DAF-FM DA is widely used as a live staining compound to show the presence of nitric oxide (NO) in cells. Applying this stain to live zebrafish embryos is known to indicate early centers of bone formation, but the precise (cellular) location of the signal has hitherto not been revealed. Using sections of zebrafish embryos live-stained with DAF-FM DA, we could confirm that the fluorescent signals were predominantly located in areas of ongoing bone formation. Signals were observed in the bone and tooth matrix, in the notochord sheath, as well as in the bulbus arteriosus. Surprisingly, however, they were exclusively extracellular, even after very short staining times. Von Kossa and Alizarin red S staining to reveal mineral deposits showed that DAF-FM DA stains both the mineralized and non-mineralized bone matrix (osteoid), excluding that DAF-FM DA binds non-specifically to calcified structures. The importance of NO in bone formation by osteoblasts is nevertheless undisputed, as shown by the absence of bone structures after the inhibition of NOS enzymes that catalyze the formation of NO. In conclusion, in zebrafish skeletal biology, DAF-FM DA is appropriate to reveal bone formation in vivo, independent of mineralization of the bone matrix, but it does not demonstrate intracellular NO.

• Klíčová slova
• bulbus arteriosus, nitric oxide, notochord sheath, ossification, osteoblasts, zebrafish,
• MeSH
• barvení a značení MeSH
• barvicí látky metabolismus   MeSH
• dánio pruhované * metabolismus   MeSH
• kosti a kostní tkáň metabolismus   MeSH
• osteogeneze * MeSH
• oxid dusnatý metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• barvicí látky MeSH
• oxid dusnatý MeSH

Ergosterol, a principal compound of the fungal plasma membrane, is regarded as a pathogen-associated molecular pattern. In the present study, the role of salicylic acid (SA), jasmonic acid (JA) and spermine signaling pathways after ergosterol elicitation were evaluated. SA, JA and spermine production, as well as accumulation of transcripts for a lipoxygenase (NaLOX3) gene, the phenylalanine-ammonia lyase gene, selected pathogenesis-related genes (PR1, PR5), and peroxidase tPOXC1 were determined in tobacco (Nicotiana tabacum L. cv. Xanthi) in response to ergosterol elicitation. To understand the sequence of the signaling cascade, several representative steps involved in the synthesis of crucial signaling molecules were targeted using specific inhibitors. SA signaling pathway, together with calmodulin-dependent protein kinases and nitric oxide, was demonstrated to play an important role in the induction of defense-related genes following ergosterol treatment. The results suggested that nitric oxide participates in defense-related gene activation following ergosterol treatment but does not directly participate in activation of reactive oxygen species production. The induction of PR5 and tPOXC1 transcripts was found to be not fully dependent on calmodulin/Ca2+ and SA signaling, contrary to the PR1a transcript. A possible candidate for this SA-independent pathway is the spermine pathway, as elevated spermine levels were detected following ergosterol treatment.

• Klíčová slova
• 2-(4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate, Chole, DAF-FM DA, Defense, Ergo, Ergosterol, JA, Jasmonic acid, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide, N-methyl-l-arginine acetate salt, NO, NOS, PAL, PLA2, PR, ROS, RR, RT-qPCR, SA, Salicylic acid, Signaling, Spermine, Tobacco, W7, cPTIO, cholesterol, ergosterol, jasmonic acid, l-NMMA, nitric oxide, nitric oxide synthase, pathogenesis-related, phenylalanine-ammonia lyase, phospholipase A(2), reactive oxygen species, real-time quantitative polymerase chain reaction, ruthenium red, salicylic acid, tPOXC1, tobacco peroxidase C1,
• MeSH
• cyklopentany metabolismus   MeSH
• ergosterol metabolismus   MeSH
• exprese genu MeSH
• houby metabolismus   MeSH
• kalmodulin metabolismus   MeSH
• kyselina salicylová metabolismus   MeSH
• nemoci rostlin mikrobiologie   MeSH
• odolnost vůči nemocem genetika   MeSH
• oxid dusnatý metabolismus   MeSH
• oxylipiny metabolismus   MeSH
• proteinkinasy závislé na vápníku a kalmodulinu metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulace genové exprese u rostlin * MeSH
• rostlinné geny MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• signální transdukce MeSH
• spermin metabolismus   MeSH
• tabák enzymologie   genetika   metabolismus   mikrobiologie   MeSH
• vápník metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklopentany MeSH
• ergosterol MeSH
• jasmonic acid MeSH Prohlížeč
• kalmodulin MeSH
• kyselina salicylová MeSH
• oxid dusnatý MeSH
• oxylipiny MeSH
• proteinkinasy závislé na vápníku a kalmodulinu MeSH
• reaktivní formy kyslíku MeSH
• rostlinné proteiny MeSH
• spermin MeSH
• vápník MeSH

BACKGROUND AND AIMS: Nitric oxide (NO) is involved in the signalling and regulation of plant growth and development and responses to biotic and abiotic stresses. The photoperiod-sensitive mutant 7B-1 in tomato (Solanum lycopersicum) showing abscisic acid (ABA) overproduction and blue light (BL)-specific tolerance to osmotic stress represents a valuable model to study the interaction between light, hormones and stress signalling. The role of NO as a regulator of seed germination and ABA-dependent responses to osmotic stress was explored in wild-type and 7B-1 tomato under white light (WL) and BL. METHODS: Germination data were obtained from the incubation of seeds on germinating media of different composition. Histochemical analysis of NO production in germinating seeds was performed by fluorescence microscopy using a cell-permeable NO probe, and endogenous ABA was analysed by mass spectrometry. KEY RESULTS: The NO donor S-nitrosoglutathione stimulated seed germination, whereas the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) had an inhibitory effect. Under WL in both genotypes, PTIO strongly suppressed germination stimulated by fluridone, an ABA inhibitor. The stimulatory effect of the NO donor was also observed under osmotic stress for 7B-1 seeds under WL and BL. Seed germination inhibited by osmotic stress was restored by fluridone under WL, but less so under BL, in both genotypes. This effect of fluridone was further modulated by the NO donor and NO scavenger, but only to a minor extent. Fluorescence microscopy using the cell-permeable NO probe DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) revealed a higher level of NO in stressed 7B-1 compared with wild-type seeds. CONCLUSIONS: As well as defective BL signalling, the differential NO-dependent responses of the 7B-1 mutant are probably associated with its high endogenous ABA concentration and related impact on hormonal cross-talk in germinating seeds. These data confirm that light-controlled seed germination and stress responses include NO-dependent signalling.

• MeSH
• biologické modely MeSH
• cyklické N-oxidy farmakologie   MeSH
• donory oxidu dusnatého farmakologie   MeSH
• fluoresceiny analýza   MeSH
• fyziologický stres * účinky léků   účinky záření   MeSH
• imidazoly farmakologie   MeSH
• kinetika MeSH
• klíčení * účinky léků   účinky záření   MeSH
• kyselina abscisová metabolismus   MeSH
• mutace MeSH
• osmóza účinky léků   účinky záření   MeSH
• oxid dusnatý farmakologie   MeSH
• pyridony farmakologie   MeSH
• regulace genové exprese u rostlin účinky léků   účinky záření   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• S-nitrosoglutathion farmakologie   MeSH
• scavengery volných radikálů farmakologie   MeSH
• semena rostlinná účinky léků   genetika   fyziologie   účinky záření   MeSH
• signální transdukce účinky léků   účinky záření   MeSH
• Solanum lycopersicum účinky léků   genetika   fyziologie   účinky záření   MeSH
• světlo * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide MeSH Prohlížeč
• 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate MeSH Prohlížeč
• cyklické N-oxidy MeSH
• donory oxidu dusnatého MeSH
• fluoresceiny MeSH
• fluridone MeSH Prohlížeč
• imidazoly MeSH
• kyselina abscisová MeSH
• oxid dusnatý MeSH
• pyridony MeSH
• regulátory růstu rostlin MeSH
• S-nitrosoglutathion MeSH
• scavengery volných radikálů MeSH