Carter Clay, Healy Rosanne, O'Tool Nicole M, Naqvi S M Saqlan, Ren Gang, Park Sanggyu, Beattie Gwyn A, Horner Harry T, Thornburg Robert W
Department of Biochemistry, Biophysics, and Molecular Biology , Iowa State University, Ames, Iowa 50011, USA.
Plant Physiol. 2007 Jan;143(1):389-99. doi: 10.1104/pp.106.089326. Epub 2006 Nov 17.
Hydrogen peroxide produced from the nectar redox cycle was shown to be a major factor contributing to inhibition of most microbial growth in floral nectar; however, this obstacle can be overcome by the floral pathogen Erwinia amylovora. To identify the source of superoxide that leads to hydrogen peroxide accumulation in nectary tissues, nectaries were stained with nitroblue tetrazolium. Superoxide production was localized near nectary pores and inhibited by diphenylene iodonium but not by cyanide or azide, suggesting that NAD(P)H oxidase is the source of superoxide. Native PAGE assays demonstrated that NADPH (not NADH) was capable of driving the production of superoxide, diphenyleneiodonium chloride was an efficient inhibitor of this activity, but cyanide and azide did not inhibit. These results confirm that the production of superoxide was due to an NADPH oxidase. The nectary enzyme complex was distinct by migration on gels from the leaf enzyme complex. Temporal expression patterns demonstrated that the superoxide production (NADPH oxidase activity) was coordinated with nectar secretion, the expression of Nectarin I (a superoxide dismutase in nectar), and the expression of NOX1, a putative gene for a nectary NADPH oxidase that was cloned from nectaries and identified as an rbohD-like NADPH oxidase. Further, in situ hybridization studies indicated that the NADPH oxidase was expressed in the early stages of flower development although superoxide was generated at later stages (after Stage 10), implicating posttranslational regulation of the NADPH oxidase in the nectary.
花蜜氧化还原循环产生的过氧化氢被证明是抑制花蜜中大多数微生物生长的主要因素；然而，花卉病原体梨火疫病菌可以克服这一障碍。为了确定导致过氧化氢在蜜腺组织中积累的超氧化物来源，用氮蓝四唑对蜜腺进行染色。超氧化物的产生定位于蜜腺孔附近，并且被二亚苯基碘鎓抑制，但不被氰化物或叠氮化物抑制，这表明NAD(P)H氧化酶是超氧化物的来源。非变性聚丙烯酰胺凝胶电泳分析表明，NADPH（而非NADH）能够驱动超氧化物的产生，氯化二亚苯基碘鎓是这种活性的有效抑制剂，但氰化物和叠氮化物没有抑制作用。这些结果证实超氧化物的产生是由于NADPH氧化酶。蜜腺酶复合物在凝胶上的迁移与叶酶复合物不同。时间表达模式表明，超氧化物的产生（NADPH氧化酶活性）与花蜜分泌、花蜜中一种超氧化物歧化酶Nectarin I的表达以及从蜜腺中克隆并鉴定为rbohD样NADPH氧化酶的蜜腺NADPH氧化酶推定基因NOX1的表达相协调。此外，原位杂交研究表明，NADPH氧化酶在花发育的早期阶段表达，尽管超氧化物在后期阶段（10期之后）产生，这意味着蜜腺中NADPH氧化酶存在翻译后调控。
