Phospholipase Is Involved In The Allelopathy Of Oridonin In Arabidopsis Thaliana

In order to investigate the allelopathic mechanism of diterpenoid Oridonin to Arabidopsis thaliana,the germination rate,seedling growth,electrolyte leakage?EL?,malondialdehyde?MDA?,and three phospholipase activities?PLA₁,PLA₂ and PLC?of four Arabidopsis thaliana lines?the Colombia ecotype WT,and three phospholipase deletion mutants pla₁,pla₂ and plc1?were measured under different Oridonin concentrations(10 and 60?mol·L^-1).After that,the G protein activity,the relative expression levels of GPA1,AGB1 and AGG2 in WT,the root net growth rate and PLC activity of four A.thaliana lines?WT,and G protein three subunit G?G?and G?deletion mutants gpa1,agb1 and agg2?were measured under 10 and 60?mol·L^-1Oridonin treatment.Subsequently,PLC activity,Ca²⁺content,PA content,H₂O₂ level and root net growth rate of WT and plc1 were detected under 10 and 60?mol·L^-1Oridonin treatment to explore the effect of PLC on the root regulation under Oridonin treatment in A.thaliana and the relationship of PLC,Ca²⁺and H₂O₂ in this process.This work was designed to explain part of the possible allelopathic mechanism of Oridonin to A.thaliana.1.The seed germination was inhibited by Oridonin,and PLA₁ and PLA₂ participated in this process.The growth of root and hypocotyl showed a“low-promotion and high-inhibition”effect.PLC was involved in the growth regulation of roots and hypocotyl.The fresh weight was inhibited by Oridonin,and PLA₁,PLA₂ and PLC were all involved in this process.2.The activities of different phospholipases changed great differently under different Oridonin concentrations,which PLA₁ activity was promoted,PLA₂ activity was increased under low-concentration(10?mol·L^-1)while declined under high-concentration(60?mol·L^-1),and PLC activity was repressed.The EL and MDA increased and were positively correlated with treatment concentration and duration.The stimulation of EL by low-concentration of oridonin was regulated by PLA₂.The stimulation of EL by high-concentration of oridonin was co-regulated by PLA₁ and PLA₂.The MDA accumulation was also co-regulated by PLA₁ and PLA₂ under oridonin treatment.3.The G protein activity was promoted by low-concentration Oridonin and inhibited by high-concentration Oridonin.The results of relative expression of GPA1,AGB1and AGG2 showed that G protein reponsed to the Oridonin,and G?had crucial role in it.The root net growth rate in WT,gpa1,agb1 and agg2 showed that G?and G?subunit protein affected the root growth regulation under Oridonin.The PLC activity in mutants was higher than that in WT,and changed in different ways,suggesting G protein was involved in regulating PLC by Oridonin.4.The Ca²⁺content was raised in WT but reduced in plc1,and Ca²⁺level was regulated by PLC.The PA content showed a"low-inhibition and high-promotion"effect in WT.In plc1,PA content was decreased by low-concentration but promoted by high-concentration,suggesting PLC regulated PA level under low-concentration The H₂O₂ level in WT and plc1 was upgraded and the elevated degree in plc1 was higher than that in WT,and H₂O₂ was mainly generated by NADPH oxidase pathway.The activities of SOD,CAT and POD in WT and plc1 were all promoted under Oridonin.However,these enzyme activities were lower in plc1 than that in WT,suggesting that PLC was involved in the H₂O₂ scanvging by improving the antioxidant enzyme activity.Both PA and Ca²⁺promoted the accumulation of H₂O₂.The concentration of H₂O₂ was closely related to root length growth,and excessive accumulation of H₂O₂ inhibited root length growth.5.Applying DPI to block the production of H₂O₂ by NADPH oxidase,the roots couldn't grow under Oridonin treatment.The root growth,apical meristem cell length and mitotic index were regulated by H₂O₂.Oridonin influenced apical meristem cell length and mitotic index to affect root growth as a“low-promotion and high-inhibition”effect with different level of H₂O₂,this process was regulated by PLC which impact production of Ca²⁺and PA to change the accumulation of H₂O₂.These results showed that the allelopathic effect of Oridonin on plants was related to phospholipase.Plants responsed to partial allelopathic effect of Oridonin by regulating phospholipase,and different phospholipases had different effects in regulating plant growth and development responding to Oridonin.G protein was involved in regulation of the PLC by Oridonin.PLC regulated the H₂O₂ level through changing the content of Ca²⁺and PA,so as to mediate the potential allelopathy of Oridonin of“low-promotion and high-inhibition”effect on root growth in A.thaliana.