Fig. 4: ZmSnRK2.9 and ZmSnRK2.10 phosphorylate ZmHAK4 then increase its Na⁺ transport activity.

ZmSnRK2.9 (a) and ZmSnRK2.10 (b) phosphorylate ZmHAK4^N but not ZmHAK4^N(S5A) in vitro. The phosphorylated ZmHAK4^N was detected by autoradiography (top) and Coomassie brilliant blue (CBB) staining (bottom) was shown as a loading control. c In-gel kinase assay of ZmSnRK2.9 and ZmSnRK2.10 under salt stress. Total proteins were extracted from 7-day-old wild type and ZmSnRK2.9^crispr ZmSnRK2.10^crispr plants treated with salt for 0 and 2 h. Recombinant GST-ZmHAK4^N was used as the substrate. ZmSnRK2.9 and ZmSnRK2.10 kinase activity was detected by autoradiography. Coomassie Brilliant Blue (CBB) staining provided a loading control. The red arrow indicates the target protein bands. d The phosphorylation of ZmHAK4^N by ZmSnRK2.9 and ZmSnRK2.10 enhances its Na⁺ transport activity in yeast. The yeast strain ant5 transformed with the indicated plasmids and grown on medium supplied with 1 mM KCl and the indicated concentrations of NaCl were shown. Similar results were seen in three independent experiments. e The results of the Na⁺-uptake assay. The ant5 cells transformed with the indicated plasmids were cultured in the liquid medium with 20 mM Na⁺ for the indicated duration, and then the yeast cells were harvested for measurement of Na⁺ content. Data in (e) represent the mean values ± SD of six independent replicates. Statistical significance was determined using a two-way ANOVA test. Different letters represent a significant difference at P < 0.05. Similar results in (a–d) were observed in three independent experiments. Source data are provided as a Source Data file.