Integration of genome wide association studies and co-expression networks reveal roles of PtoWRKY 42-PtoUGT76C1-1 in trans-zeatin metabolism and cytokinin sensitivity in poplar.

Abstract

Cytokinins are important for in vitro shoot regeneration in plants. Cytokinin N-glucosides are produced via an irreversible glycosylation pathway, which regulates the endogenous cytokinin content. Although cytokinin N-glucoside pathways have been uncovered in higher plants, no regulator has been identified to date. We performed a metabolome genome-wide association study (mGWAS), weighted gene co-expression network analysis (WGCNA), and expression quantitative trait nucleotide (eQTN) mappings to build a core triple genetic network (mGWAS-gene expression-phenotype) for the trans-zeatin N-glucoside (ZNG) metabolite using data from 435 unrelated Populus tomentosa individuals. Variation of the ZNG level in poplar is attributed to the differential transcription of PtoWRKY42, a member of WRKY multigene family group IIb. Functional analysis revealed that PtoWRKY42 negatively regulated ZNG accumulation by binding directly to the W-box of the UDP-glycosyltransferase 76C 1-1 (PtoUGT761-1) promoter. Also, PtoWRKY42 was strongly induced by leaf senescence, 6-BA, wounding, and salt stress, resulting in a reduced ZNG level. We identified PtoWRKY42, a negative regulator of cytokinin N-glucosides, which contributes to the natural variation in ZNG level and mediates ZNG accumulation by directly modulating the key glycosyltransferase gene PtoUGT76C1-1.