Exogenously applied putrescine and chitosan–putrescine nanocomposite alleviate the negative effects of chilling stress on iceberg lettuce seedlings

Abstract

Climate change-induced stressors such as extreme temperatures negatively affect plant growth and development. Therefore, methods are needed to improve plant tolerance to abiotic stresses. The aim of this study was to evaluate whether the foliar application of putrescine (Put) and a chitosan–putrescine nanocomposite (Ch–Put) can modulate the antioxidant response and membrane properties of lettuce under chilling stress. In this study, Put and Ch–Put were applied via foliar spraying at two concentrations (1 mM and 2.5 mM), and after treatment, the plants were placed in a phytotron at 4 °C and 20 °C. Changes in the properties of the cell membranes were assessed in the seedlings. Additionally, the antioxidant enzymatic activity and content of nonenzymatic bioactive compounds were evaluated. The results indicated that the use of Put and Ch–Put influenced the permeability and fluidity of the lipid membranes, which also depended on the treatment temperature. An increase in A_lim (all treatments) and a decrease in C_s⁻¹ values (Put and Ch–Put at a concentration of 2.5 mM) were observed at 4 °C, suggesting looser packing and increased elasticity of cell membranes, facilitating the metabolic and physiological adaptation of plants to stress. The treatment of chilled plants with Put and Ch–Put resulted in increased contents of proline, carbohydrates, glutathione, phenolics and L-ascorbic acid, as well as increased activity of several antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX), which could contribute to increased tolerance to stress. The strongest effects were observed for Put at concentrations of 1 mM and 2.5 mM and Ch–Put at the 2.5 mM concentration, suggesting the potential use of these substances in strategies to increase plant tolerance to chilling stress.

Introduction

Climate change is increasing the frequency and intensity of abiotic stresses, such as drought, salinity, and extreme temperatures, which significantly disrupt plant growth, development, and productivity¹. The production of reactive oxygen species (ROS), including superoxide anions, H₂O₂, hydroxyl radicals, and singlet oxygen, and the disruption of the plasma membrane are common plant responses to various stress stimuli. The excessive production of ROS can damage molecular and cellular components through the oxidation of biomolecules (lipids, carbohydrates, proteins, enzymes, and DNA), causing cellular damage and programmed cell death (PCD)². Lipids within the plasma membrane (PM) are preferential targets of ROS. The PM plays a central role in the perception and transmission of information about external stimuli to different subcellular compartments. Low-temperature stress and subsequent oxidative stress drastically change the levels of unsaturated fatty acids and, consequently, the membrane integrity from a fluid-like structure to a rigid or gel-like structure³. This process decreases membrane transport and photosynthetic efficiency. Other effects of oxidative stress include changes in membrane permeability and stability, disrupting electron transport chains and ion homeostasis⁴. Thus, maintaining the proper fluidity and permeability of cell membranes in plants is crucial for coping with abiotic stress.

Plants have developed effective mechanisms to protect against the harmful effects of ROS based on the actions of antioxidant enzymes and the biosynthesis of nonenzymatic antioxidants, as described in several review articles^5,6. The operation of antioxidant mechanisms leads to a balance between ROS production and ROS scavenging in the cell, but the antioxidant defence capacity varies among plant genotypes and depends on the stress type and duration⁶.

Rapidly implementable strategies are needed to increase crop stress tolerance, indicating the need for further innovation in agriculture. One such strategy may be the use of polyamines during agricultural production, as these compounds participate in many important processes occurring in plants, starting from the initiation of the cell cycle, gene expression, signalling, and plant growth and development to plant acclimatization to abiotic stresses^7,8. Polyamines (PAs) are ubiquitous polycations that include the most common molecules spermidine (Spd, a triamine), spermine (Spm, a tetramine), and their obligate precursor putrescine (Put, a diamine), which can be found in free and conjugated forms⁹. Among them, Put is the central product of the polyamine biosynthetic pathway and the most abundant PA in nature; it is synthesized via two main pathways derived from ornithine or arginine¹⁰. Put appears to be involved in a wide range of physiological processes, including shoot and root growth, flowering, photosynthesis, cell proliferation embryogenesis, leaf senescence, and fruit maturation, but also plays important roles in the amelioration of biotic and abiotic stresses^11,12. According to these authors, Put plays a key role in stabilizing cell membranes; scavenging free radicals; preventing lipid peroxidation; influencing DNA replication, transcription, and translation; affecting the activity of enzymes; maintaining osmotic balance by regulating ion and water transport through potassium channels and stomata; and interacting with some phytohormones. Several reports have confirmed the role of Put in mitigating abiotic stress, especially low-temperature stress^11,13. For example, Song et al.¹⁴ showed that Put increased tomato tolerance by reducing H₂O₂ and malondialdehyde (MDA) levels under chilling stress. Furthermore, Sun et al.¹⁵ noted that exogenous Put application imparts chilling stress-induced oxidative stress tolerance to anthurium by regulating the activities of the antioxidant enzymes peroxidase (POX), catalase (CAT) and superoxide dismutase (SOD).

Our intention was to analyse whether the effectiveness of Put in mitigating abiotic stress in plants increases as a result of the use of chitosan-coated nanoparticles loaded with putrescine. Chitosan (Ch), a biopolymer derived from chitin deacetylation, is used in agriculture, food packaging, cosmetics, textiles, paper, sludge dewatering, and wastewater treatment¹⁶. The application of Ch alone may increase antioxidant enzymatic activity, antioxidant compound contents, defence-related gene expression, phytohormone levels, and osmotic regulator levels in plants subjected to stress conditions¹⁷. Chitosan has active functional groups, amino (-NH₂) and hydroxyl (-OH) groups; thus, it can be chemically modified with other biomaterials or biopolymers and metal ions to improve its physicochemical, mechanical, and biological properties¹⁸ and to form Ch-based nanocomposites. Chitosan nanoparticles have been investigated as smart carriers for the delivery of various ingredients owing to their biocompatibility, biodegradability, high permeability, cost-effectiveness, and non-toxicity¹⁹. Compared with free encapsulated compounds, the biopolymer matrix of chitosan can protect putrescine from rapid degradation, enhancing its stability and controlled release; such nanocomposites offer better penetration (through improved adhesion and electrostatic forces) and sustained availability in plant tissues²⁰. Chitosan combined with putrescine may trigger a stronger synergistic induction of antioxidant systems and stress-related pathways²¹. Considering the recent advances in Ch-related nanocarriers or nanoencapsulation and the high demand for green strategies, the exploration of this topic is gaining importance in plant science^22,23.

For this reason, we focused our study on the application of putrescine (Put) and its nanocomposite form with chitosan (Ch–Put). We examined the activities or contents of several antioxidants together with the properties of the lipid membranes of plant cells.

Our hypothesis assumes an increase in the activities of several enzymes and in the contents of other bioactive compounds involved in the defence against ROS as a result of priming plants with Put and Ch–Put applied at two different concentrations, just before plant exposure to chilling conditions. We also expect that Put and Ch–Put may protect the structure of membranes at low temperatures. Understanding these effects may contribute to the development of novel foliar treatments to mitigate temperature stress in crop production.

Results and discussion

Membrane fractionation

The extracted lipids from the plant material were separated into fractions, and then, the PLs were used to create model membranes (Fig. 1).

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Fig. 1

Examples of π-A isotherms and C_s^-1 compression modulus profiles as a function of surface pressure for phospholipid fraction monolayers isolated from iceberg lettuce plants caused by treatments with different concentrations of putrescine and the chitosan–putrescine nanocomposite (Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control).

Based on the obtained Langmuir monolayers, physicochemical parameters (A_lim and C_s^-1_max) illustrating the state of the lipid membranes of the studied objects were determined (Fig. 2). The experiments were conducted with the same putrescine (Put) and nanocomposites (Ch–Put) but at different concentrations to determine the relationship between the amount of the tested factor and the level of modification of the lipid membranes of the studied plant cells. The A_lim parameter, which represents the limiting molecular area in Langmuir monolayers, exhibited temperature- and treatment-dependent changes. At 20 °C, among all the tested treatments, putrescine (Put) and chitosan–putrescine nanocomposites (Ch–Put) applied at concentrations of 1 mM and 2.5 mM reduced the A_lim values, indicating tighter molecular packing at the air–water interface. Notably, the magnitude of this reduction was comparable across all the treatments, suggesting a uniform condensation effect under moderate-temperature conditions. At 4 °C, the A_lim values were higher in all the treatment groups than in the control group but showed much greater variability. Specifically, higher concentrations of these compounds led to the greatest increase in the A_lim values, indicating looser lipid packing under chilling conditions. The C_s⁻¹ parameter, which reflects the compressibility of molecular layers such as Langmuir monolayers at the air–water interface, exhibited both temperature- and concentration-dependent responses to treatments with putrescine (Put) and a chitosan–putrescine nanocomposite (Ch–Put). At 20 °C, the application of Put at 2.5 mM resulted in a decrease in C_s⁻¹, indicating that the layer is more flexible and fluid than the control group. In contrast, treatment with Ch–Put at the same concentration (2.5 mM) led to a significant increase in C_s⁻¹, suggesting reduced compressibility and a more rigid monolayer structure. Interestingly, this pattern was reversed at the lower temperature (4 °C): at this temperature, higher concentrations of both Put and Ch–Put caused a decrease in C_s⁻¹. Therefore, the monolayer is more susceptible to compression and is less rigid and organized.

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Fig. 2

Changes in the A_lim and C_s⁻¹_max characteristics of phospholipid monolayers extracted from iceberg lettuce plants caused by treatment with different concentrations of putrescine and the chitosan–putrescine nanocomposite (Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control) at two temperatures (4 °C and 20 °C). Different letters assigned to individual parameters for temperatures of 20 °C and 4 °C indicate significant differences according to Duncan’s test (p ≤ 0.05). The values presented here are the means ± SEs (n = 3).

The observed changes in A_lim and C_s⁻¹ suggest that putrescine (Put) and the chitosan–putrescine nanocomposite (Ch–Put) affect the physical state of the plasma membrane. A lower A_lim reflects tighter lipid packing, whereas a higher C_s⁻¹ indicates greater membrane rigidity; both are linked to changes in membrane fluidity, which is essential for transport, signalling, and protein function²⁴. The extent of these modifications depends on both the temperature and the compound concentration. At 20 °C, all the treatments led to a decrease in A_lim and an increase in C_s⁻¹ (Ch–Put 2.5 mM treatment). At 4 °C, both parameters showed the opposite trend in some cases: an increased A_lim (all treatments) and decreased C_s⁻¹ (Put and Ch–Put at a concentration of 2.5 mM). These opposing effects likely reflect the temperature-dependent behaviour of lipid membranes: a low temperature reduces membrane fluidity and impairs several cellular processes, prompting adaptive changes such as increased incorporation of unsaturated fatty acids to restore membrane functionality³. The observed responses may thus represent a form of membrane remodelling, helping cells maintain membrane-associated processes under stress²⁵. At 4 °C, treatment with Put at a relatively high concentration (2.5 mM) appeared to have a beneficial effect on the state of the cell membrane, as it increased membrane elasticity (higher values of A_lim and lower values of C_s^-1 than those in the control group). Similarly, the chitosan–putrescine nanocomposite (Ch–Put) applied at the same high concentration also led to looser lipid packing (higher A_lim) and simultaneously decreased membrane stiffness (lower C_s⁻¹). Both of these treatments may improve the effective adaptation of cell membranes to chilling stress. Through appropriate membrane remodelling, cells “adjust” their membranes so that the processes they perform (e.g., signalling and transport) can continue to function correctly^26,27. Therefore, treatments that help maintain or restore membrane flexibility under stress conditions could promote better communication within the cell and activate antioxidant responses more effectively²⁸. Thus, the improved antioxidant enzyme activity observed with some treatments could be related to these effects on the membranes. Higher fluidity may facilitate faster signal transduction and better availability of substrates for enzymes such as APX, CAT and SOD^29,30. In the case of Ch–Put, the chitosan matrix may also prolong the release and availability of putrescine on the leaf surface, thereby enhancing its effects.

In the present study, both temperature changes and the presence of the tested Put and Ch–Put compounds influenced the state of the cell membranes, and their effects were the sum of the processes in which the plants had adapted to the environment to continue functioning correctly.

Carbohydrates and starch

The level of sugars in plant cells, as well as their transport, storage and consumption, are determined by the physiological activity of the cells, the type of tissue, the environmental conditions and the stage of plant development during ontogeny³¹. In this study, differences in the amount of sugars were a consequence of reduced temperature and/or the use of additional compounds for spraying plants (Table 1). The highest concentration of carbohydrates was found in the plants subjected to 4 °C and sprayed with putrescine at concentrations of 1 mM and 2.5 mM. Control plants grown at 4 °C had a significantly lower level of carbohydrates than plants subjected to the abovementioned treatments. At 20 °C, the plants treated with 1 mM and 2.5 M Put presented 50.9% and 178.6% lower carbohydrate contents, respectively, when compared to seedlings subjected to those treatments but grown at 4 °C. Putrescine application also increased the carbohydrate content in the plants grown at 20 °C when compared to control lettuce; however, significant differences were observed only among the control and Put 1 mM and among the control and Ch–Put 2.5 mM. According to El-Bassiouny and Bekheta³², putrescine treatment caused an accumulation of total carbohydrates in wheat plants. Putrescine seemed to be actively involved in the glycolytic pathway and the Krebs cycle by inhibiting carbohydrate over-accumulation in leaves and improving energy production to mitigate abiotic stress-induced damage³³.

Table 1. Changes in the carbohydrate contents of the iceberg lettuce plants caused by treatments with different concentrations of putrescine and the chitosan‒putrescine nanocomposite (Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control) under two temperatures (4 °C and 20 °C).

Treatments	Carbohydrates (µM/gfw)
	20 °C	4 °C
Control	1.618 ± 0.497a	1.486 ± 0.530a
Put 1 mM	2.928 ± 0.128b,c	4.419 ± 0.220d
Ch–Put 1 mM	2.296 ± 0.003a,b	1.741 ± 0.160a
Put 2.5 mM	2.176 ± 0.520a,b	6.062 ± 0.059e
Ch–Put 2.5 mM	3.701 ± 0.215c,d	2.342 ± 0.484a,b

Different letters next to a particular parameter indicate significant differences according to Fisher’s LSD test (p ≤ 0.05). The values presented here are the means ± SEs (n = 3).

No significant effects of temperature or the application of the tested compounds to the lettuce leaves were observed for starch (Table 2). In their review, Thalmann and Santelia³⁴ stated that the majority of the studies reported a decrease in leaf starch content in response to abiotic stress, and this remobilization led to the provision of energy and carbon at times when photosynthesis may be potentially limited. In the present study, a decreasing tendency in the starch content parallel to decreasing temperatures was observed for the control group; however, putrescine and chitosan–putrescine nanocomposite application altered the responses of the other plants. In the treatments where the plants were sprayed with Put or Ch–Put, slightly more starch was observed in the plants subjected to 4 °C than in those subjected to 20 °C; moreover, at 4 °C, less starch was present in the control plants than in the lettuce treated with Put and Ch–Put. Unfortunately, these changes could not be confirmed statistically, which was due to the large dispersion of data around their means. Similarly, Zhao et al.³⁵ observed an increase in the soluble starch content when Vitis vinifera seedlings were sprayed with putrescine.

Table 2. Changes in the starch content in iceberg lettuce plants caused by treatments with different concentrations of putrescine and chitosan–putrescine nanocomposite (Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control) at two temperatures (4 °C and 20 °C).

Treatments	Starch (µM/g fw)
	20 °C	4 °C
Control	8.310 ± 0.459a	5.693 ± 0.107a
Put 1 mM	5.317 ± 0.716a	7.529 ± 2.261a
Ch–Put 1 mM	5.051 ± 0.641a	7.607 ± 2.062a
Put 2.5 mM	5.920 ± 1.145a	6.406 ± 0.243a
Ch–Put 2.5 mM	5.596 ± 0.051a	5.763 ± 0.101a

Different letters for a particular parameter indicate significant differences according to Fisher’s LSD test (p ≤ 0.05). The values presented here are the means ± SEs (n = 3).

Antioxidant enzymatic activity

Similar to Kalisz et al.³⁶, two forms of SOD, iron superoxide dismutase (FeSOD) and copper/zinc superoxide dismutase (Cu/ZnSOD), were identified in extracts from the leaves of iceberg lettuce seedlings. The activity levels of the forms of SOD are shown in Fig. 3. FeSOD and Cu/ZnSOD are evolutionarily conserved proteins in higher plant chloroplasts^37,38. In plants, FeSOD is most frequently reported to be active in plastids^39,40. However, a cytosolic localization of some FeSOD isoforms has also been suggested^41,42. Iron superoxide dismutase 1 (FSD1) was recently characterized as a plastidial, cytoplasmic, and nuclear enzyme with osmoprotective and antioxidant functions^43,44. Cu/ZnSOD occurs mainly within chloroplasts and the cytosol^40,45,46.

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Fig. 3

Native PAGE analysis of SOD forms in iceberg lettuce plants treated with different concentrations of putrescine and the chitosan–putrescine nanocomposite: Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control; temperatures 4 °C and 20 °C. Only the upper and lower parts of the gel, which did not show any bands, were cropped. The original gels are shown in the supplementary figure (Figure S1).

No significant alteration in SOD activity was observed among the control plants subjected to chilling conditions (4 °C) and those grown at a higher temperature (20 °C), although the low-temperature treatment tended to slightly increase the activity of this enzyme (Fig. 4A). Putrescine treatment resulted in a sharp decrease in SOD activity in lettuce seedlings grown at 4 °C compared with those grown at 20 °C (by 27.7% and 56.1% for Put 1 mM and Put 2.5 mM, respectively), and a much greater decrease in the activity of this enzyme was caused by a higher concentration of applied amine. According to Verma and Mishra⁴⁷, Put treatment had no influence on SOD activity in Brassica juncea plants under control conditions or at low salinity levels, but it considerably increased the enzyme activity in plants exposed to high salinity stress. On the other hand, Çakmak and Atıcı⁴⁸ showed that upon cold treatment, Put increased SOD activity at the beginning of the experiment but later caused a decrease in SOD activity in winter wheat; however, in the cold-sensitive cultivar, putrescine treatment caused important increases in SOD activity during the cold period. Thus, possible decreases in SOD activity under the influence of putrescine remain to be explained. In the present study, we observed that the exogenous application of Ch–Put at both concentrations (1 mM and 2.5 mM) increased the activity of SOD in the plants subjected to 4 °C compared with that in the plants exposed to 20 °C (by 17.4% and 61.4%, respectively). These results may be further supported by the observations of Bahmani et al.⁴⁹, who noted that a chitosan‒putrescine nanocomposite treatment ensured high SOD activity in strawberry stored at 4 °C. The contrasting effects of putrescine (Put) and chitosan–putrescine (Ch–Put) nanocomposite on SOD activity can be attributed to the more controlled and sustained release of putrescine from Ch–Put. Moreover, chitosan itself elicits plant defence responses, stimulating ROS signalling and the upregulation of antioxidant enzymes. Similar synergistic effects of chitosan-based nanocarriers on enhancing antioxidant defences in bell pepper, strawberry, and lettuce have been reported^{50, 51–52}.

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Fig. 4

Changes in the activities of SOD (A) and CAT (B) in iceberg lettuce plants caused by treatment with different concentrations of putrescine and the chitosan–putrescine nanocomposite (Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control) at two temperatures (4 °C and 20 °C). Different letters next to a particular parameter indicate significant differences according to Fisher’s LSD test (p ≤ 0.05). The values presented here are the means ± SEs (n = 3).

The activity of CAT increased by 78.9% in the control plants due to chilling (Fig. 4B). Many studies have confirmed an increase in CAT activity because of plant exposure to abiotic stress⁵. Under chilling conditions, CAT activity increased the most (94.8%) in plants treated with putrescine at a concentration of 2.5 mM (Put 2.5 mM) compared with that at 20 °C. Sun et al.¹⁵ noted that exogenous 0.5–2.0 mM Put applied to anthurium seedlings promoted CAT activity under chilling conditions. However, lettuce seedlings sprayed with Put 1 mM or Ch–Put 2.5 mM nanocomposite presented a decrease in CAT activity at 4 °C (40.4% and 55.6%, respectively) compared with plants grown at a higher temperature (20 °C). The Ch–Put 1 mM treatment did not significantly affect the activity of this enzyme in the plants grown at 4 °C or 20 °C. In conclusion, increased CAT activity can be obtained by applying putrescine at a concentration of 2.5 mM (in our study), whereas lower concentrations and Ch–Put nanocomposite are not effective at modulating CAT activity. A probable explanation is that putrescine applied at higher concentrations is more rapidly oxidized by amine oxidases, generating H₂O₂ as a byproduct, which in turn activates CAT to prevent oxidative damage⁵³, whereas Ch–Put may limit the availability of putrescine for oxidation due to its encapsulation in the chitosan matrix, thereby reducing H₂O₂ production and the need for high CAT activity. Moreover, H₂O₂ in plant cells is reduced by several other key antioxidants, and attention should be given to glutathione, the content of which was very high in plants treated with Ch–Put 2.5 mM (Fig. 6).

In summary, at a higher concentration of 2.5 mM putrescine (Put), high CAT activity and lower SOD activity were observed, indicating efficient H₂O₂ removal and well-balanced ROS detoxification. However, chitosan–putrescine (Ch–Put) induced high SOD activity but low CAT activity, leading to H₂O₂ accumulation and stronger stress signalling, possibly by inhibiting or delaying CAT activation.

The control plants exposed to chilling conditions had higher activity of APX (by 77.4%) than plants exposed to 20 °C (Fig. 5A). APX plays important roles in both ROS scavenging and manipulating the activity of various cell signalling pathways. Under abiotic stress, the activity of APX increases rapidly in response to adverse environmental conditions⁵⁴, as observed in the results of this work. The exogenous application of Put (both concentrations: Put 1 mM and Put 2.5 mM) and Ch–Put 2.5 mM nanocomposite increased the activity of APX, with the greatest increase occurring in the plants treated with Put 1 mM and Put 2.5 mM (by 56.1% and 50.6%, respectively). A significant increase in APX activity was noted by Verma and Mishra⁴⁷ in B. juncea plants previously treated with putrescine and then subjected to salinity stress and by Li et al.⁵⁵ in melon seedlings sprayed with this amine under low-temperature conditions. Ch–Put (1 mM) did not significantly affect APX activity. In the case of the Ch–Put nanocomposite, a concentration of 1 mM was apparently too low to cause changes in the activity of this enzyme, but the application of Ch–Put 2.5 mM may alleviate chilling stress, which was also shown for APX by Panahirad et al.⁵⁶, who noted that the Ch–Put nanocomposite alleviated the adverse effects of Cd stress on Vitis vinifera by increasing APX activity.

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Fig. 5

Changes in the activities of APX (A) and GPOX (B) in iceberg lettuce plants caused by treatment with different concentrations of putrescine and the chitosan–putrescine nanocomposite (Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control) at two temperatures (4 °C and 20 °C). Different letters for a particular parameter indicate significant differences according to Fisher’s LSD test (p ≤ 0.05). The values presented here are the means ± SEs (n = 3).

Compared with 20 °C, chilling slightly increased the activity of GPOX in lettuce seedlings; however, the differences were not statistically significant (Fig. 5B). Although the increasing trends were similar to those of APX (except for Put 1 mM), large values of standard deviations were noted, indicating the dispersion of a dataset relative to its mean, which was one of the reasons for the lack of significant differences between the means. In the plants grown at 4 °C and sprayed previously with Ch–Put at both concentrations (1 mM and 2.5 mM), the activity of GPOX tended to be higher than that at 20 °C. This finding was still not confirmed by the statistical analysis. However, Panahirad et al.⁵⁶ observed higher guaiacol peroxidase activity in V. vinifera plants under Cd stress conditions when Ch–Put nanocomposites were used. Based on our results, we can conclude that only the Ch–Put 2.5 mM treatment caused a significant increase in GPOX activity in the plants subjected to 4 °C compared with the control plants grown at 20 °C and the plants treated with Put 1 mM at 4 °C, Ch–Put 1 mM at 20 °C and Put 2.5 mM at 20 °C.

L-ascorbic acid and reduced glutathione

In the present study, under chilling conditions, the AsA content was significantly increased in the leaves of the lettuce plants in all the treatment groups compared with those of the plants grown at 20 °C (Fig. 6A). Ascorbate is a very important antioxidant that plays a crucial role in scavenging ROS and effectively regulates the cellular redox status⁵⁴, the content of which usually increases under the influence of stress factors, which was already observed in previous experiments with lettuce³⁶, similar to the results of this study. When 2.5 mM Ch–Put was applied to the plants maintained at a relatively high temperature of 20 °C, the concentration of AsA increased by 28.6% compared with that of the control group. When 1 mM Put was applied to the leaves of the plants subjected to 4 °C, the AsA content increased the most (196.2%) compared with that at 20 °C. An increase in the content of AsA in plants exposed to abiotic stress and treated with Put was also reported by Zhao et al.⁵⁷ for wheat seedlings under salinity conditions. At temperature of 4 °C, application of Put at 1 mM concentration caused a significant increase in the content of this compound in comparison with that in the control plants and Ch–Put 1 mM-treated plants.

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Fig. 6

Changes in the contents of L-ascorbic acid (A) and glutathione (B) in iceberg lettuce plants caused by treatments with different concentrations of putrescine and the chitosan–putrescine nanocomposite (Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control) at two temperatures (4 °C and 20 °C). Different letters next to a particular parameter indicate significant differences according to Fisher’s LSD test (p ≤ 0.05). The values presented here are the means ± SEs (n = 3).

In the antioxidant system, reduced GSH is one of the major antioxidants⁵⁸, whose content usually increases in plants subjected to abiotic stress⁵. In our study, lettuce seedlings subjected to chilling (4 °C) presented, however, a significantly lower content of GSH (by 28.4%) than did plants grown at 20 °C (Fig. 6B), which was somewhat surprising, especially since, in earlier studies³⁶, the glutathione level was almost twice as high in iceberg lettuce subjected to chilling than at 20 °C. The older plants used in the present study (30-day-old vs. 25-day-old in the previous work³⁶) likely presented another type of plasticity in an adaptive capacity, or the presence of more leaves resulted in greater ROS production due to an increased surface area, which caused faster glutathione consumption. In this study, the application of Put and the Ch–Put nanocomposite (Put 1 mM, Put 2.5 mM, and Ch–Put 2.5 mM) increased the concentration of GSH in the plants, with the exception of the Ch–Put 1 mM treatment. The greatest increase in the amount of GSH in the plants was observed after spraying the plants with Ch–Put 2.5 mM, and the increase in the content of this component was 280.2% greater in the plants exposed to 4 °C than in those exposed to 20 °C. The level of GSH in these plants was significantly higher than that all other treatment groups. The findings of Zhao et al.⁵⁷ indicated that Put increased the GSH content in wheat seedlings under saline stress. The increases in GSH levels under the influence of chitosan itself at 4 °C and 20 °C revealed a role for this compound in the plant defence system as an elicitor of GSH biosynthesis; however, Ch nanocomposites may have different effects because of the modification of their physicochemical properties³⁶. These authors reported significantly higher concentrations of GSH but not proline in lettuce sprayed with nanocomposites of Ch with L‑cysteine and glycine betaine in chilled plants than in those grown at 20 °C. In conclusion, the results indicate that the Ch–Put nanocomposite applied at a concentration of 2.5 mM strongly affects the level of GSH in lettuce plants.

Total phenolics and carotenoids

No significant differences in the total phenolic contents were observed in control lettuce seedlings exposed to 4 °C and 20 °C; however, their levels tended to be slightly higher in plants cultivated at the higher temperature (Fig. 7A). This finding is inconsistent with the findings of Lee and Oh⁵⁹, who reported a relatively high total phenolic content in Brassica oleracea var. acephala even 1 day after exposure to 4 °C. Our previous study⁶⁰ showed an increase in the phenolic contents in basil plants, but this effect of chilling was associated mainly with the lettuce leaf basil, whereas other cultivars responded rather negligibly to low-temperature stimuli in terms of phenolic concentrations, the application of the Ch–Put nanocomposite at a 1 mM concentration (Ch–Put 1 mM) and Put at a 2.5 mM concentration to the plants caused a significant increase in the total phenolic content at 4 °C compared with that at 20 °C (32.3% and 21.6%, respectively). Bahmani et al.⁴⁹ observed that strawberries treated with Ch–Put nanocomposites and Put (2.0 mM) presented increased levels of anthocyanins and flavonoids; these authors explained the increased contents of these compounds by the induction of the activity of enzymes involved in the biosynthesis of phenols. In our study, these treatments, together with Ch–Put 2.5 mM, resulted in higher concentrations of phenolics than did the control treatment at 4 °C, whereas plants grown at 20 °C presented similar contents of these compounds, regardless of Put or Ch–Put application. The application of Ch–Put and Put at both concentrations caused a change in the phenol content, but this change occurred mostly in plants stressed by lower temperatures.

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Fig. 7

Changes in the contents of total phenolics (A) and carotenoids (B) in iceberg lettuce plants caused by treatments with different concentrations of putrescine and the chitosan–putrescine nanocomposite (Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control) at two temperatures (4 °C and 20 °C). Different letters next to a particular parameter indicate significant differences according to Fisher’s LSD test (p ≤ 0.05). The values presented here are the means ± SEs (n = 3).

A temperature of 4 °C (chilling conditions) caused an increase in the carotenoid content of the iceberg lettuce seedlings (Fig. 7B). Carotenoids protect the photosynthetic apparatus from overexcitation in the presence of strong light and dissipate excess absorbed energy, which might also protect plants from oxidative stress through the scavenging of oxy radicals⁶¹. Their concentrations often increase in the presence of chilling conditions⁶². Significant differences at 4 °C and 20 °C were detected for the control plants and the plants treated with Put and the Ch–Put nanocomposite, but these differences were detected only after the application of the lower concentration (1.0 mM). The foliar application of Put was reported to increase the content of carotenoids in periwinkle plants in a concentration-dependent manner⁶³. The greatest increase was caused by the application of Ch–Put at a 1 mM concentration, and this increase between plants grown at 4 °C and 20 °C reached 163.9%. Panahirad et al.⁵⁶ observed an increased content of carotenoids under nonstressed conditions when Ch–Put nanoparticles were used; moreover, 0.1% Ch–Put nanoparticles contributed more to the carotenoid content in plants under either Cd-induced stress or nonstress conditions. In our study, no significant changes were observed for these compounds applied at a 2.5 mM concentration because the large standard deviation indicated that the data were more spread out around the means.

Proline and malondialdehyde

The biosynthesis of osmoprotectants, such as proline, is considered an effective strategy for plant acclimatization to stress conditions⁶⁴. Zhu et al.⁶⁵ postulated that proline may act as a hydroxyl radical scavenger, energy absorber, and buffer of the cellular redox potential, thereby alleviating oxidative stress in maize. In the present study, the increased accumulation of proline in the plants subjected to 4 °C may be related to the activation of processes leading to the synthesis of this metabolite; proline is also an important regulator that counteracts osmotic changes in plant cells induced by chilling stress (Fig. 8A). Compared with the chilled control plants, the plants grown at 4 °C and treated with Put (1 mM and 2.5 mM) presented the highest proline content, which indicated that these treatments were the most effective at counteracting chilling stress. When the conditions were optimal (20 °C), only the application of Put 2.5 mM increased the proline content compared with that of the control lettuce, whereas the nanocomposites decreased the proline content in both cases (Ch–Put 1 mM and Ch–Put 2.5 mM), similar to when putrescine was applied at a 1 mM concentration. High doses of Put (2.5 mM) can induce mild metabolic stress, e.g., by increasing the production of H₂O₂ as a byproduct of amine oxidation and shifting metabolic pathways towards proline synthesis⁶⁶. The decrease in proline levels compared with those of the control group after the application of Put (1 mM) and Ch–Put (1 mM and 2.5 mM) indicates that the plant not only did not increase the proline content but also likely activated proline decomposition or limited its biosynthesis, which may indicate a strong stabilizing and antistress effect of the tested substances.

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Fig. 8

Changes in the contents of proline (A) and malondialdehyde (B) in iceberg lettuce plants caused by treatments with different concentrations of putrescine and the chitosan–putrescine nanocomposite (Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control) at two temperatures (4 °C and 20 °C). Different letters next to a particular parameter indicate significant differences according to Fisher’s LSD test (p ≤ 0.05). The values presented here are the means ± SEs (n = 3).

The results revealed that a temperature of 4 °C significantly increased the content of malondialdehyde (MDA) in control lettuce (by 45.5%) and in plants treated with Put 1 mM (by 61.8% in comparison to Put 1 mM at 20 °C) or Put 2.5 mM (by 41.1% in relation to Put 2.5 mM at 20 °C) (Fig. 8B). At a low temperature, MDA, which is a product of the peroxidation of unsaturated fatty acids, is a good indicator of free radical damage to cell membranes. Its level often increases in plants subjected to various abiotic stress factors, especially in silage corn following chilling⁶⁷. In our study, low temperature (4° vs. 20 °C) was a factor that caused an increase in the MDA content in the lettuce seedlings. No significant effects of temperature on the malondialdehyde concentration in lettuce seedlings were found for the plants treated with chitosan–putrescine nanocomposite (Ch–Put 1 mM and Ch–Put 2.5 mM), regardless of the concentration used, compared with those of the plants grown at 20 °C and treated with the same concentrations of the compound. On the other hand, Panahirad et al.⁵⁶ described a reduction in the MDA content in grapevine plants grown under nonstress conditions and in plants suffering from Cd stress. In our work, the MDA level in the plants exposed to 4 °C and treated with Ch–Put 1 mM was slightly decreased compared with that in the plants exposed to 20 °C; however, after treatment with higher concentrations (2.5 mM), the opposite trend was detected, but the difference was still not significant. The results of the statistical analyses revealed no significant differences in the malondialdehyde content in the plants grown at 4 °C, with one exception: after the application of Ch–Put 1 mM, the malondialdehyde concentration decreased by 25.1% in comparison with that in the nonsprayed control plants at 4 °C. At both applied concentrations, putrescine increased the content of malondialdehyde in plants grown at 4 °C compared with that in plants treated with the same concentrations but maintained at 20 °C. The mechanism of such a plant response requires additional studies.

Fresh weight and dry weight

Only one week of cultivation at a temperature of 4 °C resulted in significant differences in fw among the control lettuce plants, with an 11.8% decrease in fw (Fig. 9A). A significantly lower fw was also detected in plants treated with Put 1 mM and Ch–Put 2.5 mM at 4 °C than at 20 °C. The reduction in seedling biomass at a low temperature can be attributed to decreased cell division and elongation, a decreased photosynthetic capacity or reduced water and nutrient uptake⁶⁷. The application of Ch–Put at 1 mM, especially Put at 2.5 mM, to the plants alleviated the effects of chilling stress; in particular, in the case of the latter treatment (Put at 2.5 mM), the difference between the plants cultivated at 20 °C and those grown at 4 °C was only 2.6%.

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Fig. 9

Changes in the fresh weight (A) and dry weight (B) of iceberg lettuce plants caused by treatments with different concentrations of putrescine and the chitosan–putrescine nanocomposite (Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control) at two temperatures (4 °C and 20 °C). Different letters next to a particular parameter indicate significant differences according to Fisher’s LSD test (p ≤ 0.05). The values presented here are the means ± SEs (n = 3).

Compared with 20 °C, chilling caused a significant increase in the dry weight (dw) of all the experimental treatment groups (Fig. 9B). A higher dw was associated with lower tissue hydration, as evidenced by the obtained results and published data, which confirmed that low-temperature stress is accompanied by a reduction in water uptake, which is even more pronounced than the reduction in leaf transpiration⁶⁸. In the present study, plants subjected to 4 °C contained 95.1% water on average, whereas seedlings grown at 20 °C contained 96.3% water. No significant differences in dw were observed among the plants treated with the tested compounds at 4 °C. Compared with the control plants, the application of Put 2.5 mM markedly increased the dw of plants grown at 20 °C.

Heatmap

An analysis of data in a heatmap allows for a holistic view of some of the metabolic pathways studied in this work (Fig. 10). The greatest stimulating effect on the antioxidant system of plants was exerted by low-temperature (4 °C) treatment and the application of Put at a concentration of 2.5 mM. These treatments caused high activities of CAT and APX and, to a lesser extent, GPOX and high concentrations of total phenolics, carbohydrates, proline, and AsA. These values were generally higher than those obtained for control plants grown at 4 °C, with the exception of GPOX activity and AsA levels, which were statistically similar in plants subjected to these experimental treatments. An interesting result was the decrease in SOD activity in the plants that received the Put 2.5 mM treatment, which was lower than that in the other treatment groups. A putrescine concentration of 1 mM also caused many compounds to have relatively high contents of metabolites or metabolic activity in plants grown at 4 °C. On the other hand, when these data were compared with the values observed for the control plants at 4 °C, these increases were significant for the AsA, GSH, and carbohydrate contents, and higher enzyme activity was noted for APX and CAT. In both treatment groups (Put 1 mM at 4 °C and Put 2.5 mM at 4 °C), an increase in the MDA concentration was observed, especially in plants sprayed with 1 mM Put and grown at 4 °C, compared with the chilled control seedlings. However, in both cases, the differences were not statistically significant. Compared with all the other treatments, the application of the Ch–Put nanocomposite at a concentration of 2.5 mM resulted in a substantial increase in the GSH content in the chilled plants. Moreover, the total phenolic content in these seedlings was higher than that in the chilled control plants. A lower concentration of the Ch–Put nanocomposite (1 mM) also resulted in higher total phenolic concentrations in plants grown at 4 °C than in the chilled control plants. The carotenoid content was slightly higher in these plants than in the chilled control plants, but the differences were not significant. The 20 °C treatment had different effects on the response of the plants to Put or the Ch–Put nanocomposite. Compared with the results for the control plants grown at 20 °C, treatment with Ch–Put at a concentration of 1 mM resulted in a slight increase in the MDA content and a significant increase in the total phenolic concentration, whereas higher concentrations of Ch–Put resulted in decreases in the SOD activity and GSH content; however, this treatment increased the GPOX and CAT activities (and, to some extent, the APX activity) and carbohydrate content in the plants. Similarly, when Put was applied at a concentration of 1 mM, an increase in CAT activity and a decrease in the amount of GSH were observed in comparison to the control plants grown at 20 °C. When Put was applied at a concentration of 2.5 mM and the plants were grown at a temperature of 20 °C, they presented a relatively high carotenoid content (not confirmed statistically) and relatively high proline content, whereas the GSH content was relatively low, and increases in SOD, CAT, APX and GPOX activities were observed compared with those of the control plants grown at 20 °C (the differences in APX and GPOX activities were not significant).

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Fig. 10

Heatmap of compound concentrations/activities in iceberg lettuce subjected to different experimental treatments: Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; deionized water served as a control—Control. The plants were grown at two temperatures: 4 °C and 20 °C.

Conclusions

Low-temperature stress significantly affected the lipid membrane properties and metabolism of iceberg lettuce plants. The plants exposed to 4 °C presented increased contents of proline, carbohydrates, AsA, GSH, and carotenoids and elevated activities of APX and CAT, which suggested important roles for these compounds in acclimatization mechanisms. At lower temperatures, the level of MDA increased, which is indirect evidence of lipid peroxidation. The biomass of the chilled plants was also lower than that of the seedlings grown at 20 °C. The application of Put and the Ch–Put nanocomposite at both concentrations had significant effects on plant responses to chilling stress. These compounds increased the activities of certain antioxidant enzymes (e.g., APX and CAT with specific Put concentrations) but influenced other enzymes differently (e.g., SOD and CAT with Ch–Put), changed the contents of nonenzymatic antioxidants (e.g., L-ascorbic acid, glutathione, total phenolics, and carotenoids, which are related to the applied compound and its concentration) and mitigated chilling-induced membrane damage. Ch–Put (2.5 mM) application prevented an increase in the MDA content and caused a significant increase in the GSH content in plants grown at 4 °C, whereas Put 2.5 mM increased the MDA content. These selected data, among other findings, indicate the protective efficacy of Ch–Put in terms of membrane integrity. Ch–Put sometimes performs differently from Put in certain aspects, which is related to the sustained release of Put from the chitosan matrix, better uptake or translocation due to the nature of the nanoparticles, or the unique elicitor properties of chitosan, which contribute synergistically or antagonistically. Our results showed that the use of Ch nanocomposites with Put or Put itself may reduce the negative effects of stress by preserving membrane function, perhaps through an optimized balance or redirection of antioxidant pathways, or by involving nonenzymatic antioxidants and altering antioxidant enzyme activity. These compounds (Ch–Put and Put) seem to be particularly important for improving plant tolerance to harsh environmental conditions and can be used as promising strategies for supporting sustainable agriculture.

Materials and methods

Plant material and culture conditions

The iceberg lettuce (Lactuca sativa L.) cv. ‘Rumours’ (Bejo Zaden) plants used in the study were purchased from Krasoń—Group of Vegetable Seedling Producers (Piaski, Poland). Seedlings were grown in cubic peat pots (4 cm × 4 cm × 4 cm, volume of 64 mL) made from the propagation substrate Potgrond 007 (Kronen, Grądy, Poland), which were grouped in plastic boxes (150 pots per box). Three-week-old seedlings were transferred to the greenhouse of the University of Agriculture in Krakow, Poland, and placed on aluminium bench tables. Flood irrigation was applied as needed without wilting shoots, and the plants were not fertilized at this time. In the greenhouse, the average air temperature was 23/19 °C (day/night), and the air humidity oscillated at approximately 75%. No artificial lighting was used, with the natural day length being 16 h. Plants were transferred to phytotron rooms in the later stage of the experiment and placed on foil with raised edges, which allowed flood watering without the need for further sprinkling of the plants. These two rooms had different temperatures: 20 °C and 4 °C. The light source used was two Plantstar high-pressure sodium lamps (400 W, 56.500 lm, 2000°K; OSRAM GmbH, Munich, Germany) per room, the daylength was 16 h, and the relative humidity of the air was approximately 75%. The plants remained in the phytotron for 7 days.

Characterization of chemicals

Putrescine was purchased from Sigma–Aldrich (sales representative: Merck Life Science Sp. z.o.o., Poznań, Poland). According to a description provided by Sigma–Aldrich, putrescine [NH₂(CH₂)₄NH₂] is a colourless solid that is completely soluble in water (dissociation constant: 9.35–10.80 at 25 °C) with a molecular weight of 88.15 g/mol (CAS 110–60-1). Putrescine has a pungent odour; the product is chemically stable under standard conditions (room temperature). Ch–Put was synthesized using the ionic gelation technique, according to the method described by Panahirad et al.⁵⁶. The structural characterization of the chitosan‒putrescine nanocomposite was investigated via FTIR spectroscopy, which revealed several characteristic peaks observed by Panahirad et al.⁵⁶: Fig. A. The morphology of Ch–Put was also studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the SEM image revealed a sphere-like structure, which was confirmed in the TEM image that displayed the formation of spherical Ch–Put with an average diameter of 100 nm, as described by Panahirad et al.⁵⁶: Fig. D and Fig. E.

Experimental design and plant sampling

When the plants had 6–7 leaves (aged ca. 30 days), they were sprayed with Put and a nanocomposite of Ch and Put. Two different concentrations were used: 1.0 mM and 2.5 mM. Deionized water was used to prepare these concentrations and to spray control plants. A hand sprayer equipped with a nozzle resulting in a fine droplet fall was used. Thus, particular suspensions were applied evenly on the leaves, and each plant received ca. 1.0 mL (150 mL per plastic box with pots). The plants were then transferred to the phytotron rooms and remained there for 7 days. The following abbreviations were used for the experimental treatments: Put 1 mM–1 mM putrescine; Ch–Put 1 mM–1 mM chitosan–putrescine nanocomposite; Put 2.5 mM–2.5 mM putrescine; Ch–Put 2.5 mM–2.5 mM chitosan–putrescine nanocomposite; Control–deionized water. The plants were also divided into two groups: those treated at 20 °C and those treated at 4 °C, depending on the ambient temperature at which they were kept in the phytotron.

After one week, 450 plants per treatment (150 plants per repetition) were sampled and transferred to the laboratory for analysis. The fresh weight, dry weight, L-ascorbic acid content and glutathione content were determined based on fresh material, and the remaining plant samples were frozen in liquid nitrogen, stored at -40 °C, and then lyophilized for further analyses.

Membrane fractions

The lipid fraction of native cells was isolated using a chloroform/isopropanol mixture, followed by chloroform alone, based on a modified Bligh and Dyer⁶⁹ method. Some lipids were separated into phospholipid (PL) and galactolipid fractions via column chromatography on silica gel under a nitrogen atmosphere and further purified by thin-layer chromatography, as described by Block et al.⁷⁰. The PL fraction was chosen for further research because it serves as a model for the plasmalemma. The experiments were conducted using the Langmuir technique (Minitrough, KSV, Finland). Monolayers of PL were prepared by spreading chloroform solutions on the water surface and compressing them at a rate of 3.5–4.6 Ų/molecule × min. The relationship between the surface pressure (π) and the area per lipid molecule (A) was used to derive parameters characterizing the lipid monolayer structure, such as A_lim, the smallest area occupied by a lipid molecule in a layer; π_coll, the pressure at which the monolayer collapses; and C_s⁻¹, the static compressive modulus, which indicates mechanical resistance to layer compression. The surface pressure was measured with an accuracy of ± 0.1 mN/m using a Platinum Wilhelmy plate. All the experiments were conducted at 25 °C and repeated three or four times to obtain isotherms with an accuracy of ± 0.1–0.3 Ų.

Proline content determination

The amount of proline was determined using the protocol outlined by Ábrahám et al.⁷¹. One gram of leaf tissue was homogenized in 5 mL of 3% sulfosalicylic acid. The mixture was then centrifuged at 14,500 rpm for 5 min at room temperature. Following centrifugation, the reaction mixture was prepared by combining 100 μL of 3% sulfosalicylic acid, 200 μL of glacial acetic acid, 200 μL of acidic ninhydrin, and 100 μL of the supernatant. The resulting mixture was incubated at 96 °C for 60 min. After cooling, 3 mL of toluene was added to the reaction mixture, and the samples were vortexed for 30 s. The samples were then allowed to stand for 30 min to separate the organic and aqueous phases. The absorbance was measured at 520 nm, and toluene was used as a reference. The concentration of proline was determined using a standard concentration curve and was calculated on a fresh weight basis.

Analysis of soluble carbohydrates and starch

The total sugar concentration, which includes both soluble sugars and starch, was measured using the anthrone method as outlined by Maness et al.⁷². The sugars were extracted by homogenizing 1 g of leaves in 5 mL of 95% (v/v) ethanol. Following centrifugation, the supernatant was collected for the analysis of the soluble sugar contents, while the pellet underwent starch hydrolysis through an incubation with 35% HClO₄ at 4 °C for 24 h. The samples were then centrifuged again, and the resulting supernatant was utilized for further analysis.

The sugar content in both the ethanol phase and the supernatant obtained after starch hydrolysis was assessed using an anthrone reagent, which was prepared by dissolving 0.2 g of anthrone in 1 L of 96% H₂SO₄. The samples were heated at 100 °C for 10 min. After cooling, the absorbance was measured using a UV‒VIS Helios Beta spectrophotometer at a wavelength of 620 nm and Evolution™ 201 (Thermo Scientific).

Antioxidant enzymatic activities and analysis of SOD activity by native PAGE

The plant material (2 g fresh weight) was homogenized at 4 °C in 5.0 ml of phosphate buffer (0.1 M KH₂PO₄/Na₂HPO₄) at pH 7.5 containing 3 mM ethylenediaminetetraacetic acid (EDTA), and the homogenate was subsequently centrifuged at 12,000 × g for 5 min at 4 °C to isolate the soluble protein fraction, and the obtained supernatant was used to measure the antioxidant enzyme activity. The catalase (CAT, EC, 1.11.1.6) activity was determined according to the Aebi⁷³ method, which is based on the amount of hydrogen peroxide decomposed by the enzyme. One unit of CAT activity was defined as 1 μmol of H₂O₂ decomposed during a minute. The rate of changes in absorbance was monitored at 240 nm, and the extinction coefficient of H₂O₂ of 42.6 (μmol/L)/cm was used.

The activity of peroxidases (ascorbate peroxidase: APX, EC 1.11.1.11 and guaiacol peroxidase: GPOX, EC 1.11.1.7) was assessed using a procedure in which 2 g of samples were homogenized at 4 °C in 10 mL of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA (ethylenediaminetetraacetic acid), 1% PVP (polyvinylpyrrolidone), and 1 mM PMSF (phenylmethylsulfonyl fluoride). The mixture was centrifuged at 13,968 × g for 15 min at 4 °C, and the resulting supernatant was used as the enzyme extract. The activity of APX was determined according to the methods of Nakano and Asada⁷⁴. The reaction was performed in a mixture containing 1.85 mL of 50 mM potassium phosphate buffer (pH 7.0), 0.5 mL of 0.5 mM ascorbate (AsA), 0.5 mL of 0.1 mM H₂O₂, and 0.15 mL of the enzyme extract. Ascorbate oxidation in the presence of H₂O₂ was monitored by a decrease in absorbance at 290 nm (UV‒VIS Helios Beta spectrophotometer). The enzyme activity was calculated using the extinction coefficient ε = 2.8 mM/cm, and the results are reported as μmol AsA/min/g fresh weight (FW). GPOX activity was assessed according to the method of Zhang et al.⁷⁵, with guaiacol used as the substrate. The reaction mixture contained 1.4 ml of 50 mM phosphate buffer (pH 7.0), 0.2 mL of 4% guaiacol, and 1 mL of 1% H₂O₂. The reaction was initiated by adding 0.4 mL of the enzyme extract. The absorbance of tetraguaiacol was measured at 470 nm (UV‒VIS Helios Beta spectrophotometer), with an extinction coefficient of ε = 26.6 mM/cm. The activity of guaiacol peroxidase was reported as μmol tetraguaiacol/min/g fresh weight (FW).

For the analysis of superoxide dismutase (SOD, EC 1.15.11) activity, polyacrylamide gel electrophoresis (PAGE) was conducted at 4 °C at 180 V in a Laemmli⁷⁶ buffer system without sodium dodecyl sulfate (SDS) (native). The samples containing 8 μg of protein were loaded on a gel. For the determination of SOD activity, 12% native polyacrylamide gels were used, and the bands corresponding to enzyme activity were visualized according to the methods of Beauchamp and Fridovich⁷⁷. SODs were identified as described by Ślesak and Miszalski⁷⁸. The densitometric analysis of SOD bands was performed with ImageJ 2 (GPL licence). The total protein concentration in the samples was determined according to the Bradford⁷⁹ method using Coomassie Brilliant Blue G-250 dye. The absorbance was measured at 595 nm, and the protein concentration was calculated based on a standard curve prepared with bovine serum albumin (BSA).

Determination of L‑ascorbic acid, glutathione and total phenolic contents

The L-ascorbic acid (AsA) content was determined using the iodometric method described by Ikewuchi and Ikewuchi⁸⁰. A 2.5 g plant sample was homogenized in 10 mL of a 1% oxalic acid solution and then centrifuged for 10 min at 3,492 × g and 18 °C. After centrifugation, 5 mL of the resulting extract was mixed with 1 mL of a 1% starch solution in water. The mixture was then titrated with an iodine solution in potassium iodide. The ascorbic acid present in the sample reacts with iodine, and the starch solution remains colourless. Once all the AsA is oxidized, iodine forms a blue complex with the starch, indicating the end of the titration. The AsA content was calculated based on the amount of iodine in the potassium iodide solution used for titration. The strength of the iodine solution was determined by the amount of titrant used to titrate a 1% ascorbic acid solution.

The Guri⁸¹ method was used for the quantitative determination of the glutathione (GSH, reduced form) content, with slight modifications. First, 1 g of fresh leaves was finely chopped and then ground in a mortar on ice at 4 °C with 10.0 mL of 0.5 mM EDTA dissolved in 3% trichloroacetic acid (TCA). The resulting extract was centrifuged at 13,968 × g for 10 min at 4 °C. Then, 5 mL of K-phosphate buffer (pH 7.0) was added to 2 mL of the supernatant until the pH reached approximately 7.0. Two millilitres of this mixture was transferred to a test tube, and an additional 1 mL of the same K-phosphate buffer was added. Next, 0.1 mL of Ellman’s reagent (5,5-dithiobis-2-nitrobenzoic acid) was introduced into the sample. The GSH content was determined by measuring the absorbance at 412 nm and comparing it with that of the K-phosphate buffer using a Helios Beta UV‒VIS spectrophotometer. For each analytical sample, a control sample was prepared similarly but with 1.1 mL of K-phosphate buffer and without Ellman’s reagent. The GSH content was calculated using a standard curve created for the pure compound.

The concentration of phenolic compounds was determined using the Folin‒Ciocalteu method, according to the procedure described by Djeridane et al.⁸². The plant samples (2.0 g) were finely chopped and homogenized with 10 mL of 80% methanol and then centrifuged for 10 min at 3492 × g and 18 °C. To the resulting supernatant (0.1 mL), 2 mL of 2% sodium carbonate (0.1 mL) was added. After 5 min, 0.1 mL of Folin–Ciocalteu reagent (F–C), diluted with deionized water at a 1:1 v/v ratio, was added to each sample. All the samples were incubated in the dark for 45 min at approximately 20 °C. The absorbance was measured at 750 nm using a UV‒VIS Helios Beta spectrophotometer, with a blank sample (0.1 mL of 80% methanol, 2 mL of 2% Na₂CO₃, 0.1 mL of F–C with water 1:1 v/v) used as the reference. The total phenolic content (TP) was reported as gallic acid equivalents (GAEs) per 1 g of fresh weight (FW).

Analysis of the malondialdehyde content

The malondialdehyde content was determined using the method described by Dhindsa and Matowe⁸³. In this procedure, 1.0 g of each plant sample was chopped and homogenized with 10 mL of 0.1% trichloroacetic acid at 4 °C, followed by centrifugation (13,968 × g, 10 min, 4 °C). Afterwards, 0.5 mL of the obtained extract was combined with 0.5 mL of phosphate buffer (pH 7.6) and 1 mL of 0.5% thiobarbituric acid (TBA), which was previously dissolved in 20% trichloroacetic acid (TCA). The control sample consisted of 0.5 mL of TCA, 0.5 mL of phosphate buffer (pH 7.6), and 1 mL of 0.5% TBA dissolved in 20% TCA. All the mixtures were then incubated in a water bath at 95 °C for 30 min and subsequently cooled to room temperature. The absorbance was measured initially at 532 nm (peak for the MDA–TBA complex) and then at 600 nm using a UV‒VIS Helios Beta spectrophotometer. A molar absorption coefficient (ε) of 155 mM⁻¹ cm⁻¹ was used to calculate the MDA content, which was reported as μmol per gram of fresh weight (FW).

Fresh and dry weight determination

Fresh weight was determined for 15 plants per repetition (45 plants per treatment group in total). Each plant was weighed with an Ohaus PA214CM/1 balance (OHAUS Europe GmbH, Nanikon, Switzerland), the data were summarized, and the means were calculated. The dry weight was estimated by placing the plants in an oven at 65 °C until a constant weight was achieved, after which the dry material was reweighed. The fresh weight was reported in grams per plant, while the dry weight was reported as a percentage of the FW.

Statistical analysis

The statistical significance of the effects of the treatments was evaluated by one-way analysis of variance (ANOVA) using Statistica version 13.3 (TIBCO Software Inc., Palo Alto, CA, USA). After one-way ANOVA, homogenous groups were evaluated using Fisher’s LSD or Duncan post hoc tests at p ≤ 0.05. The results are presented as the means of at least 3 repetitions plus the standard error (SE).

A heatmap with the hierarchical clustering analysis was generated using the online MetaboAnalyst software (ver. 6.0). The uploaded files contained data (concentrations or activities of the determined chemical compounds) as a dot-separated value (CSV) table or descriptors of the samples. The data were autoscaled (mean-centred and divided by the standard deviation of each variable) before generating a heatmap, and the Euclidean distance measurement and Ward clustering algorithms were subsequently applied. A colour scale from light green (lower values) through black to light red (higher values) was used.

Acknowledgements

This work was financially supported by the Ministry of Education and Science of the Republic of Poland (subsidy for the University of Agriculture in Krakow, Poland, 2021; subsidy of the coordinator of agricultural and horticultural discipline).

Author contributions

A.Ka. and G.G. designed the experimental setup and provided commercial materials for the experiments. A.Ka., A.Ko., J.G., E.R.-S., K.G. and A.Si. performed the experiments and relevant laboratory analyses of the plant samples. G.G. synthesized and analysed the noncommercial nanocomposites. A.Ka., A.Ko. and J.G. performed the software and statistical analyses of the data. A.Ka., A.Ko., G.G. and J.G. performed data curation and visualization. A.Ka., J.G. and A.Ko. wrote the first draft. A.Ka., A.Ko., A.Se., D.H., J.G., M.K., G.G. and V.F. consulted on, wrote and edited the final version of the manuscript. All the authors reviewed the manuscript.

Data availability

Data is provided within the manuscript or supplementary information files. The datasets used and/or analyzed during the current study are available from the corresponding authors upon reasonable request.

Competing interests

The authors declare no competing interests.

Ethical approval

All plant-related experimental studies, including the collection of plant material, were conducted in accordance with relevant institutional, national, and international guidelines and legislation. The collection of plant material was performed legally in a dedicated plant factory, with appropriate permissions obtained where necessary. The study did not involve any protected or endangered species requiring special permits.

Abbreviations

A_lim

The area that was occupied by a single molecule in a completely packed layer

APX

Ascorbate peroxidase

AsA

L-ascorbic acid

CAT

Catalase

Ch

Chitosan

Ch–Put

Chitosan‒putrescine nanocomposite

C_s⁻¹

Surface compressional modulus

DW

Dry weight

FW

Fresh weight

GPOX

Guaiacol peroxidase

GSH

Glutathione reduced

MDA

Malondialdehyde

PL

Phospholipid

PM

Plasma membrane

POX

Peroxidase

Put

Putrescine

ROS

Reactive oxygen species

SOD

Superoxide dismutase

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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