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

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.