Present address: Molecular Genetics Section, Agriculture and Agri-Food Canada Research Station, 107 Science Place, Saskatoon, Saskatchewan, Canada, S7N 0X2.
C6-volatiles derived from the lipoxygenase pathway induce a subset of defense-related genes
Article first published online: 25 DEC 2001
The Plant Journal
Volume 16, Issue 5, pages 561–569, December 1998
How to Cite
Bate, N. J. and Rothstein, S. J. (1998), C6-volatiles derived from the lipoxygenase pathway induce a subset of defense-related genes. The Plant Journal, 16: 561–569. doi: 10.1046/j.1365-313x.1998.00324.x
- Issue published online: 25 DEC 2001
- Article first published online: 25 DEC 2001
- Received 3 July 1998; revised 8 October 1998; accepted 13 October 1998.
Six-Carbon (C6-) volatiles, including the aldehydes trans-2-hexenal, hexanal and cis-3-hexenal, as well as their corresponding alcohols, are produced from damaged or wounded plant tissue as a product of the enzymatic activity of hydroperoxide lyase (HPL), a component of the lipoxygenase (LOX) pathway. Aerial treatment ofArabidopsisseedlings with 10 μM concentrations of trans-2-hexenal induces several genes known to be involved in the plant’s defense response, including phenylpropanoid-related genes as well as genes of the LOX pathway. Genes encoding the pathogenesis-related proteins PR-1 or PR-2, however, were not induced. Trans-2-hexenal induction thus closely mimics the group of genes induced by methyl jasmonate (MeJA), also a LOX-derived volatile. However, unlike MeJA, trans-2-hexenal did not induce hydroxymethylglutaryl-coenzyme A reductase (HMGR) or thionin2–1. The inductive effect seemed to be limited to C6-related volatiles, as C8-, C9- and other related volatiles did not induce LOX mRNA levels. As has been demonstrated for MeJA, trans-2-hexenal quantitatively reduced wild-type seed germination. Trans-2-hexenal also reduced the germination frequency of the MeJA resistantArabidopsismutant, jar1–1, supporting the notion that trans-2-hexenal and MeJA are recognized via different mechanisms. In addition, trans-2-hexenal had a moderate inhibitory effect on root length relative to similar concentrations of MeJA and was approximately 10-fold less effective than MeJA at inducing anthocyanin accumulation inArabidopsisseedlings. These results suggest that C6-volatiles of the LOX pathway act as a wound signal in plants, but result in a moderate plant response relative to MeJA at both the physiological and molecular level.
Wounded plants must invoke protective measures to effectively seal off damaged tissue before opportunistic microbes invade the wounded site. Wounding results in the activation of genes responsible for the synthesis of structural compounds such as lignin ( Capellades et al. 1996 ) and cell wall proteins ( Bradley et al. 1992 ) and also initiates repair mechanisms such as cell division ( Hemerly et al. 1993 ). Wounding can be caused by different sources, including herbivorous insects or animals, mechanical wounding, as well as fungal or bacterial pathogens. The plant response to wounding requires a signal, presumably from the damaged site, to induce defense and repair mechanisms that invoke common responses as well as stress-specific mechanisms depending on the source of the damage. Furthermore, the type of damage will determine whether distal tissues are signalled or the defense response remains localized. Plants have evolved a number of strategies to signal each of these different types of disruptive stresses. For example, both mechanical wounding and herbivorous insect attack require common responses of tissue repair and a basal level of defense gene activation at the wound site to ward off opportunistic microbial infections. However, wounding from insects also induces other response pathways, both locally and distally, that are specific to insect-related damage such as the induction of proteinase inhibitor (pin) genes ( Farmer & Ryan 1990). Thus, the plant must possess a number of distinct, yet overlapping, responses that have the capability of inducing general and specific response pathways ( Baron & Zambryski 1995).
A possible source of signal molecules that alert plants to tissue damage are volatile compounds produced from the wound site. A number of volatiles are released upon wounding including ethylene, terpenoids and phenylpropanoids, as well as LOX-derived volatiles such as six Carbon (C6) volatiles and methyl jasmonate (MeJA). Volatiles are produced rapidly at the wound site and travel through the air thereby potentially serving as signal molecules to distal sites in the plant. Ethylene and MeJA are two volatile compounds released upon wounding that are associated with a wound-specific induction of pin gene expression ( Farmer & Ryan 1990;O’Donnell et al. 1996 ). Ethylene induces a number of genes and produces several physiological effects including the promotion of senescence ( Reid 1987). Similarly, MeJA induces a large number of genes, signalling the production of secondary metabolites ( Choi et al. 1994 ), stress-response genes ( Creelman & Mullet 1995) and defense genes such as thionins ( Epple et al. 1995 ). MeJA also has a number of physiological effects including induction of anthocyanins, stunting growth and senescence ( Anderson 1989;Staswick 1992).
One class of volatiles present in all green plant tissue ( Hatanaka et al. 1987 ) is a group of C6-volatiles produced from the catalytic activity of hydroperoxide lyase (HPL, Fig. 1). Depending upon the substrate, HPL produces either cis-3-hexenal or hexanal. The activity of alcohol dehydrogenase and an isomerization factor produce the remainder of the C6-volatiles including trans-2-hexenal, trans-2-hexenol, cis-3-hexenol and hexenol. This group of volatiles is among the earliest to be released from damaged tissue ( Hatanaka et al. 1987 ;Turlings et al. 1995 ) and dominate the profile of volatile compounds released from Arabidopsis (Zhuang et al. 1997, Bate et al. 1998a ). There is some evidence that these compounds have biological activity at low concentrations when released from the plant, suggesting that C6-volatiles are capable of performing a role in signalling. For example, LOX-derived C6-volatiles form the basis for ‘green note’ flavour recognition by herbivores (including humans) indicating that they act as signal molecules to animals, possess the general physical characteristics of a volatiles signal molecule and are released in sufficient quantity to be detected by animals ( Hatanaka 1993). In addition, there is some evidence to suggest that the presence of these compounds in the air surrounding plants triggers the production of phytoalexins ( Zeringue 1992) and that they reduce insect feeding rates ( Hildebrand et al. 1993 ), reduce germination frequency in soybean ( Gardener et al. 1990 ) and have antimicrobial activity ( Croft et al. 1993 ). Taken together, this body of evidence suggests that these compounds have the capability of playing a signalling role in plants.
The presence of these volatiles immediately following wounding, coupled with their biological activity, led us to determine if they act as signal molecules to induce genes associated with the defense response. By treating Arabidopsis tissue with LOX-derived C6-volatiles we show that a subset of defense genes are induced, suggesting that these compounds may play a role in alerting plants to tissue damage. A lower level of gene induction, the relatively small number of genes induced, and the attenuated biological effect relative to MeJA suggest that the C6-volatiles play a role in more general wound responses.
The potential role for LOX-derived C6-volatiles in the activation of plant defense-related genes was determined by exposing plants to a commercially available C6-aldehyde, trans-2-hexenal, for 4 h and 24 h and monitoring the induction of a number of defence-related genes by Northern blot analysis. We monitored the induction of a range of genes known to be induced by treatment with MeJA as well as genes known to play a role in plant defense, specifically: (i) genes involved in secondary metabolism, including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), dihydroflavonol reductase (DFR) and hydroxymethylglutaryl-coenzyme A reductase (HMGR); (ii) LOX pathway genes, including LOX and allene oxide synthase (AOS); (iii) pathogenesis-related proteins, including PR-1 and PR-2; and (iv) MeJA induced genes, including vegetative storage protein (VSP) and thionin2–1 (Thi2–1).
The presence of trans-2-hexenal clearly induced phenylpropanoid-related genes ( Fig. 2). The largest induction was observed with mRNA for the flavonoid branch pathway enzyme CHS and for the anthocyanin branch pathway enzyme DFR which were both induced by 4 h of treatment. CHS mRNA levels, however, continued to increase through 24 h. In contrast, PAL was only moderately induced; mRNA levels slowly increased over 24 h. In contrast to phenylpropanoid related genes, HMGR-1, which encodes the enzyme that serves as an entry point into isoprenoid biosynthesis, is not induced by trans-2-hexenal after 24 h. This is in contrast to treatment with MeJA, which actively induces the expression of HMGR in a variety of species ( Burnett et al. 1993 ;Choi et al. 1994 ).
The LOX pathway ( Fig. 1) has been previously shown to be induced by a variety of biotic and abiotic stresses ( Rosahl 1996), including pathogens ( Croft et al. 1990 ;Keppler & Novacky 1987), wounding ( Bell & Mullet 1993;Melan et al. 1993 ), and MeJA ( Avdiushko et al. 1995 ;Bell & Mullet 1991;Grimes et al. 1992 ). Treatment of Arabidopsis plants with trans-2-hexenal induces LOX mRNA accumulation ( Fig. 2) indicating that LOX responds to a variety of signals, including the presence of C6-volatiles. The LOX gene probe used in this study corresponds to LOX-2 which has been shown to be inducible by MeJA within a similar time as demonstrated here for trans-2-hexenal ( Bell & Mullet 1993).
Allene oxide synthase (AOS) is the first enzyme in the biosynthetic pathway that leads to MeJA, catalyzing the conversion of fatty acid hydroperoxides to unstable allene epoxides that subsequently give rise to jasmonic acid (JA) and its volatile methyl ester (MeJA) ( Laudert et al. 1996 ). AOS has recently been cloned from flax ( Song et al. 1993 ) and Arabidopsis ( Laudert et al. 1996 ). AOS enzyme activity ( Avdiushko et al. 1995 ) and mRNA ( Bate et al. 1998b ) are induced by MeJA and by wounding treatment ( Laudert et al. 1996 ). Thus, AOS appears to be responsive to external signals in the wound response cascade at the transcriptional level. Figure 2 shows that AOS mRNA is also induced within 4 h of treatment with trans-2-hexenal. We recently characterized an Arabidopsis HPL ( Bate et al. 1998b ) that is not inducible by treatment with 10 μm concentration of MeJA or trans-2-hexenal ( Bate et al. 1998b ; data not shown).
Pathogenesis-related proteins are a group of proteins that have been shown to play a significant role in the plant’s defense response to pathogens ( Bol et al. 1990 ). In Arabidopsis, PR-genes are rapidly induced following pathogen infection or treatment with salicylate or its analog INA ( Uknes et al. 1992 ). Treatment of Arabidopsis tissue with trans-2-hexenal did not induce the accumulation of mRNA for PR-1 or PR-2 within 24 h of treatment as has been observed with MeJA treatment ( Epple et al. 1995 ). The thionin class of antimicrobial proteins, thought to play a role in plant defense ( Bohlmann 1994), are, however, inducible by MeJA in Arabidopsis ( Epple et al. 1995 ). This argues for two distinct signal transduction pathways that lead to the induction of the PR or thionin class of antimicrobial proteins. Figure 2 shows that over a 24 h period, no induction of Thi2–1 mRNA was observed when Arabidopsis were treated with trans-2-hexenal. Trans-2-hexenal therefore does not appear to provide a signal to activate transduction pathways that leads to the induction of either class of antimicrobial protein.
The vegetative storage proteins (VSPs) are a class of protein that accumulate in a developmental stage and tissue-specific manner and, in Arabidopsis, VSP mRNA is wound and MeJA inducible ( Berger et al. 1995 ). VSP mRNA is also inducible by treatment with trans-2-hexenal ( Fig. 2).
Since the HPL pathway is responsible for the production of a group of C6-volatiles, we determined the relative efficacy of each isomer for their ability to induce defense-gene mRNA and compared them to the inductive properties of MeJA ( Fig. 3). Individual C6-isomers have been shown to have a range of efficacy on the inhibition of seed germination ( Gardener et al. 1990 ) and inducing phytoalexin production ( Zeringue 1992). Commercial C6-isomers, as well as MeJA, were diluted to equivalent concentrations and used to treat Arabidopsis plants for 24 h. Since cis-3-hexenal is not commercially available, we used alcohol dehydrogenase in a crude reaction to convert cis-3-hexenol into its aldehyde isomer. ADH treatment results in only partial conversion of cis-3-hexenol into cis-3-hexenal (J.C.M. Riley and J. Thompson, personal communication), but we reasoned that any difference between the inductive efficacy of cis-3-hexenol and the ADH reaction mixture was probably due to the presence of cis-3-hexenal produced from the ADH reaction.
Fig. 3 shows that the isomers are not equivalent in their ability to induce LOX mRNA accumulation. Hexanol and trans-2-hexenal possess equivalent inductive properties and a similar induction level was observed with ADH conversion of cis-3-hexenol into cis-3-hexenal. Hexanal, trans-2-hexenol and cis-3-hexenol did not significantly induce LOX mRNA levels. The ability to induce LOX mRNA by hexanol and trans-2-hexenal appeared to be somewhat lower than the induction by MeJA, suggesting that at similar molar concentrations the C6-volatiles are less effective than MeJA. A similar pattern of gene induction was observed with CHS (data not shown). Thionin2–1 was only induced by MeJA; none of the C6-volatiles had inductive activity over a 24 h period.
To further explore the range of compounds that induce LOX mRNA accumulation, we exposed Arabidopsis seedlings to similar concentrations of plant-related volatiles and assessed their ability to induce LOX mRNA accumulation ( Fig. 4). Hexenyl acetate had similar inductive properties as trans-2-hexenal whereas benzoic acid, octenol and methyl salicylate did not induce LOX mRNA. Nonenol, a C9-LOX derived alcohol, resulted in a slight induction in LOX mRNA levels ( Fig. 4, lane 6).
Molecular evidence ( Figs 2, 3) suggests that trans-2-hexenal and MeJA induce many of the same genes, but that the C6-volatiles induce fewer genes and to lower levels than MeJA, arguing that C6-volatiles have an attenuated effect relative to MeJA. In order to determine whether these effects were manifest at the morphological and physiological level, Arabidopsis plants and seed were treated with trans-2-hexenal and scored for effects associated with MeJA treatment.
Treatment of Arabidopsis with aerial trans-2-hexenal had a less severe effect on plant morphology than treatment with similar concentrations of MeJA ( Fig. 5). As has been previously demonstrated in a number of species, seedlings grown in the presence of MeJA are stunted and show a range of developmental and morphological effects ( Sembdner & Parthier 1993;Staswick 1992). Arabidopsis seedlings grown in the presence of 10 μm MeJA were delayed in the development of secondary leaves and had reduced root growth ( Fig. 5). In contrast, growth in the presence of 10 μm trans-2-hexenal had only a moderate effect on shoot and root morphology. At 100 μm concentrations, both treatments severely affected root growth, but trans-2-hexenal had a less severe effect on shoot development than MeJA.
To further quantify the effect of trans-2-hexenal on root growth, germinated Arabidopsis seedlings were exposed to a range of trans-2-hexenal concentrations. As is seen in Fig. 6, trans-2-hexenal had a moderate effect on root growth, and only when the seedlings were exposed to high concentrations was root length significantly affected. In contrast, MeJA has a severe effect on root growth and inhibition of root growth ( Fig. 6), and MeJA-related root inhibition has been used to isolate the MeJA insensitive mutant jar1–1 ( Staswick et al. 1992 ). The jar1–1 mutant, which is partially insensitive to MeJA treatment ( Staswick et al. 1992 ), rescues some of the MeJA effect on root growth but has no significant effect on the inhibition of root growth by trans-2-hexenal ( Fig. 6).
The genes responsible for anthocyanin production (e.g. PAL, CHS, DFR) were induced by trans-2-hexenal treatment in young Arabidopsis seedlings. Thus, we studied the effects of trans-2-hexenal treatment on anthocyanin accumulation. Figure 7 demonstrates that, as might be expected, anthocyanins accumulate following trans-2-hexenal treatment in a quantitative fashion. One μM concentrations of volatile MeJA induced anthocyanins to the same level as 10 μm trans-2-hexenal, suggesting that MeJA is 10-fold more efficacious at inducing anthocyanin biosynthesis.
As has been previously shown with soybean ( Gardener et al. 1990 ), treatment of Arabidopsis seeds with trans-2-hexenal inhibits germination frequency in a quantitative manner. Figure 7(a) shows that the germination rate was decreased to approximately 50% of the untreated control in seeds treated with 10 μm trans-2-hexenal and no germination was observed at concentrations of 100 μm. Figure 7(b) demonstrates that the inhibition of germination is not significantly different in similar tests with the Arabidopsis mutant jar1–1.
Volatiles released from damaged plant tissue serve as candidates to signal a wound response and invoke mechanisms that protect the plant ( Bruin et al. 1995 ). Volatiles are released rapidly and directly from the wound site, their presence is transient, and they have the capability to travel through the air to trigger defense mechanisms at distal sites. A number of volatiles are known to play a role in plant signalling. Ethylene, a simple gas, is released upon wounding and has been extensively studied for its ability to signal a variety of developmental and morphological effects ( Reid 1987). Similarly, MeJA induces a number of responses in plants and is thought to play a central role in the wound response ( Anderson 1989;Sembdner & Parthier 1993). Recently, methyl salicylate has been implicated as an airborne signal molecule important to plant pathogen resistance ( Shulaev et al. 1997 ). In this report, we suggest that another class of volatile compounds, the C6-volatiles, are used by plants to signal the activation of wound-related pathways.
C6-volatiles are produced rapidly upon wounding, have demonstrated biological effect, and signal the activation of gene expression. The biosynthesis of C6-aldehydes is relatively simple, involving only two enzymes (LOX and HPL;Fig. 1). The remaining C6-volatiles are produced by the action of ADH and an isomerization factor. Although both LOX ( Bell & Mullet 1993;Melan et al. 1993 ) and HPL ( Bate et al. 1998b ) are wound inducible, it is unclear whether the release of C6-volatiles requires the activation of gene expression. Tissue damage may simply allow the mixing of enzymes and substrates normally compartmentalized in the intact cell or membrane damage may release fatty acid substrates for LOX, thus rapidly producing C6-volatiles without requiring gene expression. Insect herbivory in cotton, for example, releases C6-volatiles without de novo biosynthesis ( Paré & Tumlinson 1997). Exogenous application of linolenic or linoleic acid has been shown to induce the expression of pin genes ( Farmer & Ryan 1992), suggesting that the production of MeJA from the LOX pathway is substrate limited. By association, production of C6-volatiles would tend to be dependent upon substrate availability.
The presence of C6-volatiles induces the expression of phenylpropanoid-related genes associated with the wound response and plant defense. Phenylpropanoid compounds include antibiotic phytoalexins as well as monomers for the structural polymer lignin. Since phenylpropanoid genes are wound inducible ( Lawton & Lamb 1987;Mehdy & Lamb 1987), low levels of C6-volatiles released during wounding may serve to signal simple repair mechanisms such as lignin production, and induce phytoalexins for a general protection against opportunistic microbes.
However, a key enzyme in isoprenoid biosynthesis (HMGR) was not induced by trans-2-hexenal treatment, suggesting that isoprenoid compounds are excluded from the response induced by trans-2-hexenal. It is possible, however, that the HMGR mRNA detected with the probe used in this study is not responsive to trans-2-hexenal but that other members of the HMGR gene family in Arabidopsis are inducible. In potato, hmg1 and hmg2, two HMGR family members, are regulated differently by MeJA ( Choi et al. 1994 ) suggesting that individual members of the HMGR gene family are subject to different regulatory pathways. In Arabidopsis, the inducibility of individual HMGR genes has not been studied, although it is clear that the two HMGR genes in Arabidopsis are regulated differently ( Enjuto et al. 1995 ).
Treatment with C6-volatiles also induces the expression of LOX and, although the role for LOX in plant defense and wounding responses is less clear, it is induced upon wounding and pathogen challenge ( Bell & Mullet 1991;Bell & Mullet 1993;Melan et al. 1993 ;Saravitz & Siedow 1996). It is thought that the role of LOX in wounding and plant defense could be to produce highly reactive lipid hydroperoxides as a chemical defense against pathogens ( Rogers et al. 1988 ) or that LOX may be induced by stresses to produce signal molecules such as MeJA ( Bell et al. 1995 ).
An interesting feature of the LOX pathway is that it exhibits positive feedback regulation. Treatment of plants with MeJA induces several of the enzymes in the LOX pathway as well as their corresponding genes ( Avdiushko et al. 1995 ). Similarly, treatment of plants with trans-2-hexenal induces LOX, the penultimate enzyme in the production of C6-volatiles. Positive feedback appears to be a regulatory feature of this pathway, and may reflect the role that LOX is thought to play in terminal developmental stages such as senescence ( Lynch & Thompson 1984) or in hypersenitive-related cell death ( Croft et al. 1990 ).
Trans-2-hexenal failed to induce antibiotic proteins that are thought to play significant roles in plant defense. The PR-proteins and thionins represent two classes of proteins that are induced through different signal transduction pathways as a direct response to the presence of pathogens. The failure of trans-2-hexenal to induce either of these specific pathogen-associated pathways suggests that C6-volatiles play a role in general defense mechanisms, either for tissue repair or to reduce the plant’s susceptibility to microbial infection, rather than inducing specific pathogen-related defense mechanisms.
A number of compounds are involved in signalling a wound-related response, including the peptide signal molecule systemin ( Pearce et al. 1991 ), oligosaccharides ( Bishop et al. 1984 ), as well as ethylene ( Reid 1987) and MeJA ( Anderson 1989). The overall contribution that each of these compounds plays is difficult to assess since they may trigger specific pathways in response to a particular stress or, as is more likely, they form the basis for overlapping and synergistic pathways ( Hildmann et al. 1992 ;Lee et al. 1996 ;O’Donnell et al. 1996 ) that, in combination, result in the activation of an appropriate response. The ability of C6-volatiles derived from the LOX pathway to signal the activation of genes associated with the wound response is interesting in light of the ability of LOX precursors to stimulate pin gene expression ( Farmer & Ryan 1992) and the finding that transgene-mediated suppression of LOX impairs wound induction of VSP mRNA ( Bell et al. 1995 ). Although both of these experiments are clearly associated with altered MeJA function, it is also possible that provision of LOX substrates or inhibition of LOX has an effect on the signalling ability of LOX-derived C6-volatiles as well.
C6-volatiles are less severe in their effect on several aspects of plant morphology than exposure to similar levels of MeJA. Increasing concentrations of trans-2-hexenal have only a moderate effect on root growth until high concentrations are used, i.e. 100 μm. Similarly, the development of secondary leaves is less severely affected in the presence of trans-2-hexenal relative to MeJA and trans-2-hexenal is approximately 10-fold less efficacious at inducing anthocyanin accumulation. The effect on seed germination was more severe than observed for root growth or shoot development, with even low concentrations of trans-2-hexenal (i.e. 5 μm) having a significant inhibitory effect on germination. The possibility exists that plants are much more sensitive to MeJA than they are to the presence of C6-volatiles and that MeJA is physiologically active at much lower concentrations than are commonly used in this kind of study. Alternatively, MeJA may activate a larger number of genes and trigger a larger number of responses than C6-volatiles and thus has a greater physiological effect.
Quantitative increases in trans-2-hexenal concentration translate into corresponding increases in physiological effect as demonstrated for root growth ( Figs 5, 6), anthocyanin accumulation ( Fig. 7) and germination frequency ( Fig. 8). However, incremental increases in physiological effect are observed with additive increases in trans-2-hexenal and MeJA concentration (e.g. Figure 7) that could reflect a limited range of dose response. We have also observed variation in inductive effect from experiment to experiment with both MeJA and trans-2-hexenal treatment (data not shown) suggesting that subtle differences in environmental conditions or plant developmental state translate into variations in response.
The inhibitory effect of trans-2-hexenal on seed germination and root growth was not rescued in jar1–1 mutant plants. The jar1–1 mutant is partially insensitive to MeJA inhibition of root growth and fails to accumulate VSP when exposed to MeJA, presumably resulting from an inability to perceive or transduce the MeJA signal ( Staswick et al. 1992 ). This provides further evidence that despite the similar biochemical origin of trans-2-hexenal and MeJA, as well as a similar profile of genes induced by treatment, that the two signal pathways of gene induction are distinct.
A likely role for C6-volatiles is to activate a low level of defense response and wound repair mechanisms. It is clear that plants need to differentiate between mechanical damage and damage from insect pests or pathogens as well as between degrees of damage. Repeated or extensive damage as a result of pathogen infection or insect pest herbivory would presumably trigger additional signal transduction pathways. The significance of the C6-volatile response pathway to the plant probably lies in the fact that the plant must take defensive measures, even at the slightest damage, to protect itself against opportunistic infection at a time when plant cells are exposed and without many of the normal physical defences. It would be presumably inefficient, however, to invoke comprehensive measures, including the production of PR-proteins and proteinase inhibitors, either locally or systemically, to simple mechanical abrasion. Furthermore, the more moderate physiological effects of the C6-volatiles, relative to MeJA, allow for the induction of moderate defense responses without having a severe effect on plant growth and development.
Arabidopsis (ecotype Columbia) seeds were surface sterilized, imbibed at 4°C for 48 h in 0.1% agar, and dispersed onto MS plates containing 2% sucrose. Plants were grown in environmental growth chambers with a 16 h day at 23°C and an 8 h night at 21°C. Unless otherwise stated, plants were grown for 21 days under these conditions before pre-treatment. To pre-treat plant tissue, the tops of the Petri plates were removed, the plants placed into sealable canning jars (1 l) and incubated as above for 24 h before the addition of volatile compounds. MeJA or C6-volatiles were diluted to 0.1 m in Methanol and 10 μl of cold (4°C) diluted compound were added to a sterile cotton swab suspended above the plants and the sealed containers returned to the environmental growth chamber. Control treatments (methanol) were performed first and treatments were staggered and performed in a fume hood to avoid cross-contamination of volatile treatments. Since cis-3-hexenal is not commercially available, we used purified alcohol dehydrogenase (SIGMA chemicals, St. Louis, MO, USA) in a simple enzyme reaction to produce this volatile. Briefly, in a 100 μl volume, ADH (20 U) was incubated with 0.1 m cis-3-hexenol (10 μl), 15 μl NAD (5 mg ml–1), 10% glycerol and 10 m m Tris, pH 8.9. This enzyme mixture was added to a small reaction vessel at the bottom of the canning jar, with the plants suspended above. The cis-3-hexenol treatment was identical except that the cis-3-hexenol was not mixed with the other components included in the reaction. To assess effects of chemical treatment on root growth, seeds were sterilized, imbibed for 3 days and placed onto MS containing 2% sucrose. Following germination, seedlings were transferred into a horizontal slit cut into the agar of an MS plate and treated as above with 10 μm MeJA or trans-2-hexenal. Petri plates were mounted in a vertical position and root lengths measured after 7 days. To determine anthocyanin quantity, germinated Arabidopsis seedlings (2 d post-imbibition) were grown in the presence of 10 μm MeJA or trans-2-hexenal for 5 days following germination. Anthocyanins were extracted in 0.1% HCl in methanol for 48 h at 4°C, measured in a spectrophotometer and the quantity calculated by the formula A530-(0.3A657).
Chemicals and gene probes
NAD was obtained from Boehrringer Mannheim. All other chemicals were obtained from SIGMA/Aldrich Canada, purity was greater than 95% for all compounds used. The gene probe for CHS ( Feinbaum & Ausubel 1988) was obtained from the lab of Fred Ausubel (Massachusetts General Hospital, Cambridge, MA, USA), DFR ( Shirley et al. 1992 ) was obtained from Brenda Shirley (Virginia Technical Institute), PR-1 and PR-2 ( Uknes et al. 1992 ) were obtained from Robin Cameron (University of Toronto, Toronto, ON, Canada). PAL, HMGR, LOX-1, VSP and β-ATPase were obtained from the ABRC at Ohio State University, following BLAST searches ( Altschul et al. 1990 ) using known sequence information and correspond to the following ABRC stock numbers, respectively: CD3–19; 114 m5T7; 106C8T7; ATTS1295; and G3H9T7. To obtain a gene probe for AOS ( Laudert et al. 1996 ), Arabidopsis genomic DNA was used as a template in a PCR reaction containing the following AOS gene-specific primers: 5′-CTT-TTC-ACC-GGT-ACT-TAC-ATG-CCG-3′ and 5′-GAG-CTT-GTA-TCT-GCG-GGA-TTC-GTC-3′, corresponding to bases 447–470 and 723–746, respectively ( Laudert et al. 1996 ). Similarly, a thionin clone was amplified using primers 5′-GAG-TCT-GGT-CAT-GGC-ACA-AGT-TC-3′ and 5′-CTT-GGC-ACA-TTG-TTC-CGA-CGC-TC-3′ specific to Ath2–1 ( Epple et al. 1995 ). Both PCR fragments were cloned into pGEM-T (Promega Biotech). Clones obtained from the ABRC and cloned PCR fragments were sequenced before use.
RNA was isolated by the Guanidium Isothiocyanate method ( Ausubel et al. 1987 ), and 10 μg was separated on a formaldehyde agarose gel. RNA was transferred to a nylon membrane in 20×SSC and UV cross-linked using a Stratalinker (Stratagene). Blots were pre-hybridized, hybridized and washed at 65°C according to Church & Gilbert (1984).
The research presented here was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to S.J.R. The authors are grateful to the Arabidopsis Biological Resource Center at Ohio State University for providing clones. The authors would also like to thank Dr Fred Ausubel and Dr Brenda Shirley for providing clones, and Dr Shoba Sivasankar, Dr Margie Gruber and Dr Peter Constabel for critical reading of the manuscript.
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