Regulation Of Root Growth And Root Hair Development In Lactuca Sativa L. Seedlings By The Ent-kaurane Diterpenoid Rabdosin B

Isodon plants are important genus of the Labiatae family and are perennial herbs,shrubs, or sub-shrubs. The genus is composed of approximately 150 species thatare widely distributed in tropical and subtropical regions. Ent-kauranediterpenoids are the main natural constituent in this genus and are especiallyabundant in fresh branches and leaves. To date, over 400 ent-kaurane diterpenoidshave been isolated from these plants. Being abundant and varied secondarymetabolites formed during the long evolutionary process, may have specialmeaning to the survival of species themselves. However, whether thesediterpenoids mediate interactions in natural surroundings is still unknown.In order to evaluate the phytotoxicity of the ent-kaurane diterpenoids, determinewhether they may play important ecological roles in natural surroundings andprovide foundations for the further systematic allelopathic research, rabdosin B,one of the abundant and structurally representative ent-kaurane diterpenoids fromIsodon japonica var. galaucocalyx obtained from Gansu, was adopted and theeffects on root growth and on root hair development were investigated in lettuceseedlings by bioassay method. Possible action mode of rabdosin B was alsoexplored in our research. The results are given as follows:(1) Rabdosin B, an ent-kaurane diterpenoid purified from the air-dried aerialparts of Isodon japonica var. galaucocalyx, showed a biphasic, dose-dependenteffect on root growth and a strong inhibitory effect on root hair development inlettuce seedlings (Lactuca sativa L.). Lower concentrations of rabdosin B (20μM,40μM, 80μM) significantly promoted the root growth, but its higher levels at120-200μM, by contrast, had inhibitory effects. Additionally, all of the testedconcentrations (10μM, 20μM, 40μM) inhibited root hair development of lettuceseedlings in a dose-dependent manner.(2) Different concentrations of rabdosin B affected both the size of mature cellsat the root-hair-forming regions and the mitotic activity of meristematic cells inlettuce seedlings'root tips. Regression analysis revealed a positive correlationbetween NGR and cell length and the mitotic index. Based on the results, weconcluded that the promotion effect of rabdosin B at the lower concentrationsresulted from increasing the cell length in the mature region and enhancing the mitotic activity of meristematic cells in lettuce seedlings'root tips.(3) Rabdosin B at higher concentrations inhibited the root growth by stronglyaffecting both the cell length in the mature region and the division of meristematiccells. Comet assay and cell cycle analysis demonstrated that the decrease ofmitotic activity of root meristematic cells was due to DNA damage induced cellcycle retardation of G2 phase and S phase at different times.(4) Both rabdosin B and Ag+ (an ethylene antagonist) inhibited root hairdevelopment of lettuce seedlings and showed antagonistic activity to ethephonenhanced root hair length. Seedlings with suppressed root hairs that were treatedwith rabdosin B could not be reversed by exogenous application of ethephon,suggesting that rabdosin B may interfere with the ethylene response pathwayinstead of ethylene biosynthesis.(5) Addition of rabdosin B did not overcome the inhibitory effect on rootgrowth caused by ethephon as Ag+ did. It is possible that rabdosin B interfereswith the downstream portion of the ethylene pathway, thus, specificallycontrolling root hair development instead of affecting root growth. However, Ag+may interact with an ethylene receptor at a point upstream in the ethylene pathway,thus, both affecting root hair development and root growth.Our research demonstrated the dual stimulatory and inhibitory effects ofrabdosin B on root growth and inhibitory effects on root hair development inlettuce seedlings. Further investigations on the underlying mechanism revealedthat the promotion of root growth resulted from enhanced cell length and increasedmitotic activity. In contrast, rabdosin B at higher concentrations inhibited rootgrowth by affecting both the cell length and cell division. Also, rabdosin B at 200μM could induce DNA damage in lettuce root tips, which resulted in the cell cycleretardation, and thus decreased the mitotic activity. In addition, we presumed thatrabdosin B inhibited root hair development through the mechanism of action as apotential ethylene action antagonist. Further studies need to be carried out toconfirm the role of rabdosin B in mediating the ecological interactions in naturalsurroundings.