Single-cell transcriptomics and time-series metabolite profiling reveal the spatiotemporal regulation of flavonoid biosynthesis genes and phytohormone homeostasis by PAP1 in Arabidopsis.

Background: Understanding the spatiotemporal regulation of specialized metabolism in plants is critical for advancing both basic plant biology and biotechnological applications. PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) is a well-known transcription factor that plays a key regulatory role in the biosynthesis pathway of plant flavonoids. Similar to other secondary metabolites, flavonoid biosynthesis displays cell heterogeneity. However, the cell-specific regulation network of the flavonoid biosynthetic pathway remains unclear.

Results: In this study, we utilized single-cell RNA sequencing (scRNA-seq) and time-series metabolite profiling to investigate the regulation of flavonoid biosynthesis and phytohormone homeostasis in Arabidopsis thaliana by PAP1. By comparing single-cell transcriptomes of the pap1-D mutant and wild-type plant leaves, we constructed a cell-type-specific atlas of gene expression and high-resolution dynamics of metabolites across developmental stages. Our findings reveal that PAP1 overexpression induces distinct spatiotemporal regulation of phenylpropanoid pathway genes in different cell types and widespread upregulation of glycosylation processes. Metabolomic profiling validated these transcriptional patterns and showed significant changes of metabolites in phenylalanine metabolic processes as pap1-D leaf matures. Additionally, PAP1 overexpression leads to significant changes in phytohormone levels, particularly jasmonate and salicylate, indicating complex crosstalk between flavonoid biosynthesis and hormone homeostasis.

Conclusions: This integrated multi-omics approach provides unprecedented insights into the cell-specific regulatory networks controlling specialized metabolism and establishes a valuable framework for optimizing metabolic engineering strategies to enhance the production of bioactive plant compounds.