Extremely low light promotes adventitious shoot regeneration via RpPHYA/RpHYH-antagonized auxin biosynthesis and transport through dual regulation of RpPIN1 in Robinia pseudoacacia.

Abstract

Extremely low light (ELL) is widely applied as a strategy to enhance adventitious shoot (AS) regeneration in woody plants. However, how ELL acts as an environmental signal to reprogram regenerative competence remains unclear. Here, we show that ELL for 14 days (ELL14) markedly promotes AS regeneration across multiple genotypes of Robinia pseudoacacia. Transcriptomic and functional enrichment analyses revealed that auxin biosynthesis, transport, and signaling constitute the core pathways activated under ELL14. Consistently, auxin levels increased, and the inhibition of biosynthesis or transport largely abolished the regeneration-promoting effect. Integrative weighted gene co-expression network analysis and regulatory analyses identified RpPHYA as a key integrator of ELL-responsive networks. Under ELL14, the RpPHYA-associated module promotes auxin biosynthesis by upregulating RpAMI1 and coordinates auxin transport through RpPIN1 via transcriptional regulation and RpROS1-mediated CHG demethylation, forming a dual regulatory mechanism. In contrast, under normal light, RpHYH antagonizes this regulatory program, thereby restricting regenerative capacity. Overexpression of RpPHYA, RpAMI1, RpPIN1, and RpROS1 significantly enhanced AS regeneration, functionally validating this ELL14-induced module. Collectively, our findings demonstrate that ELL functions as an environmental signal that promotes AS regeneration in R. pseudoacacia through coordinated transcriptional and DNA demethylation of auxin transport, providing mechanistic insights for improving regeneration in woody plants.