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The freezing temperature greatly limits the growth, development and productivity of plants. The C-repeat/DRE binding factor (CBF) plays a major role in cold acclimation, enabling plants to increase their freezing tolerance. Notably, the INDUCER OF CBF EXPRESSION1 (ICE1) protein has garnered attention for its pivotal role in bolstering plants' resilience against freezing through transcriptional upregulation of DREB1A/CBF3. However, the research on the interaction between ICE1 and other transcription factors and its function in regulating cold stress tolerance is largely inadequate. In this study, we found that a R2R3 MYB transcription factor CDC5 interacts with ICE1 and regulates the expression of CBF3 by recruiting RNA polymerase II, overexpression of ICE1 can complements the freezing deficient phenotype of cdc5 mutant. CDC5 increases the expression of CBF3 in response to freezing. Furthermore, CDC5 influences the expression of CBF3 by altering the chromatin status through H3K4me3 and H3K27me3 modifications. Our work identified a novel component that regulates CBF3 transcription in both ICE1-dependent and ICE1-independent manner, improving the understanding of the freezing signal transduction in plants.
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Asparagus (Asparagus officinalis L.) is an important vegetable crop in southern Ontario, Canada, where winter air and soil temperatures below 0°C are common. Consequently, cultivars growing in this area must possess winterhardiness and freezing tolerance for survival. Asparagus acquires freezing tolerance in the fall through cold acclimation and loses freezing tolerance in the spring through deacclimation. To understand the molecular bases of these processes, transcriptomic analysis (RNA-Seq) was conducted on two cultivars, one adapted, 'Guelph Millennium' (GM), and one unadapted, 'UC157' (UC), to the winter conditions of southern Ontario. RNA extracted from bud and rhizome tissues, sampled on three dates during early spring and late fall, was subjected to sequencing. In the fall, the numbers of differentially expressed (DE) genes at the second and third harvests increased, relative to the first harvest, in dormant buds and rhizomes as freezing tolerance of cultivars increased, and the majority of DE genes were downregulated. In spring, freezing tolerance decreased as plants deacclimated and most genes DE at second and third harvests were upregulated in both cultivars. GM had lower LT50 (lethal temperature at which 50% of plants die) values and hence higher freezing tolerance than UC on specific sampling dates during both spring and fall, and expression patterns of specific genes were correlated with LT50 differences. Functional analysis revealed that these genes were involved in carbohydrate metabolic process, plant hormone signal transduction (auxin and gibberellin), proline metabolism, biosynthesis of secondary metabolites, circadian rhythm, and late embryogenesis abundant proteins and could be associated with cold acclimation and deacclimation processes. These findings will help researchers understand the molecular mechanisms of freezing tolerance in asparagus, leading to breeding and genetic strategies to improve the trait.
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The present study analyzed four cations (K+, Ca2+, Mg2+, Fe2+) in leachate from freeze-injured spinach (Spinacia oleracea L. 'Reflect') leaves exposed for four freezing-durations (FDs) (0.5, 3.0, 5.5, 10.5 h) at -4.8 °C. Comparison of electrolyte leakage from right-after-thaw with that after 6-d recovery revealed that injury at 0.5 or 3 h FDs was recoverable but irreversible at 5.5 or 10.5 h FDs. Data suggests leakage of K+, the most abundant cation in leachate, can serve as a proxy for total electrolyte-leakage in determining plant freezing-tolerance and an ionic marker discerning moderate vs. severe injury. Quantitative correspondence between Ca2+- and K+-leakage supports earlier proposition that leaked K+ induces loss of membrane-Ca2+, which, in turn, promotes further K+-leakage due to weakened membrane. Reduced/undetectable Fe2+ in leachate at longer FDs suggests activation of Fenton reaction converting soluble Fe2+ into insoluble Fe3+. Enhanced Mg2+-leakage at greater freeze-injury suggests structural/functional impairment of chlorophyll/chloroplast complex.
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BACKGROUND AND AIMS: As winter and spring temperatures continue to increase, the timing of flowering and leaf out is advancing in many seasonally cold regions. This advancement could put plants that flower early in the spring at risk of decreased reproduction in years when there are late freeze events. Unfortunately, relatively little is known about floral freezing tolerance in forest communities. In this study, we examined the impact of freezing temperatures on the flowers of woody plants in a region where there is rapid winter warming in North America. METHODS: We subjected the flowers of twenty-five woody species to a hard (-5ºC) and a light freeze (0ºC). We assessed tissue damage using electrolyte leakage. In a subset of species, we also examined the impact of a hard freeze on pollen tube growth. To determine if the vulnerability of flowers to freezing damage relates to flowering time and to examine the responsiveness of flowering time to spring temperature, we recorded the date of first flower for our study species for three years. KEY RESULTS AND CONCLUSIONS: Across species, we found that floral freezing tolerance was strongly tied to flowering time with the highest freezing tolerance occurring in plants that bloomed earlier in the year. We hypothesize that these early blooming species are unlikely to be impacted by a false spring. Instead, the most vulnerable species to a false spring should be those that bloom later in the season. The flowering time in these species is also more sensitive to temperature, putting them at a great risk of experiencing a false spring. Ultimately, floral damage in one year will not have a large impact on species fitness, but if false springs become more frequent, there could be long-term impacts on reproduction of vulnerable species.
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The INDUCER OF CBF EXPRESSION 1/C-REPEAT BINDING FACTOR (ICE1/CBF) pathway plays a crucial role in plant responses to cold stress, impacting growth and development. Here, we demonstrated that ATBS1-INTERACTING FACTOR 2 (AIF2), a non-DNA-binding basic helix-loop-helix transcription factor, positively regulates freezing tolerance through the ICE1/CBF-induced cold tolerance pathway in Arabidopsis. Cold stress transcriptionally upregulated AIF2 expression and induced AIF2 phosphorylation, thereby stabilizing the AIF2 protein during early stages of cold acclimation. The AIF2 loss-of-function mutant, aif2-1, exhibited heightened sensitivity to freezing before and after cold acclimation. In contrast, ectopic expression of AIF2, but not the C-terminal-deleted AIF2 variant, restored freezing tolerance. AIF2 enhanced ICE1 stability during cold acclimation and promoted the transcriptional expression of CBFs and downstream cold-responsive genes, ultimately enhancing plant tolerance to freezing stress. MITOGEN-ACTIVATED PROTEIN KINASES 3 and 6 (MPK3/6), known negative regulators of freezing tolerance, interacted with and phosphorylated AIF2, subjecting it to protein degradation. Furthermore, transient co-expression of MPK3/6 with AIF2 and ICE1 downregulated AIF2/ICE1-induced transactivation of CBF2 expression. AIF2 interacted preferentially with BIN2 and MPK3/6 during the early and later stages of cold acclimation, respectively, thereby differentially regulating AIF2 activity in a cold acclimation time-dependent manner. Moreover, AIF2 acted additively in a gain-of-function mutant of BRASSINAZOLE-RESISTANT 1 (BZR1; bzr1-1D) and a triple knockout mutant of BRASSINOSTEROID-INSENSITIVE 2 (BIN2) and its homologs (bin2bil1bil2) to induce CBFs-mediated freezing tolerance. This suggests that cold-induced AIF2 coordinates freezing tolerance along with BZR1 and BIN2, key positive and negative components, respectively, of brassinosteroid signaling pathways.
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Proanthocyanidins (PAs) are important metabolites that enhance freezing tolerance of plants. Actinidia arguta, especially freezing-tolerant germplasms, accumulate abundant PAs in dormant shoots and thereby enhance freezing tolerance, but the underlying mechanism is unknown. In this study, we used two A. arguta with contrasting cold-resistant phenotypes, KL and RB, to explore the mechanisms in response to cold tolerance. We determined that a leucoanthocyanidin reductase gene (AaLAR1) was more highly expressed in freezing-tolerant KL than in freezing-sensitive RB. Moreover, overexpressing AaLAR1 in kiwifruit promoted PAs biosynthesis and enhanced cold tolerance. The AaLAR1 promoters of various A. arguta germplasms differ due to the presence of a 60-bp deletion in cold-tolerant genotypes that forms a functional binding site for MYC-type transcription factor. Yeast one-hybrid and two-hybrid, dual-luciferase reporter, bimolecular fluorescence complementation and coimmunoprecipitation assays indicated that the AaMYC2a binds to the MYC-core cis-element in the AaLAR1 promoter with the assistance of AaMYB5a, thereby promoting PAs accumulation in the shoots of cold-tolerant kiwifruit. We conclude that the variation in the AaLAR1 promoter and the AaMYC2a-AaMYB5a-AaLAR1 module shape freezing tolerance in A. arguta. The identification of a key structural variation in the AaLAR1 promoter offers a new target for resistance breeding of kiwifruit.
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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.
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Elevation gradients provide powerful study systems for examining the influence of environmental filters in shaping species assemblages. High-mountain habitats host specific high-elevation assemblages, often comprising specialist species adapted to endure pronounced abiotic stress, while such harsh conditions prevent lowland species from colonizing or establishing. While thermal tolerance may drive the altitudinal segregation of ectotherms, its role in structuring aquatic insect communities remains poorly explored. This study investigates the role of thermal physiology in shaping the current distribution of high-mountain diving beetles from the Sierra Nevada Iberian mountain range and closely related lowland species. Cold tolerance of five species from each altitudinal zone was measured estimating the supercooling point (SCP), lower lethal temperature (LLT) and tolerance to ice enclosure, while heat tolerance was assessed from the heat coma temperature (HCT). Alpine species exhibited wider fundamental thermal niches than lowland species, likely associated with the broader range of climatic conditions in high-mountain areas. Cold tolerance did not seem to prevent lowland species from colonizing higher elevations, as most studied species were moderately freeze-tolerant. Therefore, fundamental thermal niches seem not to fully explain species segregation along elevation gradients, suggesting that other thermal tolerance traits, environmental factors, and biotic interactions may also play important roles.
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Altitud , Termotolerancia , Animales , Escarabajos/fisiología , Ecosistema , Aclimatación , FríoRESUMEN
Freezing stress can seriously affect plant growth and development, but the mechanisms of these effects and plant responses to freezing stress require further exploration. Here, we identified a NAM, ATAF1/2, and CUC2 (NAC)-family transcription factor (TF), NAC056, that can promote freezing tolerance in Arabidopsis. NAC056 mRNA levels are strongly induced by freezing stress in roots, and the nac056 mutant exhibits compromised freezing tolerance. NAC056 acts positively in response to freezing by directly promoting key C-repeat-binding factor (CBF) pathway genes. Interestingly, we found that CBF1 regulates nitrate assimilation by regulating the nitrate reductase gene NIA1 in plants; therefore, NAC056-CBF1-NIA1 form a regulatory module for the assimilation of nitrate and the growth of roots under freezing stress. In addition, 35S::NAC056 transgenic plants show enhanced freezing tolerance, which is partially reversed in the cbfs triple mutant. Thus, NAC056 confers freezing tolerance through the CBF pathway, mediating plant responses to balance growth and freezing stress tolerance.
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Proteínas de Arabidopsis , Arabidopsis , Congelación , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nitrato-Reductasa/genética , Nitrato-Reductasa/metabolismo , Plantas Modificadas Genéticamente/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Overwintering plants survive subzero temperatures by cold acclimation (CA), wherein they acquire freezing tolerance through short-term exposure to low temperatures above 0°C. The freezing tolerance of CA plants increases when they are subsequently exposed to mild subzero temperatures, a phenomenon known as second-phase cold hardening (2PH). Here, we explored the molecular mechanism and physiological conditions of 2PH. The results show that, compared with supercooling, a freezing treatment during 2PH after CA enhanced the freezing tolerance of Arabidopsis. This required CA as a pretreatment, and was designated as second-phase freezing acclimation (2PFA). Light increased the effect of 2PFA to enhance freezing tolerance after CA. C-repeat binding factor and cold-regulated genes were downregulated by light during the 2PFA treatment, a different transcription profile from that during CA. The freezing tolerance of 2PFA plants was decreased by the presence of the photosynthetic electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea during the 2PFA treatment. Compared with wild-type plants, phototropin1,2 and phyb mutants showed lower freezing tolerance after 2PFA treatment. These results show that exposure to freezing after CA increases freezing tolerance as a secondary process, and that freezing under light conditions further increases freezing tolerance via pathways involving photoreceptors and photosynthetic electron transfer.
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Aclimatación , Proteínas de Arabidopsis , Arabidopsis , Congelación , Regulación de la Expresión Génica de las Plantas , Luz , Arabidopsis/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fitocromo B/metabolismo , Fitocromo B/genética , Mutación , FríoRESUMEN
Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas Intrínsecamente Desordenadas , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/genética , Congelación , Modelos Moleculares , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
Amphibian diversity is most prominent in the warm and humid tropical and subtropical regions across the globe. Nonetheless, amphibians also inhabit high-altitude tropical mountains and regions at medium and high latitudes, exposing them to subzero temperatures and requiring behavioural or physiological adaptations to endure freezing events. While freeze tolerance has been predominantly reported in high-latitude zones where species endure prolonged freezing (several weeks or months), less is known about mid-latitudes amphibians exposed to occasional subzero temperatures. In this study, we employed a controlled ecological protocol, subjecting three frog species from the Iberian Peninsula (Rana parvipalmata, Epidalea calamita, and Pelobates cultripes) to a 2-h exposure to temperatures of -2 °C to investigate the accumulation of urea and glucose as physiological mechanisms associated with survival at freezing temperatures. Our results revealed a moderate response in the production of cryoprotectant metabolites under experimental freezing conditions, particularly urea, with notable findings in R. parvipalmata and E. calamita and no response in P. cultripes. However, no significant alterations in glucose concentrations were observed in any of the studied frog species. This relatively weak freezing tolerance response differs from the strong response exhibited by amphibians inhabiting high latitudes and enduring prolonged freezing conditions, suggesting potential reliance on behavioural adaptations to cope with occasional freezing episodes.
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Anuros , Congelación , Glucosa , Urea , Animales , Anuros/fisiología , Anuros/metabolismo , Urea/metabolismo , Glucosa/metabolismo , Aclimatación , Ranidae/fisiología , ClimaRESUMEN
Freezing temperature during overwintering often kills plants; plants have thus, developed a defense mechanism called 'cold acclimation', in which a number of genes are involved in increasing cell protection and gene expression. Mitogen-activated protein kinase (MAPK) controls proteins' activities by phosphorylation and is involved in numerous metabolic pathways. In this study, we identified the protein interaction between TaMAPK3 and the proteins in the cold response pathway, ICE41, ICE87, and CBFIVd-D9. The subcellular localization and bimolecular fluorescence complement (BiFC) assays revealed that these proteins interact in the nucleus or in the plasma membrane. Furthermore, MAPK3-mediated phosphorylation of ICE41, ICE87, and CBFIVd-D9 was verified using an in vitro phosphorylation assay. TaMAPK3-overexpressing transgenic Brachypodium showed a lower survival rate upon freezing stress and lower proline content during cold acclimation, compared to wild-type plants. Furthermore, cold response gene expression analysis revealed that the expression of these genes was suppressed in the transgenic lines under cold treatment. It was further elucidated that MAPK3 mediates the degradation of ICE and CBF proteins, which implies the negative impact of MAPK3 on the freezing tolerance of plants. This study will help to elucidate the molecular mechanisms of cold tolerance and the activity of MAPK3 in wheat.
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Proteínas de Arabidopsis , Triticum , Congelación , Triticum/genética , Triticum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Frío , Fosforilación , Regulación de la Expresión Génica de las Plantas , Aclimatación/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
Subzero temperatures are often lethal to plants. Many temperate herbaceous plants have a cold acclimation mechanism that allows them to sense a drop in temperature and prepare for freezing stress through accumulation of soluble sugars and cryoprotective proteins. As ice formation primarily occurs in the apoplast (the cell wall space), cell wall functional properties are important for plant freezing tolerance. Although previous studies have shown that the amounts of constituent sugars of the cell wall, in particular those of pectic polysaccharides, are altered by cold acclimation, the significance of this change during cold acclimation has not been clarified. We found that ß-1,4-galactan, which forms neutral side chains of the acidic pectic rhamnogalacturonan-I, accumulates in the cell walls of Arabidopsis and various freezing-tolerant vegetables during cold acclimation. The gals1 gals2 gals3 triple mutant, which has reduced ß-1,4-galactan in the cell wall, exhibited impaired freezing tolerance compared with wild-type Arabidopsis during initial stages of cold acclimation. Expression of genes involved in the galactan biosynthesis pathway, such as galactan synthases and UDP-glucose 4-epimerases, was induced during cold acclimation in Arabidopsis, explaining the galactan accumulation. Cold acclimation resulted in a decrease in extensibility and an increase in rigidity of the cell wall in the wild type, whereas these changes were not observed in the gals1 gals2 gals3 triple mutant. These results indicate that the accumulation of pectic ß-1,4-galactan contributes to acquired freezing tolerance by cold acclimation, likely via changes in cell wall mechanical properties.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Congelación , Proteínas de Arabidopsis/metabolismo , Plantas/metabolismo , Pared Celular/metabolismo , Galactanos/metabolismo , Aclimatación/genética , Azúcares/metabolismo , Frío , Regulación de la Expresión Génica de las PlantasRESUMEN
This study explores and compares the limits for photosynthesis in subzero temperatures of six Antarctic lichens: Sphaerophorus globosus, Caloplaca regalis, Umbilicaria antarctica, Pseudephebe minuscula, Parmelia saxatilis and Lecania brialmontii combining linear cooling and chlorophyll fluorescence methods. The results revealed triphasic S-curves in the temperature response of the maximum quantum yield (FV/FM) and effective quantum yield of photosystem II (ΦPSII) for all species. All investigated species showed a high level of cryoresistance with critical temperatures (Tc) below -20 °C. However, record low Tc temperatures have been discovered for L. brialmotii (-54 °C for FV/FM and -40 °C for ΦPSII) and C. regalis (-52 °C for FV/FM and -38 °C for ΦPSII). Additionally, the yield differentials (FV/FM - ΦPSII) in functions of temperature revealed one or two peaks, with the larger one occurring for temperatures below -20 °C for the above-mentioned species. Finally, Kautsky kinetics were measured and compared at different temperatures (20 °C, 10 °C, 0 °C and -10 °C and then -10 °C after 1 h of incubation). This research serves as a foundation for further developing investigations into the biophysical mechanisms by which photosynthesis is carried out at subzero temperatures.
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Clorofila , Líquenes , Congelación , Temperatura , Líquenes/fisiología , Complejo de Proteína del Fotosistema II , Fluorescencia , FotosíntesisRESUMEN
Cold stress is a serious threat to global crop production and food security, but plant cold resistance is accompanied by reductions in growth and yield. In this study, we determined that the novel gene BcGSTF10 in non-heading Chinese cabbage [NHCC; Brassica campestris (syn. Brassica rapa) ssp. chinensis] is implicated in resistance to cold stress. Biochemical and genetic analyses demonstrated that BcGSTF10 interacts with BcICE1 to induce C-REPEAT BINDING FACTOR (CBF) genes that enhance freezing tolerance in NHCC and in Arabidopsis. However, BcCBF2 represses BcGSTF10 and the latter promotes growth in NHCC and Arabidopsis. This dual function of BcGSTF10 indicates its pivotal role in balancing cold stress and growth, and this important understanding has the potential to inform the future development of strategies to breed crops that are both climate-resilient and high-yielding.
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Arabidopsis , Brassica , Respuesta al Choque por Frío , Glutatión Transferasa/genética , Fitomejoramiento , Brassica/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
Many plants cope with cold stress by developing acquired freezing tolerance (AFT) through cold acclimation (CA), and some species have strong basal freezing tolerance (BFT) independent of CA. Although CA has been extensively studied, its potential in agricultural applications is still unclear. Here, carbohydrate metabolism and transcriptome in AFT plant Arabidopsis and BFT plant Chorispora bungeana were compared with each other. The results showed that, although both species were able to accumulate soluble sugars during CA, leaf starch accumulation in the daytime was almost blocked in Arabidopsis while it was greatly enhanced in C. bungeana, revealing that Arabidopsis experienced carbohydrate shortage during CA. Transcriptome and pathway enrichment analysis found that genes for photosynthesis antenna proteins were generally repressed by cold stress in both species. However, cold-up-regulated genes were enriched in protein translation in Arabidopsis, whilst they were enriched in carotenoid biosynthesis, flavonoid biosynthesis, and beta-amylases in C. bungeana. Furthermore, weighted gene co-expression network analysis (WGCNA) showed that the inhibition of starch accumulation was associated with down-regulation of genes for photosynthesis antenna proteins and up-regulation of genes for protein translation, DNA repair, and proteasome in Arabidopsis but not in C. bungeana. Taken together, our results revealed that over-activation of common tolerant mechanisms resulted in insufficient carbohydrate supplies in Arabidopsis during CA, and photoprotective mechanisms played important roles in cold adaptation of C. bungeana. These findings uncovered the drawback of CA in improving freezing tolerance and highlighted photoprotection as a possible solution for agricultural applications.
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The purpose of this study was to identify proteins associated with differences in the freezing tolerance of sheep sperm and to analyze their functions. Qualified fresh semen from four breeds of rams, the Australian White, white-head Dorper, Black-head Dorper, and Hu sheep breeds, were selected for cryopreservation. The sperm freezing tolerance was investigated by evaluation of the overall vitality, progressive vitality, and rapidly advance vitality of the sperm. A differential model of sperm freezing tolerance was constructed for sheep breeds showing significant differences. Differentially expressed proteins associated with sperm freezing tolerance were identified using iTRAQ and the protein functions were analyzed. It was found that sperm freezing tolerance was best in Hu sheep and worst in white-head Dorper sheep. These two breeds were used for the construction of a model based on differences in freezing tolerance and the identification of sperm proteins expressed differentially before freezing and after thawing. A total of 128 differentially expressed proteins (88 up-regulated and 40 down-regulated) were identified before freezing and after thawing in Hu sheep sperm (fresh/frozen Hu sheep sperm referred to as HL vs. HF), while 219 differentially expressed proteins (106 up-regulated and 113 down-regulated) were identified in white-head Dorper sheep (fresh/frozen white-head Dorper sheep sperm referred to as WL vs. WF). A comparison of these differentially expressed proteins showed that 57 proteins overlapped between the two breeds while 71 were only expressed in Hu sheep and 162 were only expressed in white-head Dorper sheep. Functional annotation and enrichment analyses of differentially expressed proteins down-regulated in Hu sheep involved in phosphorylation of phosphatidylinositol phosphate kinases, regulation of GTPase activity and glycolysis/gluconeogenesis signaling pathway. Up-regulated proteins of Hu sheep participated in oxidoreductase activity and oxidative phosphorylation process of sperm freezing. Furthermore, down-regulated in white-head Dorper sheep involved in the metabolic regulation of carbohydrate and nuclear sugar metabolism. Up-regulated proteins of white-head Dorper sheep involved in the ferroptosis and oxidative phosphorylation pathways. Collectively, These proteins were found to participate mainly in oxidative phosphorylation as well as phosphorylation and metabolic processes in the mitochondria to affect the freezing tolerance of sheep sperm.
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Semen , Espermatozoides , Masculino , Ovinos , Animales , Congelación , Australia , Oveja Doméstica , Fosforilación OxidativaRESUMEN
Cold acclimation is a crucial biological process that enables conifers to overwinter safely. The late embryogenesis abundant (LEA) protein family plays a pivotal role in enhancing freezing tolerance during this process. Despite its importance, the identification, molecular functions and regulatory networks of the LEA protein family have not been extensively studied in conifers or gymnosperms. Pinus tabuliformis, a conifer with high ecological and economic values and with high-quality genome sequence, is an ideal candidate for such studies. Here, a total of 104 LEA genes were identified from P. tabuliformis, and we renamed them according to their subfamily group: PtLEA1-PtLEA92 (group LEA1-LEA6), PtSMP1-PtSMP6 (group seed maturation protein) and PtDHN1-PtDHN6 (group Dehydrin). While the sequence structure of P. tabuliformis LEA genes are conserved, their physicochemical properties exhibit unique characteristics within different subfamily groupings. Notably, the abundance of low-temperature responsive elements in PtLEA genes was observed. Using annual rhythm and temperature gradient transcriptome data, PtLEA22 was identified as a key gene that responds to low-temperature induction while conforming to the annual cycle of cold acclimation. Overexpression of PtLEA22 enhanced Arabidopsis freezing tolerance. Furthermore, several transcription factors potentially co-expressed with PtLEA22 were validated using yeast one-hybrid and dual-luciferase assays, revealing that PtDREB1 could directly bind PtLEA22 promoter to positively regulate its expression. These findings reveal the genome-wide characterization of P. tabuliformis LEA genes and their importance in the cold acclimation, while providing a theoretical basis for studying the molecular mechanisms of cold acclimation in conifers.
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Arabidopsis , Pinus , Pinus/genética , Pinus/metabolismo , Proteínas de Plantas/metabolismo , Frío , Arabidopsis/genética , Aclimatación/genética , Desarrollo Embrionario/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
Cold stress is a major abiotic stress that adversely affects plant growth and crop productivity. The C-REPEAT BINDING FACTOR/DRE BINDING FACTOR 1 (CBF/DREB1) transcriptional regulatory cascade plays a key role in regulating cold acclimation and freezing tolerance in Arabidopsis (Arabidopsis thaliana). Here, we show that max (more axillary growth) mutants deficient in strigolactone biosynthesis and signaling display hypersensitivity to freezing stress. Exogenous application of GR245DS , a strigolactone analog, enhances freezing tolerance in wild-type plants and strigolactone-deficient mutants and promotes the cold-induced expression of CBF genes. Biochemical analysis showed that the transcription factor WRKY41 serves as a substrate for the F-box E3 ligase MAX2. WRKY41 directly binds to the W-box in the promoters of CBF genes and represses their expression, negatively regulating cold acclimation and freezing tolerance. MAX2 ubiquitinates WRKY41, thus marking it for cold-induced degradation and thereby alleviating the repression of CBF expression. In addition, SL-mediated degradation of SMXLs also contributes to enhanced plant freezing tolerance by promoting anthocyanin biosynthesis. Taken together, our study reveals the molecular mechanism underlying strigolactones promote the cold stress response in Arabidopsis.