Expression Of Phenylalanine Ammonia-lyase Research Articles

Salicylic acid-, but not cytokinin-induced, resistance to WClMV is associated with increased expression of SA-dependent resistance genes in Phaseolus vulgaris

Two-week-old Phaseolus vulgaris plants, wick-fed with 1 mmol/L salicylic acid (SA) or 50 nmol/L dihydrozeatin (DHZ), showed partial inhibition of the accumulation of white clover mosaic virus (WClMV) in infected primary leaves. This inhibition was measured as a decrease in the accumulation of both viral mRNA and viral coat protein, especially at the early stages of infection. Salicylic acid treatment resulted in moderately increased expression of phenylalanine ammonia lyase (PAL), NPR1, PR1 and HSP70 genes that participate in resistance to pathogens in plants. In contrast, DHZ treatments did not induce significant changes in expression of these genes. The expression of the P. vulgaris alternative oxidase (AOX) gene homolog, an enzyme implicated in plant resistance to viruses, showed low constitutive expression during the first 11 days post-infection and was not affected by either SA or DHZ. It appears that, while SA induced the NPR1-PR1 pathogen defense pathway genes, both SA and DHZ may use a different pathway to induce resistance to WClMV infection in P. vulgaris plants.

The Canadian Phytopathological Society Annual Meeting, Montréal, Quebec, 2003

The Canadian Phytopathological Society Annual Meeting, Montréal, Quebec, 2003

Abscisic acid and 2,4-dichlorophenoxyacetic acid affect the expression of anthocyanin biosynthetic pathway genes in ‘Kyoho’ grape berries

SummaryThe effects of abscisic acid (ABA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on the expression of seven anthocyanin biosynthetic pathway genes in ‘Kyoho’ grape berries were investigated. In untreated berries, the expression of the UDP-glucose-flavonoid: 3-O-glucosyltransferase (UFGT) gene was detected only at 42 d after full bloom (DAB), whereas the phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin dioxygenase (LDOX) genes were expressed throughout the growing period. ABA increased anthocyanin content in the skin and the expression of PAL, CHS, CHI, DFR and UFGT genes at 7 d after treatment. In contrast, 2,4-D inhibited the accumulation of anthocyanin and the expression of all the genes examined. The results clearly show that the anthocyanin levels resulting from the application of ABA and 2,4-D were correlated with the expression of anthocyanin biosynthetic pathway genes.

Phenylalanine ammonia lyase gene expression during abscission in citrus.

Phenylalanine ammonia lyase (PAL) gene expression was examined in fruit and leaf abscission zones of Valencia orange for periods up to 72 h after induction of abscission with Ethrel(R) (CEPA) or the citrus mature fruit-specific abscission material 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMN-P). PAL gene expression was detected in mature fruit abscission zones beginning 6 and 24 h after CEPA and CMN-P application, respectively, and continued up to 72 h. PAL gene expression was detected in leaf abscission zones 6 h after CEPA application and continued throughout the remainder of the study. In contrast, PAL gene expression was not detected in leaf abscission zones treated with CMN-P. 2-aminoindan-2-phosphonic acid (AIP), a specific inhibitor of PAL enzyme activity, decreased CMN-P-induced PAL expression in fruit abscission zones, and this was accompanied by a lack of fruit drop. PAL transcripts were low in abscission zones of immature fruit; however, the transcript was induced by CMN-P but less by CEPA application. The data suggest that downstream products of PAL activity may be important not only for wound healing and defense reactions that occur at the abscission zone fracture plane, but also for regulation of PAL gene expression during abscission.

The tobacco bZIP transcription factor BZI-1 binds to G-box elements in the promoters of phenylpropanoid pathway genes in vitro, but it is not involved in their regulation in vivo

Screening of a tobacco (Nicotiana tabacum) cDNA library resulted in the isolation of a clone encoding the bZIP transcription factor BZI-1. With respect to amino acid sequence, conservation of protein domains, genomic exon-intron structure and expression pattern, BZI-1 is closely related to CPRF2, OHP1/2, BLZ1 and REB, a group of bZIP proteins which have been described in a number of dicot and monocot species. BZI-1 exhibits the characteristics of a transcription factor. It binds to G-box and C-box cis-elements in vitro, it is localised in the nucleus, and the N-terminal region of BZI-1 functions as an activation domain in both yeast and plant cells. Since BZI-1-related transcription factors have been isolated from dicots by in vitro binding to G-box elements in the chalcone synthase ( CHS) promoter, it has been suggested that phenylpropanoid pathway genes, such as CHS and PAL (phenylalanine ammonia-lyase), are targets of these proteins in vivo. However, after infection with Pseudomonas syringae or Tobacco Mosaic Virus, no changes in pathogen-induced PAL expression were observed in transgenic plants expressing increased levels of BZI-1 or a dominant negative form of the protein, BZI-1-DeltaN. In contrast to the tissue-specific expression of CHS and PAL, BZI-1 was found to be ubiquitously expressed in tobacco plants. Furthermore, no changes in the tissue-specific expression of PAL or CHS were observed in plants that were transgenic for BZI-1-DeltaN. Expression of a VP16-BZI-1 fusion protein would be expected to result in constitutive activation of the BZI-1 target genes. However, tetracycline-dependent expression of a VP16-BZI-1 protein in tobacco plants did not result in activation of CHS or PAL. On the basis of these data, we conclude that the phenylpropanoid pathway genes analysed are not targets of BZI-1 in vivo. Thus, the pattern of in vitro DNA binding of transcription factors need not always reflect their in vivo function.

Study of Defense-related Gene Expression in Grapevine Leaves and Berries Infected with Botrytis cinerea

Defense responses of grapevine towards Botrytis cinerea were investigated. The expression of genes coding for proteins involved in defense were studied: (a) phenylalanine ammonia-lyase (PAL) and stilbene synthase (StSy), (b) an acidic chitinase (VCH3) and a basic chitinase (VCHIT1b), and (c) a polygalacturonase inhibitor protein (PGIP). Since no PGIP was known in grapevine, a complete cDNA sequence was first characterized by PCR and RACE-PCR amplifications. RNAs isolated from infected leaves and infected berries were analysed by semi-quantitative and real-time RT-PCRs. In infected leaves, the expression of PAL, StSy, PGIP and VCH3 genes occurred 6 hours post inoculation (hpi). Increase of VCHIT1b gene expression was delayed (24 hpi). Maximum levels of induction of these genes were observed at 48 hpi, except for the VCH3 gene (24 hpi). Activation of these defense responses was not sufficient to stop B. cinerea spread. In berries, no VCH3 gene expression was detected. Maximum levels of induction were observed in stage 3 (loss of berry colour and abundant production of conidia) for the PAL and PGIP genes, and in stage 4 (shrivelled berry) for the StSy and VCHIT1b genes.

Study on a novel strategy to treatment of phenylketonuria.

To replace the low phenylalanine (phe) diet treatment and to improve the quality of life of the phenylketonuria (PKU) patients, A phenylalanine ammonia-lyase (PAL) cDNA from petroselinum crispum was subcloned into expression vectors, pMG36e and pNZ8048, and transformed L. lactis by electroporation. The pMG36e PAL/L. lactis and pNZ8048 PAL/L.L. were screened and characterized by using PCR and HPLC, and prepared as a liquid type preparation that were given orally to treat hyperphenylalaninemia-rats. The phe levels of the rat plasma sample were determined by HPLC. The data showed that the plasma phe levels in hyperphenylalanemia (HPA) rats receiving preparations made from the engineered L. lactis were significantly reduced compared with non-treated HPA rats. The effect of the pNZ8048PAL/L.L. showed a higher expression of PAL and better cure results than pMG36ePAL/L.L. These results point a potential way for PKU treatment.

Molecular evidence for induction of phenylalanine ammonia-lyase during Puccinia graminis infection and elicitation in wheat

Degenerate primers corresponding to the conserved sequences in phenylalanine ammonia-lyase (PAL) genes cloned from rice and parsley were used to amplify segments of the PAL genes from wheat (Triticum aestivum). More than 76% base sequence identity with the homologous genes confirmed that a 437 base pairs (bp) fragment was derived from the coding region of PAL. Using this fragment as probe, two PAL genes linked in tandem, Wpalt1 and Wpalt2, were isolated from a wheat genomic library. Northern blot and RNA dot blot analyses using a probe for Wpalt1 demonstrated that Wpalt1 expression was organ specific. Wpalt1 expression was high in roots and moderate in stems, but no detectable expression was found in leaves. Infection of leaves with Puccinia graminis f. sp. tritici (Pgt) induced, within 4 to 8 d, a high level of Wpalt1 transcripts in the Pgt resistant near-isogenic Prelude Sr6 line, while a delayed induction was observed in the Pgt susceptible isogenic Prelude sr6 line. In cell suspension cultures, treatment with chitin oligomers or an elicitor derived from P. graminis germ tube walls activated Wpalt1 gene expression. The Pgt-derived elicitor activated PAL expression earlier than did chitin oligomers, whereas chitin oligomers appeared to be more potent inducers of PAL expression than was the Pgt-derived elicitor.

Novel anther-specific myb genes from tobacco as putative regulators of phenylalanine ammonia-lyase expression.

Two cDNA clones (NtmybAS1 and NtmybAS2) encoding MYB-related proteins with strong sequence similarity to petunia (Petunia hybrida) PhMYB3 were isolated from a tobacco (Nicotiana tabacum cv Samsun) pollen cDNA library. Northern blot and in situ hybridization revealed that NtmybAS transcripts are specifically expressed in both sporophytic and gametophytic tissues of the anther including tapetum, stomium, vascular tissue, and developing pollen. Random binding site selection assays revealed that NtMYBAS1 bound to DNA sequences closely resembling consensus MYB binding sites MBSI and MBSIIG, with a higher affinity for MBSI. Transient expression analyses of the N-terminal MYB domain demonstrated the presence of functional nuclear localization signals, and full-length NtMYBAS1 was able to activate two different phenylalanine ammonia-lyase promoters (PALA and gPAL1) in tobacco leaf protoplasts. Similar analysis of truncated NtmybAS1 cDNAs identified an essential, C-terminal trans-activation domain. Further in situ hybridization analyses demonstrated strict co-expression of NtmybAS and gPAL1 in the tapetum and stomium. Despite abundant expression of NtmybAS transcripts in mature pollen, gPAL1 transcripts were not detectable in pollen. Our data demonstrate that NtMYBAS1 is a functional anther-specific transcription factor, which is likely to be a positive regulator of gPAL1 expression and phenylpropanoid synthesis in sporophytic, but not in gametophytic, tissues of the anther.

Bacterio-opsin gene overexpression fails to elevate fungal disease resistance in transgenic poplar (Populus)

Overexpression of the bacterio-opsin (bO) gene in tobacco had previously been shown to induce hypersensitive-response-like lesions, increase viral and bacterial disease resistance, and stimulate pathogenesis-related gene expression. To see if this gene enhanced resistance to fungal pathogens of poplar, we generated a total of 35 transgenic lines in two clones of Populus trichocarpa Torr. & A. Gray × Populus deltoides Bartr. ex Marsh. and one clone of P. trichocarpa × Populus nigra L. and challenged them with the fungal pathogens Melampsora occidentalis H. Jack (leaf rust), Venturia populina (Vuill.) Fabric. (leaf and shoot blight), Septoria musiva Peck, and Septoria populicola Peck (leaf spot and stem canker) in greenhouse, field, or laboratory inoculations. Northern analysis showed that the bO gene was expressed in the transgenic poplars; however, no increase in expression of phenylalanine ammonia-lyase (PAL1) or two wound-inducible poplar chitinase genes (WIN6 and WIN8) were observed, even in one line that showed very high bO expression, intensive lesion development, and retarded growth. Poplars required a high threshold of bO expression for lesion development, and susceptibility to all of the pathogens tested was unaffected by bO overexpression.

Expression of plant defence-related (PR-protein) transcripts during hardening and dehardening of winter wheat

The pattern of expression of the pathogenesis-related (PR) proteins PR-1, chitinase, β-glucanase, and peroxidase, and phenylalanine ammonia lyase (PAL) transcripts was studied in five winter wheat cultivars differing in resistance to snow mould, under controlled conditions following growth at 20°C, during hardening at 2°C and dehardening at 20°C. The expression of these transcripts was also studied in two cultivars of winter wheat under natural conditions during autumn, winter, and early spring of 1997–98 at a field site in Lethbridge. The relative abundance of transcripts was similar in both the field and controlled environment studies: chitinase was the most abundant transcript followed by PAL, β-1,3-glucanase, PR-1, and peroxidase. Under field conditions, all PR-protein transcripts exhibited the same basic pattern of expression during the autumn, winter and spring sampling dates; transcripts were expressed during the late autumn, reached high levels by mid-winter, then decreased before reaching maximum levels during the spring. Conversely, PAL expression was low or absent in autumn, reached the highest levels by mid-winter, and then gradually decreased during the spring. Under controlled environment conditions, transcripts encoding for PAL and the PR-proteins, except chitinase, were constitutively expressed to varying extents in the unhardened treatments. The general pattern of expression fell into two groups: (1) transcripts of chitinase, β-1,3-glucanase, and PAL, were weakly expressed in the unhardened treatments, strongly up-regulated following exposure to hardening conditions, and disappeared or remained stable following dehardening; and, (2) PR-1 and peroxidase transcripts were down-regulated upon initial exposure to hardening, increased slightly following prolonged exposure to hardening conditions, and remained stable during dehardening. Under controlled conditions, the pattern of expression of any of the PR-proteins did not appear to be associated with known genotypic levels of snow mould resistance among cultivars but, under field conditions, expression levels were generally higher for all PR-proteins in the snow mould resistant cultivar, Cl14106 than in the moderately susceptible cultivar, Norstar. These results demonstrate that different PR-protein and PAL transcripts in winter wheat are expressed in the field throughout the winter and are differentially induced in response to exposure of plants to low, hardening temperatures under both field and controlled environment conditions.

Post-transcriptional regulation of phenylalanine ammonia-lyase expression in tobacco following recovery from gene silencing.

Introduction of a bean phenylalanine ammonia-lyase (PAL) transgene into tobacco plants results in epigenetic post-transcriptional gene silencing which is unstable, such that after self-pollination first generation progeny may become PAL over-expressors. The change from gene silencing to PAL over-expression is accompanied by a loss of cytosine methylation of the PAL transgene and reduced methylation of the endogenous tobacco PAL2 gene, but not the PAL1 gene. These changes are associated with the appearance of high levels of bean PAL and tobacco PAL2 transcripts in the total RNA fraction from PAL over-expressing plants. However, tobacco PAL2 transcripts are inefficiently recruited into polysomes, and tobacco PAL2 protein is not detected in leaves of PAL over-expressing or wild-type lines. Thus, in spite of the post-transcriptionally controlled increase in tobacco PAL2 transcripts in PAL over-expressors, the increased PAL activity is primarily the result of the increase in bean PAL transcripts and corresponding enzymatic activity. These results reveal a complex cross-talk between expression of the PAL transgene and the corresponding endogenous PAL genes at the levels of transcription, transcript stability and polysomal recruitment during sense transgene-mediated silencing and subsequent over-expresson of PAL in tobacco.

N-Acylethanolamines in signal transduction of elicitor perception. Attenuation Of alkalinization response and activation of defense gene expression

In a recent study of N-acylphosphatidylethanolamine (NAPE) metabolism in elicitor-treated tobacco (Nicotiana tabacum L.) cells, we identified a rapid release and accumulation of medium-chain N-acylethanolamines (NAEs) (e.g. N-myristoylethanolamine or NAE 14:0) and a compensatory decrease in cellular NAPE (K.D. Chapman, S. Tripathy, B. Venables, A.D. Desouza [1998] Plant Physiol 116: 1163-1168). In the present study, we extend this observation and report a 10- to 50-fold increase in NAE 14:0 content in leaves of tobacco (cv Xanthi) plants treated with xylanase or cryptogein elicitors. Exogenously supplied synthetic NAE species affected characteristic elicitor-induced and short- and long-term defense responses in cell suspensions of tobacco and long-term defense responses in leaves of intact tobacco plants. In general, synthetic NAEs inhibited elicitor-induced medium alkalinization by tobacco cells in a time- and concentration-dependent manner. Exogenous NAE 14:0 induced expression of phenylalanine ammonia lyase in a manner similar to fungal elicitors in both cell suspensions and leaves of tobacco. NAE 14:0, but not myristic acid, activated phenylalanine ammonia lyase expression at submicromolar concentrations, well within the range of NAE 14:0 levels measured in elicitor-treated plants. Collectively, these results suggest that NAPE metabolism, specifically, the accumulation of NAE 14:0, are part of a signal transduction pathway that modulates cellular defense responses following the perception of fungal elicitors.

Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory

Pre-inoculation of plants with a pathogen that induces necrosis leads to the development of systemic acquired resistance (SAR) to subsequent pathogen attack [1]. The phenylpropanoid-derived compound salicylic acid (SA) is necessary for the full expression of both local resistance and SAR [2,3]. A separate signaling pathway involving jasmonic acid (JA) is involved in systemic responses to wounding and insect herbivory [4,5]. There is evidence both supporting and opposing the idea of cross-protection against microbial pathogens and insect herbivores [6, 7]. This is a controversial area because pharmacological experiments point to negative cross-talk between responses to systemic pathogens and responses to wounding [8–10], although this has not been demonstrated functionally in vivo. Here, we report that reducing phenylpropanoid biosynthesis by silencing the expression of phenylalanine ammonialyase (PAL) reduces SAR to tobacco mosaic virus (TMV), whereas overexpression of PAL enhances SAR. Tobacco plants with reduced SAR exhibited more effective grazing-induced systemic resistance to larvae of Heliothis virescens, but larval resistance was reduced in plants with elevated phenylpropanoid levels. Furthermore, genetic modification of components involved in phenylpropanoid synthesis revealed an inverse relationship between SA and JA levels. These results demonstrate phenylpropanoid-mediated cross-talk in vivo between microbially induced and herbivore-induced pathways of systemic resistance.

Temporal and genotypic variation of wound-induced gene expression in bark of Populus tremuloides and P. grandidentata

In two related experiments, total RNA was extracted from wounded and unwounded bark of young aspen ramets for Northern and dot blot analyses. Wound-inducible genes isolated from other plant species were hybridized to blots, and mRNA levels were estimated. Analyses of variance (ANOVA) were used to examine the significance of experimental factors (wounding, time after wounding, and genotype) affecting variation in mRNA levels. The first experiment examined the timing (0.5-96 h after wounding) of expression of wound-inducible genes in bark tissue of a single Populus tremuloides Michx. genotype. Wounding and variation among RNA samples significantly (p < 0.05) affected mRNA levels of two chitinases (win6, win8) and phenylalanine ammonia-lyase (PAL). The second experiment examined interclonal variation of wound-induced win6 and PAL expression in aspen bark. Ramets of four P. tremuloides and one Populus grandidentata Michx. genotypes were wounded and bark was collected 4, 8, or 12 h later. Genotype, wounding, and time after wounding all significantly affected win6 and PAL mRNA levels, with levels increasing as a result of wounding.

Plant defence genes are induced in the pathogenic interaction between bean roots and Fusarium solani, but not in the symbiotic interaction with the arbuscular mycorrhizal fungus Glomus mosseae

Plant roots enter symbiotic as well as pathogenic interactions with fungi in the rhizosphere. We studied the response of bean (Phaseolus vulgaris L. cv. Saxa) roots to infection by the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe and the pathogen Fusarium solani (Mart.) Sacc. f. sp. phaseoli (Burkholder) Snyder & Hansen. In a time‐course study of the symbiotic interaction between bean roots and G. mosseae, covering all stages of mycorrhiza development, we detected little change in the expression of the defence‐related genes chitinase, β‐1,3‐glucanase and phenylalanine ammonia‐lyase compared with non‐mycorrhizal control roots. The only difference observed was a transient increase in chalcone synthase transcripts at later stages of mycorrhizal root colonization. In interactions with the pathogen, a marked induction of chitinase and phenylalanine ammonia‐lyase expression was observed at the level of both the transcripts and enzyme activities. Class I β‐1,3‐glucanase levels strongly increased at the transcript level, but there was little change in the overall β‐1,3‐glucanase enzyme activity. In the non‐host interaction between common bean and Fusarium solani (Mart.) Sacc. f. sp. pisi (Linford) Snyder & Hansen defence responses increased only slightly and transiently, if at all.

Do Plant Phenolics Confer Resistance to Specialist and Generalist Insect Herbivores?

The impact of phenolics on a specialist herbivore, Manduca sexta, and a generalist herbivore, Heliothis virescens, was investigated using transgenic tobacco with differential expression of phenylalanine ammonia-lyase. Foliar phenolics such as chlorogenic acid, rutin, and total flavonoids differentially accumulated in the respective transgenic tobacco lines; the amount of chlorogenic acid ranged from 201 to 2202 μg g-1 of fresh leaf, that of total flavonoids from 211 to 500 μg g-1 of fresh leaf, and that of rutin from 73 to 172 μg g-1 of fresh leaf. However, the levels of the phenolics and larval growth of M. sexta or H. virescens were not significantly correlated. Likewise, phenolic levels were not correlated with larval survival of M. sexta. These results suggest that plant phenolics are not a determining factor for host plant resistance against these insects in this system. Keywords: Phenylalanine ammonia-lyase; phenolics; host plant resistance; Manduca sexta; Heliothis virescens

Evidence for some signal transduction elements involved in UV-light-dependent responses in parsley protoplasts

The signalling pathways used by UV-light are largely unknown. Using protoplasts from a heterotrophic parsley (Petroselinum crispum L.) cell culture that exclusively respond to UV-B light between 300 and 350 nm with a fast induction of genes encoding flavonoid biosynthetic enzymes, information was obtained about the UV-light signal transduction pathway for chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL) gene expression. Pharmacological effectors which influence intracellular calcium levels, calmodulin and the activity of serine/ threonine kinases also changed the UV-light-dependent expression of these genes. This evaluation indicated the participation of these components on the UV-B-mediated signal transduction cascade to CHS. In contrast, neither membrane-permeable cyclic GMP nor the tyrosine kinase inhibitor genistein affected CHS or PAL expression. Similar results were obtained in protoplasts, which have been transiently transformed with CHS-promoter/GUS β-glucuronidase) reporter fusion constructs. The involvement of calcium and calmodulin was further indicated in a cell-free light-responsive in vitro transcription system from evacuolated parsley protoplasts. In conclusion, there is evidence now that components of the UV-light-dependent pathway leading to the CHS-promoter are different from the previously characterized cGMP-dependent pathway to CHS utilized by phytochrome in soybean (Glycine max) and tomato seedlings (Lycopersicon esculentum).

Differential utilization of alternate initiation sites in a plant defense gene responding to environmental stimuli.

The upstream regulatory region of an efficiently expressed phenylalanine ammonia-lyase (PAL5) gene from tomato (Lycopersicon esculentum) was characterized by DNA sequencing and S1-nuclease-protection studies. Two transcription initiation sites giving rise to a long and a short transcript were identified, each corresponding to a different putative TATA sequence element with multiple conserved upstream regulatory motifs. When tomato plants were challenged with three different environmental stimuli, wounding, light and Verticillium infection, this gene was observed to utilize the alternate initiation sites differentially, in a tissue-dependent and stimulus-dependent manner. In general, basal levels of the shorter transcript were very low but preferentially and strongly stimulated by the environmental factors. In contrast, the longer transcript was less affected, essentially representing a constitutive expression of this gene. The results suggest that the multiple regulatory sites in the PAL5 gene promoter provide for both an essential constitutive expression of phenylalanine ammonia-lyase as well as an appropriate response to different environmental stresses.

A phenylalanine ammonia-lyase gene from melon fruit: cDNA cloning, sequence and expression in response to development and wounding

Phenylalanine ammonia-lyase (PAL) is the first enzyme of phenylpropanoid biosynthesis involved in the synthesis of a multiplicity of plant natural products. We have isolated and characterized a nearly full-length cDNA clone (pmPAL-1) corresponding to a melon fruit (Cucumis melo L. var. reticulatus) gene coding for a protein which is highly similar to PAL from other plants. Melon fruit PAL is transcriptionally induced both in response to fruit ripening and wounding. PAL gene expression follows the kinetics of expression of the ethylene biosynthetic genes during fruit development. In contrast, ethylene biosynthetic genes show different induction kinetics compared to PAL expression in response to wounding. Similar results have been found for two other genes coding for enzymes involved in flavonoid biosynthesis (chalcone synthase, CHS; chalcone isomerase, CHI). Our results imply that regulation of defense gene expression in melon is a co-ordinated process in response to both ethylene and an ethylene-independent wound signal.