Overexpression of a Zea mays Brassinosteroid-Signaling Kinase Gene ZmBSK1 Confers Salt Stress Tolerance in Maize

Abstract

Salinity has become a crucial environmental factor seriously restricting maize (Zea mays L.) growth, development and productivity. However, how plants respond to salt stress is still poorly understood. In this study, we report that a maize brassinosteroid-signaling kinase gene ZmBSK1 plays a significant role in salt stress response. Expression pattern analysis revealed that the transcript level of ZmBSK1 was upregulated by NaCl treatment both in maize leaves, roots, and stems. Phenotypic and physiological analysis showed that overexpression of ZmBSK1 in maize improved salt tolerance by reducing the malondialdehyde (MDA) content, the percentage of electrolyte leakage, O₂ ^- and H₂O₂ accumulation under salt stress, relying on the increases of antioxidant defense enzyme activities and proline content. qRT-PCR analysis showed that overexpression of ZmBSK1 also positively modulated the expression levels of reactive oxygen species (ROS)-scavenging and proline biosynthesis-related genes under salt stress. Moreover, immunoprecipitation-mass spectrometry (IP-MS) assay and firefly luciferase complementation imaging (LCI) assay showed that ZmBSK1 could associate with heat shock protein ZmHSP8 and 14-3-3-like protein ZmGF14-6, and their gene expression levels could be significantly induced by NaCl treatment in different maize tissues. Our findings unravel the new function of ZmBSK1 in salt stress response, which provides the theoretical bases for the improvement of maize salt resistance.

Keywords: ZmBSK1; antioxidant defense enzyme; maize; protein interaction; reactive oxygen species; salt tolerance.

Figure 1

Expression patterns of ZmBSK1 gene in maize. (A) qRT-PCR analysis of the expression levels of ZmBSK1 in different maize tissues (roots, stems, leaves, pollens, and pistils). (B-D) The expression levels of ZmBSK1 in maize leaves (B), roots (C), and stems (D) exposed to salt stress. About 10-day-old wild type seedlings were treated with 200 mM NaCl for indicated times, and the expression was measured by qRT-PCR. ZmActin2 served as an internal control. Error bars represent ±SD (n = 3). Different letters indicate significant differences at p < 0.05 according to two-way ANOVA (Duncan’s multiple range test).

Figure 2

Effect of ZmBSK1 on root growth of maize seeds under salt stress. (A) The expression levels of ZmBSK1 in OE-ZmBSK1 and wild type (WT) plants. The expression of ZmBSK1 was measured by qRT-PCR, and ZmActin2 served as an internal control. (B) The protein levels of ZmBSK1 in OE-ZmBSK1 and WT plants. Total proteins extracted from the leaves were used for immunoblotting with anti-Flag antibody. β-actin was used as a loading control. (C) The root growth phenotypes of OE-ZmBSK1 and WT maize seeds under salt stress. The maize seeds were spread on paper containing 200 mM NaCl for 4 days during germination. Scale bar = 1 cm. (D) Statistical analysis of root length in (C). Error bars in (A,D) represent ±SD (n = 3). Different letters indicate significant differences at p < 0.05 according to one-way or two-way ANOVA (Duncan’s multiple range test).

Figure 3

Overexpression of ZmBSK1 improves salt tolerance in maize. (A) The phenotypes of OE-ZmBSK1 and WT maize plants under salt stress. About 10-day-old maize seedlings were treated with 200 mM NaCl for 14 days, and then recovered for 5 days. Scale bar = 5 cm. (B) Survival rate (%) of maize plants in (A). At least 45 seedlings of each line per replicate were used for survival rate analysis. The malondialdehyde (MDA) content (C) and the percentage of electrolyte leakage (D) in leaves of OE-ZmBSK1 and WT maize plants under salt stress. About 10-day-old maize seedlings were treated with 200 mM NaCl for 2 days, and then the physiological indexes as indicated were measured. Error bars in (B–D) represent ±SD (n = 3). Different letters indicate significant differences at p < 0.05 according to one-way or two-way ANOVA (Duncan’s multiple range test).

Figure 4

Effect of ZmBSK1 on reactive oxygen species (ROS) levels, antioxidant defense enzymes activity and proline content under salt stress. Nitroblue tetrazolium (NBT) staining of O₂⁻ (A) and 3,3′-diaminobenzidine (DAB) staining of H₂O₂ (B) in leaves of OE-ZmBSK1 and WT maize plants under salt stress. Scale bar = 1 cm. Statistical analysis of O₂⁻ production rates (C) and H₂O₂ content (D) in leaves of OE-ZmBSK1 and WT maize plants under salt stress. Ascorbate peroxidase (APX) activity (E), catalase (CAT) activity (F), superoxide dismutase (SOD) activity (G), and proline content (H) in leaves of OE-ZmBSK1 and WT maize plants under salt stress. About 10-day-old maize seedlings in (A–H) were treated with 200 mM NaCl for 2 days, and then the above physiological indexes as indicated were measured. Error bars in (C–H) represent ±SD (n = 3). Different letters indicate significant differences at p < 0.05 according to two-way ANOVA (Duncan’s multiple range test).

Figure 5

ZmBSK1 positively regulates the expression of stress-related genes in maize under salt stress. The expression levels of stress-related genes ZmcAPX (A), ZmCAT1 (B), ZmCSD5 (C), ZmMSD2 (D), ZmP5CS2 (E), and ZmP5CS1 (F) in OE-ZmBSK1 and WT maize plants exposed to salt stress. About 10-day-old maize seedlings were treated with 200 mM NaCl for 6 h, and the expressions were measured by qRT-PCR. ZmActin2 served as an internal control. Error bars in (A–F) represent ±SD (n = 3). Different letters indicate significant differences at p < 0.05 according to two-way ANOVA (Duncan’s multiple range test).

Figure 6

ZmBSK1 interacts with its target proteins ZmHSP8 and ZmGF14-6. (A) Venn diagram shows the number of ZmBSK1-interacting proteins in OE-ZmBSK1 maize plants with or without salt stress. About 10-day-old maize seedlings were treated with 200 mM NaCl for 0 or 10 h. Total proteins extracted from leaves were immunoprecipitated by anti-Flag antibody and the interacting proteins of ZmBSK1 were identified by immunoprecipitation-mass spectrometry (IP-MS). (B) The candidate target proteins of ZmBSK1 upon mass spectrometry analysis only after salt treatment. Firefly luciferase complementation imaging (LCI) assays confirm the interactions of ZmBSK1 with ZmHSP8 (C) and ZmGF14-6 (D). The combinations of ZmBSK1-nLUC and cLUC-ZmHSP8, ZmBSK1-nLUC and cLUC-ZmGF14-6 were co-transformed into tobacco leaves. The combinations of ZmBSK1-nLUC and cLUC, nLUC and cLUC-ZmHSP8, nLUC and cLUC-ZmGF14-6, nLUC and cLUC were used as negative controls. nLUC and cLUC represent the N-terminal and C-terminal fragments of firefly luciferase, respectively. Images were collected from the detached leaves after infiltration for 3 days. Scale bar = 1 cm.

Figure 7

Gene expression patterns of ZmHSP8 and ZmGF14-6 under salt stress. (A–C) The expression levels of ZmHSP8 in maize leaves (A), roots (B), and stems (C) exposed to salt stress. (D–F) The expression levels of ZmGF14-6 in maize leaves (D), roots (E), and stems (F) exposed to salt stress. About 10-day-old wild type seedlings were treated with 200 mM NaCl for indicated times, and the expression was measured by qRT-PCR. ZmActin2 served as an internal control. Error bars represent ±SD (n = 3). Different letters indicate significant differences at p < 0.05 according to two-way ANOVA (Duncan’s multiple range test).