Rice transcription factor OsMADS25 modulates root growth and confers salinity tolerance via the ABA-mediated regulatory pathway and ROS scavenging

Abstract

Plant roots are constantly exposed to a variety of abiotic stresses, and high salinity is one of the major limiting conditions that impose constraints on plant growth. In this study, we describe that OsMADS25 is required for the root growth as well as salinity tolerance, via maintaining ROS homeostasis in rice (Oryza sativa). Overexpression of OsMADS25 remarkably enhanced the primary root (PR) length and lateral root (LR) density, whereas RNAi silence of this gene reduced PR elongation significantly, with altered ROS accumulation in the root tip. Transcriptional activation assays indicated that OsMADS25 activates OsGST4 (glutathione S-transferase) expression directly by binding to its promoter. Meanwhile, osgst4 mutant exhibited repressed growth and high sensitivity to salinity and oxidative stress, and recombinant OsGST4 protein was found to have ROS-scavenging activity in vitro. Expectedly, overexpression of OsMADS25 significantly enhanced the tolerance to salinity and oxidative stress in rice plants, with the elevated activity of antioxidant enzymes, increased accumulation of osmoprotective solute proline and reduced frequency of open stoma. Furthermore, OsMADS25 specifically activated the transcription of OsP5CR, a key component of proline biosynthesis, by binding to its promoter. Interestingly, overexpression of OsMADS25 raised the root sensitivity to exogenous ABA, and the expression of ABA-dependent stress-responsive genes was elevated greatly in overexpression plants under salinity stress. In addition, OsMADS25 seemed to promote auxin signaling by activating OsYUC4 transcription. Taken together, our findings reveal that OsMADS25 might be an important transcriptional regulator that regulates the root growth and confers salinity tolerance in rice via the ABA-mediated regulatory pathway and ROS scavenging.

Fig 1. Root growth of OsMADS25 transgenic lines is correlated with the ROS levels.

A. Seedlings of wild type and OsMADS25 transgenic lines grown in standard 1/2 MS medium for 5 days. Scale bars, 2 cm. B. Relative transcript levels of OsMADS25 in OsMADS25 transgenic lines by qPCR analysis. C. Primary root length of 5–day–old wild type and OsMADS25 transgenic seedlings. D. NBT and DAB staining for O₂^– and H₂O₂, respectively, in the root tips of wild type and OsMADS25 transgenic lines shown in image A. Scale bars, 1 mm. E. Quantification of H₂O₂ content in the roots of wild type and OsMADS25 transgenic plants shown in image A. WT, wild type. RNAi1 and RNAi2, OsMADS25–RNAi transgenic lines. OE1 and OE2, OsMADS25 overexpression transgenic lines. NBT, nitroblue tetrazolium. DAB, 3, 3’–diaminobenzidine. Three independent experiments were performed with similar results. Data are means ± SE (n = 10). The statistical significance of the measurements using one-way analysis of variance (ANOVA) was determined using Student’s t-test. Asterisks indicate the significant difference between OsMADS25 transgenic lines and WT plants (t–test, ^*P < 0.05, ^**P < 0.01 or ^***P < 0.001).

Fig 2. Overexpression of OsMADS25 enhances the cell elongation.

A. Propidium iodide (PI)–stained root epidermal cells of 5–day–old wild type and OsMADS25 transgenic roots grown in standard 1/2 MS medium. Scale bars, 50 μm. B and C. Average epidermal cell length and cell width from 5–day–old wild type and OsMADS25 transgenic roots shown in image A, respectively. To measure cell length, germinated seeds were grown in standard 1/2 MS medium for 5 days, and the roots were stained with PI followed by washing for twice with sterile water, then imaged with a Leica SP8 confocal microscope. Epidermal cell length was averaged from at least 50 cells per root at a distance of 1 cm from the root tips from at least five roots examined for each treatment. Cell length was measured using Leica SP8 software. WT, wild type. RNAi1 and RNAi2, OsMADS25–RNAi transgenic lines. OE1 and OE2, OsMADS25 overexpression transgenic lines. Three independent experiments were performed. The statistical significance of the measurements using one-way analysis of variance (ANOVA) was determined using Student’s t–test. Asterisks indicate the significant difference between OsMADS25 transgenic lines and WT plants (^*P < 0.05, ^**P < 0.01 or ^***P < 0.001).

Fig 3. OsMADS25 confers H₂O₂ tolerance by promoting ROS-scavenging capability.

A. Root performance of wild type and OsMADS25 transgenic seedlings in modified 1/2 MS medium (without nitrate, with 5 mM glutamine as the N nutrition) with or without 10 mM H₂O₂ for 7 or 14 days. Scale bars, 1 cm. B–D. Measurement of primary root length, lateral root number and shoot length of 7–day–old seedlings shown in image A. E–G. Measurement of primary root length, lateral root number and shoot length of 14–day–old seedlings shown in image A. H–K. Activities of antioxidant enzymes of CAT, APX, GPX and GR in roots of 7–day–old seedlings shown in image A. L. Quantification of H₂O₂ content in the roots of 7–day–old seedlings shown in image A. WT, wild type. RNAi1 and RNAi2, OsMADS25–RNAi transgenic lines. OE1 and OE2, OsMADS25 overexpression lines. Three independent experiments were performed, and data are means ± SE (n = 15). The statistical significance of the measurements using one-way analysis of variance (ANOVA) was determined using Student’s t-test. Asterisks indicate the significant difference between OsMADS25 transgenic lines and WT plants (^*P < 0.05, ^**P < 0.01 or ^***P < 0.001).

Fig 4. Cis–element binding ability and transcriptional–activation assays of OsMADS25.

A. Nucleotide frequency distribution of the OsMADS25 core binding consensus sequence as determined ChIP–seq analysis. B. Schematic diagram of OsGST4 promoter region showing the CArG–box motif. C. Electrophoretic mobility shift assays (EMSA) indicating OsMADS25 binding specific CArG–box motif located in the promoter region of OsGST4. D. Schematic diagrams of the effector and reporter used in the yeast one–hybrid assay. E. Transcriptional–activation assays showing OsMADS25 having transactivation activity in yeast. Panel (I) shows yeast cells containing distinct effector and reporter constructs grown on an SD/-Ura medium without AbA (–Ura; –AbA). 1. pGADT7–p53/p53–AbAi (positive control); 2. pGADT7/p53–AbAi; 3. pAbAi–mOsGST4/pGADT–OsMADS25; 4. pAbAi–OsGST4/pGADT–OsMADS25. Panel (II) shows that yeast cells shown in panel (I) cultured on SD/–Ura medium containing 200 ng ml^-1 AbA (–Ura; +AbA). F. Schematic diagrams of the effector and reporter used for transient transactivation assay in Nicotiana benthamiana. G. Transactivation activity detected by GUS staining after reporter and effector plasmids coinfiltrated into the leaves of N. benthamiana. H. The transcript levels of OsGST4 in 7–day–old wild type and OsMADS25 transgenic roots by qPCR analysis. I. Measurement of GST activity in 7–day–old wild type and OsMADS25 transgenic roots. WT, wild type. RNAi1 and RNAi2, OsMADS25–RNAi transgenic lines. OE1 and OE2, OsMADS25 overexpression lines. Three independent experiments were performed. Data are means ± SE (n = 10). The statistical significance of the measurements using one-way analysis of variance (ANOVA) was determined using Student’s t-test. Asterisks indicate the significant difference between OsMADS25 transgenic lines and WT plants (^*P < 0.05, ^**P < 0.01 or ^***P < 0.001).

Fig 5. osgst4 mutant exhibits defective growth and reduced tolerance to oxidative stress.

A. Schematic diagram indicating the T–DNA insertion site in genomic region in osgst4. B. Genotyping of osgst4 T₂ seedlings performed via PCR analysis. C. Transcript levels of OsGST4 in wild type (DJ) and osgst4 mutant by qRT–PCR analysis. D. Seven–day–old seedlings of DJ and osgst4 mutant grown in standard 1/2 MS medium. Scale bar, 4 cm. E–G. Measurement of primary root length, lateral root number and shoot length in image D. H. Plant architecture of DJ and osgst4 mutant at mature stage. Scale bar, 10 cm. I–K. Comparison of plant height, seed setting rate and germination rate, respectively, between DJ and osgst4 mutant in image H. L and M. Quantification of H₂O₂ content in the shoot and root of 7–day–old seedlings in response to H₂O₂ or NaCl, respectively. N and O. Detached leaves from 4–week–old DJ and osgst4 plants exposed to 100 mM H₂O₂ or 150 mM NaCl for 3 days to indicate the oxidative stress tolerance. NBT, nitroblue tetrazolium. DAB, 3, 3’–diaminobenzidine. Three independent experiments were performed. Data are means ± SE (n = 30). The statistical significance of the measurements using one-way analysis of variance (ANOVA) was determined using Student’s t-test. Asterisks indicate the significant difference between OsMADS25 transgenic lines and WT plants (^*P < 0.05, ^**P < 0.01 or ^***P < 0.001).

Fig 6. OsMADS25 contributes to the salinity tolerance of rice.

A. Time course of OsMADS25 transcription induced by 100mM H₂O₂ and 150 mM NaCl via qPCR analysis. B. Comparison of seed germination rate between wild type and OsMADS25 transgenic lines in the presence of 150 mM NaCl. C. Phenotype of wild type and OsMADS25 transgenic seedlings exposed to salinity stress for 7 days. Scale bars, 5 cm. D. Measurement of chlorophyll content in wild type and OsMADS25 transgenic seedlings exposed to salinity stress for 7 days. E. The detached leaves exposed to 150 mM NaCl for 3 days to indicate the salinity tolerance of wild type and OsMADS25 transgenic lines. F–H. Measurement of the content of MDA, proline and soluble sugar in wild type and OsMADS25 transgenic seedlings exposed to salinity stress for 7 days. I. DAB and NBT staining for the leaves from wild type and OsMADS25 transgenic seedlings exposed to salinity stress for 7 days, respectively, to indicate ROS levels. Scale bars, 2 cm. J. Activities of ROS–scavenging enzymes CAT, APX, GPX and GR in wild type and OsMADS25 transgenic roots exposed to salinity stress for 7 days. WT, wild type. RNAi1 and RNAi2, OsMADS25–RNAi transgenic lines. OE1 and OE2 OsMADS25 overexpression transgenic lines. NBT, nitroblue tetrazolium. DAB, 3, 3’–diaminobenzidine. Three independent experiments were performed, and data are means ± SE (n = 15). The statistical significance of the measurements using one-way analysis of variance (ANOVA) was determined using Student’s t–test. Asterisks indicate the significant difference between OsMADS25 transgenic lines and WT plants (^*P < 0.05, ^**P < 0.01 or ^***P < 0.001).

Fig 7. OsMADS25 increases the ABA sensitivity of rice.

A and B. Phenotype of wild type and OsMADS25 transgenic seedlings grown in standard 1/2 MS medium with or without 5 μM ABA for 7 days. Scale bars, 2 cm. C–E. Statistical analysis of primary root length, shoot length and lateral root number of wild type and OsMADS25 transgenic seedlings, respectively, in images A and B. F and G. Phenotype of wild type and OsMADS25 transgenic seedlings grown in standard 1/2 MS medium with or without 5 μM ABA for 14 days. Scale bars, 2 cm. H–J. Statistical analysis of primary root length, shoot length and lateral root number of wild type and OsMADS25 transgenic seedlings, respectively, in images F and G. K. Relative transcription levels of key genes involved in ABA–dependent stress response pathway in 2–week–old wild type and OsMADS25 transgenic seedlings. To investigate the effect of ABA on rice growth, germinated seeds were grown in standard 1/2 MS medium with or without 5 μM ABA. WT, wild type. RNAi1 and RNAi2, OsMADS25–RNAi transgenic lines. OE1 and OE2, OsMADS25 overexpression transgenic lines. Three independent experiments were performed, and data are means ± SE (n = 15). The statistical significance of the measurements using one-way analysis of variance (ANOVA) was determined using Student’s t–test. Asterisks indicate the significant difference between OsMADS25 transgenic lines and WT plants (^*P < 0.05, ^**P < 0.01 or ^***P < 0.001).

Fig 8. OsMADS25 is a transcriptional activator of OsP5CR.

A. Schematic diagram of OsP5CR promoter region showing the CArG–box motifs. B. Electrophoretic mobility shift assays (EMSA) indicating OsMADS25 binding specific CArG–box motifs. C. Schematic diagrams of the effector and reporter used for transient transactivation assays in in rice protoplasts. REN, Renilla luciferase; LUC, firefly luciferase. D. Transactivation activity reflected by LUC activity of LUC/REN ratio. Data are means ± SE (n = 6). Probes P1 and P2 indicated oligonucleotides used for EMSA. Fragments F1, F2 and F3 indicated DNA fragments used for transcriptional–activation assays. The statistical significance of the measurements using one-way analysis of variance (ANOVA) was determined using Student’s t–test. Asterisks indicate the significant difference between treatment and control (^*P < 0.05, ^**P < 0.01 or ^***P < 0.001).

Fig 9. Proposed working model for OsMADS25 in the regulation of growth and salinity tolerance in rice.

Salinity stress produces ROS as well as induces OsMADS25 expression. OsMADS25 directly activates the transcription of OsGST4 and ABA–dependent OsP5CR to increase antioxidant responses and proline accumulation to fulfill ROS-scavenging, in combination with ABA–dependent abiotic stress–responsive regulatory pathway; besides, OsMADS25 might regulate the root growth via auxin signaling, which also enhances ABA signaling in oxidative stress, thereby accounting for the positive role of OsMADS25 in the growth and salinity tolerance in rice.