Strategies for Protecting Cereals and Other Utility Plants Against Cold and Freezing Conditions—A Mini-Review

Abstract

Low-temperature (LT) stresses (cold and frost) are major abiotic factors limiting plant growth and productivity. LT induces numerous physiological and biochemical changes in plants, changes hormonal balance and photosynthetic efficiency. Stress induced by LT often leads to yield losses in crops. While plants like maize and cucumber are highly sensitive to cold, winter cereals such as wheat and rye suffer mainly from severe frosts. Ongoing climate change and temperature fluctuations further increase the risk of LT-induced damage. To counteract the problems connected with LT stress, multiple strategies have been developed to enhance plant tolerance. Agrotechnical practices and biochemical treatments involving the application of phytohormones or osmoprotectants are designed to improve plant tolerance to LT. Beneficial plant–microbe interactions also contribute to alleviating LT stress. In addition, genetic engineering offers powerful tools for creating new cultivars that are more tolerant to LT. The CRISPR/Cas system, in particular, enables precise modifications and represents a promising tool for advancing sustainable agriculture. Integrated methods of protection are crucial for securing food supplies, especially under conditions of a changing climate. This mini-review summarises strategies for protecting plants against LT stress, with special attention paid to crop plants.

1. Introduction

LT stress is one of the most severe abiotic stresses. It affects many different plant species. Generally, plant species differ in the minimal, optimal and maximal temperatures at which they are able to grow and tolerate temperature stress. For some utility plants, such as maize or tomato, low but non-subzero temperatures of 5–6 °C are a serious stress that damages plants. On the other hand, in winter cereals, such as wheat or rye, exposure to temperature about 2–4 °C for 3–6 weeks leads to hardening (cold acclimation). This process prepares plants to survive frost during winter. For these species, low-temperature injuries are caused by severe freezing (reaching, for example, more than −20 °C). Moreover, winter cultivars of these cereals require a period of cold to become generatively induced in the process of vernalization [1]. Both cold acclimation and vernalization are accompanied by many physiological and biochemical changes. Those are among others, increased sugar accumulation [2] and osmoprotectants content [3], adjustment of hormonal homeostasis [4], accumulation of protective proteins [5,6] and changes in membrane lipid composition in the direction of membrane fluidisation [7,8]. Based on the roles of hormones or osmoprotectants in enhancing plant tolerance to LT, some of these compounds can be applied exogenously as protective agents.

Another problem is that due to the intensification of global climate change, during development, plants are threatened by unstable weather patterns in all seasons. This might be especially relevant to winter crops. Warm breaks, e.g., above 9 °C during late autumn, winter and early spring can result in the deacclimation of plants, which leads to a decrease in their frost tolerance. The details of the changes that are induced by deacclimation were previously reviewed by Stachurska and Janeczko [9] and Kosová et al. [10], and generally, they include the reversal of the changes that are induced by cold acclimation. This leads to a decrease in frost tolerance, which is particularly threatening when deacclimation is followed by a subsequent frost that may lead to frost injuries in plants. How to counteract the deacclimation-induced susceptibility of crop plants to low temperatures as yet remains unsolved.

2. Agrotechnical Methods That Protect Plants from Low Temperature

Agrotechnical methods are generally one of the most common ways by which plants can be protected against LT. To begin with, selecting species and cultivars that match the local climate and temperature patterns and ensuring that the key growth stages do not coincide with typical frost periods in the region are essential. Moreover, choosing an appropriate planting location is crucial [11,12]. In addition to the proper selection of cultivars and location, another way to protect plants from LT is to use different kinds of covers, irrigation, air disturbances, heaters, etc. Some of those agrotechnical methods have been successfully applied for many years in practice [13]. It is possible to use plastic mulches, row covers, grasses, plastic films, agrotextiles, irrigation techniques, etc. [11,14]. These provide an effective buffer against LT (including frost). For example, mulching with organic materials such as bran, grass and newspaper improved soil temperature and moisture as well as the yield of tomato (expressed as g plant⁻¹) [15]. Agrotextiles are developed to protect plants not only from frost, but also from snow, rain, heat or animals [16]. Row covers help shield plants from LT (including frost) and chilly winds during winter, creating a favourable microclimate that promotes quicker plant growth. However, too high temperatures inside plastic row covers in spring can result in plant injuries [17].

Moreover, man-made fog is effective in protecting plants against heat losses by forming a protective layer that keeps the temperature around the plants warmer. Unfortunately, this method is strongly dependent on actual weather conditions. Wind disperses the fog, and relatively high humidity is required to keep it and avoid evaporation [13]. Fogging seems to be a useful method, but rather on a smaller scale. Thus, together with the high impact of weather on this method, it has little application in fields and is primarily used in orchards. The use of a fogging spray system succeeded in cold-stressed mango trees, which also can suffer LT stress, especially during winter months. It resulted in, among others, increased yield (calculated on the basic of fruit weight per tree) and reduced number of damaged leaves [18].

Sprinkler irrigation is a method that sprays water mist on plant organs, forming a thin layer of ice that protects them from LT. Recently, this method was precisely reviewed by [14,19]. Briefly, sprinkler irrigation protects plants, e.g., fruit trees from LT, by utilising the latent heat released as irrigation water freezes, helping maintain plant and air temperatures near 0 °C [14]. Additionally, water mist improves soil moisture, which also contributes to better LT tolerance. Despite the popularity of this method in agricultural practices, excessive application may lead to the formation of heavy ice layers on plants and increase the risk of limb breakage [19]. A comparison of the different types of sprinkler irrigation methods and their effectiveness on different crops was previously conducted by Liu et al. [19]. This method seems to be useful on a smaller scale for protecting plants from LT stress, including fruit trees, strawberries, citrus and also in a tea fields. In cereals, such as wheat, sprinkler irrigation is used rather as a method of watering in arid areas.

Another successfully applied method of protecting plants from LT is air disturbance technology. Air disturbance machines like fans, wind machines and helicopters mix the warmer, upper layer of air with the cold one from near the ground and plants and increase their temperature [19]. This method is based on thermal inversion, when during nights, radiative cooling causes cold air to stay near the ground while warm air remains above it. A detailed comparison of the different methods of air disturbance protection and their effectiveness was previously given by Liu et al. [19]. Air disturbance technology is mainly used in orchards to protect fruit trees or tea plantations [19].

Additionally, it is possible to use anti-frost heating systems. This method requires heating devices, i.e., electric heating wires or combustion furnaces with liquid or solid fuel that will keep the temperature above 0 °C and thus protect plants from LT injuries. Such methods are used mainly to protect fruit trees. Unfortunately, furnaces emit smoke and worsen air quality in orchards [19]. Anti-frost heating systems are divided into two groups: fixed heating and mobile heating that moves across an orchard. Electrical means, such as electrically heating cables wrapped around grapevine plants were found to reduce frost damage to them by about 30% [20]. Now, precision heating strategies are applied in orchards in order to protect apple tree canopies and flower buds especially [21]. Different kinds of heating systems were compared by [19].

The aforementioned methods are used in practice, but a combination of them may protect plants from LT stress to a greater extent. However, agrotechnical methods require much work, are time-consuming or lead to increased environmental pollution. Combined approaches, for instance, heaters and fans, produced better results than heaters alone [22]. However, available data indicate that they are more useful for protecting orchards rather than large-scale fields with cereals. Interestingly, the usefulness of some of the abovementioned methods on a larger scale was proven on tea fields (e.g., sprinkler irrigation). Based on that, we can hypothesise that it could also be used to protect cereals, but this topic requires further studies.

3. Biochemical Treatments That Increase Plant Tolerance to LT

LT tolerance of plants can be improved using the different methods mentioned above. It can also be enhanced through biochemical approaches, such as exogenous application of substances from groups of phytohormones, osmoprotectants, saccharides, polyamines and others [23].

3.1. Phytohormones

Phytohormones regulate all of the physiological processes in plants and play an important role in the formation of cold stress tolerance and frost tolerance. One of the most common anti-stress hormones is abscisic acid (ABA). The endogenous level of ABA increases during LT stress as well as during cold acclimation in different plant species, e.g., wheat [4], maize [24] or oilseed rape [25]. In the case of wheat, the exogenous application of ABA on leaves increased its frost tolerance and resulted in, among others, an increased accumulation of sugars [26], an increased activity of antioxidant enzymes [27,28] and changes in the chloroplast structure [29]. In rice, exogenous ABA treatment reduced chilling-induced injuries of plants and improved the activity of the antioxidative system [30]. Similar results of ABA treatment were found for maize [31]. ABA treatment is useful not only in cereals, but also in other economically important species. For example, in the grapevine, the foliar application of ABA increased the frost tolerance of buds [32]. In chickpea, the exogenous ABA application decreased the negative effects of cold by improving the water status of the leaves and decreasing the oxidative stress-induced damage [33].

Another hormone from the group of stress-related hormones that can be used as a protection against LT is salicylic acid (SA). The exogenous application of SA in wheat enhanced its tolerance to LT, increasing, among others, the activity of the antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX), and also increased the proline concentration [34]. Another study conducted on wheat revealed that the application of SA during cold improved PSII efficiency and increased the accumulation of osmoprotectants [35]. Similarly, in barley, exogenous SA enhanced the activity of the antioxidative enzymes [36]. In maize, exogenous SA improved the chilling tolerance. It was accompanied by a decrease in malondialdehyde (MDA) and membrane injuries (electrolyte leakage) [37]. The reactive oxygen species (ROS) content was decreased, while the activity of SOD, CAT and ascorbate peroxidase (APX) was increased. SA treatment also improved the effectiveness of photosynthesis in cold-stressed maize [37]. SA treatment improved the LT tolerance in other plant species as well. Exogenous SA enhanced the freezing tolerance of spinach, which was accompanied by, among others, an increased content of proline [38]. SA also reduced the negative effects of frost in cabbage [39].

Jasmonic acid (JA), another so-called stress hormone, is also involved in responses to different abiotic stresses in plants including LT stress. Recently, a systematic review and meta-analysis of the role of different jasmonates in LT stress in plants was prepared by [40]. Generally, JA improved the survival rate of plants, enhanced the activity of antioxidative enzymes and decreased the MDA content. Moreover, a derivative of JA, methyl jasmonate (MeJa), when applied exogenously on wheat, promoted tolerance to LT. This treatment led to a partial recovery of the photosynthetic parameters, a decrease in MDA and H₂O₂ content, an increase in the activity of antioxidative enzymes, such as SOD and CAT, and an increase in the proline concentration. Additionally, the application of MeJa resulted in a tendency to increase the expression of the COR (cold-regulated genes) and the WCS (wheat cold-specific) genes [41]. Exogenous MeJa applied to tomato under cold conditions resulted in increased activity of the antioxidant enzymes and photosynthetic efficiency, which helped to protect plants against stress [42].

Not only stress hormones, but growth-promoting ones can affect plant LT tolerance. Cytokinins (CK), hormones that promote plant growth and, among others, cell differentiation and division, can also be involved in an improvement of LT tolerance. In the group of cereal plants, CK was exogenously applied, among others, in rice. In cold-treated rice, the application of 6-benzyladenine, which is a synthetic cytokinin, resulted in, among others, an improved activity of antioxidant enzymes and the delayed degradation of chlorophyll [43]. Additionally, the LT tolerance of a few utility plant species was effectively improved using different CK. For instance, in coffee seedlings under cold stress, the exogenous application of kinetin stimulated the production of non-enzymatic antioxidants, such as anthocyanins and improved photosynthesis efficiency [44]. Other growth-promoting hormones, auxins, can also be useful in mitigating the negative impact of LT. The application of auxin-like substances on oilseed rape, 1-[2-chloroethoxycarbonylmethyl]-4-naphthalenesulfonic acid calcium salt (TA-12) and 1-[2-dimethylaminoethoxycarbonylmethyl]naphthalene chloromethylate (TA-14) helped to rearrange the composition of proteins and stimulated the formation of dehydrins [45]. Limited results from both the application of auxin and gibberellin on LT-exposed plants are available.

Currently, in the plant kingdom, there is only known one group of steroid hormones, but they are quite important regulators that can improve LT tolerance. Brassinosteroids (BRs) play a role in the frost tolerance of plants via the CBF1-dependent and CBF1-independent (C-REPEAT/DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR (CBF) transcriptional regulators) pathways by controlling the expression of the cold-responsive (COR) genes [46]. According to [47], at low endogenous concentrations, BRs are responsible for growth response, while at higher concentrations, they are responsible for the stress response. In the case of cereals, in the maize that was grown under chilling stress, exogenous brassinolide enhanced the activity of SOD, CAT and peroxidase. BR also increased, among others, net photosynthesis and the content of indole-3-acetic acid, GA₃ and trans-zeatin [48]. Similarly, in rice, brassinolide mitigated the negative impact of cold and increased the activity of antioxidant enzymes and the accumulation of soluble sugars [49]. In winter wheat, the application of 24-epibrassinolide and also one of the mammalian hormones, progesterone, before cold acclimation decreased later frost injuries [50]. Moreover, the foliar application of BR, 24-epibrassinolide, had a protective effect on oilseed rape plants grown under LT [51]. Additionally, 24-epibrassinolide improved the frost tolerance of deacclimated plants, to some extent [52]. The role of exogenous 24-epibrassinolide in improving frost tolerance was also studied on winter rye [53] and perennial ryegrass [54]. The effects of BR in improving the LT tolerance in other utility plants were previously reviewed by [55,56].

Generally, a great deal of research has been devoted to the effect of phytohormones, especially the stress-related ones, on improving the LT tolerance of different crops. A comparison of effects of applying different phytohormones on various plants to improve their tolerance to LT stress was previously prepared by Raza et al. [56].

3.2. Osmoprotectants

Osmoprotectants play a crucial role in maintaining the tolerance to LT (particularly frost). For example, proline is accumulated by plants that are under LT stress [57]. Proline contributes to stress tolerance by helping to maintain osmotic balance, preserving cell turgidity and influencing the metabolism of ROS. The interaction between proline and other osmoprotectants and signalling molecules, such as glycine betaine, ABA, nitric oxide, hydrogen sulfide and soluble sugars, enhances the activation of protective responses under stress conditions [57]. The exogenous application of proline (seed priming or foliar application) counteracted the negative impact of LT stress on plants. Such treatment resulted in, among others, increased antioxidative activity [58,59]. As for cereals, in the case of maize, seed soaking in proline solution resulted in an improved germination potential, proline synthesis and hormonal balance under cold stress [60]. Moreover, supplementation of oilseed rape seedlings with proline resulted in an increase in endogenous proline content and was accompanied by an increase in the tolerance to LT stress of plants [61].

Another osmoprotectant that also plays a role in LT stress is glycine betaine. By maintaining the water balance and protecting protein structure, glycine betaine safeguards the cellular components against diverse stresses. Glycine betaine improves LT stress tolerance at numerous levels, such as increasing the activity of antioxidants or the accumulation of soluble sugars [62]. Foliar application of glycine betaine on wheat and tomato resulted in an enhancement in tolerance to LT. It was accompanied by an increase in osmolarity or the activity of the antioxidative system [63,64]. Soaking maize seeds in glycine betaine before chilling stress reduced its adverse effects and resulted in, among others, an increased germination rate, soluble sugar accumulation and antioxidant system activity [65].

The group of non-sugar osmoprotectants also include polyols (i.e., sugar alcohols) and polyamines. They play multiple roles in the protection of plants by regulating the osmotic balance, removing excess ROS, maintaining the cellular redox balance and protecting cells from osmotic stress, which is usually connected to LT stress [66]. Inositol, which is included in the group of polyols, and its derivatives are involved in signal transduction and stress adaptation [67,68]. The overexpression of gene OsIMP, encoding l-myo-inositol monophosphatase (IMPase), a part of the inositol synthesis pathway, resulted in an increased cold tolerance of rice [69]. The exogenous application of inositol to oilseed rape improved their frost tolerance by increasing the Ca²⁺ influx, which is one of the earliest signals of LT stress [70,71].

Polyamines, including putrescine, spermidine and spermine, are accumulated during LT stress in plants. They act as membrane stabilisers, modulators of hormonal pathways (ABA) and regulators of the expression of the genes associated with stress [72,73]. Pre-soaking wheat seeds in spermidine reduced the negative effects of LT stress on seed germination and increased their concentration of GAs [74]. The foliar application of different polyamines (e.g., putrescine, spermidine and spermine) on wheat seedlings improved their frost tolerance, decreased chlorophyll degradation and reduced membrane injuries (electrolyte leakage) [75]. Exogenous spermidine improved the cold tolerance of maize seedlings by regulating the expression of the genes associated with the ABA pathways and Ca²⁺ transport [76]. The exogenous spraying of polyamines (putrescine, spermidine or spermine) on oilseed rape resulted in an enhanced LT tolerance and, among others, an increased proline concentration [77]. Treating grapevine plants with putrescine decreased the negative effects of frost stress by increasing the antioxidant system efficiency including increasing the activity of CAT, GPX, APX and SOD [78].

3.3. Natural Biocompounds and Other Useful Substances

To ensure a higher LT tolerance of plants, many new solutions are being developed including natural biocompounds. One such substance is chitosan, a natural and biodegradable biopolymer that reduces the negative effects of LT stress on plant growth. Chitosan influences the defence mechanisms of plants and stimulates the activity of the antioxidative system [79]. Chitooligosaccharide, a derivative of chitosan, was successfully applied to rice under cold stress. It improved root and shoot fresh weight and root vigour, and also enhanced photosynthesis effectiveness [80]. Kociecka and Liberacki [81] reviewed other positive effects of chitosan on cereals.

Another substance that has been found to be useful in preventing plants from LT is silicone. Silicone accumulates in tissues and helps to preserve key cell wall components such as polysaccharides and lignin, thereby protecting a plant from LT-induced injuries [82]. In barley, silicone reduced the effects of LT, increased the activity of the antioxidative enzymes and the amount of soluble carbohydrates in leaves [83]. On the other hand, in soybean plants, silicone application reduced the negative effects of cold and increased the diversity of microbial species in the rhizosphere [84].

Melatonin, which is known for its numerous functions in animals, also has some potential in protecting plants against LT stress. Melatonin is involved in the processes of growth, rhizogenesis and photosynthesis [85]. Many studies have been dedicated to examining the role of exogenous melatonin in reducing abiotic stresses including the LT stress of crops [86]. Wheat seedlings that had been supplemented with melatonin and exposed to LT had higher concentrations of osmoprotectants [87]. Melatonin, together with cold priming, increased the photosynthetic rate, stomatal conductance and activity of antioxidants [88]. In rice and maize, exogenous melatonin reduced the effects of cold stress [89,90]. Oilseed rape plants that had been treated with melatonin were characterised by a higher survival rate when exposed to LT stress. Melatonin led to an increase in the proline content, soluble sugar accumulation and also an enhanced activity of antioxidant enzymes [91]. Additionally, melatonin treatment in cold-stressed tea plants helped to maintain a high efficiency of photosynthesis [92]. In tomato plants grown under cold stress, melatonin reduced cold-induced injuries, improved the antioxidant potential, carbon fixation and induced the expression of cold-responsive genes [93].

A deficiency or too low concentrations of microelements make plants less tolerant to abiotic stresses including LT stress [94], thus supplementation and even a slight excess of nutrients may help to prevent abiotic stress consequences. Exogenous supplementation with selenium on wheat resulted in a dose-dependent enhancement of LT tolerance, which was accompanied by, among other things, the activation of the antioxidant system [95]. Exogenous molybdenum increased the frost/cold tolerance of wheat, increased the activity of the antioxidative enzymes and COR15 protein accumulation [96,97]. Applying zinc after exposure to LT supported the recovery of rice tillers by enhancing the nitrogen and zinc uptake while helping to maintain the hormonal balance [98]. Moreover, exogenous calcium improved cold tolerance of tea by increasing the photochemical efficiency of PSII, the activity of protective enzymes and the ABA concentration [99]. An increased phosphorus concentration in the hydroponic medium of tobacco grown under LT stress improved, among others, the net photosynthetic rate and upregulated the SOD and CAT gene expression [100].

Unfortunately, all the abovementioned substances increase the cost of the cultivation. Also, the effectiveness of exogenous application depends on many factors, such as the time of application and the LT conditions experienced by plants. For example, exogenous use of 24-epibrassinolid on deacclimated oilseed rape plants exposed to frost improved their frost tolerance in the case of rather mild frost (−6 °C). Its effect was much weaker in −9 °C and −12 °C [52]. A summary of different methods that enable an improvement of LT tolerance is presented in Figure 1.

Many substances for improving the LT tolerance of other plant species have been tested, but only a few of them have provided promising results and the possibility of using them. A natural coating that improves the LT tolerance of plants is sodium alginate. Sodium alginate, which originates from the alginic acid of brown seaweed, is a biodegradable and non-toxic hydrogel that forms a protective, insulating layer on plants [101]. In the case of Vitis vinifera L., a species that is extremely sensitive to LT, it is also possible to apply a biodegradable liquid film, which protects it from both LT and dry, winter winds. According to the authors, the use of sodium alginate-based anti-freeze coatings may be a step towards sustainable plant protection in the future. An application of a hydrophobic kaolin particle film on leaves of lemon, potatoes and grapevine plants resulted in fewer frost injuries [102]. Moreover, a biodegradable liquid film composed of humic acid, reduced the LT-induced mortality of Vitis plants. This treatment promoted the accumulation of soluble sugars and decreased the content of superoxide anion in plants [103]. Carbohydrates play a significant role in the LT tolerance of plants and provide an opportunity to use them as protective substances. Exogenous irrigation of glucose affected the metabolism of melon seedlings under cold stress, by promoting the accumulation of soluble sugar and ABA. It also led to a reduction in cold injuries [104]. The irrigation of roots was more successful than the foliar application [104]. In cucumber seedlings, treatment with sucrose reduced cold stress and increased activity of antioxidative enzymes, proline and soluble sugars content [105]. Another group of growth regulators that can be used to protect plants against LT are strigolactones. These are carotenoid-derived substances that are produced in roots and are called a novel class of plant hormones that regulate shoot growth [106]. Exogenous treatment with strigolactones reduced LT stress in a few studied species. In peppers, it reduced the photoinhibition and increased the net photosynthetic rate and PSII efficiency [107]. On the other hand, in litchis, strigolactones reduced cold injuries and regulated the expression of the cold tolerance genes [108].

3.4. Plant Interactions with Other Organisms That Might Improve LT Tolerance

In addition to using biochemical substances, it is also worth paying attention to microorganisms. For example, arbuscular mycorrhizal fungi (AMF) are known to alleviate plant stress by minimising lipid peroxidation, maintaining membrane integrity, enhancing the antioxidative system and promoting the accumulation of osmoprotectants [109]. AMF (Rhizophagus irregularis) were used to colonise the roots of rice, where they increased proline accumulation and enhanced plant LT tolerance [110]. Colonisation of barley with AMF (Glomus versiforme and Rhizophagus irregularis) under LT had a positive effect on membrane integrity, antioxidant system and phenolic metabolism and increased the survival rate of plants [111].

Not only AMF, but other organisms might also be useful in improving the resilience of plants to LT. Another endophytic fungi, Piriformospora indica, affected the LT tolerance of plants via symbiosis with their roots—it enlarged the root zone and increased photosynthetic efficiency. When P. indica is combined with the plant growth-promoting bacteria Agrobacterium rhizogenes and Bacillus subtilis, those three organisms enhance the cold tolerance of rice. Their application increased the biomass of rice, photosynthesis efficiency and the accumulation of osmoprotectants and antioxidative enzymes. Furthermore, a reduction in membrane injuries was observed, indicated by a lower electrolyte leakage and MDA concentration [112]. Another study on rice in which a whole bacterial community that had been isolated from the rhizosphere of pea was used, showed that these microorganisms increased the cold tolerance of rice that resulted in an increased yield [113].

As the use of microorganisms is generally beneficial, there are some limitations, e.g., resulting from the temperature conditions. In LT conditions, AMF may be replaced by other fungi [113]. A detailed review of the roles of microorganisms that improve plant LT tolerance was previously provided by [114].

It is worth noting that the use of natural plant–microbe interactions to enhance plant tolerance to LT is an approach that aligns with the concept of “green” agriculture. Although recent studies have provided valuable insights into these interactions, further research is still necessary to fully understand and optimise their application. Advancing this field could significantly contribute to the development of sustainable, eco-friendly agricultural practices that reduce dependence on biochemical treatments or genetic interventions. Further interdisciplinary research integrating molecular biology, microbiology, and agronomy is needed to identify the most effective microorganisms and application methods. Expanding this knowledge could play a crucial role in developing eco-friendly solutions that will improve crop productivity under LT stress.

A simplified summary of the impact of protective methods on different aspects of the metabolism of crops is presented in Figure 2. Detailed different methods that allow for protection of plants against LT are also presented in Table S1.

4. Genetic Engineering Methods to Create More LT-Tolerant Plants

There are many methods for creating cultivars that are more tolerant to different stresses. It is possible to use, among others, conventional breeding. This method is however time-consuming. Modern molecular biology tools can greatly enhance the efficiency of this process. These techniques offer precise genome modification, which lead to the development of new plants with improved LT tolerance. Recently, detailed reviews of genetic engineering ways for enhancing the resilience of plants to LT were prepared by Kumar et al. [115] and Kumari et al. [116].

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technique has also gained increasing popularity for modifying plant genomes. The basics of this technique for editing the plant genome were described by Bortesi and Fischer [117]. The CRISPR/Cas technique has replaced previously applied methods for plant modifications such as zinc finger nucleases (ZNFs) and Transcription Activator-Like Effector Nucleases (TALENs). ZNFs are specially designed restriction enzymes with a nonspecific cleavage domain that is fused to sequence-specific DNA binding domains [118]. With ZNFs, it is possible to perform a wide range of modifications: deletions, translocations, inversions and point mutations [119]. TALENs is another method that is based on restriction enzymes—it consists of the TALE domain and a nuclease that cuts DNA [120]. The use of methods such as TALENs in plants is quite complicated and therefore is limited. Compared to CRISPR/Cas, both TALENs and ZNFs are characterised by a higher cost and a lower efficiency [121].

Using CRISPR/Cas, it is possible to examine the role of particular genes in maintaining LT tolerance as well as to introduce many modifications for improving LT resistance. Many of these were created on rice as this crop is sensitive to LT because of its tropical and subtropical origin. For example, rice plants with knockouts of the OsAnn3 and OsAnn5 genes exhibited an increased sensitivity to LT [122,123]. Those genes encode annexins, which are proteins that are involved in, among others, the protection of plants from abiotic stress. Moreover, increased sensitivity to LT was also observed in rice that had a disrupted OsNCED4 gene. OsNCED4 encodes an abscisic acid biosynthesis enzyme that is localised in the chloroplast and its expression increased after LT stress [124]. What is more, an overexpression of OsNCED5 resulted in an improved LT tolerance [125]. Additionally, a knockout of OsNAC050, a transcription factor, exhibited an enhanced LT tolerance [126]. Similarly, knockouts of OSWRKY63 exhibited an enhanced LT tolerance as OsWRKY63 negatively regulates LT tolerance [127]. In contrast, a knockout of OsPRP1, a gene encoding proline-rich proteins, exhibited an increased sensitivity to LT [128]. Additionally, in rice, the overexpression of the OsCNGC9 gene, which positively regulates LT tolerance via an increase in cytoplasmic calcium, resulted in a better LT tolerance of the mutant [129]. The overexpression of the chilling-responsive gene OsVPE2 increased the sensitivity of rice seedlings to LT, but a CRISPR/Cas9-generated knockout exhibited a higher LT tolerance [130]. A mutant of OsPIN9, which encodes an auxin efflux carrier, also exhibited an increased LT tolerance compared to wild-type rice [131].

The CRISPR/Cas method not only enables one gene to be edited, but also to simultaneously edit the genes that are associated with LT stress tolerance (OsMYB30) and those that affect the yield parameters (OsPIN5b and GS3), thereby resulting in rice with an improved yield (measured through yield-related traits, e.g., panicle number/plant) together with a better tolerance to cold stress [132].

Previously, a comprehensive summary of CRISPR/Cas9 application in different plant species along with the resulting improvements in their tolerance to different stresses, including LT, was prepared by [115]. Genetic engineering can be an effective method to develop stable, LT tolerant plants. However, this approach sometimes meets with public resistance and concerns about its environmental sustainability.

To summarise, improving plant tolerance to LT can be achieved through several complementary strategies. Agrotechnical methods, such as covers, fogging, sprinkler irrigation, heaters, etc., are mainly used in orchards. They provide immediate protection by changing the temperature near plants, but they may be costly. Biochemical treatments, including application of, among others, hormones (e.g., ABA, SA, or JA), osmoprotectants (e.g., proline, glycine betaine) or biocompounds (e.g., chitosan) can enhance LT tolerance. However, its effectiveness depends on many factors. The use of beneficial microorganisms, such as AMF or plant growth-promoting bacteria, offers a sustainable and eco-friendly approach to improving LT tolerance. Nonetheless, their efficiency may vary under field conditions. Finally, genetic engineering, including modern tools like CRISPR/Cas technique enables the development of LT-tolerant cultivars by directly modifying genes. For better results, all abovementioned approaches can be integrated, combining agrotechnical, biochemical, biological and genetic strategies to achieve complementary improvement in plant LT tolerance.

5. Conclusions

As global climate change intensifies, more frequent episodes of extreme weather conditions will continue to occur. Researchers have shown that there are many mechanical and biochemical methods for improving the LT tolerance of plants. In the future, developing novel methods to protect plants from LT and temperature fluctuations will probably be essential for improving crop LT tolerance. The integration of both agrotechnical methods and biochemical treatments (with natural compounds) is an eco-friendly solution for the problem of a low or lacking LT tolerance of plants. Particularly promising methods that meet the “green agriculture” criteria use natural plant–microbe interactions for improving plant tolerance to LT. On the other hand, genetic engineering also appears to be aneffective method for creating new, more stable LT-tolerant cultivars of important crops in some cases. The use of such cultivars in the field would limit the necessity for additional agricultural treatment. However, genetic modification is not always accepted by people and is not necessarily considered to be eco-friendly.