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SHORT COMMUNICATION Plant Signaling & Behavior 7:7, 1–3; July 2012; G 2012 Landes Bioscience Additional cause for reduced JA-Ile in the root of a Lotus japonicus phyB mutant Tamaki Shigeyama,1 Akiyoshi Tominaga,1,2 Susumu Arima,1,2 Tatsuya Sakai,3,4 Sayaka Inada,3 Yusuke Jikumaru,3 Yuji Kamiya,3 Toshiki Uchiumi,5 Mikiko Abe,5 Masatsugu Hashiguchi,6 Ryo Akashi,6 Ann M. Hirsch7 and Akihiro Suzuki1,2,* 1 Department of Environmental Sciences; Faculty of Agriculture; Saga University; Honjyo-machi, Saga, Japan; 2United Graduate School of Agricultural Sciences; Kagoshima University; Korimoto, Kagoshima, Japan; 3RIKEN Plant Science Center; Yokohama; Kanagawa, Japan; 4Graduate School of Science and Technology; Niigata University; Nishiku, Niigata, Japan; 5 Department of Chemistry and Bioscience; Faculty of Science; Kagoshima University; Korimoto, Kagoshima, Japan; 6Frontier Science Research Center; University of Miyazaki; Miyazaki; Miyazaki, Japan; 7Department of Molecular; Cell and Developmental Biology and Molecular Biology Institute; University of California-Los Angeles; Los Angeles, CA USA Keywords: symbiotic nitrogen fixation, shade avoidance syndrome, phytochrome, R/FR ratio, root nodule, nodulation, jasmonic acid Light is critical for supplying carbon for use in the energetically expensive process of nitrogen-fixing symbiosis between legumes and rhizobia. We recently showed that root nodule formation in phyB mutants [which have a constitutive shade avoidance syndrome (SAS) phenotype] was suppressed in white light, and that nodulation in wild-type is controlled by sensing the R/FR ratio through jasmonic acid (JA) signaling. We concluded that the cause of reduced root nodule formation in phyB mutants was the inhibition of JA-Ile production in root. Here we show that the shoot JA-Ile level of phyB mutants is higher than that of the wild-type strain MG20, suggesting that translocation of JA-Ile from shoot to root is impeded in the mutant. These results indicate that root nodule formation in phyB mutants is suppressed both by decreased JA-Ile production, caused by reduced JAR1 activity in root, and by reduced JA-Ile translocation from shoot to root. © 2012 Landes Bioscience. Light is an important environmental factor controlling plant growth. It is well known that plants require light for photosynthesis and are able to monitor both light quality and quantity for optimal survival. Plants have photoreceptors that sense the presence of their neighbors by monitoring the ratio of red light (R), which is absorbed by chlorophyll, and far red light (FR), which is not. A low R/FR ratio indicates the presence of neighbors that may compete for photosynthetically active radiation (PAR) and initiates the shade avoidance syndrome (SAS), causing plants to grow taller or bend to the light to avoid shade.1-4 Many leguminous plants establish a symbiosis with nitrogenfixing soil bacteria called rhizobia. Inside the root nodules, the rhizobia differentiate into nitrogen-fixing bacteroids. The bacteroids convert atmospheric nitrogen into ammonia, a source of fixed nitrogen for the host plant. Because assimilates from photosynthesis provide energy to fuel the symbiosis between legumes and rhizobia, the light conditions under which host plants grow are very important.5-8 Previously we reported that root nodule formation was suppressed in a Lotus japonicus phytochrome B (phyB) mutant having a constitutive SAS phenotype.9 In that paper, we concluded that the cause of reduced root nodule formation in low-R/FR-grown MG20 (wild-type) plants and white-light-grown phyB mutants is inhibition of JA-Ile (an active JA derivative) production in root. By using grafted plants prepared from MG20 and phyB mutant plants, we also showed that shoot genotype controls root nodule formation.9 Here we report additional data confirms that root nodulation is controlled by shoot genotype. The expression level of marker gene NIN,10 which is required for infection thread formation and nodule primordium initiation, was analyzed in the root of grafted plants by using real time RT-PCR by the methods described in Tominaga et al.11 The roots and shoots of five-day-old MG20 and phyB mutant plants were grafted in various combinations, as described by Magori et al.12 After 7 d, the grafted plants were inoculated with M. loti, and expression was analyzed 7 d after inoculation. Figure 1 shows the relative expression levels of NIN in roots of grafted plants. The mean value of expression in grafted MG20(scion)/MG20(root stock) plants was set as one. When phyB was the scion, both root nodule number and NIN expression levels were significantly lower than in the MG20/MG20 control regardless of the rootstock genotype. When MG20 was used as the scion on a phyB rootstock, no significant change in N1N expression was observed relative to the MG20/MG20 control. These results support the hypothesis that shoot genotype controls root nodule formation. Furthermore, we previously showed that levels of JAR1 gene expression and JA-Ile concentration are lower in roots of phyB than in roots of MG20.9 Because JAR1 codes for an enzyme that conjugates JA with amino acids to produce the active JA derivative [most likely jasmonoyl-isoleucine (JA-Ile)],13 we suggested that inhibition of root nodule formation in phyB mutants is caused by suppression of the conversion of JA to JA-Ile. To investigate whether JA-Ile levels decreased throughout the whole plant or only in root, we measured the endogenous concentrations of JA Do not distribute. *Correspondence to: Akihiro Suzuki; Email: [email protected] Submitted: 03/23/12; Revised: 04/13/12; Accepted: 04/16/12 http://dx.doi.org/10.4161/psb.20407 www.landesbioscience.com Plant Signaling & Behavior 1 observed in JA concentration between MG20 and phyB mutant plants (Fig. 2A); however, the concentration of JA-Ile was significantly higher in the shoot of phyB mutants than in MG20 (Fig. 2B). This result suggested that the translocation of JA-Ile from shoot to root is blocked and that JA-Ile accumulates in the shoot of phyB mutants. Taken together, these results indicate that decreased JA-Ile concentration caused reduced root nodule formation in the root of low-R/FRgrown MG20 plants, and that the phenotype of white-light-grown phyB mutants was produced both by decreased JAR1 activity in root and by decreased translocation of JA-Ile from shoots to roots. Thus, in wild-type plants exposed to low R/FR Figure 1. Relative expression of NIN gene in root of grafted plants. The mean value of light, SAS is triggered by the inactivation of expression in MG20(scion) / MG20(root stock) was set as 1.0. Transcript amounts were normalized against ATP synthase (internal control) transcripts. The data represent PHYB, and root nodule formation is suppressed the averages ± SE of three independent experiments using roots derived from 3–4 through regulation of the JA-Ile concentration. We different plants. Statistical significance in comparison to grafted MG20/MG20 is indicated conclude that this shade avoidance syndrome for by asterisks (**p , 0.01). root nodule formation is required for L. japonicus nodule development and is essential for establishing and JA-Ile in shoots of white light-grown MG20 and phyB. and maintaining a successful nitrogen-fixing symbiosis. Fifteen-day-old plants were inoculated with M. loti; after 7 d, Disclosure of Potential Conflicts of Interest endogenous JA and JA-Ile concentrations were measured by a 14 previously reported method. No significant difference was No potential conflicts of interest were disclosed. © 2012 Landes Bioscience. Do not distribute. Figure 2. Endogenous concentration of JA and JA-Ile in shoots of white light-grown MG20 and phyB. The data represent the averages ± SE of three independent experiments using shoots derived from six different plants. Statistical significance is indicated by asterisks (*p , 0.05). 2 Plant Signaling & Behavior Volume 7 Issue 7 Acknowledgments L. japonicus Miyakojima MG20 seeds were provided by the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. This work was 7. References 1. 2. 3. 4. 5. 6. supported by a Grant-in-Aid for Challenging Exploratory Research from the Japan Society for the Promotion of Science (grant no. 21657017 to A.S.) and the Kato Memorial Bioscience Foundation (grant to A.S.). Franklin KA, Quail PH. Phytochrome functions in Arabidopsis development. J Exp Bot 2010; 61:11-24; PMID:19815685; http://dx.doi.org/10.1093/jxb/erp304 Smith H, Whitelam GC. The shade avoidance syndrome: Multiple responses mediated by multiple phytochromes. Plant Cell Environ 1997; 20:840-4; http://dx.doi.org/10.1046/j.1365-3040.1997.d01-104.x Neff MM, Fankhauser C, Chory J. Light: an indicator of time and place. Genes Dev 2000; 14:257-71; PMID: 10673498 Franklin KA. Shade avoidance. New Phytol 2008; 179:930-44; PMID:18537892; http://dx.doi.org/10. 1111/j.1469-8137.2008.02507.x Bethlenfalvay GJ, Norris RF, Phillips DA. Effect of Bentazon, a Hill Reaction Inhibitor, on Symbiotic Nitrogen-fixing Capability and Apparent Photosynthesis. Plant Physiol 1979; 63:213-5; PMID: 16660682; http://dx.doi.org/10.1104/pp.63.1.213 Finn GA, Brun WA. Effect of atmospheric CO(2) enrichment on growth, nonstructural carbohydrate content, and root nodule activity in soybean. Plant Physiol 1982; 69:327-31; PMID:16662202; http://dx. doi.org/10.1104/pp.69.2.327 Bethlenfalvay GJ, Phillips DA. Effect of light intensity on efficiency of carbon dioxide and nitrogen reduction in Pisum sativum L. Plant Physiol 1977; 60:868-71; PMID:16660203; http://dx.doi.org/10.1104/pp.60.6. 868 8. Yoshida C, Funayama-Noguchi S, Kawaguchi M. plenty, a novel hypernodulation mutant in Lotus japonicus. Plant Cell Physiol 2010; 51:1425-35; PMID:20732950; http://dx.doi.org/10.1093/pcp/pcq115 9. Suzuki A, Suriyagoda L, Shigeyama T, Tominaga A, Sasaki M, Hiratsuka Y, et al. Lotus japonicus nodulation is photomorphogenetically controlled by sensing the red/far red (R/FR) ratio through jasmonic acid (JA) signaling. Proc Natl Acad Sci U S A 2011; 108:1683742; PMID:21930895; http://dx.doi.org/10.1073/pnas. 1105892108 10. Schauser L, Roussis A, Stiller J, Stougaard J. A plant regulator controlling development of symbiotic root nodules. Nature 1999; 402:191-5; PMID:10647012; http://dx.doi.org/10.1038/46058 11. Tominaga A, Nagata M, Futsuki K, Abe H, Uchiumi T, Abe M, et al. Enhanced nodulation and nitrogen fixation in the abscisic acid low-sensitive mutant enhanced nitrogen fixation1 of Lotus japonicus. Plant Physiol 2009; 151:1965-76; PMID:19776164; http:// dx.doi.org/10.1104/pp.109.142638 12. Magori S, Oka-Kira E, Shibata S, Umehara Y, Kouchi H, Hase Y, et al. Too much love, a root regulator associated with the long-distance control of nodulation in Lotus japonicus. Mol Plant Microbe Interact 2009; 22:259-68; PMID:19245320; http://dx.doi.org/10. 1094/MPMI-22-3-0259 13. Staswick PE, Tiryaki I. The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis. Plant Cell 2004; 16:2117-27; PMID:15258265; http://dx.doi.org/10.1105/tpc.104. 023549 14. Ohkama-Ohtsu N, Sasaki-Sekimoto Y, Oikawa A, Jikumaru Y, Shinoda S, Inoue E, et al. 12-oxophytodienoic acid-glutathione conjugate is transported into the vacuole in Arabidopsis. Plant Cell Physiol 2011; 52:205-9; PMID:21097476; http://dx.doi.org/ 10.1093/pcp/pcq181 © 2012 Landes Bioscience. Do not distribute. www.landesbioscience.com Plant Signaling & Behavior 3