Eﬀects of the Protein Hydrolysate Pretreatment on Cucumber Plants Exposed to Chilling Stress

is study aimed to evaluate the eﬀects of the protein hydrolysate Naturamin WSP on the antioxidant defense system and oxidation-related damage of young cucumber plants exposed to chilling stress. Low positive temperatures have a negative eﬀect on plant growth and performance, and besides visible alterations, such as inhibited growth, significant changes occur at the cellular level. Plants grown at low temperature typically suﬀer from oxidative damage, which leads to increased lipid peroxidation. Moreover, chilling-stressed plants accumulate more proline to protect their cell membranes. e application of biostimulants such as the protein hydrolysate Naturamin WSP can alleviate some of the adverse eﬀects caused by low temperature. Our results indicated an increased activity of guaiacol peroxidase (GPOD) in all plants treated with the biostimulant regardless of the temperature of cultivation. e mitigation of damages caused by chilling stress might be explained by an enhanced anti-oxidative defense, as demonstrated by the activity of guaiacol peroxidases and increased proline concentrations in Naturamin WSP-treated plants.


Introduction
Cucumber (Cucumis sativus L.) is an intensively cultivated salad crop worldwide. It is a cold-sensitive plant that suffers from low nonfreezing temperatures of approximately and below 10°C (Shibaeva et al., 2018). Under the climatic conditions of Bulgaria, cucumber plants are exposed to chilling stress during winter months. Chilling stress could severely inhibit the growth and development of cucumber. Visible symptoms of chilling injuries include wilting, leaf and internal tissue discoloration, surface pits, and leaf necrosis (Lukatkin et al., 2012). e extent of the damage caused by chilling stress depends on temperature, duration of exposure, plant developmental stage, and other factors (Borowski, 2009;Phansak et al., 2021). e most frequently reported chilling-provoked changes at the cellular level include alterations in gene expression, protein turnover, lipid and carbohydrate composition, membrane permeability, and solute leakage (Kuk & Shin, 2007). Some of these changes and damages can be attributed to chilling-induced oxidative stress in the plant cells (Gill & Tuteja, 2010;Lukatkin, 2002).
Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and their quenching in cells. Plant cells possess an effective anti-oxidative defense system that involves both nonenzymatic and enzymatic antioxidant components (Dumanović et al., 2021;Noctor & Foyer, 1998). Insufficient inactivation of ROS leads to oxidative damage, which affects macromolecules and biological structures. Furthermore, such damage oen results in enhanced lipid peroxidation and electrolyte leakage from the cells.
Currently, two main approaches are used to increase the tolerance of cucumber plants to chilling stress: (i) graing on rootstocks from more chilling tolerant plant species such as pumpkin (Fu et al., 2021;Xu et al., 2017) and (ii) application of different products such as plant growth promoters, plant growth regulators, and biostimulants, with anti-stress properties (Borowski, 2009;Ghanbari & Kordi, 2019;Zhao et al., 2017).
e European Biostimulant Industry Council (EBIC) defines biostimulants as fertilizing products used for stimulating plant nutrition, apart from the nutrient content of the product, to improve one or more of the following characteristics of plants or plant rhizosphere: (i) nutrient use efficiency, (ii) tolerance to abiotic stress, (iii) quality, or (iv) availability of confined nutrients in the soil or rhizosphere (European Biostimulants Industry Council, 2019;Ricci et al., 2019).
Protein hydrolysates (PH) are one of the most common groups of biostimulants containing "mixtures of free amino acids, low molecular weight peptides, and other nitrogen-containing organic substances" . In recent years, the number of publications on the protective effects of protein hydrolysates on plants exposed to different stress factors has increased significantly Nardi et al., 2016). However, information regarding the effects of protein hydrolysates on plants exposed to chilling stress is very limited. Recently, Cholakova-Bimbalova et al. (2019) reported that the application of a protein hydrolysate increased the photosynthetic performance of maize plants exposed to chilling stress.
One possible explanation for the protective effects of protein hydrolysates is related to the role of free amino acids and low-molecular-weight peptides as signal molecules and precursors for essential metabolites. Teixeira et al. (2017) showed that aer foliar application of free amino acids (phenylalanine, cysteine, and glycine) to young soybean plants, the activities of some key antioxidative enzymes, such as superoxide dismutase, catalase, and peroxidase, increased significantly. However, the present information regarding the mode of action of protein hydrolysates in stress-exposed plants is still insufficient. e aim of the current study was to evaluate the effects of a protein hydrolysate on cucumber plants exposed to chilling stress, with special attention paid to their antioxidant defense system and oxidation-related damages.

Growth Conditions and Experimental Design
Cucumber plants (Cucumis sativus L. 'Kaliopa') were grown in climatized growth chambers at the Department of Plant Physiology, Biochemistry, and Genetics, Agricultural University, Plovdiv. e seeds were imbibed in distilled water in a Petri dish for 24 hr and then sown in pots filled with perlite enriched with a modified Hoagland nutrient solution (1/2 strength). e volume of each pot was 2 L. e plants were cultivated in a controlled environment: photoperiod, 14/10 hr day/night; PPFD, 200 μmol m −2 s −1 ; temperature, 25 ± 1°C / 20 ± 1°C (day/night), and relative air humidity, 60% ± 5%. Half of the plants were sprayed with 0.1% water solution of the biostimulant Naturamin WSP (a protein hydrolysate; Daymsa, Spain) when the first true leaf reached full development (18 days aer germination). e concentration was chosen according to the manufacturer's recommendations. Control plants were sprayed with distilled water.
Aer treatment, the plants were transferred to two growth chambers with different temperature regimes and identical light and air humidity conditions. e experimental design of the study included four variants: e plants were grown under the aforementioned temperature regimes for 5 days and then cultivated at an optimal temperature (25°C) for 7 days. Each variant had three replicates (pots), with three plants per pot. e duration of the experiment was 12 days. Biochemical analyses were performed on the cucumber plants at the end of the chilling exposure period and aer 7 days of recovery. e experiment was repeated twice.

Plant Growth Analysis
e plants were harvested at the end of the experimental period and their fresh biomass and leaf area were determined. e leaf area of the plants was measured using a leaf area meter (NEO-2; Technical University, Bulgaria).

Soluble Protein Determination
Soluble protein content was determined according to Bradford (1976) using a standard curve prepared with bovine serum albumin and Coomassie blue, which reacts with basic amino acid residues, especially arginine, in response to protein concentration. Fresh plant samples (0.5 g) were ground with quartz sand, 5 mL ice-cold extraction buffer (pH 7.8), and 200 mg PVP (polyvinylpyrrolidone). Samples were centrifuged at 4°C at 13,500 rpm for 10 min. e clear supernatant was used for analysis immediately. Absorbance was measured spectrophotometrically at 595 nm.

Guaiacol Peroxidase (GPOD) Activity
Guaiacol peroxidase (GPOD) activity was determined spectrophotometrically according to Bergmeyer et al. (1974). e reaction mixture, which contained 2.3 mL of KH 2 PO 4 buffer (pH 7.0), 300 μL of H 2 O 2 , 300 μL of 8 mM guaiacol, and 100 μL of extract, was placed in a cuvette. Absorbance was measured at 436 nm against a blank with the same components but without the tissue extract (E = 26.6 mM cm −2 ). e results are expressed as U mg g −1 fresh weight (FW).

Lipid Peroxidation
Lipid peroxidation was estimated according to the method of Heath and Packer (1968) by determining thiobarbituric acid (TBA) reactive compounds. e plant leaves were homogenized in 0.1% (w/v) trichloroacetic acid (TCA) and the homogenate was centrifuged at 10,000 rpm for 20 min. One milliliter of the supernatant was mixed with 4 mL of TCA (20%) containing 0.5% w/v of TBA. e mixture was heated at 95°C for 30 min, cooled on ice, and centrifuged at 10,000 rpm for 15 min. e absorbance was measured at 532 nm and corrected at 600 nm. e concentration of TBA reactive compounds was calculated using the 155 mM cm −1 extinction coefficient.

Proline Content
Proline content was determined colorimetrically using Toluol according to Bates et al. (1973). One gram of the primary leaves was homogenized with 3% sulfosalicylic acid and centrifuged at 10,000 rpm for 10 min. e absorbance of each extract was measured spectrophotometrically at 520 nm.

Statistical Analysis
Statistical analysis of the data was performed using one-way ANOVA (for p < 0.05) and main comparison at a 95% confidence level was performed using Tukey's test.

Results
As expected, the cucumber plants exposed to chilling stress showed retarded growth and partial wilting. e results of the biometric parameters are presented in Table 1. e untreated plants grown under the chilling temperature regimes had the lowest fresh biomass and leaf area (Variant 2). e plants exposed to chilling stress and pretreated with Naturamin WSP (Variant 4) showed relatively higher values of fresh weight and leaf area than the plants of Variant 2. A similar trend was observed aer the recovery period. e values of the fresh biomass and leaf area of plants grown at 25°C and treated with Naturamin WSP were significantly higher (Variant 3) than those of other variants.
e soluble protein contents in the leaves of cucumber plants are presented in Table 2. Exposure of the plants to chilling stress reduced the protein content in their leaves (Variant 2) compared to the leaves of control plants (Variant 1). e reductions were 6% and 9% during the chilling stress and recovery periods, respectively.
Pretreatment with the biostimulant Naturamin WSP preserved, to some extent, the protein content in the leaves of plants exposed to chilling stress (Variant 4). eir protein content was significantly higher (by 10%) compared to that of untreated plants exposed to chilling stress (Variant 2) at the end of the stress period. However, pretreatment with the biostimulant increased the protein content in the leaves of plants grown at 25°C (Variant 3) by 7%-19%.
Chilling stress significantly increased the content of proline ( Pretreatment with Naturamin WSP increased the proline content in plants exposed to chilling stress (Variant 4). It was 17% higher than that of plants exposed to chilling stress without Naturamin WSP treatment (Variant 2). e proline content in the leaves of nonstressed plants pretreated with the biostimulant (Variant 3) was higher than that in the control plants but lower than that in plants exposed to chilling stress. e proline accumulation observed in the leaves of cucumber plants during the chilling period was similar to that observed at the end of the recovery period.    e effects of chilling and biostimulant pretreatment on lipid peroxidation in leaves are presented in Table 4. e lowest content of TBA-reactive compounds was found in the leaves of the control plants (Variant 1). Aer the 5-day chilling period, the content of TBA-reactive compounds in the leaves was 75% higher (Variant 2) than that in the control (Variant 1). In the plants exposed to chilling stress, a higher level of TBA-reactive compounds was observed during the recovery period.
e content of TBA reactive compounds was higher in the leaves of plants exposed to chilling stress and pretreated with Naturamin WSP (Variant 4) than that in the leaves of control plants but was significantly lower (23%) than that in the leaves of Variant 2. Interestingly, the application of the biostimulant enhanced the content of TBA reactive compounds in the leaves of plants grown at 25°C by 26% (Variant 3), but the effect reduced during the recovery period (11%).
e GPOD activity in the leaves of cucumber plants is presented in Table 5. Chilling stress more than doubled the GPOD activity of Variant 2 compared to that of the control (Variant 1). is effect was maintained during the recovery period. Interestingly, the foliar application of Naturamin WSP considerably enhanced GPOD activity compared to the variants without Naturamin WSP treatments (Variants 1 and 2) regardless of the temperature regime (Variants 3 and 4).

Discussion
is study aimed to evaluate the effects of the protein hydrolysate Naturamin WSP on the growth and performance of cucumber plants exposed to chilling stress. e results showed that a 5-day-long chilling stress caused significant growth retardation (Table 1) and oxidation-related damage to the leaves of the plants. Lower biometric parameters (Table 1), reduced leaf protein content (Table 2), and enhanced lipid peroxidation rate (Table 4) were indicators of this damage in cucumber plants exposed to chilling stress. ese results agree with earlier data on the effects of chilling stress on cucumber plants (Borowski, 2009;Ghanbari & Kordi, 2019;Shibaeva et al., 2018). In addition, our study demonstrated that the pretreatment of the cucumber plants with the biostimulant Naturamin WSP alleviated, to some extent, the negative effects of chilling stress on plant growth (Table 1) and affected several biochemical parameters ( Table 2-Table 5). ese observations corroborate the results of Botta (2013) and Cholakova-Bimbalova et al. (2019), who reported a positive influence of foliar amino acid application on lettuce and maize plants exposed to chilling stress, respectively.
Chilling stress can cause several morphological and functional disorders in plants. e most frequently reported negative effects of chilling stress are loss of membrane integrity and solute leakage, which are common consequences of oxidative stress in plant cells (Gill & Tuteja, 2010;Lukatkin, 2002). erefore, it seems reasonable to explain the observed positive effects of the biostimulant Naturamin WSP, mainly through the support of the cellular antioxidative defense system of cucumber plants exposed to chilling stress.
Peroxidases are well-known enzymes whose activities are enhanced in plants under oxidative stress. ey detoxify H 2 O 2 using a vast array of aromatic substrates. e GPOD activity in the leaves of cucumber plants exposed to chilling stress was strongly enhanced (Table 5). is stimulation may be explained by the possible upregulation of enzyme gene expression, which has been recently demonstrated in cucumber plants exposed to chilling stress (Pan et al., 2020).
Different plant biostimulants, including protein hydrolysates, have antioxidant properties (Tkaczewska et al., 2020), and they can modulate plant anti-oxidative defense network. Teixeira et al. (2017) revealed that the foliar application of single and combined amino acids can act as signaling molecules in soybean plants, and the application of small doses was sufficient to increase the activity of antioxidative enzymes. Ertani et al. (2013) and Rouphael et al. (2017) reported that protein hydrolysates can modulate peroxidase activity, mitigating the growth inhibition of salt-and alkaline-stressed plants. erefore, the biostimulant-induced increase in GPOD activity in the cucumber plants could be explained by the aforementioned properties of protein hydrolysates.
Plants accumulate water-soluble compounds with low molecular weights, called osmolytes, to overcome the negative effects of different environmental stress factors. is group of substances includes betaines, sugars, polyols, polyamines, and amino acids (mostly proline) (Giri, 2011). Proline has multifunctional roles: not only is it a compatible osmolyte, it is also an effective scavenger of reactive oxygen species. It is also used as a metabolite that stabilizes subcellular structures, modulates cell redox homeostasis, supplies energy, and can act as a signaling molecule in a range of metabolic pathways under stress conditions (Ejaz et al., 2020). erefore, we assumed that chilling stress could lead to a significant increase in proline accumulation in the leaves of plants exposed to chilling stress (Table 3).
Plant biostimulants, including protein hydrolysates, have been shown to modulate proline accumulation in plants exposed to different stress factors Ertani et al., 2013). Other studies have revealed that the application of protein hydrolysates could stimulate carbon and/or nitrogen metabolism, even in oxidative stressed plants . erefore, we support the report of Omoarelojie et al. (2021) that biostimulants may contribute to proline accumulation in plants exposed to stress by providing the necessary metabolites (glutamate or ornithine) for its biosynthesis.

Conclusion
Protein hydrolysates are a promising group of substances that can mitigate the adverse effects of low-temperature stress in susceptible plant species such as cucumber. e plant-derived product named Naturamin WSP can alleviate, to some extent, the negative impact of oxidative stress caused by low temperatures. Our results indicate that the activation of the antioxidant enzyme system in the biostimulant-treated cucumber plants, as demonstrated by the enhanced activity of guaiacol peroxidases and the higher content of the amino acid proline, have a role in the mitigation of chilling stress damage in young cucumber plants.