Exogenous SA Affects Rice Seed Germination under Salt Stress by Regulating Na+/K+ Balance and Endogenous GAs and ABA Homeostasis

Abstract

Salinity reduces agricultural productivity majorly by inhibiting seed germination. Exogenous salicylic acid (SA) can prevent the harm caused to rice by salinity, but the mechanisms by which it promotes rice seed germination under salt stress are unclear. In this study, the inhibition of germination in salt-sensitive Nipponbare under salt stress was greater than that in salt-tolerant Huaidao 5. Treatment with exogenous SA significantly improved germination of Nipponbare, but had little effect on Huaidao 5. The effects of exogenous SA on ion balance, metabolism of reactive oxygen species (ROS), hormone homeostasis, starch hydrolysis, and other physiological processes involved in seed germination of rice under salt stress were investigated. Under salt stress, Na⁺ content and the Na⁺/K⁺ ratio in rice seeds increased sharply. Seeds were subjected to ion pressure, which led to massive accumulation of H₂O₂, O₂^-, and malonaldehyde (MDA); imbalanced endogenous hormone homeostasis; decreased gibberellic acid (GA1 and GA4) content; increased abscisic acid (ABA) content; inhibition of α-amylase (EC 3.2.1.1) activity; and slowed starch hydrolysis rate, all which eventually led to the inhibition of the germination of rice seeds. Exogenous SA could effectively enhance the expression of OsHKT1;1, OsHKT1;5, OsHKT2;1 and OsSOS1 to reduce the absorption of Na+ by seeds; reduce the Na⁺/K⁺ ratio; improve the activities of SOD, POD, and CAT; reduce the accumulation of H₂O₂, O₂^-, and MDA; enhance the expression of the GA biosynthetic genes OsGA20ox1 and OsGA3ox2; inhibit the expression of the ABA biosynthetic gene OsNCED5; increase GA1 and GA4 content; reduce ABA content; improve α-amylase activity, and increase the content of soluble sugars. In summary, exogenous SA can alleviate ion toxicity by reducing Na⁺ content, thereby helping to maintain ROS and hormone homeostasis, promote starch hydrolysis, and provide sufficient energy for seed germination, all of which ultimately improves rice seed germination under salt stress. This study presents a feasible means for improving the germination of direct-seeded rice in saline soil.

Keywords: germination; hormone homeostasis; ion balance; rice; salicylic acid; salt stress.

Figure 1

Effects of different concentrations of SA on the germination of rice seeds (Nipponbare and Huaidao 5) under salt stress. Distilled water was used in the CK treatment; the concentration of NaCl was 150 mM (NaCl), and the dosage of salicylic acid (SA) was 0.1, 0.5, or 1 mM (NaCl + SA). The seeds were incubated in a 90 mm culture dish with two layers of filter paper for 5 days, followed by calculation of (A) germination rate, (B) germination percentage, and (C) germination index. Data are presented as means ± SE. Different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 2

Effects of NaCl and SA on root and shoot growth of Nipponbare and Huaidao 5.

Figure 3

Effects of exogenous salicylic acid (SA) on ion accumulation in rice seeds under salt stress. Seeds of Nipponbare were incubated in distilled water (CK), 150 mM NaCl (Salt), and 150 mM NaCl + 0.1 mM SA (Salt + SA) for 1, 3, and 5 days. The amount of Na⁺ (A) and K⁺ (B) accumulated in seeds from the different treatments and incubation times are compared, along with the Na⁺/K⁺ ratio (C). Data are presented as means ± SE. Different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 4

Effects of exogenous salicylic acid (SA) on the expression of OsHKT1;1 and OsHKT1;5 in rice seed under salt stress. Seeds of Nipponbare were incubated in distilled water (CK), 150 mM NaCl (Salt), and 150 mM NaCl + 0.1 mM SA (Salt + SA) for 1, 3, and 5 days. The expression of OsHKT1;1 (A) and OsHKT1;5 (B) in seeds subjected to the different treatments and incubation times are compared. Data are presented as means ± SE. Different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 5

Effects of exogenous salicylic acid (SA) on the accumulation of H₂O₂, O₂⁻, and malonaldehyde (MDA) in rice seeds under salt stress. Seeds of Nipponbare were incubated in distilled water (CK), 150 mM NaCl (Salt), and 150 mM NaCl + 0.1 mM SA (Salt + SA) for 1, 3, and 5 days. The accumulation of H₂O₂ (A), O₂⁻ (B), and MDA (C) in seeds subjected to the different treatments and incubation times are compared. Data are presented as means ± SE. Different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 6

Effects of exogenous salicylic acid (SA) on superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities in rice seeds under salt stress. Seeds of Nipponbare were incubated in distilled water (CK), 150 mM NaCl (Salt), and 150 mM NaCl + 0.1 mM SA (Salt + SA) for 1, 3, and 5 days. The activity of SOD (A), POD (B), and CAT (C) in seeds subjected to the different treatments and incubation times are compared. Data are presented as means ± SE. Different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 7

Effect of exogenous salicylic acid (SA) on endogenous hormones in rice seeds under salt stress. Seeds of Nipponbare were incubated in distilled water (CK), 150 mM NaCl (Salt), and 150 mM NaCl + 0.1 mM SA (Salt + SA) for 1, 3, and 5 days. The content of abscisic acid (ABA) (A), GA1 (B), and GA4 (C) in seeds from the different treatments and incubation times are compared, along with values of (GA1 + GA4)/ABA (D). Data are presented as means ± SE. Different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 8

Effects of exogenous salicylic acid (SA) on gibberellic acid (GA) biosynthesis and inactivation of associated gene expressions under salt stress. Seeds of Nipponbare were incubated in distilled water (CK), 150 mM NaCl (Salt), and 150 mM NaCl + 0.1 mM SA (Salt + SA) for 1, 3, and 5 days. The expressions of GA biosynthetic genes (A) OsGA20ox1 and (B) OsGA3ox2 and inactivation genes (C) OsGA2ox1 and (D) OsGA2ox3 were compared. Data are presented as means ± SE. Different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 9

Effects of exogenous salicylic acid (SA) on abscisic acid (ABA) biosynthesis and expression of ABA catalyzing genes under salt stress. Seeds of Nipponbare were incubated in distilled water (CK), 150 mM NaCl (Salt), and 150 mM NaCl + 0.1 mM SA (Salt + SA) for 1, 3, and 5 days. The expressions of ABA biosynthetic genes (A) OsNCED1, (B) OsNCED3, and (C) OsNCED5 and catalytic genes (D) OsABA8′OH1, (E) OsABA8′OH2, and (F) OsABA8′OH3 were recorded. Data are presented as means ± SE. Different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 10

Effects of exogenous SA on α-amylase activity (A) and soluble sugar content (B) under salt stress. Seeds of Nipponbare were incubated in distilled water (CK), 150 mM NaCl (Salt), and 150 mM NaCl + 0.1 mM SA (Salt + SA) for 1, 3, and 5 days. Data are presented as means ± SE. Different lowercase letters indicate significant differences between treatments (p < 0.05).