MdMKK9-Mediated the Regulation of Anthocyanin Synthesis in Red-Fleshed Apple in Response to Different Nitrogen Signals

Abstract

The mitogen-activated protein kinase (MAPK) signaling cascade is a widely existing signal transduction system in eukaryotes, and plays an important role in the signal transduction processes of plant cells in response to environmental stress. In this study, we screened MdMKK9, a gene in the MAPK family. This gene is directly related to changes in anthocyanin synthesis in the 'Daihong' variety of red-fleshed apple (Malus sieversii f neidzwetzkyana (Dieck) Langenf). MdMKK9 expression was up-regulated in 'Daihong' tissue culture seedlings cultured at low levels of nitrogen. This change in gene expression up-regulated the expression of genes related to anthocyanin synthesis and nitrogen transport, thus promoting anthocyanin synthesis and causing the tissue culture seedlings to appear red in color. To elucidate the function of MdMKK9, we used the CRISPR/Cas9 system to construct a gene editing vector for MdMKK9 and successfully introduced it into the calli of the 'Orin' apple. The MdMKK9 deletion mutants (MUT) calli could not respond to the low level of nitrogen signal, the expression level of anthocyanin synthesis-related genes was down-regulated, and the anthocyanin content was lower than that of the wild type (WT). In contrast, the MdMKK9-overexpressed calli up-regulated the expression level of anthocyanin synthesis-related genes and increased anthocyanin content, and appeared red in conditions of low level of nitrogen or nitrogen deficiency. These results show that MdMKK9 plays a role in the adaptation of red-fleshed apple to low levels of nitrogen by regulating the nitrogen status and anthocyanin accumulation.

Keywords: CRISPR/Cas9; MdMKK9; anthocyanin; nitrogen signals; red-fleshed apple.

Figure 1

The phenotypes and anthocyanin contents of ‘Gala’ and ‘Daihong’ apples at different stages of growth. (A): Color changes in the skin and flesh of ‘Gala’ (GL) and ‘Daihong’ (DH) apples during the S1, S2, and S3 stages of fruit development. (B): Anthocyanin contents of the skin and flesh of ‘Gala’ and ‘Daihong’ apples during the S1, S2, and S3 stages. The bars represent means ± SD (n = 3). Asterisks indicate statistically significant differences (*** p < 0.001; **** p < 0.0001).

Figure 2

Relative expression levels of genes in the MAPK gene family at different developmental stages in ‘Gala’ and ‘Daihong’. (A) MdMKK2 (a, Leaf, b, Stem, c, Skin, d, Flesh, the same below). (B) MdMKK3. (C) MdMKK4. (D) MdMKK5. (E): MdMKK6. (F): MdMKK9. The relative gene expression was calculated according to the 2^−ΔCT method. The bars represent means ± SD (n = 3). Asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 3

The phenotype, nitrogen content, anthocyanin content, and relative expression level of the MdMKK9 gene in ‘Daihong’ tissue culture seedlings grown in culture mediums with different nitrogen concentrations. (A) Phenotype. (B) Relative expression levels of MdMKK9. The relative level of MdMKK9 in ‘Dahong’ tissue culture seedlings at 0 h was set to 1. Asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). (C) Nitrogen content. (D) Anthocyanin content. In (C,D), different letters above the bars indicate significant differences (p < 0.05). The bars represent means ± SD (n = 3).

Figure 4

Relative expression of anthocyanin synthesis-related genes in ‘Daihong’ tissue culture seedlings grown in mediums with 4 and 0 mM NO₃⁻. (A) Relative expression of structural genes involved in anthocyanin synthesis (a: MdPAL, b: MdCHS, c: MdCHI, d: MdF3H, e: MdDFR, f: MdANS, g: MdANR, h: MdUFGT). (B) Relative expression of regulatory genes related to anthocyanin synthesis (a: MdMYB10, b: MdbHLH33, c: MdbHLH3, d: MdWD40). The relative level of anthocyanin synthesis-related genes in ‘Dahong’ tissue culture seedlings at 0 h was set to 1. The bars represent means ± SD (n = 3). Asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 5

Relative expression of nitrogen transport-related genes in tissue cultured seedlings of ‘Daihong’ cultured in mediums with 4 and 0 mM NO₃⁻. (A). MdNPF6.8, (B). MdNPF6.9, (C). MdNRT2.4, (D) MdNRT2.7, (E) MdAMT1.5, (F) MdNMT3.1. The relative level of nitrogen transport-related genes in ‘Dahong’ tissue culture seedlings at 0 h was set to 1. The bars represent means ± SD (n = 3). Asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; **** p < 0.0001).

Figure 6

Mutagenesis of the MdMKK9 gene based on the CRISPR/Cas9 system. (A) Expression cassette of the gene-editing vector derived from pHDE-35S-Cas9-mCherry-UBQ. The red box indicates the target site. (B) MdMKK9 mutations resulting from four positive lines of gene editing. WT, wild-type. MdMKK9-MUT-site1 and MdMKK9-MUT-site2 represent two different clones. The blue dots indicate the missing bases. Red letters indicate the protospacer adjacent motifs. (C) Sequencing chromatogram of partial genomic DNA of MdMKK9 corresponding to the mutations. The orange frames indicate target sites for gene editing.

Figure 7

The phenotype, anthocyanin content, and MdMKK9 expression levels of calli from wild-type (WT) ‘Orin’ plants, transformed CRISPR/Cas9-MdMKK9 (MUT) lines, and transformed pRI101-MdMKK9 (OE) lines grown in mediums with different nitrogen concentrations. (A) Phenotypes of the calli. (B) Anthocyanin content. (C) Relative expression levels of MdMKK9. The relative level of MdMKK9 in 40 mM NO₃⁻ treated WT calli was set to 1. The bars represent means ± SD (n = 3). Asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 8

Expression levels of structural genes related to anthocyanin synthesis in the calli of wild-type (WT) plants, transformed CRISPR/Cas9-MdMKK9 (MUT) lines, and transformed pRI101-MdMKK9 (OE) lines grown in mediums with different nitrogen concentrations. (A) Relative expression of structural genes involved in anthocyanin synthesis (a: MdPAL, b: MdCHS, c: MdCHI, d: MdF3H, e: MdDFR, f: MdANS, g: MdANR, h: MdUFGT). (B) Relative expression of regulatory genes related to anthocyanin synthesis (a: MdMYB10, b: MdbHLH3, c: MdWD40). The relative level of anthocyanin synthesis-related genes in 40 mM NO₃⁻ treated WT calli was set to 1. The bars represent means ± SD (n = 3). Asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 9

Expression levels of nitrogen regulatory genes in the calli of wild-type (WT) plants, transformed CRISPR/Cas9-MdMKK9 (MUT) lines, and transformed pRI101-MdMKK9 (OE) lines grown in mediums with different nitrogen concentrations. The relative level of nitrogen transport-related genes in 40 mM NO₃⁻ treated WT calli was set to 1. The bars represent means ± SD (n = 3). Asterisks indicate statistically significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).