OAGL6 and PaSEP1/3 cooperate in SP and L complexes of P code model to specify perianth identity in Phalaenopsis orchids.

The orchid perianth typically consists of three sepals and three petals, with the median petal transformed into a distinct structure lip. In our previously found P code model in orchids, A/E protein OAGL6 interacting with B proteins to form L (OAGL6-2/OAP3-2/OPI) and SP (OAGL6-1/OAP3-1/OPI) complexes in determining lip and sepal/petal identities. This study demonstrates that OAGL6-1 and OAGL6-2 function redundantly with the E-class genes PaSEP1/3 in regulating sepal/petal and lip identities. Single-virus-induced gene silencing (VIGS) of OAGL6-1 resulted in mild morphological alterations in sepals/petals, while silencing PaSEP1 or PaSEP3 individually caused little to no effect. However, triple silencing of OAGL6-1/PaSEP1/PaSEP3 led to additive and severe phenotypes, including greenish sepals/petals with leaf-like epidermal features, indicating a collapse of the SP complex. Similarly, triple silencing of OAGL6-2/PaSEP1/PaSEP3 resulted in the transformation of the lip into a leaf-like structure, demonstrating their redundant roles in lip specification through the advanced L complex. Protein interaction assays confirmed strong interactions between OAGL6-1/2 and PaSEP1/3, supporting the formation of novel, advanced SP and L complexes. Additionally, PaSEP1 and PaSEP3 contribute to pedicel abscission and anthocyanin pigmentation, although their effects are weaker compared to OAGL6-1. Collectively, these findings highlight OAGL6-1 and OAGL6-2 as central regulators of perianth identity through interactions with PaSEP1/3 in the SP and L complexes, respectively. PaSEP1/3 function as minor yet essential partners that help maintain distinct floral organ identities and prevent reversion to leaf-like characteristics. This work expands the P code model and deepens our understanding of the unique mechanisms governing perianth specification in orchids.