The Structure,composition And Function Of Spore/pollen Wall

Haploid gametophytes-spores,which produced by meiosis,are a major means of the reproduction and dispersal of spore bearing plants.Pollen are the homologues of spore in seed plants.Pollen also delivers male gametes to the stigma,which is essential for sexual reproduction.Spore/pollen are also the critical innovations contributing to the colonization of land by plants.Compared to their aquatic ancestors,spore/pollen of land plants face a wide range of threats,particularly desiccation,ultraviolet(UV)radiation and wide fluctuations of temperature.Spore/pollen have evolved some special defense mechanisms,including the special wall and the secondary metabolites.Although the research on the structure and chemical composition of spore/pollen has lasted for a hundred years,little is known about its protective mechanism as the lack of systematic chemical analysis,functional verification and special wall structure observation.In previous studies,we show that the pollen wall can be subjected to thioacidolysis.The phenylpropanoid dervatives and naringin were identified in the resulting lysate by 2D NMR analysis.Genetic evidence of the phenylpropane synthetic mutants in Arabidopsis showed that phenylpropanes are essential for the formation of the sporopollenin wall and provided UV-B protection.In this study,we analyzed whether land plants resist the terrestrial UV-B radiation by accumulating phenylpropanes in spore/pollen wall.In addition,flavonoids,another kind of substance in the degradation product of sporopollenin,were also analyzed,including the composition,distribution,and function.Through the work above,the chemical basis and molecular mechanism of spore/pollen adaptation to the terrestrial environment were clarified.In addition,based on our research on the chemical composition of different wall layers of spore/pollen,we screened the specific fluorescent labeled dye solution and established the corresponding microscopic observation system.The specific results are as follows:(1)In previous studies,we showed that phenylpropanes were identified in the degradation products of sporopollenin,and it was the critical substances for the formation of the spore/pollen wall.Phenylpropanes can protect pollen from UV-B damages by converting the UV-B into visible light.Further,we explored whether this UV resistance mechanism is the key factor for spore/pollen adaptation to the terrestrial environment.Firstly,we found that the content of phenolic substances in pollen wall of Arabidopsis ecotypes was significantly positively correlated with its UV resistance.Then,the spore/pollen of 69 common bryophytes,ferns,and seed plants in East China were analyzed.Our results reveal that the content of phenols in the sporopollenin wall of seed plants is the largest,with intermediated pteridophytes,and the least bryophytes.In seed plants,species that flower during high solar UV irradiation period show significantly higher phenolic level than other species.Thus,we propose that the phenolics in the sporopollenin wall may protect spore/pollen from UV-B damage,and facilitate plant adaptation and evolution in terrestrial environments.The genes involved in the phenylpropanoid phenolic synthesis pathway may be an important target of natural selection for the UV-B defense in spore/pollen.(2)The flavonoid – Naringin was also identified in the degradation products of sporopollenin.Metabolic analysis revealed the distribution pattern of flavonoids in pollen.Further,functional analysis shows that: the ultraviolet absorbing substancenaringin accumulates in the pollen wall,and cooperates with the phenolic substances in the sporopollenin to prevent UV-B radiation from entering the pollen protoplasm;while protoplasmic flavanols eliminate ROS accumulation further enhancing the resistance of pollen or spore to environmental stress.The glycoside modification of flavanol reduces its antioxidant activity and enhances its water solubility,which is accumulated in the pollen protoplasm as a reserve resource.Besides that,naringin was the only flavonoid detected in spores of bryophytes;Flavonol was detected in the spores of pteridophyte Lycopodium;Monoglycoside-modified flavonols appear in gymnosperms pollen;The types of flavonol and glycoside modification in angiosperm pollen become more diversified.These results indicate that the reactive oxygen species explosion caused by high temperature may be another important stress faced by land plants.The appearance of flavonol and glycoside modification endows land plants with the ability to cope with high temperature stress in the process of longdistance transmission.(3)At present,the structure of spore/pollen wall are imaged by electron microscopy.However,it is limited to distinguish the different layers of spore/pollen wall,and is not possible to perform living observation.In this study,based on the chemical differences of different layers of spore/pollen,we screened fluorescent dyes and explore the dye combinations that can simultaneously stain the different layers.We finally established the co-staining method of Sexine & Intine and Sexine & Pollen coat.And it can be widely used for the spore/pollen wall observation in spore plants and seed plants.The excitation and emission spectra of the dye,which used in this costaining method,do not have crosstalk.And these dyes are compatible with fluorescent protein GFP/YFP.The co-staining of Sexine & Pollen coat revealed very small cavities in the exine both in the Oryza sativa and Zea mays,and the pollen coat was filled in the cavities.Further,the pollen grains of GRP19-GFP transgenic plants were co-stained with the Sexine & pollen coat dye combination.GRP19 protein form granular structure that is peripheral to the pollen coat lipids on the pollen surface.These results show that this method can be widely used for uncovering the structure of the pollen coat and investigating the mechanism underlying the interaction between pollen coat and stigma.In addition,a dye combination of Sexine & Intine was established.The Sexine and Intine can be labeled specifically,while Nexine can be recognized by negative staining.Dyeing and chemical analysis show that Sexine and Nexine are two layers,which have different physical and chemical properties.These results show that the staining method can accurately and sensitively identify the different layer of spore/pollen,and it will become an important tool in the study of palynology.In summary,on the one hand,we found the important protective substances(phenols and flavonoids)for spore/pollen to resist the terrestrial environments.The relevant results can be used to guide the adjustment of pollen stress resistance according to demand in agricultural production,including enhancing the stress resistance of crop pollen to improve yield or create related male sterile lines.On the other hand,we screened and established a specific fluorescent labeling system,which can mark the different layer of spore/pollen.Using this method,we can get a full view of the different layers of spore/pollen wall with the help of optical sectioning and 3D reconstruction technology.This method provides a new and important tool for palynology research.