AtZIK4, a MAPK kinase kinase, positively regulates phosphate uptake gene expression to modulate phosphate starvation tolerance in Arabidopsis.

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signal transduction module in eukaryotes. We investigated the function of ZIK4, a MAPK kinase, in the phosphate starvation response of Arabidopsis. Phenotypic analysis revealed that the zik4 mutant had stronger tolerance to phosphate starvation than that to the wild type. However, the kinase domain was still expressed in zik4. The CRISPR/Cas9 system was used to construct two independent knockout mutants zik4-c3 and zik4-c15 by targeting two different sites in the AtZIK4 kinase domain. Physiological measurements showed that the phosphate content in zik4-c3 and zik4-c15 was lower than that in the wild type. Expression pattern analysis revealed that ZIK4 was induced to express under phosphate starvation. Quantitative real-time PCR results showed that the expression levels of multiple key phosphate uptake-related genes were significantly up-regulated in the zik4 mutant. These results indicate that ZIK4, as a phosphate starvation-induced MAPKKK, promotes phosphate accumulation in Arabidopsis seedlings by upregulating the expression levels of phosphate uptake-related genes. These findings not only elucidate the regulatory role of AtZIK4 kinase during abiotic stress and expand the low-phosphate (LP) signaling cascade in Arabidopsis thaliana, but also providing promising genetic targets to enhance crop performance under phosphorus-limited conditions.