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.

• Klíčová slova
• NADPH oxidase, NOX, Rboh, olive, pollen, sexual plant reproduction,
• Publikační typ
• časopisecké články MeSH

Hydrogen peroxide (H2O2) is known to be generated in Photosystem II (PSII) via enzymatic and non-enzymatic pathways. Detection of H2O2 by different spectroscopic techniques has been explored, however its sensitive detection has always been a challenge in photosynthetic research. During the recent past, fluorescence probes such as Amplex Red (AR) has been used but is known to either lack specificity or limitation with respect to the minimum detection limit of H2O2. We have employed an electrochemical biosensor for real time monitoring of H2O2 generation at the level of sub-cellular organelles. The electrochemical biosensor comprises of counter electrode and working electrodes. The counter electrode is a platinum plate, while the working electrode is a mediator based catalytic amperometric biosensor device developed by the coating of a carbon electrode with osmium-horseradish peroxidase which acts as H2O2 detection sensor. In the current study, generation and kinetic behavior of H2O2 in PSII membranes have been studied under light illumination. Electrochemical detection of H2O2 using the catalytic amperometric biosensor device is claimed to serve as a promising technique for detection of H2O2 in photosynthetic cells and subcellular structures including PSII or thylakoid membranes. It can also provide a precise information on qualitative determination of H2O2 and thus can be widely used in photosynthetic research.

• Klíčová slova
• EPR-spin trapping, amperometric biosensor, hydrogen peroxide, photosystem II, reactive oxygen species, superoxide anion radical,
• Publikační typ
• časopisecké články MeSH

Disentanglement of functional complexity associated with plant mitogen-activated protein kinase (MAPK) signaling has benefited from transcriptomic, proteomic, phosphoproteomic, and genetic studies. Published transcriptomic analysis of a double homozygous recessive anp2anp3 mutant of two MAPK kinase kinase (MAPKKK) genes called Arabidopsis thaliana Homologues of Nucleus- and Phragmoplast-localized Kinase 2 (ANP2) and 3 (ANP3) showed the upregulation of stress-related genes. In this study, a comparative proteomic analysis of anp2anp3 mutant against its respective Wassilevskaja ecotype (Ws) wild type background is provided. Such differential proteomic analysis revealed overabundance of core enzymes such as FeSOD1, MnSOD, DHAR1, and FeSOD1-associated regulatory protein CPN20, which are involved in the detoxification of reactive oxygen species in the anp2anp3 mutant. The proteomic results were validated at the level of single protein abundance by Western blot analyses and by quantitative biochemical determination of antioxidant enzymatic activities. Finally, the functional network of proteins involved in antioxidant defense in the anp2anp3 mutant was physiologically linked with the increased resistance of mutant seedlings against paraquat treatment.

• Klíčová slova
• ANP2, ANP3, Arabidopsis, antioxidant defense, mitogen-activated protein kinase kinase kinase, oxidative stress, proteomics, signaling,
• MeSH
• antioxidancia metabolismus   MeSH
• Arabidopsis účinky léků   genetika   metabolismus   MeSH
• biologické modely MeSH
• chromatografie kapalinová MeSH
• herbicidy farmakologie   MeSH
• imunoblotting MeSH
• MAP kinasy kinas (kinas) genetika   metabolismus   MeSH
• mutace MeSH
• paraquat farmakologie   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• proteomika metody   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• semenáček účinky léků   genetika   metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• superoxiddismutasa metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• ANP2 protein, Arabidopsis MeSH Prohlížeč
• ANP3 protein, Arabidopsis MeSH Prohlížeč
• antioxidancia MeSH
• herbicidy MeSH
• MAP kinasy kinas (kinas) MeSH
• paraquat MeSH
• proteiny huseníčku MeSH
• proteom MeSH
• reaktivní formy kyslíku MeSH
• superoxiddismutasa 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.

• Klíčová slova
• BY-2 cells, Nicotiana tabacum, cryptogein, protein trafficking, protein trafficking., reactive oxygen species, respiratory burst oxidase homolog D (RBOHD),
• MeSH
• buněčná membrána metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• konfokální mikroskopie MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• NADPH-oxidasy genetika   metabolismus   MeSH
• Phytophthora fyziologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tabák genetika   metabolismus   mikrobiologie   MeSH
• transmisní elektronová mikroskopie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fungální proteiny MeSH
• NADPH-oxidasy MeSH
• reaktivní formy kyslíku MeSH
• rostlinné proteiny MeSH