Journal of Experimental Botany, Vol. 53, No. 367, pp. 383-385,
February 1, 2002
© 2002 Oxford University Press
Gene Note |
Pathogen-induced expression of cyclo-oxygenase homologue in hot pepper (Capsicum annuum cv. Pukang)1
2 Plant Cell Biotechnology Laboratory, Korea Research Institute of Bioscience and Biotechnology, PO Box 115, Yusung, Taejon 305–600, Korea
3 Department of Biology, College of Science, Yonsei University, Seoul 120–749, Korea
Received 11 September 2001; Accepted 19 September 2001
Abstract
The hypersensitive reaction (HR) in plants is typified by a rapid and localized cell death at the site of pathogen infection. To understand better the molecular and cellular defence mechanism controlling HR, hot pepper leaves (Capsicum annuum cv. Pukang) were inoculated with the soybean pustule pathogen Xanthomonas campestris pv. glycine 8ra. By using the DD-PCR technique, a cDNA fragment was identified that exhibited a sequence similarity to the recently identified tobacco pathogen-induced oxygenase (PIOX) with homology to animal cyclo-oxygenase (COX). Subsequently, the full-length cDNA clone, pCa-COX1, encoding the COX homologue from the pathogen-inoculated hot pepper leaf cDNA library was isolated. The deduced amino acid sequence of Ca-COX1 shares 85.8% identity with tobacco PIOX and displays a significant degree of sequence identity (21.7–23.7%) with mammalian COXs. The expression of Ca-COX1 was markedly induced at 4–12 h after pathogen infection, while HR cell death on pepper leaves appeared at approximately 15 h post-inoculation. These results are consistent with the notion that the lipid-derived signalling pathway is involved in the initial response of hot pepper plants to pathogen infection.
Key words: Cyclo-oxygenase, hot pepper, hypersensitive reaction, pathogen infection, plant defence mechanism.
The hypersensitive reaction (HR) in higher plants is typified by a rapid and localized cell death at the site of pathogen attack and is generally associated with the production of active oxygen species (AOS), cell wall strengthening and synthesis of phytoalexins and pathogenesis-related (PR) proteins (Dangl et al., 1996
; Hammond-Kosack and Jones, 1996). The HR is a genetically programmed process and results from the transcriptional and post-transcriptional activation of diverse defence-related genes in host cells (Dixon et al., 1994; Dangl et al., 1996). To understand better the molecular and cellular defence mechanism controlling HR that results from the interaction between plant pathogen and its non-host, the response of hot pepper (Capsicum annuum cv. Pukang) to inoculation with the soybean pustule pathogen Xanthomonas campestris pv. glycine 8ra was investigated. By using the differential display polymerase chain reaction (DD-PCR) technique (Liang and Pardee, 1992), a pool of genes induced or suppressed by pathogen infection in hot pepper leaf tissue was identified. About half of the isolated genes had no sequence homology in existing databases, whereas the majority of identified DNA fragments comprised enzymes which participate in the primary and secondary metabolic pathways (Suh et al., 2001). One of the DD-PCR fragments exhibited a sequence similarity to the recently identified tobacco pathogen-induced oxygenase (PIOX) with homology to animal cyclo-oxygenase (COX) or prostaglandin endoperoxide synthase (PGHS) (Sanz et al., 1998).
COX is a key enzyme in the production of lipid-derived signal molecules that regulate diverse cellular processes in vertebrates (DeWitt and Smith, 1988; O'Banion et al., 1992; Serhan et al., 1996). It catalyses the conversion of polyunsaturated fatty acids, such as arachidonic acid (20:4), to prostaglandin and other eicosanoids, and these chemicals modulate the immune response and inflammation reaction in response to pathogen infection in mammalian cells. In tobacco leaves, the PIOX gene encoding a homologue of animal COX has been found to be markedly induced by inoculation with harpin HrpN protein which elicits an HR-like cell death as well as with the HR-inducing bacterium Erwinia amylovora (Sanz et al., 1998). When tobacco PIOX was expressed in insect cells through a baculovirus expression system, it contains COX activity using arachidonic acid and other plant unsaturated fatty acids, including linolenic acid (18:3) and linoleic acid (18:2), as substrates (Sanz et al., 1998). Based on these results, it has been suggested that tobacco PIOX could be involved in the production of lipid signal molecules that are important regulators of plant defence mechanism against pathogen attack (Sanz et al., 1998).
To gain more detailed information concerning the structure of the pathogen-induced hot pepper COX homologue, a full-length cDNA clone was isolated. A cDNA fragment produced by DD-PCR was radioactively labelled and used as a probe to screen the cDNA library constructed from hot pepper leaves inoculated with X. campestris pv. glycine 8ra. Numerous putative COX homologue cDNA clones were isolated. Subsequent restriction enzyme mapping and DNA sequencing analysis indicated that these clones represented a single group of overlapping sequences. Among isolated clones, the pCa-COX1 contains the longest insert. The pCa-COX1 is 2161 bp long consisting of a 61 bp 5'-untranslated region, a 1929 bp long coding region encoding 643 amino acids and a 171 bp 3'-untranslated region (Fig. 1) (the sequence was deposited in the GenBank database under the accession number AY040869). The predicted molecular mass of the protein encoded by pCa-COX1 is 74 kDa which is similar to that of tobacco PIOX (73 kDa) (Sanz et al., 1998). The overall nucleotide sequence identity between pCa-COX1 and tobacco PIOX clone is 82.6%, while the coding region is 86.0% identical at the nucleotide level and 85.8% at the amino acid level (Fig. 1). The protein region corresponding to the putative catalytic domain of tobacco PIOX (residues 223–603) is 89.0% identical to its corresponding region of hot pepper Ca-COX1, consistent with the role of this domain for enzyme activity. In addition, Ca-COX shares 71.9% homology with Arabidopsis protein encoded by the expressed sequence tag cDNA clone N38086, which has a COX activity in insect cell (Sanz et al., 1998). In the putative catalytic domain, three amino acid residues H-167, Y-389 and H-392, which are known to be essential for enzyme activity, are exactly conserved in plant COX homologues. Moreover, the Ca-COX1 protein shares 21.7% and 23.7% sequence homology with rat and sheep COXs, respectively (DeWitt and Smith, 1988; O'Banion et al., 1992). Collectively, these results are in line with the notion that the Ca-COX1 protein may possess the COX enzyme activity in hot pepper plant.
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To analyse the induction kinetics of the Ca-COX1 transcript, hot pepper leaves were infiltrated with X. campestris pv. glycine 8ra or buffer (0.9% NaCl). Total RNAs were then prepared after different time periods of inoculation, and RNA gel blot analysis was carried out by using 32P-labelled pCa-COX1 as a probe. Figure 2A
shows that the low level of 
2.3 kb transcript corresponding to Ca-COX1 was detected in hot pepper leaves before pathogen inoculation. In a trial treatment, the level of transcript increased at 4 h after buffer infiltration and subsequently decreased to the basal level. This transient increase in mRNA amount might be due to the wounding effect of infiltration. By contrast, the expression of Ca-COX1 began to increase at 4 h, and the high level of mRNA was further enhanced at 8–12 h post-inoculation (Fig. 2A). In the duplicated blots, the changes in the level of PR-4 mRNA were assessed as a positive marker of pathogen inoculation. The PR-4 transcript was markedly induced at 8–12 h. HR cell death on pepper leaves appeared at approximately 15 h post-inoculation with the pathogen, while the Ca-COX1 gene, as shown in Fig. 2A, was induced at a considerably earlier time point after pathogen treatment. Thus, these results raise the possibility that Ca-COX1 could be functional in the early event of the pathogen-induced defence response in hot pepper plants.
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Salicylic acid (SA), jasmonic acid (JA) and hydrogen peroxide are generally regarded as cellular signals for the activation of plant defence responses (Hammond-Kosack and Jones, 1996
; Reymond and Farmer, 1998). Sanz et al. have demonstrated that the tobacco PIOX gene is induced by various defence-related signals, such as SA, JA, wounding, and AOS in tobacco leaf tissue (Sanz et al., 1998). Therefore, the possibility that these signalling molecules could also activate Ca-COX1 was considered. To investigate this possibility, hot pepper leaves were subjected to 5 mM SA, 0.1 mM methyl jasmonate (MeJA), 15 mM ethephon, 10 mM H2O2, 5 mM CuSO4 or wounding for different time periods, and the level of Ca-COX1 transcript was monitored at each time point. RNA gel blot analysis showed that the expression of Ca-COX1 mRNA was only mildly affected by these treatments (Fig. 2B
). By contrast, it was shown that the PR-4 gene was clearly induced by MeJA and ethephon treatments as well as by wounding (Park et al., 2001). To assess the copy number of the CaCOX1 gene in hot pepper plants, the Southern blot hybridization experiment was performed on pepper genomic DNA digested with EcoRI, HindIII, XbaI or XmnI using 32P-labelled pCa-COX1 cDNA as a probe. Those restriction enzyme digestions detected three to five hybridizing bands, respectively, with different intensity (data not shown). Thus, it is most likely that there are additional Ca-COX-related genes on the hot pepper genome.
In this communication, the pathogen-induced pepper COX homologue identified by the DD-PCR technique has been described. The induction of Ca-COX1 preceded the appearance of HR on hot pepper leaves, suggesting that the Ca-COX1 protein could participate in the initial response of hot pepper plants to pathogen infection. On the other hand, the Ca-COX1 gene was not activated by various defence-related signalling molecules, including SA, MeJA, ethephon, and H2O2, while the expression of PR-4 was effectively turned on by those signalling molecules. Recently, the ATP:citrate lyase (Ca-ACL1) transcript was identified, whose expression was up-regulated by pathogen infection, but not by SA, MeJA and H2O2 in pepper leaf tissue (Suh et al., 2001). Ca-ACL1 has been suggested to be involved in the formation of phytoalexins. Thus, there may exist different signal transduction pathways and regulatory mechanisms to modulate the PR-4 gene and other pathogen-induced genes, such as Ca-COX1 and Ca-ACL1, in hot pepper leaves. These results are at variance with those of Sanz et al. who found the generalized activation of the PIOX gene by SA, JA, wounding, and AOS in tobacco leaves, and that PIOX is encoded by a single-copy gene (Sanz et al., 1998). Therefore, there is now a need to isolate the Ca-COX gene family and to investigate whether their expressions are differentially regulated at distinct environmental and physiological conditions in pepper leaves. In conclusion, these results are consistent with the notion that the lipid-derived signalling pathway is involved in the early event of the pathogen-induced defence response in hot pepper plants.
Acknowledgments
This work was supported by grants from the Plant Diversity Research Center (21st Century Frontier Research Program of MOST project No. PF 003105-01) and KOSEF (Plant Metabolism Research Center) to WTK and from Plant Diversity Research Center (21st Century Frontier Research Program of MOST) and KOSEF (Plant Molecular Genetics and Breeding Research Center) to DC.
Notes
1 The nucleotide sequence data reported in this paper have been deposited in the GenBank database under the accession number AY040869. 
4 To whom correspondence should be addressed. Fax: +82 2 312 5657. E-mail: wtkim{at}yonsei.ac.kr
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