Plant Freezing Tolerance Research Articles

Sulfenylation of ENOLASE2 facilitates H2O2-conferred freezing tolerance in Arabidopsis.

H2O2 affects the expression of genes that are involved in plant responses to diverse environmental stresses; however, the underlying mechanisms remain elusive. Here, we demonstrate that H2O2 enhances plant freezing tolerance through its effect on a protein product of lowexpression of osmotically responsive genes2 (LOS2). LOS2 is translated into a major product, cytosolic enolase2 (ENO2), and sometimes an alternative product, the transcription repressor c-Myc-binding protein (MBP-1). ENO2, but not MBP-1, promotes cold tolerance by binding the promoter of C-repeat/DRE binding factor1 (CBF1), a central transcription factor in plant cold signaling, thus activating its expression. Overexpression of CBF1 restores freezing sensitivity of a LOS2 loss-of-function mutant. Furthermore, cold-induced H2O2 increases nuclear import and transcriptional binding activity of ENO2 by sulfenylating cysteine 408 and thereby promotes its oligomerization. Collectively, our results illustrate how H2O2 activates plant cold responses by sulfenylating ENO2 and promoting its oligomerization, leading to enhanced nuclear translocation and transcriptional activation of CBF1.

Light Quality Modulates Plant Cold Response and Freezing Tolerance.

The cold acclimation process is regulated by many factors like ambient temperature, day length, light intensity, or hormonal status. Experiments with plants grown under different light quality conditions indicate that the plant response to cold is also a light-quality-dependent process. Here, the role of light quality in the cold response was studied in 1-month-old Arabidopsis thaliana (Col-0) plants exposed for 1 week to 4°C at short-day conditions under white (100 and 20 μmol m−2s−1), blue, or red (20 μmol m−2s−1) light conditions. An upregulated expression of CBF1, inhibition of photosynthesis, and an increase in membrane damage showed that blue light enhanced the effect of low temperature. Interestingly, cold-treated plants under blue and red light showed only limited freezing tolerance compared to white light cold-treated plants. Next, the specificity of the light quality signal in cold response was evaluated in Arabidopsis accessions originating from different and contrasting latitudes. In all but one Arabidopsis accession, blue light increased the effect of cold on photosynthetic parameters and electrolyte leakage. This effect was not found for Ws-0, which lacks functional CRY2 protein, indicating its role in the cold response. Proteomics data confirmed significant differences between red and blue light-treated plants at low temperatures and showed that the cold response is highly accession-specific. In general, blue light increased mainly the cold-stress-related proteins and red light-induced higher expression of chloroplast-related proteins, which correlated with higher photosynthetic parameters in red light cold-treated plants. Altogether, our data suggest that light modulates two distinct mechanisms during the cold treatment - red light-driven cell function maintaining program and blue light-activated specific cold response. The importance of mutual complementarity of these mechanisms was demonstrated by significantly higher freezing tolerance of cold-treated plants under white light.

Overexpression of galactinol synthase 1 from Solanum commersonii (ScGolS1) confers freezing tolerance in transgenic potato

Potato (Solanum tuberosum L.) is the fourth largest food crop in the world. Low temperatures cause serious damage to potato plants every year, and freezing tolerance has become a hot spot in potato research. Galactinol synthase (GolS) is a key enzyme in the synthesis of raffinose family oligosaccharides (RFOs), and plays an important role in the response of plants to abiotic stress. In this study, the ScGolS1 gene from Solanum commersonii was cloned and introduced into the S. tuberosum cultivars ‘Atlantic’ and ‘Desiree’ via Agrobacterium-mediated transformation. Phenotyping assays showed that overexpression of ScGolS1 could significantly improve freezing tolerance in transgenic potato plants. Further physiological and biochemical experiments showed that the transgenic lines had lower relative conductivity, malondialdehyde content, and 3,3′-diaminobenzidine staining and a higher plant survival rate compared with wild type (WT) under cold stress. Moreover, the C-repeat binding factors (CBF1, CBF2 and CBF3), the downstream cold-responsive genes COR413 and COR47, and the ethylene-responsive factor (ERF) transcription factor genes ERF3, ERF4 and ERF6, which function in the ethylene signaling pathway, were all induced by freezing treatment and expressed at higher levels in the ScGolS1 overexpression lines compared with WT. Besides, the expression of some genes such as MIPS, STS and RS from the RFO metabolic pathway was up-regulated under cold stress, resulting in changes in the content of some soluble sugars. This indicated that ScGolS1 overexpression altered the sugar composition and enhanced freezing tolerance in transgenic potato by inducing the ethylene and CBF signaling pathways. These results provided theoretical support and genetic resources for freezing tolerance breeding in potato.

Effects of Biodegradable Liquid Film on Cabernet Sauvignon (V. vinifera L.) Grape Quality

Biodegradable liquid film (BLF) improves soil structure and increases plant freezing tolerance after spraying on the surface of soil and plant. In this study, the effects of BLF on grape composition and volatile compounds in Cabernet sauvignon (Vitis vinifera L.) grapes were determined by spraying BLF during the dormant periods over three years. The aim of the study was to evaluate the potential impact of BLF as an overwintering protection measure on grape fruit quality. In 2020 and 2021, BLF spraying increased reducing sugar content and 100-berry weight, decreased titratable acid content, and improved the maturity factor. Compared with the vines not sprayed with BLF, the content of total phenols and total anthocyanins in grape skins showed an increase over the three-year period, with the largest increases of 31.92% and 48.38%, respectively, and the content of total tannins and total flavan-3-ols increased in 2020 and 2021. BLF treatment also increased the total phenolic content in seeds for all three years, reaching a significant level in 2019, 16.38% higher than control treatment (CK). HPLC analysis showed that BLF treatment affected the content and composition of monomeric anthocyanins in grape skins, especially in 2021, BLF treatment significantly increased the content of nine monomeric anthocyanins, and the proportion of acetylated and coumaroylated anthocyanins. However, GC-MS analysis indicated that BLF had little effect on volatile compounds. These results suggest that BLF can be used as an overwintering protection measure in cold regions to promote the accumulation of sugars and polyphenolics, thereby improving overall grape quality.

Specific Changes in Arabidopsis thaliana Rosette Lipids during Freezing Can Be Associated with Freezing Tolerance.

While the roles of a few specific lipids in plant freezing tolerance are understood, the effect of many plant lipids remains to be determined. Acclimation of plants to non-freezing cold before exposure to freezing temperatures improves the outcome of plants, compared to plants exposed to freezing without acclimation. Arabidopsis thaliana plants were subjected to one of three treatments: (1) “control”, i.e., growth at 21 °C, (2) “non-acclimated”, i.e., 3 days at 21 °C, 2 h at −8 °C, and 24 h recovery at 21 °C, and (3) “acclimated”, i.e., 3 days at 4 °C, 2 h at −8 °C, and 24 h recovery at 21 °C. Plants were harvested at seven time points during the treatments, and lipid levels were measured by direct-infusion electrospray ionization tandem mass spectrometry. Ion leakage was measured at the same time points. To examine the function of lipid species in relation to freezing tolerance, the lipid levels in plants immediately following the freezing treatment were correlated with the outcome, i.e., ion leakage 24-h post-freezing. Based on the correlations, hypotheses about the functions of specific lipids were generated. Additionally, analysis of the lipid levels in plants with mutations in genes encoding patatin-like phospholipases, lipoxygenases, and 12-oxophytodienoic acid reductase 3 (opr3), under the same treatments as the wild-type plants, identified only the opr3-2 mutant as having major lipid compositional differences compared to wild-type plants.

Gold nanoparticles as adaptogens increazing the freezing tolerance of wheat seedlings.

The intensive development of nanotechnology led to the widespread application of various nanoparticles and nanomaterials. As a result, nanoparticles enter the environment and accumulate in ecosystems and living organisms. The consequences of possible impact of nanoparticles on living organisms are not obvious. Experimental data indicate that nanoparticles have both toxic and stimulating effects on organisms. In this study, we demonstrated for the first time that gold nanoparticles can act as adaptogens increasing plant freezing tolerance. Priming winter wheat (Triticum aestivum L., var. Moskovskaya 39, Poaceae) seeds for 1day in solutions of gold nanoparticles (15-nm diameter, concentrations of 5, 10, 20, and 50µg/ml) led to an increase in freezing tolerance of 7-day-old wheat seedlings. A relationship between an increase in wheat freezing tolerance and changes in some important indicators for its formation-growth intensity, the activity of the photosynthetic apparatus and oxidative processes, and the accumulation of soluble sugars in seedlings-was established. Assumptions on possible mechanisms of gold nanoparticles effects on plant freezing tolerance are discussed.

Genome-Wide Identification and Analysis of bZIP Gene Family and Resistance of TaABI5 (TabZIP96) under Freezing Stress in Wheat (Triticum aestivum).

The basic leucine zipper (bZIP) regulates plant growth and responds to stress as a key transcription factor of the Abscisic acid (ABA) signaling pathway. In this study, TabZIP genes were identified in wheat and the gene structure, physicochemical properties, cis-acting elements, and gene collinearity were analyzed. RNA-Seq and qRT-PCR analysis showed that ABA and abiotic stress induced most TabZIP genes expression. The ectopic expression of TaABI5 up-regulated the expression of several cold-responsive genes in Arabidopsis. Physiological indexes of seedlings of different lines under freezing stress showed that TaABI5 enhanced the freezing tolerance of plants. Subcellular localization showed that TaABI5 is localized in the nucleus. Furthermore, TaABI5 physically interacted with cold-resistant transcription factor TaICE1 in yeast two-hybrid system. In conclusion, this study identified and analyzed members of the TabZIP gene family in wheat. It proved for the first time that the gene TaABI5 affected the cold tolerance of transgenic plants and was convenient for us to understand the cold resistance molecular mechanism of TaABI5. These results will provide a new inspiration for further study on improving plant abiotic stress resistance.

SPL9 mediates freezing tolerance by directly regulating the expression of CBF2 in Arabidopsis thaliana

BackgroundFreezing stress inhibits plant development and causes significant damage to plants. Plants therefore have evolved a large amount of sophisticated mechanisms to counteract freezing stress by adjusting their growth and development correspondingly. Plant ontogenetic defense against drought, high salt, and heat stresses, has been extensively studied. However, whether the freezing tolerance is associated with ontogenetic development and how the freezing signals are delivered remain unclear.ResultsIn this study, we found that the freezing tolerance was increased with plant age at the vegetative stage. The expressions of microRNA156 (miR156) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9), playing roles in regulation of ontogenetic development, were induced by cold stress. Overexpression of SPL9 (rSPL9) promoted the expression of C-REPEAT BINDING FACTOR 2 (CBF2) and hereafter enhanced the freezing tolerance. Genetic analysis indicated that the effect of rSPL9 on freezing tolerance is partially restored by cbf2 mutant. Further analysis confirmed that SPL9 directly binds to the promoter of CBF2 to activate the expression of CBF2, and thereafter increased the freezing tolerance.ConclusionsTherefore, our study uncovers a new role of SPL9 in fine-tuning CBF2 expression and thus mediating freezing tolerance in plants, and implies a role of miR156-SPL pathway in balancing the vegetative development and freezing response in Arabidopsis.

Integration of light and temperature signaling pathways in plants.

As two of the most important environmental factors, light and temperature regulate almost all aspects of plant growth and development. Under natural conditions, light is accompanied by warm temperatures and darkness by cooler temperatures, suggesting that light and temperature are tightly associated signals for plants. Indeed, accumulating evidence shows that plants have evolved a wide range of mechanisms to simultaneously perceive and respond to dynamic changes in light and temperature. Notably, the photoreceptor phytochrome B (phyB) was recently shown to function as a thermosensor, thus reinforcing the notion that light and temperature signaling pathways are tightly associated in plants. In this review, we summarize and discuss the current understanding of the molecular mechanisms integrating light and temperature signaling pathways in plants, with the emphasis on recent progress in temperature sensing, light control of plant freezing tolerance, and thermomorphogenesis. We also discuss the questions that are crucial for a further understanding of the interactions between light and temperature signaling pathways in plants.

Genotype-dependent contribution of CBF transcription factors to long-term acclimation to high light and cool temperature.

When grown under cool temperature, winter annuals upregulate photosynthetic capacity as well as freezing tolerance. Here, the role of three cold‐induced C‐repeat‐binding factor (CBF1–3) transcription factors in photosynthetic upregulation and freezing tolerance was examined in two Arabidopsis thaliana ecotypes originating from Italy (IT) or Sweden (SW), and their corresponding CBF1–3‐deficient mutant lines it:cbf123 and sw:cbf123. Photosynthetic, morphological and freezing‐tolerance phenotypes, as well as gene expression profiles, were characterized in plants grown from the seedling stage under different combinations of light level and temperature. Under high light and cool (HLC) growth temperature, a greater role of CBF1–3 in IT versus SW was evident from both phenotypic and transcriptomic data, especially with respect to photosynthetic upregulation and freezing tolerance of whole plants. Overall, features of SW were consistent with a different approach to HLC acclimation than seen in IT, and an ability of SW to reach the new homeostasis through the involvement of transcriptional controls other than CBF1–3. These results provide tools and direction for further mechanistic analysis of the transcriptional control of approaches to cold acclimation suitable for either persistence through brief cold spells or for maximisation of productivity in environments with continuous low temperatures.

Investigating the Role of the Cell Wall in Plant Freezing Tolerance

Investigating the Role of the Cell Wall in Plant Freezing Tolerance

Identification of Glutathione Peroxidase Gene Family in Ricinus communis and Functional Characterization of RcGPX4 in Cold Tolerance.

Glutathione peroxidases (GPXs) protect cells against damage caused by reactive oxygen species (ROS) and play key roles in regulating many biological processes. Here, five GPXs were identified in the Ricinus communis genome. Phylogenetic analysis displayed that the GPXs were categorized into five groups. Conserved domain and gene structure analyses showed that the GPXs from different plant species harbored four highly similar motifs and conserved exon-intron arrangement patterns, indicating that their structure and function may have been conserved during evolution. Several abiotic stresses and hormone-responsive cis-acting elements existed in the promoters of the RcGPXs. The expression profiles indicated that the RcGPXs varied substantially, and some RcGPXs were coordinately regulated under abiotic stresses. Overexpression of RcGPX4 in Arabidopsis enhanced cold tolerance at seed germination but reduced freezing tolerance at seedlings. The expression of abscisic acid (ABA) signaling genes (AtABI4 and AtABI5), ABA catabolism genes (AtCYP707A1 and AtCYP707A2), gibberellin acid (GA) catabolism gene (AtGA2ox7), and cytokinin (CTK)-inducible gene (AtARR6) was regulated in the seeds of transgenic lines under cold stress. Overexpression of RcGPX4 can disturb the hydrogen peroxide (H2O2) homeostasis through the modulation of some antioxidant enzymes and compounds involved in the GSH-ascorbate cycle in transgenic plants. Additionally, RcGPX4 depended on the MAPK3-ICE1-C-repeat-binding factor (CBF)-COR signal transduction pathway and ABA-dependent pathway to negatively regulate the freezing tolerance of transgenic plants. This study provides valuable information for understanding the potential function of RcGPXs in regulating the abiotic stress responses of castor beans.

Redox-dependent structural switch and CBF activation confer freezing tolerance in plants.

The activities of cold-responsive C-repeat-binding transcription factors (CBFs) are tightly controlled as they not only induce cold tolerance but also regulate normal plant growth under temperate conditions1-4. Thioredoxin h2 (Trx-h2)-a cytosolic redox protein identified as an interacting partner of CBF1-is normally anchored to cytoplasmic endomembranes through myristoylation at the second glycine residue5,6. However, after exposure to cold conditions, the demyristoylated Trx-h2 is translocated to the nucleus, where it reduces the oxidized (inactive) CBF oligomers and monomers. The reduced (active) monomers activate cold-regulated gene expression. Thus, in contrast to the Arabidopsis trx-h2 (AT5G39950) null mutant, Trx-h2 overexpression lines are highly cold tolerant. Our findings reveal the mechanism by which cold-mediated redox changes induce the structural switching and functional activation of CBFs, therefore conferring plant cold tolerance.

The Halophyte Halostachys caspica AP2/ERF Transcription Factor HcTOE3 Positively Regulates Freezing Tolerance in Arabidopsis.

The APETALA2 (AP2) and ethylene-responsive element-binding factor (ERF) gene family is one of the largest plant-specific transcription factor gene families, which plays a critical role in plant development and evolution, as well as response to various stresses. The TARGET OF EAT3 (TOE3) gene is derived from Halostachys caspica and belongs to the AP2 subfamily with two AP2 DNA-binding domains. Currently, AP2 family mainly plays crucial roles in plant growth and evolution, yet there are few reports about the role of AP2 in abiotic stress tolerance. Here, we report HcTOE3, a new cold-regulated transcription factor gene, which has an important contribution to freezing tolerance. The main results showed that the expression of HcTOE3 in the H. caspica assimilating branches was strongly induced by different abiotic stresses, including high salinity, drought, and extreme temperature (heat, chilling, and freezing), as well as abscisic acid and methyl viologen treatments. Overexpressing HcTOE3 gene (OE) induced transgenic Arabidopsis plant tolerance to freezing stress. Under freezing treatment, the OE lines showed lower content of malondialdehyde and electrolyte leakage and less accumulation of reactive oxygen species compared with the wild type. However, the survival rates, antioxidant enzyme activities, and contents of osmotic adjustment substance proline were enhanced in transgenic plants. Additionally, the OE lines increased freezing tolerance by up-regulating the transcription level of cold responsive genes (CBF1, CBF2, COR15, COR47, KIN1, and RD29A) and abscisic acid signal transduction pathway genes (ABI1, ABI2, ABI5, and RAB18). Our results suggested that HcTOE3 positively regulated freezing stress and has a great potential as a candidate gene to improve plant freezing tolerance.

Overexpression of TaFBA-A10 from Winter Wheat Enhances Freezing Tolerance in Arabidopsis thaliana

Freezing stress is the principal abiotic stress that is not conducive to plant growth and yield. Fructose-1, 6-bisphosphate aldolase (FBA; EC 4.1.2.13) is a key metabolic enzyme, which plays an important role in Calvin cycle and glycolysis. In this study, we found that the expression of TaFBA10 from A genome in tillering nodes of strong cold resistance winter wheat cultivar Dongnongdongmai1 (Dn1) was significantly altered subjected to severe winter. In order to study the role of FBA on carbohydrate metabolism and response to freezing stress in plants, we constructed a plant overexpression vector of TaFBA-A10 and transferred it into Arabidopsis thaliana to obtain stable inherited T3 generation plants, which were treated with freezing temperatures (− 10 °C). We found that overexpressing TaFBA-A10 led to the increased activity of FBA, as well as, enhancing the expression of key enzymes in Calvin cycle and glycolysis including SBPase, FBPase, GADPH, HXK, PFK and PyK under freezing stress. Although the phenotype of TaFBA-A10 overexpressing lines was not prominent, the electrical conductivity and MDA content of TaFBA-A10 overexpressing lines were significantly lower than those of wild type (WT) and fba mutants under freezing stress. The reducing sugar content and ROS scavenging ability of TaFBA-A10 overexpressing lines were higher than those of WT and fba mutants under freezing stress. These results could be concluded that TaFBA-A10 could regulate Calvin cycle and glycolysis rate to enhance freezing tolerance of plants by increasing FBA activity.

Overexpression of the V-ATPase c subunit gene from Antarctic notothenioid fishes enhances freezing tolerance in transgenic Arabidopsis plants

Theoretical and experimental studies have demonstrated that temperature is an important environmental factor that affects the regional distribution of plants. However, how to modify the distribution pattern of plants in different regions is a focus of current research. Obtain the information of cold tolerance genes from cold tolerance species, cloning genes with real cold tolerance effects is one of the most important ways to find the genes related to cold tolerance. In this study, we investigated whether transferring the VHA-c gene from Antarctic notothenioid fishes into Arabidopsis enhances freezing tolerance of Arabidopsis. The physiological response and molecular changes of VHA-c overexpressing pedigree and wildtype Arabidopsis were studied at −20 °C. The results showed that the malondialdehyde (MDA) and membrane leakage rates of WT plants were significantly higher than those of VHA-c8 and VHA-c11 plants, but the soluble sugar, soluble protein, proline and ATP contents of WT plants were significantly lower than those of VHA-c8 and VHA-c11 plants under −20 °C freezing treatment. The survival rate, VHA-c gene expression level and VHA-c protein contents of WT plants were significantly lower than those of VHA-c8 and VHA-c11 plants under −20 °C freezing treatment. Correlation analysis showed that ATP content was significantly negatively correlated with MDA and membrane leakage rate, and positively correlated with soluble sugar, soluble protein and proline content under −20 °C freezing treatment. These results demonstrated that overexpression of the VHA-c gene provided strong freezing tolerance to Arabidopsis by increasing the synthesis of ATP and improved the adaptability of plants in low temperature environment.

The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants.

The transient elevation of cytosolic free calcium concentration ([Ca2+ ]cyt ) induced by cold stress is a well-established phenomenon; however, the underlying mechanism remains elusive. Here, we report that the Ca2+ -permeable transporter ANNEXIN1 (AtANN1) mediates cold-triggered Ca2+ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold-induced [Ca2+ ]cyt increase and upregulation of the cold-responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca2+ transport activity, thereby potentiating Ca2+ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold-induced [Ca2+ ]cyt elevation in the ost1-3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1-AtANN1 to cold-induced Ca2+ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.

Involvement of salicylic acid in cold priming-induced freezing tolerance in wheat plants

Freezing is one of the most damaging abiotic stress factors. Cold priming has been shown to enhance freezing tolerance in wheat. However, the underlying mechanisms are unclear. Salicylic acid (SA) is an important signal molecule involved in plant responses to abiotic and biotic stresses. This study aims to investigate the role of SA in cold priming-induced freezing tolerance in wheat plants. The results showed that the expression of gene encoding phenylalanine ammonia-lyase (PAL, a key enzyme involved in SA biosynthesis) and the level of endogenous SA (both free and conjugated) were significantly up-regulated following cold priming treatment. The role of SA in cold priming-induced freezing tolerance was further explored using the donor and inhibitor of endogenous SA. l-α-aminooxy-β-phenylpropionic acid (AOPP, a specific inhibitor of PAL) pretreatment reversed the increment of SA level induced by cold priming treatment, attenuated the positive effects of cold priming on antioxidant capacity (as indicated by the contents of MDA, ASA and GSH, and the activities of APX, GR, DHAR and MDHAR) and cold-responsive genes expression (such as AOX1a, IRI2 and HSP70) under the later freezing stress as well as the freezing tolerance (as indicated by the photochemical activity, electrolyte leakage and biomass). The application of exogenous SA could obviously counteract the negative effects of AOPP. Findings of the present study suggested that the roles of SA in cold priming-induced freezing tolerance in wheat might involve regulation of antioxidant capacity and cold-responsive gene expression.

Contrasting effects of freezing-stress memory on biomass production among herbaceous plant species

Prior exposure to freezing can increase the subsequent freezing tolerance of plants and reduce the severity of injury. However, it is unknown how freezing memory influences plant productivity. We investigated the effects of repeated freezing events over multiple seasons on the biomass of Bromus inermis, Lolium perenne, Festuca rubra, Plantago lanceolata, and Poa pratensis. The plants were exposed to different combinations of freezing in the early spring, late spring, or fall (2017), as well as the following spring (2018); control plants were frozen only once, along with all of the other treatments, during the spring of 2018. Bromus inermis that experienced every freeze, and the plants frozen in both the early and late spring, had higher biomass than the controls. Similarly, Poa pratensis plants frozen in both the early and late spring had higher biomass than the controls. In contrast, Festuca rubra plants frozen in early spring and fall had lower root biomass than the control plants, and Lolium perenne plants that experienced every freeze had lower root biomass than the controls. Variation among species in repeated freezing responses could have important consequences for the relative abundances of herbaceous species in northern temperate regions.

The effects of three levels of irrigation water on the improvement of cold tolerance of acclimated viola

Due to global climate changes, the growth of ornamental plants will be influenced by warming episodes in winter. The purpose of this examination was to estimate the effect of three levels of irrigation regimes on the freezing tolerance of acclimated viola (Viola × wittrockiana ‘Iona Gold with Blotch’). This experiments included three irrigation regimes (80, 60 and 40% FC) with different temperatures (20, 0, − 3, − 6, − 9, − 12, − 15, − 18, − 21 and − 24 °C). After irrigation regimes, proline and carbohydrate contents were recorded. Then, plants were moved to the thermo-gradient freezer and electrolyte leakage percentage (EL %), survival percentage (SU %), lethal temperature resulting in 50% mortality according to the electrolyte leakage percentage and survival percentage (LT50el and LT50su) were evaluated. The carbohydrate and proline content increased when plants were under drought stress conditions. Electrolyte leakage percentage across three levels of irrigation treatment increased by lowering the temperature; this increase at − 24 °C was fivefold more than the control. Survival percentage in all treatments was not affected in the thermal ranges of 0 to − 9 °C, but decreasing the temperature to − 24 °C resulted in the full loss in all treatments. Vegetative growth and reproductive components were significantly affected by drought and freezing temperature treatments. Dry weights of different parts of plants reached peak values when plants were under 60% FC at 0 °C condition. A negative correlation between LT50el with carbohydrate and proline levels was found and drought treatment significantly increased freezing tolerance (lower LT50) in plants grown under 60% FC compared to other treatments.