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.

The MdmiR156n Regulates Drought Tolerance and Flavonoid Synthesis in Apple Calli and Arabidopsis

“…After one week of natural drought for 3-week-old A. thaliana (WT and transgenic plants) grown in the same pot, the leaves were taken to extract anthocyanin using 1% ( v / v ) HCl-methanol extraction solution at 4 °C in the dark for 24 h, to determine the anthocyanin content using the pH difference method according to the previous method [ 57 ].…”

The role of environmental stress in fruit pigmentation

“…At the molecular level, studies in apple show a number of candidate genes linking N deficiency to an elevation in the anthocyanin pathway. Low N induces anthocyanin production, partly mediated by the mitogen-activated protein kinase (MAPK) signalling cascade, as shown for red-fleshed apples, where a MAPK family gene, MdMKK9, regulates the N status (Sun et al, 2022). Under low N conditions the autophagy nutrient recycling process can be activated, and studies in apple have demonstrated that the AuTophaGy (ATG) protein, MdATG18a, improves tolerance to nitrogen deficiency via upregulation of the anthocyanin pathway (Sun et al, 2018).…”

The role of environmental stress in fruit pigmentation

“…At the molecular level, studies in apple show a number of candidate genes linking N deficiency to an elevation in the anthocyanin pathway. Low N induces anthocyanin production, partly mediated by the mitogen‐activated protein kinase (MAPK) signalling cascade, as shown for red‐fleshed apples, where a MAPK family gene, MdMKK9 , regulates the N status (Sun et al, 2022). Under low N conditions the autophagy nutrient recycling process can be activated, and studies in apple have demonstrated that the AuTophaGy (ATG) protein, MdATG18a, improves tolerance to nitrogen deficiency via upregulation of the anthocyanin pathway (Sun et al, 2018).…”