Juvenilization Promoting Flavonoid Accumulation In Ginkgo Biloba Leaves By Truncating

Ginkgo biloba L.,as a gymnosperm,is an important ornamental and economic tree species.The leaf extracts of G.biloba are the main raw materials of medicines for the treatment of cardiovascular and other diseases.The main effective ingredients are flavonoids and terpene lactones,and the amounts of these active substances in leaves are related to tree age.Generally,the content of flavonoids and terpene lactones in the leaves of young seedlings is higher than that of old trees.In this study,we truncated the 5-year-old ginkgo seedlings at the height of 10 cm,35 cm,60 cm,85 cm,and 110 cm,and found that the leaves were rejuvenated,and the accumulation of active components increased significantly.Therefore,we studied the leaves of truncated plants through cytological,physiological,molecular and bioinformatics technologies.The main results are as follows.?1?Morphologic observation showed that the leaf areas in the truncated treatment group were significantly increased,which was about 2-2.5 times compared to control group.The leaves were thicker about 1.4-1.6 times than that of the control group.Moreover,the leaf was deeply lobed to the leaf base with 5-6 lobes.In addition,the leaf petiole became shorter with a larger diameter,and developed more and stronger vascular bundles and conduction tissues.These results indicate that leaves after truncation showed obvious juvenile characteristics.?2?The results of physiological investigation showed that the chlorophyll SPAD value at the early stage?S1?of the treatment group was lower than that of the control group,while at the middle and later periods?S2-S5?,the SPAD value increased rapidly and was higher than the control group.Moreover,during the S5,the SPAD value of the treatment group continued to rise while the control showed a downward trend.The differences of Fv/Fm between the two groups were small.The Fv/Fm value of the truncated group increased continuously in the S5 whereas it decreased in the control group.Furthermore,ultrastructure observation showed that more chloroplasts distributed in the treatment group leaves at S4 compared with the control group.At S5,the leaf organelles in the control group began to degrade,especially in chloroplasts,with the distorted shape,thinner cell membrane,more osmophilic globules,accumulated starch granules,indicating the ageing mesophyll cells.However,in the treatment group,the leaf organelles are abundant with the intact chloroplast.These results indicate that control leaves began to aging with declined photosynthetic capacity and degraded chloroplast in S5,whereas the treatment leaves still retain health status and delayed aging.?3?During the harvesting period?S5?,the total flavonol glycosides of treatment leaves increased significantly?except for the 60 cm treatment group?,which was 1.17-1.20 times more than the control group.Among them,the quercetin content increased 1.18-1.63 times.The total terpene lactones content in the treatment group also showed a significant increase,especially in the 85 cm and 110 cm treatment groups?increased by 32%-34%?.The contents of ginkgolide A and ginkgolide B were 1.16-1.33 times and 1.76-1.89 times more than that of the control respectively,and the contents of ginkgolide C and bilobalide were significantly increased in the 85 cm and 110 cm treatment groups,indicating that the truncated treatments can effectively improve the accumulation of flavonoids and terpene lactones in leaves of G.biloba.?4?By the method of extensive targeted metabolomics,in total,66 differential metabolites were identified,of which most were flavonoids?including 10 flavonoids,13 flavonols,4 flavanones and 4 catechins and their derivatives,and 5 anthocyanins?.Additionally,the other major metabolites were organic acids,lipid glycerophospholipids,amino acids,nucleotides,etc.The KEGG enrichment analysis of the differential metabolite revealed that they were mainly enriched in secondary metabolite biosynthesis,amino acid biosynthesis,flavonoid biosynthesis,and flavonoids and flavonols biosynthesis.Among the flavonoids,except for the 5 flavonoids such as sakuranetin,all other substances showed higher expressions.These results indicated an increase in the content of most flavonoids in the leaves after truncating treatment.?5?The analysis of differentially expressed genes?DEGs?related to secondary metabolic pathways showed that 23 DEGs were enriched in the phenylpropanoid biosynthesis pathway.Most of these genes were up-regulated except for the Gb-25040?COMT?and Gb-31013?PER53?.There were 10 DEGs enriched in the flavonoid biosynthesis pathway,and all of them were up-regulated,especially Gb 19002?CHS??2.76 times more than the control?.Two phenylpropanoids and five flavonoid synthesis related DEGs were selected to verify by qRT-PCR and the results were consistent with the results of RNA-Seq.These results suggested that the increase of flavonoid synthesis in the truncated group was mainly associated with the high expression levels of CHS,F3'H,DFR,FLS etc.in the flavonoid synthesis pathway.?6?Correlation analysis of key genes and related metabolites of flavonoid biosynthesis revealed that Gb 19002?CHS?was associated with the most metabolites.In addition,Gb 19800?F3'H?,Gb₂6459?DFR?,Gb₁8171?CCOAOMT?,Gb₂1870?ANS?also correlated well with the related metabolites.Among the related metabolites,except 3 kinds of flavonoids such as isoflavones,the other 28 differential metabolites all showed positive correlations.Furthermore,based on the KEGG pathway,the flavonoid biosynthesis pathway showed that some genes encoding key enzymes expressed increasingly in the truncated leaves.Among the metabolites of this pathway are apigenin,luteolin 7-O-glucoside,catechin,epicatechin and dihydrokaempferol.And the contents of these metabolites increased significantly in the truncated treatment group,which showed a consistent trend with related DEGs.?7?Because plant rejuvenation might be associated with hormone levels,we determined the hormone content of leaves in the S1,S3 and S5 periods.The results showed that the leaf IAA contents in the S1 and S3 were higher than the control significantly.The ABA content in the treatment group was lower in the S1,which was only 51.45%of the control group.The cytokinin content of the treatment group was lower than the control group in the S1 period,while the ZR contents in the S2 and S3 increased rapidly more than the control group.Moreover,the GA3 content was higher in each period of the treatment group than that of the control leaves.These results indicate that the leaf rejuvenation of the truncated treatment group was probably caused by the level of plant hormone.In particular,gibberellin displayed an increasing trend during the whole growth period of leaves,suggesting its important roles in the regulation of leaf rejuvenation.?8?Through the analysis of the metabolic process of gibberellin,we found that the content of most GA,including GA3,GA15 and GA20,showed an increasing trend in the truncated treatment group.Furthermore,two genes related to DELLA protein were selected and verified by qRT-PCR in S1,S3 and S5.The results showed that the expression levels of Gb₂5388 and Gb 08892 were significantly lower in the treatment group than that in the control group in each period,suggesting an inhibition expression of DELLA protein.These results further demonstrated that gibberellin may be involved in the regulation of leaf rejuvenation,and the molecular mechanisms of gibberellin regulation in ginkgo leaf rejuvenation and flavonoid accumulation need further study.