Imaging of photo-oxidative stress responses in leaves
Imaging of photo-oxidative stress responses in leaves
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DOI：
10.1093/jexbot/53.372.1249
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发表时间：
2002-05-01
影响因子：
6.9
通讯作者：
Baker, NR
中科院分区：
文献类型：
--
作者：
Fryer, MJ;Oxborough, K;Baker, NR
高分辨率数字成像被用来确定在拟南芥叶片的光氧化应激反应的非侵入性的网站，并证明使用一套成像技术在植物中的氧化代谢的研究的潜力。利用叶绿素荧光显微镜观察了叶片中单个叶绿体光合作用的组织特异性光抑制。通过对丹磺酰基-2，2，5，5-四甲基-2，5-二氢-1H-吡咯（DanePy）与单线态氧反应产生的荧光猝灭进行成像来评估单线态氧的产生。分别通过还原氮蓝四唑（NBT）形成甲瓒沉积物和通过与3，3 '-二氨基联苯胺（DAB）聚合观察超氧化物和过氧化氢积累。从拟南芥APX 2-LUC的叶片的生物发光成像的抗氧化剂代谢基因的应激诱导的表达，其中共表达抗坏血酸过氧化物酶（APX 2）与萤火虫荧光素酶。单线态氧和超氧化物的产生主要位于叶肉组织，而过氧化氢的积累和APX 2基因的表达主要定位在维管组织。
High resolution digital imaging was used to identify sites of photo-oxidative stress responses in Arabidopsis leaves non-invasively, and to demonstrate the potential of using a suite of imaging techniques for the study of oxidative metabolism in planta. Tissue-specific photoinhibition of photosynthesis in individual chloroplasts in leaves was imaged by chlorophyll fluorescence microscopy. Singlet oxygen production was assessed by imaging the quenching of the fluorescence of dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole (DanePy) that results from its reaction with singlet oxygen. Superoxide and hydrogen peroxide accumulation were visualized by the reduction of nitroblue tetrazolium (NBT) to formazan deposits and by polymerization with 3,3'-diaminobenzidine (DAB), respectively. Stress-induced expression of a gene involved with antioxidant metabolism was imaged from the bioluminescence from leaves of an Arabidopsis APX2-LUC transformant, which co-expresses an ascorbate peroxidase (APX2) with firefly luciferase. Singlet oxygen and superoxide production were found to be primarily located in mesophyll tissues whereas hydrogen peroxide accumulation and APX2 gene expression were primarily localized in the vascular tissues.