Reactive oxygen species (ROS) are produced in the olive reproductive organs as the result of intense metabolism. ROS production and pattern of distribution depend on the developmental stage, supposedly playing a broad panel of functions, which include defense and signaling between pollen and pistil. Among ROS-producing mechanisms, plasma membrane NADPH-oxidase activity is being highlighted in plant tissues, and two enzymes of this type have been characterized in Arabidopsis thaliana pollen (RbohH and RbohJ), playing important roles in pollen physiology. Besides, pollen from different species has shown distinct ROS production mechanism and patterns of distribution. In the olive reproductive tissues, a significant production of superoxide has been described. However, the enzymes responsible for such generation are unknown. Here, we have identified an Rboh-type gene (OeRbohH), mainly expressed in olive pollen. OeRbohH possesses a high degree of identity with RbohH and RbohJ from Arabidopsis, sharing most structural features and motifs. Immunohistochemistry experiments allowed us to localize OeRbohH throughout pollen ontogeny as well as during pollen tube elongation. Furthermore, the balanced activity of tip-localized OeRbohH during pollen tube growth has been shown to be important for normal pollen physiology. This was evidenced by the fact that overexpression caused abnormal phenotypes, whereas incubation with specific NADPH oxidase inhibitor or gene knockdown lead to impaired ROS production and subsequent inhibition of pollen germination and pollen tube growth.

• Keywords
• NADPH oxidase, NOX, Rboh, olive, pollen, sexual plant reproduction,
• Publication type
• Journal Article MeSH

Reactive oxygen species (ROS) produced by plant NADPH oxidases, respiratory burst oxidase homologs (RBOHs), play key roles in biotic and abiotic stress responses and development in plants. While properly controlled amounts of ROS function as signaling molecules, excessive accumulation of ROS can cause undesirable side effects due to their ability to oxidize DNA, lipids, and proteins. To limit the damaging consequences of unrestricted ROS accumulation, RBOH activity is tightly controlled by post-translational modifications (PTMs) and protein-protein interactions. In order to analyze these elaborate regulatory mechanisms, it is crucial to quantitatively assess the ROS-producing activity of RBOHs. Given the high endogenous ROS generation in plants, however, it can be challenging in plant cells to measure ROS production derived from specific RBOHs and to analyze the contribution of regulatory events for their activation and inactivation. Here we describe human embryonic kidney 293T (HEK293T) cells as a heterologous expression system and a useful tool to quantitatively monitor ROS production by RBOHs. This system permits the reconstitution of regulatory events to dissect the effects of Ca2+, phosphorylation, and protein-protein interactions on RBOH-dependent ROS production.

• Keywords
• Human embryonic kidney 293T (HEK293T), Luminol, NADPH oxidase, Respiratory oxidase homolog (RBOH),
• MeSH
• HEK293 Cells MeSH
• Kidney metabolism   MeSH
• Humans MeSH
• NADPH Oxidases * metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Gene Expression Regulation, Plant * MeSH
• Plants metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• NADPH Oxidases * MeSH
• Reactive Oxygen Species MeSH

Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), have been identified as a major source of reactive oxygen species (ROS) during plant-microbe interactions. The subcellular localization of the tobacco (Nicotiana tabacum) ROS-producing enzyme RBOHD was examined in Bright Yellow-2 cells before and after elicitation with the oomycete protein cryptogein using electron and confocal microscopy. The plasma membrane (PM) localization of RBOHD was confirmed and immuno-electron microscopy on purified PM vesicles revealed its distribution in clusters. The presence of the protein fused to GFP was also seen in intracellular compartments, mainly Golgi cisternae. Cryptogein induced, within 1h, a 1.5-fold increase in RBOHD abundance at the PM and a concomitant decrease in the internal compartments. Use of cycloheximide revealed that most of the proteins targeted to the PM upon elicitation were not newly synthesized but may originate from the Golgi pool. ROS accumulation preceded RBOHD transcript- and protein-upregulation, indicating that ROS resulted from the activation of a PM-resident pool of enzymes, and that enzymes newly addressed to the PM were inactive. Taken together, the results indicate that control of RBOH abundance and subcellular localization may play a fundamental role in the mechanism of ROS production.

• Keywords
• BY-2 cells, Nicotiana tabacum, cryptogein, protein trafficking, protein trafficking., reactive oxygen species, respiratory burst oxidase homolog D (RBOHD),
• MeSH
• Cell Membrane metabolism   MeSH
• Fungal Proteins metabolism   MeSH
• Microscopy, Confocal MeSH
• Real-Time Polymerase Chain Reaction MeSH
• NADPH Oxidases genetics   metabolism   MeSH
• Phytophthora physiology   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Nicotiana genetics   metabolism   microbiology   MeSH
• Microscopy, Electron, Transmission MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fungal Proteins MeSH
• NADPH Oxidases MeSH
• Reactive Oxygen Species MeSH
• Plant Proteins MeSH