosine residue (WWY). Conversely, in MsYTHDF, the binding pocket comprises two highly conserved tryptophan residues and either one tryptophan residue (WWW) or tyrosine residue (WWY) in MsYTHDF.Through comparative analysis of qRT-PCR data, we observed distinct expression patterns in specific genes under abiotic stress, indicating their potential regulatory roles. Notably, five genes (MsYTH2, 14, 26, 27, 48) consistently exhibit upregulation, and two genes (MsYTH33, 35) are downregulated in response to both cold and salt stress. This suggests a common mechanism among these YTH proteins in response to various abiotic stressors in alfalfa. Further, integrating qRT-PCR with RNA-seq data revealed that MsYTH2, MsYTH14, and MsYTH16 are highly expressed in leaves at various development stages, underscoring their potential roles in regulating the growth of these plant parts. The obtained findings shed further light on the biological functions of MsYTH genes and may aid in the selection of suitable candidate genes for future genetic enhancement endeavors aimed at improving salt and cold tolerance in alfalfa.
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Medicago sativa , Triptófano , Medicago sativa/genética , Triptófano/genética , Triptófano/metabolismo , ARN Mensajero/metabolismo , Tirosina/metabolismo , Regulación de la Expresión Génica de las Plantas , FilogeniaRESUMEN
Establishing causal links between adaptive mutations and ecologically relevant phenotypes is key to understanding the process of adaptation, which is a central goal in evolutionary biology with applications for conservation, medicine, and agriculture. Yet despite recent progress, the number of identified causal adaptive mutations remains limited. Linking genetic variation to fitness-related effects is complicated by gene-by-gene and gene-by-environment interactions, among other processes. Transposable elements, which are often ignored in the quest for the genetic basis of adaptive evolution, are a genome-wide source of regulatory elements across organisms that can potentially result in adaptive phenotypes. In this work, we combine gene expression, in vivo reporter assays, CRISPR/Cas9 genome editing, and survival experiments to characterize in detail the molecular and phenotypic consequences of a natural Drosophila melanogaster transposable element insertion: the roo solo-LTR FBti0019985. This transposable element provides an alternative promoter to the transcription factor Lime, involved in cold- and immune-stress responses. We found that the effect of FBti0019985 on Lime expression depends on the interplay between the developmental stage and environmental condition. We further establish a causal link between the presence of FBti0019985 and increased survival to cold- and immune-stress. Our results exemplify how several developmental stages and environmental conditions need to be considered to characterize the molecular and functional effects of a genetic variant, and add to the growing body of evidence that transposable elements can induce complex mutations with ecologically relevant effects.
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Elementos Transponibles de ADN , Drosophila melanogaster , Animales , Drosophila melanogaster/genética , Óxidos , MutaciónRESUMEN
Cooling patients to sub-physiological temperatures is an integral part of modern medicine. We show that cold exposure induces temperature-specific changes to the higher-order chromatin and gene expression profiles of human cells. These changes are particularly dramatic at 18°C, a temperature synonymous with that experienced by patients undergoing controlled deep hypothermia during surgery. Cells exposed to 18°C exhibit largely nuclear-restricted transcriptome changes. These include the nuclear accumulation of mRNAs encoding components of the negative limbs of the core circadian clock, most notably REV-ERBα. This response is accompanied by compaction of higher-order chromatin and hindrance of mRNPs from engaging nuclear pores. Rewarming reverses chromatin compaction and releases the transcripts into the cytoplasm, triggering a pulse of negative limb gene proteins that reset the circadian clock. We show that cold-induced upregulation of REV-ERBα is sufficient to trigger this reset. Our findings uncover principles of the cellular cold response that must be considered for current and future applications involving therapeutic deep hypothermia.
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Núcleo Celular/metabolismo , Cromatina/metabolismo , Ritmo Circadiano/fisiología , Frío , ARN Mensajero/metabolismo , Línea Celular , Relojes Circadianos/genética , Relojes Circadianos/fisiología , Ritmo Circadiano/genética , Técnicas de Inactivación de Genes , Heterocromatina , Humanos , Hipotermia/cirugía , Activación Transcripcional , Transcriptoma , Regulación hacia ArribaRESUMEN
Although cells are frequently maintained at cold temperatures during experiments, the effects of cold stress on cell viability and subsequent cellular conditions remain elusive. In this study, we investigated the effects of cold stress on cancer cells under various culture conditions. We showed that cold stress induces ferroptosis, a form of cell death characterized by lipid peroxidation, in sensitive cancer cell lines. High cell density and serum starvation activate the Hippo pathway and suppress cold-induced cell death. Genetic deletion of Hippo pathway components enhances cold stress susceptibility. Furthermore, the cell attachment status influences the response to cold stress, with suspended cells showing greater resistance and faster recovery than attached cells. This study highlights the importance of cellular conditions and the Hippo pathway in the handling and storage of cancer cells at cold temperatures, thereby offering insights into experimental and clinical contexts.
RESUMEN
Accumulating evidence suggests that various cellular stresses interfere with the end processing of mRNA synthesis and lead to the production of abnormally long transcripts, known as readthrough transcripts (RTTs), which extend beyond the termination sites. Small mammalian hibernators repeatedly enter a state referred to as deep torpor (DT), where the metabolic rate, respiration rate, and core body temperature become extremely low, which produces various types of cellular stresses and therefore induces RTTs. However, the types of stresses and processes around the DT that cause RTTs are unclear. In the present study, we showed that RTTs are produced from different gene loci in the livers of Syrian hamsters under DT and summer-like conditions. Moreover, in vitro analysis using hamster primary hepatocytes revealed that DT-specific RTTs are induced by a slow decline in temperature, as seen in body temperature in the entrance phase of DT, but not by rapid cold treatment or hypoxia. In addition, it was observed that RTTs were not elongated under a significantly cold temperature (4 °C). These results indicate that DT-specific RTTs are produced during the entrance phase of torpor by a slow decrease in body temperature.
Asunto(s)
Hibernación , Animales , Cricetinae , Hibernación/genética , Temperatura , Temperatura Corporal , Mamíferos , Hígado , MesocricetusRESUMEN
The popular leafy vegetable lettuce (Lactuca sativa L.) is susceptible to cold stress during the growing season, which slows growth rate, causes leaf yellowing and necrosis, and reduced yield and quality. In this study, transcriptomic and metabolomic analyses of two cold-resistant lettuce cultivars (GWAS-W42 and F11) and two cold-sensitive lettuce cultivars (S13K079 and S15K058) were performed to identify the mechanisms involved in the cold response of lettuce. Overall, transcriptome analysis identified 605 differentially expressed genes (DEGs), including significant enrichment of genes involved in the flavonoid and flavonol (CHS, CHI, F3H, FLS, CYP75B1, HCT, etc.) biosynthetic pathways related to oxidation-reduction and catalytic activity. Untargeted metabolomic analysis identified fifteen flavonoid metabolites and 28 other metabolites potentially involved in the response to cold stress; genistein, quercitrin, quercetin derivatives, kaempferol derivatives, luteolin derivatives, apigenin and their derivatives accumulate at higher levels in cold-resistant cultivars. Moreover, MYBs, bHLHs, WRKYs and Dofs also play positive role in the low temperature response, which affected the expression of structural genes contributing to the variation of metabolites between the resistant and sensitive. These results provide valuable evidence that the metabolites and genes involved in the flavonoid biosynthetic pathway play important roles in the response of lettuce to cold stress.