| The genus Isodon japonica var. galaucocalyx includes about150species, and is acosmopolitan and important genus of the family Lamiaceae. These species are widelydistributed in subtropical regions of eastern Asia and western Africa. China has about90species and25variants,^/-kaurane diterpenoids are the main constituent in thisgenus and are especially abundant in fresh branches and leaves of plants of genusIsodon and widely used in Chinese herbal medicine. Such abundant and variedsecondary metabolites have wide and signiifcant range biological activity, such asanti-tumor, anti-inflammatory and antibacterial. We choose e/?/-kaurane diterpenoidLeukamenin E which has typical strcture and isolated from Isodon racemosa (Hemsl)Hara in Gan Su. Leukamenin E is applied to rat astrocytes which has strong toxicity,and it can induce apoptosis. In addition, Leukamenin E has antagonist properties inethephon to promote A. thaliana top root hair growth. Phospholipase D (PLD) hasbeen proposed to a key enzyme in many cellular processes, such as plant growth anddevelopment, signal transduction. PLD not only hydrolyzes phospholipids toinlfuence membrane structure, function and stability but play a pivotal role inmembrane traiffcking, cytoskeleton rearrangements, membrane degradation andformation. Although the Leukamenin E and PLD have researched in their respectivefields，but its relevance has not been reported.To study the relationship between PLD and e/?/-kauranoid diterpenoidsLeukamenin E，further explore and elaborate the responses of PLD to leukamenin Etreatment, the activities and expression patterns of PLD gene in Arabidopsis thaliana(A. thaliana) callus were researched. We also measured some characters of physiologysuch as electrolyte leakage (EL), malondialdehyde (MDA) contents, proline (Pro)content, soluble sugar (SS) content and soluble protein (SP) content, activities ofsuperoxide dismutase (SOD)，peroxidase(POD), catalase(CAT), ascorbate peroxidase(APX).The results are as follows:(1) When A. thaliana callus incubated with leukamenin E at concentrations oflOOjaM and200jaM for48h，the activities of PLD in microsomal and mitochondrialmembranes exhibit up-regulation behavior, keep the highest level at24and36h， respectively. Different concentration (lOOjaM，200jaM) Leukamenin E can activatePLD activity, and there is a concentration-dependent manner. And also, the RT-PCRanalyses suggested that PLD activity was partially corresponded to the A. thalianaPLD gene transcript level, then enhancing the PLD activity. PLD is not only involvedin the induction mechanism of Leukamenin E and more positive to response lowconcentration Leukamenin E.(2) As indicators of membrane damage, EL and MDA contents increasedsigniifcantly and peaked at hour36and then decreased which were treated byleukamenin E in A. thaliana callus，this phenomenon demonstrate that there arerelationship among EL, MDA and leukamenin E. We indicated that degradation ofphospholipids through activating PLD cause membrane damage, and the EL andMDA content increased.(3) Leukamenin E treatment resulted in the increase of the Pro, SS and SP comparedto the control (P <0.05). Pro SS content increased greatly after leukamenin E addedfrom12to36h and SP content increased from12to24h. These results suggested thatthe A. thaliana callus enhanced osmotic adjustment ability and water retentioncapacity through accumulation Pro, SS and SP content after treated by Leukamenin E.(4) With the time prolong of Leukamenin E，SOD，POD, CAT and APX activityincreased at ifrst, then decreased. It indicated that Leukamenin E can influenceantioxidant enzyme activity, which responded positively to Leukamenin E stress. Thecapabilities of the antioxidant enzyme system defense are the result of co-ordinationof the activities of SOD, POD, CAT and APX. |
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