Nitrate-responsive OsMADS27 promotes salt tolerance in rice

Abstract

Salt stress is a major constraint on plant growth and yield. Nitrogen (N) fertilizers are known to alleviate salt stress. However, the underlying molecular mechanisms remain unclear. Here, we show that nitrate-dependent salt tolerance is mediated by OsMADS27 in rice. The expression of OsMADS27 is specifically induced by nitrate. The salt-inducible expression of OsMADS27 is also nitrate dependent. OsMADS27 knockout mutants are more sensitive to salt stress than the wild type, whereas OsMADS27 overexpression lines are more tolerant. Transcriptomic analyses revealed that OsMADS27 upregulates the expression of a number of known stress-responsive genes as well as those involved in ion homeostasis and antioxidation. We demonstrate that OsMADS27 directly binds to the promoters of OsHKT1.1 and OsSPL7 to regulate their expression. Notably, OsMADS27-mediated salt tolerance is nitrate dependent and positively correlated with nitrate concentration. Our results reveal the role of nitrate-responsive OsMADS27 and its downstream target genes in salt tolerance, providing a molecular mechanism for the enhancement of salt tolerance by nitrogen fertilizers in rice. OsMADS27 overexpression increased grain yield under salt stress in the presence of sufficient nitrate, suggesting that OsMADS27 is a promising candidate for the improvement of salt tolerance in rice.

Keywords: OsMADS27; grain yield; nitrate-dependent salt tolerance; salt stress.

Figure 1

OsMADS27 is specifically responsive to nitrate. (A) Time-course analyses of OsMADS27 expression in response to N and salt stress. Seven-day-old wild-type plants grown on hydroponic medium with 1.5 mM KNO₃ were transferred to hydroponic medium without N for 2 days and then transferred to hydroponic medium with 2 mM KNO₃, 2 mM NH₄Cl, 140 mM NaCl, or 2 mM KCl for 0, 0.5, 3, 12, or 24 h. RNA was extracted from whole seedlings for qRT‒PCR analyses as described in the methods. Values are the mean ± SD (n = 3). (B) Time-course analyses of OsMADS27 expression in response to KNO₃ depletion. Seven-day-old wild-type plants grown on hydroponic medium with 1.5 mM KNO₃ were treated with 2 mM KNO₃ for 0, 0.5, 1, or 3 h and then transferred to hydroponic medium without KNO₃ for 3, 6, 12, or 24 h. RNA was extracted from whole seedlings for qRT‒PCR analyses as described in the methods. Values are the mean ± SD (n = 3). (C) KNO₃-dependent induction of OsMADS27 expression by NaCl. Wild-type seedlings grown hydroponically on N-free medium for 7 days were treated with 140 mM NaCl for 0, 0.5, 1, or 3 h and then transferred to hydroponic medium with 140 mM NaCl + 2 mM KNO₃ for 0.5, 1, or 3 h. RNA was extracted from whole seedlings for qRT‒PCR analyses as described in the methods. Values are the mean ± SD (n = 3). (D and E) The response of OsMADS27pro:GUS to NaCl. Seven-day-old OsMADS27pro:GUS lines grown on N-free medium with 2 mM KCl (D) or 2 mM KNO₃ (E) were treated with 140 mM NaCl for 0.5, 1, or 3 h. Seedlings were incubated in GUS buffer for 3 h before photographs were taken. Bar represents 1 cm. (F) Nitrate plus NaCl or ABA synergistically enhances the expression of OsMADS27. Seven-day-old wild-type plants grown on hydroponic medium with 1.5 mM KNO₃ were transferred to hydroponic medium without N for 2 days and then transferred to hydroponic medium with 2 mM KCl, 2 mM KNO₃, 140 mM NaCl, 10 μM ABA, 2 mM KNO₃ plus 140 mM NaCl, or 2 mM KNO₃ plus 10 μM ABA for 3 h. Total RNA was extracted from whole seedlings for qRT‒PCR analyses as described in the methods. Values are the mean ± SD (n = 3). Different letters denote significant differences (P < 0.05) from Duncan’s multiple range tests. (G) OsMADS27 protein level in OsMADS27pro:OsMADS27-GFP plants. Two-week-old OsMADS27pro:OsMADS27-GFP seedlings grown hydroponically on medium containing different N concentrations (0.02 mM, 0.2 mM, and 2 mM KNO₃) without (control) or with 100 mM NaCl were used for the analysis of OsMADS27 protein levels by western blotting with anti-GFP antibodies. ZH11 (WT) grown on medium with 2 mM KNO₃ served as a control. The intensity of OsMADS27-GFP bands was quantified with ImageJ from three replicates, and the statistical results are shown below the gel blots. All band intensities were normalized to that of actin in WT plants grown on medium with 2 mM KNO₃ and 0 mM NaCl. Values are the mean (n = 3). Different letters denote significant differences (P < 0.05) from Duncan’s multiple range tests.

Figure 2

Nuclear localization of OsMADS27 was observed only in the presence of nitrate. OsMADS27pro:OsMADS27-GFP plants were grown on N-free MS medium supplied with 2 mM KNO₃ (A) or 2 mM KCl (C) for 10 days. The seedlings in (A) were treated with 150 mM NaCl (B) for 60 min before green fluorescence observation. The seedlings in (C) were treated with 2 mM KNO₃ (D), 2 mM NH₄Cl (E), 150 mM NaCl (F), and 150 mM NaCl + 2 mM KNO₃ (G) for 60 min before GFP observation. The green fluorescence was observed with a Zeiss 880 microscope. Scale bars represent 20 μm.

Figure 3

Nitrate-dependent salt tolerance of seedlings. (A–D) Hydroponic salt tolerance assay. Seeds of the wild type (WT), OsMADS27 knockout mutants (osmads27-1 and osmads27-2), and overexpression lines (OE7 and OE8) were germinated at 37°C for 4 days and transferred to modified hydroponic medium containing different N concentrations (0.02 mM, 0.2 mM, 2 mM KNO₃) for 7 days followed by application of 0 mM or 140 mM NaCl for 7 days before photographs were taken (A–B), and the survival rate was calculated (C–D). Values are the mean ± SD (n = 3 replicates, 32 seedlings per replicate). (E and F) Sodium (Na⁺) and potassium (K⁺) contents in the roots and shoots of the wild type (WT), OsMADS27 knockout mutant (osmads27-1), and overexpression line (OE7). Seeds were germinated at 37°C for 4 days, transferred to modified hydroponic medium containing 0.2 mM KNO₃ for 7 days, and then treated with 140 mM or 0 mM NaCl for 5 days. Na⁺ and K⁺ contents were quantified in roots and shoots (E–F) as described in the methods. Values are the mean ± SD (n = 3 replicates, 30 seedlings per replicate). Different letters denote significant differences (P < 0.05) from Duncan’s multiple range tests.

Figure 4

Transcriptomic analysis of differentially expressed genes (DEGs) affected by OsMADS27. (A) Number of DEGs. Statistical data of DEGs in the (KO vs. WT)-control, (OE vs. WT)-control, (KO vs. WT)-salt, and (OE vs. WT)-salt comparisons. (B) Venn diagram of DEGs in the (KO vs. WT)-control, (OE vs. WT)-control, (KO vs. WT)-salt, and (OE vs. WT)-salt comparisons. The numbers represent the total numbers of DEGs in different comparison groups. (C) Hierarchical clustering analysis of N- and salt stress-related genes affected by OsMADS27 among the DEGs. The heatmap shows fold changes in the abundance of gene transcripts in different comparison groups. (D) OsMADS27 broadly regulates genes involved in salt tolerance. Seven-day-old plants grown hydroponically on medium containing different N concentrations (0.02, 0.2, and 2 mM KNO₃) supplemented with either 0 mM NaCl or 150 mM NaCl were harvested for qRT‒PCR analyses of the indicated genes. Actin was used as an internal control. Different letters denote significant differences (P < 0.05) from Duncan’s multiple range tests.

Figure 5

OsMADS27 activates OsHKT1.1 and OsSPL7 by binding to the CArG motif in their promoters. (A and B) ChIP‒qPCR assay. Enrichment of fragments containing CArG motifs (marked with asterisks) in the promoters of OsHKT1.1 and OsSPL7 was examined in OsMADS27pro:OsMADS27-GFP and wild-type plants. Approximately 200-bp fragments cis1 and cis2 of the OsHKT1.1 promoter (A) and cis2 and cis3 of the OsSPL7 promoter (B) were enriched in OsMADS27pro:OsMADS27-GFP plants by anti-GFP antibodies, as shown in qRT‒PCR analyses. Values are the mean ± SD (n = 3 replicates). Different letters denote significant differences (P < 0.05) from Duncan’s multiple range tests. (C and D) Luciferase activity assay. pRI101-OsMADS27 acted as an effector. pGreenII0800-OsHKT1.1pro::LUC/OsSPL7pro::LUC functioned as reporters. -/- represents the empty pRI101 and pGreenII 0800 plasmids. -/-, OsMADS27/-, -/OsHKT1.1pro::LUC, and -/OsSPL7pro::LUC served as negative controls; OsMADS27/OsHKT1.1pro::LUC (E) and OsMADS27/OsSPL7pro::LUC (F) were experimental groups. Different constructs were separately co-infiltrated into 4-week-old tobacco leaves, and luciferase activity was detected by the luciferase assay system.

Figure 6

OsMADS27 improves NUE and grain yield in the field under different nitrogen concentrations. (A) The panicles of wild-type ZH11 (WT), osmads27-1, and OsMADS27-OE7 plants. Scale bar represents 4 cm. (B–G) Nitrogen use efficiency (NUE) (B), actual yield per plot (C), grain yield per plant (D), panicle number per plant (E), number of seeds per plant (F), and primary branch number per panicle (G) were calculated. Values are the mean ± SD (n = 3 replicates). Different letters denote significant differences (P < 0.05) from Duncan’s multiple range tests.

Figure 7

A working model for nitrate-responsive OsMADS27-promoted salt tolerance. (A) Under nitrate-sufficient conditions, nitrate induces the expression of OsMADS27, leading to a high level of OsMADS27 that directly binds to the promoters of its target genes such as OsHKT1.1 and OsSPL7, significantly enhancing their expression and improving the salt tolerance of rice. (B) Under nitrate-deficient conditions, the expression of OsMADS27 is not induced, thereby attenuating the expression of downstream salt tolerance-related genes.