B- and E-Class MADS-Box Transcription Factors Regulate the Formation of the Labellum in Cymbidium ensifolium.

The labellum, a distinctive floral organ unique to orchids, possesses significant ornamental and research value. Here, wild type plants (W1, W2), a lip-like sepal mutant (MS), a lip-like petal mutant (MP), and a peloric flower mutant (ML) of Cymbidium ensifolium were used to elucidate the molecular mechanisms underlying labellum formation. Morphological and cytological analyses revealed that MS sepals and MP petals acquired labellum-like traits (folded structures, conical papillae), whereas ML labella adopted petal-like features (flat epidermal cells). Transcriptome analysis identified seven key B- and E-class MADS-box genes (including DEF-/AP3-, SEP-, and AGL6-like genes) potentially involved in labellum development. Subsequent qRT-PCR profiling showed that gene expression dynamics closely reflect organ fate. Expression of CeAP3-3 and CeAP3-4 correlated with the establishment of inner perianth identity (petal/labellum), while CeAGL6-2 activation was specifically associated with labellum specification. Notably, CeAGL6-2 was ectopically expressed in lip-like organs of MS and MP, but absent in the petaloid labellum of ML. Conversely, expression patterns of CeAP3-1 and CeAGL6-1 suggested roles in promoting sepal/petal or non-labellum perianth fates. Protein interaction assays (Y2H, BiFC) demonstrated that CeAP3-3 interacted strongly with CeAGL6-2 and CeSEP2, while CeAP3-4 interacted with CeSEP2. Integrating these results, we propose a model in which heteromeric complexes formed by CeAP3-3, CeAGL6-2, and CeSEP2 are central to specifying labellum identity in C. ensifolium. Overall, these findings highlight the cooperative role of B- and E-class transcription factors in labellum specification through dynamic expression shifts and protein interaction networks, thereby enriching our understanding of the molecular mechanisms driving orchid labellum formation.