Research Of ROS And PLD On Oridonin's Allelopathic Regulation In Arabidopsis Thaliana

In recent years,the chemical ecological effect of diterpene compounds has gradually been confirmed.Oridonin is a kind of diterpene compound extracted from Rabdosia rubescens,which accounts for 90% of all active ingredients.As a natural resistant plant,oridonin is widely distributed and has a series of biological activities such as anti-cancer,sterilization,and immune regulation.However,the research on its allelopathy in natural habitats is blank.Some studies have reported that allelopathic effects are often accompanied with oxidative stress.So it is an interesting question that,is the diterpene allelopathic regulator related to reactive oxygen species(ROS)levels in plant cell and what are the correlations? On the other hand,the plasma membrane and some membranous structures are the major sites for plants to experience external stimuli.Phospholipase D(PLD),as a key membrane phospholipid hydrolase,plays an important role in maintaining the integrity and stability of plant cell membranes.Its downstream product PA acts as a second messenger to mediate various cellular responses.In our previous studies on the relationship between diterpenoids and phospholipase,we speculated that PLD may play a role in allelopathy and may be closely related to ROS.In this study,wild type(WT)and Atpld? mutants were used as experimental materials to detect the allelopathic potential of oridonin on Arabidopsis thaliana.The interaction relationship of ROS and PLD in oridonininduced allelopathy regulation were investigated.This research provide some theoretical references and basis for the in-depth study of diterpene compounds' allelopathic potential and practical application of chemical ecology,It also promotes the study of allelopathy from the simple description of natural phenomena to the exploration of molecular mechanism.The main results are shown as follows:1.By detecting the effect of oridonin on seed germination and seedling growth of Arabidopsis thaliana,different concentrations of oridonin(10?M,60?M,120?M)were found to inhibit the seeds germination,number of leaves and biomass accumulation in Arabidopsis thaliana.The inhibitory effect increases with the increase of oridonin treatment concentration and treatmen time;Oridonin promoted the growth of Arabidopsis roots at a low concentration(10?M),and inhibited the roots growth at ahigh concentration(60?M,120?M);Morphologically,oridonin had a more obvious allelopathic effect on Arabidopsis roots.The results of this study indicate that oridonin is an allelopathic potential substance that has an allelopathic effect on the seed germination,seedling and root growth of Arabidopsis thaliana,and this effect has a concentration variability and selectivity.2.Oridonin at 10?M and 60?M triggered the generation of H2O2 and O2.-,the malondialdehyde(MDA)content and the activities of antioxidative enzyme including superoxide dismutase(SOD),Catalase(CAT)and peroxidase(POD)in Arabidopsis thaliana seedling;The application of H2O2 inhibited the growth of Arabidopsis roots,while the ROS scavenger DMTU and Tiron effectively protected roots from the phytotoxicity of oridonin;Exogenous addition of H2O2 increased MDA content and antioxidant enzyme activity of Arabidopsis seedlings,while ROS scavengers and DPI(an inhibitor of NADPH oxidase)inhibited the increase of MDA content and antioxidant enzyme activity.The results showed that: Oridonin induced ROS production,MDA accumulation and antioxidative enzymes activity in Arabidopsis thaliana,and the allelopathy of the diterpene compound oridonin on Arabidopsis root growth is regulated by ROS,but ROS is not the only pathway.3.After treated with different concentrations of oridonin(10?M,60?M),low concentration oridonin(10?M)play an important role in promoting the activity of PLD enzyme in the early stage,but in high concentrations of oridonin(60?M)treatment,PLD enzyme activity was inhibited at the initial stage,however,the PLD enzyme activity gradually increased at 12 h,showing a significant promoting effect;PLD? gene expression increased at low concentrations of oridonin(10?M),but at a high concentration,the expression level increased only before 12 h,then it was always inhibited and was lower than the control;The application of PA significantly promoted the roots growth of Arabidopsis seedling,and the roots length of the pld? deletion mutant was longer than in wild-type.The results showed that Oridonin regulates the enzyme activity of PLD and the expression of PLD? gene in Arabidopsis thaliana seedlings,and PLD is involved in the allelopathic effect of oridonin on root growth.In addition,PLD? negatively regulates the growth of Arabidopsis roots.4.The ROS level in the pld? mutant was lower than that in the wild typeArabidopsis under different concentrations of oridonin;The application of PA significantly promoted the ROS levels and MDA content in Arabidopsis seedlings,while application of 1-butanol(a specific inhibitor of PLD to produce PA)in Arabidopsis,the ROS levels and MDA content were significantly decreased;in addition,H2O2 inhibited the PLD enzyme activity of Arabidopsis seedlings,while DPI promoted PLD enzyme activity.The results showed that under the treatment of oridonin,PLD promoted the increase of ROS level in Arabidopsis;at the same time,the increase of ROS level inhibited the activity of PLD.