The OsCBL8-OsCIPK17 Module Regulates Seedling Growth and Confers Resistance to Heat and Drought in Rice

Abstract

The calcium signaling pathway is critical for plant growth, development, and response to external stimuli. The CBL-CIPK pathway has been well characterized as a calcium-signaling pathway. However, in most reports, only a single function for this module has been described. Here, we examined multiple functions of this module. CIPK showed a similar distribution to that of CBL, and OsCBL and OsCIPK families were retained after experiencing whole genome duplication events through the phylogenetic and synteny analysis. This study found that OsCBL8 negatively regulated rice seed germination and seedling growth by interacting with OsCIPK17 with overexpression and gene editing mutant plants as materials combining plant phenotype, physiological indicators and transcriptome sequencing. This process is likely mediated by OsPP2C77, which is a member of the ABA signaling pathway. In addition, OsCBL mediated the targeting of OsNAC77 and OsJAMYB by OsCIPK17, thus conferring resistance to high temperatures and pathogens in rice. Our work reveals a unique signaling pathway, wherein OsCBL8 interacts with OsCIPK17 and provides rice with multiple resistance while also regulating seedling growth.

Keywords: CBL; CIPK; Oryza sativa; drought; growth; high temperature.

Figure 1

OsCBL and OsCIPK have specific interactions and different spatiotemporal expression patterns. (A) The interaction between OsCBL2, OsCBL8 and part of OsCIPK was confirmed by Y2H analysis. (B) The interaction between OsCBL2, OsCBL8 and OsCIPK17 was confirmed by BIFC analysis. (C) The heat maps of gene expression of OsCBL2, OsCBL8 and OsCIPK17 under different stress. (D) The heat maps of gene expression of OsCBL2, OsCBL8 and OsCIPK17 in different parts. (E) Subcellular localization of OsCBL2, OsCBL8 and OsCIPK17. Bar = 100 μm. (F) Gus tissue staining of Arabidopsis transformed with the promoter regions of OsCBL8 and OsCIPK17 under high temperature stress. The scale is 100 μm. CK, control group, without high temperature treatment; HT, experimental group, with high temperature treatment at 30 °C for one day.

Figure 2

OsCBL8 can negatively regulate rice seed germination rather than OsCBL2 and OsCIPK17. (A) Germination phenotype of OsCBL8 knockout and overexpression lines on seed germination of rice. Each petri dish contained 100 rice seeds. Photographs of 7-day-old plants are shown. Bar = 4 cm. (B) Seed germination rate of OsCBL2 knockout and overexpression lines. (C) Seed germination rate of OsCBL8 knockout and overexpression lines. (D) Seed germination rate of OsCIPK17 knockout and overexpression lines. (E) Changes in seed germination rate of knockout and overexpression lines of OsCBL8 in 7 days. Data of (B–E) are mean ± SD. from three biological replicates. Statistical analysis was performed using Šídák’s multiple comparisons test and Dunnett’s multiple comparisons test; **** p < 0.0001. (F) The vigor of seeds was demonstrated by TTC (2, 3, 5-triphenyltetrazole chloride) staining. After the seeds were immersed in TTC staining, they were cultured at 33 °C for 2 h and photographed for observation. The redder the seed color, the higher the seed vigor. Bar = 6 mm.

Figure 3

OsCBL8 and OsCIPK17 can regulate seedling growth. (A,F) Morphological phenotypes of OsCBL8 and OsCIPK17 knockout and overexpression lines on rice seedling growth. Photographs of 14-day-old plants are shown. Bar = 2 cm. (B,C) Plant height and root length of OsCBL8 knockout and overexpression lines. Data are mean ± SD from 20 biological replicates for OsCBL8. (D,E) Fresh and dry weight of knockout and overexpression lines of OsCBL8 were measured. (G,H) Plant height and root length of OsCIPK17 knockout and overexpression lines. Data are mean ± SD from 12 biological replicates for OsCIPK17. Statistical analysis was performed using Šídák’s multiple comparisons test. (I,J) Fresh and dry weight of knockout and overexpression lines of OsCIPK17 were measured. Data are mean ± SD from three biological replicates. Statistical analysis was performed using Dunnett’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Transcriptome analysis of OsCBL8 and OsCIPK17 overexpressing lines. (A) Volcano map showing the changes in gene expression in overexpression lines of OsCBL8 and NIP. (B) Volcano map showing the changes in gene expression in overexpression lines of OsCIPK17and NIP. OsCBL8 and OsCIPK17 are marked with a diamond. (C) Venn diagram showing the intersection of up- and downregulated differentially expressed gene (DEG) sets. (D) Plant Ontology (PO) enrichment analysis of OsCBL8 overexpression lines. (E) Plant Ontology (PO) enrichment analysis of OsCIPK17 overexpression lines. (F) The Venn diagram is used to show the common terms produced by different PO enrichment analysis. (G) Gene Ontology (GO) enrichment analysis of OsCBL8 overexpression lines. (H) Gene Ontology (GO) enrichment analysis of OsCIPK17 overexpression lines. Only the top 10 terms from MF, BP and CC are shown. (I) The Venn diagram is used to show the common terms produced by different GO enrichment analyses.

Figure 5

Protein–protein interaction network analysis of DEGs. (A) Protein–protein interaction networks generated by DEGs from OsCBL8 and (B) OsCIPK17 overexpression lines. The up- (square, red) and downregulated (round, blue) genes form two clusters, which are distinguished by different shapes and colored with log2(FC). Different PO terms are also mapped to proteins. (C,D) and (E) Sub-modules mined by MCODE from OsCBL8 overexpression lines. According to the score, the top three are shown. The scores of them were 6.75, 5.2 and 4, respectively. (F–H) Sub-modules mined by MCODE from OsCIPK17 overexpression lines. According to the score, the top three are shown. The scores of them were 6, 3 and 3, respectively.

Figure 6

OsCIPK17 can interact with OsPP2C77, OsJAMYB, OsDREB1H and OsNAC77. (A) The Y2H experiment verified the target protein that may have a regulatory relationship with OsCIPK17. P: positive control; N: negative control. (B,C) Y2H experiment verified that OsCIPK17 can interact with OsJAMYB. Aureobasidin A (AbA) and 3-amino-1, 2, 4-triazole (3-AT) were added to QDO medium to inhibit self-activation. (D) The Y2H experiment verified that OsCIPK17 can interact with OsDREB1H. (E) Detection of trans-activatory activity of OsDREB1H. A plus sign indicates that the ingredient has been added, and a minus sign indicates the opposite. (F) BiFC experiments verified that OsCIPK17 can interact with OsNAC77, OsJAMYB, OsPP2C77 and OsDREB1H, respectively. Bar = 100 μm.

Figure 7

ABA participates in OsCBL8–OsCIPK17 module to regulate the growth of rice seedlings. (A) Phenotypes of seeds treated with 5 mg L⁻¹ ABA and 100 mg L⁻¹ Na₂WO₄ for 1 and 2 weeks, respectively. The bars are 2 and 4, respectively. (B,C) Plant height of seeds treated with 5 mg L⁻¹ ABA and 100 mg L⁻¹ Na₂WO₄ for 2 weeks. Data are mean ± SD from 10 biological replicates. Statistical analysis was performed using Dunnett’s multiple comparisons test; **** p < 0.0001; ns: no significance. (D–G). Determination of hormone content in leaves of 2-week-old seedlings of different lines. Data are mean ± SD from three biological replicates. Statistical analysis was performed using Dunnett’s multiple comparisons test; **** p < 0.0001; ns: no significance. * p < 0.05, ** p < 0.01, *** p < 0.001; ns: no significance.

Figure 8

OsCBL8-OsCIPK17 can regulate the resistance to high temperature and drought. (A) Phenotype of 2-week-old seedlings after 9 days of high temperature treatment. Bar = 8 cm. (B–D) Measurement of indexes after high temperature stress. The contents of ROS, PRO and SOD activity were determined. ROS: reactive oxygen species; PRO: proline; SOD: superoxide dismutase. Data are mean ± SD from three biological replicates. Statistical analysis was performed using Dunnett’s multiple comparisons test; **** p < 0.0001, ** p < 0.01, * p < 0.05. (E) Phenotype of 22-day-old rice seedlings after 12 days of drought treatment. Bar = 10 cm.

Figure 9

Potential regulatory mechanism of the OsCBL8-OsCIPK17 module. Calcium signaling begins with OsCBL8, which is located in the cell membrane, binds to calcium and then enforces OsCIPK17 and binds to it. OsCIPK17 may further regulate the growth and development of seedlings by interacting with some unknown target genes. This pathway is also regulated by ABA. ABA initially binds to the corresponding receptor protein (PYR/PYL/RCAR). The receptor protein with ABA further binds to OsPP2C77 and transmits the signal to it. Subsequently, OsPP2C77 dephosphorylates OsCIPK17 to inhibit its regulation of downstream targets. OsCBL8-OsCIPK17 can also endow seedlings with high temperature resistance. OsCIPK17 can phosphorylate the transcription factor OsNAC77 involved in stress response. OsNAC77 combines with the DNA-specific sequences that are located in the promoter region of OsCLPD1 and OsOAT to give seedlings the ability to resist heat stress. Meanwhile, OsCIPK17 can phosphorylate the JA-mediated transcription factor OsJAMYB. OsJAMYB can bind to the promoter of OsAGO18 to activate its transcription, so as to promote rice antiviral defense. In addition, OsCBL8 may regulate seed germination by interacting with other OsCIPK. The dotted line indicates that this process is speculative.