Development of semi-dwarf Echinacea purpurea by inducing silencing of the endogenous brassinosteroid- biosynthetic gene DWF4

Brassinosteroids (BRs) are plant steroidal hormones that are crucial for plant growth and development, with dwarfism being a hallmark of BR deficiency. DWARF4 (DWF4) is a key enzyme that catalyzes the rate-limiting C22 oxidation step in the BR biosynthetic pathway. Understanding its role in ornamental plants is crucial for developing dwarf varieties that can significantly enhance their aesthetics and broaden their applications. This study aimed to provide the functional analysis of the DWF4 gene isolated from Echinacea purpurea . We found that the EpDWF4 gene comprised a 1458 bp open reading frame that encodes 486 amino acids and is highly expressed in leaves. EpDWF4 showed high homology to AtCYP90B1, a steroid 22-alpha-hydroxylase involved in the BR biosynthetic pathway in Arabidopsis thaliana . Transgenic E. purpurea plants with suppressed EpDWF4 expression were generated using an RNA interference vector that produced hairpin-looped double-stranded RNAs, leading to sequence-specific RNA silencing of EpDWF4 . This resulted in a dwarf phenotype. The physiological function of EpDWF4 was evaluated by complementation in Arabidopsis plants. EpDWF4 completely rescued the extremely dwarf phenotype of the A. thaliana dwf4-102 mutant. In addition, transgenic homozygous A. thaliana plants containing a copy of 35S: EpDWF4 in the intergenic region significantly increased in height. Moreover, steroid 22-alpha-hydroxylase activity, which converts campestanol to 6-deoxocathasterone, increased by 43% in these transgenic plants compared to wild-type A. thaliana . Our results indicate that the knockdown of BR biosynthesis-related genes by RNA interference (RNAi) supports valuable dwarf plant production for commercial horticultural or agricultural applications.