CsSPX3-CsPHL7-CsGS1/CsTS1 module mediated pi-regulated negatively theanine biosynthesis in tea (Camellia sinensis)

Phosphorus (P) is the macronutrients essential for the development and growth of plants, but how external inorganic phosphate (Pi) level and signaling affect tea plant growth and characteristic secondary metabolite biosynthesis are not understood. Theanine is major secondary metabolites, and its contents largely determine tea favor and nutrition qualities. Here, we found theanine contents in tea leaves and roots declined as Pi concentration increased in tea plants after Pi feeding. The transcriptome analysis of global gene expression in tea leaves under Pi feeding suggested a wide range of genes involved in Pi/N transport and responses were altered. Among them, CsSPX3 and CsPHL7 transcript levels in response to Pi feeding to tea plants, their expression patterns were generally opposite to these of major theanine biosynthesis genes, indicating possible regulatory correlations. Biochemical analyses showed that CsSPX3 interacted with CsPHL7, and CsPHL7 negatively regulated theanine biosynthesis genes CsGS1 and CsTS1. Meanwhile, VIGS and transient overexpression systems in tea plants verified the functions of CsSPX3 and CsPHL7 in mediating Pi-feeding-repressed theanine biosynthesis. This study offers fresh insights into the regulatory mechanism underlying Pi repression of theanine biosynthesis, and the CsSPX3-CsPHL7-CsGS1/CsTS1 module plays a role in high Pi-inhibition of theanine production in tea leaves. It has an instructional significance for guiding the high-quality tea production in tea garden fertilization.