A β-Carotene Ketolase Gene NfcrtO from Subaerial Cyanobacteria Confers Drought Tolerance in Rice

doi:10.1016/j.rsci.2023.10.002

Abstract

Abstract:

Nostoc flagelliforme is a terrestrial cyanobacterium that can resist many types of stressors, including drought, ultraviolet radiation, and extreme temperatures. In this study, we identified the drought tolerance gene NfcrtO, which encodes a β-carotene ketolase, through screening the transcriptome of N. flagelliforme under water loss stress. Prokaryotic expression of NfcrtO under 0.6 mol/L sorbitol or under 0.3 mol/L NaCl stress significantly increased the growth rate of Escherichia coli. When NfcrtO was heterologously expressed in rice, the seedling height and root length of NfcrtO-overexpressing rice plants were significantly higher than those of the wild type (WT) plants grown on ½ Murashige and Skoog solid medium with 120 mmol/L mannitol at the seedling stage. Transcriptome analysis revealed that NfcrtO was involved in osmotic stress, antioxidant, and other stress-related pathways. Additionally, the survival rate of the NfcrtO-overexpression lines was significantly higher than that of the WT line under both hydroponic stress (24% PEG and 100 mmol/L H₂O₂) and soil drought treatment at the seedling stage. Physiological traits, including the activity levels of superoxide dismutase, peroxidase, catalase, total antioxidant capacity, and the contents of proline, trehalose, and soluble sugar, were significantly improved in the NfcrtO-overexpression lines relative to those in the WT line under 20% PEG treatment. Furthermore, when water was withheld at the booting stage, the grain yield per plant of NfcrtO-overexpression lines was significantly higher than that of the WT line. Yeast two-hybrid analysis identified interactions between NfcrtO and Dna J protein, E3 ubiquitin-protein ligase, and pyrophosphate-energized vacuolar membrane proton pump. Thus, heterologous expression of NfcrtO in rice could significantly improve the tolerance of rice to osmotic stress, potentially facilitating the development of new rice varieties.

Key words: antioxidant enzyme, β-carotene ketolase, drought resistance, Nostoc flagelliforme, osmotic stress, rice, transcriptome analysis

Gao Ningning, Ye Shuifeng, Zhang Yu, Zhou Liguo, Ma Xiaosong, Yu Hanxi, Li Tianfei, Han Jing, Liu Zaochang, Luo Lijun. A β-Carotene Ketolase Gene NfcrtO from Subaerial Cyanobacteria Confers Drought Tolerance in Rice[J]. Rice Science, 2024, 31(1): 62-76.

Figures/Tables 8

Fig. 1. Transcriptome and NfcrtO expression profile analyses of Nostoc flagelliforme under drought stress. A, Reads per kilobase per million mapped reads (RPKM) data of NfcrtO from a transcriptome analysis of N. flagelliforme under drought stress. B, Expression profile analysis of NfcrtO under drought stress. NC, Normal conditions; D1-D5, RNA sequencing of N. flagelliforme under water loss levels of 10%, 30%, 50%, 70%, and 90%. Data are Mean ± SD (n = 3). *, P < 0.05; **, P < 0.01, by the Student’s t-test.

Fig. 2. Heterologous expression of NfcrtO in Escherichia coli. A and B, Protein expression of NfcrtO under different concentrations (A) and times (B) of isopropyl- β-d-1-thiogalactopyranosid (IPTG) induction. Arrow indicates expressed NfcrtO. C-F, Growth charts of NfcrtO-expressing E. coli under normal conditions (C), 0.4 mmol/L IPTG induction (D), 0.6 mol/L sorbitol treatment and 0.4 mmol/L IPTG induction (E), and 0.3 mol/L NaCl treatment and 0.4 mmol/L IPTG induction (F). Data are Mean ± SD (n = 3). *, P < 0.05; **, P < 0.01, by the Student’s t-test.

Fig. 3. NfcrtO-overexpressing rice seedlings on ½ Murashige and Skoog (MS) solid medium under mannitol treatment. A and B, Phenotypes of NfcrtO-overexpression (OE1-OE4) and wild type (WT) lines under normal (A) and 120 mmol/L mannitol (B) treatments on ½ MS solid medium. Scale bars, 5 cm. C and D, Seedling plant heights of NfcrtO-overexpression and WT lines under normal (C) and 120 mmol/L mannitol (D) treatments on ½ MS solid medium. E and F, Seedling root lengths of NfcrtO-overexpression and WT lines under normal (E) and 120 mmol/L mannitol (F) treatments on ½ MS solid medium. Data are Mean ± SD (n = 3). *, P < 0.05; **, P < 0.01, by the Student’s t-test.

Fig. 4. RNA-sequencing of NfcrtO-overexpression seedlings in rice on ½ Murashige and Skoog (MS) solid medium under mannitol stress. A and B, Up-regulated (A) and down-regulated (B) genes as determined by comparative transcriptome analysis between the wild type (WT) and overexpression (NfcrtO) lines. WT-CK, NfcrtO-CK, WT-T, and NfcrtO-T represent WT and overexpression lines under normal conditions (CK) and mannitol stress treatment (T), respectively. C, Enriched Gene Ontology (GO) terms in the comparative transcriptome analysis between the WT and NfcrtO-overexpression lines. FDR, False discovery rate. D, Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the comparative transcriptome analysis between the WT and NfcrtO- overexpression lines. E and F, Validation by qRT-PCR of up-regulated (E) and down-regulated (F) genes. Leaves of rice seedlings on ½ MS culture flasks with and without 120 mmol/L mannitol for 7 d were sampled for transcriptome sequencing analysis. RNA was used for qRT-PCR, and Actin gene was used as the reference gene to normalize the target gene expression. OE1, OE2, and OE3 represent NfcrtO-overexpression lines. Data are Mean ± SD (n = 3). **, P < 0.01, by the Student’s t-test.

Fig. 5. NfcrtO-overexpression lines of rice under polyethylene glycol (PEG) and H2O2 stresses. A and B, Phenotypes of rice NfcrtO-overexpression (OE1, OE3, and OE4) and wild type (WT) lines under 24% PEG (A) and 100 mmol/L H2O2 (B) treatments. Scale bars, 5 cm. C and D, Survival rates of NfcrtO-overexpression and WT lines under 24% PEG (C) and 100 mmol/L H2O2 (D) treatments. Data are Mean ± SD (n = 3). *, P < 0.05; **, P < 0.01, by the Student’s t-test.

Fig. 6. Physiological indices of NfcrtO-overexpression lines of rice under 20% polyethylene glycol (PEG) treatment. A, Total antioxidant capacity (T-AOC) activity. B, Superoxide dismutase (SOD) activity. C, Peroxidase (POD) activity. D, Catalase (CAT) activity. E, Soluble sugar content. F, Trehalose content. G, Proline content. NC, Normal conditions; PEG(2) and PEG(3), PEG osmotic stress for 2 and 3 d, respectively; WT, Wild type; OE3 and OE4, NfcrtO-overexpression lines. Data are Mean ± SD (n = 3). *, P < 0.05; **, P < 0.01, by the Student’s t-test.

Fig. 7. Rice seedlings of NfcrtO-overexpression lines at the seedling stage in soil under drought treatment. A, Phenotypes of NfcrtO-overexpression rice seedlings (OE1, OE3, and OE4) in soil under drought stress. Scale bar, 5 cm. B, Survival rates of NfcrtO-overexpression rice seedlings under drought stress. WT, Wild type. Data are Mean ± SD (n = 3). *, P < 0.05; **, P < 0.01, by the Student’s t-test.

Fig. 8. NfcrtO-overexpression lines of rice at the booting stage under drought treatment. A, Phenotypes of NfcrtO-overexpression lines (OE3 and OE4) at the booting stage under drought treatment. Scale bar, 20 cm. B, Seed-setting rate of NfcrtO-overexpression lines. C, Grain yield per plant of NfcrtO-overexpression lines. WT, Wild type. Data are Mean ± SD (n = 3). **, P < 0.01, by the Student’s t-test.

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