CHOLINE TRANSPORTER-RELATED 4 (CTR4) Is Involved in Drought and Saline Tolerance in Rice

doi:10.1016/j.rsci.2024.10.002

Abstract

Abstract:

The tolerance of rice to drought and saline stress is crucial for maintaining yields and promoting widespread cultivation. From an ethyl methanesulfonate (EMS)-mutagenized mutant library, we identified a mutant that is susceptible to osmotic stress, named Osmotic Stress Sensitivity 1 (Oss1). Using MutMap sequencing, we characterized the role of a choline transporter-related family gene, CTR4 (Choline Transporter-Related 4), in rice’s tolerance to drought and salt stress. CTR4 plays a critical role in regulating membrane lipid synthesis. In knockout mutants, the total membrane lipid content, especially unsaturated fatty acids, was significantly reduced. Compared with the wild type, knockout mutants exhibited decreased membrane lipid stability under drought and salt stress, faster water loss, higher relative electrolyte leakage, and lower levels of proline and soluble sugars, leading to impaired tolerance to drought and salt stress. In contrast, the overexpression of CTR4 enhanced seedling tolerance to drought and saline stress. The overexpression lines displayed lower malondialdehyde levels, reduced relative electrolyte leakage, and slower rates of leaf water loss under stress conditions, thereby improving seedling survival rates during stress. Moreover, lipid synthesis gene expression was down-regulated in CTR4 mutants, potentially exacerbating membrane permeability defects and further compromising stress resistance. These findings suggest that CTR4 mediates choline transport and influences cell membrane formation, thereby enhancing rice defenses against drought and salt stress by maintaining lipid homeostasis.

Key words: Oryza sativa, drought stress, salt stress, choline transporter, lipid homeostasis

Yu Shicong, Luo Ruxian, Zheng Shuqin, Ning Jing, Shi Yuanzhu, Guo Daiming, Jia Liangmeng, Wang Sen, Xiao Guizong, Guo Pengwang, Li Yang, Ma Xiaoding. CHOLINE TRANSPORTER-RELATED 4 (CTR4) Is Involved in Drought and Saline Tolerance in Rice[J]. Rice Science, 2025, 32(1): 52-66.

Figures/Tables 7

Fig. 1. Drought and osmotic sensitivity phenotypes of wild type Yixiang 1B (YX1B) and Oss1 mutant. A-C, Phenotypes of YX1B and Oss1 after treatment with 200 g/L PEG6000 (A), 150 mmol/L NaCl (B), and drought for 5 d with sandy moisture at 8% (C). Scale bars, 5 cm. D-F, Recovery rates of YX1B and Oss1 after treatment with 200 g/L PEG6000 (D), 150 mmol/L NaCl (E), and drought for 5 d with sandy moisture at 8% (F). G, Nitroblue tetrazolium (NBT) and 3,3′-diaminobenzidine (DAB) staining of YX1B and Oss1 to visualize reactive oxygen species (ROS) accumulation. Blue spots originate from the reaction of superoxide anions with NBT, indicating ROS accumulation. Brown spots result from peroxidase breaking down hydrogen peroxide (H2O2), oxidizing DAB to produce a brown precipitate. Scale bars, 5 cm. H and I, Malondialdehyde (MDA) and H2O2 contents in YX1B and Oss1 after treatment with 200 g/L PEG6000 for 24 h. In D-F, H, and I, data are Mean ± SD (n = 3). **, P < 0.01 by the Student’s t-test.

Fig. 2. Mapping and cloning of CTR4 by MutMap sequencing and its expression levels under different treatments. A, Distribution of ∆index of single nucleotide polymorphisms (SNPs) across the 12 chromosomes. ∆SNP-index means the absolute value of the difference in SNP index between the phenotypic bulks of wild type Yixiang 1B (YX1B) and the Oss1 mutant. B, Mutation sites in the Oss1 alleles within the genome. ATG and TGA represent the translation start and stop codons, respectively. Black lines indicate introns, black boxes represent exons, and white boxes denote the 5′ untranslated region (5′-UTR) and 3′ untranslated region (3′-UTR). C, Relative expression levels of CTR4 in Dongjin and Yixiang 1B after treatment with 200 g/L PEG6000 and 150 mmol/L NaCl. OsActin (LOC_Os03g50885) was used as the internal reference, and the expression level of CTR4 in untreated seedlings at 0 h was used as the normalized reference value. Data are Mean ± SE (n = 3).

Fig. 3. Drought and saline sensitivity phenotype of CTR4. A, Phenotypes of wild type Dongjin (DJ), knockout mutant line CTR4-KODJ, and overexpression line CTR4-OEDJ after treatment with drought (8% moisture content in the sandy soil), 200 g/L PEG600, and 150 mmol/L NaCl for 0 and 5 d, followed by 10 d of recovery. Scale bars, 5 cm. B, Recovery rates of DJ, CTR4-KODJ, and CTR4-OEDJ after treatment with drought (D), 200 g/L PEG6000 (E), and 150 mmol/L NaCl (F). Data are Mean ± SE (n = 3). * and **, P < 0.05 and P < 0.01, respectively, by the Student’s t-test.

Fig. 4. CTR4 was constitutively expressed in rice tissues and induced by abiotic stress. A, Relative expression levels of CTR4 in different tissues, including flag leaf (FL), second leaf from the top (2ndL), third leaf from the top (3ndL), fourth leaf from the top (4thL), fifth leaf from the top (5thL), root, panicle, culm, and leaf sheath, of wild type Yixiang 1B (YX1B). Actin1 was used as the internal reference gene. Data are Mean ± SD (n = 3). B-F, CTR4PRO-GUS plant staining in young leaf (B), node (C), spikelet (D), panicle (E), and seedlings (F). Scale bars in B-F, 1 cm, 2 mm, 2 mm, 1 cm, and 1 cm, respectively. G, Staining of GUS transgenic plants after 24 h of 200 g/L PEG6000 and 150 mmol/L NaCl treatments. Scale bar, 2 cm. H-I, Subcellular localization of CTR4 in tobacco leaves (H) and rice protoplasts (I) with eGFP used for subcellular localization control. Green represents the fluorescence signal from the green fluorescent protein (GFP), red represents the fluorescence signal from the plasma membrane (PM) protein, and yellow indicates where the signals overlap in the combined image. Scale bars in H and I, 25 μm and 10 μm, respectively.

Fig. 5. Accumulation of reactive oxygen species (ROS) in rice leaves of wild type Dongjin (DJ), knockout mutant line CTR4-KODJ, and overexpression line CTR4-OEDJ. A, 3,3′-Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining for ROS accumulation. Brown spots result from peroxidase breaking down H2O2, oxidizing DAB to produce a brown precipitate. Blue spots originate from the reaction of superoxide anions with NBT, generating a blue precipitate used to label ROS accumulation. Scale bars, 5 cm. B-G, H2O2 contents after NaCl (B) and drought (E) treatments, malondialdehyde (MDA) contents after NaCl (C) and drought (F) treatments, and proline contents after NaCl (D) and drought (G) treatments. Data are Mean ± SE (n = 3). * and **, P < 0.05 and P < 0.01, respectively, by the Student’s t-test.

Fig. 6. Transcriptional analysis of genes regulated in drought and saline tolerance in roots of wild type Dongjin (DJ) and knockout mutant line (CTR4-KODJ) and overexpression line (CTR4-OEDJ). A-F, Relative expression of WR1 (A), WS1 (B), CATB (C), DMS2 (D), ERF71 (E), MRLK63 (F) under 200 g/L PEG6000 at 0.5, 1, 2, 4, 8, and 12 h after treatment. G-L, Relative expression of WR1 (G), WS1 (H), HKT1;5 (I), DREB6 (J), NAC6 (K), and SOS1 (L) under NaCl stress at 0.5, 1, 2, 4, 8, and 12 h after treatment. Actin1 was used as the internal reference gene. Data are Mean ± SE (n = 3). **, P < 0.01 by the Student’s t-test.

Fig. 7. CTR4 is involved in regulation of rice cell membrane homeostasis. A and B, Relative conductivity rates of wild type Dongjin (DJ), knockout mutant line (CTR4-KODJ), and over expression lines (CTR4-OEDJ under drought (A) and 150 mmol/L NaCl (B) treatments. Data are Mean ± SE (n = 3). The different lowercase letters on the bars denote the significant difference at P < 0.05. C and D, Choline (C) and phosphatidylcholine (D) contents in leaves of untreated DJ, CTR4-KODJ, and CTR4-OEDJ. Data are Mean ± SE (n = 3). * and **, P < 0.05 and P < 0.01, respectively, by the Student’s t-test between wide-type and mutants. E, Water loss rates of DJ, CTR4-KODJ, and CTR4-OEDJ at 25 ºC. F, Total fatty acid content of membrane lipids in DJ and CTR4-KODJ seedlings after 5 d of 200 g/L PEG6000 treatment. Data are Mean ± SE (n = 3). The different lowercase letters on the bars denote the significant difference at P < 0.05. G and H, Percentage of total fatty acids content of membrane lipids (G) and fatty acid content of membrane lipids (H) in DJ and CTR4-KODJ seedlings after 5 d of 200 g/L PEG6000 treatment. Data are Mean ± SE (n = 3). The different lowercase letters on the bars denote the significant difference at P < 0.05 within each fatty acid group.

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