A K+-Efflux Antiporter is Vital for Tolerance to Salt Stress in Rice

Abstract

Salt damage significantly affects rice growth and development, posing a threat to food security. Understanding the mechanisms underlying rice's response to salt stress is crucial for enhancing its tolerance. This study aimed to elucidate the genetic and physiological mechanisms of rice adaptation to salt stress. We found that the expression of OsKEA1, a potassium (K⁺)-efflux antiporter gene in rice, was induced by salt. Both genetic and physiological experiments demonstrated that the mutation in OsKEA1 disrupted the Na⁺/K⁺ balance under salt stress conditions. Furthermore, OsKEA1 mutation exacerbated reactive oxygen species (ROS) accumulation, disrupted the antioxidant enzyme system, and compromised chloroplast integrity under salt stress. This study unveils the adaptive mechanisms of rice to salt damage and highlights the critical role of OsKEA1 in managing salt stress.

Keywords: OsKEA1; Chloroplast integrity and DNA damage; Na+/K+ and ROS balance; Rice; Salt stress.

Fig. 1

Phenotypic comparison of WT, oskea1, cas9-3 and cas9-8 seedlings grown under salt stress. A–C Phenotypic characterization in different plants under normal condition (A), 6 d for 180 mM NaCl treatment (B) condition. C, D Fresh weight (C) and dry weight (D) in different plants under 180 mM NaCl treatment for 6 d. E Survival rates of different plants recoverd for 7 days. Data are means ± SD (n = 3). Asterisks show statistical significances at **p ≤ 0.01 according to Student’s t-test

Fig. 2

The function of OsKEA1 in ion homeostasis. A Changes in expression level of OsKEA1 after 180 mM NaCl treatment. B–D Ion contents in leaves of 2-week-old plants after 6 d salt treatment (180 mM NaCl). The ion content of normal and NaCl treated leave samples was analyzed. The values of Na⁺ (B) and K⁺ (C) content together with the ratio of Na⁺/K⁺ (D) are presented. DW, dry weight. Data are means ± SD (n = 3). Asterisks show statistical significances at *p ≤ 0.05, **p ≤ 0.01 according to Student’s t-test

Fig. 3

ROS accumulation in the WT, oskea1, cas9-3 and cas9-8 plants under different conditions. A DAB staining of WT, oskea1, cas9-3 and cas9-8 leaves under normal (left) and 180 mM NaCl treatment (right) conditions. B NBT staining of WT, oskea1, cas9-3 and cas9-8 leaves under normal (left) and 180 mM NaCl (right) treatment conditions. c-3, cas9-3; c-8, cas9-8.C, D H₂DCFDA probe labeling of WT, oskea1, cas9-3 and cas9-8 leaves under normal (C) and 180 mM NaCl treatment (D) conditions. Green signal, oxidized H₂DCFDA; Red signal, chlorophyll. Scale bars = 1 cm in A, B; Scale bars = 100 μm in C, D.

Fig. 4

Effect of WT, oskea1, cas9-3 and cas9-8 plants on the cellular redox homeostasis under different conditions. A–C Statistical analysis contents of (A) hydrogen peroxide (H₂O₂), B superoxide anion (O₂⁻) and C malondialdehyde (MDA) in different plants under normal and 180 mM NaCl treatment conditions. (D-F) Statistical analysis activity of D catalase (CAT), E peroxidase (POD) and F superoxide dismutase (SOD) in different plants under normal and 180 mM NaCl treatment conditions. Data are means ± SD (n = 3). Asterisks show statistical significances at **p ≤ 0.01 according to Student’s t-test

Fig. 5

Determination of pigment content and ultrastructural observation of chloroplasts of different plants under normal and 180 mM NaCl treated conditions. A, B Pigment content under normal (A) and 180 mM NaCl treated (B) conditions. Chl, chlorophyll. Car, carotenoids. C, D Ultrastructure of chloroplasts in mesophyll cells of different plants under normal (C) and 180 mM NaCl treated (D) conditions. Og, osmiophilic granule; Dc, degradation of chloroplast. Scale bars = 10 μm in C, 6 μm in D, 1 μm in enlarged part of C and D; Data are means ± SD (n = 3). Asterisks show statistical significances at **p ≤ 0.01 according to Student’s t-test

Fig. 6

Determination of expression levels of photosynthetic genes and accumulation levels of photosynthetic proteins. A–F Relative expression levels of chlorophyll synthesis and photosynthesis-related genes in different plants under normal and 180 mM NaCl treated conditions. G Accumulations of photosynthetic proteins detected in total proteins in WT, oskea1, cas9-3 and cas9-8 plants under normal (left) and 180 mM NaCl treated (right) conditions. PS, photosystem. β-actin was used as a loading control

Fig. 7

Determination of DNA damage degree and expression levels of DNA replication and repair-associated genes. A TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) signals in different plants under normal (left) and 180 mM NaCl treated (right) conditions. Red, TUNEL-positive signals; Blue signal, DAPI staining. Scale bars = 50 μm. B–D Relative expression levels of DNA replication and repair-associated genes in different plants under normal and 180 mM NaCl treated conditions. Data are means ± SD (n = 3). Asterisks show statistical significances at *p ≤ 0.05, **p ≤ 0.01 according to Student’s t-test