Studies On The Synthetic Regulatory Mechanism Of Flavonols In The Induction Of Adventitious Roots In Arabidopsis Cuttings

Cutting is considered as a commonly used asexual reproduction method in agriculture,forestry and horticulture,which is of great importance.The key to the survival of cutting depends on'whether it can produce adventitious roots.However,its formation is mostly influenced by many external and internal factors.Previous studies from our laboratory have demonstrated that H2O2 bursts rapidly at the cutting excision and transmits to the upper section after cutting the primary roots,and then leads to the initiation of adventitious roots through regulating the auxin synthesis and polar transport,and auxin plays an important role.H2O2,as a free radical,exerts the stress effect with high concentration,so it needs to be precisely regulated in plant.Flavonols are an important secondary metabolites in plants,and have strong antioxidant activity,which can regulate the homeostasis of H2O2.Moreover,the studies have shown it can also regulate the production of primary and lateral roots.So whether flavonols could involve in the formation of adventitious roots or not?If it involves,what is the synthetic regulation mechanism?Here,the Arabidopsis thaliana seedlings with long hypocotyls were used as experimental materials,and then the synthesis and regulation mechanisms of flavonols in the induction of adventitious roots of Arabidopsis thaliana were studied by means of pharmacology,fluorescence detection and gene expression analysis.The conclusions are as followed below:1.After cutting the primary root of Arabidopsis thaliana,H2O2 in the excision rapidly produced and transmited upwards.Flavonols had the similar generation and distribution with H2O2.It appeared and gradually rose,reached the highest level at 2-6 h after cutting,and progressively decreased at 6-12 h,and its distribution showed a more level in the base,a less level in the middle,and second level in the upper section.The exogenous treatment with quercetin could inhibit the H2O2 accumulation,while H2O2 scavenger DMTU reduced the level of H2O2 in the cuttings,thus inhibiting the production of flavonols.The results indicated that the synthesis of flavonols depends on the injury signal H2O2 production during the induction stage of adventitious root formation,and its accumulations may play a role in regulating H2O2 levels in the cuttings.2.In order to understand the mechanism of flavonol accumulation in the cuttings,we divided the cuttings into base,middle and upper sections according to the different levels of flavonols,and detected the expression of related genes in the flavonol synthesis pathway at different times after cutting.The results revealed that the expressions of CHS,CHI,F3H,F3'H and FLS1 in different parts of the cuttings showed significant changes.In the base and middle of the cuttings,the expression levels of CHS,CHI and FLS1 increased greatly at 6 h after cutting,and gradually decreased at 12 h.These genes increased to a lesser extent in the upper section of the cuttings and occurred a little later.The expression of F3H and F3'H increased sharply at 12 h after excision,and the upper section showed a trend similar to or even stronger than the base and middle.Besides,the expression of encoding and catalyzing flavonol glycoside hydrolase BGLU15 gene,exhibited a significant enhancement at the base of the cuttings at 0.5 h after excision,and a gradual decrease at 6 h,but the enhancement in the middle and upper was not obvious.The flavonol accumulation in the deletion mutant bglu15-1 obviously reduced at 2 h,indicating that BGLU15 might be involved in the early flavonol accumulation.Moreover,the accumulation of H2O2 rose greatly,and the number of adventitious roots also increased,indicating that BGLU15 participated in the formation of adventitious roots by mediating the early flavonol synthesis of cuttings.The results suggested that the synthesis pathway of flavonols is different at varied time after cutting,which may mainly involve the hydrolysis of existing flavonol glycosides to release flavonol monomers at 0-2 h,while the de novo synthesis of flavonols is dominated at 6-12 h.3.To further investigate the regulation mechanism of flavonol synthesis in cuttings,we tested the expression levels of MYB11MYB12 and MYB111 that regulated flavonol synthesis at different time after cutting,in Arabidopsis.The results showed that MYB11 slightly increased in the base of the cuttings at 2-6 h after cutting and its change in the middle section was not obvious,and the cutting upper section increased at 12 h.The expression of MYB12 in the base and middle section of the cuttings was markedly increased at 2 h after cutting,and the upper section increased obviously at 12 h.The MYB111 in the base gradually increased at 1-12 h,and the middle of the cuttings slightly enhanced at 1 h,The expression of MYB11 decreased at other time,and increased remarkably at 6-12 h in the upper section of the cuttings.Compared with the wild-type cuttings after cutting for 2 h,the production of endogenous flavonols in myb11 myb12-1f and mybl1myb12 deletion mutant had minor reduction,and flavonols of myblll deletion mutant and myb1mybl2myb111 triple mutant significantly decreased.The endogenous H2O2 levels of these mutant were even stronger than the wild type,and the root numbers of cuttings also evidently increased.The results indicated that MYB11,MYB12 and MYB111 transcription factors participate in the regulation of H2O2 homeostasis in the cuttings by positively regulating the synthesis of flavonols during the induction stage in the adventitious root of Arabidopsis thaliana cuttings,and negatively regulating the induction process of adventitious roots.4.The expression level of transcription factor HY5 gene increased obviously at 2 h after cutting,and then gradually decreased.The expression in the middle part of cuttings was similar to its base section,but the increase was small.The expression in the upper part of cuttings gradually increased after cutting at 0.5 h,and exhibited the largest increase at 12 h.Compared with the wild type,the flavonols and H2O2 levels showed that the accumulation of flavonols in the hy5 deletion mutant apparently reduced;the accumulation of H2O2 was remarkbly increased;the number of adventitious roots was significantly enhanced.However,the HY5-OX mutant endogenous flavonols,H2O2 levels and adventitious root numbers showed an opposite change.The results indicated that HY5 can positively regulate the flavonols synthesis in the induction stage of adventitious roots in Arabidopsis thaliana,and then participate in the regulation of the adventitious roots formation process.5.The expression level of transcription factor MYBL2 showed an obvious change after cutting,and it exhibited similar regularity in the base,middle and upper parts.They all decreased distinctly at 0.5-1 h,increased slightly at 2 h,and then gradually reduced.Compared with wild type,the endogenous flavonols and H2O2 levels of myb12 deletion mutant increased and decreased,respectively,and the number of adventitious roots evidently reduced.These results suggested that MYBL2 may increase the accumulation of H2O2 by inhibiting the flavonols synthesis during the induction stage of adventitious roots in Arabidopsis thaliana cuttings,thus promoting the beginning of adventitious roots.6.To explore the effect of miR858a on the flavonol synthesis,we examined the change in endogenous flavonols and H2O2 levels of over-expression mutants MIR858a-0X#2,MIR858a-OX#5 and expression-reduced mutants STTM858#1,STTM858#2.Compared with the wild type,the flavonol levels of MIR858a-0X#2 and MIR858a-OX#5 cuttings slightly enhanced,and H2O2 levels and the number of adventitious roots reduced.However,STTM858#1 and STTM858O2 mutants showed the decreasing accumulation of flavonol,the raising level of H2O2,and the number of increasing adventitious roots.These results suggested that miR858a may promote the flavonol synthesis by reducing the H2O2 level during the induction stage of adventitious roots in Arabidopsis thaliana,then inhibiting the induction process of adventitious root.In summary,flavonols are rapidly synthesized as the scavengers of H2O2 to regulate its level during the induction stage of adventitious root in Arabidopsis cuttings.The synthesis of flavonols in the early stage is related to BGLU15 gene.However,its synthesis in the later stage is related to the increasing expression of CHS,CHI,F3H,F3 'H and FLS1,and this process is regulated by transcription factors including MYB11,MYB12,MYB111,HY5,MYBL2 and miR858a.