Effects And Mechanisms Of Stem Removal And Defloration On Root Growth And Quality In Bupleurum Chinense DC.

Radix Bupleuri is one of the most important major medicines in the world.In recent years,the cultivated germplasms of Bupleurum chinense DC.has become to be the main source of Radix Bupleuri.However,the yield of B.chinense DC.was low.Due to its limited biomass of roots compared with that of aerial part,and the serious imbalance of root/shoot ratio,high degree of closeness and plant mortality often occur in the second and the third year,planting density is also unable to improve,which results in the inhibition of both benefit and enthusiasm for planting B.chinense DC..Although removing stems and flowers were found to contribute to yield,to date,the physiological and molecular mechanism of the above agronomic measures remain poorly described in B.chinense DC.Here,the main variety 'Zhongchai No.2' was used as material;through dynamic analysis of growth and development,GC-MS and transcriptome sequencing analysis,we notably uncovered the distinct mechanisms of removing stems and flowers on root growth and quality formation of B.chinense DC..The main results were as follows:1.The biomass of the aerial part and root of B.chinense DC.plant increased gradually along with growth,but the biomass of the aerial part was larger than that of the root.The root-shoot ratio of B.chinense DC.plant decreased gradually along with growth and reached the largest decreasing at the flowering stage with the value of 53.9%.The proportion of xylem in the total root area of B.chinense DC.gradually increased.HPLC detection showed that the contents of saponin a and d in xylem were lower than that in cortex of B.chinen.se DC.roots from seedling stage to withering stage,and the contents of saponin a and d in cortex of B.chinense DC.reached highest at flowering stage.The results of GC-MS showed that,the species of volatiles in the xylem of root decreased gradually from seedling stage to withering stage,while the species of volatiles in the cortex increased gradually,and the species of volatiles in the cortex of root were always more than that in the xylem.2.The fresh weight(FW)and dry weight(DW)of root after removing the stem increased from 3.19±2.75 g to 5.28 ± 2.58 g,and from 1.55±1.26 g to 1.63±0.62 g,respectively,in the first year.In the second year,FW and DW of root increased from 4.99± 1.52 g to 8.94±2.77 g,and from 1.63±0.62 g to 3.22 ± 0.98 g,respectively.The contents of saponin a and d increased with no significance.The results of GC-MS showed that 26 volatiles increased in the root of treatment group.The results of RNA-Seq showed that differentially expressed genes were found in ABA pathway(PYL4),ethylene pathway(ERF1),and jasmonic acid(TIFY 10A)pathway.Saponin synthesis genes(?-AS,Galactinol synthase 1,Beta-fructofuranosidase,TPPJ and RHM1)and root growth-associated genes(GAPDH,CYP735A,TIFY 10A,CAD and Prx)were significantly up-regulated in stem removal group.The results showed that stem removal measures can improve the yield and quailty of B.chinense DC.,and the treatment might trigger the biosynthesis of ABA and ethylene,and the response of injury stress.3.The FW and DW of root after defloration increased from 2.61 ± 0.99 g g to 7.08 ±2.53 g,and from 1.01 ± 0.31 g to 2.80±1.03 g,respectively,in the first year.In the second year,the fresh and dry weight of root increased from 5.26±1.53 g to 12.62±3.93 g,and from 1.92±0.53 g to 4.66 ± 1.51 g,respectively.The content of saponin a and d increased by 25.61%and 34.10%,respectively.The results of GC-MS showed that 5 volatiles increased in the root of treatment group.The results of RNA-Seq showed that differentially expressed genes were found in auxin pathway(TIR,ARF),brassinolide pathway(cyclin D3),cytokinin pathway(ARR),ABA(PYR/PYL?ABI5?ABI7?PP2C?SnRK2)pathway,ET(ERFI)pathway.Saponin biosynthesis genes(AACT,HMGS,HMGR,DXS)and root growth-associated genes(endoglocanase,PL,CRF2,SAUR41,TIR1,MYB59,SCARECROW-like 28)were significantly up-regulated in the treatment group.The results showed that defloration could promote root growth,development,biomass accumulation and saponin biosynthesis,through regulating several key enzymes in hormone pathway and saponin synthesis pathway.4.The annual average increase of FW and DW after removing the stem the stems were 67.62±3.31%and 58.96±11.71%,respectively.The average annual increase of FW and DW after defloration were 155.57±22.20%and 159.20±24.10%,respectively,compared with the stem removal.The increase of contents of saponins a and d were about 4 times and 1.7 times after defloration than removing the stem,respectively.Eleven candidate genes including HRD3A,PTB3,26S,RAP2.2,UVR8,DSK2a,SERINC,GDI,SKP1,UBC9,ACT7 and one common gene ?-tubulin were obtained from the transcriptome database.12 candidate genes were detected in control and treatment groups of stem removal and defloration.The results showed that 12 primers exhibited high specificity and amplification efficiency.In the stem removal group,26S and PTB3 were the most stable internal reference genes,however,PTB3 and SERINC showed most stability in the defloration group.The expression patterns of 13 target genes analyzed by qRT-PCR were consistent with the result of RNA-Seq,so the sequencing analysis was reliable.The effects and mechanisms of stem removal and defloration on the yield and quality of roots were systematacially compared.Removing stems reduced vegetative growth and reproductive growth,which triggered the increasing levels of ABA and ethylene,and decreasing level of auxin.The reponse of injure stress was induced,so the increase in biomass and saponin contents was not significant.B.chinense DC.exhibited vigorous flowering and long flowering period,so defloration showed higher increasement in yield and quality of root,compared with that of stem removal group.The levels of ABA and ethylene decreased,while the levels of auxin,cytokinin and the expression of key genes-associated saponin biosynthesis increased,the root biomass and the effective component content increased by 2-4 times as much as that of stem removal group.It is speculated that defloration measures delayed the senescence process in plant,and prolonged the formation of root yield and quality.In the future,we would further explore why stem removal and defloration measures exhibited different mechanisms in root growth and plant development process.Then,optimize the operation plan of stem removal and defloration measures,so as to provide a good reference for the high-yield and high-quality cultivation of root herbs.