Studies of vitality/mortality of cortex cells, as well as of the concentrations of ethylene (ETH), gibberellins (GAs), indolic compounds/auxins (ICs/AUXs) and cytokinins (CKs), were undertaken to explain the hormonal background of kinetin (Kin)-regulated cell death (RCD), which is induced in the cortex of the apical parts of roots of faba bean (Vicia faba ssp. minor) seedlings. Quantification was carried out with fluorescence microscopy, ETH sensors, spectrophotometry and ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC‒MS/MS). The results indicated that Kin was metabolized to the transport form, i.e., kinetin-9-glucoside (Kin9G) and kinetin riboside (KinR). KinR was then converted to cis-zeatin (cZ) in apical parts of roots with meristems, to cis-zeatin riboside (cZR) in apical parts of roots without meristems and finally to cis-zeatin riboside 5'-monophosphate (cZR5'MP), which is indicated to be a ligand of cytokinin-dependent receptors inducing CD. The process may be enhanced by an increase in the amount of dihydrozeatin riboside (DHZR) as a byproduct of the pathway of zeatin metabolism. It seems that crosstalk of ETH, ICs/AUXs, GAs and CKs with the cZR5'MP, the cis-zeatin-dependent pathway, but not the trans-zeatin-dependent pathway, is responsible for Kin-RCD, indicating that the process is very specific and offers a useful model for studies of CD hallmarks in plants.

• MeSH
• Cell Death MeSH
• Cytokinins metabolism   MeSH
• Kinetin pharmacology   MeSH
• Indoleacetic Acids MeSH
• Seedlings metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Vicia faba * metabolism   MeSH
• Zeatin metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokinins MeSH
• Kinetin MeSH
• Indoleacetic Acids MeSH
• Zeatin MeSH

BACKGROUND AND AIMS: Cytokinins are positive regulators of shoot development. However, it has previously been demonstrated that efficient activation of the cytokinin biosynthesis gene ipt can cause necrotic lesions and wilting in tobacco leaves. Some plant pathogens reportedly use their ability to produce cytokinins in disease development. In response to pathogen attacks, plants can trigger a hypersensitive response that rapidly kills cells near the infection site, depriving the pathogen of nutrients and preventing its spread. In this study, a diverse set of processes that link ipt activation to necrotic lesion formation were investigated in order to evaluate the potential of cytokinins as signals and/or mediators in plant defence against pathogens. METHODS: The binary pOp-ipt/LhGR system for dexamethasone-inducible ipt expression was used to increase endogenous cytokinin levels in transgenic tobacco. Changes in the levels of cytokinins and the stress hormones salicylic, jasmonic and abscisic acid following ipt activation were determined by ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). Trends in hydrogen peroxide content and lipid peroxidation were monitored using the potassium iodide and malondialdehyde assays. The subcellular distribution of hydrogen peroxide was investigated using 3,3'-diaminobenzidine staining. The dynamics of transcripts related to photosynthesis and pathogen response were analysed by reverse transcription followed by quantitative PCR. The effects of cytokinins on photosynthesis were deciphered by analysing changes in chlorophyll fluorescence and leaf gas exchange. KEY RESULTS: Plants can produce sufficiently high levels of cytokinins to trigger fast cell death without any intervening chlorosis - a hallmark of the hypersensitive response. The results suggest that chloroplastic hydrogen peroxide orchestrates the molecular responses underpinning the hypersensitive-like response, including the inhibition of photosynthesis, elevated levels of stress hormones, oxidative membrane damage and stomatal closure. CONCLUSIONS: Necrotic lesion formation triggered by ipt activation closely resembles the hypersensitive response. Cytokinins may thus act as signals and/or mediators in plant defence against pathogen attack.

• Keywords
• Cytokinin, Nicotiana tabacum, abscisic acid, hydrogen peroxide, hypersensitive response, jasmonic acid, lipid peroxidation, non-photochemical quenching, pathogenesis-related proteins, photosynthesis, salicylic acid, stomatal conductance,
• MeSH
• Alkyl and Aryl Transferases genetics   MeSH
• Cell Death MeSH
• Chlorophyll metabolism   MeSH
• Chloroplasts genetics   metabolism   MeSH
• Cytokinins genetics   metabolism   MeSH
• Dexamethasone pharmacology   MeSH
• Photosynthesis genetics   MeSH
• Plants, Genetically Modified MeSH
• Host-Pathogen Interactions * MeSH
• Plant Leaves cytology   genetics   physiology   MeSH
• Necrosis genetics   MeSH
• Oxidative Stress genetics   MeSH
• Hydrogen Peroxide metabolism   MeSH
• Lipid Peroxidation MeSH
• Plant Stomata physiology   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Plant Growth Regulators genetics   metabolism   MeSH
• Nicotiana genetics   microbiology   physiology   MeSH
• Gene Silencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• adenylate isopentenyltransferase MeSH Browser
• Alkyl and Aryl Transferases MeSH
• Chlorophyll MeSH
• Cytokinins MeSH
• Dexamethasone MeSH
• Hydrogen Peroxide MeSH
• Plant Growth Regulators MeSH