| Lotus(Nelumbo nucifera Gaertn),belong to Nelumbonaceae and Nelumbo,is a perennial economic crop with ornamental value,which is both medicine and food.The various tissues of lotus can be used as medicine and have different medicinal effects.Among them,lotus seed,lotus plumule,Nelumbinis receptaculum,Nelumbinis stamen,lotus leaf and lotus rhizome are all Chinese medicines,which also included in the Chinese Pharmacopoeia.Different medicinal tissues of lotus have different pharmacological activities.It is reported in the literatures that the alkaloids and flavonoids contained in lotus are the most important active ingredients present in various tissues,among which benzyl isoquinoline alkaloids have a wide range of pharmacological activities.However,it has not been reported whether the metabolism of these alkaloids during the growth of lotus are related to the functional differentiation of different tissues of lotus.Therefore,it is necessary to study the biosynthetic pathways and metabolism of these alkaloids in plants,which can provide a reference for revealing the material basis of "homologous and different effect".In addition,the flavonoids in lotus are closely related to flower color.However,the correlation between flavonoids and flower color phenotype is still unclear,and the composition and distribution of flavonoids in lotus need to be further studied.In this article,focusing on the active ingredients of alkaloids and flavonoids in lotus,the metabolomics analysis method of ultra-high performance liquid and quadrupole tandem time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS)was applied to explore the metabolic differences of its medicinal components in different types of tissues,and further deduce their biosynthetic pathways.The main results and conclusions of the study are as follows:1.Based on the morphological characteristics,the metabolic accumulation research of the apophine alkaloids in the different developmental stages of the lotus leaf and the different developmental time points of the lotus leaf maturity stage was conducted.The results showed that with the growth and development of lotus leaves,the content of apophine alkaloids in lotus leaves showed an upward trend,and the content of alkaloids in different stages was significantly different,indicating that the accumulation of apophine alkaloids in lotus leaves was influenced by the growth and development of lotus leaves.When the lotus leaf was fully expanded,the yield of alkaloids from the lotus leaf was the highest,and the content of apophine alkaloids in lotus leaves tended to be stable during maturity.The research results can provide a basis for determining the most suitable harvest stage of lotus leaves.In addition,the content of alkaloids in lotus leaves of different varieties and genotypes was quite different,suggesting that genetic background is an important factor affecting the metabolism and distribution of alkaloids in lotus leaves.The experimental results revealed the changes in the metabolic accumulation of apophine alkaloids in the lotus leaf during the growth and development of the lotus leaf.These findings will help clarify the biosynthesis and regulation of alkaloids in lotus leaves at the entire plant level,and lay a solid foundation for further research on the metabonomics of the effective components of lotus.2.Based on the bisbenzylisoquinoline alkaloids(liensinine,isoliensinine and neferine),the main alkaloid components in the lotus plumule,the alkaloid components in the 155 sequenced varieties of lotus plumule were analyzed for the first time,and the alkaloids in the upper embryo and hypocotyl of lotus plumule were investigated respectively.The results showed that the total alkaloids in the upper embryo of lotus plumule were about 2 to 3 times that of the total alkaloids in the hypocotyl,but the content distribution of alkaloids in the two parts of the upper embryo and hypocotyl were similar,indicating that the lotus plumule don’t need to be divided into upper and lower parts in clinical application.Lotus plumule can be used as a whole to ensure the efficacy,saving time and effort and avoiding waste of resources.The experimental results provided a scientific theoretical basis for the better clinical application of lotus plumule.In addition,among 155 different lotus varieties,the alkaloid content was significantly different.Cultivar 122(rhizome lotus"SZO")had the highest total alkaloid content(997.25 ug/g),while cultivar 103(flower lotus "JLD")had the lowest total alkaloid content(322.73 ug/g).3.Based on apophine alkaloids(N-nornuciferine,O-nornuciferine,annonine,nuciferine and roemerine),the main alkaloids in lotus leaves,the alkaloid composition of 155 different sequenced varieties of lotus leaves at the maturity stage was analyzed and studied,and the results found that the content of lotus leaf alkaloids in different varieties was significantly different,and the overall variation range of lotus leaf total alkaloids was 181.72 ug/g-3304.58 ug/g.In most varieties,nuciferine and N-nornuciferine are the most important alkaloid compounds.Judging from the content of alkaloids in each variety,cultivar 121(rhizome lotus "SLH1"),cultivar 142(seed lotus "XFR2"),cultivar 56(flower lotus "ZGGDL"),and cultivar 23(flower lotus"SN")are the preferred materials for studying apophine alkaloids in lotus.4.Correlation analysis of the alkaloid components in lotus plumule and lotus leaves of 155 different sequenced varieties was performed.The results showed that the total alkaloids of lotus leaf and the total alkaloids of lotus plumule were not significant(P>0.05),but the total alkaloids of lotus leaf,the content of nuciferine and N-nornuciferine were all negatively correlated with liensinine,and positively correlated with isoliensinine(P<0.01),indicating that the biosynthetic pathway of N-nornuciferine and nuciferine may be in a homologous competition relationship with that of liensinine.The analysis of the correlation between the alkaloids in the lotus plumule and the lotus leaf provided a reference for further deriving the biosynthetic pathway of alkaloid compounds in lotus,and also provided a scientific theoretical basis for further exploring the regulation mechanism of alkaloid biosynthesis and breeding lotus clutivars with high alkaloid content.5.The difference in alkaloids in lotus leaf and lotus plumule showed that its synthesis and accumulation in the whole lotus plant had tissue specificity.Thus,UPLC-Q-TOF-MS/MS metabolomics techniques was applied to characterize as many alkaloid compounds in lotus leaves and lotus plumule as possible,and for the first time identified and analyzed the alkaloids in lotus milky sap,to gain more in-depth understanding of the metabolic rule of the alkaloids.Results showed that a total of 43 alkaloid compounds were identified,including 27 species in lotus leaf,29 species in lotus plumule,and 30 species in lotus milky sap.And there were 16 kinds of alkaloids identified in lotus for the first time,including norcoclaurine isomer,3’-hydroxy-N-methylcoclaurine,4’-O-methylarmepavine,(S)-reticuline,nuciferine-O-methanol,betaine,N-benzylidenemethylamine,N-oleoylethanolamine,tyramine,4-hydroxymandelonitrile,L-tyrosine,trigonelline,vasconine,dopamine,serotonin and tryptophan.Most of these alkaloids in lotus possess good biological activity,laying a foundation for the further development and utilization of lotus alkaloids.Besides,the alkaloids in different tissues of lotus was further compared.It was found that the alkaloids in the lotus milky sap had a higher similarity with the ingredients in the lotus plumule,especially the higher content of bisbenzylisoquinoline alkaloids.And we further deduced the biosynthetic pathway of the alkaloids in lotus,combined with the structural characteristics of the lotus plant,we believed that the lotus milky sap may act as a bridge.Due to the effects of various enzymes in plants and the differences of external environments,the alkaloids synthesized in different plant tissues had distinctive structures,which resulted in the variety of the chemical composition in different plant tissues,and thus resulted in different medicinal effects.The derived lotus alkaloid synthesis pathway diagram greatly enriched our understanding of the metabolism and biosynthesis of lotus alkaloids,and provided a scientific theoretical basis for further studying the metabolic flux of alkaloids in the whole lotus plant.However,the metabolic pathways of alkaloids in lotus still need to be studied in depth.Next,we will combine the association analysis of the transcriptome and the genome to mine the key functional genes in the lotus alkaloid biosynthesis pathway.6.Research on the correlation between flavonoids and flower color phenotypes of lotus was performed.And a method that can simultaneously detect anthocyanins and flavonols within 25 minutes was established,which was highly sensitive and stable,and greatly shortened the separation time,with great accuration and efficient.In addition,UHPLC-ESI-QTOF-HRMSn technology was applied to simultaneously characterize 5 anthocyanin compounds and 18 flavonol compounds in lotus petals.Among them,4 flavonols were identified in lotus for the first time,namely quercetin 3-O-glucuropyranosyl-rhamnopyranoside,quercetin 7-O-glucoside,laricitrin 3-O-hexose,and laricitrin 3-O-glucuronide,respectively.What’s more,the flavonoids and their color parameters in 207 different varieties of lotus petals were statistically analyzed.The quantitative results of flavonoids in lotus petals showed that the content and composition of anthocyanins and flavonols in different varieties were quite different.The statistical results of the color parameters of 207 different lotus varieties showed that the brightness of yellow and white varieties was higher,and the saturation of purple-red varieties was higher.The correlation analysis of the color parameters,anthocyanin content and flavonol content of different varieties of lotus showed that there was a certain correlation among the three ones.A significant correlation was observed among different color parameters,and color parameters were also significantly related to most flavonoids.For example,anthocyanins were significantly negatively correlated with L*,b*and CI values(P<0.01),but positively related with a*,C*and h values(P<0.01).Besides,most flavonols are negatively correlated with a*,C*and h values,but positively correlated with L*,b*and CI values.Multiple linear regression results showed that reducing the content of total anthocyanins and increasing the content of Myr-3-G1cA would make the petals lighter and brighter;increasing the content of total anthocyanins and Kae-3-Rut and decreasing the content of Qc-3-Sam and the total flavonols would make the petals red;while increasing the content of Qc-3-Neo,Myr-3-G1cA,and Syr-3-G1cA and reducing the content of Pn-3-Glc and Syr-3-Hex would make the petals yellow.According to the experimental results,the biosynthetic pathway of flavonoids in lotus was further deduced,and candidate genes were inferred based on mGWAS technology.The research results provided a scientific basis for studying the relationship between flavonoids and lotus petal color,and exploring the molecular mechanism of lotus petal coloring.These findings will also promote our understanding of the biosynthesis of flavonoids and provide a theoretical basis for the development of lotus petals as natural sources of anthocyanins and flavonols.In the later stage of the study,the predicted candidate genes will be verified by molecular biology based on the results of mGWAS. |
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