HetSI Mechanism Of The Heteromorphic Plant Plumbago Auriculata Lam. By Untargeted Metabolomics

Plumbago auriculata Lam.native to southern Africa,it is now imported in many places in China,with good adaptability.The unique light blue flowers have excellent ornamental value and deeply loved by the consumer market.Plumbago auriculata is a typical plant with two different morphs:long-styles and short-styles.Heterostyly is a genetic polymorphism while heterostyly plants are usually associated with self-incompatibility(SI)acts as a safeguard against self-fertilization and inbreeding depression.The self-incompatibility mechanism comprises the homomorphic(Hom SI)and the heteromorphic(HetSI)self-incompatibility,discovered in Darwin’s time.Indeed,there is little evidence established the metabolic mechanism of the HetSI.Although previous studies on the HetSI mechanism have made some progress,there are still deficiencies,the HetSI mechanism still needs to be supplemented by more experimental evidence of heteromorphic plant from different levels.For instance,the contribution of stigma-stamen polymorphism to fertilization disorder in the HetSI is still controversial and the metabolites that directly or indirectly cause the incompatibility reaction are not clear which leading to a lack of evidence at the metabolomics level.Previous metabolomics studies on the Hom SI reaction have shown that the lack of energy may be one of the causes of fertilization failure.Then,is the HetSI mechanism also related to the lack of energy? To address the problem,this study combined morphological evidence with UPLC-MS/MS and GC-MS to analyze the main metabolites involved in the HetSI mechanism and their correlation with the HetSI reaction from the level of metabolomics,in order to supplement the evidence of the HetSI mechanism.The main results and conclusions of the study are as follows:1.P.auriculata has reciprocally arranged female and male reproductive organs,dense clusters of papilla cells on styles and protuberant pollen ornamentation are the factors that promote cross-pollinations.(1)P.auriculata has long and narrow flower tube and reciprocally arranged female and male reproductive organs,which can effectively promote pollen transfer and cross-pollinations.(2)Pollens and styles of different morphs showed distinct characters.The stigmatic papillaes on the S style are more numerous has a higher density on the claw structured stigmas,which are more beneficial to grasp pollens.(3)P.auriculata has olive-like pollens.The L pollens have dense verrucous structures,each verrucous structure is born with small bumps.However,the verrucous structures on the S stigma pollens are relatively rounder.2.The concentration of main metabolites in the S style have higher levels than that in the L styleMetabolic results showed that higher levels of amino acids,flavonoids,other organic acids,carbohydrates and glucosides in the S styles(based on UPLC-MS/MS).Abundant energy-related nutrients accumulate in the S style,providing enough energy for faster pollen germination and pollen tube growth.SC pollens had a higher germination rate and faster growth rate on the S style.3.The key time node of the HetSI mechanism of P.auriculata was 2 h after pollinations,and no pollen tube germination was observed on the surface of stigma after SI pollinations.(1)According to fluorescence microscope(FM)observation,pollen tube germination was observed about 2 h after cross-pollinations,indicating that identification between pollen and stigma had been completed before this time point.4 h later,the pollen tubes extended into the stigmas,8 h later,pollen tubes were successfully reached the ovarys and completed the whole fertilization process.Incompatible pollen was observed and no pollen tube was found germinated on the stigmas or even adhered on the stigmas.It is worth mentioning that the SC pollens on the S stigmas have a higher the germination rates,a faster growth rates and a higher efficiency of successful fertilizations,which is closely related to the sufficient supply of energy-related nutrients in the S styles.(2)Through scanning electron microscope(SEM)observations,a large number of "foot" structures were observed in the S×L pollinations which "flow" out of the germination groove and connected to the papillaes on the stigma,it is even not necessary for the germination groove to touch the stigmatic papillaes.However,in L×S pollinations,the connection mode between pollen and stigma was not the same,papillaes on stigma was directly connected to the germination groove on the SC pollens.Interestingly,the stigmatic papillae around the germinated pollen became less globular.While no similar structures mentioned above have been observed in the SI pollinations,the SI pollens on the stigmas never successfully rehydrated and germinated or even adhered on the stigmas.The experiment indicated that 2 h was the key time node for the HetSI mechanism and the HetSI mechanism was happened on the stigma surfaces,.This results provides an ideal sampling time for follow-up metabolomics studies.4.UPLC-MS/MS and GC-MS metabolites detection methods are feasible,with different focuses and complementary.(1)A total of 142 metabolites are detected by UPLC-MS/MS,while 53 metabolites are detected by GC-MS.The amount of metabolites detected by UPLC-MS/MS is larger.UPLC-MS/MS has a strong ability to detect secondary metabolites.Although the number of GC-MS detection is less,it has a strong ability to detect primary metabolites.The two methods complement each other.The compounds can be classified into amino acids,other organic acids,fatty acids and lipids,sugars and glycosides,flavonoids and other metabolites.(2)The statistical results show that there are significant differences between different pollinations(VIP>1 and P value<0.05).In the L×S and L×L comparison groups,there are 67 metabolites with significant differences in UPLC-MS/MS results,and 24 metabolites were significantly changed in GC-MS detection.In the S×L and S×S comparison groups,there are 67 metabolites that significantly changed that detected by UPLC-MS/MS,and 41 metabolites with significant difference in the GC-MS detection.5.The lack of energy nutrients is not the reason for the HetSI mechanism,and the specific identification of pollen-style on early stage may be one of the reasons for the fertilization failures.In SI pollination,metabolites related to energy supply were abundant,such as amino acids and carbohydrates.The lack of energy supply for pollen tube growth is not one of the reasons leading to the HetSI mechanism.The tricarboxylic acid cycle(TCA cycle)and glycolysis are the main carbon sources of plants that supply energy during the growth of pollen tubes.This fully indicates that insufficient energy supply do not leading the HetSI mechanism.6.The variation of pollen tube growth rate is one of the reasons cause the reverse trends of style-metabolites.The changes of style-metabolites of P.auriculata are complex,and the differences of germination and growth rates of the SC pollens on different types of styles may be one of the reasons for the diversity of metabolic trends.UPLC-MS/MS detect a large number of metabolites such as other organic acids and alcohols that show an opposite trend in the L×S and S×L pollinations.These changes may be closely related to the complex mechanism of plants and the rates of pollen germination and tube growth.Through the morphological study,the SC pollen germinations on the L stigmas are relatively delayed.After pollen hydration,enzymes became active through phosphorylation,enriched metabolites rapidly in to supply pollen autotrophy.However,the growth of non-autotrophic pollen tubes in S×L pollination may consume energy-related nutrients in the style which may cause the decline of metabolites.In conclusion,this paper discussed the characteristics of the HetSI mechanism of P.auriculata from the perspective of morphological structures and metabolomics.The conclusion is that P.auriculata is a typical HetSI plant,and the content of energy metabolites varies greatly with different types of styles.The HetSI mechanism of P.auriculata occurred in the stage of stigma-pollen identification,and the key time node for the occurrence was 2 h after successful pollinations.The lack of energy substances in the style may directly affect the germination and growth rate of pollen tubes,but the lack of energy-related nutrients is not one of the reasons for the HetSI mechanism.The HetSI mechanism of P.auriculata may be directly related to the stigma-pollen specific recognitions.