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First published online April 23, 2004; 10.1104/pp.103.035436 Plant Physiology 135:561-573 (2004) © 2004 American Society of Plant Biologists
Capsicum annuum Tobacco Mosaic Virus-Induced Clone 1 Expression Perturbation Alters the Plant's Response to Ethylene and Interferes with the Redox Homeostasis1School of Life Sciences and Biotechnology, Korea University, Seoul 136–701, Republic of Korea (R.S., J.-M.A., C.-J.P., Y.J.K., K.-H.P.); and Department of Biology, Yonsei University, Seoul 120–749, Republic of Korea (S.J., W.T.K.)
Capsicum annuum tobacco mosaic virus (TMV)-induced clone 1 (CaTin1) gene was expressed early during incompatible interaction of hot pepper (Caspsicum annuum) plants with TMV and Xanthomonas campestris. RNA-blot analysis showed that CaTin1 gene was expressed only in roots in untreated plants and induced mainly in leaf in response to ethylene, NaCl, and methyl viologen but not by salicylic acid and methyl jasmonate. The ethylene dependence of CaTin1 induction upon TMV inoculation was demonstrated by the decrease of CaTin1 expression in response to several inhibitors of ethylene biosynthesis or its action. Transgenic tobacco (Nicotiana tabacum) plants expressing CaTin1 gene in sense- or antisense-orientation showed interesting characteristics such as the accelerated growth and the enhanced resistance to biotic as well as abiotic stresses. Such characteristics appear to be caused by the elevated level of ethylene and H2O2. Moreover, in transgenic plants expressing antisense CaTin1 gene, the expression of some pathogenesis-related genes was enhanced constitutively, which may be mainly due to the increased ethylene level. The promoter of CaTin1 has four GCC-boxes, two AT-rich regions, and an elicitor-inducible W-box. The induction of the promoter activity by ethylene depends on GCC-boxes and by TMV on W-box. Taken together, we propose that the CaTin1 up-regulation or down-regulation interferes with the redox balance of plants leading to the altered response to ethylene and biotic as well as abiotic stresses.
Multiple preformed antimicrobial compounds contribute to the constitutive defense machinery of plants against pathogenic organisms. In addition, plants can trigger the inducible defense programs upon the perception of invaders. The early signaling molecules leading to R-gene-mediated resistance are ion fluxes, GTP-binding proteins, protein kinases, phosphatases, and phospholipases (Hammond-Kosack and Jones, 1996  ). Plants also respond to external stimuli such as microbial elicitors of cell death and/or defense responses by changing the calcium influx of the cell (Ebel and Scheel, 1997; Higgins et al., 1998). The increase of cytosolic calcium leads to cell death, which seems necessary for hypersensitive cell death triggered by rust fungi (Xu and Heath, 1998). The calcium channel blocker La3+, on the other hand, prevents the bacteria-induced hypersensitive response (HR) in soybean (Glycine max) leaves (Levine et al., 1996). The burst in the oxidative metabolism generated within minutes of infection leads to the accumulation of the reactive oxygen intermediates (ROI) such as H2O2 and superoxide anion (O2 –), involving NADPH-dependent oxidase. ROI serve as signaling molecules, for example in the recognition of the attack by fungal pathogens (Lamb and Dixon, 1997).
Ethylene is widely known to modulate the organ senescence induced by various stress factors such as plant pathogens (Moore et al., 2000
The TMV-induced hot pepper (Caspsicum annuum) gene, CaTin1 gene, was isolated as an incompatible interaction-specific gene through the differential screening between TMV inoculated and mock inoculated hot pepper plants (Shin et al., 2001
CaTin1 gene was induced by ethylene and methyl viologen (MV). The promoter of CaTin1 gene has four GCC-boxes and one W-box. The analysis of PR gene promoters has led to the identification of the11-bp ethylene-responsive element TAAGAGCCGCC, which has been referred as the GCC-box (Ohme-Takagi and Shinshi, 1995
The CaTin1 Gene Has the PLAT Domain and Is Expressed Only in Roots
In the previous study, we isolated N6-11 gene that was one of the genes induced specifically during the incompatible interaction between hot pepper plants and TMV (Shin et al., 2001
Based on the high stringent Southern-blot analysis, a few copies of CaTin1 gene were detected in pepper genome (data not shown). To examine the steady state transcription level of CaTin1 gene in various organs of pepper plants, the total RNA was extracted from the red ripe fruits, green unripe fruits, young leaves, old leaves, roots, stems, and flowers. As shown in Figure 2A , the transcription of CaTin1 was detected only in roots and not in any other organs.
The CaTin1 Expression during the Incompatible Interaction with TMV and Xanthomonas campestris
The expression pattern of CaTin1 gene was monitored in hot pepper cv Bugang plants after TMV-P0 (avirulent strain) inoculation. A mock inoculation treatment was carried out as a control to rule out any CaTin1 gene expression that might arise from wounding due to rubbing with carborundum. As shown in Figure 2B, the accumulation of CaTin1 transcripts was barely detected in mock-inoculated or TMV-P1.2 (virulent strain) inoculated leaves. However, CaTin1 transcripts started to accumulate from 24 h after the inoculation with TMV-P0 and increased until 72 h after the inoculation. As a positive control, the expression pattern of the CaPR1 gene, one of the PR genes isolated from hot pepper, was also monitored (Shin et al., 2001
We also examined whether CaTin1 could be induced during the systemic acquired resistance (SAR) after TMV inoculation. The transcripts corresponding to CaTin1 gene were accumulated in the distant uninoculated leaves 3 d after TMV inoculation (Fig. 2C). The systemic induction of the CaTin1 gene was earlier than CaPR1 gene that is known to be induced during SAR (Sticher et al., 1997 It was investigated whether the TMV-P0-inducible CaTin1 gene in plants is also involved in the defense against other pathogens. Pepper leaves were challenged with a pathogenic bacterium Xanthomonas campestris pv vesicatoria (Xcv). When the leaves of the Xcv resistant cv ECW-20R were infiltrated with Xcv, the leaves became dark purple within 24 h of inoculation and subsequently became necrotic. In contrast, within 48 h after the infiltration, the susceptible cv ECW leaves were damaged severely and exhibited chlorosis (data not shown). Northern-blot analysis carried out with the CaTin1 probe revealed that the transcripts of CaTin1 gene accumulated only in ECW-20R. This suggested HR-specific induction of CaTin1 by viral and bacterial challenges (Fig. 2D). Thus, the CaTin1 expression upon biotic stresses had a tendency of earlier induction than that of well-known CaPR1 gene.
To examine whether CaTin1 gene can be induced by other stimuli, plants were treated with various abiotic inducers and the expression of CaTin1 gene was examined at the times indicated. In pepper leaves sprayed with SA, the transcripts of CaTin1 gene were not detected. This is clearly different from the induction of CaPR1 and CaPR4 transcripts. Also CaTin1 gene was not induced by MeJA treatment but the CaPR4 transcripts were well induced. CaPR1 was known to be not induced by MeJA (Park et al., 2001a; Fig. 3A ). On the other hand, CaTin1 gene expression started to increase 2 h after ethylene treatment, and then maintained until 8 h at similar level. Compared to the CaPR1 gene expression, the CaTin1 gene expression started very early and quickly disappeared (Fig. 3A).
To see whether other abiotic inducers induce the CaTin1 expression, ABA at a concentration of 10 µM, 250 mM NaCl, or 0.5 mM MV was applied to the unrooted pepper plants. MV induces H2O2 (Dodge, 1994 ). NaCl induced the expression of CaTin1 gene slightly. MV, on the other hand, induced the expression of CaTin1 gene significantly (Fig. 3B). In pepper plants, MV induced the expression of CaTin1 gene more efficiently than the expression of Capsicum annuum ascorbate peroxidase (CaAPX1) gene that was used as the positive control (Yoo et al., 2002).
In regard to the response to various stimuli, the expression of CaTin1 was different from previously characterized PR genes that are responsive to ethylene and MV only. We thus examined the role of ethylene and H2O2 in the induction of the CaTin1 gene expression in response to biotic stresses. The leaves of hot pepper inoculated with TMV were treated with the ethylene biosynthesis inhibitors aminoethoxyvinylglycine (AVG) and
To examine whether the expression of CaTin1 gene is induced by incision-wounding, leaves were cut randomly with a pair of scissors and their expression of CaTin1 gene was examined. Control was the untreated upper leaves of the same plant. The proteinase inhibitor II gene (CaPinII) that has been reported to be induced by local and systemic wounding was used as the positive control (Shin et al., 2001
To understand the biological function of CaTin1 in planta, we generated transgenic tobacco plants harboring the sense CaTin1 gene or the anti-sense CaTin1 gene. Although CaTin1 gene is a hot pepper gene, we used the tobacco system because of the difficulty of obtaining a large number of transgenic pepper plants (Shin et al., 2002 As we observed that ethylene was the key regulator of the CaTin1 gene expression, we examined specifically whether the expression of CaTin1 gene is induced by the ethylene-dependent but SA-independent signal transduction pathway. Since the expression of CaTin1 was quite fast, it was possible that this gene might be regulated earlier than other PR genes in defense signal transduction pathway. At first, we compared the level of protein expression in transgenic plants and control plants. The expression level in transgenic plants harboring the sense CaTin1 gene (sense transgenic plants) was higher than control plants. The expression level in control plants, in turn, was higher than in transgenic plants harboring the antisense gene (antisense transgenic plants). In fact, CaTin1 protein in antisense transgenic plants was barely detectable (Fig. 4A ). And after TMV inoculation, CaTin1 protein level was high in sense transgenic plants but very low in antisense transgenic plants even though the plants were infected by TMV. We also investigated the expression of PR genes in these transgenic plants. NtPR1, NtPR2, and NtPR3 show 100% homology to X06361, M59442, and S44869 at NCBI database and the full-length NtPR1, the 3' 1 kb region of NtPR2, or NtPR3 were used as probes. As shown in Figure 4B, NtPR2 and NtPR3 but not NtPR1 gene were expressed in the untreated antisense transgenic plants. Acidic NtPR1 was known to show SA-dependent gene expression pattern but basic NtPR2 and NtPR3 ethylene-dependent expression pattern. The above results suggest the expression of NtPR2 and NtPR3 might have been related to ethylene dependent CaTin1 expression change. To investigate the possible relationship of their expressions with ethylene, transgenic plants were treated with ethylene blocker (STS) and the expression of CaTin1 was examined (+ signs in Fig. 4B). The expression of NtPR2 and NtPR3 gene in the antisense transgenic plants disappeared or decreased. From this result, we speculated that the expression of NtPR2 and NtPR3 gene in the antisense transgenic plants is regulated mainly by ethylene. Interestingly, both the sense and antisense CaTin1 transgenic plants flowered earlier than control plants. Furthermore, the sense transgenic plants were taller than controls, whereas the antisense transgenic plants were shorter than control plants (Fig. 4C). The data suggest that the expression of CaTin1 may be an important factor of the ethylene signal transduction pathway.
The Alteration of the HR Lesion Formation and Ion Conductivity in CaTin1 Transgenic Plants after TMV Inoculation
The HR response elicited by avirulent pathogens is rapid cell death. We noticed that the sense transgenic plants showed the irregular and bigger HR lesion formation after TMV inoculation. As shown in Figure 5A
, 10 d after TMV inoculation, the HR lesions of the sense transgenic plants were over 40% bigger than control plants (a versus b). The lesion in the antisense plants was smaller than control (b versus c). The correlation between the cell death severity and the ion leakage level has been reported (Greenberg and Ausubel, 1993
The Elevated Level of Ethylene and H2O2 in CaTin1 Transgenic Plants When ethylene blockers were applied to the transgenic plants, the expression of CaTin1 as well as PR genes was decreased or disappeared (Figs. 3C and 4B). In addition, the sense transgenic plants showed the significant progression of the HR lesion and increased ion conductivity in response to TMV inoculation (Fig. 5). Based on these results, we postulated that the ethylene and/or H2O2 level may be elevated in the transgenic plants. Hence, in addition to measuring the in vivo ethylene and H2O2 level directly, we evaluated the expression of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene and glutathione-S-transferase (GST) gene in the reverse transcription (RT)-PCR analysis. The former gene is the marker for the ethylene level and the latter gene is the marker for the H2O2 level (Fig. 6 ). In the antisense transgenic plants, the expression of the ACC oxidase gene and the ethylene level were higher than control plants. In the sense transgenic plants, the levels were slightly elevated (Fig. 6A). In contrast, the H2O2 level and the GST expression level were higher in the sense transgenic plants than control plants. The levels in the antisense plants were slightly higher than control plants not undergoing HR (Fig. 6B). However, the H2O2 level of TMV inoculated control plants undergoing HR was about 5 times higher than that of sense transgenic plants.
CaTin1 Transgenic Plants Are Enhanced in Tolerance against Virus Infection, Drought, and Salt Treatments Our data show that the sense as well as the antisense CaTin1 transgenic plants have the higher ethylene and H2O2 levels than control plants (Fig. 6). We thus examined whether CaTin1 sense and antisense transgenic plants showed altered tolerance against biotic and/or abiotic stresses. To determine whether CaTin1 transgenic plants showed enhanced resistance to pathogens, we inoculated control and transgenic plants with the virulent viral pathogen cucumber mosaic virus Y (CMV-Y) that infects hot pepper systemically. Uninoculated upper leaves were taken for infection analysis after 21 d of virus inoculation. As shown in Table I, resistance to CMV-Y of the sense as well as the antisense transgenic plants was increased by 19% to 73%.
We also examined whether the higher levels of ethylene and H2O2 in transgenic plants alter their tolerance to drought or salt. The leaf discs of transgenic tobacco plants were immersed in 6% mannitol or 400 mM NaCl for 7 d. Subsequently the chlorophyll content in transgenic tobacco and control plants was compared. As shown in Table I, the tolerance in the sense and the antisense transgenic plants to drought and salt was increased by 2.8 times and from 1.5 times to the maximum 11 times, respectively. The data demonstrated that the resistance to various stresses in the sense transgenic plants might be caused mainly by the elevated H2O2 level. The increased resistance in the antisense transgenic plants may be due to the higher ethylene level resulting in the PR gene expression.
To characterize the sequences involved in the CaTin1 transcriptional regulation, we generated the deletions of the full-length promoter region of CaTin1, connected them to GUS coding gene, and produced transgenic tobacco plants. The CaTin1 promoter has four ethylene responsive GCC-boxes, two AT-rich regions known to be responsible for the root-specific expression, and an elicitor-induced WRKY transcription factor-binding W-box. The activity of each deletion construct was measured by using the leaf samples, which showed almost no expression of CaTin1 without any treatment. The activity was not detected by the histochemical GUS staining in the leaf even when the full-length promoter construct (p-1434Tin1) was used. However, constructed with the AT-rich regions, the GUS activity could be detected specifically in the apical meristem of roots without any treatment (data not shown). When the transgenic tobacco plants of the deletion constructs of CaTin1 promoter were inoculated with TMV, they had higher GUS activity compared to the promoterless GUS construct as long as they had a W-box. In contrast, the GUS activity of the deletion promoters treated with ethylene was dependent on the GCC-boxes as expected. The construct with one GCC-box showed weak activity. The constructs with three or four GCC-boxes showed strong activity (Fig. 7 ).
The expression of CaTin1 gene containing the PLAT domain is induced by avirulent pathogens, ethylene, NaCl, and MV. SA or MeJA is ineffective. Many genes containing the PLAT domain, such as lipoxygenase and lipase, have other functional domains in addition to the PLAT domain. CaTin1 and its homologs of other plants have only PLAT domain (Fig. 1). Although PKD1 and PKD2 appear to form homo- and heterodimer with each other (Tsiokas et al., 1997 ), we found that CaTin1 does not form homodimers based on the yeast-two-hybrid interaction experiment (data not shown). CaTin1 gene may be different from other known genes containing the PLAT domain in terms of not having other functional domain such as the lipoxygenase domain of lipoxygenase gene. However, our results together with previous reports suggest that the function of CaTin1 gene may be to help the communication between the ethylene signal pathway and the abiotic/biotic stress-related pathway through the protein-protein or protein-lipid interaction in a Ca2+ dependent fashion (Gillmor et al., 1997; Tsiokas et al., 1997; Bateman and Sanford, 1998).
Several studies confirmed the importance of SA for the establishment of disease resistance (Dempsey et al., 1999
The expression of CaTin1 was induced during SAR (Fig. 2C). SAR induction was known to require the signal molecule SA. The removal of SA in transgenic plants expressing salicylate hydroxylase (nahG) prevents the establishment of SAR (Gaffney et al., 1993
The promoter of CaTin1 has bidirectional activity. In addition, another homologous gene (CaTin1-2) is located in the front of CaTin1 in the head-to-head fashion. The expression of CaTin1 and CaTin1-2 in response to several elicitors was very similar except for the slightly different induction time upon TMV challenge (Shin et al., 2003
CaTin1 antisense transgenic tobacco plants expressed some basic PR genes constitutively, especially NtPR2 and NtPR3, without any treatment (Fig. 4B). As mentioned earlier, NtPR2 and NtPR3 are SA-independent PR genes. On the other hand, NtPR1 gene belonging to SA-dependent PR genes was not expressed in these transgenic plants under the same conditions. The CaTin1 protein expression level in the sense transgenic plants was higher than the antisense transgenic plants (Fig. 4A). The CaTin1 protein level homeogenesis in planta may be important. If CaTin1 is more underexpressed than the normal biological level, plants should have to make CaTin1 and the ethylene level may be increased for producing CaTin1. Moreover, because the CaTin1 level seems to be important in planta, the promoter of CaTin1 should regulate its homolog in hot pepper at the same time (Shin et al., 2003
The CaTin1 sense transgenic plants were taller than control plants, whereas the antisense plants were shorter than control plants (Fig. 4C). Both the sense and the antisense transgenic plants flowered earlier than control plants. Although the CaTin1 sense transgenic plants grew more rapidly than control plants, the number of internodes was not changed (data not shown). After TMV inoculation, the sense transgenic plants showed bigger, and the antisense plants smaller, HR lesion than control plants (Fig. 5A). Furthermore, 10 d after the inoculation, ion conductivity was higher in the sense transgenic plants than control plants (Fig. 5B). Bent et al. (1992)
Previous studies show that WRKY transcription factors and their downstream genes such as NPR1 are associated with the pathogen- and SA-induced pathway (Chen and Chen, 2000 The CaTin1 gene expression was regulated by ethylene and the ethylene level seemed to be controlled by the CaTin1 expression level. We propose that the perturbation of the CaTin1 expression alters the response of plants to ethylene and interferes with the redox homeostasis.
Plant Cultivation and Pathogen Inoculation Hot pepper (Capsicum annuum) L. cv Bugang that is susceptible to the P1.2 pathotype of TMV but resistant to the P0 pathotype was used. Plants were grown in a growth chamber or greenhouse at 25°C with the photoperiod cycle of 16 h light and 8 h dark. Healthy and well-expanded leaves of 2-month-old plants were used. TMV-P0 and TMV-P1.2 strains were maintained in the leaves of tobacco (Nicotiana tabacum cv Burley 21) in petri dishes containing CaCl2. The leaf sap of TMV-P0 or TMV-P1.2 was prepared by grinding infected leaves in 0.25 M phosphate buffer containing 5 mM EDTA (pH 7.4). To inoculate plants, virus-containing sap was applied to the surface of 4 to 5 fully expanded leaves of hot pepper plants and rubbed with carborundum (Hayashi Chemical, Osaka). Mock-inoculated plants were rubbed with phosphate buffer and carborundum only. To assess systemic responses, 2 lower leaves were inoculated with TMV-P0 sap and the upper leaves were harvested at 0, 3, 6, 9, and 15 d after inoculation. Two cultivars of hot pepper, Early Calwonder (ECW; bs1/bs1, bs2/bs2, and bs3/bs3) and Early Calwonder-20R (ECW-20R; bs1/bs1, Bs2/Bs2, and bs3/bs3) were used for infiltration with Xcv (avrBs2). Xcv was cultured in the medium containing yeast extract, dextrose, and calcium carbonate. Cell suspensions (1 x 108 cells/mL) were infiltrated into leaves, and RNA was extracted from the infiltrated leaves.
Pepper leaves were sprayed with 5 mM SA or 10 µM MeJA solution. Control plants were sprayed with distilled water. Leaves thus treated were harvested at the times indicated, quickly frozen in liquid nitrogen, and stored at –80°C. For the treatments of abscisic acid (ABA), NaCl, or MV, unrooted pepper plants were placed in Falcon tubes filled with the chemicals or water for various durations and frozen in liquid nitrogen. The ethylene treatment was performed by keeping pepper plants in a chamber equilibrated with 50 µL/L gaseous ethylene for the durations indicated. Ethylene concentration was determined by gas chromatography. Control plants were kept in a chamber without ethylene. For the wound treatment, leaves were cut randomly with a pair of scissors. RNA was extracted from the wound-treated leaves and the nontreated upper leaves of the same plant at various times after wounding. CaPR1 was used as a control for HR, SA, or ethylene treatment. CaPR4 was used as a control for MeJA treatment. CaAPX1 was used as a control for MV treatment. Arabidopsis pyrroline-5-carboxylate synthase (AtP5CS) was used as a control for salt or ABA treatment. CaPinII was used as a control for wound treatment (Kasuga et al., 1999
Two leaves from hot pepper plants were inoculated with 1 µg/mL TMV. After 24 h incubation at 22°C, the infected leaves from 2 plants were injected with either water, 2 mM AIB, 1 mM AVG, or 0.5 mM STS. Immediately following the injection, the treated leaves were detached and immersed in the corresponding solution. The unrooted plants were soaked in 100 µM DPI solution for 4 h and were inoculated with TMV- P0 or was treated with 0.5 mM MV for 24 h (Orozco-Cárdenas et al., 2001
After 24 h incubation in various solutions, the leaves were harvested and used for RNA analysis (Guo et al., 2000
Genomic DNA and RNA were isolated from the pepper leaves by the method of Ausubel et al. (1995) Indirect ELISA was used for the detection of CMV-Y using anti-rabbit-CMV-Y antibody (3 µg/mL). For the detection of CaTin1 in transgenic plants, western-blot analysis using anti-rat-CaTin1 antibody (2 µg/mL) was performed.
Plasmids used in the transformation of tobacco were prepared with the sense and antisense orientation of the full-length CaTin1 cDNA cloned into the polylinker site of the binary vector pMBP2 (Han et al., 1999
To investigate the tolerance to the salt and drought stress, healthy and fully expanded leaves of wild-type and transgenic plants were detached and washed briefly in distilled water. The 1-cm diameter leaf disc was cut and immersed in Murashige and Skoog medium containing 400 mM NaCl or 6% mannitol for 7 d (Veena and Sopory, 1999
For the measurement of ethylene evaporation, 3 to 5 plants per transgenic T1 line were analyzed. Plants were kept in a sealed test bottle for 12 h. Each 1-mL gas sample was withdrawn from the bottle with a hypodermic syringe and ethylene was assayed on a gas chromatography equipped with an aluminum column and flame ionization detector (Yi et al., 1999
The ion leakage was measured with the Orion ion conductivity meter (model 150; Beverly, MA). Leaves of 2-month-old plants were inoculated with TMV. 10 d later, leaf discs obtained by punching with a number 4 cork borer (diameter 7.5 mm) were immersed in 2 mL of distilled water with the abaxial side down. After 2 h immersion at room temperature, the conductivity of the bathing solution was measured (Greenberg and Ausubel, 1993
By using the CaTin1 gene specific primer 1 (5'-AGCCTGAAATAGAAGAAACGGAGATGGAGATGAGA-3') and 2 (5'-GGAACCAGAATTGGTTACTCATGGCTACCTGAAC-3'), CaTin1 promoter region was obtained from the adaptor-ligated genomic DNA fragments in GenomeWalker libraries according to the manufacturer's instructions (CLONTECH, Palo Alto, CA). The deletion fragments of CaTin1 promoter were generated by PCR using the primers corresponding to serial 100-bp deletions and containing the EcoRI restriction site (forward primers) and the NcoI site (reverse primer). Amplified products were cloned into pGEM Easy Vector (Promega, Madison, WI), digested with EcoRI and NcoI, and cloned into pCambia 1305.1 (CAMBIA, Canberra, Australia) that contains a gus reporter gene and the hygromycin selectable marker flanked by T-DNA border sequence. The constructs were introduced into tobacco cv Samsun NN using Agrobacterium-mediated transformation.
For the GUS activity assay, 112.5 µL 4-methylunbelliferyl Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AF242731 for CaTin1 and AF480414 for CaTin1-2. Received October 28, 2003; returned for revision February 19, 2004; accepted February 20, 2004.
1 This work was supported by a grant (CG1223) from the Crop Functional Genomics Center of the 21st Century Frontier Research Program funded by the Korea Ministry of Science and Technology, a Biogreen grant from the Rural Development Administration, and by a grant from the Plant Signaling Network Research Center of Korea Science and Engineering Foundation. 
2 Present address: Donald Danforth Plant Science Center, 975 N. Warson Road, St. Louis, MO 63132. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.035436. * Corresponding author; e-mail khpaek95{at}korea.ac.kr; fax 82–2–928–1274.
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-aminoisobutyric acid (AIB), the ethylene action inhibitor silver thiosulfate (STS), or the H2O2 production enzyme NADPH oxidase inhibitor (diphenylene iodonium, DPI) and the induction of the CaTin1 gene was examined. Treated with various ethylene synthesis inhibitors or ethylene action inhibitors, the CaTin1 gene expression in the TMV-inoculated plants was reduced. Particularly, the expression was markedly diminished in plants treated with STS (
-D-glucuronide solution in a microtube was preincubated in a water bath for 5 min at 37°C. Then, 75 µL of cell extract in GUS/ luciferase buffer (0.1 M KPO4, pH 7.8, 2 mM Na2-EDTA, 2 mM dithiothreitol, and 5% glycerol) was added and incubated at 37°C. After 30 min or 60 min, 50 µL was removed from each assay tube and transferred to a test tube containing 1 mL stop solution (0.2 M Na2CO3). The progress of the reactions was quantitatively assessed under the emission wavelength of 455 nm. The standard curve was generated by determining the fluorescence of various concentrations of 4-methylumbelliferone standards ranged from 0 to 10 µM (Maliga et al., 1995