OsMADS23 phosphorylated by SAPK9 confers drought and salt tolerance by regulating ABA biosynthesis in rice

Abstract

Some of MADS-box transcription factors (TFs) have been shown to play essential roles in the adaptation of plant to abiotic stress. Still, the mechanisms that MADS-box proteins regulate plant stress response are not fully understood. Here, a stress-responsive MADS-box TF OsMADS23 from rice conferring the osmotic stress tolerance in plants is reported. Overexpression of OsMADS23 remarkably enhanced, but knockout of the gene greatly reduced the drought and salt tolerance in rice plants. Further, OsMADS23 was shown to promote the biosynthesis of endogenous ABA and proline by activating the transcription of target genes OsNCED2, OsNCED3, OsNCED4 and OsP5CR that are key components for ABA and proline biosynthesis, respectively. Then, the convincing evidence showed that the OsNCED2-knockout mutants had lower ABA levels and exhibited higher sensitivity to drought and oxidative stress than wild type, which is similar to osmads23 mutant. Interestingly, the SnRK2-type protein kinase SAPK9 was found to physically interact with and phosphorylate OsMADS23, and thus increase its stability and transcriptional activity. Furthermore, the activation of OsMADS23 by SAPK9-mediated phosphorylation is dependent on ABA in plants. Collectively, these findings establish a mechanism that OsMADS23 functions as a positive regulator in response to osmotic stress by regulating ABA biosynthesis, and provide a new strategy for improving drought and salt tolerance in rice.

Fig 1. Morphological phenotypes of osmads23 mutant and OsMADS23-overexpressing lines.

(A) Schematic diagram indicating the T-DNA insertion sites in genomic region in osmads23-1 mutant (M1). Black boxes represent exons; lines between black boxes are introns. The arrow indicates the transcription orientation. (B) Quantitative PCR analysis of different regions of OsMADS23 in osmads23-1. (C) and (D) Phenotypes of wild type (Z11) and osmads23-1 for 10 and 80 days, respectively. (E) Internode morphology in images in (D). (F) and (G) Quantification of shoot length and internode length in wild type and osmads23-1. (H) Quantitative PCR analysis of OsMADS23 in OsMADS23-overexpressing lines (OE13 and OE14). (I) and (J) Phenotypes of wild type (Nip) and OsMADS23-overexpressing lines for 10 and 80 days, respectively. (L) Internode morphology in images in (J). (K) and (M) Quantification of shoot length and internode length of Nip and OsMADS23-overexpressing lines. The significant difference between osmads23-1 or OsMADS23-overexpressing lines and their corresponding wild type was determined by Student’s t test. *p < 0.05, **p < 0.01 or ***p < 0.001. All data displayed as a mean ± SD. M1, osmads23-1 mutant. In (G) and (M), two-way ANOVA was performed, followed by Bonferroni’s post-hoc test. Different letters indicate significant differences (p < 0.05). Three independent experiments were performed (n = 30 plants per genotype in each independent experiment).

Fig 2. Performance of osmads23 mutant and OsMADS23-overexpressing lines under drought stress.

(A) Images showing the phenotypes of wild type (Nip) and OsMADS23-overexpressing plants (OE13 and OE14) under drought stress. Scale bars, 5 cm. (B) The survival rates of overexpression plants or osmads23-1 mutant (M1) and their corresponding wild type after drought and then rewatering. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 48 plants). (C) Images showing the phenotypes of wild type (Z11) and osmads23-1 under drought stress. Scale bars, 5 cm. In (A) and (C), 35-day-old plants were subjected to drought stress and then resumed growth. (D) and (E) Water loss rates of detached leaves from 70-day-old plants. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 5 plants). (F) DAB staining for the leaves from rice plants exposed to drought stress for 5 days to indicate H₂O₂ levels. Scale bars, 1.5 cm. (G) Expression of ROS-scavenging and proline-biosynthetic genes in plants exposed to drought stress for 3 days. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). The significant difference between OsMADS23-overexpressing lines or osmads23-1 and their corresponding wild-type plants was determined by Student’s t test. *p < 0.05, **p < 0.01. All data displayed as a mean ± SD. Three independent experiments were performed.

Fig 3. Phenotypes of OsMADS23-overexpressing lines under salt stress.

(A) Images showing the phenotypes of wild type (Nip) and OsMADS23-overexpressing lines (OE13 and OE14) under salt stress. Twenty-eight-day-old plants were subjected to 300 mM salt stress and then resumed growth. Scale bars, 5 cm. (B) The survival rates of wild type and overexpression lines after 8 days of salt stress and then 5 days of resuming growth. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 48 plants). (C) The leaves detached from 60-day-old plants were exposed to 200 mM NaCl for 3 days to indicate the salt stress tolerance. Scale bars, 2 cm. (D) DAB staining for the leaves of plants exposed to salt stress for 5 days to indicate H₂O₂ levels. Scale bars, 1.5 cm. (E) Quantification of H₂O₂ content in the leaves from plants exposed to salt stress for 5 days. (F) and (G) Activities of SOD and CAT in plants exposed to salt stress for 5 days, respectively. (H) Expression of ROS-scavenging genes in plants exposed to salt stress for 3 days. (I) and (J) Content of proline and MDA in plants exposed to salt stress for 5 days, respectively. In (E) to (J), error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). *p < 0.05, **p < 0.01 or ***p < 0.001 (Student’s t test). All data are means ± SD. Three independent experiments were performed.

Fig 4. Overexpression of OsMADS23 promoted plant adaption to oxidative stress.

(A) Performance of OsMADS23-overexpressing plants (OE13 and OE14) or osmads23-1 mutant (M1) in half-strength medium supplemented with 2 μM MV for 7 days. Two-day-old seedlings were grown in medium with or without MV. Scale bars, 2 cm. (B) and (C) Shoot length of OsMADS23-overexpressing plants and osmads23-1 mutant in the medium with or without MV for 7 days, respectively. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 20 plants). (D) The detached leaves from 60-day-old plants were exposed to 5 μM MV for 3 days to indicate the oxidative tolerance. Scale bars, 1.5 cm. (E) The chlorophyll content of 3-day-old plants growing in the medium with or without MV for 7 days, respectively. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). MV, methyl viologen. *p < 0.05, **p < 0.01 or ***p < 0.001 (Student’s t test). All data are means ± SD. Three independent experiments were performed.

Fig 5. OsMADS23 mediates ABA sensitivity and ABA-induced stomatal movement in rice.

(A) Seed germination of OsMADS23-overexpressing plants (OE13 and OE14) or osmads23-1 mutant (M1) compared to their corresponding wild type (Nip or Z11) on half-strength MS medium without or with ABA for 4 days, respectively. Scale bars, 2 cm. (B) Seed germination rates scored from day 1 to day 4 after stratification on medium supplemented without or with 1 μM ABA, respectively. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 50 seeds). (C) Plant growth in half-strength MS medium without or with ABA for 4 days, respectively. Two-day-old seedlings were transferred on medium with or without ABA. Scale bars, 2 cm. (D) Decrease rate of shoot and root length in 1 μM ABA compared to mock on day 4. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 20 plants). (E) ABA content in OsMADS23-overexpressing lines and osmads23 mutant, and their corresponding wild type after exposed to drought stress. Two-week-old plants were subjected to drought stress for 3 days, and leaves were collected for measurement of ABA content. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 5 plants). (F) Percentages of completely open, partially open, and completely closed stomata in wild type (Nip) and OsMADS23-overexpressing plants under normal and ABA treatment conditions. Results represent means ± SD (n = 300) from 10 plants per genotype. Two-way ANOVA was performed, followed by Bonferroni’s post-hoc test. Scale bars, 10 μm. (G) Relative transcription levels of key genes involved in ABA-dependent stress response pathway in 2-week-old plants under drought conditions for 3 days. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). *p < 0.05, **p < 0.01 or ***p < 0.001 (Student’s t test). Three independent experiments were performed.

Fig 6. OsMADS23 is a transcriptional activator of OsNCED2.

(A) Schematic diagram of OsNCED2 promoter region showing the positions of CArG-box motifs and P1-P5 fragments amplified by ChIP-qPCR. (B) Electrophoretic mobility shift assays (EMSA) indicating OsMADS23 binds the CArG-box motifs in OsNCED2 promoter specifically. Probe 1 (-1266 to -1226 bp), probe 2 (-1051 to -1010 bp) and probe 3 (-687 to -645 bp). (C) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay showed that ABA enhances OsMADS23 binding to the promoter regions of OsNCED2. P1-P5 represents the regions shown in (A) detected by ChIP-qPCR. The enrichment values were normalized to input. Immunoglobulin G (IgG) immunoprecipitated DNA was used as a control. Error bars indicate SD with biological triplicates. Ten-day-old rice plants overexpressing OsMADS23-GFP were treated by ABA for 16 h and used for ChIP analysis. Error bars indicate SD with biological triplicates. (D) Schematic diagrams of the effector and reporter used for transient transactivation assays in the leaves of Nicotiana benthamiana. The fragment from -1362 to -574 bp in the promoter of OsNCED2 was used for constructing reporter. (E) Transactivation activity was detected by GUS staining in N. benthamiana leaves. (F) Schematic diagrams of the effector and reporter used for transient transactivation assays in rice protoplasts. REN, Renilla luciferase; LUC, firefly luciferase. The fragment from -1362 to -574 bp in the promoter of OsNCED2 was used for construction reporter. (G) Transactivation activity reflected by LUC activity of LUC/REN ratio. Data represent the means of three independent experiments. (H) Relative transcription levels of OsNCED2 in wild type (Nip) and OsMADS23-overexpressing plants. *p < 0.05, **p < 0.01 or ***p < 0.001 (Student’s t test). Three independent experiments were performed.

Fig 7. OsMADS23 binds to the promoters of OsNCED3, OsNCED4 and OsP5CR, and activates their expression in vivo.

(A) Schematic diagrams of OsNCED3, OsNCED4 and OsP5CR showing the positions of CArG-box motifs and fragments amplified by ChIP-qPCR, respectively. (B) ChIP-qPCR analysis of the gene fragments of OsNCED3, OsNCED4 and OsP5CR enriched by OsMADS23 in rice plants, respectively. The enrichment values were normalized to input. Immunoglobulin G (IgG) immunoprecipitated DNA was used as a control. Ten-day-old rice plants overexpressing OsMADS23-GFP were used for ChIP analysis. Error bars indicate SD with biological triplicates. (C) Schematic diagrams of the effector and reporter used for transient transactivation assays in rice protoplasts. REN, Renilla luciferase; LUC, firefly luciferase. The fragments in the promoter regions of OsNCED3 (-1964 to -1740 bp), OsNCED4, (-1349 to -1100 bp) and OsP5CR (-686 to -781 bp) were used for construction of reporters. (D) Transactivation activity reflected by LUC activity of LUC/REN ratio in rice protoplasts. Error bars indicate SD with biological triplicates. *p < 0.05, **p < 0.01 or ***p < 0.001 (Student’s t test).

Fig 8. osnced2 mutants were more sensitive to drought and oxidation stress than wild type.

(A) Schematic diagram of CRISPR-Cas9-mediated target mutagenesis of OsNCED2. (B) Phenotypes of osnced2 mutants and wild type (Z11) in half-strength medium for 7 days. Scale bars, 4 cm. (C) Shoot and root length of wild type and osnced2 mutants in half-strength medium for 7 days. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 20 plants). (D) ABA content in the leaves of wild type (Z11) and osnced2 mutants growing for 20 days. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). (E) Images showing the phenotypes of wild type and osnced2 mutants under drought stress. Twenty-day-old plants were subjected to drought stress and then rewatering. Scale bars, 5 cm. (F) The survival rates of wild type and osnced2 mutants after drought stress and rewatering. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 48 plants). (G) The detached leaves from 70-day-old plants were exposed to 5 μM MV for 3 days to indicate the oxidative tolerance. Scale bars, 2 cm. (H) The chlorophyll content of wild type and osnced2 mutants in MV for 7 days. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). MV, methyl viologen. *p < 0.05, **p < 0.01 (Student’s t test). Three independent experiments were performed.

Fig 9. OsMADS23 physically interacts with SAPK9.

(A) Yeast two-hybrid assays of OsMADS23 and SAPK9. SD, synthetic dropout medium. DDO, SD/-Leu-Trp. QDO, SD/-Ade-His-Leu-Trp. (B) OsMADS23 interacts with SAPK9 in the in vitro pull-down assay. GST-tagged OsMADS23 (GST-OsMADS23) was used as a bait, and pull-down of His-SAPK9 was detected by anti-His antibody (Proteintech). (C) The interaction of OsMADS23 and SAPK9 by bimolecular fluorescence complementation (BiFC) in rice protoplasts. YFP, Yellow fluorescent protein. Scale bars, 5 μm. (D) OsMADS23-SAPK9 interaction detected by coimmunoprecipitation (CoIP) assay. Total proteins from Nicotiana benthamiana leaves coexpressing OsMADS23-GFP with SAPK9-3×FLAG or 3×FLAG were used. Proteins before (input) and after IP were detected by the anti-GFP and anti-FLAG antibodies (Proteintech), respectively. Three independent experiments were performed.

Fig 10. SAPK9 phosphorylates OsMADS23 in vitro and in vivo.

(A) Thr-20 and Ser-36 in OsMADS23 are main sites of SAPK9-mediated phosphorylation. Kinase assays in vitro were performed by coexpressing His-SAPK9 with the mutated form of GST-OsMADS23 containing both putative phosphorylation sites substituted with Ala in Escherichia coli BL21 (DE3). GST-OsMADS23 and phosphorylated GST-OsMADS23 (GST-OsMADS23-P) were purified and detected using a Phos-tag gel by anit-GST (top panel). An equal amount of each recombinant protein was separated on the gel without the Phos-tag as a loading control, detected by anti-GST (middle panel) and anti-His (bottom panel), respectively. (B) Serine 176 is the key phosphorylation site of SAPK9 on OsMADS23. Kinase assays were performed by coexpressing GST-OsMADS23 with His-SAPK9 or His-SAPK9^S176A in BL21 (DE3). GST-OsMADS23 and GST-OsMADS23-P were purified and detected using a Phos-tag gel by anit-GST (top panel). An equal amount of each recombinant protein was separated on the gel without the Phos-tag as a loading control, detected by anti-GST (middle panel) and anti-His (bottom panel), respectively. (C) OsMADS23-GFP was phosphorylated by SAPK9-3×FLAG in Nicotiana benthamiana leaves. OsMADS23-GFP was transiently coexpressed with empty FLAG protein (3×FLAG), SAPK9-3×FLAG, or SAPK9^S176A-3×FLAG in 3-week-old N. benthamiana leaves by Agrobacterium infiltration. An equal amount of each total proteins from tobacco leaves was detected using a Phos-tag gel by anit-GFP (top panel). An equal amount of each protein was separated on the gel without the Phos-tag, detected by anti-GFP (middle panel) and anti-FLAG (bottom panel), respectively. Coomassie brilliant blue (CBB) staining indicates similar amounts of proteins were loaded. (D) Phosphorylation of OsMADS23-GFP in DJ (wild type) and sapk9 mutant. OsMADS23-GFP was transiently expressed in the protoplasts of DJ or sapk9 mutant, and then was immunoprecipitated with anti-GFP and detected with biotinylated Phos-tag (Phosbind biotin BTL-104, APE×BIO). An equal amount of protein extracts were separated on the gel without the Phos-tag and detected by anti-GFP (middle panel). Coomassie brilliant blue (CBB) staining indicates similar amounts of proteins were loaded. To avoid protein degradation, MG132 and a cocktail of proteinase inhibitors were added. (E) Immunoprecipitated OsMADS23-GFP protein from OsMADS23-GFP rice plants was treated with or without Lambda protein phosphatase (λ PP). The phosphorylated proteins were detected with biotinylated Phos-tag (Phosbind biotin BTL-104, APE×BIO) (top panel). An equal amount of immunoprecipitated OsMADS23-GFP protein was separated on the gel without the Phos-tag as a loading control, and detected by anti-GFP (bottom panel). Red arrows represent retarded phosphorylated protein. To avoid protein degradation, MG132 and a cocktail of proteinase inhibitors were added. In these west blot experiments, ani-His, anti-GST, anti-GFP and anti-FLAG antibodies (Proteintech) were used. Three independent experiments were performed. Red arrows indicate phosphorylated proteins.

Fig 11. The phosphorylation of OsMADS23 by SAPK9 is required for its stability and transcriptional activity in an ABA-dependent manner.

(A) Cell-free degradation assays of GST-OsMADS23 or its different mutated versions (GST-OsMADS23^{T20A S36A} and GST-OsMADS23^{T20D S36D}) in wild type (WT) or sapk9 mutant with or without ABA treatment. GST-OsMADS23 and its mutated versions were detected by western blotting using anti-GST antibody. The Coomassie blue–stained ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. GST-OsMADS23 and its mutated versions were expressed in BL21 (DE3) and purified; an equal amount of each was incubated for different times at 30°C with equal amount protein extracts from leaves of 10-day-old wild-type and sapk9 plants, with or without 50 μM ABA treatment for 16 h. (B) and (C) Quantification analysis of the results described in (A). The relative levels of GST-OsMADS23 and its mutated versions in different protein extracts at 0 h were defined as 1. Data represent the means of three independent experiments. (D) Cell free degradation of GST-OsMADS23 or its different mutated versions with 50 μM MG132. GST-OsMADS23 and its mutated versions were detected by western blotting using anti-GST antibody. The Coomassie blue-stained ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. (E) Time course of OsMADS23-GFP degradation when the protein extracts from OsMADS23-GFP plants were incubated with His or His-SAPK9. Equal amounts of plant crude extracts were added to equal amounts of the recombinant proteins in the in vitro cell-free degradation assays. OsMADS23-GFP was detected by western blotting using anti-GFP antibody. The Coomassie blue-stained ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. (F) Quantification analysis of the results in (E). The relative levels of OsMADS23-GFP at 0 h were defined as 1. Data represent the means of three independent experiments. (G) Schematic diagram of the constructs used in the transient transactivation assay. (H) SAPK9 as well as ABA can increase the transactivation activity of OsMADS23 in Nicotiana leaves. One half of the infiltrated leaves were incubated with 50 μM ABA for 16 h. Data represent the means of three independent experiments. *p < 0.05, **p < 0.01 (Student’s t test). These cell-free degradation assays were performed in the presence of ATP.

Fig 12. Working model of OsMADS23 conferring the osmotic stress tolerance via the ABA signaling in rice.

ABA, which is induced by abiotic stress such as drought or salt stress, inhibits the activity of OsPP2Cs to release the kinase activity of SAPK9 for further activation of OsMADS23 through phosphorylation. OsMADS23 could promote the ABA levels through directly activating the expression of OsNCEDs. Meanwhile, OsMADS23 also enhanced proline content by targeting proline synthesis gene OsP5CR. The circled “P” indicates phosphorylation (+P).