Study On The Effect And Relationship Between Flavonols And H₂O₂ In The Adventitious Root Induction Stage Of Cuttings

The mechanism of adventitious roots formation is an important issue in developmental biology.The research could provide a theoretical basis for the propagation of seedlings in agroforestry,Cuttings are classical systems for the study of adventitious roots formation.Mechanical injury results in the disruption of root-to-ground material transport and the generation of jasmonic acid,ethylene and reactive oxygen species?ROS?.Our previous research in the laboratory showed that the injury signal ROS burst rapidly at the cuttings incision and was transmitted upward to the tip to induce adventitious roots formation by regulating auxin levels and polar auxin transport?PAT?.What is the spatio-temporal change of the signal ROS during the induction of adventitious roots?Flavonols are important ROS scavengers in plants.Whether it participates in the regulation of the level of signal ROS during the induction of adventitious roots?For this reason,the Arabidopsis thaliana wild-type and related mutants were used as materials to study the effects of flavonols and wound signal H₂O₂ during the induction of adventitious roots in Arabidopsis cuttings by using pharmacology,molecular genetics,and gene expression analysis methods.The role and relationship in the induction process are as follows:1.After the main root of Arabidopsis was removed,the burst of H₂O₂ could be rapidly occurred at the incision of the cuttings,and it could spread rapidly between cells and conduct rapidly through hypocotyls.During the upward conduction process,the intensity and conduction rate of H₂O₂ gradually weakened,showing the strongest fluorescence in the base of the cuttings,followed by the middle,and the weakest distribution of fluorescence in the upper,the tip and leaf.Being cut after 2 h,H₂O₂ reached the highest level and was transmitted to the entire cuttings,and the fluorescence no longer increased after 2 h,and remained at the high level until about 6 h after being cut,and then decreased gradually.Thus,H₂O₂ presented a parabolic pattern throughout the induction period of adventitious roots.H₂O₂ scavengers ASA and DMTU can significantly inhibit the generation of H₂O₂ fluorescence in the base of wild-type cuttings and inhibit its upward transmission through the hypocotyls.Meanwhile,H₂O₂ can significantly promote the rooting of Arabidopsis cuttings and promote the elongation of adventitious roots.After H₂O₂ scavengers ASA,DMTU treating Arabidopsis cuttings,the number and length of adventitious roots was significantly inhibited.These results indicate that the injury signal H₂O₂ participates in the induction of adventitious root in Arabidopsis cuttings.2.Detection of auxin levels by the DR5rev::GFP strain revealed that the auxin levels in the tip,the leaf,especially the leaf margin,gradually increased,and then the auxin levels in the base of the cuttings also gradually increased.The auxin levels in the base of cuttings was significantly increased at 6 h after being cut,and this high levels continued until 12 h after being cut,and the levels of auxin decreased after 24 h.H₂O₂ scavengers ASA and DMTU could strongly inhibit the increase of auxin levels in the tip,the leaf and the base of DR5rev::GFP cuttings,indicating that the injury signal H₂O₂ may be involved in the regulation of auxin levels and PAT during the induction of adventitious roots.Gene expressions results showed that the expressions of auxin synthesis genes YUCCA2 and YUCCA6 in the cuttings exhibited a weak tendency to increase at the early stage of adventitious root induction.The expressions levels of IA A-AH genes ILL1,ILL2,ILL3 and IAR3 increased significantly.The expression levels of IAR3 genes was significantly increased 1 h after being cut.The expression levels of PIN3,an polar auxin transport gene,increased significantly at 2 h after being cut.Subsequently,the expression levels of PIN1,ABCB19,AUX1,and PIN7 gradually increased,suggesting that IAR3 and PIN3 may play an important role in auxin hydrolysis and PAT,respectively.3.After the primary root of Arabidopsis was removed,flavonols were gradually produced at the incisions of the cuttings.The flavonols were detected in the base,the middle,the upper,the tip and leaf of the cuttings at 1 h after being cut,and the flavonols reached the highest level after 2 h.Then it remained at a high level until about 6 h after being cut,and then gradually decreased.In addition,the distribution of flavonols showed the strongest in the base of the cuttings,followed by the tip and leaf,and then the upper,and the middle fluorescence was the weakest.Exogenous quercetin and kaempferol can significantly inhibit the production of injury signal H₂O₂ in the cuttings.At the same time,the flavonol-over-production mutant tt3-1 had a significantly higher flavonol levels than the wild type at 2 h after being cut and the injury signal H₂O₂ levels were lower than the wild type,while the flavonol-deletion mutant tt4-4 had little flavonols almost 2 h after being cut and the H₂O₂ levels were significantly higher than that of the wild type,indicating that the levels of flavonol were negatively correlated with the H₂O₂ levels.At the same time,quercetin and kaempferol significantly inhibited the formation of adventitious roots in Arabidopsis cuttings.In addition,the phenotypes of the flavonol-over-production mutant tt3-1 and the flavonol-deletion mutant tt4-4 and tt4-5 were the opposite,showing the inhibition of the number and the length of adventitious roots and the promotion of the number and length of adventitious roots,respectively.The results shows that flavonols participate in the rooting induction process of Arabidopsis cuttings by regulating the homeostasis of ROS.4.Similar to H₂O₂ scavengers,quercetin can significantly inhibit the increase of auxin levels in the tip,the leaf and the base of DR5rev::GFP cuttings;H₂O₂ can significantly increase the expressions of auxin synthesis genes,IAA-AH genes and PAT related genes in wild-type cuttings.DMTU and quercetin significantly inhibited the expressions of these genes.The flavonol-deletion mutant tt4-4 showed enhanced expressions of these auxin-related genes,while the expressions of these genes in flavonol-over-production mutant tt3-1 were significantly reduced.These results confirms that flavonols act as scavengers of the injury signal H₂O₂ in Arabidopsis cuttings and participate in the regulation of the injury signal H₂O₂ levels,which in turn regulate the auxin levels and PAT during the adventitious roots induction stage-In summary,in the induction process of adventitious roots in Arabidopsis thaliana cuttings,flavonols as a scavenger of the injury signal H₂O₂ participates in the regulation of the levels of injury signal H₂O₂ to negetively regulate the spatio-temporal distribution of auxins in the cuttings so that it participates in the initiation of adventitious roots.