Fluorescence lifetime imaging of sporopollenin: An alternative way to improve taxonomic level of identifying dispersed pollen and spores

Abstract

Spores/pollen records abundant information of biodiversity, evolution, and climate condition. The exine of spores/pollen is mainly composed of sporopollenin, which is chemically stable over geological time. Morphological study of the exine is a classical method used in palynology. In this study, we developed a fluorescence lifetime imaging (FLIM) method to investigate the numbers and lifetimes of fluorophores in sporopollenin. We used 20 pollen grains of each species to study sporopollenin FLIM. The sporopollenin was purified using the acetolysis method. Laser scanning confocal microscopy (LSCM) was used to study the autofluorescence of sporopollenin. The sporopollenin FLIM of pollen grains collected in different years/geographical populations showed no significant difference. Three fluorophores were estimated in the sporopollenin of all the tested plants, including angiosperms (Oryza sativa and Zea mays, Brassica napus and Arabidopsis thaliana), gymnosperms (Cryptomeria fortune and Metasequoia glyptostroboides), ferns (Lygodium japonicum and Ceratopteris thalictroides), and lycophyte (Lycopodium clavatum). Quantitative analysis demonstrated that the lifetimes of fluorophores varied in different species, showing that FLIM provides additional information to distinguish spores/pollen. Further analysis showed significant differences in sporopollenin fluorescence lifetimes between morphologically similar pollen grains of cultivated crops and their wild relatives of the genera Oryza and Triticum. These results show that sporopollenin FLIM offers a new dimension of information on spores/pollen. This new technique has a big potential to fill a gap for traditional morphological comparison to identify dispersed fossil pollen and spores.