Foliar spraying with amino acids and their chitosan nanocomposites as promising way to alleviate abiotic stress in iceberg lettuce grown at different temperatures

Abstract

We analyzed the effects of foliar spraying with amino acids, chitosan (CHS) and nanocomposites (NCs) of chitosan with the amino acids proline, L-cysteine and glycine betaine (CHS-Pro NCs; CHS-Cys NCs, CHS-GB NCs, respectively) on the changes in the physiological and biochemical parameters of iceberg lettuce grown at the control temperature (20 °C) and under chilling conditions (4 °C). The physicochemical parameters of the phospholipid monolayers (PLs) extracted from plants showed the effects of the treatments on the properties of the monolayers, namely, the packing density and flexibility. We observed increased accumulation of proline at 4 °C, and differences in the concentrations of sugars in most of the analyzed variants were a consequence of the lowered temperature and/or the use of organic compounds. A temperature of 4 °C caused a significant increase in the L-ascorbic acid level compared with that at 20 °C. Differences were also found in glutathione (GSH) content depending on the temperature and treatment with the tested organic compounds. CHS NCs loaded with Pro and GB were effective at increasing the amount of phenols under stress temperature conditions. We noted that a significant increase in the antioxidant activity of plants at 4 °C occurred after priming with Cys, CHS-Cys NCs, Pro and CHS-Pro NCs, and the CHS nanocomposites were more effective in this respect. Both low-temperature stress and foliar spraying of lettuce with various organic compounds caused changes in the activity of antioxidant enzymes. Two forms of dismutase (SOD), iron superoxide dismutase (FeSOD) and copper/zinc superoxide dismutase (Cu/ZnSOD), were identified in extracts from the leaves of iceberg lettuce seedlings. The application of the tested organic compounds, alone or in combination with CHS, increased the amount of malondialdehyde (MDA) in plants grown under controlled temperature conditions. Chilling caused an increase in the content of MDA, but some organic compounds mitigated the impact of low temperature. Compared with that of plants subjected to 20 °C, the fresh weight of plants exposed to chilling decreased. However, the tested compounds caused a decrease in fresh weight at 4 °C compared with the corresponding control samples. An interesting exception was the use of Cys, for which the difference in the fresh weight of plants grown at 20 °C and 4 °C was not statistically significant. After Cys application, the dry weight of the chilled plants was greater than that of the chilled control plants but was also greater than that of the other treated plants in this group. To our knowledge, this is the first report demonstrating that engineered chitosan-amino acid nanocomposites could be applied as innovative protective agents to mitigate the effects of chilling stress in crop plants.

Keywords: Antioxidants; Catalase; Low-molecular weight compounds; Plasma membrane; Polyphenols; Superoxide dismutases.

Figure 1

Exemplary π–A isotherms for PL fractions isolated from plants treated with chitosan, amino acids and their derivatives and nanocomposites of chitosan with the mentioned compounds; deionized water served as a control (for abbreviations, see the Materials and methods).

Figure 2

Exemplary courses of the C_s⁻¹ compression modulus as a function of surface pressure for PL lipid monolayers extracted from plants treated with chitosan, amino acids and their derivatives and nanocomposites of chitosan with the mentioned compounds; deionized water served as a control (for abbreviations, see the Materials and methods).

Figure 3

Changes in the A_lim (A) and C_s⁻¹_max (B) characterizing phospholipid monolayers extracted from iceberg lettuce plants caused by the treatments with chitosan, amino acids and their derivatives (blue columns) and nanocomposites of chitosan with the mentioned compounds (red columns); deionized water served as a control (for abbreviations, see the Materials and methods).

Figure 4

Changes in the proline contents in the leaves of iceberg lettuce seedlings caused by treatments with chitosan, amino acids and their derivatives (green columns) and nanocomposites of chitosan with the mentioned compounds (orange columns); deionized water served as a control (for abbreviations, see the Materials and methods). Means followed by different lower-case letters, separately for temperature groups, are significantly different at the 0.05 level according to Duncan’s multiple range test. Different capital letters show a comparison of the influence of temperature on the examined parameter according to the t test (p < 0.05). The values are the means of three replications (± SDs).

Figure 5

Changes in the contents of total phenolics in the leaves of iceberg lettuce seedlings caused by the treatments with chitosan, amino acids and their derivatives (green columns) and nanocomposites of chitosan with the mentioned compounds (orange columns); deionized water served as a control (for abbreviations, see the Materials and methods). Means followed by different lower-case letters for each temperature group are significantly different at the 0.05 level according to Duncan’s multiple range test. Different capital letters show a comparison of the influence of temperature on the examined parameter according to the t test (p < 0.05). The values are the means of three replications (± SDs).

Figure 6

Changes in the total antioxidant capacity of the leaves of iceberg lettuce seedlings caused by treatment with chitosan, amino acids and their derivatives (green columns) and nanocomposites of chitosan with the mentioned compounds (orange columns); deionized water served as a control (for abbreviations, see the Materials and methods). Means followed by different lower-case letters for each temperature group are significantly different at the 0.05 level according to Duncan’s multiple range test. Different capital letters show a comparison of the influence of temperature on the examined parameter according to the t test (p < 0.05). The values are the means of three replications (± SDs).

Figure 7

Changes in the activity of CAT (A) and SOD (B) in the leaves of iceberg lettuce seedlings caused by treatments with chitosan, amino acids and their derivatives (green columns) and nanocomposites of chitosan with the mentioned compounds (orange columns); deionized water served as a control (for abbreviations, see the Materials and methods). Means followed by different lower-case letters for each temperature group are significantly different at the 0.05 level according to Duncan’s multiple range test. Different capital letters show a comparison of the influence of temperature on the examined parameter according to the t test (p < 0.05). The values are the means of three replications (± SDs).

Figure 8

Native PAGE analysis of SOD forms.

Figure 9

Changes in the activity levels of APX (A) and GPOX (B) in the leaves of iceberg lettuce seedlings caused by the treatments with chitosan, amino acids and their derivatives (green columns) and nanocomposites of chitosan with the mentioned compounds (orange columns); deionized water served as a control (for abbreviations, see the Materials and methods). Means followed by different lower-case letters for each temperature group are significantly different at the 0.05 level according to Duncan’s multiple range test. Different capital letters show a comparison of the influence of temperature on the examined parameter according to the t test (p < 0.05). The values are the means of three replications (± SDs).

Figure 10

Changes in the MDA contents in the leaves of iceberg lettuce seedlings caused by treatments with chitosan, amino acids and their derivatives (green columns) and nanocomposites of chitosan with the mentioned compounds (orange columns); deionized water served as a control (for abbreviations, see the Materials and methods). Means followed by different lower-case letters for each temperature group are significantly different at the 0.05 level according to Duncan’s multiple range test. Different capital letters show a comparison of the influence of temperature on the examined parameter according to the t test (p < 0.05). The values are the means of three replications (± SDs).

Figure 11

(a) SEM image and (b) DLS result of the chitosan-cysteine nanocomposite (CHS-Cys NCs).

Figure 12

Experimental design of the study.