Regulatory network mediated by CmMKK5–CmMPK13 cascade response to phosphorus starvation in chrysanthemum

Abstract

Chrysanthemum (Chrysanthemum morifolium Ramat.) is an important autumnal ornamental and economically important flower with numerous cultivars and history of medicinal use. Phosphorus (P) is an essential nutritional element for chrysanthemum growth and development. This study analyzed the mitogen-activated protein kinase (MAPK) cascade pathway CmMKK5–CmMPK13 in response to low-phosphate (LP) stress in chrysanthemum. CmMPKK5 and CmMPK13 induced up-regulation in the roots, whereas CmMPK13 decreased in the leaves following 2 days of LP treatment. Subcellular localization analysis of transient expression systems in tobacco leaves revealed that CmMPKK5 and CmMPK13 proteins were localized to the entire cell structure. Mutants T200A, Y202A, and T200A-Y202A of the CmMPK13 (Cmmpk13s) post-translational modification (PTM) phosphorylation sites were obtained by site-directed mutagenesis. Subcellular localization analysis revealed that the altering the phosphorylation sites did not affect their distribution in cells. Phosphorylation analysis revealed CmMPK13 and T200A could be phosphorylated by CmMKK5 or undergo autophosphorylation. However, Y202A and T200A-Y202A could not be phosphorylated by CmMKK5 or be autophosphorylated. In Arabidopsis, CmMKK5 overexpression in transgenic lines (Oe) could complement the growth of mutant mkk5 and cause early flowering under LP stress. CmMPK13-Oe and T200A caused early flowering of mutant mpk13 under both LP and HP conditions, whereas Y202A and T200A-Y202A did not. In chrysanthemum, the phosphate (Pi, H2PO4-) concentration, root structure, height, and leaf weight of CmMPK13-Oe were significantly higher than those of wild-type (WT) under LP treatment. Phosphoproteome analysis showed that CmMPK13 could promote responsiveness to P deficiency by altering transcription and translation in the chrysanthemum. This work promotes our understanding of the molecular mechanism of MAPK and provides genetic resources for improving the efficiency of P utilization in chrysanthemum.