| Ginkgo biloba L.,a perennial tree of Gymnospermae,which is an important economic tree species in China.Leaves of G.biloba contain many secondary metabolites such as flavonoids and terpenoids,which have important medicinal value.Because of the high content of medicinal active ingredients in the leaves of young ginkgo trees,Leaf-Oriented ginkgo is often cultivated by sowing.Generally,the leaves of 1-4 years old ginkgo trees can be used as raw materials for medicine.With the increase of tree age,the medicinal active ingredients in leaves decrease significantly,leading to the deterioration of leaf quality.Therefore,it is of great significance for the development of ginkgo industry to explore the accumulation of medicinal active ingredients in ginkgo leaves at different ages.In this study,1-7 years old ginkgo leaves were used as experimental materials.Through observing the plant growth trend,leaf morphology and structure,as well as measuring the content of main metabolites in leaves,the growth and accumulation rules of secondary metabolites of different age trees were determined.Through transcriptome and metabonomics analysis,we further explored the molecular regulation mechanism of secondary metabolite accumulation in leaves of ginkgo trees at different ages.The main results are as follows:(1)By measuring the growth of ginkgo trees of different ages,we found that the growth rate of 1-2 years old ginkgo trees was relatively slow,with the characteristics of short plant,less branches and shallow root distribution.At the fourth year,the main stem of the plant was significantly thickened and the height of the plant was up to 2 m,but the stratification between the trunk and the crown was not obvious.After the fifth year,the plant entered the rapid growth period,and grown to more than 4 m,the crown of the tree was concentrated in the middle and upper part,which was clearly distinguished from the trunk,showing the shape of an arbor.These results showed that the seedlings of ginkgo were saplings in 1-4 years and the plants were relatively short,from the 5th year the plant with tall tree body began to enter the rapid growth period.(2)The leaf shape of ginkgo changed obviously with the increase of tree age.1-4 years old ginkgo trees were with larger leaf area,heavier fresh leaf weight and more splits.In particular,the juvenile characteristics of the leaves were most obvious in 2-3 years old plants.However,the leaves of ginkgo trees began to become smaller and thinner,the number of leaf splits also decreased or even disappeared after 5 years.In addition,SEM observation showed that the epidermis of young ginkgo leaves was slightly wrinkled,and the cells were closely arranged.With the increase of age,the cell arrangement became loose and the stomata on the lower epidermis gradually caved in and were covered by the surrounding subsidiary cells.(3)The contents of flavonoids and terpenoids in the leaves of ginkgo trees at different ages were analyzed.It was found that the contents of flavonoids and terpenoids were higher in the leaves of ginkgo trees under 5 years old.Among them,the total flavonoid content of 1-2 years old ginkgo leaves reached 32.4±1.96 mg/g and 40.96±3.9 mg/g,respectively,which was more than twice that of 7 years old ginkgo leaves.The content of flavonol glycoside also presents the same trend.Total flavonol glycoside in 1-year old ginkgo leaves was 9.88±0.27 mg/g,which was significantly higher than that of 4 years old and 7 years old ginkgo leaves,and the difference of quercetin content was the most obvious.The content of quercetin in 7 years old ginkgo leaves was 38.7%lower than that of 1 year old.These results indicated that flavonoids and terpenoid lactones were more accumulated in 1-4 years old leaves of ginkgo,while the accumulation of active components in leaves decreased significantly when the trees entered the rapid growth phase.(4)Furthermore,the components of the leaves at 1,4 and 7 years ginkgo trees were analyzed by widely targeted metabolomics.A total of 11 different metabolites,including flavonoids,lipids,amino,organic acids were identified,among which flavonoids accounted for the largest proportion,82%of flavonoids showed a decreasing trend in content with the increase of tree age.In particular,the difference of flavonoid metabolite content between 1 and 7-year-old ginkgo leaves was the most significant.57 flavonoids were discovered altogether,including flavonols,flavonoid,flavanols,isoflavones,anthocyanins,biflavones in 7 categories.The contents of them decreased significantly except for 5 substances.In addition,nine terpenoid differential metabolites were identified,the contents of most terpenoids differential metabolites were the highest in the 4-year-old ginkgo leaves.(5)Transcriptome sequencing was performed on the leaves at 1,4 and 7 year ginkgo trees 26 differentially expressed genes(DEGs)were enriched in the phenylpropanoid biosynthesis pathway.21 of these genes had the highest gene expression in 1-year-old trees and were down-regulated in 4,7 year trees.There were 27 DEGs enriched in the flavonoid biosynthesis pathway,among them,the expression of 21 genes decreased with the increase of tree age,especially FLS,which is closely related to kaempferol and quercetin synthesis,9 flavonoid synthesis related DEGs were selected to verify by qRT-PCR and the results were consistent with the results of RNA-Seq.Metabolome and transcriptome conjoint analysis showed that down-regulation of key structural genes in the flavonoid synthesis pathway might be the main reason for the decrease of flavonoid with the increase of age.(6)20 different photosynthesis-related genes were identified,and most of them tended to be down-regulated with the increase of age.The expression levels of 15 differential genes identified in chlorophyll synthesis pathway were all down-regulated with age,while two chlorophyll degradation related genes were highly expressed in 1-year-old leaves.By analyzing three categories of sugar related metabolic pathways,we found that most of the DEGs were also down regulated with the increase of age.These results indicated that chlorophyll synthesis efficiency in young leaves of Ginkgo biloba was higher,photosynthesis was stronger,and primary metabolites of sugars were more produced,which may also promote the accumulation of secondary metabolites in young leaves.(7)12 DEGs related to JA synthesis were identified in hormone synthesis and signaling pathways,of which 11 genes were down-regulated with increasing age.In addition,11 DEGs involved in SA synthesis also showed the same expression trend.These stress-related hormone expression trends indicated that young ginkgo trees had weaker ability to resist adversity and more stress-related hormones were synthesized,which may be one of the factors promoting the accumulation of secondary metabolites such as flavonoids in young trees. |
