JXB Advance Access published online on December 7, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm263
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Jasmonates and its mimics differentially elicit systemic defence responses in Nicotiana attenuata
1Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena, Germany
2Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena, Germany
3University of Warmia and Mazury in Olsztyn, Faculty of Biology, Chair of Plant Physiology and Biotechnology, Oczapowskiego 1A, 10-719 Olsztyn, Poland
* To whom correspondence should be addressed. E-mail: Baldwin{at}ice.mpg.de
Received 18 August 2007; Accepted 24 September 2007
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Abstract |
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Coronalon (6-ethyl indanoyl isoleucine), a synthetic jasmonate mimic, is known to regulate levels of transcripts and secondary metabolites that are commonly elicited by methyl jasmonate (MeJA) in a variety of plants. The ability of coronalon and its derivative (In-L-Ile-Me) to elicit MeJA-activated transcriptional and defence responses [nicotine and trypsin proteinase inhibitors (TPIs)] was compared in treated and systemic untreated tissues of wild-type (WT) and NaLOX3-silenced Nicotiana attenuata plants which are unable to activate either local or systemic defence responses. Coronalon and its derivative significantly regulated 71% and 86% of genes up-regulated by MeJA and 53% and 66% of the genes down-regulated by MeJA in the treated leaves, but only 3% and 7% of all regulated genes in untreated, but phylotactically connected, leaves of WT plants. Consistent with their ability to elicit transcriptional responses in treated tissues, coronalon and In-L-Ile-Me increased nicotine and TPIs when applied to the tissues in which these metabolites are produced, namely roots and leaves. However, treating roots elicited TPI activity in leaves in both WT and NaLOX3-silenced plants, suggesting that mimics can be transported apoplastically from roots to leaves in the xylem. This response was lower in NaLOX3-silenced plants, suggesting that the ability of coronalon and In-L-Ile-Me to elicit TPI responses in leaves after root treatments requires intact jasmonic acid (JA) signalling. Treating leaves did not elicit detectable changes in endogenous JA levels but did decrease free salicylic acid contents. It is concluded that coronalon and In-L-Ile-Me elicit jasmonate responses in treated tissues and could be valuable tools for dissecting local and systemic jasmonate signalling networks in plants.
Key words: Coronalon, 6-ethyl indanoyl isoleucine, indanoyl isoleucine conjugates, MeJA-induced response, Nicotiana attenuata, nicotine, trypsin protease inhibitors
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Introduction |
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Jasmonic acid (JA) and its volatile methyl ester (MeJA) or amino acid conjugates are known as jasmonates. Synthesized via the octadecanoid pathway, these fatty acid derivatives mediate plant defence responses to herbivores and pathogens, and are involved in developmental processes, senescence, and abiotic stress responses (Creelman and Mullet, 1997; Muller, 1997; Halitschke and Baldwin, 2005). For example, in sweet basil (Ocimum basilicum L.), the total phenolic content increased significantly after plants were treated with MeJA (Kim et al., 2006). MeJA treatment increased the production of terpenoid indole alkaloids in Catharanthus and Cinchona (Aerts et al., 1994) and enhanced the production of these secondary metabolites in the treated tissues of tomato (Chen et al., 2006). Jasmonates are known to control the production and the emission of volatiles from many plants (Boland et al., 1995).
Recently many new synthetic functional mimics of octadecanoid-derived signals have been created to investigate their effects on plant secondary metabolite biosynthesis and on plant stress physiology. Fluoro- and hydroxyl-containing jasmonates have been used to elicit Taxus cell cultures (Qian et al., 2004, 2005), and coronalon, a structural mimic of coronatine, and its derivative were used as elicitors in seven species including the agronomically important tobacco, tomato, barley, and soybean (Schüler et al., 2001, 2004; Hu et al., 2005; Mithöfer et al., 2005). If synthetic jasmonate mimics and endogenous jasmonates elicit similar responses and are not catabolized in the way that endogenous jasmonates are, these jasmonate mimics can be used as tools to examine the role jasmonates play in eliciting systemic responses in unelicited tissues.
Plants have evolved an array of defences to thwart attack from herbivores. Defence responses are activated not only in the attacked tissues but also in distal, unwounded parts of the plant. These systemic defence responses have been thoroughly studied in tomato (Solanum esculentum), where an 18 amino acid peptide, named ‘systemin’ for its role in activating systemic responses (Pearce et al., 1991), is processed from a larger precursor, prosystemin, in vascular phloem parenchyma cells (Narvaez-Vasquez and Ryan, 2004). When applied to unwounded tomato plants at very low levels (fmol per plant), systemin elicits the accumulation of proteinase inhibitor (PI) I and II. These PIs act as anti-nutritive defence compounds. Data from grafting experiments between wild-type tomato plants and mutants that are either deficient in JA production (acx1), JA insensitive (jai1), or defective in systemin activity (spr1), as well as from transgenic tomato plants that constitutively express prosystemin, demonstrate that systemic signalling requires the plant to produce JA at the site of wounding and to be able to perceive a JA-based signal in the distal leaf (Howe, 2004; Schilmiller and Howe, 2005). The mobile signal that elicits PI activity in distal, unwounded tomato leaves is clearly JA based, but which jasmonate is transported from damaged to undamaged tissues to activate defence responses? Determining which jasmonate mediates systemic signalling is complicated by the fact that plants produce many biologically active jasmonates.
Herbivore attack and exogenous application of JA and MeJA to the native tobacco Nicotiana attenuata increase the accumulation of two important kinds of defence metabolites, nicotine and trypsin proteinase inhibitors (TPIs), in treated leaves and systemically in untreated leaves. Nicotine, which is synthesized in the roots after leaf damage and is subsequently transported to leaves in the xylem stream, is a systemically elicited response (Baldwin, 1999; Ohnmeiss and Baldwin, 2000; Winz and Baldwin, 2001). Jasmonate treatments and herbivore attack also increase the systemic emission of volatiles (Halitschke and Baldwin, 2003) as well as dramatically altering gene expression in both attacked and unattacked leaves: housekeeping genes mainly involved in photosynthesis are down-regulated; JA-responsive genes mainly involved in defence signalling processes [lipoxygenase (LOX); ACC oxidase (ACO)] and secondary metabolism [e.g. a suite of PI genes; polyphenol oxidase (PPO); S-adenosylmethionine decarboxylase (SAMDC)] (Lou and Baldwin, 2004) are up-regulated. All of these systemic responses appear to require intact JA signalling in N. attenuata, as they are not found in plants which have been transformed to silence the particular lipoxygenase (NaLOX3) that supplies fatty acid hydroperoxides for JA biosynthesis (Halitschke et al., 2004). Because exogenous jasmonate treatments are thought to elicit endogenous jasmonate production in a number of plants (Wasternack, 2004), how to interpret the results from exogenous jasmonate treatments is unclear.
Here the effect of jasmonates (JA and MeJA) and of coronalon (a synthetic 6-ethyl indanoyl isoleucine conjugate) and its derivative (the unsubstituted indanoyl isoleucine: In-L-Ile-Me) on local and systemic defence responses and transcriptional changes was analysed in wild-type (WT) and NaLOX3-silenced N. attenuata plants.
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Materials and methods |
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Plant growth
WT N. attenuata Torr. ex Wats (synonymous with Nicotiana torreyana Nelson and Macbr.) seeds, collected in Utah, USA, and selfed for 17 generations, were germinated in smoke-treated soil. Germinated plants were grown in an individual 1-litre hydroponic chamber as described by Hermsmeier et al. (2001). After 10–14 d of growth in 1-litre hydroponic chambers, plants received an additional 28 mg of N as KNO3. All plants were grown in a growth chamber under the following conditions: 28 °C/16 h light, 25 °C/8 h dark.
Treating the plants and harvesting the samples
For microarray analysis, 20 µl of lanolin containing 100 µg of MeJA or jasmonate mimics were applied to leaves at nodes 0, +1, and +2, where leaves at node 0 are undergoing the source–sink transition and leaves at node +2 are fully expanded source leaves of rosette stage N. attenuata plants; controls were treated with 20 µl of pure lanolin (see Supplementary Fig. S1 at JXB online for the complete hybridization scheme). After 24 h, two treated leaves (0 and +1) and two younger leaves (–1 and –2) were harvested, flash-frozen in liquid nitrogen, and stored at –80 °C until RNA extraction.
To analyse secondary metabolites, 100 µg of MeJA, In-L-Ile-Me, or 6-ethyl-In-L-Ile-Me were applied in 20 µl of lanolin (leaf treatments) or in 1 ml of 10% ethanol solution (root treatments) in different experiments to elicit different tissues. Lanolin paste containing MeJA or lanolin-soluble jasmonate mimics were applied to the first fully expanded rosette stage leaf of N. attenuata (leaf +1); control plants were treated with 20 µl of pure lanolin. Ethanol solutions were applied to the hydroponic medium when N. attenuata plants were in the rosette-stage; control plants were treated with 10% ethanol. In experiments with polar elicitors, 20 µl of aqueous solution of elicitors were immediately applied to puncture wounds on the leaf lamina produced by rolling a pattern wheel across twice the length of the leaf parallel to the midrib. After 96 h, treated leaves (+1) and roots were harvested, flash-frozen in liquid nitrogen, and stored at –80 °C until analysis. To measure TPI activity in systemic leaves, one younger (–2) leaf and one older (+6) leaf were compared with the treated leaf. These leaves are all connected to the treated leaves via orthostichous vascular connections (Schittko et al., 2001; Schittko and Baldwin, 2003). The level of nicotine in the systemic response was measured in leaf +1. When roots were treated, leaf +2 was analysed for systemic responses.
Microarray hybridization and RNA extraction
RNA was extracted as described in the TRI reagent protocol (Sigma, Taufkirchen, Germany). Isolating poly(A)+ RNA from total RNA, generating cDNAs, fluorescent labelling, hybridization, scanning, and quantifying hybridized arrays were performed as described by Halitschke et al. (2004). In all cases, RNA from treated plants was hybridized against RNA from control plants grown at the same time and at the same location, and harvested from leaves growing at the same nodes. The cDNAs hybridized to an individual array were produced from RNA extracted individually from the four plants. The microarray analysis was performed with a custom microarray which contained 50mer oligonucleotides from herbivore-regulated genes. The normalized data of the two microarrays per treatment were analysed. The mean expression ratio (ER) was calculated from the two microarrays, and transcripts were defined as being significantly regulated when the following criteria were fulfilled: (i) the ER was significantly different from 1 as determined by a Student's t-test (P <0.05); and (ii) the ER exceeded the thresholds of –1.5 and 1.5 for down- and up-regulation, respectively.
Analysis of nicotine and TPIs
Nicotine was extracted and quantified by HPLC as described in Keinanen et al. (2001). Trypsin proteinase activity was analysed by radial diffusion activity as described in van Dam et al. (2001).
Analysis of JA and SA levels
About 200 mg of tissues from each sample was homogenized on a FastPrep homogenizer (Thermo Electron) with 1 ml of ethyl acetate spiked with 200 ng of 1,2-[13C]JA and D4-salicylic acid (SA) in FastPrep tubes. After being centrifuged at 16 000 g for 10 min at 4 °C, the supernatants were transferred to fresh 2 ml Eppendorf tubes, and the pellet was re-extracted with 1 ml of ethyl acetate and centrifuged. The supernatants were combined and evaporated completely on a vacuum concentrator. The residue was resuspended in 0.5 ml of 70% methanol (v/v) and centrifuged at 20 000 g for 5 min. The supernatants were then used to analyse JA and free SA using the 1200L LC/MS system (Varian, Palo Alto, CA, USA). A 15 µl aliquot of each sample was injected onto a Pursuit C8 column (3 µm, 150x2 mm, Varian) at a flow rate of 0.1 ml min–1. A gradient of mobile phase composed of solvent A (0.05% formic acid) and solvent B (0.05% formic acid in methanol) was used for separation and a negative ESI mode was used for detection. Ions with m/z at 209 and 211 generated from endogenous JA and internal standard, respectively, were fragmented under a 12 V collision energy. The ratios of ion intensities of their respective daughter ions, m/z 59 and 61, were used to quantify endogenous JA. Ions with m/z at 137 and 141 generated from endogenous SA and an internal standard, respectively, were fragmented under a 15 V collision energy. The ratios of ion intensities of their respective daughter ions, m/z 93 and 97, were used to quantify endogenous SA.
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Results |
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The N. attenuata system was used to investigate the response induced by jasmonates (MeJA and JA) and their two mimics, coronalon (6-ethyl-In-L-Ile-Me, Fig. 1) and the unsubstituted indanoyl isoleucine (In-L-Ile-Me, Fig. 1); both the synthesis of nicotine and the activity of TPIs are well understood. Elicitors were applied to the leaves (using a lanolin paste and an aqueous solution, respectively) of plants grown in soil or in liquid medium and to the roots (aqueous solution) of hydroponically grown plants. Local responses were elicited in treated leaves (TPIs and transcript accumulation) and in treated roots (nicotine; Fig. 2A); systemic responses were elicited in treated leaves (nicotine accumulation) and untreated leaves (TPIs and transcript accumulation; Fig. 2B). Experiments were first conducted on WT plants whose jasmonate signalling was intact and subsequently on plants expressing NaLOX3 in an antisense orientation (asLOX3), which are deficient in jasmonate signalling (Halitschke and Baldwin, 2003).
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The local effects of coronalon and its derivative on defence metabolites
Levels of nicotine and TPIs in soil-grown and hydroponic plants were measured 96 h after treatment. To establish if coronalon and its derivative can easily enter the plant cell, those compounds were applied as a lanolin paste (Fig. 3A; ANOVA: for leaves at node +1, F3,16=10.0130, P=0.0006) and as an aqueous solution (Fig. 3B; ANOVA: for leaves at node –2, F5,18=3.6644, P=0.0184; for leaves at node +1, F5,18=7.8350, P=0.0005). The level of defence metabolites in the local response was established by measuring TPI activity and nicotine in treated tissues (in leaf +1 and in the roots, respectively). Because nicotine is transported from the roots to the leaves after being synthesized, it was also measured in the leaves in the case of root treatment (Fig. 3C; ANOVA: for leaves at node +2, F4,15=24.9915, P <0.0001; for root, F4,15=3.8715, P=0.0236). TPIs and nicotine were induced after treatment with MeJA and jasmonate mimics (Fig. 3). Jasmonate mimics appear to induce TPIs more strongly than does MeJA. Among the jasmonate treatments, the most TPI activity was obtained when JA was applied as an aqueous solution to puncture wounds. The largest increase in nicotine was obtained after roots were elicited with MeJA.
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The systemic effects of coronalon and its derivative on defence metabolites
Initially, the elicitation of MeJA-induced defence metabolites was explored in untreated (systemic) leaves after leaf treatment. In leaves, which were connected to the treated leaves via orthostichous vascular bundles, only treatment with MeJA significantly elicits TPI activity in an upward direction toward younger leaves (at node –2) and downward in the plant toward older leaves (at node +6) (Fig. 3A; ANOVA: for leaves at node –2, F3,16=4.987, P=0.0125; and for leaves at node +6, F3,16=7.406, P=0.0025). When the elicitors were applied in lanolin paste, treatment with In-L-Ile-Me, but not coronalon, also elicited systemic increases in TPIs in untreated younger leaves (at node –2) (Fig. 3A). However significant changes in TPI activity, in younger systemic leaves, were not observed when the jasmonates were applied as aqueous solution to the puncture wounds. In this case, increased TPI activity could result from an increased pool of JA after leaf damage (Fig. 3B).
To explore more thoroughly whether coronalon and its derivative elicit the same systemic responses as MeJA, nicotine (Fig. 4A; ANOVA, F3,16=103.7648, P <0.0001, and 4B; ANOVA, F5,24=58.741, P <0.0001) and TPI levels (Fig. 4C; ANOVA, F4,15=13.728, P <0.0001) were measured in leaves after leaf or root treatments. As shown in Fig. 4A and B, nicotine levels increased only when the plants were treated with MeJA or JA. Coronalon and its derivative did not increase the level of nicotine when either a lanolin paste or an aqueous solution was applied to leaves. This demonstrates that although coronalon and its derivative can elicit nicotine when applied directly to roots (Fig. 3C), these compounds are not transported to the roots when applied to leaves and do not elicit an endogenous signal that is transported to the roots to increase nicotine production. Strongly increased TPI activity was observed in the leaves after root treatment with coronalon and its derivative (Fig. 4C), demonstrating that either these compounds or another elicited signal are readily transported from the treated roots to the leaves. However, after treatment of the roots with MeJA, TPI levels increased only marginally.
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Transcriptional responses elicited by coronalon, its derivative, and MeJA in treated and systemic leaves
A majority of the genes examined were commonly regulated by MeJA and coronalon (6-ethyl-In-L-Ile-Me) or the unsubstituted indanoyl isoleucine conjugate (In-L-Ile-Me) in treated leaves (Fig. 5). Some genes, such as 5-epi-aristolochene synthase and threonine deaminase, and thionin were highly elicited by the jasmonate mimics (see Supplementary Fig. S2 at JXB online). In the systemic response, transcript accumulation was mainly regulated by MeJA elicitation (Fig. 5).
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Of the 1404 oligonucleotides present on the microarray, a total of 305 were significantly regulated (–1.5 > mean ratios > 1.5; P <0.05) in treated leaves after at least one of three treatments (Fig. 6A). Locally, 37% (52 genes) and 14% (24 genes) of the significantly regulated genes were up- or down-regulated by MeJA and jasmonate mimics. In treated leaves, In-L-Ile-Me regulated mRNA levels of more genes than did MeJA and coronalon treatments. In-L-Ile-Me induced 120 genes and suppressed 124 genes. Among those, 29 and 59 genes were uniquely induced or suppressed by In-L-Ile-Me (Fig. 6A). In treated leaves, genes such as XTH (xyloglucan endotransglycosylase), RALF precursor (rapid alkalization factor), and S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase (SAMT) were significantly up-regulated by In-L-Ile-Me. In addition, ACO was up-regulated by both mimics. In plants treated with jasmonate mimics, genes involved in plant signalling, such as
-DOX (-dioxygenase) and HPL (hydroperoxide lyase), were induced only in treated leaves; in plants treated with MeJA, those genes were elicited both in treated and in untreated leaves (see Supplementary Fig. S3 at JXB online).
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In systemic untreated leaves, only half of the genes regulated in treated leaves were significantly regulated (148 genes; –1.5 > mean ratios > 1.5; P <0.05; Fig. 6B). Almost all of them were regulated by MeJA. Only some of the PIs and PPOs were induced in untreated leaves as well as by In-L-Ile-Me.
Mimics of jasmonates do not require the de novo synthesis of JA; however, they do suppress free SA pools
To determine if the responses induced by the mimics require de novo biosynthesis of JA, the levels of JA were analysed in WT and asLOX3 plants (Fig. 7A; n=5 pairs, paired t-test, t=2.796; P=0.0117) and the levels of secondary metabolites in asLOX3 plants (Fig. 8). LOX3 is known to be required for JA biosynthesis. Only in leaves treated with MeJA was JA detectable 24 h after elicitation (Fig. 7A). Unlike MeJA elicitation, elicitation with coronalon and unsubstituted indanoyl isoleucine conjugates decreased free SA levels (Fig. 7B; ANOVA: for WT plants, F3,12=14.178, P=0.0003; for asLOX3 plants, F3,12=5.044, P=0.0173) in leaves of WT and as well as of asLOX3 plants. The decreases in the free SA pool could be the consequence of methyl salicylate emissions, which are known to be elicited by coronalon treatment in some species (Schüler et al., 2004).
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Elicited TPI and nicotine levels of asLOX3 plants (Fig. 8) were very similar to those of WT plants (Figs 3A, C, 4C
). Like WT plants (Fig. 8A inset; ANOVA: for leaves at node +1, F3,8=11.227, P=0.0031; for leaves at node –2, F3,8=28.589, P=0.0001), jasmonate mimics elicit more TPI activity in treated leaves of asLOX3 plants than does MeJA treatment (Fig. 8A; ANOVA: for leaves at node +1, F3,8=56.579, P <0.0001; for leaves at node –2, F3,8=11.554, P=0.0028). However, unlike the elicitation of TPIs in treated leaves, the response in untreated leaves (node –2) was greater in WT plants than in asLOX3 plants (Fig. 8A and 8A inset) for all elicitors, highlighting the importance of intact jasmonate signalling in the systemic between-leaf response. It appears that intact jasmonate signalling is necessary to induce fully nicotine (Figs 3C, 8B; ANOVA: for leaves at node + 2, F4,15=8.237, P=0.0010; for root, F4,15=14.080, P <0.0001) and TPI activity in leaves after root treatments (Figs 4C, 8C; ANOVA, F4,15=14.373, P <0.0001). |
Discussion |
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Using a native tobacco as a model system, the induction of defence metabolites, nicotine and TPIs, as well as the elicitation of transcript accumulation with microarrays, were examined to compare the responses induced by coronalon, its derivative, and MeJA. The induction of secondary metabolites by these elicitors has been well documented; however, all previous studies were carried out with suspension cultures or on the treated parts of plants (Schüler et al., 2004; Berim et al., 2005). The data indicate that coronalon and its derivative mimic the jasmonate response but only in treated tissues. In N. attenuata, MeJA and its mimics induce both TPIs after leaf elicitation and nicotine after elicitors are applied to the roots (Fig. 3). The level of detected JA (Fig. 7A) suggests that coronalon and its derivative are functional mimics of JA. The jasmonate mimics did not elicit JA (Koch et al., 1999) as has been reported for coronatine (Weiler et al., 1994). The increased JA level detected in leaves treated with MeJA was probably a consequence of either the demethylation of MeJA or the induction of endogenous JA synthesis (Bell and Mullet, 1993). Staswick's pioneering work on JAR1 in Arabidopsis (Staswick et al., 1992; Staswick and Tiryaki, 2004) suggests that exogenously applied MeJA first needs to be demethylated into JA to be activated. Indeed, a MeJA esterase (MJE) which hydrolyses MeJA has recently been purified from tobacco (Stuhlfelder et al., 2004). High enzymatic levels of MJE in roots and flowers corresponded to high levels of RNA in plant organs. Coronalon and its derivative can induce secondary metabolites (TPIs and nicotine) without de novo synthesis of JA (Koch et al., 1999). However, mimics do not appear to be metabolized in the same manner as endogenous jasmonates and do not lead to the synthesis of a mobile signal other than JA. Here it is shown that the jasmonate response in systemic untreated tissue is different from that elicited by mimics and this is most clearly seen in the lack of significant regulation of gene expression in untreated tissues (Fig. 6). Higher JA levels (Fig. 7A) and increased TPI activity in untreated leaves at node –2 in the WT (Figs 3A, 8A inset) compared with asLOX3 (Fig. 8A) plants suggests that for the full jasmonate response, an intact JA pathway is necessary.
Unlike after MeJA treatment, treatments of the roots with both mimics were able to induce TPI activity in the leaves (Fig. 4C). This suggests that coronalon and its derivative were probably transported from roots to the leaves where TPI activity was induced. JA has been detected in phloem sap (Anderson, 1985), and LOX and allene oxide cyclase, the enzymes of the JA synthesis pathway, have been found in the phloem of cucumber (Avdiushko et al., 1994) and tomato (Hause et al., 2003), which suggests that JA is probably the phloem-based mobile signal (Ryan and Moura, 2002). JA, like other phytohormones such as SA and indole acetic acid, may travel toward the shoot's tip and toward the roots through the phloem, which carries assimilates to the growing parts of plants and their storage organs (van Bel and Gaupels, 2004). Analysis of TPI activity in leaves after treatments of roots suggests that the mimics can be apoplastically transported from roots in the xylem to elicit responses in the leaves.
The xylem of a living plant is an interconnected, water-containing apoplastic system of tubes held together by cohesion. The main portion of the water is taken up by young roots and transported through the roots to the shoot. In the leaves, water is released by transpiration. The water can travel apoplastically (through the cell walls) or symplastically (from protoplast to protoplast via plasmodesmas). However, the solutes have to be taken up into the roots symplastically before they can enter the xylem (Tester and Leigh, 2001). MeJA or its derivatives may not be able to cross the endodermal cell layer, which acts as a barrier preventing apoplastic diffusion into the vascular system. Dathe and colleagues demonstrated that the plasma membrane of mesophyll cells was nearly impermeable to the JA anion (Dathe et al., 1993), which could explain why TPI levels do not change after root elicitation by MeJA. Furthermore, MeJA is known to affect guard cell potassium channels and potassium fluxes that promote stomatal closure (Evans, 2003). Exogenous JA treatment, which can lower CO2 and H2O exchanges, affected not only transpiration but also assimilation (Herde et al., 1997; Filella et al., 2006). In contrast, a recent study has shown that coronatine, a bacterial phytotoxin which mimics the jasmonate response, opens stomata and facilitates the penetration of the plant's leaves by bacteria (Melotto et al., 2006; Schulze-Lefert and Robatzek, 2006). Coronalon, a synthetic structural mimic of coronatine, may also be able to open stomata. Increased transpiration might increase the absorption of both water and coronalon and their transport to leaves, where high levels of TPIs are elicited.
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Supplementary material |
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Supplementary material is available at JXB online.
Fig. S1. Outline of experimental design used for microarray analysis depicting plant treatments, tissue collection, and microarray replication.
Fig. S2. Transcriptional response of genes highly elicited by mimics of jasmonates (In-L-Ile-Me and 6-ethyl-In-L-Ile-Me) in treated leaves. Genes which are significantly regulated in two microarray replicates in at least one treatment are shown: 5-epi-aristolochene synthase (EAS; 780—N. tabacum), threonine deaminase (TD; 288—N. attenuata), and flower-specific thionin (FST; 37—N. tabacum).
Fig. S3. Transcriptional response of genes involved in plant signalling elicited by MeJA, In-L-Ile-Me, and 6-ethyl-In-L-Ile-Me in treated leaves. Genes which are significantly regulated in two microarray replicates in at least one treatment are shown.
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Acknowledgements |
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Supported by the Max Planck Society. We thank E Wheeler for editorial comments, W Kroeber, S Allmann, and T Hahn for assistance with the microarray hybridization and scanning, and Dr A Berg and E Rothe for their help with the phytohormone analysis.
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References |
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