Commercial biostimulants products from aquatic environments
As a marine representative, the brown macroalga Ascophylum nodosum is the species most used in commercial biostimulant products. Products derived from A. nodosum (alone or together with other bioactive compounds) have been tested for their ability to induce chilling and freezing tolerance in plants (Table 1).
| Biostimulants Source | Tested plant | Temperature | Effect |
| Powdered alkali extract of Ascophyllum nodosum | Arabidopsis thaliana | -7.5℃/ -5.5℃ | Reduced 70% chlorophyll damage due to decreased expression of the chlorophyllase genes |
| Arabidopsis thaliana | -2℃ | Augmented chlorophyll content, possibly due to the downregulation of chlorophyll degradation genes, and upregulation in genes encoding cryoprotection of chloroplast stromal protein | |
| Tobacco( Nicotiana tabacum) | 0℃, -3℃, -5℃ | Improved cell growth, membranestability, and nuclear integrity, andreduced cell death, due to theupregulation of key freezing tolerance genes | |
| Ascophyllum nodosum, Fucus spp., Laminaria spp. | Maize( Zea mays) | 12 – 14℃ | Reduction of leaf necrosis, positiveeffects on plant growth and root development due to the increased superoxide dismutase activity |
| Garlic (Allium sativum) and powdered extract of Ascophyllum nodosum | Orange fruits( Citrus X sinensis) | 5土1℃ | Induces lower percentage of fruitdecay, total acidity and highest totalsugar content |
The application of a powdered alkaline extract of A. nodosum in Arabidopsis thaliana significantly increased tolerance to freezing temperatures both in both in vitro and in vivo assays. Extract-treated plants recovered from freezing temperatures of -7.5℃ in vitro and -5.5℃ in in vivo. The plants exhibited reduced expression of the chlorophyllase genes AtCHL1 and AtCHL2 during freezing stress, resulting in a 70% reduction in chlorophyll damage (Rayirath et al., 2009). This product also protected A. thaliana plants from induced cold stress (-2℃) by enhancing chlorophyll content, possibly due to downregulation of chlorophyll degradation genes (AtCLH1 and AtCLH2) and the upregulation of the transcription factor DREB1A and the COR78/RD29A genes, which encode cryoprotective chloroplast stromal protein – key regulators of cold stress tolerance.
It also improved the survival of in vitro tobacco cells (BY-2) after exposure to freezing temperatures (0, -3, and -5℃). The treatment enhanced cell growth, membrane stability, and nuclear integrity, while reducing cell death of cold-stressed BY-2 tobacco cell lines. This seaweed extract influenced cellular and molecular regulation, triggering mechanisms such as osmolyte accumulation and antioxidant activity to combat freezing stress. This response was associated with the upregulation of key freezing tolerance genes, including galactinol synthase 2, pyrroline 5-carboxylate synthase, and acetyl-CoA carboxylase.
Another alkaline powdered extract of the brown seaweeds A. nodosum, Fuccus spp., and Laminaria spp., demonstrated positive effects on maize plants subjected to low temperatures (12 – 14℃). The combination of these two products reduced leaf necrosis in maize (with only 0-15% of the leaf area affected) and promoted plant growth, particularly in root development. The beneficial effect of the Zn/Mn treatments and seaweed extracts were associated with increased superoxide dismutase (SOD) activity in the root and leaf tissues, playing a key role in defense against oxidative stress, with Zn, Mn, Cu, and Fe serving as essential enzymatic co-factors.The use of a product combining A. nodosum extract with garlic oil improved the quality of Valencia orange fruits during cold storage at 5℃. Specifically, a solution of 1 g/l Cytolan extract combined with 0.5% garlic oil resulted in the lowest percentage of fruit decay, the smallest reduction in total acidity, and the highest total sugar content. Additionally, treating the fruits with a mixture of 0.5% or 1% garlic oil and 3 g/l Cytolan led to the lowest loss of weight (%).
Commercial biostimulants products from terrestrial sources
Biostimulants from terrestrial sources (e.g., plant extracts, agricultural residues, soil microorganisms, and animal protein hydrolysates) have also been tested in plants to induce tolerance to low temperatures (Table 2).
| Biostimulants Source | Tested plant | Temperature | Effect |
| Fermented compounds of vegetables, meltedrice, seaweed, yeast extract and minerals | Leek ( Alliumampeloprasum), Celery( Aptum graveolens), and Parlsey( Petroselinum crispum) | 10 and 15℃ | Increased seed germination |
| Smoked wheat straw | Tomato ( Solanum lycopersicum) | – | Improved tomato growth and yield by regulating nutritional uptake, leaf temperature, photosynthesis, ROS scavenging, and CBF transcriptional activation |
| Enzymatic hydrolysisof porcine hemoglobin – PHH | Lettuce ( Lactuca sativa) | -3℃ | Increased mean fresh and dry weights |
| Tomato ( Solanum lycopersicum) | 11.0℃ to 12.6℃ | Improved root growth due to the extra amino acid availability and the biosynthesis of salicylic acid | |
| Enzymatic hydrolysisof porcine hemoglobin – PHH | Strawberry ( FragariaX ananassa) | -6℃ | More biomass of newly formed roots, earlier flowering and fruit production |
| Amino acid product obtained by enzymatic hydrolysis | Lettuce ( Lactuca sativaL. var. capitata) | Root zone: 6℃, and diurnal colds: 4℃ | Higher recovery of fresh weights, increased plants weight, stomatal openingand transpiration |
| Maize ( Zea mays) | 10℃ | Increased plant performance: leaf gas exchange, photosynthetic pigment content, and chlorophyll fluorescence | |
| Sorghum sp. and Mortnga Sp. | Maize ( Zea mays) | 8-13℃ | Increased crop growth rate, leaf area index, leafarea duration, plant height, grain yield, andtotal dry matter accumulation |
| Piriformospora indica | Arabidopsis thaliana | -6℃ / 4℃ | Reduced negative effects of freezing, increased the amounts of soluble proteins, proline andascorbic acid and stimulated the expression ofseveral genes involved in the CBF-dependent pathway |
| Coffee refuse | Japanese pear cultivars ( Pyrus Spp) | -3℃ | At 2 ppm delayed flower bud freezing by 8hours (vs. 3 hours in controls) and preservedpollen germination under cold stress (-3℃) insensitive cultivars (e.g., ‘Kosui’) |
An enzymatic hydrolyzed porcine hemoglobin – PHH was tested in crop species such as tomato, lettuce, and strawberry plants. In the lettuce experiment, the mean fresh and dry weights of plants subjected to intense cold (-3℃, for 4h) and then treated with PHH at concentrations of 0.4, 0.8, and 1.6 mg/mL H2O were significantly greater than those of untreated plants. In tomato plants, Pepton (430 mg PHH/container) in a hydroponic system at low temperature (water temperature between 11.0℃ and 12.6℃) promoted root growth by day 4 of the experiment, likely due to increased amino acid availability for growth and/or stimulation of specific hormonal pathways, such as salicylic acid. Strawberry plants were exposed to nighttime temperatures below zero, reaching -6℃, showed that the highest concentration of PHH (4 L/ha) resulted in a greater biomass of newly formed roots compared to the 2 L/ha treatment. Furthermore, both PHH treatments (2 and 4 L/ha) stimulated early flowering and significantly enhanced initial fruit production.
Other commercial products found to induce cold tolerance in plants. In lettuce, a foliar application of 3 mL/L led to a greater recovery of fresh weight in both the root zone (at 6℃) and during daytime cooling (at 4℃). Additionally, treated lettuce exhibited significantly higher stomatal conductance at the onset of cold stress, resulting in enhanced stomatal opening and increased transpiration, which promotes the flow of water and nutrients from roots to shoots – an effect likely due either to the direct action of amino acids on stomata or to improved plant water status via stimulated root growth. When applied to maize plants grown at 10℃, Foliar improved leaf gas exchange parameters by 22-23% and increased photosynthetic pigment content by around 15%, further supporting its potential to enhance recovery and overall cold tolerance in crops.
A fermented product of vegetables, seaweed, and yeast, when applied in combination with humic acid, it resulted in enhanced seed germination of leek, celery, and parsley across all temperature treatments, including 10℃ and 15℃. In addition to commercial products, other plant extracts have also been tested for chilling and freezing tolerance. A mix of two plant extracts (sorghum and moringa) combined with salicylic acid and thiourea was tested in maize plants and demonstrated stress-alleviating effects by improving physiological and biochemical attributes, as well as overall maize growth, under suboptimal temperature stress (8-13℃). Furthermore, the co-cultivation of the endophytic root fungus Piriformospora indica with A. thaliana, which was exposed to -6℃ for 6 h, showed reduced negative effects of freezing. The plants exhibited higher levels of soluble proteins, proline, and ascorbic acid during the post-thaw recovery period at 4℃ for 12 h.
A coffee-derived extract with anti-ice-nucleation properties, was evaluated at 2 ppm for mitigating cold-induced damage in the Japanese pear cultivars (Pyrus spp.) ‘Kosui’ and ‘Hosui’. When applied during the scale-separation stage (from pollen mother cell to tetrad phase), the extract delayed the onset of flower bud freezing by 8h, compared to just 3h in water-treated controls. This intervention preserved pollen germination rates under low-temperature stress (-3℃ for 10h), attributed to reduced cellular dehydration and membrane disruption. Notably, ‘Hosui’ cultivar exhibited inherent cold tolerance, maintaining germination rates even at -6℃, highlighting its potential as a cost-effective commercial solution for cold stress management in orchards.
An interesting group of natural plant growth regulators called karrakins (KAR), found in smoke from wheat straw, also has the ability to induce tolerance to low temperatures in plants. When applied alongside smoked water (SW) on tomato plants, KAR improved growth and yield under suboptimal temperature conditions by regulating nutrient uptake, leaf temperature control, photosynthetic defense mechanisms, reactive oxygen species scavenging, and the transcriptional activation of C-repeat binding factors (CBF). Thus, SW, which operates through the KAR-mediated strigolactones (SLs) and ABA signaling network, holds potential for enhancing cold tolerance in tomato production.
Non-commercial biostimulants products
In addition to established commercial formulations, scientific research has increasingly explored naturally derived and non-commercial blends for their potential to enhance cold tolerance in plants (Table 3). A recent review highlighted the use of nanobiotechnology and bacteria to promote cold tolerance in crop species. To better understand the current landscape of developing new products that induce cold tolerance in plants, it is relevant to expand the focus include other alternatives, such as crude algal extracts, plant-based extracts, and multi-component botanical and/or microbial mixtures. Although these formulations are not yet standardized or commercially available, they have been investigated for their ability to induce cold tolerance in crops.
| Biostimulants | Source | Tested plant | Temperature | Effect |
| Brown seaweed extract | Non-identified brown seaweed | Tomato (Solanum lycopersicum) | 4℃ | Higher stomatal conductance, net photosynthesis, and yield in plants treated with the biostimulant. Biochemical improvements were corroborated bymolecular data |
| Hot water | Water | Tomato fruits (S. lycopersicum) | 5℃ | The treatment with HWT(42℃/5 min) induced better metabolic performance oftomato fruits under cold storage. The reported effeect was associated with a higher accumulation of antioxidants and osmolytes |
| CRDAP-ZNC composite | Diammonium phosphate and Paecilomyces wariofii extract | Winter wheat (Triticum aestivum L.) | Near 5℃ (most of the time in the first 10 days) | Enhanced cold tolerance, with 22% highersoil available phosphorus, 30% increased root growth, up to 17% higher photasynthesis, and 8.6% reduction inH2O2 content |
| Moringa leaf extract | Moringa oleijera | Spring hybridnaize (Zeamays L.) | 5 – 10℃ | The seed priming enhanced stand establishment and reduced chilling damage. The 20-day-old transplanted seedlings showed improved agronomic traits, yield, and quality compared to direct sowing and 30-day-old transplants |
| Trichoderma harzianum seed inoculant | T.harzianum Rifai strain T.22 (American Type) | Tomato ( S.tycoperstcum) cv. Jubilee | 10 土 0.2 ℃ | Accelerated germination speed of seedsunder chilling stress |
| T. harzianum strain AK20G isolated from soil | Tomato (S. tycopersicumL) Cv.CaljN3 | 8℃ | Inoculated plants restored the photochemical PS Il efficiency, plant growth and electrolyte leakage percentage under cold stress. Relative expression rates of Tas14 and PSCS genes were significantly higher in treated plantsthan control | |
| T.harzianum | Maize (Zea mays L.) | 5℃ | Seed priming enhanced cold tolerance, improved seedling emergence, root dry weight, and catalase activity, particularly inthe cold-resistant cultivar AR68. | |
| Various fungal isolates | Brassica oleracee endophytic fungal | Kale (B. oleracea var. acephala) | 12℃ | Inoculation with Phialocephala sp., Fusariums., and Acrocalymma sp. increased plantgrowth and weight. |
Studies have investigated the role of Bacillus spp. in mitigating cold stress, demonstrating their ability to enhance plant resilience through multiple mechanisms. For instance, cold-tolerant strains such as Bacillus GBAC46 and RJGP41 promote growth by increasing the IAA biosynthesis and the activity of enzymes like SOD, CAT, and APX. Additionally, their volatile organic compounds (VOCs) improve root development and upregulate stress-related genes. Similarly, Lysinibacillus fusiformis and L. sphaericus enhance phosphorus solubilization and osmolyte accumulation (proline and glycine betaine), which improves photosynthesis and membrane stability under low temperatures. Furthermore, the synergistic action of Bacillus subtilis and Piriformospora indica further strengthens cold tolerance by enhancing biomass production, enzymatic defenses, and stress-related gene expression. These findings highlight Bacillus spp. as key contributors to plant adaptation under cold stress conditions.
In the study, a biostimulant derived from an unspecified brown alga species was evaluated in tomatoes exposed to low temperatures. Physiological and molecular analyses were conducted, revealing that stomatal conductance, net photosynthesis, and yield were significantly higher in the plants treated with the biostimulant compared to the untreated plants. Additionally, the molecular analysis indicated that the extract resulted in increased cellular contents of proline, polyphenols, flavonoids, tannins, and carotenoids.
In a postharvest study, Delgado-Vargas et al. evaluated the effects of hot water treatment (HWT – 45℃/5 min) on tomato fruits under chilling conditions (5℃/20 days). HWT-treated fruits exhibited reduced chilling injury (CI), higher total phenolics (TP), and greater antioxidant activity (AoxA) compared to controls. They also showed increased accumulation of phenolics, sugars, and certain alkaloids, potentially mediated by azelaic acid, glutamine, and tryptophan. Contrarily. N-feruloylputrescine, esculeoside AII, and hydroxy-α-tomatine II contents decreased. The improved metabolic performance of HWT-treated tomatoes during cold storage was linked to the accumulation of antioxidant and osmolyte compounds. Identifying metabolites associated with CI reduction enhances the understanding of tolerance mechanisms and offers targets for CI prevention, such as genetic improvements or direct metabolite application.
Chen et al. investigated the cold tolerance response of winter wheat (Triticum aestivum L.) treated with a composite of controlled-release diammonium phosphate (CRDAP) and the extract of the fungus species Paecilomyces variotii (ZNC). Under spring low-temperature stress, the composite synchronized phosphorus release with crop demand, boosting soil available phosphorus by 22% in the top 40 cm, enhancing root growth by nearly 30%, and elevating photosynthetic rates by up to 17% compared to conventional fertilizers. Moreover, there was an increased biosynthesis of IAA in roots and strengthened antioxidant defenses, with an 8.6% reduction in hydrogen peroxide (H2O2) concentration, further improving cold resilience.
Mastouri et al. investigated the effects of Trichoderma harzianum T22 seed treatment on tomato seeds under chilling stress. The results showed that T22-treated seeds germinated significantly faster than untreated seeds, even when exposed to chilling stress at 10℃ for up to 3 days. Although T22 treatment accelerated germination speed, the final germination percentage did not differ significantly from that of the control seeds. This positive effect of T22 on germination under thermal stress suggests that the application of this fungal species may trigger a physiological response in seeds, enhancing germination speed under cold conditions without negatively affecting the final germination rate.
In the study conducted by Poveda et al., the inoculation of kale plants with various fungal isolates, including Pyrenophora sp., Fusarium sp., Phialocephala sp., Chaetomium sp., Diaporthe sp., and Acrocalymma sp., significantly increased plant growth and tolerance under cold stress (12℃). Inoculation with Phialocephala, Fusarium sp., and Acrocalymma sp. notably enhanced plant weight, with an almost twofold increase compared to the control. Similarly, dry weight also increased in the inoculated plants, highlighting the positive impact of fungal inoculation on cold tolerance.
Afrouz et al. determined the effect of seed biopriming with Trichoderma harzianum on maize tolerance to cold stress. The results showed that biopriming significantly enhanced seedling emergence and physiological parameters, particularly under cold conditions (5℃). Among the pretreatments, T. harzianum resulted in improved root dry weight and increased catalase activity in the maize cultivars investigated, with the highest root dry weight detected in the cultivar AR68. These findings suggest that T. harzianum priming effectively enhances tolerance in maize.
In the study by Ghorbanpour et al., the inoculation of T. harzianum AK20G strain in tomato seeds was investigated for its role inducing cold tolerance in plantlets exposed to 8℃ for 6 days. The results showed that T. harzianum effectively mitigated the adverse effects of cold stress by enhancing photosynthesis and growth rates, reducing lipid peroxidation, and minimizing electrolyte leakage. Additionally, this fungal species improved leaf water content and proline accumulation while promoting the expression of genes involved in cold stress tolerance, such as TAS14 and P5CS.
Junaid et al. evaluated the effect of moringa leaf extract (MLE – Moringa oleifera) in maize (Zea mays L.) exposed to low temperatures (5℃-10℃) during early seedling development. Seed priming (24 h) with 3% MLE improved chilling tolerance by enhancing the stand establishment and increasing both emergence percentage and speed. The greatest improvements in agronomic traits, yield, and quality were observed in 20-day-old transplanted seedlings, while direct sowing and 30-day-old transplants showed inferior performance. These results highlight the potential of MLE priming to mitigate chilling stress and enhance maize productivity under temperature fluctuations.
Other exogenously applied cold and chilling tolerance inducers
Since commercial products or natural extracts that effectively induce tolerance to cold, chilling, or freezing effects are scarce, some isolated compounds with the intended biological effect have been investigated, yielding interesting results (Table 4).
| Compounds | Tested plant | Temperature | Effect |
| ABA | Rice (Oryza sativa) and weedy rice (O.sativa f. spontanea) | 5℃ | Reduced the chilling damage by activating antioxidant enzymes |
| Grapevine (Vitis vinifera) | 4℃ and 14℃ | Increase the expression of promote freezing tolerant proteins | |
| Mangrove (Kandelia obovata) | 9.7℃, -4.1℃ and -5.3℃ | Alleviated the effects of freezing stressdue to the enrichment of arginine and proline, starch and sucrose metabolism | |
| Banana (Musa spp.) and tomato (Solanum lycopersicum) | 4℃ | Alleviated chilling injury byaccumulation of endogenous A BA, unsaturated fatty acids, and flavonoid content, and reduced the saturated fatty acid content | |
| ABA and GB | Arabidopsis thaliana | -3.1℃ to -4.5℃ | Increased freezing tolerance and endogenous glycine betaine(GB) content |
| GB | Maize (Zea mays) | 15℃ | Improved germination scores, solublesugars and antioxidants |
| Tomato (S. lycopersicum) | 14℃ | Increased the seed germination scoresand regulate gibberellin (GA) andabscisic acid (ABA) | |
| SA | Maize (Zea mays) | 2℃ | Upregulation of antioxidant enzymes |
| Tomato (Solanum lycopersicum) | 5℃ | Induced the synthesis of stress-related proteins | |
| Maize (Zea mays) | 5℃ | Inhibition of catalase activity | |
| Peach (Prunus persica) | 0℃ | Chilling tolerance by upregulation of aheat shock protein (sHSP) expression | |
| Anthurium andraeanum | 4℃ and 12℃ | Suppressed chilling injury by maintaining high contents of antioxidants and membrane integrity | |
| Lemon fruit (Citrus limon) | -2.5, 2℃ and 4.5℃ | Increased total phenolics and antioxidant enzyme activity | |
| Apple (Malus domestica) | 8℃ | Enhanced antioxidant enzymes expression and endogenous SA | |
| Wheat (Triticum aestivum) | -7℃ | Increased freezing tolerance by improved in photochemical efficiency, accumulation of osmo-protectant and antioxidant compounds | |
| Maize (Zea mays) | 4℃ | Increased cold tolerance by lowering MDA concentration and increasing antioxidant contents, besides osmotic adjustment associated with augmented proline amounts | |
| SA, AsA and H2O2 | Maize (Zea mays) | ＞2.9℃ (early stages) | Improved seed growth, leaf relativewater, chlorophyll b contents, membrane stability, and enzymatic antioxidant activities |
| MT | Bermudagrass (Cynodon dactylon) | 4℃/-5℃ and -5℃ | Increased cold tolerance by lowering MDA and electrolyte leakage (EL) and increase antioxidant enzymes |
| Maize (Zea mays) | 5℃ | Improved germination under coldstress, proteomic changes linked tostress tolerance | |
| Waxy maize (Zea mays) | 13℃ | Enhanced seed vigor, root growth, antioxidant enzyme activity, and starch metabolism while reducing oxidative damage | |
| Tea (Camellia sinensis) | 4℃ | Enhances cold tolerance by lowering MDA level, increasing photosynthetic efficiency, alleviating ROS burst and up-regulate expression of antioxidant enzyme | |
| Barley (Hordeum vulgare) | 5℃ and 15℃ | Alleviate growth inhibition of seedlings, restored circadian rhythmic oscillation and lowers MDA and soluble sugars level | |
| PA | Wheat (T. aestivum L.) | 4℃ | Shoot and root dry weight, chlorophyll, carotenoid, proline contents, catalase and ascorbate peroxidase activities were improved. |
| Apple (M. domestica) | -10℃ to -25℃ | Spermidine improved cold tolerance by stabilizing tissues, reducing ROS effects through increased proline content and antioxidant enzyme activity, besides enhancing polyamine metabolism |
Table 4. Compounds with biostimulants effect that induces chilling or frozen tolerance in plants.
The plant phytohormone ABA has been demonstrated to be a key regulator of plant responses to abiotic stresses, including chilling and freezing. ABA application reduced the chilling damage (5℃) in four rice genotypes. Pre-treatment with ABA decreased the levels of superoxide anion (O2-), H2O2 and malondialdehyde (MDA) caused by chilling stress through increasing the activities of SOD, CAT, APX, glutathione reductase and the contents of ascorbic acid and glutathione. ABA also alleviated chilling injury of banana fruits, by inducing the accumulation of endogenous ABA, (un)saturated fatty acids, and flavonoids, by upregulating the transcription levels of MaABI5-like, fatty acid desaturation genes, and flavonoid synthesis-related genes during cold storage. The application of ABA (26 mg/mL) to dormant buds of grapevine (Vitis vinifera) increased the expression of the CBF/DREB1, VvCBF2, VvCBF3, VvCBF4, and VvCBF6 transcription factors. Others describe that applying ABA in the mangrove species Kandelia obovata under natural frost conditions at approximately 32°N latitude effectively alleviated the adverse effects of freezing stress. This was achieved by activating antioxidant enzyme activity and increasing the accumulation of osmolytes, such as proline. Notably, the effectiveness of these responses was proportional to the concentration of ABA applied.
Another interesting effect of the exogenous application of ABA is the increase in endogenous concentrations of glycinebetaine (GB). GB is an amino acid that helps protect plants against abiotic stresses through osmoregulation or osmoprotection, and it contributes to the differential expression of stress tolerance-related genes. When applied in isolation, GB has demonstrated various effects on cold or freezing tolerance in plants. For instance, a 10 mM GB foliar spray on Arabidopsis tahaliana increased the freezing tolerance of plants to -4.5℃. Similarly, maize seeds treated with GB significant improvements in germination rate, root and shoot length, seedling fresh and dry weight, leaf and root scores, soluble sugars, α-amylase activity, and total antioxidants capacity compared to untreated seeds under both optimal (27℃) and stress conditions (15℃). GB-treated tomato seeds (10 mmol/L) also exhibited better germination rates under cold stress (14℃). Analysis of gene expression and metabolism revealed that GB positively regulated endogenous gibberellin content, while the opposite effect was observed for abscisic acid content. Moreover, GB reduced the starch content in the tomato seeds by upregulating amylase gene expression.
Another important compound with recognized tolerance-inducing activity in plants is SA. Under low-temperature stress conditions (2℃), the application of 0.5 mM SA led to the upregulation of antioxidant enzymes, including APX, SOD, guaiacol peroxidase (GPOX), and GR. It also inhibited the activity of isozymes CAT-1 and CAT-2 in Z. mays. The application of SA at 50 mg/L on the leaves of Z. mays seedlings under 4℃ increased the antioxidant activities of APX, CAT, SOD, and peroxidase (POD), which decreased relative electrolyte conductivity (REC) and the levels of MDA and ROS (H2O2 and O2–). Additionally, there was an increase in proline content and relative water content (RWC) in the maize seedlings, enhancing their osmotic adjustment capacity. High activities of CAT and SOD were also found in cut anthurium flowers (Anthurium andraeanum) subjected to cold stress (4℃ to 12℃), resulting in detoxification of ROS and maintenance of membrane integrity.
In apple plants (Malus domestica), SA increased the expression of cytosolic malate dehydrogenase and improved the redox state of the plant cell. Furthermore, transgenic plants that overexpressed cytosolic malate dehydrogenase (MdcyMDH) demonstrated greater tolerance to cold stress compared to the wild type, as they produced higher amounts of free and total amino acids.
The application of SA also aids in post-harvest activities by increasing the chilling tolerance of fruits during cold storage through the upregulating of stress-related proteins. Low concentrations of methyl salicylate (MeSA), i.e., 0.01 mM, helped protect tomato fruits (S. lycopersicum) in cold storage (5℃) by inducing the synthesis of certain stress-related proteins, such as the PR proteins PR-2b and PR-3a mRNAs, while also slightly increasing PR-3b mRNA accumulation. MeSA treatment on peaches fruits (Prunus persica) during cold storage (0℃) helped overcome chilling stress by upregulating the expression of small heat shock protein (sHSP) genes, ultimately leading to the production of sHSP, particularly HSP17.6. Furthermore, a concentration of 2.0 mM SA-mediated enhanced the synthesis of total phenolics and increased the activity of phenylalanine ammonia-lyase, thereby improving chilling tolerance in cold-stored lemon fruit.
The study by Wang et al. investigated the effects of cold and SA on the accumulation of osmolytes in wheat leaves under freezing stress. The results indicated that both treatments significantly increased the levels of sucrose and free proline, improving the leaf’s water potential and reducing cell death, which enhanced tolerance to freezing. The accumulation of proline was promoted by both increased synthesis and inhibition of its degradation. Additionally, cold and SA stimulated the synthesis and hydrolysis of sucrose, positively regulating glucose catabolism and ammonia assimilation. These findings suggest that both treatments favor the accumulation of proline and sucrose, coordinating carbon and nitrogen metabolism to confer resistance to freezing stress.
The combination of SA with other compounds has also yielded interesting results. When SA was applied alongside AsA and H2O2, it improved seedling growth, leaf relative water content, chlorophyll b levels, membrane stability, and enzymatic antioxidant activities in pot-cultivated maize. In field experiments, the application of these substances – either through seed priming or foliar spray – enhanced morphological and yield-related attributes, as well as grain yield of spring maize under suboptimal temperatures in the early stages (>2.9℃). Notably, seed priming proved to be more effective than foliar application.
Priming treatments have also been shown to induce cold tolerance in plants. For instance, melatonin (MT) priming in maternal wheat plants (T. aestivum) during the grain filling stage promoted seed germination at 10℃ in offspring. This effect was attributed to accelerated starch degradation and improved cold tolerance of the seedlings (10℃ day; 6℃ night) through the activation of antioxidant enzymes and enhancement of photosynthetic electron transport efficiency.
The application of MT has been linked to specific mechanism of cold tolerance, such as a decrease in MDA levels and electrolyte leakage (EL). Additionally, it has associated with increased amounts of chlorophyll and enhanced enzymatic activity (e.g., SOD and POX), as well as changes in 46 metabolites when exogenously applied to bermudagrass plants (Cynodon dactylon). Among the measured metabolites, five sugars (arabinose, mannose, glucopyranose, maltose, and turanose) and one organic acid (propanoic acid) were significantly increased. Tea plants (Camellia sinensis) treated with MT on their leaves exhibited a reduction in ROS burst, decreased MDA levels, and maintained high photosynthetic efficiency. Moreover, these tea plants demonstrated elevated levels of GSH and AsA, along with increased activities of SOD, POX, CAT, and APX under cold stress (4℃). Notably, MT treatments can positively upregulate the expression of genes involved in the biosynthesis of antioxidant enzyme, specifically CsSOD, CsPOX, CsCAT, and CsAPX.
In a similar study, maize seed priming with MT (50 and 500 μM) enhanced stress tolerance by improving germination under suboptimal thermal conditions (i.e., 5℃) and modifying the seed proteome. Proteomic analysis revealed MT-induced changes in protein expression related to stress response, suggesting a biochemical mechanism for enhanced chilling tolerance. These findings indicate that MT priming can be an effective strategy to improve maize resilience under low-temperature conditions.
In another priming study, Cao et al. demonstrated that MT (50 and 100 μM) improved maize germination under chilling stress at 13℃ by increasing germination potential, seed vigor, and root growth. MT-treated seeds exhibited reduced oxidative damage (lower H2O2 and MDA levels) and enhanced antioxidant enzyme activity (SOD, POX, CAT, APX), along with improved starch metabolism, further supporting its role in cold stress tolerance.
Additionally, the exogenous application of MT could restore the rhythmic circadian oscillation of clock genes, such as HvCCA1 and HvTOC1, in barley (Hordeum vulgare) seedlings, whose rhythmic phenotypes were disrupted due to environmental cold stress (5℃ and 15℃). The results also confirmed that exogenous MT reduced the accumulation of key physiological indicators under cold stress, including MDA and soluble sugars.
Polyamines represent an important group of compounds known for their effects on enhancing cold tolerance in plants. A recent review summarized the benefits of exogenous polyamine applications on plant stress responses to cold. Here, we briefly highlight a few recent studies. For instance, Gholizadeh et al. reported increases in morphophysiological and biochemical parameters in two wheat cultivars (‘Mihan’ and ‘Rakhshan’) grown under cold stress (4℃) when treated with putrescine and spermidine. The authors observed augmented activity of antioxidant-related enzymes such as catalase (CAT) and ascorbate peroxidase (APX), with concomitant upregulation of genes involved in polyamine synthesis (e.g., TaADC, TaSAMDC, and TaSPDS) and catabolism (TaPAO11-7B and TaPAO11-7D). Similarly, He et al. demonstrated that exogenous spermidine enhanced cold resistance in 1-year-old apple branches by stabilizing tissue, reducing reactive oxygen species through increased proline content, and boosted antioxidant enzyme activity. They also noted the upregulation of key polyamine metabolism genes (MdADC1, MdSAMDC1, and MdSPDS1), alongside the cold-responsive genes (MdCBF1/2/3, MdCOR47, and MdKIN1). Collectively, these findings indicate that exogenous polyamines modulate metabolism and enhance antioxidant defenses, thereby improving cold tolerance in diverse crops.
Conclusion
Bioactive elicitors biostimulants from macro- and microalgae enhance plant resilience to cold by activating natural defense mechanisms, promoting the expression of cold tolerance genes, and improving physiological processes. Commercial products derived from macroalgae, particularly Ascophyllum nodosum, are among the most widely used and show promise in boosting cold tolerance by increasing gene expression, reducing chlorophyll damage, and stimulating plant growth. Similarly, plant-derived biostimulants, such as extracts and soil microorganisms, can promote resilience in crops, by enhancing nutrient uptake, improving overall plant health, and stimulating natural defense mechanisms.
However, several potential research gaps remain regarding broader applicability, long-term effects, optimal dosages, and practical implementation in agricultural settings. Further investigation of biostimulants in this area is essential to develop more effective strategies for mitigating cold stress in crop systems. This research could ultimately bridge the gap between laboratory findings and practical agricultural solutions for challenging climates.
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