Background: Glutathione S-transferases (GSTs) mediate vacuolar sequestration of anthocyanins and are key determinants of flower color. Several Phi (GSTF) and Tau (GSTU) members have been linked to anthocyanin transport, but how GST repertoires in woody ornamentals respond to light quality is largely unknown in Rhododendron . Results: We identified 87 GST genes in the Rhododendron simsii genome and classified them into seven subfamilies, with Tau and Phi predominating. Segmental duplication was the main driver of family expansion, and most promoters carried multiple light-responsive cis-elements. Twelve representative RsGST homologues were mapped to Rhododendron hybridum and showed distinct tissue- and stage-specific expression, including a group ( RsGSTF2 , RsGSTL7 , RsGSTU1 ) preferentially expressed in floral organs. Under defined light-quality treatments, GST transcripts in buds were rapidly induced by UV-A/blue light, whereas red/far-red light elicited stronger and more sustained induction in fully open flowers. Total petal anthocyanin content increased markedly under red and far-red light, broadly paralleling the expression of RsGSTF2 , RsGSTL7 and RsGSTU1 . Conclusions: Our integrative genomic and expression analyzes indicate that the GST family in Rhododendron is tightly connected to light signaling and floral development, and highlight flower-preferential, light-quality-responsive Tau and Phi members as candidate vacuolar anthocyanin transporters for light-dependent flower color formation.

Background Glutathione S-transferases (GSTs) mediate vacuolar sequestration of anthocyanins and are key determinants of flower color. Several Phi (GSTF) and Tau (GSTU) members have been linked to anthocyanin transport, but how GST repertoires in woody ornamentals respond to light quality is largely unknown in Rhododendron . Results We identified 87 GST genes in the Rhododendron simsii genome and classified them into seven subfamilies, with Tau and Phi predominating. Segmental duplication was the main driver of family expansion, and most promoters carried multiple light-responsive cis-elements. Twelve representative RsGST homologues were mapped to Rhododendron hybridum and showed distinct tissue- and stage-specific expression, including a group ( RsGSTF2 , RsGSTL7 , RsGSTU1 ) preferentially expressed in floral organs. Under defined light-quality treatments, GST transcripts in buds were rapidly induced by UV-A/blue light, whereas red/far-red light elicited stronger and more sustained induction in fully open flowers. Total petal anthocyanin content increased markedly under red and far-red light, broadly paralleling the expression of RsGSTF2 , RsGSTL7 and RsGSTU1 . Conclusions Our integrative genomic and expression analyzes indicate that the GST family in Rhododendron is tightly connected to light signaling and floral development, and highlight flower-preferential, light-quality-responsive Tau and Phi members as candidate vacuolar anthocyanin transporters for light-dependent flower color formation.

The flower color of Rhododendron is primarily determined by anthocyanin biosynthesis, with cytochrome P450 CYP75 family members, particularly flavonoid 3′,5′-hydroxylase (F3′5′H), playing a central role. However, the composition and functional characterization of CYP75 genes in Rhododendron remain insufficiently explored. This study performed genome-wide identification of the CYP75 gene family using the Rhododendron simsii reference genome and functionally characterized the corresponding F3′5′H homolog cloned from Rhododendron × hybridum petals (red cultivar and pink cultivar). Seven RsCYP75 genes were identified, categorized into two subfamilies: RsCYP75A (A1–A5) and RsCYP75B (B1–B2), with a prominent cluster on chromosome 13. All encoded proteins contained a conserved cytochrome P450 domain and typical heme-binding motifs. Among these, RhCYP75A2 showed the highest expression level in red petals at full blooming period and was designated as RhF3′5′H. RhF3′5′H encodes a basic membrane protein with the characteristic F3′5′H motif, with its transcript most abundant in flowers. Transient overexpression of RhF3′5′H in red R. × hybridum petals resulted in a 9.74-fold increase in its transcript levels and a 1.25-fold increase in anthocyanin content compared to that in the control accompanied by the up-regulation of CHS, F3H, DFR and ANS. Conversely, RhF3′5′H silencing reduced anthocyanin accumulation but increased CHS and F3H transcript levels, suggesting a compensatory transcriptional response in the upstream anthocyanin pathway. Moreover, RhF3′5′H was heterologously expressed in E. coli Rosetta as an MBP fusion protein, purified, and identified by LC-MS/MS and ELISA. The protein showed the ability to promote anthocyanin accumulation. Molecular docking analysis demonstrated that RhF3′5′H can bind to naringenin and dihydrokaempferol. These results confirm that RhF3′5′H is a functional F3′5′H-type CYP75A enzyme and a positive regulator of anthocyanin accumulation in Rhododendron petals. This work enriches the CYP75 gene catalog in Rhododendron and provides candidate genes for future studies on flower color regulation and molecular breeding.