Combined metabolomic and transcriptomic analysis reveals key components of OsCIPK17 overexpression improves drought tolerance in rice

Abstract

Oryza Sativa is one of the most important food crops in China, which is easily affected by drought during its growth and development. As a member of the calcium signaling pathway, CBL-interacting protein kinase (CIPK) plays an important role in plant growth and development as well as environmental stress. However, there is no report on the function and mechanism of OsCIPK17 in rice drought resistance. We combined transcriptional and metabonomic analysis to clarify the specific mechanism of OsCIPK17 in response to rice drought tolerance. The results showed that OsCIPK17 improved drought resistance of rice by regulating deep roots under drought stress; Response to drought by regulating the energy metabolism pathway and controlling the accumulation of citric acid in the tricarboxylic acid (TCA) cycle; Our exogenous experiments also proved that OsCIPK17 responds to citric acid, and this process involves the auxin metabolism pathway; Exogenous citric acid can improve the drought resistance of overexpression plants. Our research reveals that OsCIPK17 positively regulates rice drought resistance and participates in the accumulation of citric acid in the TCA cycle, providing new insights for rice drought resistance.

Keywords: CIPK; Oryza sativa; auxin; citric acid; drought; metabolome; transcriptome.

Figure 1

Drought tolerance was increased in OsCIPK17-OE plants but decreased in OsCIPK17-Mutant plants. (A) Growth phenotype of 3-week-old transgenic OsCIPK17-OE, OsCIPK17-Mutant, and WT plants at different stages of drought stress; bar = 7cm. The upper and middle channels in a have the same results as Gao et al. (2022a) (Published in the International Journal of Molecular Science on 18 October 2022). (B) Relative water content of drought-treated plants. Parameters of water status in 3-week-old OsCIPK17-OE, OsCIPK17-Mutant and WT plants were measured at 10 days after drought stress treatment (n=20). (C) Survival rates of each line after recovery were measured at 2 days after re-watering (n=60). (D) Growth phenotypes of 5-week-old transgenic OsCIPK17-OE, OsCIPK17-Mutant and WT plants under drought stress and normal water conditions. (E) Root length of each line in (D) was measured (n=6). Experiments in (A, D) were repeated three times, and similar results were obtained. Data in (B, C, E), are presented as mean ± SD of three independent experiments, and the significant differences between OsCIPK17-OE/OsCIPK17-Mutant and WT plants according to one-way ANOVA are indicated by asterisks. Asterisks indicate significance (**p < 0.01, ***p < 0.001).

Figure 2

Drought-induced changes in proline, soluble sugar, and ROS levels and SOD activity in OsCIPK17-OE and OsCIPK17-Mutant plants. (A) In situ detection of H₂O₂ and superoxide anion in leaves using DAB and NBT staining, respectively. (B) Proline content, (C) ROS content, (D) SOD activity. (E) Soluble sugar content. Leaf samples were collected from 3-week-old OsCIPK17-OE, OsCIPK17-Mutant, and WT plants grown under normal water or drought conditions (at 5 days after water withholding) and subjected to physiological measurements. Data in (B–E) are presented as mean ± SD of three independent experiments, and significant differences between OsCIPK17-OE/OsCIPK17-Mutant and WT plants according to one-way ANOVA are indicated by asterisks. Asterisks indicate significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

PC biplot of the transcriptome and metabolome of rice seedlings under drought stress and identification of differentially expressed genes and metabolites. (A) PC biplot of transcriptome. (B) PC biplot of metabolome. (C) Volcano plot of differentially expressed genes between NIP and OsCIPK17-OE9 under drought treatment. (D) Volcano plot of differentially expressed metabolites between NIP and OsCIPK17-OE9 under drought treatment. (E) KEGG analysis of differentially expressed genes and metabolites between NIP and OsCIPK17-OE9 under drought treatment. CK means control check, which means growth under normal conditions without drought treatment. D means under drought treatment. OE9 means OsCIPK17 overexpression line 9. NIP-D, NIP-CK, OE9-D, and OE9-CK mean combination of lines and treatment methods. The same meaning as the following figure.

Figure 4

Sugar content in the control and drought treatment with metabolomics analysis. Data are presented as mean ± SD of six independent experiments, and significant differences between NIP-CK and OE9-CK/NIP-D or OE9-D and OE9-CK/NIP-D plants according to one-way ANOVA are indicated by asterisks. Asterisks indicate significance (*p < 0.05, **p < 0.01, ***p < 0.001). CK: NIP or OE materials without drought treatment. OE9: OsCIPK17-OE9. D: drought treatment (with 20% PEG treatment of 3-week-old rice seedlings for 5 days).

Figure 5

Metabolites and enzymes related to trehalose metabolism. (A) Contents of metabolites in the trehalose pathway. Black boxes indicate the substances detected by the metabolomic analysis, and red boxes indicate the substances not detected by the metabolomic analysis. (B) Expression of enzymes involved in trehalose pathway detected in the transcriptome. Data are presented as mean ± SD of six independent experiments, and significant differences between NIP-CK and OE9-CK/NIP-D or OE9-D and OE9-CK/NIP-D plants according to one-way ANOVA are indicated by asterisks. CK: NIP or OE9 materials without drought treatment. OE9: OsCIPK17-OE9. D: drought treatment. Asterisks indicate significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6

Changes in the contents of intermediate metabolites in the TCA cycle in rice roots under drought. Asterisks indicate significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7

Exogenous citric acid stimulates the growth of OsCIPK17-OE plants. (A) Growth performance of OsCIPK17-OE, OsCIPK17-Mutant, and WT seedlings grown in clear water with or without 25 μmol·L^-1. (B) Relative shoot length and (C) relative root length of OsCIPK17-OE, OsCIPK17-Mutant, and WT seedlings grown in clear water with or without 25 μmol·L^-1 at 14 days after germination. (D–F) are the expression levels of citric acid treated and controlled with auxin-related genes in (A). Relative expression levels of the gene were normalized to the internal control of the OsACTIN-1 (LOC_Os05g36290) gene. Data in (B–F) are presented as mean ± SD of three independent experiments, and significant differences between sCIPK17-OE/OsCIPK17-Mutant and WT plants according to one-way ANOVA are indicated by asterisks. Asterisks indicate significance (*p < 0.05, **p < 0.01, ****P<0.0001).

Figure 8

Exogenous citric acid treatment enhanced the drought tolerance of OsCIPK17-OE, while it did not affect OsCIPK17-Mutant and WT. (A) Twenty-two days after germination, OsCIPK17-OE, OsCIPK17-mutant, and WT seedlings were grown in clear water with or without 25 μmol·L^-1. Water isolation treatment was performed on day 23 and watering was stopped, and photographs were taken and recorded at different intervals. The basic number of seedlings per pot is 30. (B) The survival of each strain after rehydration in (A) was counted, and the survival was considered when the leaves were unfolded and green. Survival rate = number of survivors/total number of seedlings. bar = 7cm. (C) It is the magnified field of view of OsCIPK17-OE surviving seedlings in (A). bar = 1cm. Data in (B) are presented as mean ± SD of three independent experiments, and significant differences between CIPK17-OE and WT plants according to one-way ANOVA are indicated by asterisks. Asterisks indicate significance (**p < 0.01).

Figure 9

Correlation network map. Expression patterns of genes related to sugar, amino acid, nucleotide, glutathione, quinoline, and flavone metabolism and hormone signaling.

Figure 10

Summary of major metabolic events observed, which confer drought tolerance through OsCIPK17-OE in rice. The features described in this work are marked with gray ellipses. Possible connections are connected by dashed lines.