小麦抗条锈反应中活性氧及细胞质钙离子的作用

 为探讨活性氧和细胞质钙离子在小麦抗条锈病反应中的作用,以小麦品种洛夫林13与具有不同致病力的单孢锈菌CY29、CY25的互作体系为平台,对锈菌侵染后小麦叶片中活性氧(ROS)积累、保护酶系(SOD、CAT和APX)活性变化动态、细胞质膜的透性改变及细胞质钙离子浓度变化做了研究。结果表明,不亲和锈菌CY25侵染可引起小麦叶片内2次ROS的爆发,第1次出现在接种后前期(接种后第2天),强度较小,第2次出现在接种后期(接种后第5天),强度较大;亲和锈菌CY29侵染只引起1次ROS爆发,出现在接种后期(接种后第5和第6天之间),但强度极大。过敏性坏死反应HR只出现在不亲和互作小麦叶片上前期ROS爆发之后,表明前期ROS爆发与HR的产生有关。伴随着后期小麦叶片中强度极高的ROS的爆发,叶片细胞原生质膜遭到了破坏,细胞内物质外渗,细胞不久便死亡,表明高强度的ROS爆发会导致细胞死亡。根据不同互作体系ROS爆发时期SOD、CAT和APX等保护酶的活性变化分析,不亲和互作体系前期强度较小的ROS爆发主要成分是H₂O₂,后期强度较高的ROS爆发主要成分是O₂^-_·和H₂O₂;亲和互作体系强度极高的ROS爆发主要成分是O₂^-_·和H₂O₂。由此说明H₂O₂是引起小麦抗病反应HR发生的因素,而O₂^-_·则是引起细胞死亡的因素。细胞质钙离子浓度变化研究表明,HR的发生与细胞质钙离子浓度增加相关。细胞质钙离子浓度的降低推迟了HR的发生,这说明小麦叶片细胞内细胞质钙离子浓度的增加是HR的必要条件,同时也说明Ca²⁺是植物HR的胞内第二信使参与植物抗病防卫反应。

Reactive oxygen species and cytosolic free calcium in the process of wheat resistance to stripe rust

To understand the role of reactive oxygen species (ROS) and cytosolic free calcium in plant defense responses to pathogen infection, changes of ROS, activities of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX), the enzymes known to detoxify ROS in plant, permeability of cell plasma membrane and concertration changes of cytosolic free calcium were investigated in the incompatible and compatible interaction systems, Lovrin13/CY25 and Lovrin13/CY29, respectively. The time course of ROS detected by ESR showed that two peaks of ROS production were observed in leaves inoculated with avirulent or incompatible rust strain CY25. The first peak occurred on the second day whereas the second peak was on the fifth day after inoculation. However, only one single peak of ROS production was observed in leaves on the sixth day after inoculation with virulent or compatible strain CY29. HR occurred in the incompatible inte-raction system was associated only with the early oxidative burst of ROS. This implicated that early oxidative burst could mediate the occurrence of HR. Accompanied with a big amount of ROS production in the infected wheat leaves at late oxidative burst period, plasma membrane was damaged, cell substances leaked out and the cell died soon. The results indicated that strong ROS could cause cell death. According to the activity changes of SOD, CAT and APX at the two periods of oxidative burst, the main component of ROS was H₂O₂ at the early period in incompatible interaction and O₂^-_· and H₂O₂ at the late period in both incompatible and compatible interactions. This suggested that H₂O₂ was the signaling substance in HR of wheat to avirulent or incompati-ble rust strains, whereas O₂^-_· was responsible for cell death. Detecting of cytosolic free calcium in wheat leaves inoculated with stripe rust through laser scanning confocal microscope (LSCM) showed that the occurrence of HR was associated with an increase ofCa²⁺]_cyt in cellular matrix. Decline ofCa²⁺]_cyt in cellular matrix could delay the occurrence of HR. It implied that increase in cytosolic calcium was necessary for hypersensitive cell death, and Ca²⁺ was involved in pathogen defense processes as a vital intracellular second messenger.