Combination of β-Aminobutyric Acid and Ca2+ Alleviates Chilling Stress in Tobacco (Nicotiana tabacum L.)

Abstract

Chilling is a major abiotic factor limiting the growth, development, and productivity of plants. β-aminobutyric acid (BABA), a new environmentally friendly agent, is widely used to induce plant resistance to biotic and abiotic stress. Calcium, as a signaling substance, participates in various physiological activities in cells and plays a positive role in plant defense against cold conditions. In this study, we used tobacco as a model plant to determine whether BABA could alleviate chilling stress and further to explore the relationship between BABA and Ca²⁺. The results showed that 0.2 mM BABA significantly reduced the damage to tobacco seedlings from chilling stress, as evidenced by an increase in photosynthetic pigments, the maintenance of cell structure, and upregulated expression of NtLDC1, NtERD10B, and NtERD10D. Furthermore, 0.2 mM BABA combined with 10 mM Ca²⁺ increased the fresh and dry weights of both roots and shoots markedly. Compared to that with single BABA treatment, adding Ca²⁺ reduced cold injury to the plant cell membrane, decreased ROS production, and increased antioxidant enzyme activities and antioxidant contents. The combination of BABA and Ca²⁺ also improved abscisic acid and auxin contents in tobacco seedlings under chilling stress, whereas ethylene glycol-bis (β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) reversed the effects of BABA. These findings suggested that BABA enhances the cold tolerance of tobacco and is closely related to the state of Ca²⁺ signaling.

Keywords: calcium ion; chilling stress; membrane lipid damage; oxidative stress; β-aminobutyric acid.

FIGURE 1

Effect of BABA on the chloroplast ultrastructure in tobacco under cold stress. Ultrastructural changes with control treatment (A,D,G,J), cold treatment (B,E,H,K), and cold + 0.2 mM BABA treatment (C,F,I,L). V, vacuolar; Chl, chloroplast; T, thylakoid; N, nuclear; Nm, nuclear membrane; Pm, plasma membrane; p, plastoglobule.

FIGURE 2

Relative expression of genes in response to cold stress in tobacco. Gene expression was analyzed by qRT-PCR. Seedlings were pre-treated with β-aminobutyric acid (BABA) for 3 days prior to cold stress. The L25 gene was used as an internal control. The gene level in the control treatment group was given an arbitrary value of 1. Gene expression data were normalized to levels in the control group. Data are representative of three biological replicate experiments. Different letters indicate a significant difference between means (P < 0.05).

FIGURE 3

Effects of BABA, Ca²⁺, BABA + Ca²⁺, and BABA + EGTA on electrolyte leakage (A) and MDA (B) of tobacco under cold stress. Data are expressed as mean value ± SE, n = 3. Mean values in columns with different letters are significantly different at the 0.05 level according to Duncan’s test.

FIGURE 4

Effects of BABA, Ca²⁺, BABA + Ca²⁺, and BABA + EGTA on H₂O₂ (A), O₂^– (B), GSH (C), and AsA (D) of tobacco under cold stress. Data represent means ± SE (n = 3). Different letters denote significant differences according to Duncan’s test (P < 0.05).

FIGURE 5

Effects of BABA, Ca²⁺, BABA + Ca²⁺, and BABA + EGTA on SOD (A), POD (B), and CAT (C) of tobacco under chilling stress. Data is the mean value ± SE from three replicates. The values with the same letter are not significantly different at P < 0.05 according to Duncan’s test.

FIGURE 6

Schematic representation of mechanism through which BABA or BABA + Ca²⁺ protects tobacco from chilling.