The first two authors contributed equally to this work.
Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles
Article first published online: 21 APR 2002
The Plant Journal
Volume 24, Issue 1, pages 113–126, October 2000
How to Cite
Hause, B., Stenzel, I., Miersch, O., Maucher, H., Kramell, R., Ziegler, J. and Wasternack, C. (2000), Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles. The Plant Journal, 24: 113–126. doi: 10.1046/j.1365-313x.2000.00861.x
- Issue published online: 21 APR 2002
- Article first published online: 21 APR 2002
- Received 8 May 2000; revised 24 July 2000; accepted 27 July 2000.
- tomato (Lycopersicon esculentum);
- allene oxide cyclase;
- oxylipin signature;
- vascular bundles;
A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor, cis(+)12-oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato . In stems, young leaves and young flowers, AOC mRNA accumulates to a low level , contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g−1 fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue-specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue-specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink–source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin-mediated wound response of tomato.
Jasmonic acid (JA) and its methyl ester (JAME), collectively named jasmonates, are ubiquitously occurring plant growth regulators. They act as signals in various plant responses to biotic and abiotic stresses, as well as in distinct stages of plant development (reviewed in Creelman and Mullet, 1997; Wasternack and Parthier, 1997). An endogenous rise of jasmonates occurs upon wounding ( Conconi et al., 1996a ; O'Donnell et al., 1996 ; Peña-Cortés et al., 1995 ), elicitation of cell suspension cultures ( Gundlach et al., 1992 ), osmotic stress ( Kramell et al., 2000 ), pathogen attack ( Penninckx et al., 1996 ), burning and electric current application ( Herde et al., 1996 ), chitosan and oligogalacturonide treatment ( Bowles, 1990; Doares et al., 1995 ) or UV stress ( Conconi et al., 1996b ). Recent GC–MS analysis revealed that the rise in JA is preceded by a more pronounced rise in octadecanoids such as 12-oxophytodienoic acid (OPDA), as shown for elicitation of cell suspension cultures ( Parchmann et al., 1997 ), osmotic stress ( Kramell et al., 2000 ), wounding ( Ziegler et al., 2000 ) or touch of tendrils ( Stelmach et al., 1998 ). The rise in the levels of jasmonates and OPDA is followed by expression of specific genes. These up-regulated genes include those coding for proteinase inhibitors (reviewed by Ryan, 1992), thionins ( Bohlmann et al., 1998 ) and defensins ( Thomma et al., 1998 ), as well as for enzymes involved in the biosynthesis of phytoalexins ( Blechert et al., 1995 ), alkaloids ( Baldwin et al., 1997 ) and monoterpenes ( Koch et al., 1999 ). Enzymes involved in the biosynthesis of JA are also induced by exogenous application of JA ( Feussner et al., 1995 ; Heitz et al., 1997 ; Laudert and Weiler, 1998 ; Maucher et al., 2000 ). Another interesting aspect of JA action is the down-regulation of house-keeping proteins such as Rubisco ( Reinbothe et al., 1993 ; Roloff et al., 1994 ), which may be part of its senescence-promoting effect.
Apart from jasmonates, OPDA has also been shown to possess signalling properties, e.g. in volatile formation ( Koch et al., 1999 ), in tendril coiling ( Blechert et al., 1999 ; Stelmach et al., 1998 ), or in the expression of specific genes ( Kramell et al., 2000 ). The profiles of various oxylipins including octadecanoids were found to be different in various plants. This ‘oxylipin signature’ was suggested to mediate plant-specific JA/OPDA-related responses ( Farmer et al., 1998 ; Weber et al., 1997 ).
There is currently great interest in cloning all genes coding for enzymes of JA biosynthesis. The biosynthesis of JA elucidated by Vick and Zimmerman (1983) originates from α-linolenic acid (α-LeA), thus representing one branch within the oxylipin pathway (reviewed in Blée, 1998 ; Hamberg and Gardner, 1992). The α-LeA might be released from membrane-located phospholipids by phospholipase A2 activity as shown recently ( Narváez-Vásquez et al., 1999 ). In the subsequent step, a 13-lipoxygenase (LOX) catalyses the regio-selective and stereo-specific dioxygenation of α-LeA to (13S,9Z,11E,15Z)-13-hydroperoxy-(9,11,15)-octadecatrienoic acid (13-HPOT) which may be metabolized into leaf aldehydes, divinyl ether, or products of the peroxygenase as well as of the reductase branch of the LOX pathway. However, 13-HPOT is also the substrate for the JA-specific branch of the oxylipin pathway, leading in a first step to the formation of an unstable allene oxide, catalysed by allene oxide synthase (AOS). In aqueous media, the allene oxide is converted non-enzymatically into α- and γ-ketols and a minor amount of a cyclopentenone compound, racemic OPDA, whereas the enzymatic formation of OPDA by allene oxide cyclase (AOC) leads to the exclusive formation of its cis-(9S,13S) enantiomer. Finally, the reduction of OPDA and three β-oxidation steps lead to (+)-7-iso-(3R,7S)-JA, which isomerizes into the more stable (–)-(3R,7R)-JA ( Figure 1).
With the exception of the enzymes catalysing the β-oxidation steps, all the enzymes of JA biosynthesis have been cloned in recent years. For 13-lipoxygenases, several cDNAs coding for different isoenzymes were characterized for tomato ( Heitz et al., 1997 ), potato ( Royo et al., 1996 ), soybean ( Bunker et al., 1995 ) , Arabidopsis ( Bell and Mullet, 1993), rice ( Peng et al., 1994 ), tobacco ( Véronési et al., 1996 ) and barley ( Holtman et al., 1997 ; Vörös et al., 1998 ). However, the first clues as to which isoform functions specifically in JA biosynthesis were obtained only recently ( Bell et al., 1995 ; Heitz et al., 1997 ; Royo et al., 1999 ). Most of the 13-LOXs are wound- and JA-inducible and are located within the chloroplasts ( Bell et al., 1995 ; Feussner et al., 1995 ). Some of them were found to be expressed specifically in flower development ( Rodriguez-Concepción and Beltrán, 1995) . cDNAs coding for the AOS have been characterized from flax ( Song et al., 1993 ), Arabidopsis ( Laudert et al., 1996 ), guayule ( Pan et al., 1995 ), tomato ( Howe et al., 2000 ; Sivasankar et al., 2000 ) and barley ( Maucher et al., 2000 ), and the AOS protein has been shown to reside in the chloroplast ( Harms et al., 1995 ; Maucher et al., 2000 ). AOS has been suggested to be a control point in JA biosynthesis ( Laudert et al., 1998 ). However, it is not yet known how much racemic OPDA and α- and γ-ketols, which were detected recently in Lemna paucicostata ( Yokoyama et al., 2000 ) and barley (O. Miersch, unpublished data), could bypass a regulatory role of AOS. Furthermore, the recently purified AOS of corn is irreversibly inactivated by its substrate 13-HPOD ( Utsunomiya et al., 2000 ). If this effect occurred in vivo, the positive feedback regulation of JA biosynthesis would be hampered. A recent isotopic dilution experiment including GC–MS analysis suggested that there is no positive feedback of JA biosynthesis in tomato leaves ( Miersch and Wasternack, 2000). The AOC-catalysed reaction produces cis(9S,13S)-OPDA, which is, according to present knowledge, the unique precursor of the naturally occurring stereoisomer of JA. AOC was first purified to apparent homogeneity from corn ( Ziegler et al., 1997 ). Its importance in JA biosynthesis is reflected by its exclusive specificity for fatty acid derivatives carrying an epoxide group in the n-6,7 position and a double bond in the n-3 position, whose cyclization only leads to JA-like structures ( Ziegler et al., 1999 ). Two OPDA reductases (OPR1, OPR2) were purified recently which differed in their substrate specificity ( Schaller et al., 1998 ). OPR1 was cloned from Arabidopsis and did not use the 13-S-configurated isomer of OPDA which is the intermediate in JA biosynthesis ( Schaller and Weiler, 1997). Among the OPRs known so far, only the recently characterized OPR3 was shown to act in JA biosynthesis due to its exclusive formation of the naturally occurring cis(+)-stereoisomer ( Schaller et al., 2000 ).
The increased knowledge on JA biosynthesis and the availability of cDNAs coding for its enzymes allow analysis of the physiological effects of JA on senescence, root growth or germination. For AOS, a specific up-regulation was found in distinct tissues of developing barley seedlings and was correlated with elevated JA levels ( Maucher et al., 2000 ). In A. thaliana, the AOS promoter has been shown to be preferentially active in distinct tissues such as the abscission zone of flowers ( Kubigsteltig et al., 1999 ). Although it remains to be determined whether this activity leads to increased JA levels, these data on AOS point to a tissue-specific function for JA biosynthesis. Due to instability of the AOS product, tissues and even organelles with AOS activity should contain AOC activity.
Recently, a full-length cDNA clone coding for AOC was isolated from tomato ( Ziegler et al., 2000 ). Over-expression of a truncated version of the protein showed that it catalysed the exclusive formation of cis(9S,13S)-OPDA. In this paper, we have analysed AOC expression during development. Most surprisingly, organ- and tissue-specific expression was detected within vascular bundles of roots, stems and leaves. Expression also occurred within flower stalks and ovules of flower buds and of young flowers. This tissue-specific expression was tightly correlated with elevated levels of JA, JAME, JA amino acid conjugates and octadecanoids, and the various flower tissues contained unique ratios of the various jasmonates and octadecanoids. This suggests a specific role of AOC and octadecanoid/jasmonate profiles during flower development and stress responses.
The AOC mRNA accumulates tissue-specifically during flower development
Analysis of the organ-specific expression of AOC showed a weak constitutive accumulation of AOC mRNA in the stem, young leaves and young flowers ( Figure 2). A detailed inspection of the various parts of flowers revealed a significant accumulation in sepals and the pistil. The most abundant AOC mRNA accumulation was in the flower stalks and flower buds ( Figure 2). Also, a remarkable amount of AOC mRNA accumulated in roots. No accumulation was detected in fruits, seeds or later leaf stages including senescent (yellow) leaves. The occurrence and amount of AOC protein correlated only partially with that of AOC mRNA. Most interestingly, a high level of AOC protein was detected in the pistil. Even red fruits, old flowers and senescent leaves, which were devoid of detectable amounts of AOC mRNA, contained significant amounts of AOC protein.
The AOC protein accumulates in ovules, and in parenchymatic cells of vascular bundles of flower stalks, petioles, stems and roots, and is accompanied by high AOC activity
The different levels of AOC expression in various flower organs and its specific occurrence in flower buds prompted us to examine the presence of the AOC protein in different floral tissues and in tissues with vascular bundles using an immunocytochemical approach. For this we chose flower buds which allowed inspection of several flower organs in the same section. If probed with a pre-immune serum, transverse sections of flower buds (6–9 mm in length) exhibited very faint staining due to some non-specific cross-reactions ( Figure 3a–f). However, specific labelling could be shown with an anti-AOC antibody. The strongest signal was found within the pistil ( Figure 3a′,a′′,b′). Here, the transmission tissue of the style ( Figure 3a′,a′′,a′′′) as well as the ovules within the ovary ( Figure 3b′,b′′) exhibited strong labelling. In contrast, the petals showed only a weak staining, whereas sepals and stamens were free of label ( Figure 3a′). Moreover, the parenchymatic cells of vascular bundles of a receptacle ( Figure 3c′,c′′) as well as of the flower stalk above ( Figure 3e′,e′′) or below ( Figure 3d′) the abscission region were also labelled. Longitudinal sections of the flower stalk indicated that, with the exception of the vascular bundles, no label occurred even in the abscission region ( Figure 3f′). The tissue-specific occurrence of AOC protein in flowers probed immunocytochemically corresponds to immunoblot analysis and Northern blot analysis ( Figure 2).
Of the flower tissues exhibiting high AOC protein levels, flower stalks were chosen for AOC activity testing. In crude extracts of this tissue, AOC activity of about 8 nmol OPDA formed per mg protein was measured, whereas in crude extracts of leaves no AOC activity could be detected. In AOC expression studies involving different treatments of leaves, AOC mRNA was found to accumulate abundantly upon glucose treatment (data not shown). Together with the fact that ovule tissues are regarded to be sink tissues, this prompted us to determine whether levels of the AOC transcript can be up-regulated in flower buds by glucose. As shown in Figure 4, freshly harvested or water-floated flower buds exhibited AOC mRNA levels comparable to those in Figure 2. However, AOS mRNA accumulated strongly upon glucose treatment and weakly upon JAME treatment, but not upon treatment with 6-deoxyglucose, indicating that AOC gene expression in flower buds is glucose-responsive.
In addition to the analysis of flower tissues, the high expression of AOC in flower stalks, stems and roots prompted us to conduct immunocytochemical analysis on these tissues, as well as on petioles. Immunostaining of hand sections of roots, stems and petioles revealed the presence of AOC protein in parenchymatic cells of all vascular bundles ( Figure 5a–c′′). Only hand sections that were treated with the anti-AOC antibody exhibited the characteristic colour given by the product of the alkaline phosphatase reaction ( Figure 5a′–c′, a′′–c′′) , whereas hand sections treated with the pre-immune serum showed only a green colour due to chlorophyll ( Figure 5a,b,c) . Even the bicollateral organization of the tomato vascular bundles can be seen by this staining procedure, preferentially in hand sections of the petiole (arrows in Figure 5c′′), and specific labelling of parenchymatic cells of the vascular bundles is clear. To inspect the obviously cell-specific occurrence of AOC protein in the vascular bundles more clearly, we compared semi-thin cross-sections of the petiole ( Figure 5d–d′′), a middle vein of a leaflet ( Figure 5e–e′′) and the intercostal region of a leaflet ( Figure 5f–f′′). In all three tissues, AOC protein was clearly detectable in parenchymatic cells of the vascular bundles (arrows in d′′ and e′′) . In the leaf cross-section, AOC protein was found in the chloroplasts of cells of the bundle sheath ( Figure 5f′′).
Jasmonates and octadecanoids accumulate tissue-specifically during flower development
To determine whether the tissue-specific accumulation of AOC mRNA and AOC protein is accompanied by elevated levels of octadecanoids and jasmonates, these compounds were analysed quantitatively by GC–MS analysis ( Figure 6).
With the exception of ripe flowers, the accumulation of AOC mRNA and AOC protein was accompanied by a remarkable elevation of jasmonates/octadecanoids ( Figure 6d versus Figure 2). In all tissues, the JA content was about one order of magnitude higher than the methyl ester, reaching 5–7 nmol g−1 fresh weight in buds (< 6 mm), stalks and sepals ( Figure 6a). Remarkably high amounts of the isoleucine conjugate of JA were found in buds (< 6 mm), ripe flowers, stalks and pistils ( Figure 6b). Interestingly, in pistils, the methyl ester of the isoleucine conjugate of JA accumulated in similar amounts as the free acid of the conjugate, whereas in buds a greater than fivefold higher level of the methyl ester of the isoleucine conjugate of JA was found. Buds, ripe flowers and pistils contained more conjugates than JA. Surprisingly, in the various organs, the OPDA levels are not reflected in the levels of JA. In pistils, OPDA accumulated up to 7 nmol g−1 fresh weight, which is about fourfold higher than the JA level in this tissue. However, in sepals, for example, up to 5 nmol JA g−1 fresh weight and almost no OPDA could be detected. Despite the metabolic link between OPDA, JA and the isoleucine conjugate of JA, the ratios of their levels are significantly different in different tissues.
In contrast to the altered jasmonate levels that occur in response to external stimuli, little is known about jasmonate levels during development. Such levels might be sustained by substrate availability in the JA pathway, and/or activity of the enzymes of the pathway. The first possibility was suggested by the linolenic acid (18:3) deficiency of the mutant fad3–2 fad7–2 fad8 of Arabidopsis thaliana ( McConn and Browse, 1996). The unique deficiency of linolenic acid in the tapetum, the pollen-feeding tissue of flowers, led to male sterility ( McConn and Browse, 1996), thereby demonstrating the essential role of JA in flower development. Furthermore, this mutant exhibits JA deficiency in the leaves ( McConn et al., 1997 ).
Jasmonate levels may be taken as an indication of the activity of the JA pathway. In soybean seedlings, a gradient of jasmonates was found, with higher levels in young rapidly dividing tissues ( Creelman and Mullet, 1995). This agrees with the observation that in barley seedlings a gradient of jasmonate was found in the root, increasing towards the tip (Hause et al., unpublished data), and in the leaf, increasing towards the base ( Maucher et al., 2000 ). Flowers of dicotyledonous plants are known to contain high levels of jasmonates including amino acid conjugates or amides ( Miersch et al., 1997 ; Schmidt et al., 1990 ). However, the correlation of jasmonate content in distinct plant tissues with the corresponding occurrence and activity of JA biosynthetic enzymes is not yet known, but a correlation between the levels of AOS mRNA, AOS protein and JA was recently observed during barley seedling development in specific tissues such as the scutellar nodule and the leaf base ( Maucher et al., 2000 ).
AOC is expressed preferentially in vascular bundles and in ovule tissues, and its occurrence correlates with elevated levels of jasmonates and octadecanoids
The tissue-specific AOC mRNA accumulation occurring abundantly in roots, flower buds and flower stalks is not completely reflected in AOC protein accumulation, which could be observed in tissues with undetectable amount of AOC mRNA. This might be caused by instability of AOC mRNA in some tissues and/or post-transcriptional control of AOC protein levels. In general, tissues with high AOC expression such as flower buds, flower stalks and pistils ( Figure 2) also exhibited elevated levels of octadecanoids and jasmonates ( Figure 6). Furthermore, high AOC activity was detectable in crude extracts of flower stalks but not in crude extracts of leaf tissue. The activity in flower stalks is about twofold higher than in corn seeds, in which the highest AOC activity so far has been measured ( Ziegler et al., 1997 ). This correlation between AOC expression and jasmonate/octadecanoid levels points to a regulatory link between both events. Also, AOC expression and accumulation correlate temporally upon external stimuli such as wounding ( Ziegler et al., 2000 ). A similar link was found between wound-induced AOS expression and JA levels in Arabidopsis ( Laudert and Weiler, 1998), as well as transgenic potato and tobacco plants over-expressing the flax AOS ( Harms et al., 1995 ; Wang et al., 1999 ). Moreover, the recently cloned tomato AOS is highly expressed in flower buds and mature unopened flowers as well as in leaves upon wounding or treatment with systemin, OPDA or JAME ( Howe et al., 2000 ; Sivasankar et al., 2000 ). These data correspond with the high AOC expression shown here for flower buds and flower stalks and upon treatment of tomato leaves by wounding ( Ziegler et al., 2000 ) or with systemin, OPDA or JAME (Stenzel et al., unpublished data) . The expression of AOS and AOC might be coordinately regulated. However, the intriguing role of AOC is due to its ability to catalyse the exclusive formation of cis(+)OPDA, as shown recently with recombinant AOC as well as in vivo upon wounding of tomato leaves ( Ziegler et al., 2000 ).
Two types of tissues exhibited the presence of AOC protein: (i) the parenchymatic cells of vascular bundles of various organs such as root, stem, petiole or flower stalk, and (ii) the ovules and the transmission tissue of the style. Interestingly, the prosystemin gene coding for the polypeptide precursor of the systemic wound signal systemin ( McGurl et al., 1992 ) is specifically expressed in vascular bundles of stems, petioles or petiolules, the region of attachment of the leaflet to the petiole ( Jacinto et al., 1997 ). Furthermore, systemin is transported in vascular bundles ( Narváez-Vásquez et al., 1995 ). Recently, the following scenario was suggested ( Jacinto et al., 1997 ; Ryan, 2000). Prosystemin is expressed when a vein is wounded, followed by its processing to systemin. Systemin may activate wound signal pathway genes in the vascular bundle and is rapidly loaded into the phloem, where it can be distributed throughout the plant, thereby functioning systemically in wound-responsive gene expression in the mesophyll cells of a leaf. The data presented here extend this scenario: The tissue-specific expression of AOC in the vascular bundles ( Figure 5) is reinforced by up-regulation of AOC upon wounding ( Ziegler et al., 2000 ), and in the wounded leaf, the levels of cis(+)OPDA and the corresponding enantiomer of JA will rise, presumably mainly in the veins ( Ziegler et al., 2000 ). This local rise of octadecanoids and jasmonates may potentiate the JA-responsive prosystemin expression ( Jacinto et al., 1997 ), leading to an increased systemic response. Furthermore, octadecanoids and jasmonates may diffuse into the surrounding tissues, thus leading to the local wound response seen for many wound-inducible genes.
The specific AOC expression in certain flower tissues and the accumulation of octadecanoids and jasmonates shown here suggest a specific function in development. Interestingly, a number of JA-responsive genes are also specifically expressed in ovules and flower stalks. Among them are those coding for histones ( Kim et al., 1998 ). As evidenced by in situ hybridization with a leucine amino peptidase (LAPA) antisense RNA probe or promoter activity tests in LapA1:GUS tomato plants ( Chao et al., 1999 ) and pin2:GUS tomato plants ( Peña-Cortés et al., 1991 ), both genes are highly expressed in ovules. These data correspond to the high trypsin inhibitory activity of ovaries of Nicotiana tabacum and N. plumbaginifolia, where pin2 is highly expressed ( Atkinson et al., 1993 ; Ausloos et al., 1995 ). Another JA-responsive gene specifically expressed in the pistils of potato is a dioxygenase suggested to be involved in the biosynthesis of deterrent alkaloids ( Lantin et al., 1999 ). Pathogenesis-related (PR) proteins including chitinase, β-1,3-glucanases and osmotin are also developmentally up-regulated in flower tissue ( Lotan et al., 1989 ; Neale et al., 1990 ), and are at least partially JA-responsive ( Chao et al., 1999 ). So far, the signals that contribute to the tissue-specific expression of all these plant defence proteins are unknown. It is tempting to speculate that JA might be such a signal, since AOC expression and elevated JA levels occur in these tissues exhibiting JA-responsive gene expression.
Another known effect of exogenously applied JA/JAME is the redistribution of nutrients ( Creelman and Mullet, 1997). This effect suggests that an analysis of the JA responsiveness of genes coding for enzymes with a function in sink–source relationships such as invertases would be worthwhile. Interestingly, one of the extracellular tomato invertases (LIN6) known to supply a sink tissue with glucose is specifically expressed in flower buds ( Godt and Roitsch, 1997). Such a tissue-specific increase in glucose could supply energy to a non-photosynthetic tissue such as the ovule. Consequently, ovule-specific AOC expression may occur due to its high glucose responsiveness as shown in Figure 4. This in turn may elevate OPDA/JA levels which may contribute to the protection of these tissues against pathogens by expression of various defence proteins such as pin2, LAPA or PR proteins.
An equivalent scenario was recently described in which glucose was shown to simultaneously induce sink-specific genes and defence-related genes, accompanied by down-regulation of photosynthetic genes ( Ehness et al., 1997 ). The elevation of jasmonates in a sink tissue such as ovules following the glucose-inducible AOC expression shown here would synergistically potentiate the effect of glucose. The down-regulation of photosynthetic genes by jasmonate is a well-known phenomenon (cf. Creelman and Mullet, 1997).
An oxylipin signature occurs tissue-specifically in tomato flowers
JA and JAME are considered to be signals in various stress responses. This is based on the observation that an external stimulus leads to an endogenous rise of jasmonates followed by the expression of a distinct set of genes which is similar or even identical to that expressed upon JA treatment of a plant tissue. Also JA amino acid conjugates ( Kramell et al., 1997 ) and structurally related indanone derivatives ( Krumm et al., 1995 ) were shown to be independent signals. Furthermore, the JA precursor OPDA was found to accumulate upon touch ( Stelmach et al., 1998 ), osmotic stress ( Kramell et al., 2000 ), elicitation of cell cultures ( Parchmann et al., 1997 ) or wounding of leaves ( Parchmann et al., 1997 ; Ziegler et al., 2000 ), and was identified as JA-independent signal in JA-related responses ( Blechert et al., 1995 ; Blechert et al., 1999 ; Kramell et al., 2000 ). The term ‘oxylipin signature’ was proposed after the observation that different plants exhibit distinct oxylipin profiles upon wounding ( Weber et al., 1997 ). Such an individual oxylipin pattern upon external stimuli may be caused by a shift in the various branches of the LOX pathway as was recently shown for barley leaves treated with salicylate ( Weichert et al., 1999 ).
Much less is known about oxylipin patterns during development. Here, we detected for the first time the occurrence of OPDA, JA isoleucine conjugate and its methyl ester in flowers. Most interestingly, during flower development, the different tissues exhibited different ratios of JA, JAME, OPDA and JA isoleucine conjugates. For leaves, JA, OPDA, the structural related coronatine , isoleucine conjugates of JA, and structural analogues such as indanone derivatives were shown to induce gene expression per se , if applied exogenously ( Blechert et al., 1995; Blechert et al., 1999 ; Kramell et al., 1997 ; Kramell et al., 2000 ; Wasternack et al., 1998 ; Koch et al., 1999 ). Due to the preferential accumulation of one or more jasmonate and octadecanoid compound(s) in the various tissues of tomato flowers ( Figure 6), it is tempting to speculate that this tissue-specific oxylipin signature might contribute to the expression of individual sets of genes.
Furthermore, JA amino acid conjugates seem to possess a specific function in pollen development. These conjugates are the unique JA compounds accumulating in pollen ( Knöfel and Sembdner, 1995), sometimes existing as special derivatives such as [(–)-jasmonoyl]-tyramine ( Miersch et al., 1997 ). The ultimate role of jasmonates in flower development is also supported by three Arabidopsis mutants which are disturbed in fatty acid synthesis and degradation as well as in biosynthesis of jasmonates. The triple mutant fad3-2 fad7-2 fad8 is unable to form linolenic acid, the essential precursor of JA biosynthesis ( McConn and Browse, 1996). Male sterility and impairment of pollen development were the strongest alterations of the phenotype. The recently identified aim1 mutant of Arabidopsis was shown to be deficient in the enoyl-CoA hydratase, an essential enzyme of β-oxidation. As a consequence, abnormal inflorescence development and altered fatty acid composition occurred, which led to the suggestion that lipid-derived signals including jasmonates might be altered in aim1 ( Richmond and Blecker, 2000). Mutants in biosynthesis of jasmonates in Arabidopsis found recently exhibit a block in OPR3 and are male-sterile ( Sanders et al., 2000 ; Stintzi and Browse, 2000).
So far we have no data on the preferential expression of AOC and elevated levels of jasmonates and octadecanoids in tomato anthers, but the data discussed above suggest expression of plant defence genes via facilitation of jasmonate biosynthesis in another tissue of the tomato flower, the ovules.
(±)-JAME ([3R,7R]/[3S,7S]-JAME) was purchased from Firmenich (Geneva, Switzerland) and used to prepare JA by saponification. (±)-JAME contained less than 10% of (±)-7-iso-JAME ([3R/7S]/[3S/7R]-JAME) as determined by GC–MS analysis. [2H6]-JA was synthesized as described previously ( Miersch, 1991). JA-[2H3]-leucine was synthesized chemically from (±)-JA and [2H3]-leucine as described for the isoleucine conjugate of JA ( Kramell et al., 1997 ).
Plant materials and treatments
Lycopersicon esculentum Mill. cv. Moneymaker was grown as described previously ( Wasternack et al., 1998 ). Whole flowers before opening were floated on water (control), 0.5 m glucose, 0.5 m 6-deoxyglucose or 50 µm JA, for 4 h. Plant material was frozen in liquid nitrogen and stored at −80°C until use.
Extraction of RNA and Northern blot analysis
Total RNA was extracted from frozen tissues by the use of phenol:chloroform:isoamyl alcohol 25:24:1 (v/v/v) according to the method described by Sambrook et al. (1989) . If not otherwise indicated, 20 µg total RNA per lane was subjected to electrophoresis, and Northern blot analysis was performed according to the method described by Sambrook et al. (1989) . Blots were hybridized at 65°C for 16 h with a 32P-labelled fragment of tomato AOC cDNA encompassing the full-length cDNA sequence. Gel loading was checked by probing with a 600 bp fragment of a cDNA homologous tomato ADP/ATP carrier nucleotide sequence. In cases of different levels of occurrence of ADP/ATP carrier mRNA in flower organs, ethidium bromide staining of the rRNA was used as a control.
Extraction of proteins, immunoblot analysis and assays of AOC activity
Proteins extracted according to the method described by Meyer et al. (1988) were solubilized in SDS sample buffer, subjected to SDS–PAGE and used in immunoblot analysis. A rabbit polyclonal antibody raised against the recombinant tomato AOC ( Ziegler et al., 2000 ) was used as primary antibody at a dilution of 1:5000, and anti-rabbit IgG conjugated with alkaline phosphatase (Boehringer Mannheim) was used as secondary antibody. Staining of immunodecorated AOC was done with p-nitroblue tetrazoline chloride (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP). The anti-AOC antibody was free of cross-reactivity in an immunoblot analysis of a total protein extract of tomato leaves. AOC activity was measured in 0.5 g flower stalks or 0.5 g leaf tissues as described previously ( Ziegler et al., 1997 ; Ziegler et al., 1999 ).
Tissues were fixed, embedded in PEG and cut as described previously ( Hause et al., 1996 ). Sections of 2 µm thickness were immunolabelled with the rabbit anti-AOC antibody raised against recombinant tomato AOC (diluted 1:2000 in PBS containing 5% w/v BSA). Subsequently, an anti-rabbit IgG antibody conjugated with alkaline phosphatase (Boehringer Mannheim, Germany) was used according to the supplier's instructions. After staining with NBT and BCIP, the slides were analysed by bright-field microscopy with a Zeiss ‘Axioskop’ microscope (Zeiss, Jena, Germany) equipped with a CCD camera (Sony, Japan).
Hand sections (0.5 mm thickness) from roots, stems and petioles were fixed with 4% paraformaldehyde in PBS at room temperature for 4 h. After washing with PBS and blocking with 5% BSA in PBS, sections were immediately incubated with the rabbit anti-AOC antibody (diluted 1:5000 in PBS containing 5% BSA) at 4°C overnight. Subsequently, incubations were performed as described above with the secondary antibody and staining with NBT and BCIP. Sections were analysed with a stereomicroscope (Stemi 2000, Zeiss) equipped with a CCD camera. In all experiments, controls were performed by using the pre-immune serum.
Extraction, isolation and quantification of jasmonates, JA amino acid conjugates and octadecanoids
Tissues (1 g fresh weight) were frozen in liquid nitrogen, homogenized in a mortar and extracted with 5 ml 80% v/v methanol. For quantification of JA and JAME, appropriate amounts of (2H6) JA were added to the extract, whereas in the case of the isoleucine conjugate of JA and its methyl ester, JA-[2H3]-Leu was used as the internal standard.
The methanolic extracts were purified by chromatographic steps as described for the isolation of JA and JAME ( Kramell et al., 2000 ). The final separation was performed by RP –HPLC (column: LiChrospher 100, RP-18, 250 mm × 4 mm , length 5 m; flow rate: 1 ml min−1; UV detection at 210 nm) using a 70:30 v/v mixture of methanol and water (containing 0.2% v/v acetic acid) as the mobile phase. The fractions corresponding to authentic JA (4–5 min) and the leucine and isoleucine conjugates of JA (6–8 min) were concentrated in vacuo. The content of the isoleucine conjugate of JA and its methyl ester was calculated on the basis of a calibration curve recorded with methylated JA-[2H3]-Leu. The intensities of the molecular ions at m/z 340 for the deuterated compound and m/z 337 for the non-labelled compounds were compared. For quantification of OPDA, 0.5 g plant material was extracted and prepared for GC–MS analysis according to the method described by Müller and Brodschelm (1994). GC–MS was performed with a Finnigan GCQ: 70 eV, NCl , ionization gas NH3, source temperature 140°C, column Rtx-5 (30 m × 0.25 mm, 0.25 µm film thickness), injection temperature 250°C, interface temperature 275°C, helium 40 cm sec−1; splitless injection; column temperature programme: 1 min at 60°C, 25°C per min to 180°C, 5°C per min to 270°C, 1 min at 270°C, 10°C per min to 300°C, 25 min at 300°C. Retention times were 9,10,dihydro-JA-pentafluorobenzyl ester: 15.13 min ; OPDA-pentafluorobenzyl ester: 25.21 min and 25.88 min using fragments m/z 211 (standard) and m/z 291 for quantification.
We thank M. Krohn, S. Vorkefeld and C. Kuhnt for technical assistance in quantitative measurements of jasmonates and octadecanoids, J. Page for critical reading and C. Dietel for typing the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft SFB 363/C5.
- 1993) Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors. Plant Cell, 5, 203 213. , , , , (
- 1995) Proteinase inhibitor II is developmentally regulated in Nicotiana flowers. Physiol. Plant. 94, 701 707. , , , (
- 1997) Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris. Planta, 201, 397 404.DOI: 10.1007/s004250050082 , , , , , , (
- 1993) Characterization of an Arabidopsis lipoxygenase gene responsive to methyl jasmonate and wounding. Plant Physiol. 103, 1133 1137. & (
- 1995) A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis. Proc. Natl Acad. Sci. USA , 92, 8675 8679. , , (
- 1995) The octadecanoic pathway: signal molecules for the regulation of secondary pathways. Proc. Natl Acad. Sci. USA , 92, 4099 4105. , , , , , , , (
- 1999) Structure–activity analyses reveal the existence of two separate groups of active octadecanoids in elicitation of the tendril-coiling response of Bryonia dioica Jacq. Planta, 207, 470 479.DOI: 10.1007/s004250050506 , , , , , , (
- 1998) Phytooxylipins and plant defense reactions. Prog. Lipid Res. 37, 33 72.DOI: 10.1016/s0163-7827(98)00004-6 (
- 1998) Wounding and chemicals induce expression of the Arabidopsis thaliana gene Thi2.1 encoding a fungal defense thionin, via the octadecanoid pathway. FEBS Lett. 437, 281 286.DOI: 10.1016/s0014-5793(98)01251-4 , , , , , (
- 1990) Defense-related proteins in higher plants. Annu. Rev. Biochem. 59, 873 907. (
- 1995) Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low. Plant Cell, 7, 1319 1331. , , , , , (
- 1999) Leucine aminopeptidase RNAs, proteins, and activities increase in response to water deficit, salinity, and the wound signals systemin, methyl jasmonate, and abscisic acid. Plant Physiol. 120, 979 992. , , , , (
- 1996a) Intracellular levels of free linolenic and linoleic acids increase in tomato leaves in response to wounding. Plant Physiol. 111, 797 803. , , , (
- 1996b) The octadecanoid signalling pathway in plants mediates a response to ultraviolet radiation. Nature, 383, 826 829. , , , (
- 1995) Jasmonic acid distribution and action in plants: regulation during development and response to biotic and abiotic stress. Proc. Natl Acad. Sci. USA , 92, 4114 4119. & (
- 1997) Biosynthesis and action of jasmonates in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 355 381. & (
- 1995) Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway. Proc. Natl Acad. Sci. USA , 92, 4095 4098. , , , (
- 1997) Glucose and stress independently regulate source and sink metabolism and defense mechanisms via signal transduction pathways involving protein phosphorylation. Plant Cell, 9, 1825 1841. , , , (
- 1998) Fatty acid signaling in Arabidopsis. Planta, 206, 167 174.DOI: 10.1007/s004250050388 , , (
- 1995) Jasmonate-induced lipoxygenase forms are localized in chloroplast of barley leaves (Hordeum vulgare cv, Salome). Plant J. 7, 949 957. , , , , (
- 1997) Regulation and tissue-specific distribution of mRNAs for three extracellular invertase isoenzymes of tomato suggests an important function in establishing and maintaining sink metabolism. Plant Physiol. 115, 273 282. & (
- 1992) Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc. Natl Acad. Sci. USA , 89, 2389 2393. , , , (
- 1992) Oxylipin pathway to jasmonates: biochemistry and biological significance. Biochim. Biophys. Acta , 116, 1 18. & (
- 1995) Expression of a flax allene oxide synthase cDNA leads to increased endogenous jasmonic acid (JA) levels in transgenic potato plants but not to a corresponding activation of JA-responding genes. Plant Cell, 7, 1645 1654. , , , , , , (
- 1996) Developmental and tissue-specific expression of JIP-23, a jasmonate-inducible protein of barley. Plant Cell Physiol. 37, 641 649. , , , , (
- 1997) A gene encoding a chloroplast-targeted lipoxygenase in tomato leaves is transiently induced by wounding, systemin, and methyl jasmonate. Plant Physiol. 114, 1085 1093. , , (
- 1996) Localized wounding by heat initiates the accumulation of proteinase inhibitor II in abscisic acid-deficient plants by triggering jasmonic acid biosynthesis. Plant Physiol. 112, 853 860. , , , , , (
- 1997) Lipoxygenase-2 oxygenates storage lipids in embryos of germinating barley. Eur. J. Biochem. 248, 452 458. , , , (
- 2000) Cytochrome P450-dependent metabolism of oxylipins in tomato. Cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase. Plant Physiol. 123, 711 724. , , , , (
- 1997) Tomato prosystemin promoter confers wound-inducible, vascular bundle-specific expression of the β-glucuronidase gene in transgenic tomato plants. Planta, 203, 406 412.DOI: 10.1007/s004250050207 , , , , (
- 1998) Induction of reproductive organ-preferential histone genes by wounding or methyl jasmonate. Mol. Cells , 8, 669 677. , , , (
- 1995) Jasmonates from pine pollen. Phytochemistry, 38, 569 571.DOI: 10.1016/0031-9422(94)00748-i & (
- 1999) Differential induction of plant volatile biosynthesis in the lima bean by early and late intermediates of the octadecanoid-signaling pathway. Plant Physiol. 121, 153 162. , , , , (
- 1997) Amino acid conjugates of jasmonic acid induce jasmonate-responsive gene expression in barley (Hordeum vulgare L.) leaves. FEBS Lett. 414, 197 202.DOI: 10.1016/s0014-5793(97)01005-3 , , , , , (
- 2000) Octadecanoid-derived alteration of gene expression and the ‘oxylipin signature’ in stressed barley leaves – implications for different signalling pathways. Plant Physiol. 123, 177 186. , , , , (
- 1995) Induction of volatile biosynthesis in the Lima bean (Phaseolus lunatus) by leucine- and isoleucine conjugates of 1-oxo- and 1-hydroxyindan -4-carboxylic acid: evidence for amino acid conjugates of jasmonic acid as intermediates in the octadecanoid signalling pathway to jasmonates . FEBS Lett. 377, 523 529.DOI: 10.1016/0014-5793(95)01398-9 , , (
- 1999) Structure and regulation of the Arabidopsis thaliana allene oxide synthase gene. Planta, 208, 463 471.DOI: 10.1007/s004250050583 , , (
- 1999) Pollination, wounding and jasmonate treatments induce the expression of a developmentally regulated pistil dioxygenase at a distance, in the ovary, in the wild potato Solanum chacoense Bitt. Plant Mol. Biol. 41, 371 386. , , (
- 1998) Allene oxide synthase: a major control point in Arabidopsis thaliana octadecanoid signalling. Plant J. 15, 675 684. & (
- 1996) Cloning, molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP 74), the first enzyme of the octadecanoid pathway to jasmonates . Plant Mol. Biol. 31, 323 335. , , , , (
- 1989) Pathogenesis-related proteins are developmentally regulated in tobacco flowers. Plant Cell, 1, 881 887. , , (
- 1996) The critical requirement for linolenic acid is pollen development, not photosynthesis, in an Arabidopsis mutant. Plant Cell, 8, 403 416. & (
- 1997) Jasmonate is essential for insect defense in Arabidopsis. Proc. Natl Acad. Sci. USA , 94, 5473 5477.DOI: 10.1073/pnas.94.10.5473 , , , , (
- 1992) Structure, expression, and antisense inhibition of the systemin precursor gene. Science, 255, 1570 1573. , , , (
- 2000) Allene oxide synthases of barley (Hordeum vulgare cv. Salome) – tissue specific regulation in seedling development. Plant J. 21, 199 213.DOI: 10.1046/j.1365-313x.2000.00669.x , , , , (
- 1988) Preparation by two-dimensional electrophoresis of proteins for antibody production: antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts. Electrophoresis, 9, 704 712. , , , (
- 1991) Synthesis of (±)-(10-2H,11-2H2,12-2H3) jasmonic acid . Z. Naturforsch. 46b, 1724 1729. (
- 2000) Octadecanoid and jasmonate signalling in tomato leaves (Lycopersicon esculentum Mill.) – endogenous jasmonates do not induce jasmonate biosynthesis. Biol. Chem. 381, 715 722. & (
- 1997) A jasmonic acid conjugate N-[(–)-jasmonoyl]-tyramine from Petunia pollen. Phytochemistry, 47, 327 329.DOI: 10.1016/s0031-9422(97)00617-1 , , , , (
- 1994) Quantification of jasmonic acid by capillary gas chromatography–negative chemical ionization–mass spectrometry. Anal. Biochem. 218, 425 435.DOI: 10.1006/abio.1994.1202 & (
- 1999) Positional specificity of a phospholipase A activity induced by wounding, systemin, and oligosaccharide elicitors in tomato leaves. Plant Cell, 11, 2249 2260. , , (
- 1995) Autoradiographic and biochemical evidence for the systemic translocation of systemin in tomato plants. Planta, 195, 593 600. , , , , (
- 1990). Chitinase, β-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation. Plant Cell, 2, 673 684. , , , , , , , (
- 1996) Ethylene as a signal mediating the wound response of tomato plants. Science, 274, 1914 1917. , , , , , (
- 1995) The major protein of guayule rubber particles is a cytochrome P450. J. Biol. Chem. 270, 8487 8494. , , , , , , (
- 1997) Induction of 12-oxo-phytodienoic acid in wounded plants and elicited plant cell cultures. Plant Physiol. 115, 1057 1064. , , (
- 1991) Abscisic acid mediates wound induction but not developmental-specific expression of the proteinase inhibitor II gene family. Plant Cell, 3, 963 972. , , (
- 1995) Signals involved in wound-induced proteinase inhibitor II gene expression in tomato and potato plants. Proc. Natl Acad. Sci. USA , 92, 4106 4113. , , (
- 1994) A novel lipoxygenase from rice. Primary structure and specific expression upon incompatible infection with rice blast fungus. J. Biol. Chem. 269, 3755 3761. , , , , , (
- 1996) Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. Plant Cell, 8, 2309 2323. , , , , , , , , (
- 1993) Methyl jasmonate represses translation initiation of a specific set of mRNAs in barley. Plant J. 4, 459 467. , , (
- 2000) A defect in β-oxidation causes abnormal inflorescence development in Arabidopsis. Plant Cell. 11, 1911 1923. & (
- 1995) Repression of the pea lipoxygenase gene loxg is associated with carpel development. Plant Mol. Biol. 27, 887 899. & (
- 1994) Relationship between degradation of ribulose-bisphosphate carboxylase/oxygenase and synthesis of an abundant protein of 23 kDa of barley leaves (Hordeum vulgare cv. Salome) induced by jasmonates. J. Plant Physiol. 143, 39 46. , , (
- 1996) Characterization of three potato lipoxygenases with distinct enzymatic activities and different organ-specific and wound-regulated expression patterns. J. Biol. Chem. 271, 21012 21019. , , , , , , (
- 1999) Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests. Proc. Natl Acad. Sci. USA , 96, 1146 1151. , , , , , , , (
- 1992) The search for the proteinase inhibitor inducing factor, PIIF. Plant Mol. Biol. 19, 123 133. (
- 2000) The systemin signaling pathway: differential activation of plant defensive genes. Biochim. Biophys. Acta , 1477, 112 121. (
- 1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. , , (
- 2000) The Arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway. Plant Cell, 12, 1041 1061. , , , , , , (
- 1997) Molecular cloning and characterization of 12-oxophytodienoate reductase, an enzyme of the octadecanoid signaling pathway from Arabidopsis thaliana. J. Biol. Chem. 272, 28066 28072. & (
- 1998) 12-Oxophytodienoate-19,11-reductase: occurrence of two isoenzymes of different specificity against stereoisomers of 12-oxophytodienoic acid. Plant Physiol. 188, 1345 1351. , , (
- 2000) 12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis. Planta, 210, 979 984. , , , , (
- 1990) Gas chromatographic/mass spectrometric and tandem mass spectrometric investigations of synthetic amino acid conjugates of jasmonic acid and endogenously occurring related compounds from Vicia faba L. Biomed. Environ. Mass Spectrom. 19, 327 338. , , , , , , , (
- 2000) Expression of allene oxide synthase determines defense gene activation in tomato. Plant Physiol. 122, 1335 1342. , , (
- 1993) Molecular cloning of an allene oxide synthase. A cytochrome P-450 specialized for metabolism of fatty acid hydroperoxides. Proc. Natl Acad. Sci. USA , 90, 8519 8523. , , (
- 1998) Quantitation of the octadecanoid 12-oxo-phytodienoic acid, a signalling compound in plant mechanotransduction. Phytochemistry, 47, 539 546. , , , , , (
- 2000) Restoration of male-fertility by jasmonic acid. In 6th International Congress on Plant Molecular Biology, Quebec, 18–24 June 2000,(Abstract) S25 S31. & (
- 1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc. Natl Acad. Sci. USA , 95, 15107 15111. , , , , , , (
- 2000) Purification and inactivation by substrate of an allene oxide synthase (CYP74) from corn (Zea mays L.) seeds. Phytochemistry, 53, 319 323. , , , , , , (
- 1996) Lipoxygenase gene expression in the tobacco–Phytophthora parasitica nicotianae interaction. Plant Physiol. 112, 997 1004. , , , , (
- 1983) The biosynthesis of jasmonic acid: a physiological role for plant lipoxygenase. Biochem. Biophys. Res. Commun. 111, 470 477. & (
- 1998) Characterization of methyljasmonate-inducible lipoxygenase from barley (Hordeum vulgare cv. Salome) leaves. Eur. J. Biochem. 251, 36 44. , , , , , , , (
- 1999) Overexpression of a cytoplasm-localized allene oxide synthase promotes the wound-induced accumulation of jasmonic acid in transgenic tobacco. Plant Mol. Biol. 40, 783 793. , , (
- 1997) Jasmonate-signalled plant gene expression. Trends Plant Sci. 2, 302 307. & (
- 1998) Diversity in octadecanoid-induced gene expression of tomato. J. Plant Physiol. 152, 345 352. , , , , , , , , , , (
- 1997) Dinor -oxo-phytodienoic acid: a new hexadecanoid signal in the jasmonate family . Proc. Natl Acad. Sci. USA , 94, 10473 10478. , , (
- 1999) Metabolic profiling of oxylipins upon salicylate treatments in barley leaves – preferential induction of the reductase pathway by salicylate. FEBS Lett. 464, 133 137. , , , , (
- 2000) Stress-induced factor involved in flower formation of Lemna is an α-ketol derivative of linolenic acid . Plant Cell Physiol. 41, 110 113. , , , , , , , , , (
- 1997) Purification and characterization of allene oxide cyclase from dry corn seeds. Plant Physiol. 114, 565 573. , , , (
- 1999) On the specificity of allene oxide cyclase. Lipids, 34, 1005 1015. , , (
- 2000) Molecular cloning of allene oxide cyclase: the enzyme establishing the stereochemistry of octadecanoids and jasmonates. J. Biol. Chem. 275, 19132 19138. , , , , , , , , (