# Genome-Wide Characterisation of the AP2/ERF Family in Salvia miltiorrhiza Identifies Hormone-Responsive Candidates Associated with Phenolic Acid Accumulation

## TL;DR

This study identifies AP2/ERF genes in Salvia miltiorrhiza linked to hormone responses and phenolic acid production, offering insights for metabolic engineering.

## Contribution

The first genome-wide analysis of AP2/ERF genes in Salvia miltiorrhiza, identifying hormone-responsive candidates tied to phenolic acid accumulation.

## Key findings

- 169 SmAP2/ERF genes were identified and classified into five subfamilies.
- Four SmAP2/ERF genes showed distinct hormone responsiveness and tissue-specific expression.
- Phytohormone treatments altered key phenolic-acid pathway genes and compound accumulation.

## Abstract

APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors integrate phytohormone signalling with developmental programmes and specialised metabolism, yet their family-wide features and potential contributions to phenolic-acid biosynthesis remain to be systematically clarified in Salvia miltiorrhiza. In this study, we conducted a comprehensive genome-wide analysis and identified 169 SmAP2/ERF genes, which were classified into five subfamilies (AP2, ERF, DREB, RAV and Soloist). SmAP2/ERFs were unevenly distributed across chromosomes and expanded predominantly through tandem and segmental duplication, and Ka/Ks analysis indicated that tandem-duplicated pairs have mainly undergone purifying selection. Promoter analysis revealed abundant cis-acting elements related to light, phytohormones and stress responses, indicating extensive regulatory potential. Comparative phylogenetic analysis with Arabidopsis thaliana prioritised four candidates (SmAP2/ERF88, SmAP2/ERF110, SmAP2/ERF121 and SmAP2/ERF122) closely associated with specialised-metabolism regulators. These genes exhibited distinct tissue-preferential expression patterns and divergent hormone responsiveness: SmAP2/ERF88/110 were broadly inducible, whereas SmAP2/ERF121/122 responded mainly to abscisic acid and were repressed by brassinosteroids. Confocal imaging of GFP fusions confirmed nuclear localisation of all four proteins. Phytohormone treatments differentially regulated key phenolic-acid pathway genes (PAL, C4H, 4CL, TAT, HPPR, RAS and CYP98A14) and altered rosmarinic acid and salvianolic acid B accumulation. These results broaden the genome-wide understanding of the SmAP2/ERF family in Salvia miltiorrhiza. Hormone-responsive SmAP2/ERFs show expression patterns associated with hormone-dependent transcriptional changes in phenolic-acid pathway genes and with RA and SAB accumulation, providing candidates for future functional validation and metabolic engineering.