MicroRNA408 negatively regulates salt tolerance by affecting secondary cell wall development in maize

Abstract

Although microRNA408 (miR408) is a highly conserved miRNA, the miR408 response to salt stress differs among plant species. Here, we show that miR408 transcripts are strongly repressed by salt stress and methyl viologen treatment in maize (Zea mays). Application of N, N1-dimethylthiourea partly relieved the NaCl-induced down-regulation of miR408. Transgenic maize overexpressing MIR408b is hypersensitive to salt stress. Overexpression of MIR408b enhanced the rate of net Na+ efflux, caused Na+ to locate in the inter-cellular space, reduced lignin accumulation, and reduced the number of cells in vascular bundles under salt stress. We further demonstrated that miR408 targets ZmLACCASE9 (ZmLAC9). Knockout of MIR408a or MIR408b or overexpression of ZmLAC9 increased the accumulation of lignin, thickened the walls of pavement cells, and improved salt tolerance of maize. Transcriptome profiles of the wild-type and MIR408b-overexpressing transgenic maize with or without salt stress indicated that miR408 negatively regulates the expression of cell wall biogenesis genes under salt conditions. These results indicate that miR408 negatively regulates salt tolerance by regulating secondary cell wall development in maize.

Figure 1.

Regulation of ZmmiR408 by salt stress. A, Expression patterns of ZmmiR408 in maize leaves as determined by in situ hybridization analysis. Sense probes were used as negative controls. Scale bars = 50 µm. Scale bars in magnified images = 25 µm. B, Assay of ZmmiR408 responses to salt stresses by small RNA northern blot. C, Effects of salt stress on MIR408 precursor expression in maize seedlings. Quantifications were normalized to the expression of ZmACTIN1. Error bars represent the standard errors of three independent experiments. Means with the same letter are not significantly different at P < 0.05 according to the least significant difference (LSD) test. D, Effects of N, N¹-dimethylthiourea (DMTU) treatment on miR408 expression in maize seedlings. The maize seedlings were subjected to NaCl stress for 12 h. Two independent experiments were performed. E, Effects of methyl viologen on miR408 expression in maize seedlings. Three independent experiments were performed. In (B), (D), and (E), U6 was used as the loading control. Numbers below each lane indicate the expression level of miR408 relative to U6.

Figure 2.

Responses of ZmMIR408b-overexpressing transgenic maize to salt stress. A, ZmmiR408 levels in ZmMIR408b-overexpressing transgenic maize as indicated by small RNA northern blot. U6 was used as the loading control. B, Representative images of first leaves from the bottom of ZmMIR408b-overexpressing transgenic maize cultured in hydroponic solutions supplied with 150 mM NaCl for 7 d. Scale bars = 1 cm. C, ROS accumulation in WT plants and ZmMIR408b-overexpressing transgenic maize labeled with H₂DCFDA (n = 10). The plants were subjected to 150 mM NaCl for 7 d. Representative plants were photographed. Scale bars = 50 μm. D, Effects of salt stress on MDA concentrations in WT plants and ZmMIR408b-overexpressing transgenic maize. E, Effects of salt stress on relative electrolyte leakage in WT plants and ZmMIR408b-overexpressing transgenic maize. Error bars in (D) and (E) represent standard errors of three independent experiments. Means with the same letter in (D) and (E) are not significantly different at P < 0.05 according to one-way ANOVA followed by Tukey's multiple comparison test.

Figure 3.

Na⁺ distribution in ZmMIR408b-overexpressing transgenic maize. A, Effects of salt stress on Na⁺ and K⁺ concentrations in WT plants and ZmMIR408b-overexpressing transgenic maize. Error bars represent standard errors of three independent experiments. Means with the same letter are not significantly different at P < 0.05 according to one-way ANOVA followed by Tukey's multiple comparison test. B, Effects of salt stress on net Na⁺ fluxes in the second leaf from the base of WT plants and ZmMIR408b-overexpressing transgenic maize. A continuous flux recording of 12 to 13 min was conducted. Each point represents the mean of four individual leaves. Error bars represent standard errors of three independent experiments. C, X-ray fluorescence-based Na⁺ visualization in mesophyll cells of WT plants and ZmMIR408b-overexpressing transgenic maize. At least five leaves were observed for each line. Representative plants were photographed. Scale bars = 2 μm. CW, cell wall; Vac, vacuole.

Figure 4.

Development of vascular tissues in ZmMIR408b-overexpressing transgenic maize. A, Phloroglucinol staining of the second leaf from the base of WT plants and ZmMIR408b-overexpressing transgenic maize cultured in hydroponic solutions containing 150 mM NaCl for 7 d. Representative plants were photographed. Scale bars = 50 μm. The red arrows show hyper-lignified fibers. B, Effects of salt stress on the thickness of pavement cell walls in WT plants and ZmMIR408b-overexpressing transgenic maize (n of border cell wall = 30, n of interior cell wall = 15). Error bars represent standard errors of three independent experiments. Means with the same letter are not significantly different at P < 0.05 according to one-way ANOVA followed by Tukey's multiple comparison test. C, Transmission electron micrographs of the second leaf from the base of WT plants and ZmMIR408b-overexpressing transgenic maize under salt conditions. Scale bars = 30 μm. Red dotted lines indicate the vascular region.

Figure 5.

Responses of the mir408a/b mutant to salt stress. A, Alignment of the nucleotide sequence between mir408a/b loss-of-function mutants generated by the CRISPR/Cas9 system and the corresponding wild-type (WT) at positions edited by CRISPR/Cas9. B, miR408 levels in mir408a/b loss-of-function mutants as indicated by small RNA northern blots. U6 was used as the loading control. C, Representative images of the second leaf from the base of WT plants and mir408a/b loss-of-function mutants cultured in hydroponic solutions supplied with 150 mM NaCl for 4 d. Scale bars = 5 cm. D, Phloroglucinol staining of the second leaf from the base of WT plants and mir408a/b loss-of-function mutants cultured in hydroponic solutions containing 150 mM NaCl for 4 d. Representative plants were photographed. Scale bars = 20 μm. The arrows show hyper-lignified fibers. E, Effects of salt stress on the thickness of pavement cell walls in WT plants and mir408a/b loss-of-function mutants (n = 30). F, Effects of salt stress on vascular cell number of WT plants and mir408a/b loss-of-function mutants (n = 15). Error bars in (E) and (F) represent standard errors of three independent experiments. Means with the same letter in (E) and (F) are not significantly different at P < 0.05 according to one-way ANOVA followed by Tukey's multiple comparison test.

Figure 6.

Expression pattern of ZmLAC9. A, mRNA abundance of ZmLAC9 and ZmLAC18 in ZmMIR408b-overexpressing transgenic maize and mir408a/b loss-of-function mutants. B, ZmLAC9 mRNA cleavage sites detected by 5′ RACE. Numbers indicate the frequency of cleavage at the site. C, Effects of salt stress on the expression of ZmLAC9. Expression levels in (A) and (C) were normalized to the expression levels of ZmACTIN1. Error bars in (A) and (C) represent the standard errors of three independent experiments. Means with the same letter in (A) are not significantly different at P < 0.05 according to the least significant difference (LSD) test. Asterisks in (C) indicate significant differences between control and salt treatments at P < 0.01 according to t-tests. D, Expression patterns of ZmLAC9 in maize leaves as determined by in situ hybridization analysis. Sense probes were used as negative controls. Scale bars = 50 µm. Scale bars in insets = 25 µm.

Figure 7.

Responses of ZmLAC9-overexpressing transgenic maize to salt stress. A, mRNA abundance of ZmLAC9 in ZmLAC9-overexpressing transgenic maize. Expression levels detected by RT-qPCR were normalized to the expression levels of ZmACTIN1. B, Phloroglucinol staining of the second leaf from the base of WT plants and ZmLAC9-overexpressing transgenic maize cultured in hydroponic solutions supplied with 150 mM NaCl for 4 d. Representative plants were photographed. Scale bars = 20 μm. The arrows show hyper-lignified fibers. C, Effects of salt stress on the thickness of pavement cell walls in WT plants and ZmLAC9-overexpressing transgenic maize (n = 25). D, Effects of salt stress on vascular cell number of WT plants and ZmLAC9-overexpressing transgenic maize (n = 15). Error bars in (A), (C), and (D) represent standard errors of three independent experiments. Means with the same letter in (A), (C), and (D) are not significantly different at P < 0.05 according to one-way ANOVA followed by Tukey's multiple comparison test. E, Representative images of the second leaf from the base of WT plants and ZmLAC9-overexpressing transgenic maize cultured in hydroponic solutions containing 150 mM NaCl for 4 d. Scale bars = 5 cm.

Figure 8.

Genes down-regulated by salt stress in WT plants and ZmMIR408b-overexpressing transgenic maize. A, Venn diagram showing the overlap of genes down-regulated by salt stress in WT plants and ZmMIR408b-overexpressing transgenic maize. B, GO classifications of genes specifically affected by miR408 abundance. The point size represents the number of genes in the terms; the point color represents -log₁₀ (P-value). C, A proposed model for the role of ZmmiR408 in maize salt tolerance. Arrows indicate positive regulation and blunt-ended bars indicate inhibition.