The Mechanisms Of Rabdosin B Mediating Polar Auxin Transport Of Arabidopsis Thaliana Root Gravitropism

Terpenoids are a huge group of plant secondary metabolites and widely distributed in nature.Ent-kaurane diterpenoids are abundant in fresh branches and leaves of Isodon plants(Labiatae)which more than 400 have been extracted.Abundant and various ent-kaurane diterpenoids are mainly released from leaves of plants into soil,suggesting that they have an important impact on the ecological environment.Studies have shown that these diterpenodis show significant allelopathic potential for other plants and can be involved in regulating plant growth and development,promoting or inhibiting root growth of plant seedlings.We found that these compounds also significantly influence root gravitropism.In order to explore the mechanisms of these compounds influence root gravitropism,structurally representative ent-kaurane diterpenoids rabdosin B and model plant Arabidopsis thaliana were used in this study.The experiment selected plant seedlings placed vertically(V),cotyledons are placed horizontally to the left(LH)and cotyledons are placed horizontally to the right(RH),to test rabdosin B interfere Arabidopsis thaliana root gravitopism as well as the elongation of cell in the curved site.The effect of rabdosin B on the distribution of auxin were observed by DR5::DR5:GFP transgenic strain,and observed the effect of NAA or 2,4-D combined with rabdosin B on Arabidopsis thaliana seedlings,to verify that the interference of rabdosin B on root gravitropism was associated with auxin polar transport.Furthermore,the mutants deficient in auxin polar transport carriers,fusion protein marker strain and transgenic reporter strain were used to investigate the effect and mechanism of rabdosin B on the auxin polar transport carriers and auxin distribution of Arabidopsis thaliana root gravitropism.The main results are given as follows:(1)Arabidopsis thaliana seedlings under vertical growth,LH and RH conditions,20 ?M,40 ?M,60 ?M,80 ?M rabdosin B had an effect on the root gravitropism,causing the root deflected to the left.Arabidopsis thaliana seedlings under LH condition,40 ?M,60 ?M,80 ?M rabdosin B significantly increased the curvature of root to gravitational response,and the ratio of cells length between the upper and lower sides of the curved site is increased.Arabidopsis thaliana seedlings under RHcondition,the same concentration of rabdosin B notable reduced the curvature of root to gravitational response and the ratio of cells length between the upper and lower sides of the curved site is reduced.(2)Arabidopsis thaliana seedlings under LH condition,the accumulation of auxin in the epidermal cells at the lower side of the root after 40 ?M and 60 ?M rabdosin B treatment was significantly higher than the control group(after gravistimulation).Arabidopsis thaliana seedlings under RH condition,the accumulation of auxin in the epidermal cells at the lower side of the root after same concentration of rabdosin B treatment was less than the control group(after gravistimulation).The results showed that rabdosin B interferes with the root gravitropism by regulate the asymmetric distribution of auxin in root of Arabidopsis thaliana seedlings.(3)Arabidopsis thaliana seedlings under LH and RH conditions,0.1 ?M NAA or0.01 ?M 2,4-D had no significant effect on the seedlings root gravitropism,whereas the interference of the seedlings root gravitropism was treated by 40 ?M rabdosin B could be reversed by 0.1 ?M NAA or 0.01 ?M 2,4-D,suggesting that rabdosin B interfered with Arabidopsis thaliana root gravitropism most likely by mediating the polar auxin transport.(4)Arabidopsis thaliana seedlings under LH and RH conditions,the effect of 40?M and 60 ?M rabdosin B on the root gravitropism of pin1 mutant was not significantly different from wild type.40 ?M and 60 ?M rabdosin B significantly enhance the angle to the left bend of pin2 mutant and the angle of non-directional bend of aux1 mutant,confirming that PIN2 did not contribute to the left bend of rabdosin B on root.(5)Arabidopsis thaliana seedlings under LH condition,40 ?M rabdosin B treatment had no effect on the root bending angle of the pin3-4 and pin4-3 mutant compared to the control group,but the ratio of its bending angle is significantly lower than the ratio of bending angle between 40 ?M rabdosin B and 0 ?M rabdosin B in wild type.Arabidopsis thaliana seedlings under RH condition,there was no significant difference in the angle ratio between the pin3-4 and pin4-3 mutanttreatment group and the control group compared with the angle ratio between the wild type treatment group and the control group.This suggests that PIN3 and PIN4 are likely to be involved in rabdosin B-induced bending factors.Importantly,40 ?M rabdosin B treatment of pin2,3,4 mutant was notable weaker than pin2 mutant treated group in root deflected to the left.This strongly suggests that rabdosin B causes Arabidopsis thaliana seedlings to left bend most likely by inducing the synergistic effect of PIN3 and PIN4.(6)The expression of PIN1,PIN2,PIN3,PIN4 and AUX1 were increased in different degree after treatment with 40 ?M and 60 ?M rabdosin B for 1-4 h,and PIN3 as well as PIN4 showed the distribution enhancement at the lower side of the root under LH condition and the distribution enhancement at the upper side of the root under RH condition,suggesting that asymmetric distribution of PIN3 and PIN4 may have important value in rabdosin B-induced bending effects.(7)The transcription levels of PIN1,PIN2,PIN3,PIN4,PIN7 and AUX1 were not affected after treatment with 40 ?M and 60 ?M rabdosin B for 4 h.In summary,ent-kaurane diterpenoids rabdosin B causes the Arabidopsis thaliana root to bend to the left and interfere with the root gravitropism.The compound is likely to regulate the distribution of PIN3 and PIN4 in column cells of Arabidopsis thaliana root,and then change the distribution of auxin,resulting in the cells length at the right side of root longer than the cells length at the left side of root and deflecting to the left.