Liquid Profiling in Plants: Identification and Analysis of Extracellular Metabolites and MiRNAs in Pollination Drops of Ginkgo Biloba

The pollination drop (PD), also known as an ovular secretion, is a critical feature of most wind-pollinated gymnosperms and function as an essential component of pollination systems. However, the metabolome and small RNAs of gymnosperm PDs are largely unknown. We employed gas chromatography-mass spectrometry to identify a total of 101 metabolites in Ginkgo biloba L. PDs. The most abundant metabolites were sugars (45.70%), followed by organic acids (15.94%) and alcohols (15.39%) involved in carbohydrate metabolism, glycine, serine and threonine metabolism. Through pollen culture of the PDs, we further demonstrated that the metabolic components of PDs are indispensable for pollen germination and growth; in particular, organic acids and fatty acids play defensive roles against microbial activity. In addition, we successfully constructed a small RNA library and detected 45 known and 550 novel miRNAs in G. biloba PDs. Interestingly, in a comparative analysis of miRNA expression between PDs and ovules, we found that most of the known miRNAs identified in PDs were also expressed in the ovules, implying that miRNAs in PDs may originate from ovules. Further, combining with potential target prediction, degradome validation and transcriptome sequencing, we identified that the interactions of several known miRNAs and their targets in PDs are involved in carbohydrate metabolism, hormone signaling and defense response pathways, consistent with the metabolomics results. Our results broaden the knowledge of metabolite profiling and potential functional roles in gymnosperm PDs and provide the first evidence of extracellular miRNA functions in ovular secretions from gymnosperms.