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First published online September 29, 2006; 10.1104/pp.106.085258 Plant Physiology 142:1202-1215 (2006) © 2006 American Society of Plant Biologists OPEN ACCESS ARTICLE
Contribution of Ethylene Biosynthesis for Resistance to Blast Fungus Infection in Young Rice Plants1,[OA]National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305–8602, Japan (T.I., S.S., Y.O.); Miyagi Prefectural Agriculture and Horticulture Research Center, Takadate-kawakami, Natori, Miyagi 981–1243, Japan (T.I.); and National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305–8518, Japan (A.M.)
The role of ethylene (ET) in resistance to infection with blast fungus (Magnaporthe grisea) in rice (Oryza sativa) is poorly understood. To study it, we quantified ET levels after inoculation, using young rice plants at the four-leaf stage of rice cv Nipponbare (wild type) and its isogenic plant (IL7), which contains the Pi-i resistance gene to blast fungus race 003. Small necrotic lesions by hypersensitive reaction (HR) were formed at 42 to 72 h postinoculation (hpi) in resistant IL7 leaves, and whitish expanding lesions at 96 hpi in susceptible wild-type leaves. Notable was the enhanced ET emission at 48 hpi accompanied by increased 1-aminocyclopropane-1-carboxylic acid (ACC) levels and highly elevated ACC oxidase (ACO) activity in IL7 leaves, whereas only an enhanced ACC increase at 96 hpi in wild-type leaves. Among six ACC synthase (ACS) and seven ACO genes found in the rice genome, OsACS2 was transiently expressed at 48 hpi in IL7 and at 96 hpi in wild type, and OsACO7 was expressed at 48 hpi in IL7. Treatment with an inhibitor for ACS, aminooxyacetic acid, suppressed enhanced ET emission at 48 hpi in IL7, resulting in expanding lesions instead of HR lesions. Exogenously supplied ACC compromised the aminooxyacetic acid-induced breakdown of resistance in IL7, and treatment with 1-methylcyclopropene and silver thiosulfate, inhibitors of ET action, did not suppress resistance. These findings suggest the importance of ET biosynthesis and, consequently, the coproduct, cyanide, for HR-accompanied resistance to blast fungus in young rice plants and the contribution of induced OsACS2 and OsACO7 gene expression to it.
In monocot plants, the mechanism of disease resistance, including the roles of defense signal compounds for resistance (R)-gene-mediated resistance, such as salicylic acid (SA), ethylene (ET), and jasmonic acid (JA), has not been well elucidated. To study the mechanism, the Japonica rice (Oryza sativa) cv Nipponbare is an attractive model because of recent developments in genomic and molecular information, such as the Rice Genome Research Program (http//rgp.dna.affrc.go.jp; International Rice Genome Sequencing Project, 2005  ). Rice blast fungus (Magnaporthe grisea) is an extensively studied pathogen whose infection seriously affects rice yields worldwide. Genetic studies have identified 13 major R genes to blast fungi in rice plants, and standard rice cultivars with individual R genes and corresponding standard blast fungal races have been prepared to identify the race of blast fungus and R genes in a rice cultivar (Yamada et al., 1976; Kiyosawa, 1984). In Japan, attempts have been made to generate individual isogenic lines that contain a specific R gene with the same genetic background as practical rice cultivars, such as cv Nipponbare (Ise and Horisue, 1988). Actually, multilines containing compatible and incompatible lines were reported to be effective for disease control in the field (Browning and Frey, 1969) and, to our knowledge, there is no information to date on the breakdown of disease resistance by blast fungus races that have newly acquired the ability to infect all lines composing a multiline.
To analyze the resistant mechanism of rice plants to blast fungus infection, we used an isogenic line containing the R gene Pi-i to blast fungus race 003 in the background of cv Nipponbare and first focused on the levels of defense signal compounds after blast fungus infection. Koga (1994)
On resistance with the HR in dicot plants such as Tobacco mosaic virus (TMV) in tobacco (Nicotiana tabacum; De Laat and van Loon, 1983
On the other hand, the role of ET emission on resistance with the HR was poorly understood. Therefore, we were especially interested in the ET level after blast fungus inoculation in both susceptible and resistant rice lines. We studied the role of ET emission using susceptible wild-type Nipponbare and its isogenic resistant line, IL7, which contains R gene Pi-i to blast fungus race 003 (Ise and Horisue, 1988
Transient Increase in ET Emission during the Formation of HRLs in IL7
First, we established an experimental system to analyze the mechanism of resistance to pathogen infection in rice plants. We used young rice plants at the four-leaf stage of wild-type Nipponbare and its isogenic line, IL7, which contains the R gene Pi-i (Ise et al., 1988
We determined the level of ET emitted from the fourth leaves after blast fungus inoculation, using 16 fourth leaves as one sample (Fig. 2A ). One gram of healthy leaf from both IL7 and wild type emitted about 0.9 nL of ET per hour just after detaching. In both mock- and fungus-inoculated IL7 plants, the first peak in ET emission was found at 24 hpi, which was also the case in wild type. This peak by mock inoculation may be caused by the inoculation itself, which involved incubation of plants under high humidity in the dark for 20 h just after spraying the inoculum. The peak at 24 hpi was higher after blast fungus inoculation than mock inoculation in both IL7 and wild type (Fig. 2A), indicating an additive enhancement of ET emission by fungal infection. The second peak of ET emission was detected only in fungus-inoculated IL7 plants at 48 hpi. The enhanced rate of ET emission by fungal inoculation at 48 hpi was 6.4 nL g–1 h–1 fresh weight in inoculated IL7 and 0.36 nL g–1 h–1 fresh weight in inoculated wild type, respectively. Because primary HRLs found at 42 hpi (black arrow) were mature by 63 hpi, the significant enhancement of ET emission found in IL7 was thought to be related to the formation of HRLs. During 63 to 96 hpi, emitted ET from inoculated IL7 was maintained at a high level (4 nL g–1 h–1 fresh weight). In inoculated wild-type leaves, the level of ET emission was clearly lower than that in inoculated IL7 and similar to that in mock-inoculated wild-type leaves from 36 to 62 hpi. In wild type, a slight increase was detected at 72 to 96 hpi at which time the development of ELs had started (white arrow). Independent triplicate experiments with similar results confirmed that the data obtained here were reproducible.
The major pathway of ET biosynthesis contains two catalytic steps involving ACS and ACO, producing equal moles of ET and cyanide (Peiser et al., 1984 ). To study the mechanism of enhanced ET biosynthesis in the resistant response, we determined the levels of emitted ET (Fig. 2A), ACC (Fig. 2B), and ACO activity (Fig. 2C) in blast fungus-inoculated rice leaves at the same time. High levels of ACC were accumulated in both IL7 and wild type at 24 to 36 h after both fungus and mock inoculation. ACC content was 1.5-fold higher after fungus inoculation than mock inoculation, indicating that the fungal infection and/or spraying of the conidial suspension further enhanced ACC accumulation in both rice plants. The peaks at 24 hpi seem to be related to the first peaks in ET emission in Figure 2A. At 48 to 72 hpi during which HRLs were formed and completed, the ACC content of inoculated IL7 leaves was about 1.5- to 1.7-fold that of mock-inoculated leaves, whereas no significant difference in ACC levels was found between mock- and fungus-inoculated wild-type leaves (Fig. 2B). These results suggested that enhanced ACC synthesis and/or suppressed ACC degradation were more strongly induced in resistant IL7 than in susceptible wild type after blast fungus inoculation. In wild type, the ACC level was increased at 96 hpi (Fig. 2B) when EL formation was started. These results indicated that ACC synthesis was accompanied by the formation of both HRLs and ELs. In inoculated IL7 leaves, ACO activity was dramatically increased at 48 to 63 hpi and remained at a considerable level at 72 to 96 hpi, about 4 nL g–1 h–1 fresh weight, which was 2.5-fold higher than that in mock-inoculated leaves. In wild-type plants, the profile of ACO activity was quite different from that in IL7. The level was only slightly higher than in mock-inoculated leaves at and after 48 hpi with no clear peak. The time-course profile of ACO activity in inoculated IL7 leaves resembled that of the ET emission profile at 36 to 96 hpi (Fig. 2A), suggesting that increased ACO activity leads to enhanced ET emission in incompatible interaction at and after 48 hpi. At 63 hpi, ACO activity in inoculated IL7 leaves had a peak instead of a rapid decrease in ET emission, probably because of decreased ACC content and/or possible negative regulation by elevated ET.
What kinds of ACS and ACO contribute to ET biosynthesis in blast fungus-infected rice plants? We searched for ACS and ACO genes from expressed sequence tags, full-length cDNAs, and genome databases of rice cv Nipponbare (http://riceblast.dna.affrc.go.jp/; http://cdna01.dna.affrc.go.jp/cDNA).
At least five rice ACS genes are reported to exist in the rice genome (Zarembinski and Theologis, 1993
A phylogenetic analysis with ACS proteins from rice, Arabidopsis (Yamagami et al., 2003 ), tobacco (Liu and Zhang, 2004), and wheat (Subramaniam et al., 1996) revealed that the genes fall into three groups (Fig. 3B). ACSs from Arabidopsis in groups I and II exhibited ACS activity, but not in group III, which contains AtACS10 and 12 similar to Ala or Asp aminotransferases in Arabidopsis (Yamagami et al., 2003). Thus, OsACS1 to OsACS5 in groups I or II, but not III, might work in rice. To elucidate the genomic organization of ACS in rice, genomic DNA from rice cv Nipponbare (wild type) was subjected to Southern-blot analysis with mixed probes containing the catalytic domain of ACS, which were prepared by PCR amplification using OsACS1, OsACS2, and OsACS5 as templates (Fig. 3C). Because the highest homology based on nucleotide sequence between the probe and OsACS1 to OsACS6 is 100%, 100%, 76%, 73%, 100%, and 51%, respectively, the mixed probes would detect five signals corresponding to OsACS1 to OsACS5 in the digests by EcoRI whose internal site was not found in OsACSs. These results suggested that five rice ACSs in groups I and II compose a functional ACS gene family.
As an ACO gene in deepwater rice, OS-ACO1 (X85747) has been reported in relation to submergence (Mekhedov and Kende, 1996
OsACO genes were classified into three major groups in a phylogenic tree based on putative amino acid sequences (Fig. 4B). OsACO1, OsACO2, and OsACO3 are classified in group I, which contains NtACO1 and NtACO2, whose gene expression was accompanied by the formation of HRLs in tobacco (Liu and Zhang, 2004 ). OsACO7 is classified in group II, which contains StACO3, which was induced in the potato (Solanum tuberosum) tuber by inoculation with Fusarium eumartii and treatment of SA and indole acetic acid (Zanetti et al., 2002), and LEACO5, whose expression was anaerobically induced (Sell and Hehl, 2005). Genomic Southern-blot analysis with mixed probes containing the catalytic domain of ACO, which were prepared by PCR amplification using OsACO1 to OsACO5 and OsACO7 as templates, detected six signals in both EcoRI and HindIII digests and seven signals in NcoI digests, indicating that seven OsACO genes, including the OsACO6 pseudogene, compose a gene family in rice (Fig. 4C).
As described, our results suggested the importance of ET biosynthesis for resistance to blast fungus infection. When the data on ACC content and ACO activity after blast fungus inoculation in Figure 2 were reconstructed in Figure 5A , it became clearer that the levels before and during the formation of HRLs (36–63 hpi) were significantly higher in IL7 than those in wild type, suggesting an additive effect on ET biosynthesis in IL7 leaves. Thus, we studied the expression profiles of ACS and ACO genes in inoculated IL7 and wild-type plants.
The time-course expression profiles of six ACS genes were studied by one-step reverse transcription (RT)-PCR with specific primers for each ACS gene (Fig. 5B). Transient OsACS1 expression was found in mock-inoculated leaves at a low level and in blast fungus-inoculated leaves at a high level at 24 hpi in both plants, indicating OsACS1 may function for the first peak of ET emission at 24 hpi (Fig. 2A). OsACS2 was transiently induced at 48 hpi in fungus-inoculated IL7 plants, which is likely related to increased ACC content and dramatic ET emission at 48 hpi (Fig. 2A). A considerable level of OsACS2 transcript was found at 96 hpi in wild type, but not IL7. It may be accompanied by the formation of ELs in wild-type plants. OsACS3, OsACS4, and OsACS6 were almost constitutively expressed in mock- and blast fungus-inoculated IL7 and wild-type leaves. No signal for the OsACS5 transcript was found in fourth leaves under the same conditions (data not shown). These results suggest that OsACS1 contributes to dark- and high humidity-induced ET emission found at 24 hpi, and OsACS2 mainly acts to increase the ACC level during HRL formation at 48 hpi in IL7 and EL formation at 96 hpi in wild type. Next, expression profiles of the six ACO genes, except for the pseudogene OsACO6, were determined using one-step RT-PCR with specific primers for each gene (Fig. 5B). OsACO1 was detected at 24 to 48 hpi in mock- and fungus-inoculated IL7 and wild-type plants. OsACO1 may be expressed under dark and high humidity conditions during inoculation, probably contributing to the first peak of ET emission at 24 hpi (Fig. 2A). OsACO2 was constitutively expressed in both plants, but down-regulated in blast fungus-inoculated IL7 at 72 and 96 hpi. OsACO3 was constitutively expressed in both plants not affected by the treatments. No signal for the OsACO4 transcript was found in either plant (data not shown). Expression of both OsACO5 and OsACO7 was inducible and enhanced by infection in both plants. Notably, expression of OsACO7 was very transient and strong at 48 hpi in IL7, but not wild-type plants. Whereas OsACO5 was also transiently enhanced to express at 48 hpi in IL7, the transcript was found in wild type at 48 to 96 hpi as well. From these results, transient expression of OsACO7 is thought to be most important for the increase of ACO activity at 48 to 72 hpi in infected IL7 leaves, possibly in cooperation with OsACO5 expression.
Blast fungus-induced OsACSs and OsACOs expression profiles were compared with control defense-related genes, such as OsPR1a (AJ278436; Agrawal et al., 2000
Enhanced ET emission during the formation of HRLs in IL7 depends on ET biosynthesis. To confirm the contribution of ET biosynthesis to resistance, the effect of aminooxyacetic acid (AOA), which is an inhibitor of pyridoxal-5'-P-related enzymes, such as ACS (Yu et al., 1979
To confirm the effect of AOA on resistance to blast fungus in rice, infected leaf pieces were treated at 96 hpi with lactophenol-trypan blue, which stains the mycelium blue. In 0.1 mM AOA-treated IL7 leaf pieces, HRLs remained dark brown and were not stained blue as in water-treated leaves (Fig. 6C), indicating that 0.1 mM AOA could not entirely inhibit the formation of HRLs, whereas 63% of ET emission at 48 to 51 hpi was inhibited. However, treatment with 0.5 and 1.0 mM AOA, which almost completely suppressed ET emission, resulted in larger lesions with blue-stained mycelia in IL7 plants. Thus, reduced ET emission possibly led to a breakdown of HRL formation, permitting enhanced mycelial growth in the inoculated IL7 leaves. In fungus-inoculated wild-type leaves treated with 0.1 to 1.0 mM AOA, similar ELs were formed in the water-treated control wild type and 0.5 to 1 mM AOA-treated IL7 leaves.
AOA is known as an inhibitor of ACS, but also that of deaminases and transaminases containing PAL (Amrhein and Gerhardt, 1979
Does the necessity of ET biosynthesis, which accompanies the production of equal moles of cyanide for resistance, mean the importance of ET signaling? To elucidate, we examined the effect of the inhibitors for ET action, 1-methylcyclopropene (1-MCP) and silver thiosulfate (STS). Fungus-inoculated rice plants at the four-leaf stage were treated with gaseous 1-MCP 24 hpi for 16 h at 2 µL L–1, which is enough concentration to disturb accelerated ripening and softening of climacteric fruit or accelerated senescence of cut flowers (Serek et al., 1995 ). Phenotypes of formed HRLs and ELs in the treated IL7 and wild-type leaves were very similar to these in nontreated control leaves at 96 hpi, respectively (Fig. 7B). Treatment with 2 mM STS solution through the base of detached leaves at 24 hpi for 1 h resulted in very similar phenotypes of HRLs and ELs compared with nontreated IL7 and wild-type leaves, respectively (Fig. 7B). These results indicated that inhibitors for ET action would not affect resistance to blast fungus infection, at least in our experimental condition, and the importance of cyanide production via ET biosynthesis.
We demonstrated here that ET biosynthesis, but not ET itself, is necessary for resistance to blast fungus infection in young rice plants. In Pi-i-mediated resistance in IL7 plants, enhanced ET emission at 48 hpi is distinctive because the phenomenon was not found in fungus-infected wild-type plants or mock-inoculated IL7 and wild-type plants. AOA treatment, which induced remarkable inhibition of ET emission, broke down resistance in IL7, preventing the formation of HRLs. Addition of ACC compromised the AOA-induced breakdown of resistance, indicating ACC synthesis by ACS is critical for resistance. Treatment of 1-MCP and STS, the inhibitors of ET action, did not induce the breakdown of resistance. From the characterization of the rice ACS and ACO gene families, we found that enhanced ET emission during the formation of HRLs would be supported by specific ACS and ACO genes at the transcriptional level.
In our experimental system, the resistant cultivar IL7 exhibits strong resistance to blast fungus race 003; saturated numbers of solid HRLs small in size and dark brown in color were detected at about 63 hpi, with no remarkable enlargement thereafter. On the other hand, typical susceptibility is found in wild type, which lacks the Pi-i gene in almost the same genetic background; whitish ELs were first visualized at 96 hpi, and they rapidly develop in size with vigorous conidiation in the center. In this system, the time point around 48 hpi is crucial to detect the difference in resistance to blast fungus because, at this time, HRLs have just started to form in IL7 leaves, whereas no clear phenotype is found in inoculated wild-type leaves (Fig. 1). Two peaks of ET emission were observed in IL7 and wild-type plants infected with blast fungus race 003. The first peak at 24 hpi was likely a result of the darkness and high humidity in both host plants, and the second peak at 48 hpi was specific for the formation of HRLs in infected IL7 plants (Fig. 2A).
The level of the second peak in ET emission was almost proportional to the number and size of developing HRLs. When the conidial suspension (1 x 105 conidia mL–1) was sprayed onto IL7 plants at the four-leaf stage, about 150 HRLs were detected on the fourth leaf accompanied by ET emission at the rate of 7 nL g–1 h–1 fresh weight at 48 hpi. This level of ET emission is similar to the formation of HRLs mediated by the N gene (18 nL g–1 h–1fresh weight) in TMV-infected tobacco plants (De Laat and Van Loon, 1981
In deepwater rice, OS-ACS1, whose product shares 99% homology with OsACS1 from Nipponbare wild type, was induced by partial submergence at the uppermost elongating internode and involved in stem elongation (Zarembinski and Theologis, 1993 Maintenance of a high level of ACO activity at 48 to 96 hpi may guarantee a considerable level of ET biosynthesis during this period. Two newly characterized rice ACO genes, OsACO5 and OsACO7, out of seven ACO members, were transiently induced by blast fungus in IL7 plants (Fig. 5B). Because OsACO5 lacks one of nine conserved amino acid residues, it might confer weaker or no ACO activity (Fig. 4A). OsACO5 expression was induced by blast fungus infection in both IL7 and wild type in a similar manner, suggesting no relation to resistance. Interestingly, the maximal level of the OsACO7 transcript was found at 48 hpi in infected IL7, but not wild type, indicating a transcriptional contribution of OsACO7 to ET emission during the formation of HRLs in IL7. It was also increased at 24 hpi in both IL7 and wild type, possibly indicating a contribution to the first peak of ET emission at 24 hpi. OsACO7 belongs to group II, as well as StACO3, which was induced by infection with F. eumartii in potato (Fig. 4B). On the other hand, information on transcriptional regulation of plant ACO genes is limited and we could find no evidence of posttranscriptional regulation of ACO in plants. Thus, the mechanism on the regulation of ACO for disease resistance remains to be solved.
In this article, we proposed the involvement of ET biosynthesis, but not ET itself, in resistance to blast fungus infection in rice plants. This indicates the positive role of ET biosynthesis in blast fungus-dependent HRL formation and suppression of subsequent fungal growth. AOA treatment at 24 hpi clearly inhibited ACC synthesis and subsequent ET emission in IL7 at 48 hpi, inducing EL-like lesions with vigorous hyphal growth instead of HRLs, which are observed in the absence of AOA (Fig. 6, B and C). Addition of ACC canceled the inhibitory effect of AOA, recovering enhanced ET emission and formation of HRLs in IL7 (Fig. 7A). These results indicate that ET biosynthesis is essential for Pi-i (R-gene)-mediated resistance. In the cytological studies reported by Koga (1994) The analysis of rice ACS and ACO gene families pointed out that specific members, such as OsACS2 and OsACO7, are transcriptionally activated during the formation of HRLs in fungus-infected IL7 leaves. The expression profile of a gene often suggests its function, but the studies about modification of the gene products such as dynamic activation/inhibition or stabilization/unstabilization would also be important. Loss- or gain-of-function studies about OsACS2 and OsACO7 would clearly indicate the roles of these genes in the defense against blast fungus in relation to ET biosynthesis. Such experiments have been started in our laboratory.
Plant Materials
Rice (Oryza sativa cv Nipponbare) and the isogenic line IL7 (Ise and Horisue, 1988
Blast fungus race 003 was grown on oatmeal medium (Difco) for 2 weeks at 26°C in the dark, and then spores were induced to form under a 20-W BLB light (FL20S BLB; Toshiba) for 2 to 3 d at 24°C. A spore suspension (1 x 105 conidia mL–1) containing 0.05% (w/v) Tween 20 was sprayed onto rice plants. The inoculated plants were incubated at 25°C with high humidity in the dark for 20 h and then moved to a greenhouse.
Blast fungus-inoculated leaves, cut 0.5 cm in length, were vacuum infiltrated with water and then stained with a lactophenol-trypan blue solution containing 10 mL of lactic acid, 10 mL of glycerol, 10 g of phenol, and 10 mg of trypan blue dissolved in 10 mL of distilled water (Koch and Slusarenko, 1980). Leaf segments were boiled for 3 min in the stain solution and decolorized in a chloral hydrate solution containing 2.5 g of chloral hydrate dissolved in 1 mL of distilled water for at least 24 h. They were mounted in the chloral hydrate solution and viewed under a microscope.
AOA was purchased from Sigma-Aldrich. AOA was dissolved in water and the pH adjusted to 7.0 with NaOH solution. 1-MCP was provided by Rohm and Haas. Two millimolar S TS solution was prepared by adding 20 mL of 0.01 M silver nitrate (Sigma-Aldrich) solution to 80 mL of 0.01 M STS (Sigma-Aldrich) solution. Inoculated fourth leaves were detached at 24 hpi and fed with solutions of 0.1, 0.5, and 1.0 mM AOA, 1 mM ACC, both 0.5 mM AOA and 1 mM ACC, or 2 mM STS, respectively, from the cut base of freshly detached leaf blades. Inoculated rice plants at the four-leaf stage were treated with gaseous 1-MCP in airtight containers at 24 hpi for 16 h, following the manufacturer's instructions.
At 0, 24, 36, 48, 63, 72, and 96 h after the inoculation, fourth leaves were detached from the base of the leaf blade. Sixteen leaves were put into 52-mL glass vials with 5 mL of water, sealed with a gas-proof septum, and left in a growth cabinet at 24°C for 3 h under light. One milliliter of gas was withdrawn from the airspace of each tube using a gas-tight syringe (Hamilton) and injected into a gas chromatograph (Shimadzu GC-14B) equipped with an aluminum column (Shumpak-A; Shimazu) and a flame-ionization detector for ET determination.
Leaf material frozen in liquid nitrogen was ground with a mortar and pestle and stirred with a 5% (w/v) sulfosalicylic acid solution (2 mL g–1 fresh weight) for 30 min at room temperature. The concentration of ACC in the supernatant after centrifugation at 30,000g for 30 min was determined directly by chemical conversion to ET according to Lizada and Yang (1979)
The ACO assay was performed as described by Mekhedov and Kende (1996)
DNA- and RNA-blot analyses were performed using the digoxigenin nonradioactive nucleic acid labeling and detection system (Roche), following the manufacturer's instructions.
Genomic DNA was isolated from rice seedlings as described by Murray and Thompson (1980)
Total RNA was extracted from rice leaves using the auxin tricarboxylic acid method described by Nagy et al. (1988)
Total RNA was extracted from inoculated rice leaves using TRIzol Reagent (Invitrogen). Also, 0.5 µg of total RNA as template was supplied for amplification of rice ACO and ACS genes using the SuperScript One-Step RT-PCR system with Platium Taq (Invitrogen). Specific primers were designed based on DNA sequences reported to GenBank and displayed the primer DNA sequence listed in Table I . The specificity of the primers was checked by excising the RT-PCR products after electrophoresis. Among 0.01 to 1 µg of total RNA as a template for RT-PCR, amplification products for each gene increased linearly. PCR amplification conditions were 50°C for 30 min, 96°C for 2 min, followed by 30 cycles of 96°C for 30 s, 56°C for 30 s, and 72°C for 1 min, and then one cycle of 72°C for 5 min. The relative transcript amounts were visualized by using a luminescent image analyzer LAS-1000plus (Fujifilm) from the images of agarose gels after electrophoresis.
Accession numbers of each gene are described in parentheses: OsACS1 (AK071011), OsACS2 (AK064250), OsACS3 (P0617H07.9 in AC135427), OsACS4 (OSJNBb0006B22.3 in AC136224), OsACS5 (D46839), OsACS6 (AK065212), OsACO1 (AK058296), OsACO2 (AK071557), OsACO3 (AK065039), OsACO4 (AK105491), OsACO5 (AK061064), OsACO6 (OJ1504_G04.8 in AC105772), OsACO7 (AK102472), OsPR1a (AJ278436; Agrawal et al., 2000
We would like to thank Dr. Tokio Imbe and Dr. Ikuo Ando of the National Agriculture and Food Research Organization for providing IL7. We also thank Drs. Naoki Midoh and Mitiaki Iwata of Meiji Seika Kisha Ltd. and Dr. Eiichi Minami of the National Institute of Agrobiological Sciences for providing the PBZ1 and PAL clones, respectively. We are grateful to Dr. Hisatoshi Kaku for advice on experiments, to Dr. Shigeo Nakamura and Dr. Ichiro Mitsuhara for helpful suggestions, and to Hisako Ochiai for technical support. Received June 16, 2006; accepted September 5, 2006; published September 29, 2006.
1 This work was partially supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yuko Ohashi (yohashi{at}affrc.go.jp).
[OA] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.085258 * Corresponding author; e-mail yohashi{at}affrc.go.jp; fax 81–298–38–7469.
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