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

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.

Ammonium and nitrate regulate NH4+ uptake activity of Arabidopsis ammonium transporter AtAMT1;3 via phosphorylation at multiple C-terminal sites.

In plants, nutrient transporters require tight regulation to ensure optimal uptake in complex environments. The activities of many nutrient transporters are post-translationally regulated by reversible phosphorylation, allowing rapid adaptation to variable environmental conditions. Here, we show that the Arabidopsis root epidermis-expressed ammonium transporter AtAMT1;3 was dynamically (de-)phosphorylated at multiple sites in the cytosolic C-terminal region (CTR) responding to ammonium and nitrate signals. Under ammonium resupply rapid phosphorylation of a Thr residue (T464) in the conserved part of the CTR (CTRC) effectively inhibited AtAMT1;3-dependent NH4+ uptake. Moreover, phosphorylation of Thr (T494), one of three phosphorylation sites in the non-conserved part of the CTR (CRTNC), moderately decreased the NH4+ transport activity of AtAMT1;3, as deduced from functional analysis of phospho-mimic mutants in yeast, oocytes, and transgenic Arabidopsis. Double phospho-mutants indicated a role of T494 in fine-tuning the NH4+ transport activity when T464 was non-phosphorylated. Transient dephosphorylation of T494 with nitrate resupply closely paralleled a transient increase in ammonium uptake. These results suggest that T464 phosphorylation at the CTRC acts as a prime switch to prevent excess ammonium influx, while T494 phosphorylation at the CTRNC fine tunes ammonium uptake in response to nitrate. This provides a sophisticated regulatory mechanism for plant ammonium transporters to achieve optimal ammonium uptake in response to various nitrogen forms.