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ISSN 0435-1096 Gamma Field Symposia

Gamma Field Symposia

Number 42




Ohmiya-machi, Naka-gun, Ibaraki-ken Japan


Report of Symposium held on July 16-17, 2003

Institute of Radiation Breeding NIAS

Ohmiya-machi, Naka-gun, Ibaraki-ken 319-2293 Japan


The lecturers and the members of the Symposium Committee

General discussion


List of Participants

(42nd GF Symposium)

ABE, F. AJIRO, T. ARAI, T. ASAUMI, H. ASHIKARI, M. DEGI, K. EHIRA, M. ENDO, T. EZURA, H. FUJITA, M. FUJITA, Y. FUKATSU, E. FUWA, N. GONAI, T. GOTO, Y. GOTO, Y. HARA, H. HASEGAWA, H. HATASHITA, M. HATTORI, E. HATTORI, K. HATTORI, T. HAYAKAWA, Y. HAYASHI, Y. HIGO, K. HIRAI, T. HIRATA, Y. HIRAYAMA, T. HOBO, T. HOSOYA, T. INAGAKI, H. INOUE, E. ISHII, T. ISHIMARU, K. ITO, A. ITO, Y. IWABUCHI, M. IYOZUMI, H. KADOTA, N. KAMIYA, Y. National Institute of Crop Science Tokyo University of Agriculture and Technology Gifu Research Institute for Agricultural Sciences Ehime Agricultural Experiment Station Bioscience and Biotechnology Center, Nagoya University Institute of Radiation Breeding, National Institute of Agrobiological Sciences Tokyo University of Agriculture and Technology Miyagi Furukawa Agricultural Experiment Station University of Tsukuba RIKEN Tsukuba Institute Japan International Research Center for Agricultural Sciences Forest Tree Breeding Center Snow Brand Seed Co., Ltd. Ibaraki Plant Biotechnology Institute Forest Tree Breeding Center Hitachi High-Technologies Ibaraki University University of Shiga Prefecture The Wakasawan Energy Research Center Toyota Motor Corporation Graduated School of Bioagricultural Sciences, Nagoya University Bioscience and Biotechnology Center, Nagoya University Fukui Prefectural University Fukushima Agricultural Experiment Station National Institute of Agrobiological Sciences Tokyo University of Agriculture Tokyo University of Agriculture and Technology RIKEN Central Institute RIKEN Laboratory of Plant Molecular Biology High School of Agriculture, Mito Ibaraki Shizuoka Agricultural Experiment Station Ibaraki University Ibaraki Agricultural Center University of Tsukuba National Institute of Fruit Tree Science Institute of Radiation Breeding, National Institute of Agrobiological Sciences National Institute of Agrobiological Sciences Shizuoka Agricultural Experiment Station Takii & Co., LTD RIKEN Yokohama Institute


KANEKO, T. KARITA, E. KASHIMA, M. KATAOKA, H. KATO, K. KATO, M. KAWAKATSU, M. KOBAYASHI, M. KOBAYASHI, S. KUBOTA, K. KUBOYAMA, T. KUDO, S. KUJIOKA, H. KURODA, K. KUSABA, M. KUSANO, M. KUSANO, T. KUSHIRO, T. KUZUYA, M. MATSUBAYASHI, Y. MATSUI, H. MIYANO, S. MORISHITA, T. MORITA, M. MORITA, R. MORITA, R. MORITA, Y. MOTODA, J. NAGATO, Y. NAGATOMI, S. NAITO, K. NAITO, T. NAITO, Y. NAKAJIMA, I. NAKAMURA, S. NAKANO, T. NARA, Y. NARUKAWA, M. NIKI, T. NISHIMURA, M. NISHIMURA, S. NONAKA, S. NOZAWA, G.T. OBARA, N. Plant Bioengineering Research Laboratories, Sapporo Breweries Tokyo University of Science Ibaraki Agricultural Center University of Tsukuba Shizuoka Agricultural Experiment Station Takii Plant Breeding & Experiment Station Institute of Radiation Breeding, National Institute of Agrobiological Sciences RIKEN Tsukuba Institute National Institute of Fruit Tree Science Nagano Agricultural Experiment Station Ibaraki University Graduate school of Agricultural Science, Tohoku University High School of Agriculture, Mito Ibaraki National Institute of Agrobiological Sciences Institute of Radiation Breeding, National Institute of Agrobiological Sciences Kaisui Chemical Industry Co., LTD. Graduate School of Life Sciences, Tohoku University RIKEN Plant Science Center Ibaraki Agricultural Center Graduated School of Bioagricultural Sciences, Nagoya University Kyoto University Keisei Rose Nurseries, LTD. Institute of Radiation Breeding, National Institute of Agrobiological Sciences Kyoto University Institute of Radiation Breeding, National Institute of Agrobiological Sciences Tokyo University of Agriculture The University of Tokyo Graduated School of Bioagricultural Sciences, Nagoya University Graduate School of Agricultural and Life Science, The University of Tokyo Institute of Radiation Breeding, National Institute of Agrobiological Sciences Institute of Radiation Breeding, National Institute of Agrobiological Sciences School of Agriculture, Meiji University Tokyo University of Agriculture National Institute of Fruit Tree Science National Institute of Crop Science RIKEN Central Institute Tokyo University of Science Tokyo University of Science National Institute of Floricultural Science Institute of Radiation Breeding, National Institute of Agrobiological Sciences University of Tsukuba University of Tsukuba Tokyo University of Agriculture and Technology VISTA, LTD


OHMIYA, Y. OHSAWA, K. OHTA, K. OHTA, Y. OHTSUBO, N. OKA, A. OKA, S. OKAMURA, J. OKAMURA, M. OKAZAKI, K. OKUDAIRA, M. OKUMOTO, Y. ONO, Y. ONOZAKI, T. RIKIISHI, K. SAISHO, D. SAITO, K. SAITO, M. SANADA, T. SANO, Y. SATO, M. SATO, M. SATO, T. SATO, Y. SEKIGUCHI, F. SHIMOMURA, S. SOEJIMA, J. SUGIYAMA, K. SUNOHARA, H. SUZUKI, A. SUZUKI, Y. TAJI, T. TAKABATAKE, R. TAKAHASHI, H. TAKAHASHI, M. TAKAHASHI, T. TAKANO, T. TAKASAKI, T. TAKATSU, Y. TAKEUCHI, S. TAKYU, T. TANAKA, M. TANIGUCHI, T. TANISAKA, T. Forest Tree Breeding Center Nagano Agricultural Research Center Tokyo University of Agriculture and Technology Tokyo University of Agriculture and Technology Ministry of Education, Culture, Sports, Science and Technology Institute for Chemical Research, Kyoto University National Institute of Agrobiological Sciences Sakata Seed Corp. Plant Lab., Kirin Brewery Co., Ltd. Niigata University Iwate Agricultural Research Center Kyoto University Bio-oriented Technology Research Advancement Institution National Institute of Floricultural Science Okayama University Okayama University Saitama Agriculture and Forestry Research Center Fukui Agriculture Experiment Station National Institute of Fruit Tree Science Graduate School of Agriculture, Hokkaido University Oita Agricultural Research Center Japan International Research Center for Agricultural Sciences Akita Agricultural Experiment Station Toyota Motor Corporation Japan Women's University National Institute of Agrobiological Sciences National Institute of Fruit Tree Science Shizuoka Citrus Experiment Station Nagoya University Iwate University National Institute of Crop Science RIKEN Laboratory of Plant Molecular Biology National Institute of Agrobiological Sciences Tokyo University of Science Forest Tree Breeding Center Watanabe Seed Co., LTD. Institute of Radiation Breeding, National Institute of Agrobiological Sciences Tochigi Agricultural Experiment Station Ibaraki Agricultural Center Keisei Rose Nurseries LTD. Institute of Radiation Breeding, National Institute of Agrobiological Sciences Nagasaki Fruit Tree Experiment Station Forest Tree Breeding Center Graduate School of Agriculture, Kyoto University



National Institute of Fruit Tree Science Graduate School of Agricultural and Life Science, The University of Tokyo Hamamatsu Photonics, LTD High School of Agriculture, Mito Ibaraki Japan Atomic Energy Research Institute-Takasaki Iwate University Institute of Radiation Breeding, National Institute of Agrobiological Sciences Japan Seed Trade Association Institute of Radiation Breeding, National Institute of Agrobiological Sciences Ibaraki Agricultural Center Yokohama Nursery Co,. LTD. Saitama Flower & Garden Center Takii & Co., LTD. Kyoto University National Institute of Crop Science University of Tsukuba Sapporo Breweries



Forty years have passed since full-scale mutation breeding research began with the founding of the Institute of Radiation Breeding (IRB). During this time, many mutation varieties bred in Japan have been accepted as familiar agricultural products. Mutation breeding is expected to play an increasingly important role in Japan for improving plants. In this sense, this symposium provides a vital opportunity for exchanging research information and engaging in much-needed discussion. Dramatic progress has been made during the last decade in the study of biosynthesis, perception, and signal transduction of plant hormones. Due to the importance of recent basic molecular knowledge of plant hormones to future research in mutation breeding, we have selected the theme "Plant hormone research and mutation" for the 42nd Gamma Field Symposium. We have invited eight lectures on this subject, including a special lecture entitled, "Biosyntheses and regulation of plant hormones" on the physiological action of plant hormones to be given by Dr. Yuji Kamiya of the Institute of Physical and Chemical Research (RIKEN). The IRB has economically evaluated mutation breeding in agricultural production and prepared pamphlets to introduce mutation varieties released in Japan. As of March 2003, 320 mutant varieties have served in developing 50 kinds of crops. The cumulative cultivation of mutation varieties has occupied 5.5 million hectares, corresponding to 1.1-fold of the total area under cultivation per year in Japan. The cumulative production value of such agricultural products reached 7 trillion yen, which corresponds to total annual domestic agricultural production. Mutagens (agents of mutation induction) used for these 320 mutant varieties included gammarays at 72%, tissue culture at 11%, chemical substances at 10%, and X-rays and others at 7%. Among mutation varieties, 145 were used as is, obtained directly from mutagen processing. Of these 74 varieties used IRB facilities, accounting for 51%. The number of indirectly used varieties obtained by hybridizing mutants or the progeny of mutants was 175, demonstrating how the use of mutated genes had advanced. In closing, we thank the lecturers who have so kindly taken time out from busy schedules to prepare for this symposium and to all who have provided their unstinting support in making this symposium a success.


The Symposium Committee Shigeki NAGATOMI, Chairperson Yuji ITO Toshikazu MORISHITA Yasuo NAGATO Minoru NISHIMURA Seibi OKA Tetsuro SANADA Yoshio SANO Takatoshi TANISAKA Nobuhiro TSUTSUMI



Opening address : S. NAGATOMI Congratulatory address : M. IWABUCHI

Special lecture Chairperson : S. TANO Biosyntheses and Regulation of Plant Hormones Session ¿ Chairperson : S. OKA Molecular Mechanisms for Auxin Responce and Signal Transduction Session À Chairperson : Y. NAGATO Gibberellin Responce and Signal Transduction Session Á Chairperson : T. TANISAKA Cytokinin Signal Transduction and Two-Component Regulatory System Session  Chairperson : N. TSUTSUMI Molecular Mechanisms for ABA Responce and Signal Transduction Session à Chairperson : T. SANADA Molecular Mechanisms for Ethylene Perception and Signal Transduction Session Ä Chairperson : H. HASEGAWA Mechanism of Brassinosteroid Signaling Session Å Chairperson : K. HATTORI Peptide Plant Hormone, Phytosulfokine Session Æ Chairperson : Y. SANO General discussion Closing address : Y. NAGATO











Y. KAMIYA M. ASHIKARI H. ITOH M. UEGUCHI M. MATSUOKA A. OKA T. HIRAYAMA T. UGAJIN T. NAKANO S. YOSHIDA T. ASAMI Y. MATSUBAYASHI Biosyntheses and Regulation of Plant Hormones Gibberellin Responce and Signal Transduction 1 13

Cytokinin Signal Transduction and Two-Component Regulatory System Molecular Mechanisms for Ethylene Perception and Signal Transduction Mechanism of Brassinosteroid Signaling


41 53

Peptide Plant Hormone, Phytosulfokine


General discussion (in Japanese


Gamma Field Symposia, No. 42, 2003 Institute of Radiation Breeding NIAS, Japan




Yuji KAMIYA RIKEN Plant Science Center 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045

Introduction Plant hormones are signal molecules, present in trace quantities. Changes in hormone concentration and tissue sensitivity mediate a whole range of developmental process in plants, many of which involve interactions with environmental factors. So far seven hormones are considered as major plant hormones, namely auxin, gibberellins, cytokinins, abscisic acid, ethylene, brassinosteroids and jasmonic acid. Each of these hormones has its own particular properties, so the pathways regulating their production and degradation are quite diverse and have been elucidated by use of chemistry, biochemistry, plant physiology, genetics and molecular genetics. Recently the genomic sequence of Arabidopsis is available for hormone study and that of the rice is also in hand. Now it is possible to study hormone biosyntheses and their regulation by reverse genetic approach. In this symposium I focused on biosyntheses of gibberellins, cytokinins and absisic acid, which we are now studying in my laboratories. Part of this paper involves some new results and data, which are now provisionally accepted in some journals. Therefore this paper is neither a review nor an original paper. However, this paper describes what I talked during the symposium. In order to give credits to the researchers in the three different topics, major researchers are listed in parentheses.

Regulation of GA biosynthesis by cold temperature in Arabidopsis germinating seeds (Yukika YAMAUCHI and Shinjiro YAMAGUCHI) Gibberellins (GA)s are involved in many processes of plant development, such as seed germination, stem elongation, leaf expansion, flowering, and seed development (DAVIES 1995). GAs are synthesized from geranylgeranyl diphosphate (GGDP), which is sequentially converted to biologically active GAs by terpene cyclases, cytochrome P450 monooxygenases and 2-oxoglutaratedependent dioxygenases (Fig. 1) (HEDDEN and KAMIYA 1997). Most of the genes encoding GA biosynthesis and catabolism enzymes have now been identified (OLSZEWSKI et al. 2002).



Fig. 1. Gibberellin biosynthesis in Arabidopsis.

It has been known that GA promotes seed germination in many plant species. In Arabidopsis, severe GA deficient-mutants, such as ga1-3 and ga2-1, are defective in seed germination (KOORNNEEF and VAN DER VEEN 1980), and chemical inhibitors of GA biosynthesis inhibit germination (NAMBARA et al. 1991). These observations indicate that de novo GA biosynthesis is necessary for seed germination in Arabidopsis (HEDDEN and KAMIYA 1997). Light is a critical environmental factor for seed germination in some small-seeded plants such as lettuce, tomato and Arabidopsis (SHINOMURA, 1997). The effect of light on seed germination is primarily mediated by phytochromes (BORTHWICK et al. 1952; BUTLER et al. 1959). Genes encoding GA 3-oxidases, which convert inactive precursor to active GAs, are regulated by phytochromes in germinating lettuce and Arabidopsis seeds (TOYOMASU et al. 1998; YAMAGUCHI et al. 1998). Temperature is another crucial external cue that controls seed germination (BEWLEY and BLACK 1982). In many plant species, exposure of seeds to low temperature (typically 2 -5 ) immediately after imbibition is effective to promote germination. (SHROPSHIRE et al. 1961; CONE and SPRUIT 1983). This treatment is called "stratification". Although this method is widely used to improve the frequency and synchronization of germination, the mechanism for the thermoregulation of seed germination has been unclear. The effect of cold treatment on GA content has been reported in the 1970s, based on semiquantitative analysis of endogenous GAs using -amylase bioassay and/or gas chromatographymass spectrometry (GC-MS) (ROSS and BRADBEER 1971; SINSKA et al. 1973; WILLIAMS et al. 1974). DERKX et al. (1994) analyzed the effect of pre-chilling of Arabidopsis seeds and reported that bioactive GA4 was detectable only in pre-chilled seeds, but not in dark-imbibed seeds. It has not been clear whether the effect of pre-chilling is a direct response to low temperature because pre-



chilling treatment in published studies also involved a longer imbibition period. Yukika YAMAUCHI and Shinjiro YAMAGUCHI have worked intensively about the effect of cold treatment. We first carried out large-scale expression analysis during imbibition of after-ripened Arabidopsis dry seeds at 4 . This investigation indicates that a number of GA-related genes are differentially expressed between dry and cold-treated seeds. Our GC-MS and reverse transcriptionPCR analyses show that GA biosynthesis is activated in response to low temperature in darkimbibed seeds, and that the effect of temperature is targeted to particular GA biosynthesis genes. Using a loss-of-function mutant of the cold-inducible AtGA3ox1 gene, we show that this gene is required for cold-promoted synthesis of active GAs and seed germination. Our results suggest that germination of Arabidopsis seeds is stimulated in response to low temperature in part through modulating GA biosynthesis (Fig. 2). Furthermore, we show that cold treatment increases the number of cell types accumulating the AtGA3ox1 transcript detectable by in situ hybridization analysis, suggesting a complex regulatory mechanism by which the spatial distribution of GA biosynthesis is determined (YAMAUCHI et al. 2004).

Biosynthesis of prenyl side chain of cytokinins (Hiroyuki KASAHARA, Kentaro TAKEI, Shinjiro YAMAGUCHI and Hitoshi SAKAKIBARA) Cytokinins (CKs) have many physiological roles in plants, such as promotion of cell division and shoot formation in the presence of auxin, release of lateral buds from apical dominance, stimulation of chloroplast development, and delay of senescence. The biological activity, biosynthesis and metabolism of CKs have been well studied (MOK and MOK, 2001). Recently, a CK receptor has been identified in Arabidopsis (INOUE et al. 2001; YAMADA et al. 2001). Functional analysis

Fig. 2. In Arabidopsis germinating seeds, GA3ox1 gene is regulated by light, temperature and negative feed back. Red and blue and arrows indicate effects of light and cold temperature respectively.



using the receptor protein expressed in Escherichia coli and yeast have indicated that free CKs, transzeatin (tZ) and isopentenyl adenine (iP), are active CK species in Arabidopsis (INOUE et al. 2001). Most of CKs identified from plants are derivatives of N6-prenylated adenine. Plants have two possible biosynthetic pathways for the production of the side chain of CKs, namely mevalonate (MVA) pathway in the cytosol and methylerythritol phosphate (MEP) pathway in plastids (LICHITENTHALER 1999; ROHMER 2003). Both pathways supply common precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Although the MVA and MEP pathways are localized in different subcellular compartments, there is some exchange of common precursor(s) between the two pathways (KASAHARA et al. 2002). Therefore, application of a precursor specific to the MEP pathway can suppress the growth inhibition caused by a block in the MVA pathway, and vice versa (HEMMERLIN et al. 2003). Hiroyuki KASAHARA and Shinjiro YAMAGUCHI in collaboration with Kentaro TAKEI and Hitoshi SAKAKIBARA worked about the prenylation of CK side chain. CK biosynthesis pathway in plants has initially been deduced on the basis of bacterial enzymes. The formation of N6-prenylated adenine from AMP was first demonstrated using cellfree extracts of a slime mold, Dictyostelium discoideum (TAYA et al. 1978). A CK biosynthesis gene TMR, which encode AMP:DMAPP-isopentenyltransferase (AMP:DMAPP-IPT), was isolated from Ti-plasmid of Agrobacterium tumefaciens (BARRY et al. 1984; AKIYOSHI et al. 1984). Because CK levels were elevated in transgenic plants that overproduce bacterial AMP: DMAPPIPT, the formation of isopentenyl adenosine monophosphate (iPRMP) from AMP and DMAPP is likely to be a committed step in CK biosynthesis. Subsequent formation of iP from iPRMP requires 5'-nucleotidase and adenosine nucleosidase. The conversion of iP into tZ is catalyzed by trans-hydroxylase, which is probably a P450 monooxygenase (CHEN and LEISNER 1984). Recently, a search for Arabidopsis genes that are homologous to bacterial AMP:DMAPP-IPT has identified nine AtIPT genes (TAKEI et al. 2001; KAKIMOTO 2001). Unlike bacterial IPTs, seven AtIPTs were able to transfer DMAPP not only to AMP, but also to ADP and ATP, to give corresponding nucleotide CKs in vitro. Thus, the AMP/ADP/ATP-dependent pathway has been proposed for the biosyntheses of iP/tZ in plants. On the other hand, the tRNA-dependent pathway has been also proposed for the biosynthesis of CKs in plants because tRNAs in bacteria, yeast and plants contain a N6-prenylated adenine moiety, which, by hydrolysis, is capable of forming CKs (MURAI 1994). Among nine IPT-related sequences in Arabidopsis, two AtIPT genes encode (putative) tRNA-isoprenyltransferases (tRNAIPTs). The prenylated adenine element in tRNA is usually consisted of iP and cis-zeatin (cZ), the cis-isomer of tZ. Therefore, the tRNA-dependent pathway has not been considered as the main route to tZ. However, the occurrence of cis-trans isomerase activity in Phaseolus vulgaris immature seeds suggested that the tRNA-dependent pathway might also contribute to the biosynthesis of tZ through cZ (BASSIL et al. 1993). The MVA pathway had been considered as the sole route providing DMAPP to CKs until the



MEP pathway was uncovered recently. The incorporation of 14C-labeled MVA into the iP element of tRNA in vivo has been demonstrated in tobacco pith tissue (CHEN and HALL 1969). Also, 13Clabeled MVA was incorporated into iP and trans-zeatin riboside (tZR) in vitro in the endosperm of Sechium edule (PIAGGESI et al. 1997). In addition, there are some reports that indicate that CK levels are reduced in plants when the MVA pathway is limited (ÅSTOT et al. 2000). On the other hand, the contribution of the MEP pathway to CK biosynthesis has never been issued before. It should be noted that the incorporation of MVA does not exclude a potential role of the MEP pathway in the biosynthesis of CKs because it has often been observed that isoprene units from both MVA and MEP pathways are incorporated into a single downstream isoprenoid (KASAHARA et al. 2002; HEMMERLIN et al. 2003). Thus, a possible contribution of the MEP pathway to the biosynthesis of CKs needs to be examined to better understand how CKs are synthesized in plant. In order to selectively label metabolites from the MVA or MEP pathways with 13C in vivo, we have previously carried out feeding of [1-13C] 1-deoxy-D-xylulose (DX) or [2-13C]mevalonolactone (MVL) to Arabidopsis seedlings. DX is converted into an MEP pathway intermediate 1-deoxy-Dxylulose 5-phosphate (DXP) by phosphorylation. Therefore, exogenous DX is able to complement the albino phenotype of the cla1-1 mutant (ESTEVEZ et al. 2000), which is defective in DXP in the MEP pathway. Similarly, the growth inhibition due to a block in the MVA pathway by mevastatin (an inhibitor of HMG-CoA reductase) is rescued by exogenous application of MVL (KASAHARA et al. 2002). Efficient 13C-labeling of metabolites from the MVA or MEP pathways was thus achieved by feeding 13C-labeled DX and MVL to the cla1-1 mutant and mevastatin-treated plants, respectively. These 13C-labeling systems allowed us to determine contribution of the MVA and MEP pathways to the biosynthesis of GAs by gas chromatography-mass spectrometry (KASAHARA et al. 2002). We studied the biosynthesis route for the prenyl moiety of CKs using the 13C-labeled tracers in Arabidopsis seedlings. Our data demonstrate that the prenyl side chain of tZ- and iP-type CKs are mainly produced through the MEP pathway (Fig. 3), whereas a large fraction of cZ derivatives is synthesized through the MVA pathway. We also show the subcellular location of AtTPTs produced as GFP-fusion proteins. Based on these data, we proposed a crucial role of the plastid-localized MEP pathway in CK biosynthesis (KASAHARA et al. 2004).

Cloning of P450 genes involved in abscisic acid degradation (Tetsuo KUSHIRO, Masanori OKAMOTO, Kazumi NAKABAYASHI and Eiji NAMBARA) Abscisic acid (ABA) controls numerous aspects of plant life cycle including seed dormancy, germination and adaptive responses to environmental stresses (ZEEVAART and CREELMAN, 1988). ABA-deficient mutants from several plant species show reduced seed dormancy and wilty phenotype (MCCARTY 1995). ABA content increases during seed development or when a plant is subjected to various stresses such as osmotic stress, while it rapidly decreases during subsequent



Fig. 3. Origin of side chains of trans-zeatin and cis-zeatin in Arabidopsis. cZ, cis-zeatin; cZR, cis-zeatin riboside; cZRMP, cis-zeatin ribosidemonophosphate; DMAPP, dimethylallyl diphosphate; iP, isopentenyladenine; iPR, isopentenyladenine riboside; iPRMP, isopentenyladenine riboside monophosphate; MEP, methylerythritol phosphate; MVA, mevalonate

germination or the recovery from stress. ABA content is determined by the balance between biosynthesis and catabolism. When endogenous ABA levels is maintained high, both ABA biosynthesis and catabolism are active (HARRISON and WALTON 1975; ZEEVAART 1980; PIERCE and RASCHKE, 1981). Constitutive expression of ABA biosynthetic gene in transgenic plants exhibits a more prominent accumulation of the catabolites compared to a moderate increase in ABA contents (QIN and ZEEVAART, 2002). Recently, most of ABA biosynthetic genes have been identified (SCHWARTZ et al. 2003; SEO and KOSHIBA 2002). However, molecular mechanisms underlying ABA catabolism remain poorly understood. ABA is catabolized into inactive forms either by oxidation or conjugation (MILBORROW 1969; MILBORROW 1975; WALTON and SONDHEIMER 1972; SONDHEIMER et al. 1974; XU et al. 2002; see review; CUTLER and KROCHKO 1999) (See Fig. 4). The predominant pathway for ABA catabolism is the oxidative pathway, which is triggered by hydroxylation at C-8' to produce 8'hydroxy ABA. The 8'-hydroxy ABA is subsequently isomerized spontaneously to form phaseic acid (PA) (MILBORROW et al. 1988). Biological activity of PA is significantly less than that of ABA, therefore, the major regulatory step in inactivation is likely to be 8'-hydroxylation of ABA (ARAI et al. 1999). This reaction is known to be catalyzed by a cytochrome P450 monooxygenase (P450) (GILLARD and WALTON, 1976; KROCHKO et al. 1998). However, the gene encoding ABA 8'-hydroxylase remained elusive. It is necessary to identify this gene to understand the molecular mechanism controlling the hormonal level of ABA. Tetsuo KUSHIRO, Masanori OKAMOTO (Ph.D. student from the Tokyo Metropolitan Univ., Prof. Tomokazu KOSHIBA), Kazumi NAKABAYASHI



Fig. 4. Abscisic acid catabolism. 8'-Hydroxylation is catalyzed by CYP707A1-A4 in Arabidopsis.

and Eiji NAMBARA have worked intensively about the cloning and characterization of the ABA catabolic enzyme. P450s are large family of enzymes that catalyze the oxidation of various low molecular weight compounds. They have been conserved for billions of years and exist in most of the organisms on the earth ranging from bacteria to mammals. In mammals, P450s play a major role in drug metabolism, and have been a center of research in the pharmaceutical field. In plants, P450s participate in numerous aspects of plant metabolism, which include phytohormones and secondary metabolites (SCHULER 1996; CHAPPLE 1998). Completion of the Arabidopsis genome sequencing has revealed that there are at least 272 P450 genes in this single organism. In the following completion of the rice genome sequencing, nearly 450 P450 genes have been identified ( These numbers obviously indicate how widely these genes have evolved and deeply rooted in the plant life cycle. The genome sequencing efforts were successful in accumulating sequence information, however, it is still a major challenge to identify the function of each gene. This will be a major task in the post genomic era, and will require novel approaches as well as systematic analysis of the gene. P450s are especially challenging since in most cases, the substrate of the enzyme cannot be easily predicted. Furthermore, the number of possible steps where P450 participates along the metabolic pathways is largely unknown. In order to identify the P450 gene for ABA 8'-hydroxylase, we have set out to search for the gene of our interest among hundreds of candidate genes. Once the gene for ABA 8'-hydroxylase is identified, it would be possible to fine-tune the level of ABA in plants, and thus, would expect to improve drought tolerance as well as to prevent precocious germination in crops. Therefore, the identification of this gene would have an enormous impact on the agricultural industry. Our extensive and careful prediction led to the first successful identification of the members of CYP707A family as ABA 8'-hydroxylase genes. Expression analysis and genetic analysis demonstrated that CYP707 genes play a regulatory role in vivo to define the ABA level during these processes (KUSHIRO et al. 2004).




AKIYOSHI, D.E., KLEE, H., AMASINO, R.M., NESTER, E.W. and GORDON, M.P. (1984) T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc. Natl. Acad. Sci. USA 81: 5994-5998. ARAI, S., TODOROKI, Y., IBARAKI, S., NAOE, Y., HIRAI, N. and OHIGASHI, H. (1999) Synthesis and activity of 3'-chloro, -bromo, and -iodoabscisic acids, and biological activity of 3'-fluoro-8'-hydroxy abscisic acid. Phytochemistry 52: 1185-1193. ÅSTOT, C., DOLEZAL, K., NORDSTRÖM, A., WANG, Q., KUNKEL, T., MORITZ, T., CHUA, N.H. and SANDBERG, G. (2000) An alternative cytokinin biosynthesis pathway. Proc. Natl. Acad. Sci. USA 97: 14778-14783. BARRY, G.F., ROGERS, S.G., FRALEY, R.T. and BRAND, L. (1984) Identification of a cloned cytokinin biosynthetic gene. Proc. Natl. Acad. Sci. USA 81: 4776-4780. BASSIL, N.V., MOK, D. and MOK, M.C. (1993) Partial Purification of a cis-trans-Isomerase of Zeatin from Immature Seed of Phaseolus vulgaris L. Plant Physiol. 102: 867-872. BEWLEY, J.D. and BLACK, M. (1982) Physiology and biochemistry of seeds. (Volume 2). Viability, dormancy and environmental control. (Berlin, Germany: Springer-Verlag). BORTHWICK, H.A., HENDRICKS, S.B., PARKER, M.W., TOOLE, E.H. and TOOLE, V.K. (1952) A reversible photoreaction controlling seed germination. Proc. Natl. Acad. Sci. USA 38: 662-666. BUTLER, W.L., NORRIS, K.H., SIEGELMAN, H.W. and HENDRICKS, S.B. (1959) Detection, assay, and preliminary purification of the pigment controlling phtoresponsive development of plants. Proc. Natl. Acad. Sci. USA 45: 1703-1708. CHAPPLE, C. (1998) Molecular genetics analysis of plant cytochrome P450-dependent monooxygenases. Annu. Rev. Plant Physiol. Mol. Biol. 49: 311-343. CHEN, C.M. and HALL, R.H. (1969) Biosynthesis of N6-(d2-isopentenyl) adenosine in the transfer ribonucleic acid of cultured tabacco pith tissue. Phytochemistry 8: 1687-1696. CHEN, C.M. and LEISNER, S.M. (1984) Modification of cytokinins by cauliflower microsomal enzymes. Plant Physiol. 75: 442-446. CONE, J.W. and SPRUIT, C.J.P. (1983) Imbibition conditions and seed dormancy of Arabidopsis thaliana. Physiol. Plant. 59: 416-420. CUTLER, A.J. and KROCHKO, J.E. (1999) Formation and breakdown of ABA. Trends Plant Sci. 4: 472-478. DAVIES, P.J. (1995) Plant Hormones: Physiology, Biochemistry and Molecular Biology. (Dordrecht, The Netherlands: Kluwer Academic Publishers). DERKX, M.P.M., VERMEER, E. and KARSSEN, C.M. (1994) Gibberellins in seeds of Arabidopsis thaliana: Biological activities, identification and effects of light and chilling on endogenous levels. Plant Growth Regul. 15: 223-234. ESTEVEZ, J.M., CANTERO, A., ROMERO, C., KAWAIDE, H., JIMENEZ, L.F., KUZUYAMA, T., SETO, H., KAMIYA, Y. and LEON, P. (2000) Complementation of albino mutant cla1-1 of Arabidopsis thaliana by 1-deoxy-Dxylulose: A CLA1 gene require for the non-mevalonate pathway. Plant Physiol. 124: 95-104. GILLARD, D.F. and WALTON, D.C. (1976) Abscisic acid metabolism by a cell-free preparation from Echinocystis lobata liquid endosperm. Plant Physiol. 58: 790-795. HARRISON, M. A. and WALTON, D.C. (1975) Abscisic acid metabolism in water-stressed bean leaves. Plant Physiol. 56: 250-254. HEDDEN, P. and KAMIYA, Y. (1997) Gibberellin biosynthesis: Enzymes, genes and their regulation. Annu. Rev. Plant Phys. Plant Mol. Biol. 48: 431-460. HEMMERLIN, A., HOEFFLER, J. F., MEYER, O., TRITSCH, D., KAGAN, I.A., GROSDEMANGE-BILLIARD, C., ROHMER, M. and BACH, T.J. (2003) Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco Bright Yellow-2 cells. J. Biol. Chem. 278: 26666-26676.



HOLTON, T.A. and CORNISH, E.C. (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7: 1071-1083. INOUE, T., HIGUCHI, M., HASHIMOTO, Y., SEKI, M., KOBAYASHI, M., KATO, T., TABATA, S., SHINOZAKI, K. and KAKIMOTO, T. (2001) Identification of CRE1 as a cytokinin receptor from Arabidopsis. Narure 409: 1060-1063. KAKIMOTO, T. (2001) Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyltransferases. Plant Cell Physiol. 42: 677-685. KASAHARA, H., HANADA, A., KUZUYAMA, T., TAKAGI, M., KAMIYA, Y. and YAMAGUCHI, S. (2002) Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis. J. Biol. Chem. 277: 45188-94. KASAHARA, H., TAKEI, K., UEDA, N., HISHIYAMA, S., YAMAYA. T., KAMIYA, Y., YAMAGUCHI, S. and SAKAKIBARA, H. (2004) Distinct isoprenoid origins of cis-and trans-Zeatin biosyntheses. J. Biol. Chem. 279: 14049-14054. KOORNNEEF, M. and VAN DER VEEN, J.H. (1980) Induction and analysis of gibberellin sensitive mutants in Arabidopsis thaliana (L.) Heynh. Theor. Appl. Genet. 58: 257-263. KROCHKO, J.E., ABRAMS, G.D., LOEWEN, M.K., ABRAMS, S.R. and CUTLER, A.J. (1998) (+)-Abscisic acid 8'hydroxylase is a cytochrome P450 monooxygenase. Plant Physiol. 118: 849-860. KUSHIRO, T., OKAMOTO, M., NAKABAYASHI, K., KITAMURA, S., ASAMI, T., HIRAI, N., KOSHIBA, T., KAMIYA, Y. and NAMBARA, E. (2004) The Arabidopsis cytochrome P450 CYP707A encodes ABA8' hydoroxylases: Key enzyme in ABA catabolism. The EMBO Jornal 23: 1647-1656. LICHITENTHALER, H.K. (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 47-65. MCCARTY (1995) Genetic control and integration of maturation and germination pathways in seed development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 71-93. MILBORROW, B.V. (1969) Identification of "Metabolite C" from abscisic acid and a new structure for phaseic acid. Chem. Commun. 966-967. MILBORROW, B.V. (1975) The absolute configuration of phaseic and dihrdrophaseic acids. Phytochemistry 14: 1045-1053. MILBORROW, B.V., CARRINGTON, N.J. and VAUGHAN, G.T. (1988) The cyclization of 8'-hydroxy abscisic acid to phaseic acid in vivo. Phytochemistry 27: 757-759. MOK, D.W.S. and MOK, M.C. (2001) Cytokinin metabolism and action. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 89-118. NAMBARA, E., AKAZAWA, T. and MCCOURT, P. (1991) Effects of the gibberellin biosynthetic inhibitor uniconazol on mutants of Arabidopsis. Plant Physiol. 97: 736-738. PIAGGESI, A., PICCIARELLI, P., CECCARELLI, N. and LORENZI, R. (1997) Cytokinin biosynthesis in endosperm of Sechium edule Sw.. Plant Sci. 129: 131-140. PIERCE, M. and RASCHKE, K. (1981) Synthesis and metabolism of abscisic acid in detached leaves of Phaseolus vulgaris L. after loss and recovery of turgor. Planta 153: 156-165. QIN, X. and ZEEVAART, J.A.D. (1999) The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean. Proc. Natl. Acad. Sci. USA 96: 15354-15361. ROHMER, M. (2003) Mevalonate-independent methylerythritol phosphate pathway for isoprenoid biosynthesis. Elucidation and distribution. Pure Appl. Chem. 75: 375-387. ROSS, J.D. and BRADBEER, J.W. (1971) Studies in seed dormancy. V. The content of endogenous gibberellins in seeds of Corylus avellana L. Planta 100: 288-302. SCHULER, M.A. (1996) Plant cytochrome P450 monooxygenases. Crit. Rev. Plant Sci. 15: 235-284. SCHWARTZ, S.H., QIN, X. and ZEEVAART, J.A.D. (2003) Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol. 131: 1591-1601. SEO, M. and KOSHIBA, T. (2002) Complex regulation of ABA biosynthesis in plants. Trends Plant Sci. 7: 41-48.



SHINOMURA, T. (1997) Phytochrome regulation of seed germination. J. Plant Res. 110: 151-161. SHROPSHIRE, J.W., KLEIN, W.H. and ELSTAD, V.B. (1961) Action spectra of photomorphogenic induction and photoinactivation of germination in Arabidopsis thaliana. Plant Cell Physiol. 2: 63-69. SINSKA, I., LEWAK, S., GASKIN, P. and MACMILLAN, J. (1973) Reinvestigation of apple-seed gibberellins. Planta 114: 359-364. SONDHEIMER, E., GALSON, E.C., TINELLI, E. and WALTON D.C. (1974) The metabolism of hormones during seed germination and dormancy. Plant Physiol. 54: 803-808. TAKEI, K., SAKAKIBARA, H. and SUGIYAMA, T. (2001) Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana. J. Biol. Chem. 276: 26405-26410. TAYA, Y., TANAKA, Y. and NISHIMURA, S. (1978) 5'-AMP is a direct precursor of cytokinin in Dictyostelium discoideum. Nature 271:545-547. TOYOMASU, T., KAWAIDE, H., MITSUHASHI, W., INOUE, Y. and KAMIYA, Y. (1998) Phytochrome regulates gibberellin biosynthesis during germination of photoblastic lettuce seeds. Plant Physiol. 118: 1517-1523. WALTON, D.C., and SONDHEIMER, E. (1972) Metabolism of 2-14C-( )-abscisic acid in excised bean axes. Plant Physiol. 49: 285-289. WILLIAMS, P.M., BRADBEER, J.W., GASKIN, P. and MACMILLAN, J. (1974) Studies in seed dormancy VIII. The identification and determination of gibberellins A1 and A9 in seed of Corylus avellana L. Planta 117: 101-108. XU, Z-J., NAKAJIMA, M., SUZUKI, Y. and YAMAGUCHI, I. (2002) Cloning and characterization of the abscisic acid-specific glucosyltransferase gene from Adzuki bean seedlings. Plant Physiol. 129: 1285-1295. YAMADA, H., SUZUKI, T., TERADA, K., TAKEI, K., ISHIKAWA, K., MIWA, K., YAMASHINO, T. and MIZUNO, T. (2001) The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant Cell Physiol. 42: 1017-1023. YAMAGUCHI, S., SMITH, M.W., BROWN, R.G., KAMIYA, Y. and SUN, T.-P. (1998) Phytochrome regulation and differential expression of gibberellin 3b-hydroxylase genes in germinating Arabidopsis seeds. Plant Cell 10: 2115-2126. YAMAUCHI, Y., OGAWA, M., KUWAHARA, A., HANADA, A., KAMIYA, Y. and YAMAGUCHI, S. (2004) Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds. Plant Cell 16: 367-378. ZEEVAART, J.A.D. (1980) Changes in the levels of abscisic acid and its metabolites in excised leaf blades of Xanthium strumarium during and after water stress. Plant Physiol. 66: 672-678. ZEEVAART, J.A.D. and CREELMAN, R.A. (1988) Metabolism and physiology of abscisic acid. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39: 439-473.





Stratification AtGA3ox1 MS AtGA3ox1 AtGA3ox1 T-DNA AtGA3ox1 MVA MEP cla1 MEP MEP transciscisMVA transMEP 2 AtGA3ox2 GA3 GCGA4


12 8' 273 4 CYP707A


8' P450 NADPH 8'

Gamma Field Symposia, No. 42, 2003 Institute of Radiation Breeding NIAS, Japan




Motoyuki ASHIKARI, Hironori ITOH, Miyako UEGUCHI and Makoto MATSUOKA Bioscience and Biotechnology Center, Nagoya University Furoucho, Chikusaku, Nagoya, 464-8601 E-mail : [email protected]

Introduction GAs are a large family of tetracyclic diterpenoid plant growth regulators and have been reported to be associated with a number of plant growth and development processes such as seed germination, stem elongation, flowering and fruit development (Reid 1993; Hooley 1994; Ross et al. 1997). GA-related mutants in plants show dwarf or elongated phenotypes, and these mutants are crucial for elucidating the regulatory mechanisms governing the GA biosynthetic and signal transduction pathways. Because dwarf characteristics are favored in plant breeding, the study of these characteristics has applications not only for understanding basic plant biology but also for molecular breeding. Many GA-related mutants have been isolated from numerous plant species (Reid 1993; Hooley 1994; Ross et al. 1997) and can be roughly classified into 2 categories: GAsensitive and GA-insensitive. A GA-sensitive mutant responds to exogenous GA because it cannot produce GA, or it produces insufficient GA due to a deficiency in genes encoding GA catalytic enzymes. On the other hand, a GA-insensitive mutant does not respond to exogenous GA, and gene related to GA-insensitivity may be associated with GA signal transduction (Hedden and Phillips 2000; Olszewski et al. 2002). Here shows the rice GA insensitive mutants and their gene functions. slender rice 1 (slr1) mutant The slender rice 1 (slr1) mutant show a slender phenotype with an elongated stem and leaf and reduced root number and length, which is similar to that of rice plants treated with GA3 (Fig. 1) (Ikeda et al. 2001; Itoh et al. 2002). The slr1 mutant was first identified on the basis of its abnormal elongation phenotype at the seedling stage, which is similar to the appearance of wildtype rice plants infected by "Bakanae-disease". In fact, it is difficult to distinguish between slr1 and "Bakanae- disease" plants. The slr1 phenotype seems to be the result of saturation with GAs, however, the levels of endogenous GAs (GA19, GA20 and GA1) in slr1 are actually lower than in the wild-type. Also, GA-inducible -amylase (Ramy1A) is produced in the aleurone cells in the absence of GA application. However, the GA-saturation phenotype of slr1 is not affected by


Motoyuki ASHIKARI, Hironori ITOH, Miyako UEGUCHI and Makoto MATSUOKA

Fig. 1 Gross morphology of slender1 (slr1) and domain structure of SLR1

treatment with uniconazole, a GA biosynthesis inhibitor (Ikeda et al. 2001). These results indicate that slr1 is a constitutive GA response mutant and that the SLR1 protein may be associated with GA signal transduction as a negative regulator (Ikeda and et al. 2001; Itoh et al. 2002). The SLR1 gene has been isolated by linkage analyses between a rice gene homologous to Arabidopsis GAI and the slender phenotype. Some slr1 alleles contain a nucleotide substitution or deletion that disrupts the open reading frame, and therefore these are considered to be loss-of-function alleles. Actually, the introduction of the wild-type SLR1 gene complements the slender mutation (Ikeda et al. 2001). On the basis of these findings, the SLR1 gene is regarded to be homologous to Arabidopsis GAI, which encodes a putative repressor protein for the GA signaling pathway. The SLR1 protein shares high amino acid identity with Arabidopsis GAI (47.2 %), RGA (41.2 %), wheat RHT-D1a (77.2 %) and d8 (80.3 %). The SLR1 gene is located on the long arm of rice chromosome 3, a region which shows the genome synteny with the wheat Rht locus of chromosome 4 and maize D8 locus of chromosome 1, confirming that these genes of grass species are orthologous (Peng et al. 1999; Ikeda et al. 2001). The deduced SLR1 protein has 625 amino acid residues and contains the DELLA, TVHYNP domain (called regions I and II in GAI) in the N-terminal region which is conserved among Arabidopsis GAI and RGA, wheat RHT and maize d8 (Peng et al. 1999) (Fig. 2). SLR1 also contains other consensus domains at the C-terminal region, such as a leucine heptad repeat, NLS, VHIID, PFYRF and SAW, which belong to the GRAS family (Pysh et al. 1999). Since proteins in the GRAS family, including Arabidopsis SCR (Laurenzio et al. 1996), are considered to function as transcriptional factors, SLR1 may have a similar role. Biochemical analyzes of SLR1, namely nuclear localization and transcriptional activity, support this idea (Itoh et al. 2002; Ogawa et al. 2000). To investigate the function of SLR1 in plants, we have generated transgenic rice plants that



Fig. 2 Schematic structure of SLR1

constitutively produce the SLR1-GFP protein under the control of the rice Actin1 promoter. These transgenic plants show the dwarf phenotype, supporting the idea that SLR1 functions as a negative regulator of GA signaling (Itoh et al. 2002). The GFP signal is localized in the nucleus but disappears following treatment with GA3; this effect is accompanied by leaf and stem elongation. The disappearance of SLR1 in response to GA3 treatment has been confirmed by immunoblot analysis using an anti-SLR1 antibody (Itoh et al. 2002). Based on these results, we have proposed a model for SLR1 function whereby, in the absence of a GA signal, the SLR1 protein localized in the nucleus suppresses GA activity as a transcriptional regulator, but SLR1 rapidly degrades in response to a GA signal, thereby releasing the suppression of GA action (Itoh et al. 2002). Similar findings have also been reported for SLR1 homologous proteins: the Arabidopsis RGA protein and barley SLN protein are localized in the nuclei (Dill and Sun 2001; Silverstone et al. 2001; Gubler et al. 2002) and RGA and SLN disappear following the application of GA3 (Dill and Sun 2001; Silverstone et al. 2001; Fu et al. 2002; Gubler et al. 2002). This suggests that the suppressive action of SLR1, SLN1, and RGA in rice, barley, and Arabidopsis, respectively, is similar in the regulation of GA signaling. Unlike SLR1, RGA and SLN1 proteins, the GAI and RGL1 (RGA-like1) proteins in Arabidopsis are not degraded by the GA treatment (Fleck and Harberd 2002; Wen and Chang 2002). There are two classes of the SLR1 orthologous proteins in Arabidopsis, one of which (RGA) disappears from the nucleus in response to GA-treatment, the other (GAI and RGL1) does not (Fleck and Harberd 2002). Dominant alleles in the Arabidopsis gai, wheat Rht-B1/Rht-D1, and maize D8 loci confer GAinsensitive mutants with the dwarf phenotype (Koornneef et al. 1985; Peng et al. 1993; Peng et al. 1997; Harberd and Freeling 1989; Winkler and Freeling 1994). Molecular cloning of Arabidopsis GAI has demonstrated that the in-frame deletion of its N-terminal domain, DELLA (region I), induces the gai mutant (Peng et al. 1997). Similarly, wheat Rht-B1/Rht-D1 and maize D8 have mutations in their N-terminal domains, DELLA (region I) and TVHYNP (region II), as in GAI (Peng et al. 1999). Transgenic plants that overproduce a SLR1 protein truncated in the DELLA domain have a dominant dwarf phenotype similar to Arabidopsis gai (Ikeda et al. 2001; Itoh et al. 2002). Interestingly, all of these mutants and transgenic plants that overproduce the truncated form of SLR1 show GA-insensitive characteristics. These results suggest that the N-terminal region involving the DELLA and TVHYNP domains may function as a receptor for upstream GA signals.


Motoyuki ASHIKARI, Hironori ITOH, Miyako UEGUCHI and Makoto MATSUOKA

To examine the function of the conserved domain of SLR1, we have performed a domain analysis of SLR1 using transgenic plants that overproduce various truncated SLR1 proteins. Transformants overproducing DELLA or TVHYNP show a severe dwarf phenotype and lack GA-responsiveness. Correspondingly, the DELLA and TVHYNP proteins do not degrade following GA treatment. These results strongly suggest that these N-terminal domains are involved in the perception of GA signals. In contrast to the N-terminal proteins, the C-terminal region containing the VHIID, PFYRE, and SAW domains is involved in the suppressive function of SLR1. This is supported by the finding that the null alleles of slr1 often contain nucleotide substitutions or deletions in the C-terminal region. Domain analysis has also revealed that there are an additional two functional domains in SLR1, that is, a dimer formation domain and a regulatory domain. As its name suggests, the dimer domain is important for formation of a dimer of SLR1, and proteins lacking this domain ( LZ) do not retain their repressive function. Conversely, if a truncated SLR1 protein containing the dimer domain, but not the suppressive domain ( C-ter protein), is overproduced in the wild-type, the transformants show the slender phenotype, demonstrating the dominant negative function of the truncated SLR1 containing the dimer domain. The regulatory domain, which is rich in serine/threonine residues, may be involved in the regulation of SLR1 repression activity (Itoh et al. 2002). In fact, it has been proposed that the activity or stability of SLR1 is regulated by O-GluNAcylation or phosphorylation via the action of the SPINDLY protein (Thornton et al. 1999) or kinase, with the serine/threonine residues as the target site Dill et al. (2001) have also performed a domain analysis of RGA in Arabidopsis using transgenic plants overproducing truncated RGA proteins. Transgenic plants with DELLA show the GA-insensitive severe dwarf phenotype and the protein is resistant to degradation following GA treatment. This also demonstrates that the DELLA motif is essential for GA-induced RGA degradation. Why do the loss-of-function alleles of RGA or GAI show an almost normal phenotype, even though rice slr1 and barley sln1 show the GA-constitutive response phenotype? For example, gait6, the loss-of-function allele of gai has wild-type features but has slightly increased resistance to paclobutrazol (PAC), an inhibitor of GA biosynthesis. This has been explained by a functional redundancy of GAI, RGA and other orthologous proteins. Indeed, RGA has a highly similar structure to that of GAI, and also works as a negative regulator of GA signaling (Silverstone et al. 1998). Consequently, the loss-of-function of RGA does not result in a typical constitutive GA response phenotype but rather a partial suppression of the dwarf phenotype conferred by the GAdeficient mutation, ga1-3 (Silverstone et al. 1997; Silverstone et al. 1998). Double mutants gai/gai, rga/rga do not show the slender phenotype, but it slightly higher than wild. It is probably due to the presence of redundant genes, RGLs (RGL1, RGL2 and RGL3) (Gill and Sun 2001). However, recently it is reported that RGL1 and RGL2 play a larger role in seed germination than does GAI or RGA which are mainly associate with stem elongation (Wen and Chang 2002; Lee et al. 2002). In contrast, the barley sln1 mutant has the slender phenotype (Foster 1977) and induces -amylase



expression without GA treatment, as is the case in rice (Chandler 1988; Lanahan and Ho 1988; Croker et al. 1990). The rice and barley genomes have only one gene encoding an orthologous protein to GAI/RGA (Chandler et al. 2002). Such non-redundancy of GA-related genes in rice should provide an advantage for studying the GA signal transduction pathway. gid2 mutant The gid2 mutant lines show a severe dwarf phenotype with wide leaf blades and dark green leaves (Fig. 3), which are features of GA-related mutants such as d1 and d18 (Ashikari et al. 1999; Itoh et al. 2001). gid2 does not show any GA-responsiveness when measured against the three criteria as follows, second leaf sheath elongation, -amylase induction in aleurone, and feed-back expression of GA20 oxidase. Moreover, even though the gid2 mutants have severe dwarfism, they accumulate more than 150 times the level of bioactive GA1 than that in wild-type plants. Given the GA-insensitivity of the gid2 mutant, we expect that the GID2 gene encodes a positive regulator of GA signaling. To elucidate the molecular function of GID2, the gene has been isolated by positional cloning. Genetic analysis enabled us to narrow-down the gid2 mutation to a 13kb region on rice chromosome 2. A comparison of the nucleotide sequence of this region between gid2 and the wildtype revealed that all three gid2 alleles have nucleotide substitutions or deletions in one putative gene that introduce novel stop codons, suggesting that these are null alleles. Introduction of a wild DNA fragment spanning the entire region of the candidate gene into the gid2 mutant rescues the

Fig. 3 Gross morphology of gibberellin-insensitive dwarf 1 and 2 mutants (gid1 and gid2) Left: wild-type, Right: gid2


Motoyuki ASHIKARI, Hironori ITOH, Miyako UEGUCHI and Makoto MATSUOKA

gid2 phenotype to normal. The GID2 gene encodes a 636bp open reading frame, capable of producing a polypeptide of 212 amino acid residues. The deduced amino acid sequence of GID2 contains an F-box domain, which is a conserved motif of F-box proteins that form a component of an E3 ubiquitin-ligase complex. The F-box sequence in GID2 is well conserved in other F-box proteins from Arabidopsis, yeast, mold, and humans. Many F-box proteins contain a proteinprotein interaction domain, such as leucine-rich repeat (LRR) or WD-40 repeat sequences outside the F-box (Dashaies 1999; Yang et al. 1999; Li and Jonston 1997; Skowyra et al. 1997; Winston et al. 1999). However, we have not found any conserved motifs outside the F-box in the GID2 structure, but the structure of GID2 is similar to that of Arabidopsis SLY1 protein which is considered to be a positive regulator of GA signaling in Arabidopsis (McGinnis et al. 2003) It is very likely that the rice GID2 and Arabidopsis SLY1 are orthologous proteins. As described above, the SLR1 protein functions as a repressor of GA signaling in rice and its degradation is essential for the downstream action of GA. Since the GID2 gene encodes a F-box protein, which is a component of a SCF complex (E3 ubiquitin-ligase complex), we thought that the SLR1 protein might be targeted for degradation by the SCF complex in a GA-dependent manner. Immunoblot analysis with an anti-SLR1 antibody has revealed that the SLR1 protein accumulates at a high level in the gid2 mutant, whereas it is only present at low levels in the wildtype. The immunoreactive SLR1 protein in the wild-type is degraded following GA3 treatment, but this does not occur in the gid2 mutant. These findings indicate that the GA-dependent degradation of SLR1 is defective in gid2 and therefore SCFGID2 may directly target the SLR1 protein for degradation through ubiquitination. Interestingly, there are two immunoreactive bands with different mobilities on SDS-PAGE in the gid2 mutant whereas only one band is detected in the wild-type. (Sasaki et al. 2003). In gid2, the band with higher mobility (Form I) has the same mobility as the protein synthesized in E. coli, indicating that this band corresponds to the nascent protein of SLR1. We suspect that the band with lower mobility (Form II) may be an intermediate in the SLR1 degradation process (Sasaki et al. 2003). Actually, the band with higher mobility is not detected under natural SCFGID2 functional conditions. The appearance of a band with higher mobility has also been noted in the barley sln1d mutant (Gubler and others 2002), and therefore may be a common part of the degradation process of the SLR1/ RGA/ SLN1 proteins. Treatment of a crude extract of gid2 with calf intestine alkaline phosphatase (CIP) prior to immunoblotting leads to the disappearance of Form II SLR1.This suggests that Form II is a phosphorylated form of the SLR1 protein. Phosphorylation of SLR1 has also been examined by in vivo labeling with radioactive phosphate, 32PO4- (Sasaki et al. 2003). When the wild-type plants were treated with 32PO4-, we detected one faint radioactive SLR1 band, which disappeared following GA3 treatment. In contrast, one strong radioactive band was observed when the gid2 plants were treated with 32PO4-, and its intensity was increased by GA3 treatment. These results suggest to us that GA increases the phosphorylated form of SLR1 and leads to its degradation by



interacting with the SCFGID2 complex. In gid2, the phosphorylation of SLR1 also occurs following GA3 treatment but the degradation does not occur due to the loss-of-function of the GID2 protein, and consequently the SLR1 protein is accumulated (Sasaki et al. 2003). This model is consistent with previous findings in yeast, mammals and plant, that is, phosphorylation of a target protein triggers the degradation process (Deshaies 1999). A recent publication describes the inhibition of barley SLN1 protein degradation by a proteasome inhibitor (Fu et al. 2002). This supports the notion that the SLR1 protein is degraded through the proteasome. Conclusions and Prospects Based on the results described in this review, we conclude that SLR1 functions as a molecular switch in GA signaling in rice plants. Actually, whether GA activity occurs or not is readily determined by the absence or presence, respectively, of the functional SLR1 protein in the nucleus. GID2 encodes an F-box protein that may be a component of an SCF ubiquitin-ligase complex. The fact that GID2 encodes an F-box protein and SLR1 is highly accumulated in the gid2 mutant led us to speculate that GA-dependent degradation of SLR1 is mediated by the SCFGID2 complex. This is supported by the finding that a phosphorylated form of the SLR1 protein is also accumulated in gid2. So far, there are previous reports that phosphorylation of target proteins triggers SCF-mediated degradation, our results also indicate that GA-dependent phosphorylation of SLR1 triggers the ubiquitin-mediated degradation (Fig. 3), in a similar manner to the SCFmediated pathway in plant, yeast and animals. On the other hand, the mechanism by which SLR1 perceives the GA signal is still unknown. It is possible that the other GA-insensitive dwarf gene, GID1, modifies the molecular structure of the SLR1 protein. Unlike other plant hormones, the GA receptor has not yet been identified. Identification of new mutants associated with GA signaling will be important for elucidating the mechanism of the GA signal transduction pathway, including identification of the GA receptor. As in the case of

Fig. 4 Putative model for the GA signal transduction pathway in rice.


Motoyuki ASHIKARI, Hironori ITOH, Miyako UEGUCHI and Makoto MATSUOKA

SLR1, there is a tendency for the rice genome to have a single gene associated with GA signaling. This non-redundant relationship of GA signal-related genes in rice plants should facilitate the study of the GA signal transduction pathway.


1. Ashikari, M., Wu, J., Yano, M., Sasaki, T. and Yoshimura, A. (1999) Rice gibberellin-insensitive dwarf mutant gene Dwarf 1 encodes the alpha-subunit of GTP-binding protein. PNAS 96: 10284-10289. 2. Chandler, P. M. (1988) Hormonal regulation of gene expression in the "slender" mutant of barley (Hordeum vulgare L.). Planta 175: 115-120. 3. Chandler, P. M., Marion, P. A, Ellis, M. and Gubler, F. (2002) Mutants at the Slender1 locus of barley cv Himalaya. Molecular and physiological characterization. Plant Physiol 129: 181-190. 4. Croker, S. J., Hedden, P., Lenton, J. R. and Stoddart, J. (1990) Comparison of gibberellin in normal and slender barley seedlings. Plant Physiol 94: 194-200. 5. Dashaies, R. J. (1999) SCF and Cullin/Ring H2-based ubiquitin ligases. Annu. Rev. Cell Dev. Biol. 15: 435-467. 6. Dill, A., Jung, H. S., Sun, T. P. (2001) The DELLA motif is essential for gibberellin-induced degradation of RGA. PNAS 98: 14162-14167. 7. Fleck, B. and Harberd, N. P. (2002) Evidence that the Arabidopsis nuclear gibberellin signaling protein GAI is not destabilized by gibberellin. Plant Journal. 32: 935-947. 8. Foster, C. A. (1977) Slender: an accelerated extension growth mutant of barley. Barley Genetics Newsletter 7: 24-27. 9. Fu, X., Richards, D. E., Ait-Ali, T., Hynes, L. W., Ougham, H., Peng, J. and Harberd, N. P. (2002) Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 repressor. Plant Cell 14: 3191-3200. 10. Gubler, F., Chandler, P. M., White, R. G., Llewellyn, D. J. and Jacobsen, J. V. (2002) Gibberellin signaling in barley aleurone cells. Control of SLN1 and GAMYB expression. Plant Physiol 129: 191-200. 11. Harberd, N. P. and Freeling, M. (1989) Genetics of dominant gibberellin-insensitive dwarfism in maize. Genetics 121: 827-838. 12. Hedden, P. and Phillips, A. L. (2000) Gibberellin metabolism: new insights revealed by the genes. Trend Plant Sci 5: 523-530. 13. Hooley, R. (1994) Gibberellin: perception, transduction and responses. Plant Mol Bio 26: 1529-1555. 14. Ikeda, A., Ueguchi-Tanaka, M., Sonoda, Y., Kitano, H., Koshioka, M., Futsuhara, Y., Matsuoka, M. and Yamaguchi, J. (2001) slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8. Plant Cell 13: 999-1010. 15. Itoh, H., Ueguchi-Tanaka, M., Sentoku, N., Kitano, H., Matsuoka, M. and Kobayashi, M. (2001) Cloning and functional analysis of two gibberellin 3 beta -hydroxylase genes that are differently expressed during the growth of rice. PNAS 98: 8909-8914. 16. Itoh, H., Ueguchi-Tanaka, M., Sato, Y., Ashikari, M. and Matsuoka, M. (2002) The gibberellin signaling pathway is regulated by the appearance and disappearance of SLENDER RICE1 in nuclei. Plant Cell 14: 57-70. 17. Koornneef, M., Elgersma, A., Hanhart, C. J., van Loenen-Martinet, E. P., van Rign, L., and Zeevaart, J. A. D. (1985) A gibberellin insensitive mutant of Arabidopsis thaliana. Physiol Plant 65: 33-39. 18. Lanahan, M. B. and Ho, T. H. D. (1988) Slender barley: A constitutive gibberellin-response mutant. Planta 175: 107-114. 19. Laurenzio, D. L., Wysocka-Diller, J., Malamy, J. E., Pysh, L., Helariutta, Y., Freshour, G., Hahn, M. G., Feldmann, K. A. and Benfey, P. N. (1996) The SCARECROW gene regulates an asymmetric cell division



that is essential for generating the radial organization of the Arabidopsis root. Cell. 86:423-33. 20. Lee, S., Cheng, H., King, K. E., Wang, W., He, Y., Hussain, A., Lo, J., Harverd, N. P. and Peng J. (2002) Gibberellin regulates Arabidopsis seed germination via RGL2, a GAI/RGA-like gene whose expression is up-regulated following imbibition. Gene Develop. 16 : 646-658. 21. Li, F. N. and Johnston, M. (1997) Grr1 of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skp1: coupling glucose sensing to gene expression and the cell cycle. EMBO J. 16: 5629-5638. 22. McGinnis, K. M., Thomas, S. G., Soule, J. D., Strader, L. C., Zale, J. M., Sun, T. P. and Steber, C. M. (2003) The Arabidopsis SLEEPY1 gene encodes a putative F-BOX subunit of an E3 ubiquitin ligase. Plant Cell 15: 1120-1130. 23. Ogawa, M., Kusano, T., Katsumi, M. and Sano, H. (2000) Rice giberellin-insensitive gene homolog, OsGAI, encodes a nuclear-localized protein capable of gene activation at transcriptional level. Gene 245: 21-29. 24. Olszewski, N., Sun, T. P. and Gubler, F. (2002) Gibberellin signaling: biosynthesis, catabolism, and response pathway. Plant Cell 14: 61-80. 25. Peng, J. and Harberd, N. P. (1993) Derivative alleles of the Arabidopsis gibberellin-insensitive (gai) mutation confer a wild-type phenotype. Plant Cell 5: 351-360. 26. Peng, J., Carol, P., Richards, D. E., King, K. E., Cowling, R. J., Murphy, G. P. and Harberd, N. P. (1997) The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses. Gene Develop. 11: 3194-3205. 27. Peng, J., Richards, D. E., Hartley, N. M., Murphy, G. P., Devos, K. M., Flintham, J. E., Beales, J., Fish, L. J., Worland, A. J., Pelica, F., Sudhakar, D., Christou, P., Snape, J. W., Gale, M. D. and Harberd, N. P. (1999) `Green revolution' genes encode mutant gibberellin response modulators. Nature 400: 256-261. 28. Pysh, L. D., Wysocka-Diller, J. W., Camilleri, C., Bouchez, D. and Benfey, P. N. (1999) The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROWLIKE genes. Plant J. 18: 111-119. 29. Reid, J. B. (1993) Plant hormone mutants. J Plant Growth Regul. 12: 207-226. 30. Ross, J. J., Murfet, I. C. and Reid, J. B. (1997) Gibberellin mutants. Physiol. Plant 100: 550-560. 31. Sasaki, A., Itoh, H., Gomi, K., Ueguchi-Tanaka, M., Ishiyama, K., Kobayashi, M., Jeong, D-H., An, G., Kitano, H., Ashikari, M. and Matsuoka, M. (2003) Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant. Science 299: 1896-1898. 32. Silverstone, A. L., Mak, P. Y., Martinez, E. C. and Sun, T. P. (1997) The new RGA locus encodes a negative regulator of gibberellin response in Arabidopsis thaliana. Genetics. 146: 1087-1099. 33. Silverstone, A. L, Ciampaglio, C. N. and Sun, T. S. (1998) The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway. Plant Cell 10: 155-169. 34. Silverstone, A. L, Jung, H. S., Dill, A., Kawaide, H., Kamiya, Y. and Sun, T. S. (2001) Repressing a repressor: Gibberellin-induced rapid reduction of the RGA protein in Arabidopsis. Plant Cell 13: 1555-1566. 35. Skowyra, D., Craig, K. L., Tyers, M., Elledge, S. J. and Harper, J. W. (1997) F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91: 209-219. 36. Thornton, T. M., Swain, S. M. and Olszewski, N. E. (1999) Gibberellin signal transduction presents...the SPY who O-GlcNAc'd me. Trends Plant Sci. 4: 424-428. 37. Wen, C. K. and Chang, C. (2002) Arabidopsis RGL1 encodes a negative regulator of gibberellin response. Plant Cell. 14:87-100. 38. Winkler, R. G. and Freeling, M. (1994) Physiological genetics of the dominant gibberellin-nonresponsive maize dwarf, Dwarf8 and Dwarf9. Planta 193: 341-348. 39. Winston, J. T., Strack, P., Beer-Romero, P., Chu, C. Y., Elledge, S. J. and Harper, J. W. (1999) The SCFTRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IB and catenin and stimulates IB ubiquitination in vitro. Genes Dev. 13: 270-283. 40. Yang, M., Hu, Y., Lodhi, M., McCombie, W. R. and Ma, H. (1999) The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation. PNAS 96: 11416-11421.


Motoyuki ASHIKARI, Hironori ITOH, Miyako UEGUCHI and Makoto MATSUOKA

464-8601 GA: gibberellin GA






gid1 gid1 slender slender slender slender

slr1 gid2 gid1 gid2


D1 G D1 D1 100 G


D1 100 D1 D1

slr1 gid2 gid2 slender slender







Motoyuki ASHIKARI, Hironori ITOH, Miyako UEGUCHI and Makoto MATSUOKA

gid2 10 D1

gid1 gid2




gid2 5 10

Gamma Field Symposia, No. 42, 2003 Institute of Radiation Breeding NIAS, Japan




Atsuhiro OKA Institute for Chemical Research, Kyoto University Uji, Kyoto 611-0011, Japan

Introduction Expression of bacterial genes is frequently modulated in response to specific environmental stimuli (e.g., the genes involved in Escherichia coli chemotaxis, Rhizobium nitrogen fixation, and Agrobacterium pathogenicity). In most such cases, several functionally related genes are collectively governed by a single regulatory system, thus constituting a regulon. Many of these regulons have a common regulatory feature, termed the two-component regulatory system (STOCK et al. 2000). This type of system consists of two signal transducers, a sensor protein histidine kinase anchored at the cell membrane and a response regulator present in the cytoplasm. The twocomponent regulatory system appears to be a powerful device for a wide variety of adaptive responses in bacterial cells, as the genome of each bacterial species encodes mostly 30-60 different pairs of sensor histidine kinases and response regulators. Although this regulatory system was initially thought to be specific to prokaryotes, many instances have since been uncovered in diverse eukaryotic species, including higher plants, yeast, fungi, and slime molds. Cytokinins are a class of plant hormones that are responsible for a variety of physiological events. The first cytokinin identified, kinetin, was purified from an autoclaved DNA sample through tracing its ability to promote cell division of tobacco parenchymal cells, and its chemical structure was identified as 6-furfurylaminopurine (for a history of cytokinin research, see OKA 2003). Both natural and synthetic cytokinins are now known, and chemically they are either N6substituted aminopurines (e.g., trans-zeatin, isopentenyladenine, kinetin, 6-benzylaminopurine) or diphenylurea derivatives (e.g., thidiazuron). Along with identification of these compounds, a great deal of physiological information on cytokinins has been accumulated. In addition to promoting cell division, cytokinins induce chloroplast development, seed germination, sink nutritional enhancement, release of lateral bad inhibition, inhibition of root elongation, formation of vascular bundles, increase of pollination efficiency, stomatal opening, cotyledon and leaf development, delay of leaf senescence, shoot formation from calli, etc. (MOK and MOK 2001). For more than two decades, a number of attempts were made to elucidate how cytokinins are recognized by plant cells


Atsuhiro OKA

and how their signals are transduced intracellularly. However, no accurate answers to these questions were obtained until 2001. Using both forward and reverse genetics approaches, two protein species, each resembling the bacterial histidine kinases and response regulators, were found to sense and to be activated by cytokinins, respectively (INOUE et al. 2001; SAKAI et al. 2001). This review will briefly summarize the characteristics of the prokaryotic two-component regulatory system, and then present the results of recent research on cytokinin signaling in Arabidopsis, focusing on the similarities and dissimilarities between the plant and prokaryotic two-component regulatory systems. For further information regarding the observations and experimental results presented without citations in the text to save space, the reader should refer to the appropriate reviews together with references therein (AOYAMA and OKA 2003; HABERER and KIEBER 2002; HWANG et al. 2002; OKA et al. 2002; STOCK et al. 2000; WURGLER-MURPHY and SAITO 1997).

Phosphorelay signaling by bacterial two-component regulatory systems The two-component regulatory system was so named because of a set of two protein components that collectively control expression of the member genes of a regulon (Fig. 1). One component, a sensor protein histidine kinase, is generally composed of an individual N-terminal periplasmic domain with membrane-anchored regions, and a common C-terminal transmitter/kinase (HK) domain, which extends into the cytoplasm. The N-terminal domain together with the neighboring

Fig. 1. Phosphorelay and the domain architecture of protein components involved in the two-component regulatory system. The arrow indicates transfer of the phosphoryl group after autophosphorylation of sensor histidine kinase. "H" within the HK (brown) and HPt (blue) domains and "D" within the RR domain (yellow) are histidine and aspartate residues that are targeted by the phosphoryl group. (a) represents the most popular phosphorelay example of the bacterial two-component regulatory system. Instances corresponding to (b), (c), and (d) are the Agrobacterium VirA-VirG pathogenicity system, the yeast Sln1-Ypd1-Ssk1 osmosis system, and the B. subtilis KinBSpo0F-Spo0B-Spo0A sporulation system, respectively.



regions are thought to be involved in monitoring environmental stimuli either directly or indirectly, whereas the HK domain phosphorylates its own specific histidine residue and then transfers the phosphoryl group to the other component, a cytoplasmic response regulator. It also consists of two domains. The common N-terminal region is called the signal receiver (RR) domain that contains, as a hallmark, two aspartates and one lysine residue (D-D-K) separated by invariant distances. The central aspartate acquires the phosphoryl group from the phospho-histidine on the HK domain. The RR domain is followed by an individual output domain, which is involved mainly in binding to DNA and activating transcription. Some sensor kinases, such as VirA (Agrobacterium phenolic compound sensor) and ArcB (E. coli anaerobic sensor), are called hybrid-type sensor kinases, in which an extra region resembling the RR domain of the cognate response regulator follows the HK domain. Bacterial two-component regulatory systems usually involve no additional component for signal transduction. However, the phosphoryl group is sometimes transferred through a bridge component that carries the histidine-containing phosphotransfer (HPt) domain (e.g., Bacillus subtilis Spo0B and E. coli ArcB), which exists either alone or as a portion of the sensor kinase. Another bridge component is the polypeptide molecule that harbors the RR domain without any obvious output domain (e.g., B. subtilis Spo0F). The RR domain on hybrid-type sensor kinases might be included in this category. Phosphotransfer always occurs as either His-to-Asp or Asp-toHis (Fig. 1). Thus, the two-component regulatory system is also called the His-Asp phosphorelay signal transduction system.

A brief history of the early research on plant two-component regulatory systems By 1993, it was evident that a considerable number of bacterial two-component regulatory systems function in adaptive responses, and that the underlying molecular mechanism involves a unique His-Asp phosphorelay. At about the same time, one of the first eukaryotic sensor histidine kinases, Sln1, was discovered in the budding yeast Saccharomyces cerevisiae (OTA and VARSHAVSKY 1993). Sln1 is an osmosensor, the architecture of which closely resembles that of the bacterial hybrid-type kinase. Subsequently, two downstream components, Ypd1 of an HPt protein and Ssk1 of a response regulator, were identified. Phosphorelay in this system occurs in the order Sln1 (His)>Sln1 (Asp)>Ypd1 (His)>Ssk1 (Asp). The Sln1 RR domain and the Ypd1 HPt protein act as bridge components between Sln1 HK and Ssk1 RR. An interesting feature is that the Ssk1 response regulator is not located at the end of the signal flow, further modulating the downstream Hog1 MAP kinase cascade, the underlying mechanism of which is not phosphorelay but consecutive protein-protein interactions. Exposure to high osmolarity inhibits Sln1 autophosphorylation, and the resulting accumulation of non-phosphorylated molecules of Ssk1 activates the Ssk2 MAPKKK (POSAS and SAITO 1998). Bacterial sensor kinases are generally active under conditions that are not conducive to survival, but the opposite is true in the case of Sln1. Furthermore, the genome sequence indicates that S. cerevisiae harbors no other typical two-component regulatory system.


Atsuhiro OKA

At almost the same time as the identification of Sln1, the Arabidopsis ETR1 gene encoding a hybrid-type histidine kinase was identified as the causative gene of a dominant ethylene-insensitive mutant (CHANG et al. 1993). It was later shown that the ETR1 protein actually binds to ethylene, and acts as an ethylene receptor (SCHALLER and BLEECKER 1995). Another histidine kinase gene, CKI1, was identified using an activation-tagging procedure with hypocotyl explants (KAKIMOTO 1996). Overexpression of CKI1 promoted greening and shoot formation from calli in the absence of exogenous cytokinin, which is usually required for wild-type explants to generate green shoots. Therefore, the CKI1 protein was presumed to be a cytokinin sensor, although its involvement in cytokinin signaling is still not clear. The view was thus established in 1996 that the plant histidine kinases participate in the first step of some phytohormone signaling pathways, as in the case of bacterial adaptive responses. However, information regarding their expected partners, plant response regulators, was not available from studies with phytohormone-insensitive mutants. In 1996, we began in silico screening of higher plants for bacterial-like response regulators, and found that a considerable number of response regulator genes are distributed along the Arabidopsis and rice genomes. Furthermore, these loci encode proteins that can be classified into two groups. The A-type proteins are relatively small molecules consisting of only the RR domain with an extremely short stretch at the N- and/or C-termini, whereas the B-type proteins contain additional functional domains downstream of the RR domain (IMAMURA et al. 1998; SAKAI et al. 1998). As the entire genome sequence of Arabidopsis is now available (The Arabidopsis Genome Initiative 2000), we know that Arabidopsis carries eleven genes for each of the A-type and B-type response regulators (ARR1 to ARR22), eleven sensor histidine kinase genes, and five HPt genes (AHP1 to AHP5). Moreover, there are five genes encoding phytochromes for light sensing (PHYA to PHYE), which show extremely weak homology to the HK domain (SCHNEIDER-POETSCH et al. 1991), and seven genes for pseudo-response regulators (APRR), in which the aspartate phosphorylation target is substituted by glutamate. The architectures of all these components, with the exception of APRRs that are not relevant to this review, are illustrated schematically in Fig. 2.

The CRE1 histidine kinase is a cytokinin sensor In early 2001, mutants were isolated that showed impaired generation of green calli from hypocotyl explants grown in the presence of exogenous cytokinin (INOUE et al. 2001). Another mutant was also reported, in which root elongation was less inhibited by cytokinin than in wildtype plants (UEGUCHI et al. 2001). Their causative genes were identified independently and found to be the same (CRE1/AHK4). This gene encodes a hybrid-type histidine kinase with an extra, atypical, RR segment (Fig. 2). Two additional genes, AHK2 and AHK3, resembling CRE1 were also found. These gene products actually function as cytokinin sensors, as demonstrated by functional complementation with heterologous systems (INOUE et al. 2001; SUZUKI et al. 2001; UEGUCHI et al. 2001). In S. cerevisiae cells, CRE1 functions as a substitute for the Sln1



Fig. 2. Architecture of Arabidopsis proteins belonging to the family of two-component regulatory systems. The relative sizes of proteins and functional domains are drawn on an approximate scale. Vertical bars show putative membrane-spanning regions. Each functional domain is colored as in Fig. 1.

osmosensor only when cytokinin is present in medium. Similar replacements are possible for the Schizosaccharomyces pombe Phk1/Phk2/Phk3 osmosensors and the E. coli RcsC regulator for extracellular polysaccharide synthesis. The signal flow in these systems appears to occur through the His-Asp phosphorelay, as (i) mutations in the putative phosphorylation sites of CRE1 (His-459 and Asp-973) abolish the ability of CRE1 to complement the defect of Sln1, and (ii) the heterologous downstream components (Ypd1 and Ssk1) are absolutely required for complementation. Furthermore, CRE1 has the ability to bind to cytokinin chemicals, as shown using the membrane fractions derived from S. pombe synthesizing CRE1 (YAMADA et al. 2001). Therefore, CRE1 is able to deliver a signal to a non-cognate HPt protein, dependent on cytokinin, as a substitute for the respective sensor kinases in hetero-complementation analyses. As both N6-substituted aminopurines and diphenylurea derivatives are effective in this regard, CRE1 is a universal receptor for cytokinins. The wooden leg (wol) mutant, which is defective in generation of phloem and cambium cells in the root vasculature, carries a missense mutation in the CRE1 gene. This mutant protein cannot bind cytokinin. Cytokinin detected by CRE1 is thus implicated in asymmetrical cell


Atsuhiro OKA

division for xylem and phloem development during early embryogenesis (MÄHÖNEN et al. 2000). The visible wol phenotype indicates limited functional redundancy among CRE1, AHK2, and AHK3, although their expression patterns macroscopically overlap in the roots and other adult tissues. Thus, it is obvious that CRE1, AHK2, and AHK3 recognize cytokinin in a partly overlapping manner followed by initiation of His-Asp phosphorelay to HPt factors.

The B-type response regulators are transcription factors activated by cytokinin The B-type response regulators have been identified utilizing sequence similarity to known RR domains (SAKAI et al. 1998). The ARR1 gene was the first to be cloned and has been examined extensively. Its translation product has an architecture similar to that of the typical bacterial response regulators. The RR domain is located at the N-terminal end, and is followed by the ARRM (or GARP) and Q domains (Fig. 2). The ARRM domain, which faintly resembles the DNA binding domain of mammalian Myb, has the ability to bind double-stranded DNA in a sequencespecific manner in vitro (5'-AGATT-3'). NMR spectroscopy indicated that it contains three -helices, of which the latter two constitute a helix-turn-helix motif, and the most C-terminal -helix together with the N-terminal flexible arm are involved in base-pair recognition (HOSODA et al. 2002). The Q domain is rich in glutamine and proline residues, and is capable of activating transcription (SAKAI et al. 2000). In addition, there is a typical nuclear localization signal between RR and ARRM, together with a few additional elements along the ARR1 molecule. Therefore, ARR1 is equipped with all the functional domains essential for transcription factors. In fact, ARR1 and its truncated version lacking the RR domain are localized almost constantly in nuclei, and overexpression of ARR1 leads to transcriptional activation of a reporter gene preceded by the ARR1 target sequence in its promoter region. Transactivation exerted by the truncated ARR1 is much higher than that by the full-length ARR1, indicating that the RR domain masks the ability of ARR1 to activate transcription (SAKAI et al. 2000). These structural and biochemical characteristics are common to ARR2, ARR10, and ARR11, and all of the eleven B-type ARRs may be functionally redundant. Most of the B-type ARR genes are probably expressed in all adult tissues at different levels, and at significantly higher levels in roots than in other tissues, at least in the cases of ARR1, ARR2, ARR10, and ARR11. Overexpression of ARR1 does not significantly affect plant morphology except for hypertrophic cotyledons, longer cotyledonary petioles, and shorter roots than wild-type plants. Removing a DNA region corresponding to the RR domain from this ARR1 transgene however results in severe phenotypic changes, such as occasional formation of ectopic shoots on the adaxial surface of cotyledons and growth inhibition with concomitant disordered cell proliferation around the shoot apex, depending on the expression level of the transgene. These serious phenotypes probably result from the constitutive transactivating function of the truncated ARR1, independent of a signal from an upstream component. The phenomena, such as shorter



roots, ectopic shoot formation, and disordered cell division, suggest the occurrence of hypercytokinin responses. This has been verified by characterization of an ARR1 mutant and by demonstration that the cytokinin sensitivity of plants is correlated to the expression level of ARR1 (SAKAI et al. 2001). Overexpression of ARR2 shows a delayed leaf senescence phenotype (HWANG and SHEEN 2001). Definitive evidence for the involvement of B-type ARRs in cytokinin signaling has been provided by using, as molecular markers, the A-type ARR genes, expression of which is induced by cytokinin without protein synthesis (see below). Higher levels of expression of ARR1 lead to higher levels of induction of the A-type genes. Furthermore, artificial activation of ARR1 without de novo protein synthesis elevates the levels of A-type gene transcripts (SAKAI et al. 2001). Thus, ARR1 is a transcriptional activator for the A-type genes. All of these observations are consistent with the view that ARR1 and most B-type ARRs are activated by the cytokinin signal, presumably derived through the CRE1 sensor.

The A-type response regulator genes respond to cytokinin The A-type response regulators were identified both by their sequence similarity to known RR domains and by analysis of genes up-regulated upon cytokinin treatment, although no such upregulation was found for the B-type ARR genes. Subsequent studies revealed that exogenous cytokinin up-regulates the transcript levels of all the A-type genes to a greater or lesser degree depending on the gene (D'AGOSTINO et al. 2000). This up-regulation occurs without de novo protein synthesis, indicating that A-type genes are the genes primarily responsive to cytokinin. Therefore, the A-type ARRs appear to be effectors acting downstream from cytokinin signal transduction. Among the A-type genes, ARR4 has been studied most extensively. This gene product can be associated with the AtDBP1 and AtDBP2 DNA-binding proteins, and also with PhyB. The binding of phospho-ARR4 to the PhyB N-terminal portion stabilizes its active Pfr form, leading to elevation of red light sensitivity (SWEERE et al. 2001). ARR4 thus appears to modulate light signaling by PhyB, as an effector of the cytokinin signal. The expression patterns of the A-type genes significantly differ from one another, although all of these genes are expressed in all tissues. Their intracellular localization patterns are also different: some occur exclusively in the nucleus, while others have been detected in both the cytoplasm and the nucleus. These variations in the expression and intracellular localization patterns of the A-type ARRs may reflect their possible diverse functions as effectors of cytokinin signal transduction.

The AHP (HPt) proteins bridge the cytokinin signal from CRE1 to ARR1 The B-type ARRs are transcription factors that are activated by cytokinin in plant cells, while CRE1 together with its relatives are cytokinin sensors. Expression of the truncated ARR1 missing


Atsuhiro OKA

the RR domain in the wol mutant suppresses the wol phenotype, implying that ARR1 and probably also the other B-type members are actually located downstream of CRE1 in the cytokinin signaling cascade (Fig. 3). CRE1 is presumably anchored at the plasma membrane, whereas ARR1 is always localized in the nucleus, thereby preventing any direct association. Their functional relationship seems to be mediated by the HPt proteins encoded by the five AHP genes (SUZUKI et al. 2000), as (i) AHPs can potentially associate directly with several B-type and A-type ARRs, including ARR1, and some histidine kinases, such as ETR1 and CKI1, and (ii) hetero-complementation by CRE1 in yeast and eubacteria requires the respective HPt components. Although AHP molecules are small enough to diffuse through the nuclear pore complex, they are localized mainly in the cytoplasmic compartment. Some fractions might be translocated to the nucleus transiently upon cytokinin treatment. The five AHP genes have different expression patterns, which are not influenced by exogenous cytokinin. These gene products actually function as HPt factors in phosphorelay because (i) they act as substitutes for Ypd1, but interfere with the ability of CRE1 to complement the Sln1 defect in the yeast hetero-complementation system, and (ii) they have the potential to deliver the phosphoryl group to both B- and A-type ARRs under specific in vitro conditions. Based on this circumstantial evidence and analogies with bacterial systems, it is reasonable to assume that

Fig. 3. Framework of intracellular cytokinin signal transduction pathway in Arabidopsis. The hybrid-type sensor histidine kinases, CRE1, AHK2, and AHK3, perceive cytokinin and phosphorylate their own conserved histidine residues. The phosphoryl group is transferred to HPt factors (AHPs) via the C-terminal RR domain of the sensor histidine kinases. AHPs carrying the phosphoryl group move into the nucleus and transfer it to ARR1 and other B-type response regulators. ARR1 transactivates cytokinin-responsive genes including the A-type response regulator genes.



the cytokinin signal flows from CRE1 to ARR1 via AHP as the following phosphotransfer: cytokinin stimulus>CRE1 (His)>CRE1 (Asp)>AHP (His)>B-type ARR (Asp)>transactivation of Atype ARRs>cytokinin responses. It is unclear whether the A-type ARRs are directly involved in the cytokinin-dependent phosphorelay from CRE1, although they may compete with the B-type ARRs, negatively regulating the phosphorelay from CRE1 to B-type ARRs.

Two-component regulatory systems for ethylene, osmosis, and light signaling Ethylene is a well-known phytohormone that modulates a wide range of physiological actions, including apoptosis of leaves and flowers, fruit ripening, germination, and defense responses. Many ethylene-insensitive mutants have been isolated by screening for alterations in these attributes. Among the causative agents, the ETR1, ETR2, and EIN4 genes, encode the hybrid-type histidine kinase, whereas their two homologs ERS1 and ERS2, which were identified based on similarities to ETR1 and EIN4, encode prototypal histidine kinases. From the Arabidopsis genome sequence, we know that there is no additional gene encoding ethylene-related histidine kinases. As ETR1 binds to ethylene, these kinases function as ethylene receptors. Although the individual abolition of each of these five genes generally results in slight phenotypic changes, simultaneous alterations in three or more of the five genes result in constitutive ethylene responses (HUA and MEYEROWITZ 1998). Therefore, these gene products overlap functionally, and negatively control the downstream pathway for ethylene responses. CTR1, which interacts directly with ETR1 and resembles Raf1 (a member of the MAPKKK family), plays a leading role in negative regulation, because CTR1 mutants show constitutive ethylene responses. CTR1 is activated by ETR1 in the absence of ethylene to block the downstream pathway for ethylene responses, whereas CTR1 remains inactive in the presence of ethylene, thereby relieving the negative regulation of the ethylene responses. This regulatory process is similar to that of Sln1 in modulating Ssk2 MAPKKK through Ypd1 and Ssk1. No biochemical or genetic connections have been established between the ethylene sensors and the downstream HPt proteins and response regulators. As the ethylene sensors, at least ETR1, bear the enzyme activity of histidine kinase (probably in the absence of ethylene) and thus may also deliver the phosphoryl group to HPts and then to ARRs, they seem to be able to attend cytokinin signaling through the pool of five AHPs and eleven B-type ARRs (and possibly eleven A-type ARRs), generating cross-talk between ethylene and cytokinin signaling (Fig. 4). This cross-talk appears consistent with the observation that cytokinin and ethylene have roughly opposite physiological roles. Cytokinin, as a hormone involved in vitality, stimulates cell division, whereas ethylene provokes maturation and senescence and represents a hormone for aging and apoptosis. Among the eleven sensor histidine kinases of Arabidopsis, AHK1 was initially considered an osmosensor, based on both its relatively high degree of homology to the yeast osmosensor Sln1 and its potential to substitute for Sln1 in yeast cells. However, definitive evidence regarding the


Atsuhiro OKA

Fig. 4. Cross-talk between cytokinin, ethylene, and red light signal transduction systems through the pool of response regulators and HPt proteins in plant cells (green ellipse). Cytokinin signaling occurs synergistically with light signaling, but is counterbalanced by ethylene signaling. Ligand binding to AHK1, CKI1, and CKI2 might also initiate phosphorelay to AHPs and participate in the cross-talk.

role of AHK1 is currently lacking. As described above, CKI1 together with CKI2 missing the membrane-spanning regions were identified by altering cytokinin-dependence through activation tagging. Although there is no direct evidence that CKI1 and CKI2 participate in cytokinin recognition, they may contribute to cytokinin sensing in certain tissues and cells, e.g., CKI1 is expressed specifically in female gametes. There is no cognate response regulator corresponding to AHK1, CKI2, and CKI2, but they seem to have the histidine kinase activity, perhaps triggering phosphorelay to the pool of AHKs and ARRs in plant cells. Therefore, these three histidine kinases might also affect cytokinin signaling similarly to the ethylene sensors. In addition to these eleven sensor histidine kinases, five phytochromes (PHYA to PHYE) belong, in a broad sense, to this category. However, the histidine kinase-like structure located in the C-terminus of the phytochromes is considerably diverged from the typical histidine kinase, and the histidine residue that corresponds to the phosphorylation site is missing. As mentioned above, ARR4 associates specifically with PhyB to preserve the active Pfr form for longer periods. Light signaling acts synergistically with cytokinin to produce multifarious physiological effects, such as chloroplast development. As the expression of ARR4, as well as the other A-type ARR genes, is induced by cytokinin through the CRE1-ARR1 phosphorelay, it is likely that cytokinins indirectly enhance red light signaling. Therefore, light signaling is another participant in the cross-talk that occurs between the cytokinin and ethylene signal transduction systems (Fig. 4).

Summary This review has presented a framework for the Arabidopsis two-component regulatory systems that are involved in the intracellular transduction of signals from cytokinin, ethylene, and light. With regard to cytokinin signal transduction, cytokinin recognition by CRE1, AHK2, and AHK3 is followed by a phosphorelay signal transfer that involves AHPs and B-type ARRs (Fig. 3). The organization of this system, the structural characteristics of the component proteins, and their



molecular activities resemble those of bacterial systems, suggesting that the His-Asp phosphorelay signal transduction system is a powerful device in plat cells for a variety of environmental stress responses. Meanwhile, characteristics that are not seen in prokaryotes have been elucidated for plants. Three sensor kinases, five AHPs, and multiple B-type ARRs (probably almost all of the Btype ARRs) are involved in cytokinin signal transduction and transactivate multiple A-type ARR genes. In prokaryotes, a histidine kinase generally partners with a cognate response regulator in a specific one-to-one manner, and phosphorelay swapping among multiple molecular species is rare. Furthermore, there is no equivalent of ETR1 in prokaryotes, for which a partner response regulator is absent. Although the elements of the two-component regulatory systems of plants do not differ markedly from those of bacteria, the plant systems collectively employ complex cross-talk networks to govern different adaptive responses.


AOYAMA, T. and OKA, A. (2003). Cytokinin signal transduction in plant cells. J. Plant Res. 116: 221-223. CHANG, C., KWOK, S.F., BLEECKER, A.B. and MEYEROWITZ, E.M. (1993). Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators. Science 262: 539-544. D'AGOSTINO, I.B., DERUERE, J. and KIEBER, J.J. (2000). Characterization of the response of the Arabidopsis response regulator gene family to cytokinin. Plant Physiol. 124: 1706-1717. HABERER, G. and KIEBER, J.J. (2002). Cytokinins. New insights into a classic phytohormone. Plant Physiol. 128: 354-362. HOSODA, K., IMAMURA, A., KATOH, E., HATTA, T., TACHIKI, M., YAMADA, H., MIZUNO, T. and YAMAZAKI, T. (2002). Molecular structure of the GARP family of plant Myb-related DNA binding motifs of the Arabidopsis response regulators. Plant Cell 14: 2015-2029. HUA, J. and MEYEROWITZ, E.M. (1998). Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94: 261-271. HWANG, I., CHEN, H.C. and SHEEN, J. (2002). Two-component signal transduction pathways in Arabidopsis. Plant Physiol. 129: 500-515. HWANG, I. and SHEEN, J. (2001). Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413: 383-389. IMAMURA, A., HANAKI, N., UMEDA, H., NAKAMURA, A., SUZUKI, T., UEGUCHI, C. and MIZUNO, T. (1998). Response regulators implicated in His-to-Asp phosphotransfer signaling in Arabidopsis. Proc. Natl. Acad. Sci. USA 95: 2691-2696. INOUE, T., HIGUCHI, M., HASHIMOTO, Y., SEKI, M., KOBAYASHI, M., KATO, T., TABATA, S., SHINOZAKI, K. and KAKIMOTO, T. (2001). Identification of CRE1 as a cytokinin receptor from Arabidopsis. Nature 409: 1060-1063. KAKIMOTO, T. (1996). CKI1, a histidine kinase homolog implicated in cytokinin signal transduction. Science 274: 982-985. MÄHÖNEN, A.P., BONKE, M., KAUPPINEN, L., RIIKONEN, M., BENFEY, P.N. and HELARIUTTA, Y. (2000). A novel two-component hybrid molecule regulates vascular morphogenesis of the Arabidopsis root. Genes Dev. 14: 2938-2943. MOK, D.W.S. and MOK, M.C. (2001). Cytokinin metabolism and action. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 89-118. OKA, A. (2003). New insights into cytokinins. J. Plant Res. 116: 217-220.


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OKA, A., SAKAI, H. and IWAKOSHI, S. (2002). His-Asp phosphorelay signal transduction in higher plants: receptors and response regulators for cytokinin signaling in Arabidopsis thaliana. Genes Genet. Sys. 77: 383-391. OTA, I.M. and VARSHAVSKY, A. (1993). A yeast protein similar to bacterial two-component regulators. Science 262: 566-569. POSAS, F. and SAITO, H. (1998). Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 twocomponent response regulator. EMBO J. 17: 1385-1394. SAKAI, H., AOYAMA, T., BONO, H. and OKA, A. (1998). Two-component response regulators from Arabidopsis thaliana contain a putative DNA-binding motif. Plant Cell Physiol. 39: 1232-1239. SAKAI, H., AOYAMA, T. and OKA, A. (2000). Arabidopsis ARR1 and ARR2 response regulators operate as transcriptional activators. Plant J. 24: 703-711. SAKAI, H., HONMA, T., AOYAMA, T., SATO, S., KATO, T., TABATA, S. and OKA, A. (2001). ARR1, a transcription factor for genes immediately responsive to cytokinins. Science 294: 1519-1521. SCHALLER, G.E. and BLEECKER, A.B. (1995). Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene. Science 270: 1809-1811. SCHNEIDER-POETSCH, H.A.W. (1992). Signal transduction by phytochrome: phytochromes have a module related to the transmitter modules of bacterial sensor proteins. Photochem. Photobiol. 56: 839-846. STOCK, A.M., ROBINSON, V.L. and GOUDREAU, P.N. (2000). Two-component signal transduction. Annu. Rev. Biochem. 69: 183-215. SUZUKI, T., MIWA, K., ISHIKAWA, K., YAMADA, H., AIBA, H. and MIZUNO, T. (2001). The Arabidopsis sensor His-kinase, AHK4, can respond to cytokinins. Plant Cell Physiol. 42: 107-113. SUZUKI, T., SAKURAI, K., IMAMURA, A., NAKAMURA, A., UEGUCHI, C. and MIZUNO, T. (2000). Compilation and characterization of histidine-containing phosphotransmitters implicated in His-to-Asp phosphorelay in plants: AHP signal transducers of Arabidopsis thaliana. Biosci. Biotechnol. Biochem. 64: 2486-2489. SWEERE, U., EICHENBERG, K., LOHRMANN, J., MIRA-RODADO, V., BÄURLE, I., KUDLA, J., NAGY, F., SCHÄFER, E. and HARTER, K. (2001). Interaction of the response regulator ARR4 with phytochrome B in modulating red light signaling. Science 294: 1108-1111. THE ARABIDOPSIS GENOME INITIATIVE (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796-815. UEGUCHI, C., SATO, S., KATO, T. and TABATA, S. (2001). The AHK4 gene involved in the cytokinin-signaling pathway as a direct receptor molecule in Arabidopsis thaliana. Plant Cell Physiol. 42: 751-755. WURGLER-MURPHY, S.M. and SAITO, H. (1997). Two-component signal transducers and MAPK cascades. Trends Biochem. Sci. 22: 172-176. YAMADA, H., SUZUKI, T., TERADA, K., TAKEI, K., ISHIKAWA, K., MIWA, K., YAMASHINO, T. and MIZUNO, T. (2001). The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant Cell Physiol. 42: 1017-1023.






3 ii Sln1

iii CRE1 CRE1



Atsuhiro OKA









30 30 1 2 3 A 11 11 6 1

Gamma Field Symposia, No. 42, 2003 Institute of Radiation Breeding NIAS, Japan




Takashi HIRAYAMA 1), 2) and Tsutomu UGAJIN 2)


Laboratory of Plant Molecular Biology, Yokohama Institute, RIKEN 2) Graduate School of Integrated Science, Yokohama City Univ. 1-7-29, Suehiro, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan

Introduction The gaseous plant hormone ethylene is involved in a variety of growth and developmental processes including germination, cell elongation, flower and leaf senescence, sex determination and fruit ripening. To gain insight into the molecular mechanisms of ethylene action, a molecular genetic approach has been applied using the ethylene-evoked triple response phenotype of Arabidopsis seedlings. Analysis of those ethylene related mutants allowed us to draw the overall structure of the ethylene signal transduction pathway, from ethylene perception to nuclear events. Based on the similarity of the sensor proteins, the ethylene signal transduction pathway has been thought to be similar to the yeast osmosensing signaling pathway. However, recent studies have revealed that the ethylene pathway is unique and quite complicated. Although the ethylenesignaling pathway is most understood among the signal transduction pathways for plant hormones, there are still many matters to be clarified. Here, we are going to describe the recent studies on the ethylene-signaling components and discuss the possible model for the ethylene-signaling pathway.

Isolation of ethylene related mutants A gaseous hormone ethylene is involved in diverse developmental and physiological processes of plants. Treatment of etiolated seedlings with ethylene evokes dramatic morphological changes referred to as the "triple response" that includes exaggerated apical hook, radial swelling of hypocotyl and inhibition of hypocotyl and root elongation in Arabidopsis. These morphological changes are highly specific for ethylene. A genetic approach that relies on the triple response phenotype as a morphological marker has allowed the identification of several classes of mutants with impaired responses to ethylene (BLEECKER et al. 1988; GUZMAN and ECKER 1990). These mutants can be classified into several groups; ethylene insensitive mutant, etr1, etr2, ein2, ein3, ein4, ein5/ain1 and ein6; constitutive ethylene response mutant, eto1, 2, 3 and ctr1; and tissue-


Takashi HIRAYAMA and Tsutomu UGAJIN

specific ethylene response mutant hls1 and eir1; enhanced ethylene sensitive mutant, eer1; finally, altered ethylene recognition mutant, ran1. Based on the results from extensive genetic studies with these mutants, a model has been drawn for the ethylene-signaling pathway, in which identified components act in a linear pathway (Figure 1) (GUZMAN and ECKER 1990). Isolation of the corresponding genes and molecular analysis of encoded proteins have greatly facilitated our understanding of the ethylene-signaling pathway at the molecular level.

Ethylene receptors Based on the genomic sequence, Arabidopsis has five ethylene receptors, namely, ETR1, ERS1, ETR2, EIN4 and ERS2. Structure of ethylene receptor protein is reminiscent of membranespanning histidine kinase. The three N-terminal membrane-spanning domains are necessary and sufficient for ethylene binding. This region is highly conserved among ethylene receptors. Analysis of mutants such as etr1-1 and knock-out mutants have shown that ethylene receptors negatively regulate down stream components (HUA and MEYEROWITZ 1998). In the absent of ethylene molecule, ethylene receptors are active and inhibit the ethylene response. On the other hands, in the presence of ethylene, ethylene receptors become inactive and let downstream ethylene-

Figure 1 The ethylene-signaling pathway drawn based on the results obtained from genetic analyses. Genetically identified components involved in the ethylene response are shown. The allow heads indicate only the direction of the signal. RAN1 and EER1 function as modifier for ethylene receptors and CTR1, respectively (see text).



signaling pathway turns on. However, as discussed below, the biochemical property of active ethylene receptor has not been elucidated yet. As ethylene is a tiny olefin molecule, it has been proposed that transition metals such as copper or zinc ion are required for ethylene recognition. Bleecker's group has demonstrated that copper (I) ion is required for ethylene binding, and that Cys65 and His69 residues in the second membrane spanning domain are required for copper coordination and ethylene binding activities using recombinant ETR1 proteins expressed in the budding yeast cell (RODRIGUEZ et al. 1999). Based on these results, they proposed a model for ethylene recognition in which two Cys65 residues and two His69 residues of ETR1 dimer coordinate with one copper (I) ion that is able to bind one ethylene molecule (Figure 2). These results, however, did not offer any idea on the linkage between copper requirement and ethylene receptor activity. It could be speculated at this time that ethylene receptor is active without copper ion since the conversion Cys65 to Tyr (etr1-1 mutation) confers an ethylene insensitive phenotype. It is the analysis of the ran1 mutants that offered in planta functional evidence for the copper requirement for both ethylene perception and the proper conformation of ethylene receptors (HIRAYAMA et al. 1999). The ran1 mutants were isolated in a screen for mutants with altered ethylene-recognition specificities aiming to gain insight into the mechanism of ethylene

Figure 2 A proposed model for ethylene recognition. Two ethylene receptor molecules constitute a functional ethylene receptor complex. Cys65 and His69 on the second membrane-spanning domain coordinate with one copper (I) ion that binds one ethylene molecule.


Takashi HIRAYAMA and Tsutomu UGAJIN

perception. The ran1 mutants show the ethylene phenotype in response to treatment with transcyclooctene, a potent ethylene binding inhibitor, which normally inhibits the ethylene response. The ran1 mutants seem to have a relaxed ligand-specificity since they respond normally to ethylene. The RAN1 gene has been identified by map based cloning and shown to encode a P-type copper transporter similar to human Menkes or Wilson disease protein, or yeast Ccc2p. These proteins have been demonstrated to deliver copper ions to the post Golgi compartment where copper requiring proteins are modified and sorted subsequently to the plasma membrane or outside of the cell, etc. These finding lead us the idea that RAN1 delivers copper ions to ethylene receptors. Several RAN1 co-suppressed transgenic lines and the null-type ran1 mutant (ran1-3) exhibit strong ethylene constitutive responding phenotypes (HIRAYAMA et al. 1999; WOESTE and KIEBER 2000). In the ran1-3 mutant, ethylene receptors are expressed at the same level as wild type (ZHAO et al. 2002). These results strongly suggest that ethylene receptors cannot function without copper delivery. However, this conclusion seems inconsistent with the etr1-1 phenotype. As discussed above the conversion Cys65 to Tyr confers an ethylene insensitive phenotype, while the defect in RAN1 results in the constitutive activation of the ethylene response. One possible and plausible explanation for this discrepancy is that Cys65 is required not only for copper coordination but also for the proper conformation of ethylene receptor. Presumably, ethylene receptor is locked at active state without Cys65. Since both of the ran1-1 and ran1-2 mutations are missense mutations, these mutated genes presumably express mutated copper transporters. These mutations cause the conversion of important amino acid residue for copper transporting activity to another residue, indicating the mutated copper transporters have reduced activities. Actually a recombinant ran1-1 protein has a reduced copper transporting activity in the yeast cells (HIRAYAMA et al. 1999). If one copper ion is incorporated in a functional ethylene receptor as Bleecker's group proposed, the ran1-1 or ran1-2 plant would have just two types of ethylene receptors, receptor with copper or without copper. However, having these two types of ethylene receptors cannot explain the relaxed ligand specificity of the ran1-1 and ran1-2 mutants. Determination of the fine structure of ethylene recognition domain is necessary. Although all the ethylene receptors have a His-kinase like domain, the amino acid sequences are different among them. ETR1 and ERS1 have all the motifs that are required for His-kinase activity, namely H, N, G1, F and G2 motifs. By contrast, ETR2, EIN4 and ERS2 lack some or all of them, indicating these His-kinase like domains do not have His-kinase activity. Recently, Wang et al. reported that the physiological roles of His-kinase accompanied receptors, ETR1, ERS1, are different from those of ETR2, EIN4 and ERS2, and that their His-kinase activities are not required for the ethylene response (WANG et al. 2003). Furthermore, NTHK1, a tobacco ethylene receptor, was shown to have Ser/Thr kinase activity (XIE et al. 2003). Based on these reports, it is more unlikely that the ethylene-signaling pathway belongs to His-Asp phospho-relay signaling pathway.



The fact that the ethylene-signaling pathway does not require the His-kinase activity of ethylene receptors raises another question why ETR1 and ERS1 possess His-kinase activity. It is possible that the His-kinase activities of those ethylene receptors are required for functions other than the ethylene-signaling pathway. Arabidopsis has several His-kinases although their physiological roles are not fully elucidated. A cytokinin receptor, CRE1, is one of the histidine kinase signal transducers whose physiological functions have been assigned. Recent studies have demonstrated that the cytokinin signal was transduced from CRE1 to nucleus by phospho-relay components (SAKAI et al. 2001). Some of those components have the ability to interact with ETR1 (URAO et al. 2000). It might be possible that the His-kinase activity of ETR1 or ERS1 is involved in the crosstalk between the signal transduction pathways for ethylene and other stimuli or plant hormones such as cytokinin. Since ethylene molecules can pass freely through the lipid bilayer membrane, ethylene receptors do not need to localize to the plasma membrane. Schaller's group showed that ethylene receptors localized to the membrane of endoplasmic reticulum (CHEN et al. 2002). By contrast, Xie et al. demonstrated that a tobacco ethylene receptor, NTHK1, fused to green fluorescent protein localized to the plasma membrane (XIE et al. 2003). Further analysis is required for the determination of the subcellular localization of ethylene receptors.

Signaling components from receptor to nucleus How do the ethylene receptors transduce the signal downstream? The genetically identified component is CTR1, a Raf-type Ser/Thr kinase. Loss-of-function mutations of CTR1 cause recessive ethylene constitutive response phenotypes, suggesting CTR1 functions as a negative regulator like ethylene receptors. Thus ethylene receptors presumably activate CTR1. Two-hybrid analysis using budding yeast revealed that CTR1 interacted with ETR1 and ERS1 (CLARK et al. 1998). Recently, one of the mutant alleles, ctr1-8, turned out to be defective in the association with ETR1, providing in vivo evidence for the requirement of physical interaction between them for the CTR1 function (HUANG et al. 2003). Furthermore, a recent study showed that ETR1 and CTR1 colocalized to ER membrane, supporting this idea (GAO et al. 2003). Raf-1 in mammal cells functions as a MAP-kinase-kinase-kinase. It has been postulated that CTR1 is one of the components of a MAP kinase cascade that is responsible for the ethylenesignaling pathway. Ouaked et al. reported that a sort of MAP-Kinase-kinase and MAP-kinase were activated upon ethylene treatment in a CTR1-dependent manner (OUAKED et al. 2003). The authors reported overexpression of this MAPKK induced the triple response phenotype in the absence of ethylene. These kinases might constitute a MAP kinase cascade with CTR1. However these kinases are activated by ethylene while CTR1 is inactivated. Such a regulation has not been reported in other MAP kinase systems so far. Although the ctr1 mutants exhibit constitutive ethylene phenotypes, treatment with ethylene


Takashi HIRAYAMA and Tsutomu UGAJIN

enhances ethylene phenotypes. This observation suggests the existence of other CTR1 like components. There are several CTR1 like genes on the Arabidopsis genome. Although the physiological functions of those are not known yet, these kinases might function in the ethylenesignaling pathway. Recently, Larsen and Chang reported another ethylene related mutant, eer1. This mutant shows enhanced ethylene response phenotypes (LARSEN and CHANG 2001). The ctr1 eer1 double mutant exhibits stronger constitutive ethylene response phenotypes. The EER1 gene has been cloned and shown to be identical to RCN1 that encodes an A subunit of protein phosphatase 2A (PP2A) (LARSEN and CANCEL 2003). RCN1 has been reported to be involved in the responses to auxin and abscisic acid in root and in guard cell, respectively. In mammal cells, PP2A positively regulates Raf-1 and shown to interact with Raf-1 kinase directly (ABRAHAM et al. 2000). Actually, not RCN1 but a C subunit of PP2A, PP2A-1C, can interact with the N-terminal domain of CTR1 in vitro. It might be possible that RCN1/EER1 and/or other PP2As are involved in the regulation of CTR1 activity. The ein2 mutants have a semi-dominant strong ethylene insensitive phenotype, suggesting its important function in the ethylene-signaling pathway. However, little is known about EIN2. The EIN2 gene encodes a novel membrane-spanning protein (ALONSO et al. 1999). EIN2 has twelve putative membrane-spanning domains in its N-terminal half. This region has a significant similarity to Nramp divalent cation transporters. However, there is no evidence for the transporter activity of EIN2. In addition, two residues that have been shown to be required for the transporting activity of yeast Smf1p are not conserved in EIN2, suggesting that EIN2 does not have such an iontransporting activity. Although EIN2 must localize to membrane structures because of these membrane-spanning domains, the subcellular localization of EIN2 has not been determined yet. The C-terminal half seems to be a cytoplasmic domain. The function of this region is also not clarified yet since this region does not have any known motifs. However, overexpression of the Cterminal half confers constitutive ethylene response phenotypes in the absence of ethylene, suggesting that this region has a pivotal role in the activation of the down stream signal transducer. The mutation sites of dozens of the ein2 mutants have been determined. All of them except one (ein2-9) are non-sense mutations or frame-sift mutations. Given that the C-terminal domain is required for the EIN2 function, these mutant EIN2 proteins cannot activate the downstream component(s) and result in the ethylene insensitive phenotype. ein2-9 is a missense mutation that causes an amino acid conversion His1143 to Pro in the C-terminal domain. Since this residue is not conserved in the rice EIN2 homologue, it is postulated that this His residue does not have a specific function but the conversion from this His residue to Pro might disturb the functional structure of the C-terminal region. The relationship between EIN2 and Nramp seems similar to that of yeast glucose transporters (HXTs) and glucose sensors (Snf3p and Rgt2p). Snf3p and Rgt2p have a structure similar to HXTs at their N-terminal regions and a unique C-terminal cytoplasmic domain. At the beginning,



Snf3p and Rgt2p had been thought to be a kind of glucose transporter because of the similarity in their N-terminal domain. Detailed studies of those proteins have revealed that these proteins function as not glucose transporters but glucose sensors, and that the C-terminal domains of those proteins have the ability to function as a signal transducer. Overexpression of the C-terminal domain of those proteins in yeast cells activates the glucose response. Analogy to this relationship, it can be postulated that EIN2 might be a sensor for something, for example a divalent cation. So far, no missense mutation in the N-terminal region has been reported. To address this possibility, we generated dozens of the mutated ein2 genes that have an insertion of five-amino-acid in the Nterminal region. Preliminary results indicate that the N-terminal region of EIN2 is also required for the ethylene response (Ugajin and Hirayama, unpublished data). Detailed analysis of this region will reveal the EIN2 function in the ethylene response. The identification of the EIN2 function is necessary for the understanding of the ethylene-signaling pathway.

Nuclear events Ethylene treatment induces the expression of a lot of genes. The inducible expression of such genes evokes the ethylene response. The known downstream component of EIN2 is EIN3. Defect in the EIN3 gene confers a weak ethylene insensitive phenotype. The EIN3 gene encodes a novel protein. Although it does not have any known motifs, the amino acid composition of EIN3 implies its nuclear function (CHAO et al. 1997). Actually, the EIN3-GUS fusion protein expressed transiently in Arabidopsis cells localized to nucleus. It has been reported that ethylene inducible genes has a cis-element called GCC-box in their promoter regions. A screen for Arabidopsis proteins that bind GCC-box sequence identified EREBP, an AP2 transcriptional factor, functioning in the gene activation by ethylene (OHME-TAKAGI and SHINSHI 1995). EIN3 does not have an AP2like structure and the ability to bind GCC-box sequence. The level of EIN3 mRNA is not affected by ethylene treatment. Therefore, Solano et al. thought that early ethylene inducible genes could be candidates for EIN3 target, and tried to find such genes. They found that a gene encoding an EREBP-like protein, ERF1, was induced very quickly by ethylene treatment in an EIN3-dependent manner (SOLANO et al. 1998). In addition, a recombinant EIN3 protein had the ability to bind the promoter region of ERF1. Based on these results, they concluded that EIN3 activates the ERF1 gene and produced ERF1 in turn activates ethylene inducible genes through GCC-box. Overexpression of ERF1 in the ein3 mutant induced the ethylene constitutive phenotypes, confirming their idea. The mechanisms for EIN3 activation have not been clarified yet. Arabidopsis has several EIN3-like genes. Among them, at least EIL1 and EIL2 have similar function since overexpression of EIL1 or EIL2 suppresses the ein3 mutant phenotype. The eil1 mutant exhibits a very weak ethylene insensitive phenotype. Interestingly, the ein3 eil1 double mutant shows a strong ethylene insensitive phenotype similar to ein2, suggesting that the functions of EIN3 and EIL1 in the ethylene-signaling pathway are largely overlapping (ALONSO et al. 2003).


Takashi HIRAYAMA and Tsutomu UGAJIN

This idea is consistent with the weak ethylene insensitive phenotype of the ein3 mutants.

Future perspective As described above, the over all structure of the ethylene-signaling pathway, from the perception to the gene expression, has been elucidated through the studies using Arabidopsis (Figure 3). Orthologues for Arabidopsis ethylene-signaling components have been found in other plant systems, including tomato, maize and rice. Therefore the ethylene-signaling pathway described here presumably is common in large parts in plant kingdom.

Figure 3 Schematic representation of a model for the ethylene-signaling pathway. Ethylene receptor binds a copper ion presumably during protein modification in the Golgi apparatus. Copper ion is delivered by RAN1 copper transporter. Ethylene receptor seems to localize to the ER membrane although this is still controversial. The subcellular localization of EIN2 is not elucidated yet. EIN2 might sense some signals although there is no evidence. EIN3 functions in the nuclear although it is not clear if EIN3 localizes to the nuclear in the absence of ethylene or not. In the absence of ethylene, ethylene receptor and CTR1 are active and presumably inhibit EIN2. When ethylene receptor recognizes ethylene molecule, it turns off and consequently allows EIN2 to activate EIN3 somehow. Activated EIN3 induces the expressions of the ERF1 gene and some ethylene responsive genes (not illustrated). Produced ERF1, a transcriptional factor, in turn activates ethylene responsive genes.



In spite of amount of efforts made by many researchers world wide, there are still many unsolved issues. For example, how do ethylene receptors activate down stream signaling molecules?, what is the CTR1 substrate?, what is the EIN2 function?, how is EIN3 regulated?, how is EIN5 or EIN6 involved in the ethylene-signaling pathway?, etc. Additional genetic studies, such as the isolation of suppressor mutants for existing mutants, may be required to identify new components. Furthermore, describing the biochemical properties, cellular functions and threedimensional structures of known components is necessary for understanding the ethylene-signaling pathway.


1. ABRAHAM, D., PODAR, K., PACHER, M., KUBICEK, M., WELZEL, N., HEMMINGS, B.A., DILWORTH, S.M., MISCHAK, H., KOLCH, W. and BACCARINI, M. (2000). Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation. J Biol Chem. 275: 22300-22304. 2. ALONSO, J.M., HIRAYAMA, T., ROMAN, G., NOURIZADEH, S. and ECKER, J.R. (1999). EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science. 284: 2148-2152. 3. ALONSO, J.M., STEPANOVA, A.N., SOLANO, R., WISMAN, E., FERRARI, S., AUSUBEL, F.M. and ECKER, J.R. (2003). Five components of the ethylene-response pathway identified in a screen for weak ethyleneinsensitive mutants in Arabidopsis. Proc Natl Acad Sci U S A. 100: 2992-2297. 4. BLEECKER, A.B., ESTELLE, M.A., SOMERVILLE, C. and KENDE, H. (1988). Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science. 241: 1086-1089. 5. CHAO, Q., ROTHENBERG, M., SOLAN, R., ROMAN, G., TERZAGHI, W. and ECKER, J.R. (1997). Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell. 89: 1133-1144. 6. CHEN, Y.F., RANDLETT, M.D., FINDELL, J.L. and SCHALLER, G.E. (2002). Localization of the ethylene receptor ETR1 to the endoplasmic reticulum of Arabidopsis. Journal of Biological Chemistry. 277: 1986119866. 7. CLARK, K.L., LARSEN, P.B., WANG, X. and CHANG, C. (1998). Association of the Arabidopsis CTR1 Raflike kinase with the ETR1 and ERS ethylene receptors. Proc Natl Acad Sci U S A. 95: 5401-5406. 8. GAO, Z., CHEN, Y.F., RANDLETT, M.D., ZHAO, X.C., FINDELL, J.L., KIEBER, J.J. and SCHALLER, G.E. (2003). Localization of the Raf-like Kinase CTR1 to the Endoplasmic Reticulum of Arabidopsis through Participation in Ethylene Receptor Signaling Complexes. J Biol Chem. 278: 34725-34732. 9. GUZMAN, P. and ECKER, J.R. (1990). Exploiting the triple response of Arabidopsis to identify ethylenerelated mutants. Plant Cell. 2: 513-523. 10. HIRAYAMA, T., KIEBER, J.J., HIRAYAMA, N., KOGAN, M., GUZMAN, P., NOURIZADEH, S., ALONSO, J.M., DAILEY, W.P., DANCIS, A. and ECKER, J.R. (1999). RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis. Cell. 97: 383-393. 11. HUA, J. and MEYEROWITZ, E.M. (1998). Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell. 94: 261-271. 12. HUANG, Y., LI, H., HUTCHISON, C.E., LASKEY, J. and KIEBER, J.J. (2003). Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis. Plant Journal. 33: 221-233. 13. LARSEN, P.B. and CANCEL, J.D. (2003). Enhanced ethylene responsiveness in the Arabidopsis eer1 mutant results from a loss-of-function mutation in the protein phosphatase 2A A regulatory subunit, RCN1. Plant Journal. 34: 709-718.


Takashi HIRAYAMA and Tsutomu UGAJIN

14. LARSEN, P.B. and CHANG, C. (2001). The Arabidopsis eer1 mutant has enhanced ethylene responses in the hypocotyl and stem. Plant Physiology. 125: 1061-1073. 15. OHME-TAKAGI, M. and SHINSHI, H. (1995). Ethylene-inducible DNA-binding proteins that interact with an ethylene-responsive element. Plant Cell. 7: 173-182. 16. OUAKED, F., ROZHON, W., LECOURIEUX, D. and HIRT, H. (2003). A MAPK pathway mediates ethylene signaling. The EMBO Journal. 22: 1282-1288. 17. RODRIGUEZ, F.I., ESCH, J.J., HALL, A.E., BINDER, B.M., SCHALLER, G.E. and BLEECKER, A.B. (1999). A copper cofactor for the ethylene receptor ETR1 from Arabidopsis. Science. 283: 996-998. 18. SAKAI, H., HONMA, T., AOYAMA, T., SATO, S., KATO, T., TABATA, S. and OKA, A. (2001). ARR1, a transcription factor for genes immediately responsive to cytokinins. Science. 294: 1519-1521. 19. SOLANO, R., STEPANOVA, A., CHAO, Q.M. and ECKER, J.R. (1998). Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSEFACTOR1. Genes & Development. 12: 3703-3714. 20. URAO, T., MIYATA, S., YAMAGUCHI-SHINOZAKI, K. and SHINOZAKI, K. (2000). Possible His to Asp phosphorelay signaling in an Arabidopsis two-component system. FEBS Letters. 478: 227-232. 21. WANG, W., HALL, A.E., O'MALLEY, R. and BLEECKER, A.B. (2003). Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission. Proc Natl Acad Sci USA. 100: 352-357. 22. WOESTE, K.E. and KIEBER, J.J. (2000). A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype. Plant Cell. 12: 443-455. 23. XIE, C., ZHANG, J.S., ZHOU, H.L., LI, J., ZHANG, Z.G., WANG, D.W. and CHEN, S.Y. (2003). Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco. Plant Journal. 33: 385-393. 24. ZHAO, X.-C., QU, X., MATHEWS, D.E. and SCHALLER, G.E. (2002). Effect of ethylene pathway mutations upon expression of the ethylene receptor ETR1 from Arabidopsis. Plant Physiology. 130: 1983-1991.



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52 RAN1

Takashi HIRAYAMA and Tsutomu UGAJIN


etr1 EIN3 EIN2

Gamma Field Symposia, No. 42, 2003 Institute of Radiation Breeding NIAS, Japan




Takeshi NAKANO, Shigeo YOSHIDA and Tadao ASAMI Plant Functions Lab., RIKEN 2-1 Hirosawa, Wako 351-0198

Introduction Steroid hormones, compounds with a signature tetracyclic structure, are synthesized downstream of the isoprenoid pathway in many organisms. In animals, steroid hormones regulate embryonic and adult cell development, and also act on neurons, heart tissue, sperm, oocytes and other organs and cells. In insects, the steroid hormone ecdysone also controls metamorphosis and reproduction. Brassinolide is the most bioactive form in all plant steroids and the first purification and determination of the structure was done in using of chemicals from bee-collected rape pollen. Brassinosteroids, plant steroid hormones that are widely distributed throughout the plant kingdom, have biological effects on many plant growth processes, such as stem and pollen tube elongation, leaf development, and xylem development. Brassinosteroids can also regulate chloroplast that is closely associated with the unique plant organelle. As these steroids can regulate each organism specific organ development, steroids are interesting in the aspect of molecular evolution (Clouse, 2001, Fujioka and Yokota, 2003).

Brassinosteroid biosynthesis In the past decade, Arabidopsis brassinosteroid biosynthetic mutants such as det2 (Li et al., 1996), dwf4 (Choe et al., 1998) and cpd (Szekeres et al., 1996) had been identified and characterized (Fig. 1). Before the screening of these mutants, the importance of brassinosteroid for plant growth had not been confirmed yet. Brassinosteroid feeding for plant could not cause so drastic change on plant phenotype and some visible effects might be thought as similar to other known phytohormones. But, these brassinosteroid-deficient mutants have a pleiotropic dwarf phenotype with very short stem, shorten and waving leaves, and dark greened leaves. Gibberellin deficient mutants were known to similar to the dwarf phenotype, but these brassinosteroid-deficient mutants did not showed the germination inhibition and the late flowering that were observed in gibberellin deficient mutants. Then, brassinosteroids could be identified as special and unique bioactivity in comparison with another known phytohormones, and had participated in actual


Takeshi NAKANO, Shigeo YOSHIDA and Tadao ASAMI

Fig. 1 Brassinosteroid biosynthesis pathway.

Arabidopsis mutants such as det2, dwf4 and cpd clarified the detail brassinosteroid biosynthetic pathway. One possible target of Brz is the cytochrome P450 enzyme encoded by DWF4.

phytohormone group.

Brz, Brassinosteroid biosynthesis inhibitor, revealed brassinosteroid functions in plant growth The recently-synthesized compound brassinazole (Brz or Brz220) was the first specific inhibitor of brassinosteroid biosynthesis to be discovered. One possible target of Brz220 is the cytochrome P450 enzyme encoded by DWF4 (Asami et al., 2001) (Fig. 1). Brz220 treatment of



plants under dark conditions causes phenotypes similar to those displayed by brassinosteroiddeficient mutants, such as shortened hypocotyl, cotyledon opening, and de-etiolation. In the light, plants treated with the Brz displayed dwarf and highly greened leaves (Asami et al., 2000, Asami and Yoshida, 1999). In the stem tissues, development of the vascular bundle cells was inhibited by Brz. (Nagata et al., 2001) The Brz also revealed the regulation of chloroplast development by brassinosteroid. In the cotyledon of dark-germinated plant, the mRNA of chloroplast genes; i.e., cab and rbcS, did not expressed. But, in the cotyledon of dark-germinated plant with Brz, these chloroplast genes expression was detected (Asami et al., 2000, Nagata et al., 2000). After 2 hour light emission to these dark-germinated plant, the Brz-treated plant harbored very quick developed chloroplast that contained more numbered thylakoid membrane layer that Brz-untreated plant. These result revealed that plastid development was regulated brassinosteroid. (Fig. 2)

Brassinosteroid receper BRI1 The Arabidopsis bri1 mutant was also identified by its dwarf phenotype, but brassinosteroid treatment did not recover the dwarfism to wild-type phenotype and did not inhibit the elongation of the roots of this mutant (Clouse et al., 1996, Li and Chory, 1997) (Fig. 3). Study of bri1 revealed that BRI1 is a critical component in brassinosteroid signaling, and that mutation in its BRI1 gene

Fig. 2 Brz effect to the plant.

Brz treatment of plants under dark conditions causes photomorphogenesis such as shortened hypocotyl, cotyledon opening. In the light, plants treated with the Brz displayed dwarf and highly greened leaves. The Brz also revealed the regulation of chloroplast development by brassinosteroid.


Takeshi NAKANO, Shigeo YOSHIDA and Tadao ASAMI

Fig. 3 Brassinosteroid receptor mutant bri1.

The Arabidopsis bri1 mutant was identified by its dwarf phenotype. BRI1 is a member of the leucine-rich repeat (LRR) receptor kinase family.

causes a deficiency in brassinosteroids. BRI1 is a member of the leucine-rich repeat (LRR) receptor kinase family, and brassinolide binds strongly to a plasma membrane fraction purified using an anti-BRI1 antibody (Wang et al., 2001). In animal cells, steroid hormones are perceived through nuclear-localized steroid-binding proteins, but plants can perceive steroid hormones at the cell surface by the BRI1 component (Schumacher and Chory, 2000). How this signal is transduced to regulate plant nuclear gene expression is unknown.

Application of Brz (brassinosteroid biosyhthesis inhibitor) for screening of brassinosteroid signaling mutants Many research on molecular biological mechanism for plant growth has been performed using genetic methods in Arabidopsis. As initial steps in the study of the molecular genetics of a plant hormone, screens are conducted to identify phytohormone-deficient and phytohormone-insensitive mutants. These trials can identify a number of genes, but these genes are likely not all of the players in the regulation of plant growth by the phytohormone (Kende and Zeevaart, 1997). The next step to consider is a screen to identify suppressor mutants that repress phytohormone deficiency symptoms, as these mutants may be permanently activated in phytohormone signaling. In the gibberellin research field, rga was identified as a suppressor mutant of the ga1-3 gibberellin biosynthesis mutant , and the mutated gene was found to belong to the VHIID family (Silverstone et al., 2001). In addition, the spy mutant was identified on the basis of its resistance to the



gibberellin biosynthesis inhibitor paclobutrazol, and the gene was found to encode a homolog of Nacetylglucosamine transferase (Jacobsen et al., 1996). Research in the brassinosteroid signaling field is now proceeding to the second strategy for Arabidopsis mutant screening, using brassinosteroid-deficient mutants. In order to analyze in detail the mechanisms of brassinosteroid biosynthesis and signal transduction, we performed a screen for mutants with altered responses to Brz220 treatment in darkness in the germination stage (Fig. 4). A screen of 140,000 Arabidopsis seeds that had been subjected to EMS and fast neutron mutagenesis revealed several mutants that had significantly longer hypocotyls than the wild type when grown in the dark and treated with Brz220. These plants were designated bil mutants (Brz-insensitive-long hypocotyl).

bil1, Brz-insensitive-long hypocotyl1 Initially, we identified a dominant mutant, bil1-1D, from the EMS-treated lines. When grown in medium containing 3 µM Brz, wild-type plants had quite short hypocotyls, but bil1 mutants had hypocotyls as long as those of wild-type plants grown on unsupplemented medium (Fig. 5). In parallel, bzr1-1D and bes1-1D were identified as Brz-resistant and bri1-suppressor mutants, respectively (Fig. 5). Gene sequencing revealed that the bzr1-1D gene is the same gene as bil1-1D, even containing the same mutation (Wang et al., 2002). These genes are 88% identical to BES1,

Fig. 4 Strategy for new brassinosteroid signaling mutants by using of Brz.

Hopeful bil (Brz-insensitive-long hypocotyl) mutant can overcome the dwarf and shorten hypocotyl that are caused by brassinosteroid deficiency with Brz.


Takeshi NAKANO, Shigeo YOSHIDA and Tadao ASAMI

Fig. 5 Screened bil (Brz-insensitive-long hypocotyl) mutants of Arabidopsis.

bil1 mutants with 3 M Brz had hypocotyls as long as those of wild-type plants grown on unsupplemented medium.

and the bes1 mutant has the same nucleotide substitution (Yin et al., 2002). The plant-specific gene family encompassing BZR1, BES1 and BIL1 encodes novel phosphoproteins containing a putative nuclear localization signal. The BIL1/BZR1:CFP fusion protein localizes mainly to the cytoplasm and also to the nucleus at low levels, but treatment with brassinosteroids results in a significant increase of BIL1/BZR1:CFP levels in the nucleus within thirty minutes. In contrast, a BZR1:CFP protein containing the mutation localizes continuously to the nucleus (Wang et al., 2002). Then, the mutants felt that brassinosteroid signaling is on in every time, and these can showed wild-type like phenotype in brassinosteroid deficient condition. These results suggest that BIL1/BZR1 and BES1 is a key component in brassinosteroid signaling from the cell surface to the nucleus (Fig. 6). The phenotypes of the bzr1/bil1 and bes1 Brz-resistance and bri1-suppressor mutants, respectively, are very strong, with the resistant mutant just like wild-type plants in appearance, even though it is severely deficient in brassinosteroids. These mutants, the mutant alleles of which are both dominants, resulted from the substitution of just one amino acid as compared to the wild type. Overexpression of the BZR1 or BES1 wild-type genes via the CaMV 35S promoter resulted in only weak resistance against brassinosteroid deficiency (Wang et al., 2002, Yin et al., 2002). These results suggest that bzr1/bil1 and bes1 mutants would be difficult to identify from activationtagged pools of plants in the background of a mutant deficient in brassinosteroids, such as det2 or dwf4. An alternative method is to induce point mutations by chemical treatment of brassinosteroiddeficient mutants. The most widely-used method to identify point mutations is genomic walking, performed using backcrosses with another ecotype. The screening is best performed on recombinant F2 plants, to allow identification of the brassinosteroid-deficiency mutation as a



Fig. 6 Mechanism of brassinosteroid signaling conducted by BIL1/BZR1/BES1 protein.

Treatment with brassinosteroids results in movement of BIL1/BZR1/BES1 from cytosol to nucleus. BIL1/BZR1/BES1 protein containing the mutation localizes continuously to the nucleus.

homozygous det2/det2 plant, and not as det2/DET2 or DET2/DET2 plants. This is because distinguishing between putative brassinosteroid-signaling mutants with a det2/det2 homozygous background and det2/DET2 or DET2/DET2 plants with no mutation is very difficult, since they would all have a phenotype of resistance against brassinosteroid deficiency. Identifying a suppressor mutant resulting from a point mutation might also be challenging. These predictions suggest that the combination of Brz and a simple point mutation in the wild-type Colombia ecotype can allow rapid identification of crucial signaling proteins. This role for Brz will be a great contribution to plant science.

bil5, Brz-insensitive-long hypocotyl5 We have identified a recessive mutant, bil5, from seeds that received fast neutron treatment. The length of the hypocotyls of this mutant on medium containing Brz is less than that of bil1-D, but at least twice that of the wild type (Fig. 5). Interestingly, adult bil5 plants have pale green, thin stems, thin leaves and a shortened stem length. The dwarf-like phenotype is distinct from the brassinosteroid-deficient dwarf phenotype, and the pale-green leaves of bil5 are in contrast to the dark-green leaves of brassinosteroid-deficient mutants. A preliminary analysis has shown that chloroplast gene expression is lower in bil5 than in the wild type. At least in darkness, brassinosteroid is a negative regulator of chloroplast development. BIL5 may be a key protein in the brassinosteroid regulation of chloroplasts. In addition, the bil5 shortened stem phenotype can be rescued by humidity of 85% or above and light conditions of less than 25 µE, and its stomata have lowered responses to ABA and tend to stay opened. This observation suggests that the


Takeshi NAKANO, Shigeo YOSHIDA and Tadao ASAMI

mutant can be rescued by less stressful conditions, which may relate to the cross-talk between brassinosteroids and ABA. The bil5 mutation maps to the lower arm of chromosome I. Isolation of the bil5 mutant gene, by sequencing and complementation, is in progress. To screen for more bil mutants, we are starting from an activation-tagged line, in collaboration with the groups of Dr. Shinozaki and Dr. Matsui. The genes identified by analysis of these mutants may also be members of the brassinosteroid signaling cascade.

Additional new players for brassinosteroid signaling brs1 and bak1 were identified as bri1-5 dwarf suppressor mutants by activation tagging. BRS1 encodes a carboxypeptidase, and its role in BR signalling has not been defined (Li et al., 2001) (Fig. 7). BAK1, however, encodes a leucine-rich-repeat type receptor-like kinase that could interact directly with BRI1 (Li et al., 2002, Nam and Li, 2002) (Fig. 7). On the basis of their phenotypes, bak1-1D and brs1-1D mutants could potentially be Brz-insensitive mutants. Several

Fig. 7 Current key players on brassinosteroid signaling.

BIL1: Brz-insensitive-long hypocotyl, BRI1: brassinosteroid insentive1, BAK1:bri1 associated kinase, BIN: brassinosteorid insenstive, BRS1: bri1 suppressor. Brz: Brassinosteroid biosynthesis inhibitor.



brassinosteroid-insensitive dwarf mutants, bin2 and bin3/bin5, have also been identified. BIN2 encodes a cytosolic GSK-kinase (He et al., 2002), whereas BIN3 and BIN5 encode proteins of the topoisomerase family (Yin et al., 2002) (Fig. 7). These proteins could be related to brassinosteroid signaling. This idea could be investigated by examining the phenotypes of transformed plants in which expression of these genes has been modified, such as by overexpression, and monitoring the plants for a Brz-insensitive phenotype. Rop2 is a type of GTPase, and transformants in which this protein is constitutively active show hypersensitivity to BRs. (Li et al., 2001) (Fig. 7). As a dominant negative Rop2 transformant does not display BR insensitivity, the actual relationship of this gene with BRs is not yet clear. The det3 mutant, with a lesion in a gene encoding a vacuolelocalized ATPase, is less sensitive to BRs (Schumacher et al., 1999). Future studies using this mutant should help reveal the currently unknown role of the vacuole in BR signaling. These two genes have possible roles in brassinosteroid signal transduction, and transformed plants with altered expression of these genes also could be Brz-insensitive. The combined analysis of the above mutants, gene-modified plants, and Brz should give further insights into brassinosteroid signaling. In another approach toward the understanding of brassinosteroid signaling, Drs. Joanne Chory and Detlef Weigel have mapped quantitative trait loci (QTL) responsible for natural variations in hormone and light responses (Borevitz et al., 2002). They first collected 141 Arabidopsis thaliana accessions from the Northern hemisphere and analyzed the lengths of their hypocotyls in different hormone and light conditions. From these accessions, an Arabidopsis recombinant inbred line (RIL) resulting from a cross of the Cape Verde Islands (Cvi) and Landsberg erecta (Ler) accessions was chosen for detailed analysis with Brz treatment. The resulting QTL map predicted at least three strong loci that confer Brz insensitivity and long hypocotyls in darkness, and five weaker loci were also identified. As these strong Brz-insensitivity loci do not map near the already confirmed or potential Brz-insensitivity genes, a more detailed QTL analysis and more genetic screening for BR signaling mutants will be needed to clarify the mysterious mechanisms of plant growth regulation by brassinosteroids. Recently, gene chip methods have been used to predict genes induced by brassinosteroids (Mussig et al., 2002, Goda et al., 2002). However, it is difficult to determine which genes are actually involved in brassinosteroid signaling in plants under normal conditions, because these methods are based on artificial situations such as chemical stimulation. Reverse genetics approaches that study the phenotypes of transgenic plants in which brassinosteroid or Brz synthesis genes are overexpressed or suppressed should be more useful in determining which genes are truly involved in brassinosteroid signaling.

Acknowledgements We are grateful to Drs. J. Chory, Y. Yin, and S. Mora-Garcia, at Plant Biology Laboratory, Salk Institute, CA, USA for their kind help and coopolation in this research. We also thank to Ms.


Takeshi NAKANO, Shigeo YOSHIDA and Tadao ASAMI

T. Sato, S. Kanda, A. Yamagami, R. Kiuchi, and Mrs. T. Matsuyama, K. Sekimata, T. Suzuki, T.Komatsu, at Plant Functions Laboratory, RIKEN, for their valuable technical assistance. This research was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 15031229 to T.N.), and a grant from Bioarchitect Research Project of RIKEN (to T.N.).


1. ASAMI, T., MIN, Y.K., NAGATA, N., YAMAGISHI, K., TAKATSUTO, S., FUJIOKA, S., MUROFUSHI, N., YAMAGUCHI, I. and YOSHIDA, S. (2000) Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor. Plant Physiology 123: 93-99. 2. ASAMI, T., MIZUTANI, M., FUJIOKA, S., GODA, H., MIN, Y.K., SHIMADA, Y., NAKANO, T., TAKATSUTO, S., MATSUYAMA, T., NAGATA, N., SAKATA, K. and YOSHIDA, S. (2001) Selective interaction of triazole derivatives with DWF4, a cytochrome P450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in Planta. Journal of Biological Chemistry 276: 25687-25691. 3. ASAMI, T. and YOSHIDA, S. (1999) Brassinosteroid biosynthesis inhibitors. Trends in Plant Science 4: 348-353. 4. BOREVITZ, J.O., MALOOF, J.N., LUTES, J., DABI, T., REDFERN, J.L., TRAINER, G.T., WERNER, J.D., ASAMI, T., BERRY, C.C., WEIGEL, D. and CHORY, J. (2002) Quantitative trait loci controlling light and hormone response in two accessions of Arabidopsis thaliana. Genetics 160: 683-696. 5. CHOE, S.W., DILKES, B.P., FUJIOKA, S., TAKATSUTO, S., SAKURAI, A. and FELDMANN, K.A. (1998) The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22 alpha-hydroxylation steps in brassinosteroid biosynthesis. Plant Cell 10: 231-243. 6. CLOUSE, S.D. (2001) Integration of light and brassinosteroid signals in etiolated seedling growth. Trends in Plant Science 6: 443-445. 7. CLOUSE, S.D., LANGFORD, M. and MCMORRIS, T.C. (1996) A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiology 111: 671-678. 8. FUJIOKA, S. and YOKOTA, T. (2003) Biosynthesis and metabolism of brassinosteroid. Annual Review of Plant Biology 54: 137-164. 9. GODA, H., SHIMADA, Y., ASAMI, T., FUJIOKA, S. and YOSHIDA, S. (2002) Microarray analysis of brassinosteroid-regulated genes in Arabidopsis. Plant Physiology 130: 1319-1334. 10. HE, J.X., GENDRON, J.M., YANG, Y.L., LI, J.M. and WANG, Z.Y. (2002) The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 99: 10185-10190. 11. JACOBSEN, S.E., BINKOWSKI, K.A. and OLSZEWSKI, N.E. (1996) SPINDLY, a tetratricopeptide repeat protein invlovled in gibberellin signal transduction in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 93: 9292-9296. 12. KENDE, H. and ZEEVAART, J.A.D. (1997) The five "classical" plant hormones. Plant Cell 9: 1197-1210. 13. LI, H., SHEN, J.J., ZHENG, Z.L., LIN, Y. and YANG, Z. (2001) The Rop GTPase switch controls multiple developmental processes in Arabidopsis. Plant Physiology 126: 670-684. 14. LI, J., LEASE, K.A., TAX, F.E. and WALKER, J.C. (2001) BRS1, a serine carboxypeptidase, regulates BRI1 signaling in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 98: 5916-5921. 15. LI, J., WEN, J.Q., LEASE, K.A., DOKE, J.T., TAX, F.E. and WALKER, J.C. (2002) BAK1, an Arabidopsis



16. 17. 18. 19.


21. 22. 23.




27. 28.


LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell 110: 213-222. LI, J.M. and CHORY, J. (1997) A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90: 929-938. LI, J.M., NAGPAL, P., VITART, V., MCMORRIS, T.C. and CHORY, J. (1996) A role for brassinosteroids in light-dependent development of Arabidopsis. Science 272: 398-401. MUSSIG, C., FISCHER, S. and ALTMANN, T. (2002) Brassinosteroid-regulated gene expression. Plant Physiology 129: 1241-1251. NAGATA, N., ASAMI, T. and YOSHIDA, S. (2001) Brassinazole, an inhibitor of brassinosteroid biosynthesis, inhibits development of secondary xylem in cress plants (Lepidium sativum). Plant and Cell Physiology 42: 1006-1011. NAGATA, N., MIN, Y.K., NAKANO, T., ASAMI, T. and YOSHIDA, S. (2000) Treatment of dark-grown Arabidopsis thaliana with a brassinosteroid-biosynthesis inhibitor, brassinazole, induces some characteristics of light-grown plants. Planta 211: 781-790. NAM, K.H. and LI, J.M. (2002) BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell 110: 203-212. SCHUMACHER, K. and CHORY, J. (2000) Brassinosteroid signal transduction: still casting the actors. Current Opinion in Plant Biology 3: 79-84. SCHUMACHER, K., VAFEADOS, D., MCCARTHY, M., SZE, H., WILKINS, T. and CHORY, J. (1999) The Arabidopsis det3 mutant reveals a central role for the vacuolar H+-ATPase in plant growth and development. Genes & Development 13: 3259-3270. SILVERSTONE, A.L., JUNG, H.S., DILL, A., KAWAIDE, H., KAMIYA, Y. and SUN, T.P. (2001) Repressing a repressor: Gibberellin-induced rapid reduction of the RGA protein in Arabidopsis. Plant Cell 13: 15551565. SZEKERES, M., NEMETH, K., KONCZKALMAN, Z., MATHUR, J., KAUSCHMANN, A., ALTMANN, T., REDEI, G.P., NAGY, F., SCHELL, J. and KONCZ, C. (1996) Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85: 171-182. WANG, Z.Y., NAKANO, T., GENDRON, J., HE, J.X., CHEN, M., VAFEADOS, D., YANG, Y.L., FUJIOKA, S., YOSHIDA, S., ASAMI, T. and CHORY, J. (2002) Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Developmental Cell 2: 505-513. WANG, Z.Y., SETO, H., FUJIOKA, S., YOSHIDA, S. and CHORY, J. (2001) BRI1 is a critical component of a plasma-membrane receptor for plant steroids. Nature 410: 380-383. YIN, Y.H., CHEONG, H., FRIEDRICHSEN, D., ZHAO, Y.D., HU, J.P., MORA-GARCIA, S. and CHORY, J. (2002) A crucial role for the putative Arabidopsis topoisomerase VI in plant growth and development. Proceedings of the National Academy of Sciences of the United States of America 99: 10191-10196. YIN, Y.H., WANG, Z.Y., MORA-GARCIA, S., LI, J.M., YOSHIDA, S., ASAMI, T. and CHORY, J. (2002) BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109: 181-191.


Takeshi NAKANO, Shigeo YOSHIDA and Tadao ASAMI



1979 40 10

bri1 LRR BRI1 GFP BRI1 1999 Arabidopsis Brz Brz bil1 Brz-insensitive-long hypocotyl1 bil1 Arabidopsis EMS Brz Brz GFP BRI1



65 BIl1/BZR1 mRNA

30 BIL1 GSK3/Shaggy kinase BIL1/BZR1 BIL1/BZR BIN2


BIN2 kinase

Gamma Field Symposia, No. 42, 2003 Institute of Radiation Breeding NIAS, Japan




Yoshikatsu MATSUBAYASHI Graduate School of Bio-Agricultural Sciences, Nagoya University Chikusa, Nagoya, 464-8601, Japan e-mail: [email protected]

Key words: phytosulfokine, peptide hormone, receptor kinase, dedifferentiation, proliferation

Abstract Plant cells retain features characteristic of totipotent stem cells. That is, they have the potential to dedifferentiate, re-differentiate, and give rise to all the organs of a new plant. However, relative rates of these cellular processes are strictly dependent on initial cell density, suggesting that cell-to-cell communication is necessary for these processes. Recent biochemical purification studies have demonstrated that phytosulfokine (PSK), a small sulfated peptide, acts as an extracellular ligand involved in the initial step of cellular dedifferentiation, proliferation and redifferentiation. Furthermore, a 120-kD leucine-rich repeat receptor kinase, specifically interacting with PSK, has been purified from plasma membranes using ligand-based affinity chromatography. Lines of evidence suggest that this ligand-receptor pair confers competence for dedifferentiation and re-differentiation to individual cells, rather than directly determining cellular fate. In this article, I review what is known about PSK signaling.

Introduction A high proportion of plant cells, even at a fully differentiated stage, can dedifferentiate and proliferate in vitro as totipotent stem cells, forming a structure called a callus, after treatment with plant hormones such as auxin and cytokinin. (Skoog F & Miller CO 1967) Callus cells can differentiate into various organs and give rise to a new plant, indicating that plant cells from specific adult tissues are capable of differentiating into cells of all tissues. However, relative rates of cellular dedifferentiation and growth in vitro generally depend on initial cell density. Cellular dedifferentiation and growth progress efficiently under high cell density, but are significantly suppressed under low cell density. To promote cellular growth at low cell density, several researchers have used specialized culture techniques such as nurse cultures, in which target cells



are grown close to but physically separated from nurse cells (Fig. 1). (Muir WH et al. 1954; Torrey JG 1957; Raveh et al. 1973) Interestingly, growth suppression of low-density cells is also negated by addition of conditioned medium in which cells have previously been grown. (Stuart R & Street HE 1969; Somers DA et al. 1985) These observations strongly indicate that plant cells secrete an autocrine-type factor responsible for cellular dedifferentiation and growth.

Discovery of phytosulfokine We tested several cell types for use in a high-throughput bioassay system for purification of the conditioning factor. Among these, the best results were obtained with a primary culture system of mechanically dispersed mesophyll cells prepared from asparagus cladodes. Dedifferentiation and proliferation of dispersed asparagus cells was completely suppressed under low cell density, but was significantly promoted by addition of conditioned medium derived from asparagus cell culture. This bioassay system clearly responds to a small amount of crude conditioned medium, and provides reliable results within one week. (Matsubayashi Y & Sakagami Y. 1996) The activity of the conditioning factor was completely lost by the protease treatments, indicating that this factor is a peptide. Using this bioassay system, in 1996, we succeeded in isolating a growth factor from conditioned medium derived from asparagus suspension culture. (Matsubayashi Y & Sakagami Y. 1996) A purification of approximately 107-fold was achieved, with recovery of activity of about 15%. This factor is a sulfated peptide composed of only 5 amino acids (Fig. 2), and, due to the presence of sulfate esters, it was named phytosulfokine (PSK). PSK induces cellular dedifferentiation and proliferation of dispersed plant cells at concentrations as low as 1.0 nM. Sulfated tyrosines are

Fig. 1. Density effect in plant cell culture. Cellular dedifferentiation and growth are significantly suppressed under the low-density. This growth suppression is negated by the use of nurse cultures in which target cells are grown close to but physically separated from nurse cells. These phenomena strongly indicate that plant cells secrete an autocrine-type growth factor responsible for cellular dedifferentiation and growth.



Fig. 2. Chemical structure of the purified growth factor. This factor is a sulfated peptide composed of only five amino acids, and due to the presence of sulfate esters, it was named phytosulfokine (PSK).

often found in secreted peptides in animals, (Huttner WB. 1982, Niehrs C et al. 1993) but, to date, PSK is the only example of post-translational sulfation of tyrosine in plants. PSK is present, with identical structure, in conditioned medium derived from cell lines of many plants, including dicotyledons and monocotyledons, indicating that it is widely distributed among higher plants.

Possible function of PSK Initial cell density is a determining factor in in vitro cellular re-differentiation such as tracheary element (TE) formation, which occur at high frequency under high cell density but are significantly suppressed below threshold densities, suggesting that intercellular signaling is involved in initiation and/or subsequent progress in cellular re-differentiation. (Fukuda H, & Komamine A. 1980) Interestingly, PSK triggers TE differentiation of Zinnia mesophyll cells at nanomolar concentrations.(Matsubayashi Y. et al. 1999) This phenomenon is not a secondary effect caused by increased cell density, because a high proportion of TEs differentiate directly from dispersed mesophyll cells without intervening cell division. It has also been demonstrated that PSK signaling is involved in somatic embryogenesis in a carrot system (Kobayashi T. et al. 1999, Hanai H. et al. 2000), and in adventitious bud formation on callus of Antirrhinum majus. (Yang G. et al. 1999) However, neither cellular re-differentiation nor dedifferentiation can be induced by PSK alone; they require certain ratios and concentrations of auxin and cytokinin in addition to PSK. (Matsubayashi Y. et al. 1999) Whereas PSK triggers TE differentiation of Zinnia mesophyll cells without intervening cell division in a cytokinin-rich medium, it induces cellular dedifferentiation and proliferation in an auxin-rich medium. Neither dedifferentiation nor re-differentiation occurs without PSK. In this context, it is possible that PSK first confers competence to individual cell plants, and that auxin/cytokinin then determines cell fate (Fig. 3). PSK also promotes adventitious root formation on cucumber hypocotyls (Yamakawa S. et al. 1998); it is likely that this organ development is determined by the endogenous auxin/cytokinin level.

Genes for PSK precursor proteins Five paralogous genes encoding 80-amino-acid precursors of PSK have been identified in



Fig. 3. Possible mode of action of PSK. PSK confers a competence for dedifferentiation and/or re-differentiation on fully differentiated cells. In this step, no morphological change is observed. After the acquisition of competence, auxin, cytokinin, and other factors determine cell fate.

Arabidopsis. (The Arabidopsis Genome Initiative. 2000, Yang H. et al. 2001) Each predicted protein has a probable secretion signal at the N-terminus and a single PSK sequence close to the Cterminus. In addition, there are dibasic amino acid residues immediately upstream from the PSK domain. (Fig. 4) Peptide-hormones and other biologically active peptides are generally synthesized as inactive higher-molecular-weight precursors that must undergo a variety of post-translational processing steps to yield the active peptides. (Harris RB. 1989) PSK mRNAs are detected not only in dedifferentiated callus cells but also in leaves and roots

Fig. 4. Alignment of the deduced amino acid sequences of PSK precursor proteins in Arabidopsis. Red box indicate the five-amino-acid PSK domain. Predicted amino terminal signal sequences are underlined and putative processing site immediately upstream from PSK domain are boxed by yellow. Aspartic acid residues on the amino-terminal side of the first tyrosine of PSK domain, one of the most important determinant of the sulfation of PSK precursor, are boxed by blue. Identical amino acid residues are indicated by an asterisk, and similar amino acid residues are indicated by a colon.



of intact plants, indicating that PSK expression is not limited to regions in which cells actively divide and differentiate. (Yang H. et al. 2001) To study the function of PSK in plants, a large number of transformation experiments have been performed using Arabidopsis. However, loss-offunction techniques have not produced visible, directly informative phenotypes, suggesting functional redundancy between these 5 PSK genes in Arabidopsis. A definite picture will only emerge when combinations of all five knockouts are available. Overexpression of PSK slightly promotes callus formation in vitro in the presence of auxin/cytokinin, (Yang H. et al. 2001) but does not affect the growth of seedlings.

Structure-activity relationships of PSK Derivatization of peptide hormones with biochemical tags such as photoactivatable groups has been used in characterization and purification of hormone receptors. (Hazum E. 1983) A key factor in the use of such functional groups is the ability to derivatize peptides without loss of binding activity or biological activity. To identify the active core of PSK, we synthesized several PSK analogs by solid phase peptide synthesis and direct sulfation of the peptide-resin using dimethylformamide-sulfurtrioxide complex. (Matsubayashi Y. et al. 1996) As shown in Fig. 5, N-terminal tetrapeptide and tripeptide of PSK retained 8% and 20% of the activity of the parent pentapeptide, respectively, but Nterminal dipeptide showed no activity. Deletion of the sulfate groups of Tyr1 and Tyr3 resulted in compounds with 0.6% and 4% of the activity of PSK, respectively, indicating that the sulfate group of Tyr1 is more important than that of Tyr3 for activity. In contrast, the N-terminal-truncated analog and an unsulfated analog exhibited no activity. Thus, the N-terminal tripeptide fragment Tyr(SO3H)Ile-Tyr(SO3H) has been identified as the active core of PSK. A popular method for covalently linking functional groups to a peptide involves the use of activated esters of the functional groups, which react with primary amines to form amide bonds. However, modification of the N-terminal amino group of PSK by addition of Gly strongly decreases its biological activity. (Matsubayashi Y. et al. 1996) Thus, functional derivatization of PSK requires incorporation of an additional primary amino group at the C-terminal region, which is less involved in PSK activity than the N-terminal. To fulfill these requirements, several Alasubstituted PSK analogs were tested for activity, and the analog [Ala5]PSK and [Lys5]PSK was found to possess binding activity equal to that of PSK (Fig. 5). (Matsubayashi Y. et al. 1999) Interestingly, [Lys5]PSK retained significant activity after derivatization of the side chain of Lys5 by biotin, even when a very long spacer chain was inserted between the amino group of Lys5 and the carboxyl group of biotin. This finding provided the breakthrough in a series of experiments conducted with the aim of visualization and purification of PSK receptors.



Fig. 5. Structure-activity relationships of PSK. PSK analogs were prepared by solid phase peptide synthesis and direct sulfation of the peptide-resin. Relative activity of each analog was determined by the bioassay using asparagus mesophyll cells or the competitive receptor binding assay (asterisk). ND=not determined. Among these, [125I]-[N -(4-azidosalicyl)Lys5]PSK was used for photoaffinity labeling experiments, and [Lys5]PSK-Sepharose was used for purification of the PSK receptor.

PSK receptor Because of the presence of highly hydrophilic sulfate groups in PSK molecules, it is unlikely that they pass through plasma membranes and directly interact with target molecules inside cells. To determine whether a cell surface receptor for PSK exists, radiolabeled PSK was synthesized by coupling [35S]sulfuric acid with the phenolic groups of tyrosine. (Matsubayashi Y. et al. 1997) Binding of [35S]PSK was detected on the surface of suspension cultured rice cells and in the plasma-membrane-enriched fractions. The binding is reversible and saturable, and only PSK analogs that possess biological activity can effectively displace the radioligand. To further characterize the PSK receptor, [3H]PSK, which has higher specific radioactivity, was synthesized by catalytic reduction of a PSK analog containing tetradehydroisoleucine. (Matsubayashi Y & Sakagami Y. 1999) Ligand saturation analysis using [3H]PSK revealed the existence of a high-affinity binding site in microsomal fractions derived from rice, maize,



asparagus and carrot. Among these, the greatest abundance of binding sites was found in the carrot membranes: approximately 150 fmol per mg microsomal proteins, with an estimated dissociation constant (Kd) of 4.2 nM. This PSK receptor protein was visualized by photoaffinity labeling of carrot plasma membrane fractions using the photoactivable 125I-labeled PSK analog [N -(4-azidosalicyl) Lys5]PSK (Fig. 5), the binding activity of which are 10% of unmodified PSK.(Matsubayashi Y & Sakagami Y. 2000) SDS-PAGE analysis of the labeled proteins indicated that a 120-kD protein and a minor 150-kD protein specifically interact with PSK. Both proteins contain approximately 10 kD of Nlinked oligosaccharide chains that can be cleaved by treatment with peptide N-glycosidase F. We purified these PSK-binding proteins from microsomal fractions of carrot cells by Triton X100 solubilization and specific ligand-based affinity chromatography using a [Lys5]PSK-Sepharose column containing a long spacer chain between ligand and matrix. (Matsubayashi Y. et al. 2002) SDS-PAGE of the proteins in the fractions eluted by PSK showed specific recovery of a major 120kD protein and a minor 150-kD protein. Both proteins were absent in the fractions eluted by an inactive PSK analogs; this indicates that binding of the proteins is specific. Several independent purifications were performed, yielding 50 µg of the major 120-kD protein with 96,000-fold purification from 4,800 mg of microsomal proteins (corresponds to 24 L of suspension-cultured cells), with an overall recovery rate of 40%. (Matsubayashi Y. et al. 2002) Based on the internal sequence of this protein, a 3.5-kb cDNA clone was isolated from the carrot cDNA library. The cDNA encoded a 1021-amino-acid protein with a deduced molecular mass of 112 kD, with features found in several hormone receptors in plants and animals (Fig. 5). It contained an N-terminal hydrophobic signal sequence, extracellular leucine-rich repeats (LRRs), a transmembrane domain, and a cytoplasmic kinase domain. Northern blot analysis showed that mRNA of this protein accumulated ubiquitously in leaf, apical meristem, hypocotyl and root of carrot seedlings, although its expression level was far lower than in cultured carrot cells. The major extracellular domain of this protein contained 21 tandem copies of a 24-amino-acid LRR; it has been suggested that this string of LRRs plays a key role in protein-protein interactions. (Kobe B & Deisenhofer J. 1994) In addition, a 36-amino-acid island was detected in the 18th LRR. An island domain has also been found among the extracellular LRRs of the brassinosteroid receptor BRI1, and has been shown to be critical for its function. (Li J & Chory J. 1997) Transgenic carrot cells overexpressing the cDNA of the major 120-kD protein showed a significant increase in PSK binding sites in the membrane fractions and accelerated growth in response to PSK, compared with control cells. Photoaffinity cross-linking and immunoprecipitation analysis of the membrane proteins derived from the transformants revealed expression of the 150kD protein in addition to the 120-kD protein, indicating that both proteins are encoded by a single gene. In contrast, expression of antisense mRNA of the major 120-kD protein significantly inhibited callus growth. These findings strongly suggest that this receptor kinase is a component of a functional PSK receptor that directly interacts with PSK.



Fig. 6. Schematic of the 120-kD PSK receptor. The diagram shows the signal peptide (SP, red), extracellular leucine-rich repeats (LRRs, yellow), a 36-amino-acid island, a transmembrane domain (TM, blue), and a cytoplasmic kinase domain (green).

Future perspectives Now that in vitro function of PSK and the molecular basis of ligand-receptor interaction in PSK signaling have been established, the next phase of research is characterization of the in vivo role of PSK and its downstream signaling pathway in plants. The carrot PSK receptor exhibits a high percentage of amino acid identity with several LRR receptor-like kinases found in Arabidopsis. The sequencing of the Arabidopsis genome is now complete, and large collections of gene-disruption lines are available. Once PSK-binding activities of these LRR-RLKs are confirmed, direct clues to in vivo function of PSK will be provided by phenotypes of knockout mutants.


1. Beisswanger R, Corbeil D, Vannier C, Thiele C, Dohrmann U, Kellner R, Ashman K, Niehrs C, Huttner WB. (1998) Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterization of tyrosylprotein sulfotransferase-2. Proc Natl Acad Sci USA 95: 11134-11139. 2. Bundgaard JR, Vuust J, Rehfeld JF. (1997) New consensus features for tyrosine O-sulfation determined by mutational analysis. J Biol Chem 272: 21700-21705. 3. Fukuda H, Komamine A. (1980) Establishment of an experimental system for the tracheary element differentiation from single cells isolated from the mesophyll of Zinnia elegans. Plant Physiol 65: 57-60. 4. Hanai H, Matsuno T, Yamamoto M, Matsubayashi Y, Kobayashi T, Kamada H, Sakagami Y. (2000) A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation. Plant Cell Physiol 41: 27-32. 5. Hanai H, Nakayama D, Yang H, Matsubayashi Y, Hirota Y, Sakagami Y. (2000) Existence of a plant tyrosylprotein sulfotransferase: novel plant enzyme catalyzing tyrosine O-sulfation of preprophytosulfokine variants in vitro. FEBS Lett 470: 97-101. 6. Harris RB. (1989) Processing of pro-hormone precursor proteins. Arch Biochem Biophys 275: 315-333. 7. Hazum E. (1983) Photoaffinity labeling of peptide hormone receptors. Endocr Rev 4: 352-362. 8. Huttner WB. (1982) Sulphation of tyrosine residues, a widespread modification of proteins. Nature 299: 273-276. 9. Huttner WB. (1988) Tyrosine sulfation and the secretory pathway. Annu Rev Physiol 50: 363-376. 10. Kobayashi T, Eun C-H, Hanai H, Matsubayashi Y, Sakagami Y, Kamada H. (1999) Phytosulfokine- , a peptidyl plant growth factor, stimulates somatic embryogenesis in carrot. J Exp Botany 50:1123-1128. 11. Kobe B, Deisenhofer J. (1994) The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci 19: 415-421.



12. Li J, Chory J. (1997) A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90: 929-938. 13. Matsubayashi Y, Hanai H, Hara O, Sakagami Y. (1996) Active fragments and analogs of the plant growth factor, phytosulfokine: structure-activity relationships. Biochem Biophys Res Commun 225: 209-214. 14. Matsubayashi Y, Sakagami Y. (1996) Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L. Proc Natl Acad Sci USA 93: 7623-7627. 15. Matsubayashi Y, Takagi L, Sakagami Y. (1997) Phytosulfokine- , a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites. Proc Natl Acad Sci USA 94: 13357-13362. 16. Matsubayashi Y, Takagi L, Omura N, Morita A, Sakagami Y. (1999) The endogenous sulfated pentapeptide phytosulfokine- stimulates tracheary element differentiation of isolated mesophyll cells of Zinnia. Plant Physiol 120: 1043-1048. 17. Matsubayashi Y, Morita A, Matsunaga E, Furuya A, Hanai N, Sakagami Y. (1999) Physiological relationships between auxin, cytokinin, and a peptide growth factor, phytosulfokine- , in stimulation of asparagus cell proliferation. Planta 207: 559-565. 18. Matsubayashi Y, Takahata Y, Morita A, Atsumi K, Sakagami Y. (1999) Preparation and characterization of fully active biotinylated analogs of phytosulfokine- . Biosci Biotechnol Biochem 63: 1847-1849. 19. Matsubayashi Y, Sakagami Y. (1999) Characterization of specific binding sites for a mitogenic sulfated peptide, phytosulfokine- , in the plasma-membrane fraction derived from Oryza sativa L. Eur J Biochem 262: 666-671. 20. Matsubayashi Y, Sakagami Y. (2000) 120- and 160-kDa receptors for endogenous mitogenic peptide, phytosulfokine- , in rice plasma membranes. J Biol Chem 275: 15520-15525. 21. Matsubayashi Y, Ogawa M, Morita A, Sakagami Y. (2002) An LRR receptor kinase involved in perception of a peptide plant hormone, phytosulfokine. Science 296: 1470-1472. 22. Muir WH, Hildebrandt AC, Riker AJ. (1954) Plant tissue cultures produced from single isolated cells. Science 119: 877-878. 23. Niehrs C, Beisswanger R, Huttner WB. (1993) Protein tyrosine sulfation, an update. (1994) Chem Biol Interact 92: 257-271. 24. Ouyang Y, Lane WS, Moore KL. (1998) Tyrosylprotein sulfotransferase: purification and molecular cloning of an enzyme that catalyzes tyrosine O-sulfation, a common posttranslational modification of eukaryotic proteins. Proc Natl Acad Sci USA 95: 2896-2901. 25. Raveh D, Hubermann E, Galun E. (1973) In vitro culture of tobacco protoplasts: use of feeder techniques to support division of cells plated at low densities. In Vitro 9: 216-222. 26. Skoog F, Miller CO. (1967) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol. 11: 118-140. 27. Somers DA, Birnberg PR, Petersen WL, Brenner ML. (1985) The effect of conditioned medium on colony formation from Black Mexican sweet corn protoplasts. Plant Cell Rep 4: 155-157. 28. Stuart R, Street HE. (1969) Studies on the growth in culture of plant cells. IV. The initiation of division in suspensions of stationary phase cells of Acer pseudoplatanus L. J Exp Bot 20: 556-571. 29. The Arabidopsis Genome Initiative. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature ;408: 796-815. 30. Torrey JG. (1957) Cell division in single isolated cells in vitro. Proc Natl Acad Sci USA 43: 887-891. 31. Yamakawa S, Sakuta C, Matsubayashi Y, Sakagami Y, Kamada H, Satoh S. (1998) The promotive effects of a peptidyl plant growth factor, phytosulfokine- , on the formation of adventitious roots and expression of a gene for a root-specific cystatin in cucumber hypocotyls. J Plant Res 111: 453-458. 32. Yang G, Shen S, Kobayashi T, Matsubayashi Y, Sakagami Y, Kamada H. (1999) Stimulatory effects of a novel peptidyl plant growth factor, phytosulfokine- , on the adventitious bud formation from callus of Antirrhinum majus. Plant Biotechnol 16: 231-234.



33. Yang H, Matsubayashi Y, Hanai H, Nakamura K, Sakagami Y. (2000) Molecular cloning and characterization of OsPSK, a gene encoding a precursor for phytosulfokine- , required for rice cell proliferation. Plant Mol Biol 635: 635-647. 34. Yang H, Matsubayashi Y, Nakamura K, Sakagami Y. (2001) Diversity of Arabidopsis genes encoding precursors for phytosulfokine, a peptide growth factor. Plant Physiol 127: 842-851.




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sd1 BRI1





SD1 D35 KO 2 SD1 slender XA1 XA21



100 1970 entAMO1618 P450 P450 ent-

85 23






2 3








Gamma Field Symposia Number 42

97 pages

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