Improved Freezing Tolerance Research Articles

Low frequency of the wild-type freezing-tolerance LsCBF7 allele among lettuce population suggests a negative selection during domestication and breeding

Key messageSustainable winter production in lettuce requires freezing tolerant varieties. This study identified a wild-type allele of LsCBF7 that could contribute to freezing tolerance improvement in lettuce.Lettuce is one of the most consumed vegetables globally. While ideally grown in 13–21 °C, its cultivation extends into winter in milder climates. However, occasional freezing temperatures can significantly reduce yields. Therefore, the development of freezing-tolerant lettuce varieties has become a long-term goal of lettuce breeding programs. Despite its significance, our understanding of freezing tolerance in lettuce remains limited. Plants have evolved a coping mechanism against freezing, known as cold acclimation, whereby they can increase freezing tolerance when pre-exposed to low nonfreezing temperatures. The CBF pathway is well-known for its central role in cold acclimation. Previously, we identified 14 CBF genes in lettuce and discovered that one of them, LsCBF7, had a loss-of-function mutation. In this study, we uncovered that accessions from colder regions carried the wild-type allele of LsCBF7 and this allele likely contributed to increased freezing tolerance, with 14% of the lettuce population carrying this allele. Interestingly, in wild lettuce (L. serriola) that is considered a progenitor of cultivated lettuce, this wild-type allele was much more common, with a frequency of 90%. This finding suggests that this wild-type allele may have undergone negative selection during the domestication or breeding of lettuce. Our data strongly indicate that this allele could be linked to early bolting, an undesirable trait in lettuce, which may have driven the negative selection. While this wild-type allele shows promise for improving freezing tolerance in lettuce, it is crucial to decouple it from the early bolting trait to fully harness its potential in lettuce breeding.

Overexpression of TaMPK3 enhances freezing tolerance by increasing the expression of ICE-CBF-COR related genes in the Arabidopsis thaliana.

Mitogen-activated protein kinases (MAPKs) play important roles in plant stress response. As a major member of the MAPK family, MPK3 has been reported to participate in the regulation of chilling stress. However, the regulatory function of wheat (Triticum aestivum ) mitogen-activated protein kinase TaMPK3 in freezing tolerance remains unknown. Dongnongdongmai No.1 (Dn1) is a winter wheat variety with strong freezing tolerance; therefore, it is important to explore the mechanisms underlying this tolerance. In this study, the expression of TaMPK3 in Dn1 was detected under low temperature and hormone treatment. Gene cloning, bioinformatics and subcellular localisation analyses of TaMPK3 in Dn1 were performed. Overexpressed TaMPK3 in Arabidopsis thaliana was obtained, and freezing tolerance phenotype observations, physiological indices and expression levels of ICE-C-repeat binding factor (CBF)-COR -related genes were determined. In addition, the interaction between TaMPK3 and TaICE41 proteins was detected. We found that TaMPK3 expression responds to low temperatures and hormones, and the TaMPK3 protein is localised in the cytoplasm and nucleus. Overexpression of TaMPK3 in Arabidopsis significantly improves freezing tolerance. TaMPK3 interacts with the TaICE41 protein. In conclusion, TaMPK3 is involved in regulating the ICE-CBF-COR cold resistance module through its interaction with TaICE41, thereby improving freezing tolerance in Dn1 wheat.

Methyltransferase TaSAMT1 mediates wheat freezing tolerance by integrating brassinosteroid and salicylic acid signaling.

Cold injury is a major environmental stress affecting the growth and yield of crops. Brassinosteroids (BRs) and salicylic acid (SA) play important roles in plant cold tolerance. However, whether or how BR signaling interacts with the SA signaling pathway in response to cold stress is still unknown. Here, we identified an SA methyltransferase, TaSAMT1 that converts SA to methyl SA (MeSA) and confers freezing tolerance in wheat (Triticum aestivum). TaSAMT1 overexpression greatly enhanced wheat freezing tolerance, with plants accumulating more MeSA and less SA, whereas Tasamt1 knockout lines were sensitive to freezing stress and accumulated less MeSA and more SA. Spraying plants with MeSA conferred freezing tolerance to Tasamt1 mutants, but SA did not. We revealed that BRASSINAZOLE-RESISTANT 1 (TaBZR1) directly binds to the TaSAMT1 promoter and induces its transcription. Moreover, TaBZR1 interacts with the histone acetyltransferase TaHAG1, which potentiates TaSAMT1 expression via increased histone acetylation and modulates the SA pathway during freezing stress. Additionally, overexpression of TaBZR1 or TaHAG1 altered TaSAMT1 expression and improved freezing tolerance. Our results demonstrate a key regulatory node that connects the BR and SA pathways in the plant cold stress response. The regulatory factors or genes identified could be effective targets for the genetic improvement of freezing tolerance in crops.

Alkaline Invertase 2 positively modulates cold adaptive of Camellia sinensis and enhances freezing and salt tolerance in transgenic Arabidopsis thaliana

Alkaline/neutral invertase (A/N-Inv) is a type of hydrolases that irreversibly hydrolyze sucrose into glucose and fructose, thus participating in the plant morphogenesis and stress responses. In tea plant, a putative A/N-Inv gene, CsINV2, mediated the abiotic stress responses of tea plant, but the detailed molecular mechanism has not been elucidated yet. In this study, CsINV2 was verified as an alkaline invertase that can split sucrose into glucose and fructose in vitro, which showed the optimal catalytic activity at 45 °C and pH 8 conditions. Besides, the high transcription of CsINV2 contributed to improving the cold adaptation of tea plant, while silencing of CsINV2 reduced cold adaptive of tea plant. Overexpression of CsINV2 improved freezing tolerance of transgenic Arabidopsis by improving A/N-Inv activity, glucose content, hexose/sucrose ratio, and also via a feedback regulation pathway. Meanwhile, overexpression of CsINV2 promoted gemination rate and root elongation of transgenic Arabidopsis under salt stress condition. Finally, a cold-induced transcription factor CsAHL17 was confirmed to interact with the promoter of CsINV2, silencing of CsAHL17 reduced the transcription of CsINV2 both under 25 °C and 4 °C conditions. In summary, it can draw a conclusion that CsINV2 regulates the cold resistance of tea plant by influencing the contents of different soluble sugar components under the regulation of CsAHL17. This study will further promote the understanding to the role of invertase in tea plants.

Wheat MEDIATOR25, TaMED25, plays roles in freezing tolerance possibly through the jasmonate pathway

The MEDIATOR (MED) complex is a molecular bridge that regulates transcription by connecting transcription factors (TFs) and specific gene promoters. The MED subunit 25 (MED25) can interact with specific TFs in the phytohormone signaling pathways to regulate the transcription of downstream genes and affect various physiological processes in plants. Although MED25 plays an important role in adaptation to abiotic stresses, it is still unclear whether MED25 is involved in freezing tolerance in wheat. In this study, we found that the expression of TaMED25 was significantly increased under extremely low temperatures (–25 °C) in Dongnongdongmai1 (Dn1) plants, a variety of winter wheat with excellent cold-stress resistance,and hypothesized that TaMED25 may be a novel regulator of freezing tolerance in this wheat variety. To further characterize the function of TaMED25 in freezing tolerance, we cloned TaMED25 from Dn1 plants and overexpressed it in Arabidopsis thaliana. The results showed that the overexpression of TaMED25 improved freezing tolerance in Arabidopsis plants, with decreased relative conductivity, enhanced ability to effectively scavenge reactive oxygen species (ROS), and increased expression of cold-responsive genes. Furthermore, we proposed that TaMED25 mediates jasmonate-induced cold resistance in plants, possibly by interacting with TaJAZ7 and TaMYC2 of the jasmonate signaling pathway and TaICE41 of the ICE–CBF–COR pathway. Our findings reveal the functions of MED25 in plants and provide a scientific basis for an in-depth analysis of the transcriptional regulation mechanism of freezing tolerance in winter wheat.

Raising Mowing Height Improves Freeze Tolerance of Putting Green–type Bermudagrass

There is a growing trend of cultivating hybrid bermudagrass [Cynodon dactylon (L.) Pers. × Cynodon transvaalensis Burtt-Davy] on golf course putting greens in the transition zone because of its excellent quality in the summer months, coupled with less pesticide input than creeping bentgrass (Agrostis stolonifera L.). However, the long-term success of bermudagrass putting greens is hindered by low temperatures in winter months, particularly in the transition zone. To address this issue, in addition to genetic improvement for cold hardiness through the development of new cultivars, effective management approaches are necessary to enhance the winter survival of putting green–type bermudagrass. The objective of this study was to investigate the relative freeze tolerance of four bermudagrasses and the effects of raising mowing height on the freeze tolerance of putting green–type bermudagrasses. In this study, two experimental putting green–type bermudagrasses (11X2 and OKC0805) along with cultivars TifEagle and OKC3920 were tested at two mowing heights (3.2 vs. 6.4 mm) at freeze temperatures that ranged between –4 and –11 °C. The lethal temperature to kill 50% of the population (LT50) as well as regrowth vigor during recovery were evaluated. Variety ‘OKC3920’ demonstrated enhanced freeze tolerance compared with ‘TifEagle’ at both mowing heights. Increasing the mowing height from 3.2 mm to 6.4 mm improved freeze tolerance for most genotypes tested in this study. After exposing the grasses to –8 °C for 1 hour, genotypes such as 11X2 exhibited better regrowth vigor and demonstrated a faster recovery. This study suggests that golf course managers can enhance winter resilience of bermudagrass putting greens by selecting genotypes strategically with superior freeze tolerance and raising mowing heights in the fall acclimation process.

Over-activation of cold tolerance in arabidopsis causes carbohydrate shortage compared with Chorispora bungeana

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.

Comprehensive Transcriptome Analysis of Responses during Cold Stress in Wheat (Triticum aestivum L.).

Wheat production is often impacted by pre-winter freezing damage and cold spells in later spring. To study the influences of cold stress on wheat seedlings, unstressed Jing 841 was sampled once at the seedling stage, followed by 4 °C stress treatment for 30 days and once every 10 days. A total of 12,926 differentially expressed genes (DEGs) were identified from the transcriptome. K-means cluster analysis found a group of genes related to the glutamate metabolism pathway, and many genes belonging to the bHLH, MYB, NAC, WRKY, and ERF transcription factor families were highly expressed. Starch and sucrose metabolism, glutathione metabolism, and plant hormone signal transduction pathways were found. Weighted Gene Co-Expression Network Analysis (WGCNA) identified several key genes involved in the development of seedlings under cold stress. The cluster tree diagram showed seven different modules marked with different colors. The blue module had the highest correlation coefficient for the samples treated with cold stress for 30 days, and most genes in this module were rich in glutathione metabolism (ko00480). A total of eight DEGs were validated using quantitative real-time PCR. Overall, this study provides new insights into the physiological metabolic pathways and gene changes in a cold stress transcriptome, and it has a potential significance for improving freezing tolerance in wheat.

Overwintering Camelina and Canola/Rapeseed Show Promise for Improving Integrated Weed Management Approaches in the Upper Midwestern U.S.

Winter oilseed cash cover crops are gaining popularity in integrated weed management programs for suppressing weeds. A study was conducted at two field sites (Fargo, North Dakota, and Morris, Minnesota) to determine the freezing tolerance and weed-suppressing traits of winter canola/rapeseed (Brassica napus L.) and winter camelina [Camelina sativa (L.) Crantz] in the Upper Midwestern USA. The top 10 freezing tolerant accessions from a phenotyped population of winter canola/rapeseed were bulked and planted at both locations along with winter camelina (cv. Joelle) as a check. To phenotype our entire winter B. napus population (621 accessions) for freezing tolerance, seeds were also bulked and planted at both locations. All B. napus and camelina were no-till seeded at Fargo and Morris at two planting dates, late August (PD1) and mid-September (PD2) 2019. Data for winter survival of oilseed crops (plants m-2) and their corresponding weed suppression (plants m-2 and dry matter m-2) were collected on two sampling dates (SD) in May and June 2020. Crop and SD were significant (p < 0.05) for crop plant density at both locations, and PD in Fargo and crop x PD interaction in Morris were significant for weed dry matter. At Morris and Fargo, PD1 produced greater winter B. napus survival (28% and 5%, respectively) and PD2 produced higher camelina survival (79% and 72%, respectively). Based on coefficient of determination (r2), ~50% of weed density was explained by camelina density, whereas ≤20% was explained by B. napus density at both locations. Camelina from PD2 suppressed weed dry matter by >90% of fallow at both locations, whereas weed dry matter in B. napus was not significantly different from fallow at either PD. Genotyping of overwintering canola/rapeseed under field conditions identified nine accessions that survived at both locations, which also had excellent freezing tolerance under controlled conditions. These accessions are good candidates for improving freezing tolerance in commercial canola cultivars.

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.

Supplemental calcium improves freezing tolerance of spinach (Spinacia oleracea L.) by mitigating membrane and photosynthetic damage, and bolstering anti-oxidant and cell-wall status

Supplemental calcium (Ca2+) fertilization has been reported to enhance plant tolerance against various abiotic stresses, however, its effect on freezing tolerance has not been well studied. We investigated the effect of Ca2+ supplementation on freezing tolerance of spinach (Spinacia oleracea L.) at whole-plant as well as excised-leaf level. Seedlings supplemented with 20 and 25 mM Ca2+ through sub-irrigation efficiently assimilated Ca2+, evident by higher leaf-Ca2+content. Supplemental Ca2+ did not impede leaf-growth (leaf-area) but slightly increased dry weight/fresh weight ratio compared to the control. Supplemental Ca2+ significantly improved freezing tolerance as evident by reducing K+/Mg2+/total ion-leakage (i.e. indicative of less membrane damage), alleviating oxidative stress (less accumulation of O2 and H2O2), and enhancing PSII potential quantum yield /energy trapping efficiency (Fv/Fm) following a relatively moderate or severe freeze-thaw stress, i.e. −5.5 and −6.5 ˚C. Improved freezing tolerance by Ca2+ supplementation was paralleled by enhanced activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). Our data further suggests that, besides a bolstered antioxidant status, improved freezing tolerance of Ca2+-fed seedlings may also be associated with reduced loss of Ca2+ from plasma membrane (due to lower abundance of leaked K+ in extracellular spaces) as well as due to increased mechanical strength of cell wall as evident by cell wall augmentation.

Evaluating the freeze tolerance of bermudagrass genotypes

AbstractBermudagrasses (Cynodon spp.) are commonly used in golf courses, athletic fields, and home lawns in the transitional climatic region of the United States. Winterkill, however, is a major concern for bermudagrasses grown in this region. Controlled environment testing is a reliable method to evaluate freeze tolerance contributing to the selection of freeze tolerant genotypes. Therefore, this study was designed to test the freeze tolerance of two elite experimental genotypes, OKC1873 and OKC1406, along with two industry standards (‘Tifway’ and ‘Tahoma 31’), by exposing them to various freeze temperatures (−4 to −14 ℃) in a controlled environment. The freezing test was replicated in time, and the mean lethal temperature to kill 50% of the population (LT50) for each genotype was determined. Tifway (freeze‐sensitive standard) had an LT50 value of −7.0 ℃. The genotype OKC1873 (−7.2 ℃), was in the same statistical group as Tifway. Tahoma 31 was the best‐performing genotype with the lowest LT50 value of −9.1 ℃. The genotype OKC1406 (−8.8 ℃) was in the same statistical group as Tahoma 31. Top‐performing experimental genotypes will move on for further screening in replicated field trials for future consideration for commercial release based on qualities such as improved freeze tolerance, desirable turfgrass quality, and sufficient disease resistance.

A metabolomics study of ascorbic acid-induced in situ freezing tolerance in spinach (Spinacia oleracea L.).

Freeze–thaw stress is one of the major environmental constraints that limit plant growth and reduce productivity and quality. Plants exhibit a variety of cellular dysfunctions following freeze–thaw stress, including accumulation of reactive oxygen species (ROS). This means that enhancement of antioxidant capacity by exogenous application of antioxidants could potentially be one of the strategies for improving freezing tolerance (FT) of plants. Exogenous application of ascorbic acid (AsA), as an antioxidant, has been shown to improve plant tolerance against abiotic stresses but its effect on FT has not been investigated. We evaluated the effect of AsA‐feeding on FT of spinach (Spinacia oleracea L.) at whole plant and excised‐leaf level, and conducted metabolite profiling of leaves before and after AsA treatment to explore metabolic explanation for change in FT. AsA application did not impede leaf growth, instead slightly promoted it. Temperature‐controlled freeze–thaw tests revealed AsA‐fed plants were more freezing tolerant as indicated by: (a) less visual damage/mortality; (b) lower ion leakage; and (c) less oxidative injury, lower abundance of free radicals (O2·- and H2O2). Comparative leaf metabolite profiling revealed clear separation of metabolic phenotypes for control versus AsA‐fed leaves. Specifically, AsA‐fed leaves had greater abundance of antioxidants (AsA, glutathione, alpha‐ & gamma‐tocopherol) and compatible solutes (proline, galactinol, and myo‐inositol). AsA‐fed leaves also had higher activity of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, and catalase). These changes, together, may improve FT via alleviating freeze‐induced oxidative stress as well as protecting membranes from freeze desiccation. Additionally, improved FT by AsA‐feeding may potentially include enhanced cell wall/lignin augmentation and bolstered secondary metabolism as indicated by diminished level of phenylalanine and increased abundance of branched amino acids, respectively.

Tall fescue germplasm response to freezing temperatures

AbstractTall fescue [Schedonorus arundinaceus (Schreb.) Dumort., nom. cons.] is a perennial cool‐season grass in which improved freeze tolerance could result in greater winterhardiness and persistence. Our objective was to evaluate survival and freeze‐tolerance thresholds of ‘Flecha’ summer‐dormant (SD) and ‘Texoma Max QII’ summer‐active (SA) tall fescue at seedling (1–2 leaves) and tillered (5–6 leaves) growth stages using controlled environment freezing chambers. Target temperatures ranged from 0 to −9 °C (seedlings) and 0 to −12 °C (tillered plants). Predicted lethal temperature at which 50% mortality occurs (LT50) was −5.7 and −5.5 °C for SA and SD seedlings, respectively, and −6.0 and −5.7 °C for SA and SD tillered plants, respectively. Based on these results, a controlled temperature chamber appears to be a useful tool for plant breeders when evaluating survival and freeze tolerance of SA and SD tall fescue germplasm at seedling and tillered growth stages.

Biochemical and molecular responses during overwintering of red clover populations recurrently selected for improved freezing tolerance

Low freezing tolerance reduces the persistence of red clover under northern climate. The incidence of winter damages in perennial crops could increase in the future due to the adverse effects of the predicted warmer fall temperature on plant cold acclimation. To accelerate breeding progress, two cultivars of red clover Christie (C-TF0) and Endure (E-TF0) were exposed to a recurrent selection protocol for freezing tolerance performed indoor. New populations were obtained after five (C-TF5 and E-TF5), six (C-TF6 and E-TF6), and seven (C-TF7 and E-TF7) cycles of recurrent selection. These populations were overwintered under natural conditions and monitored for freezing tolerance and cold-induced molecular traits. Freezing tolerance was improved by up to 6 °C in recurrently selected populations when compared to initial cultivars confirming that further progress are achieved with advanced cycles of selection. Monthly analysis of biochemical changes shows that higher starch concentrations at the onset of the fall hardening period are contributing to the acquisition of superior freezing tolerance through its impact on sucrose accumulation. They also contribute to the vigor of spring regrowth by sustaining more pinitol and proline synthesis. Larger concentrations of these metabolites in populations with higher levels of freezing tolerance (TF7) hint at their involvement in winter survival of red clover. Among genes differentially expressed in response to both cold acclimation and recurrent selection, a concomitant cold induction of APPR9 and cold repression of 1-aminocyclopropane-carboxylate synthase suggests a link between the repression of a pathway regulated by ethylene and the improvement of freezing tolerance in red clover.

Fall Ethephon Application Enhances the Freezing Tolerance of Magnolia wufengensis During Overwintering

Magnolia wufengensis L.Y. Ma et L. R. Wang, a rare species which has been introduced and cultivated from southern China to northern China, frequently suffers from freezing injuries. To figure out the influence of ethephon (ETH) application on the cold tolerance during the natural overwintering of M. wufengensis, one-year shoots subjected to ethephon application at four concentrations (0, 700, 1000, and 1500 mg·L−1) were collected to measure the physiological and biochemical changes from September 2017 to 1 April 2018. The fall ETH application increased the freezing tolerance of M. wufengensis, and the optimum concentration for M. wufengensis was 1000 mg·L−1 (T2), which not only improved the shoot freezing tolerance by 1.4 times, but also led to a 25.0% faster cold acclimation rate and 13.7% slower de-acclimation rate. Moreover, 1000 mg·L−1 ETH delayed bud-burst in the spring by 10. 7 d, which was helpful for effectively avoiding cold spells in the spring, and improved the rates of bud survival by 47.1%. The improved freezing tolerance under exogenous ETH application was associated with an increased dehydration and accumulation of proline content. It seems that exogenous ETH application may be used on M. wufengensis grown in northern China to protect against freezing tolerance during the overwintering period.

Effect of Exogenous Salicylic Acid on the Physiological and Biochemical Processes of Ligustrum lucidum during Natural Cold Acclimation

The evergreen Ligustrum lucidum (glossy privet) suffers from freezing injury in northern China, where there are short growing seasons and early fall frost events. To investigate the influence of exogenous salicylic acid (SA) application on the natural cold acclimation of glossy privet, physiological and biochemical changes in glossy privet seedlings subjected to SA treatments at four concentrations (0, 150, 250, and 350 mg·L−1) were evaluated from Sept. to Dec. 2016. The optimum application concentrations were between 250 and 350 mg·L−1, which led to better freezing tolerance during natural cold acclimation. The improved freezing tolerance under exogenous SA application was associated with the accumulation of chlorophyll, proline, soluble protein, and soluble sugar, and the regulations of gibberellic acid (GA) and abscisic acid (ABA). Salicylic acid treatments started a cascade of steps for advancing the cold acclimation process of glossy privet. We suggest that exogenous SA application may be used on glossy privet grown in northern China.

Enhancing freezing tolerance of Brassica napus L. by overexpression of a stearoyl-acyl carrier protein desaturase gene (SAD) from Sapium sebiferum (L.) Roxb.

Sapium sebiferum (L.) Roxb. is an important woody oil tree and traditional herbal medicine in China. Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays a key role in the transformation of saturated fatty acids into unsaturated fatty acids in oil; these fatty acids greatly influence the freezing tolerance of plants. However, it remains unclear whether freezing tolerance can be regulated by the expression level of SsSAD in S. sebiferum L. Our research indicated that SsSAD expression in S. sebiferum L. increased under freezing stress. To further confirm this result, we constructed a pEGAD-SsSAD vector and transformed it into B. napus L. W10 by Agrobacterium tumefaciens-mediated transformation. Transgenic plants that overexpressed the SsSAD gene exhibited significantly higher linoleic (18:2) and linolenic acid (18:3) content and advanced freezing tolerance. These results suggest that SsSAD overexpression in B. napus L. can increase the content of polyunsaturated fatty acids (PUFAs) such as linoleic (18:2) and linolenic acid (18:3), which are likely pivotal in improving freezing tolerance in B. napus L. plants. Thus, SsSAD overexpression could be useful in the production of freeze-tolerant varieties of B. napus L.

Technology Allowing Baby Spinach Leaves to Acquire Freezing Tolerance

This study focuses on improving the freezing tolerance of spinach leaves when applying vacuum impregnation (VI) and pulsed electric fields (PEF) as pre-treatments. Changing the cultivation conditions of the spinach plants was tested for improved freezing tolerance of the harvested leaves. Spinach plants were initially cultivated at 20 °C for 5 weeks and harvested at the beginning of week 6 before the cultivation conditions were changed to 5 °C. After exposing the plants to cold stress, leaves were harvested on day 1, 8, and 20. The leaves from different cultivation temperatures were treated with VI with 30% (w/v) trehalose solution and PEF prior freezing at − 22 °C in a blast freezer. After freezing and thawing, the viability of harvested spinach leaves was 50% for the plants cultivated at 20 °C for 5 weeks. When the cultivation conditions were changed, the viability of the harvested leaves increased to 82% on day 1, 89% on day 8, and without a significant further increase on day 20. During cold cultivation, sucrose accumulated in the leaves, which might have contributed to the increased survival. The influence of impregnating the accumulated sugars instead of changing the cultivation conditions on the survival of the leaves was tested. The viability of the leaves was 75%, which was higher than the survival of the control (50%). The results indicate that it is possible to increase leaf survival after freezing and thawing by applying VI and PEF in combination with either by changing the cultivation conditions of spinach plants or by externally impregnating additional sugars to the harvested leaves.

Assessing freeze tolerance in St. Augustinegrass: II. acclimation treatment effects

The adaptation of St. Augustinegrass (Stenotaphrum secundatum [Walt.] Kuntze) in the transitional climatic zone of the United States is limited due to a lack of sufficient freeze tolerance. Lab-based freeze testing provides plant breeders with a reliable method for evaluating freeze tolerance. However, in developing protocols for freeze testing it is important to account for the underlying mechanisms that affect freeze tolerance. The objective of this research was to evaluate the effects of cold acclimation and deacclimation on nine St. Augustinegrass genotypes ranging in their levels of freeze tolerance. Results indicated that accounting for all levels of acclimation provided excellent genotype separation across freezing temperatures (− 3 and − 4 °C) and supports the hypothesis that the inclusion of acclimation response offers the best overall assessment of freeze tolerance. Genotypes with the highest known field winter survival had also the highest improved freeze tolerance upon cold-acclimation. Other genotypes did not respond to cold-acclimation which resulted in poor survival and recovery rates. Conversely, a significant loss of freeze tolerance was identified when plants were subjected to deacclimation events suggesting that St. Augustinegrass can be negatively affected by rapid temperature changes in the transitional climatic zone leading to increased sensitivity to winterkill. Overall, this study provides preliminary information regarding the complex relationships within and between mechanisms affecting freeze tolerance in St. Augustinegrass. Moreover, results presented here should aid in the development of protocols for selection of freeze tolerant breeding materials under controlled environmental conditions.