Methyl Jasmonate-Induced Cell Death in Grapevine Requires Both Lipoxygenase Activity and Functional Octadecanoid Biosynthetic Pathway

Biologia 68/5: 896—903, 2013 Section Botany DOI: 10.2478/s11756-013-0220-4

Methyl jasmonate-induced cell death in grapevine requires both lipoxygenase activity and functional octadecanoid biosynthetic pathway

Vladimír Repka,MáriaČarná &JánPavlovkin

Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9,SK-84523 Bratislava, Slovakia; e-mail: [email protected]

Abstract: Grapevine (Vitis vinifera L., cv. Limberger) leaf tissues and suspension-cultured cells were induced to undergo programmed cell death (PCD) by exogenously added methyl jasmonate (MeJA). The elicitor signaling pathway involved in MeJA-induced PCD was further investigated using pharmacological, biochemical and histological approaches. Pharmaco- logical dissection of the early events preceding the execution of MeJA-triggered PCD indicated that this process strongly depends on both, de novo protein and mRNA synthesis. Treatment of leaf discs and cell suspensions with lipase inhibitor Ebelactone B and speciﬁc lipoxygenase inhibitor Phenidone blocked MeJA-induced PCD. These results suggest that some chloroplast membrane-derived compound(s) is required for MeJA-induced PCD in grapevine. The progression of MeJA- triggered PCD may be further inhibited by the use of metabolic inhibitors of key enzymes of octadecanoid biosynthesis including AOS, AOC, and OPR indicating that the functional jasmonate biosynthetic pathway is an integral part of the MeJA-induced signal transduction cascade that results in the coordinate expression of events leading to PCD. Finally, the activation of the octadecanoid pathway, as a critical point in MeJA-induced PCD, was independently demonstrated with cellulysin, a macromolecular elicitor acting via octadecanoid signaling. The cellulysin was shown to be a very potent enhancer of MeJA-triggered PCD in grapevine cells. Key words: Vitis vinifera; cell death; oxylipin; histochemistry; inhibitors Abbreviations: AOC, allene oxide cyclase; AOS, allene oxide synthase; CHX, cycloheximide; COR, cordycepin; DIECA, diethyldithiocarbamic acid; HR, hypersensitive response; JA, jasmonic acid; LOX, lipoxygenase; MeJA, methyljasmonate; NIA, necrosis-inducing activity; OPDA, 12-oxophytodienoic acid; OPR, 12-oxophytodienoic acid reductase; qRT-PCR, quantitative real-time polymerase chain reaction; TMV, tobacco mosaic virus; TUB, tubulin.

Introduction (Repka et al. 2001, 2004) and other woody plant species (Repka 2002). Jasmonic acid (JA) and its volatile methyl ester Understanding the actual physiological roles of jas- (MeJA), collectively named “jasmonates“, are by far monates in these many diverse processes is complicated the best-studied fatty acid-derived signals in plants by the observation that intermediates in the biosyn- (Schaller & Stintzi 2009). These ubiquitously occurring thesis of jasmonate are also active in at least some of plant growth regulators are believed to act as signals these processes (Stintzi & Browse 2000). The levels of in various developmentally-regulated processes includ- JA, OPDA, and other intermediates of oxylipin synthe- ing growth inhibition, promotion of senescence and/or sis vary considerably among species, giving rise to the abscission, and induction of specific leaf proteins in suggestion that the relative and absolute concentrations monocots and dicots (Chen et al. 2011). Jasmonate sig- of different compounds – the oxylipin signature – may naling is now known to be crucial for plant stress re- provide an additional flexibility to this multifunctional sponse following wounding, chilling, ozone, UV light, in- chemical signaling system (Weber et al. 1997). sect attack, drought stress and salt stress (Weber 2002; There are a number of key players in jasmonate Schlink 2011; Ismail et al. 2012). Subsequent research signal transduction identified in Arabidopsis. The gene revealed that exogenously applied jasmonates exert dis- for the F-box protein COI1 was identified in a mu- tinct effects on gene expression and that wounding and tant screen for plants insensitive to growth inhibi- elicitors could cause JA/MeJA accumulation in plants. tion by the JA analog, coronatine, a phytotoxin pro- These results implied a role for jasmonate in plant de- duced by some pathogenic strains of Pseudomonas fense that has been confirmed (Avanci et al. 2010). More syringae (Feys et al. 1994). Mutations in the COI1 recently, a strong hypersensitive response-like inducing gene result in plants compromised in all known JA activity of MeJA has been demonstrated in grapevine responses: defense against biotic and abiotic stresses,

c 2013 Institute of Botany, Slovak Academy of Sciences Methyl jasmonate-induced cell death in grapevine cells 897 growth inhibition, and fertility (Zhang & Turner macological dissection of the octadecanoid-signaling 2008). cascade. JA-dependent gene activation involves hormone induced degradation of a transcriptional repressor, Material and methods the jasmonate ZIM/tify-domain (JAZ/TIFY) proteins (Vanholme et al. 2007). JAZ/TIFY proteins in Ara- Plant material bidopsis thaliana are encoded by 12 genes, desig- Grapevine (Vitis vinifera L. cv. Limberger) plants were grown in a growth chamber at 28 ± 1 ◦C (RH 60%) with nated JAZ1 to JAZ12 (Chini et al. 2007). JAZ/TIFY −2 −1 a14hlightperiod(30µmol m s ). Intact two-month- proteins interact with and repress activators of JA old plants, excised leaves and suspension cell cultures main- gene expression, including a transcription factor, the tained as described by Repka et al. (2000) were used for all Arabidopsis homolog of myelocytomatosis viral onco- experiments. gene (MYC), MYC2 (Lorenzo et al. 2004). Acti- vation of MYC2 induces a rapid transcription of Induction experiments early JA-responsive genes including JAZ/TIFY genes Leaf discs (d = 10 mm) from the same-aged grapevine leaves themselves. Moreover, it has been demonstrated that were cut with a cork borer and immediately transferred jasmonate and phytochrome a signaling in Arabidop- into sterile 24-well tissue culture plates (Nunc) containing sis wound and shade responses are integrated through a solution of the test substance in 25 mM HEPES-KOH JAZ1/TIFFY protein stability (Robson et al. (pH 7.6) buffer. Discs were floated with their abaxial sur- face down on 0.4 mL of test solutions. The experimental 2010). setup was maintained at 25 ± 1 ◦C (RH 60%) with a 14 h Jasmonate signaling seems to be a complex net- light period (30 µmol m−2 s−1). Methyl jasmonate (50 µM work of individual signals (jasmonates and oxylip- MeJA, Duchefa) was prepared from a stock solution in abso- ins) and recent findings on specific activities of these lute ethanol (0.1% final concentration). Commercially avail- molecules add to this picture (Weber 2002). Accord- able cellulysin (a cocktail of cellulases and endoglucanases, ingly, it is of crucial importance to identify components Sigma) was used at concentration of 50 µgmLin25mM of the jasmonate signaling pathway and especially the HEPES-KOH (pH 7.6) buffer. The inhibitors Ebelactone jasmonate receptor(s) (Yan et al. 2009). In recent years B (2-ethyl-3,11-dihydroxy-4,6,8,10,12-pentamethyl-9-oxo-6- tetradecenoic-1,3-lactone, Novabiochem GmbH, 1 mg mL), tremendous advances have been achieved. Based on the phenidone (Sigma, 10 mM), salicylic acid (Duchefa, 10 mM) structure model COI1, molecular modeling of COI1- and acetylsalicylic acid (Merck, Darmstadt, Germany, JA interactions, and the evidence that COI1 directly 10 mM) were dissolved in DMSO. Diethyldithiocarbamic binds JA-Ile/COR it was proposed that COI1 serves as acid (Sigma, 10 mM) and n-propyl gallate (Sigma, 10 mM) a receptor for jasmonate (Yan et al. 2009). The crystal were dissolved in EtOH. For experiments on effects of differ- structure of the co-receptor COI1-JAZ is also available ent inhibitors on MeJA-inducible hypersensitive response, (Sheard et al. 2010). leaf discs were pre-incubated for 17 h with the inhibitor solu- Conceptually, there are two major approaches that tions diluted to working concentration with 25 mM HEPES- are furthering our understanding of jasmonate signal- KOH (pH 7.6) buffer. ing. First, jasmonate-insensitive mutants and mutants The RNA and protein synthesis inhibitors cordycepin (COR) and cycloheximide (CHX), respectively, diluted to with a constitutive jasmonate response have given us working concentration with 25 mM HEPES-KOH (pH 7.6) new insights into the mode of action of jasmonates buffer were added to the leaf discs 4 h prior to the addition (Weber 2002). For example, the jasmonate biosynthesis of MeJA. mutant opr3 in Arabidopsis thaliana allowed the dissection of cyclopentanone and cyclopentenone signaling, Biological assays thus defining specific roles for these molecules (Stintzi Necrosis-inducing activity (NIA) of MeJA and various in- & Browse 2000). Second, jasmonate signaling and jas- hibitors and/or stimulators of the octadecanoid pathway monate biosynthesis have been studied by identification wasassayedonleafdiscsincubatedinagrowthchamber of biosynthetic enzymes, determination of endogenous under conditions described above. Routinely, MeJA was applied at the concentrations indicated as 0.01 mL droplets jasmonate levels and use of inhibitors (Kramell et al. on leaf discs (one drop per disc). As an alternative ap- 2000). proach, discs with necroses were laid flat on dark screen and The grapevine leaves and cells used in this study photographed on slide film beside a reference ruler. Slides have previously been characterised with respect to their were projected onto a digitizing tablet (model 1212, Kurta), responses to various elicitors which induced changes in and necroses were measured directly using SigmaScan soft- ion fluxes across plasma membrane, ROS production, ware (Jandel Scientific). If not stated otherwise, NIA inhibi- biosynthesis of ethylene, SA etc. (Repka et al. 2000; tion data represent results from three independent replicates Repka 2001, 2002) and methyl jasmonate was found containing 10 leaf discs per treatment. to be a potent elicitor of multiple defense responses in grapevine leaves and cell suspension cultures (Repka Tissue processing, histochemistry and microscopy Leaf material for sectioning was fixed in HistoChoice MB so- 2004). ◦ lution (Amresco) for 24 h at 4 C. The fixed materials were In the present work we focused on an attempt dehydrated with ethanol and embedded in paraffin (Para- to unravel the signal transduction pathways and com- plast + , Sigma). Embedded tissues were sectioned on a mi- ponents involved in jasmonate-induced hypersensitive crotome (Vibratome, model 3000+, The Vibratome Co.) to response-like cell death in grapevine by the use of phar- be 10 µm thick and transferred to slides coated with 0.1% 898 V. Repka et al. poly-L-lysine (Poly-Prep slides, Sigma). The serial sections aoc1-reverse (5’-CAAGAACTTTACGTACGTTATCGA on the slides were deparaffinized with HistoChoice (Am- AG-3’) and aoc1-forward (5’-dT23(C/G)(A/C/G/T)-3’) resco, Solon, USA), hydrated sequentially in 100%, 96% and GenBank accession no. BAA95763); opr3-reverse (5’-CCCG 70% ethanol (5 min per wash), followed by a final wash GGATCCGATTCAGAGGCGGGAAGAAGGAGCCAAG- (2 times for 5 min each) in 1X PBS (10X PBS = 75 mM 3’) and opr3-forward (5’-CCCGGGTCTAGAATTCATGA NaH2PO4,25mMNa2HPO4, 145 mM NaCl, pH 7.4). For CGGCGGCACAAGGAACTC-3’) (GenBank accession no. anatomical comparisons, sections were counterstained with AT2G06050). 0.5 % Nuclear Fast Red (Trevigen) for 1 min, briefly washed The temperatures were as follows: denaturation 94 ◦C with distilled water, and viewed under bright field. For for 1 min, primer annealing 56 ◦C for 2 min, polymerase ex- ◦ detection of autofluorescence, sections were processed as tension 72 C for 3 min and the MgCl2 concentration was above, the glass slides were cover-slipped with Fluoromount- 1.5 mM. The number of cycles was 20. To exclude DNA G (Merck) media, and examined using a Leica DMIRB epi- contamination, extracted poly(A)+ RNA was treated with fluorescence microscope (Leica). Images were obtained us- DNase I (Gibco). The reactions were run on an Eppendorf ing a Leica DC480 CCD camera (11.5 MP, Leica) coupled to MastercyclerTM ep realplex2 qRT-PCR cycler (Eppendorf). the microscope, and the digital pictures were processed us- Three biological replicates along with two technique repli- ing the software Adobe Photoshop CS4 (Adobe) and printed cates were conducted for each mRNA. Following qRT-PCR, with a colour printer HP DeskJet 5500C (Hewlett-Packard). differences in mRNA expression levels were assessed by an- alyzing the mean CT values. TUNEL-in situ assay For the PCR amplification and loading controls, the The embedded tissues were sectioned with a microtome (Vi- same template cDNA was amplified using primers specific bratome, model 3000+, Vibratome Co., St. Louis, USA) to for the constitutive tubulin (tub) gene based on sequences µ be 7 m thick and transferred to slides coated with 0.1% of Arabidopsis (GenBank accession no. M21415). Ten mi- poly-L-lysine (Poly-Prep slides, Sigma). The serial sections croliters of the PCR product was dotted using dot blot ap- on the slides were deparaffinized with HistoChoice (Am- paratus (PR648, Hoefer Scientific Instruments) and stained resco), hydrated sequentially in 100%, 96% and 70% ethanol with ethidium bromide. The image obtained was digitized (5 min per wash), followed by a final wash (2 times for 5 min and edited with Adobe Photoshop CS4 (Adobe). each) in 1X PBS. The samples on the slides were permeabilized with Cytonin for 30 min at 18–24 ◦C, washed with DNase-free water (2 × 2 min) and immersed for 5 min in Cell death quenching solution (3% H2O2 in absolute methanol) to re- For the assay of cell death, grapevine cell suspensions were move endogenous peroxidase. incubated for 15 min with 0.05 % Evans blue (Sigma, An in situ apoptosis detection kit apoTACS Blue (Tre- Deisenhofen, Germany) and then washed extensively to re- vigen) was used to detect the nuclear DNA fragmenta- move excess and unbound dye. Dye bound to dead cells was ◦ tion according to the protocol provided by the manufac- solubilized in 50% methanol with 1% SDS for 30 min at 50 C turer. Permeabilized sections were incubated at 37 ◦Cfor2 and quantified by absorbance according to Repka (2002). h with the reaction mixture containing terminal deoxynu- cleotidyltransferase (TdT), 1X Labeling buffer and biotiny- lated dNTP and then were rinsed with 1X TdT Stop buffer Results and discussion to stop labeling reaction. After washing the slides in deion- ized water (2 × 2 min), the section were covered with Effect of MeJA treatment on induction of the octa- ◦ streptavidin-HRP solution for 30 min at 18–24 C, devel- decanoid-signaling pathway oped with TACS Blue label (Trevigen) while the signal was The lipid-based signaling pathway is composed of at optimal and finally counterstained with Nuclear Fast Red. least four structurally different types of compounds, The positive signal showed the dark blue precipitate. The experimental positive and negative controls were the TACS- probably endowed with signaling qualities: (1) acyclic Nuclease-treated and untreated samples, respectively. All fatty acids and functionalized derivatives; (2) cyclopen- the photographs were taken with a Provis AX-70 (Olym- tanoid C18 fatty acids;(3) cyclopentanoid C12 fatty pus) microscope equipped with a Progressive-3 CCD camera acids such as (+)-7-iso-JA and (-)-JA; and (4) amino (Sony) and the digital pictures were processed using a Pho- acid conjugates of the intermediates of the Vick & Zim- toshop CS4 software (Adobe). merman cascade (Fig. 1, Vick & Zimmerman 1984). To demonstrate the induction of the octadecanoid- qRT-PCR analysis of gene expression signaling pathway in response to exogenous MeJA Total RNA was extracted from 0.5 g of leaf material µ stored in RNA Later solution by using an RNAWIZ (50 M) treatment we used qRT-PCR. Treatment of (Ambion) isolation reagent as described in Repka et al. grapevine leaves in sealed containers with MeJA stim- (2001). The poly(A)+ RNA was isolated using Oligo- ulate expression of genes encoding the key enzymes of tex suspension and mini-spun columns (Qiagen) accord- jasmonate biosynthetic pathway namely lox, aos, aoc ing to the manufacturer’s instructions. Quantitative data and opr3 (Fig. 2). The specificity of this response was for gene expression were obtained by qRT-PCR with a further verified by the quantitation of mRNA levels for SuperScript One Step qRT-PCR system (Life Technolo- the same set of genes following treatment of grapevine gies) in presence of forward and reverse primers. The leaves with cellulysin (Fig. 2), a macromolecular elici- following list of gene-specific primers were used: lox3- tor acting via octadecanoid signaling (Piel et al. 1997). reverse (5’-CAGCCATATCTCCCAAGTGAA-3’) and lox3- forward (5’-TCTCGAGGCATATGTTTT-3’)(GenBank ac- Interestingly, we found that irrespective of their molec- cession no. AJ249794); aos-reverse (5’-CTTTTACCGGTA ular structure and concentration used both, MeJA and CTTACATGCCG-3’) and aos-forward (5’-GAGCTTGTAT cellulysin upregulated the expression of either genes al- CTGCGGATTCGTC-3’) (nonhem-probe amplification); most to the same extents. Methyl jasmonate-induced cell death in grapevine cells 899

Fig. 2. Dot blot analysis of qRT-PCR products of the key jasmonate biosynthetic genes either in water-treated cells, cells pre- treated with cellulysin (macromolecular elicitor) or treated with 50 µM MeJA. Accumulation of the constitutively expressed tubulin (tub) gene was incorporated to the experiment as a control.

Jasmonate-induced octadecanoid signaling and cell Fig. 1. Simplified biosynthetic pathway and intracellular fluxes death require both, de novo RNA and protein bio- of jasmonates in the plant cell. Key enzymatic steps are shown synthesis on the left side and their putative inhibitors (hatched circles) are The mechanism by which the octadecanoid signal is shownontherightside. generated and subsequently transduced to affect cell death is not known. To begin to address this ques- tion, we studied the effect of inhibitors of RNA and protein biosynthesis on MeJA-induced cell death response. Treatment of grapevine leaf discs with two different concentrations of the cordycepin (COR), which

Fig. 3. Effect of treatment with cordycepin and cycloheximide on MeJA-stimulated cell death in excised grapevine leaf discs. A – time course of the cell death symptom appearance; B – time course of necrosis inducing activity (NIA). 900 V. Repka et al. is known to act as chain terminator of plant RNA synthesis (Seeley et al. 1992; Zhu et al. 1995), resulted in the complete blocking of MeJA-stimulated necrosis- inducing activity (NIA, Figs 3A, B). Moreover, obtained results also suggest that increased RNA turnover may be a general feature of MeJA-induced death response. We also tested the effect of protein synthesis inhibitor cycloheximide (CHX) on the induction of NIA by exogenous MeJA. Addition of CHX at 25 µM substantially reduced the extent of MeJA-triggered NIA (Fig. 3A) corresponding to 25% of that observed in MeJA-treated leaf tissue not pretreated with CHX (Fig. 3B). By contrast, at 50 µM, CHX did not attenuate MeJA-stimulated NIA by a concentration- dependent manner as it was expected and overall inhibitory effect reached the value 50% of that observed in tissues not treated with CHX prior to application of 50 µM MeJA (Fig. 3B). However, it is interesting to note that both concentration of CHX significantly delayed the MeJA-stimulated cell death response, sug- gesting the requirement de novo biosynthesis of specific protein(s). In this context it is important to note our earlier study with tunikamycin, an inhibitor of gly- cosylation, indicating that MeJA-stimulated cell death also strongly depends on a post-translational modifi- cation of some types of proteins (Repka & Fischerová 2001). Moreover, a series of recent breakthroughs has identified a set of diverse proteins, the SCF complex, Fig. 4. Pharmacological analysis of MeJA-induced cell death using leaf discs assay. Discs excised from grapevine leaves were in- as central components in the perception and transcrip- cubated with respective inhibitors for 17 h prior to treatment tional response to jasmonate (Gfeller et al. 2010). Fur- with 50 µM MeJA. Necrosis inducing activity was observed at thermore, upstream of the signaling events diverse sets indicated intervals. Preincubation with cellulysin, water, EtOH, of microRNAs negatively regulates either the produc- DMSO, and HEPES served as a control. tion of jasmonate-derived signals (Gfeller et al. 2010) or jasmonate-triggered cell death (Repka 2008). plast membranes by the DAD1-like lipase 6, galactoli- Pharmacological analysis of MeJA-induced cell death pase DONGLE (DGL) which like DAD1 is a member of In an attempt to unravel the critical points in the signal the PLA-I family of lipases (Ishiguro et al. 2001; Hyun transduction pathways leading to cell death upon elic- et al. 2008). More recently, precise analysis of Arabidop- itation with exogenous MeJA, a number of compounds sis mutants defective in the expression of lipases with known to affect subsequent steps in octadecanoid sig- predicted chloroplast localization identified a phospho- naling pathway were used. Pre-incubation of grapevine lipase A (PLA) PLA-Iγ1 as a novel target gene to ma- leaf discs in the presence of 100 µMEbelactoneB,an nipulate jasmonate biosynthesis (Ellinger et al. 2010). inhibitor of lipases and some methylesterases, inhibited Biochemical and pharmacological analyses also revealed the appearance of cell death response (Fig. 4) and the that the lipase activity of grasshopper oral secretions NIA (Fig. 5) following elicitation with 50 µMMeJA. (GS) plays a central role in the GS-induced accumu- Support for the involvement of a lipolytic activity in the lation of oxylipins, especially OPDA, which could be biosynthesis of jasmonates came from the observations fully mimicked by treating puncture wounds only with of Mueller et al. (1993) and Conconi et al. (1996) that a lipase from Rhizopus arrhizus (Schäfer et al. 2011). elicitation or wounding of plants caused rapid and tran- Phenidone (1 mM), a specific inhibitor of lipoxyge- sient changes in the lipid composition of plant cell mem- nases (LOX), completely blocked both MeJA-stimula- branes. Phospholipase A2 (PLA2, Conconi et al. 1996) ted cell death and NIA even though the leaf discs were is located in the plasma membrane and can not directly treated with MeJA for 4 h (Figs 4 and 5). Similarly, attribute to JA biosynthesis which takes place at least phenidone inhibited the induction of JAZ1/TIFY10a in the first part in chloroplasts. Moreover, characteriza- by salt stress very efficiently, especially in Vitis ru- tion of the dad1 (defective in anther dehiscence1) mu- pestris (Ismail et al. 2012). Collectively, these results tant of Arabidopsis thaliana led to the identification of suggest that JA signaling (more specifically JA biosyn- the lipase specifically involved in the biosynthesis of jas- thesis) is necessary to mediate MeJA-stimulated cell monate during plant development (Ishiguro et al. 2001). death and the induction of JAZ1/TIFY10a expression In leaves α-LeA is released from galactolipids of chloro- in response to NaCl. Several studies focused on the Methyl jasmonate-induced cell death in grapevine cells 901

Fig. 5. Comparative analysis and quantification of inhibition of necrosis inducing activity, expressed as the extent of necrotic lesion area, by respective inhibitor of the key enzymes of jasmonate biosynthetic pathway. Preincubation with cellulysin, water, EtOH, DMSO, and HEPES served as a control. Error bars represent ± SE. lipoxygenase (LOX) pathway in the wound response in way was actually fully functional in our semi-in vitro Solanaceae. The data indicate that LOX supplies fatty system. acid hydroperoxides as substrates for jasmonate synthesis (Halitschke & Baldwin 2003). Interestingly, an- Histological analysis of MeJA-induced cell death tisense depletion of a specific LOX isoform in potato To verify whether there is an absolute requirement compromised the wound-induced expression of defense for lipoxygenase activity and functional octadecanoid- genes and increased susceptibility to herbivore attack signaling pathway in MeJA-induced cell death in vivo, (Royo et al. 1999). A stroma-localized plastidial LOX the leaves of intact grapevine plants were locally pre- (LOX2) was previously proposed to be responsible for treated with water (control), Ebelactone B (100 µM) the wound-induced biosynthesis of JA in Arabidopsis or phenidone (1 mM) prior to elicitation with 50 µM (Bell et al.1995). MeJA. The current model of phyto-oxylipin synthesis de- In control, water-treated, leaves there were no vi- picts a number of different enzymes, each acting on hy- sual signs of ultrastructural changes that accompany droperoxide substrates whose activity may control the programmed cell death during hypersensitive-like re- flux into the respective metabolic pathway (Schaller sponse (Figs 6A, B). Striking differences were observed et al. 2005). Thus, the inhibitor of allene oxide syn- in sections of leaves treated locally with 50 µMMeJA thase (AOS), DIECA (1 mM) significantly affected cell for 2 h. In these leaves, cell death is initiated and prop- death response in case that leaf tissues were elicited agated through parenchyma cell layers rich in chloro- with 50 µM MeJA for 2 h but did not dramatically plasts, i.e. pallisade mesophyll and spongy mesophyll affect this response when MeJA treatment was pro- (Fig. 6C). As revealed by the UV epi-illumination, longed for 4 h (Figs 4 and 5). A similar response was dead, collapsed cells appeared as non-transparent and noticed with n-propyl gallate (1 mM), a potent in- highly fluorescent due to an accumulation of tannins hibitor of allene oxide cyclase (AOC) and with salicylic and other phenolic compounds (Fig. 6D). Administra- acid (SA, 1 mM) which blocks the conversion of 13-S- tion of 100 µM Ebelactone B for 17 h before treat- HPLA to 12-OPDA (Figs 4 and 5). Interestingly, un- ment of leaves with 50 µM MeJA substantially inhibited like SA, pretreatment of the grapevine leaf discs with the extent of jasmonate-triggered cell death (Figs 6E, acetylsalicylic acid (ASA, 1 mM) completely abolished F). A more profound effect was observed when leaves death-inducing activity of exogenous MeJA (Figs 4 and were pretreated for 17 h with a specific lipoxygenase 5). Obtained results are in a good accordance with inhibitor, phenidone (1 mM). As shown in Fig. 6G and the recently published results of Ismail et al. (2012). H, this compound completely blocked of jasmonate- They demonstrated that pre-treatment of grapevine stimulated progression of hypersensitive-like cell death. cells with 1mM ASA completely suppressed the in- To exact prove the inhibitory effect of Ebelac- duction of JAZ/TIFFY transcripts by salt stress, in- tone B and phenidone on MeJA-induced cell death, dicating that the response of transcription requires JA we have performed an in situ TUNEL assay in par- signaling. As expected, pretreatment of grapevine leaf allel experiment. Results obtained by this technique discs with cellulysin (50 µgmL−1) strongly activated clearly demonstrate no positive signal in control (un- MeJA-induced cell death by a time-dependent man- treated) leaves (Fig. 6I) while the exogenous application ner, indicating that the octadecanoid-signaling path- of MeJA induced extensive cell death (Fig. 6J). Pre- 902 V. Repka et al.

Fig. 6. Histology of control (healthy) leaf (A, B), 50 µM MeJA-treated leaf (C, D), leaf pre-treated first with Ebelactone B and then with 50 µM MeJA (E, F), leaf pre-treated first with Phenidone and then with 50 µM MeJA (G,H). Specimens were counterstained either with Nuclear Fast Red (A,C,E,G) or viewed using epifluorescence (B,D,F,H); ade, adaxial epidermis; pp, pallisade mesophyll; sm, spongy mesophyll; abe, abaxial epidermis. Scale bar = 300 µm. In situ TUNEL assay of control (healthy) leaf (I), 50 µM MeJA-treated leaf (J), leaf pre-treated first with Ebelactone B and then with 50 µM MeJA (K), leaf pre-treated first with Phenidone and then with 50 µM MeJA (L). Scale bar = 300 µm.

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