为了探讨入侵植物土荆芥的化感作用机制,以其入侵地广泛种植的农作物蚕豆叶片下表皮为受试材料,通过对保卫细胞的活性分析,研究了土荆芥挥发油及其两种主要成分α-萜品烯和对伞花素诱导保卫细胞死亡及其信号调节的机制.结果表明:土荆芥挥发油、α-萜品烯、对伞花素具有显著的细胞毒性,随着处理剂量增加,保卫细胞存活率显著下降,细胞核出现了畸形、碎裂和降解等程序性细胞死亡的典型特征;活性氧(Reactive oxygen species,ROS)、一氧化氮合酶(Nitric oxide synthetase,NOS)和Ca2+的组织化学定位显示,在土芥挥发油、α-萜品烯和对伞花素作用下,保卫细胞内ROS、NOS和Ca2+的水平明显高于对照组;活性氧清除剂(AsA)、Ca2+螯合剂(EGTA)和硝酸还原酶抑制剂(NaN3)均可有效缓解土荆芥挥发油、α-萜品烯和对伞花素的细胞毒性,显著提高了保卫细胞的存活率(P＜0.05).上述结果表明,ROS、NO和Ca2+参与了土荆芥挥发油、α-萜品烯和对伞花素诱导蚕豆保卫细胞死亡的信号调节过程.土荆芥挥发油、α-萜品烯和对伞花素诱导的保卫细胞死亡,可能是通过ROS和NO调控保卫细胞内Ca2+水平的变化而引起的.%Allelopathy is a direct or indirect effect of one plant on another and occurs by releasing allelochemicals into the surrounding environment.When allelopathy inhibits the growth and development of receptor plants,it is considered a biotic stress called " allelochemical stress." Allelopathy is one of the successful mechanisms of alien plant invasions.Allelochemicals can enhance the production of reactive oxygen species (ROS) and increase membrane lipid peroxidation.Allelochemicals can also show obvious cytotoxicity and genotoxicity on receptor plants by inducing enzyme inactivation,DNA damage,and programmed cell death (PCD).Chenopodium ambrosioides L.(Mexican tea) is one of the aromatic herbaceous species of the Chenopodium genus,native to tropical Central and South America.It has recently become a major invasive plant in China,and has strong allelopathic effects on surrounding plants.This plant is threatening the ecological security of China in more than 20 provinces and cities by crowding out and suppressing native species.The guard cells are highly specialized cells,which are sensitive to the change of environment.These specialized cells could receive and integrate a great number of external and internal stimuli to accurately respond to various physiological requirements of the plant.Therefore,stomata are considered an optimal material for studying environmental stress.This study aimed to explore the mechanism of apoptosis and its signal regulation in guard cells induced by volatile oil from C.ambrosioides and by α-terpinene and cymene.Vicia faba L.(broad bean),which is widely planted in the areas invaded by C.ambrosioides,was chosen as the receptor plant.Epidermal strips of leaves were incubated in 2-(N-morpholino) ethanesulfonic acid (MES) buffer containing volatile oil,α-terpinene and cymene for 30 minutes in light at 25℃ in an illumination incubator.After treatment,the epidermal strips were stained with acridine orange/ethidium bromide (AO/EB) to detect cell viability,and were also stained with histochemical localization to indicate intracellular reactive oxygen species (ROS),nitric oxide synthase (NOS) and calcium ion (Ca2+) levels.The results showed that volatile oil,α-terpinene,and cymene treatments resulted in a decrease in guard cell viability of broad bean leaves,and these effects were dose-dependent.Apoptosis features including nuclear malformation,karyorrhexis,and degradation were observed in guard cells under the volatile oil,α-terpinene,and cymene treatments.ROS,NOS and Ca2+ histochemical localization showed that the coloration of ROS,NOS and Ca2+ in guard cells deepened considerably under the treatments of volatile oil,α-terpinene and cymene.Meanwhile,the guard cell survival rates increased when epidermal strips were exposed to volatile oil,α-terpinene and cymene combined with different concentrations of ROS scavenger ascorbic acid (AsA),nitrite reductase inhibitor sodium azide (NaN3),or Ca2+ chelator ethylene glycol tetraacetic acid (EGTA).All results suggested that ROS,NO,and Ca2+ participate in apoptosis signal pathways induced by volatile oil,α-terpinene,and cymene in V.fava stomata guard cells.Changes in Ca2+ levels regulated by ROS and NO may mediate programmed cell death induced by volatile oil,α-terpinene,and cymene.
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