Abstract
BACKGROUND: Dendrobium species are prized for their ornamental and economic value, yet their low hybrid compatibility remains a critical challenge, potentially linked to stigma recognition mechanisms. The stigma, as the primary site for pollen interaction, undergoes dynamic developmental changes that lay the foundation for successful pollination. However, the molecular mechanisms governing stigma maturation and subsequent pollen recognition are poorly characterised. This study systematically investigated the transcriptomic, phenotypic, and physiological dynamics of Dendrobium stigma development to establish a mechanistic basis for understanding pollen-stigma recognition. RESULTS: Morphological observations of the four developmental stages of stigma maturation (day 1 post-anthesis [CK], early anthesis [T1], mid-anthesis [T2], and late anthesis [T3]) and investigations into the optimal receptivity window revealed that stigmatic mucilage secretion and viscosity peaked during the mid-anthesis phase (T2). Concurrently, stigma receptivity was highest at this stage, as evidenced by robust pollen tube elongation and ovarian enlargement. Enzyme activities linked to stigma-pollen recognition—esterase, superoxide dismutase (SOD), and peroxidase (POD)—demonstrated stage-specific dynamics, with peak activity occurring during mid-anthesis (T2). Transcriptome analysis revealed that a total of 13,209 (T1), 12,557 (T2), and 18,593 (T3) Differentially expressed genes (DEGs) were identified, predominantly enriched in plant hormone signal transduction, MAPK signaling, and plant-pathogen interaction pathways. Transcription factors (TFs) and KIN-class genes exhibited coordinated regulation of these pathways. During the peak pollination period (T2 stage), a total of 3,069 stage-specific differentially expressed genes (DEGs) were identified that were not observed in other developmental stages. Among these unique DEGs, one was found to encode superoxide dismutase (SOD) enzymes, while seventeen encoded esterases. Reactive oxygen species (ROS) levels peaked at T2, potentially implicating their involvement in MAPK signaling through Ca²⁺-CNGCs-CDPK-Rboh cascades, though this proposed mechanism requires experimental validation. O-GlcNAc glycosylation was detected in the stigma transcriptome for the first time, potentially linked to kinase activity and immune responses. CONCLUSIONS: Developmental and physiological profilings suggest that mid-anthesis represents a critical period for active pollen-stigma recognition processes, potentially driven by dynamic biochemical and transcriptional adaptations in the stigma. TFs, KIN-class genes, and ROS interactively regulate stigma maturation and pollen recognition through hormone signaling, MAPK cascades, and pathogen-like defense pathways. Key candidates, including MPK6, MYC2, ERF1, and Pti1/5, were hypothesized as critical regulators. However, functional validation of these genes and pathways remains absent, and the study’s reliance on unpollinated stigmas limits insights into pollination-specific responses. Further experimental validation is required to confirm mechanistic interactions.