The Third Korea-Japan Joint Symposium Plant Metabolism ... · PDF fileThe Third Korea-Japan...

The Third Korea-Japan Joint Symposium Plant Metabolism: from biosynthesis to signal transduction November 8 (Thur) – 11 (Sun), 2007 Venue: Paradise Hotel, Incheon, Korea Hosts: Korean Society for Plant Biotechnology

Japanese Society for Plant Cellular and Molecular Biology

Korean Organizer: Sunghwa CHOE, Professor

School of Biological Sciences College of Natural Sciences Seoul National University Seoul 151-747, Korea

Japanese Organizer: Takashi HASHIMOTO, Professor

Graduate School of Biological Sciences Nara Institute of Science and Technology 630-0192 Nara, Japan

Sponsors: Korean Science and Engineering Foundation

Japanese Society for the Promotion of Science

Proceedings of the 3rd Korea-Japan Joint Symposium on Plant Metabolisms

Contents 1. Contents --------------------------------------------------------------------------- 1 2. Welcome address (Jang R Liu) ------------------------------------------------ 2 3. Welcome address (Fumihiko Sato) -------------------------------------------- 3 4. Agenda (Sunghwa Choe & Takashi Hashimoto) ----------------------------- 4 5. Program ---------------------------------------------------------------------------- 5 6. Biosynthesis of isoflavonoids and triterpenoids in a model legume, Lotus japonicus

(Shin-ichi Ayabe) ----------------------------------------------------------------- 7 7. Molecular Genetics of Nicotine Biosynthesis and Transport (Takashi Hashimoto)

-------------------------------------------------------------------------------- 12 8. Dual biosynthetic pathways of phytosterol in plant (Toshiya Muranaka) -- 16

9. Enzymology and molecular biology of aurone biosynthesis (Toru Nakayama) ---------------------------------------------------------------------------------------- 20

10. Camptothecin Biosynthetic System – Pathway Elucidation, Gene Discovery and Self-Resistance (Kazuki Saito) --------------------------------------------------- 25

11. Comparative analysis of ABA signaling between moss and higher plants (Yoichi Sakata) ------------------------------------------------------------------------------ 29

12. Metabolic Engineering in Benzylisoquinoline Alkaloid Biosynthesis (Fumihiko Sato) ---------------------------------------------------------------------------------- 34

13. Aromatic substrate prenyltransferase involved in plant secondary metabolism (Kazufumi Yazaki) ---------------------------------------------------------------- 41

14. NMR and LC/MS as a tool for metabolomics study (Nam-In Baek) ------ 44

15. Brassinosteroid biosynthesis and its effects on other hormones (Sunghwa Choe) --------------------------------------------------------------------------------------- 48

16. Phytochromes negatively regulate their interacting bHLH transcription factors in Arabidopsis (Giltsu Choi) ------------------------------------------------------ 53

17. Regulation of Terpene Secondary Metabolism by Multiple Gene in Gymnosperms (Soo-Un Kim) --------------------------------------------------------------------- 55

18. Cooperation and Functional Diversification of Two Closely Related Galactolipase Genes for Jasmonate Biosynthesis (Ilha Lee) ----------------------------------- 59

19. Beta-glucosidase homologs play critical roles in homeostasis of ABA (Inhwan Hwang) ------------------------------------------------------------------------------- 64

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Welcome address On behalf of the Korean Society for Plant Biotechnology, I would like to invite Korean and Japanese Scientists working in the field of plant biotechnology to 2007 Korea-Japan Joint Symposium to be held in Incheon , Korea during November 8-11. Korean and Japanese scientists in the field of plant biotechnology have had joint symposia for the last 20 years. I believe that these symposia have provided scientists from both countries with opportunities to know each other better personally as well as to exchange information, ideas, and experiences. The ultimate subject of the event in this year is plant metabolomics and metabolic engineering. Plant metabolomics is a recently emerging powerful means for functional genomics and metabolic engineering is one of the most practical tools in the application of plant biotechnology. Many scientists from both countries are renowned for their tremendous contributions to this subject and some of them have been selected and invited to this event as speakers. The Joint Symposium is held at Paradise Hotel in Incheon, 40 minutes driving distance from Incheon International Airport. Incheon is one of the largest cities in Korea, only next to Seoul, located 20 km west of Seoul and the largest port in Korea. Participants will be provided with opportunities to learn Korean traditional cultures and arts. I promise that the trip to Incheon will be worthwhile, meaningful, and enjoyable. Professor Sunghwa Choe from Seoul National University has made a great effort in organizing this event. I would like to take this opportunity to appreciate his sincere endeavor. I look forward to seeing you in Incheon. Jang R Liu, Ph.D. President Korean Society for Plant Biotechnology

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Welcome address Joint Symposium for Tighter Collaboration 안녕하십니까?

It is my great pleasure to address a brief opening remark at the Korea-Japan Joint Symposium on “Plant metabolism: from biosynthesis to signal transduction”. This is the third Joint Symposium between Korean Society of Plant Biotechnology (KSPB) and Japanese Society of Plant Cellular and Molecular Biology (JSPCMB). We are so glad to see that the joint activities further deepen. After the first two Symposia on “Platform Technology for Plant Bioproducts” and “Current Status and the Future of Transgenic Crops in Japan and Korea”, this symposium is organized to clarify our roadmap of Plant Biotechnology to develop more productive plant species, which will meet the current environmental crisis. In this Symposium, two key subjects, “Molecular bases of metabolism including metabolic engineering and metabolomics” and “Signal

transduction of plant hormones” are focused. I believe that this symposium is very timely and useful for the development of Plant Biotechnology. I would like to thank Prof. Sunghwa Choe and Prof. Takashi Hashimoto, who organized this Symposium as the core members of KSPB and JSPCMB with their energetic efforts. I also appreciate many participants from the two Societies. JSPCMB was established in 1981 as Japanese Society for Plant Tissue Culture. In 1995, our society was changed to the present name to reflect broader scientific activities. As all of us notice, JSPCMB and KSPB are the sister societies that share the same interests and their contribution in this field is crucial. I sincerely hope that this joint symposium will bring the much tighter collaboration between two societies for the future development of Plant Biotechnology and for the establishment of sustainable world. 감사합니다. Fumihiko Sato, Ph.D. President Japanese Society of Plant Cell and Molecular Biology

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Agenda This joint symposium on “Plant Metabolism: from biosynthesis to signal transduction” is expected for Korean and Japanese scientists to share and exchange valuable research results and unpublished information. Through this meeting, we hope that both the two countries will maintain and further elevate the currently high standards of plant metabolic researches in the future. The symposium consists of three major sessions. 1. Metabolism and Signal Transduction of Plant Hormones Metabolic pathways and genes involved in major plant hormones are being characterized by biochemical and molecular genetic approaches in the model plants Arabidopsis thaliana and rice, and in a few other plant species. Mutants defective in hormone perceptions are used to identify critical players in the signal transduction pathways, which involve hormone receptors, F-box-containing protein degradation machinery, and downstream transcriptional repressors and activators. Current status and emerging regulatory themes will be reported and discussed in model plants and non-model plants. Applications to plant breeding will also be discussed. 2. Plant Secondary Metabolism Major plant secondary metabolite families include flavonoids, terpenoids, and alkaloids. Biosynthetic enzymes for particular groups of these useful natural products have been cloned, and their enzymology and molecular evolution have been studied. Based on these progresses, biotechnological applications of these new genes for metabolic engineering are becoming increasingly popular. Emerging new areas of interest include intracellular compartmentalization of the subset of pathways and inter-cellular and inter-organ transport of metabolite intermediates and final products. Insect hervivory, diseases from microorganisms, and other environmental factors play intricate controls over biosynthesis and transport of secondary metabolites. These basic and applied topics of diverse area will be discussed. 3. Metabolomics and Future Technologies In model plants in which genome sequences were or will soon be clarified, large-scale analyses of gene transcripts and metabolites are being applied to understand regulatory networks of complicated and seemingly unrelated metabolic pathways. These modern technologies will be useful for deciphering metabolisms of crop plants and will facilitate breeding of superior quality crops with desired metabolite profiles. Up-to-date technologies and their practical applications on metabolisms will be reported and discussed. November 9, 2007 Japanese Organizer Takashi HASHIMOTO, Ph.D. Korean Organizer Sunghwa CHOE, Ph.D.

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❏ Program ❏

<November 9, Fri>

� Opening ceremony (13:15-13:30)

� Session 1: Metabolism and Signal Transduction of Plant Hormones 1 (09:00-11:00)

Chair: Dr. Hyung-Taeg Cho (Chungnam National University) 13:30-14:00 Cooperation and Functional Diversification of Two Phospholipase A1

Genes for Jasmonate Biosynthesis (Dr. Ilha lee, Seoul National University) 14:00-14:30 Molecular genetics of plant sterol backbone biosynthesis (Dr. Toshiya

Muranaka, RIKEN Plant Science Center) 14:30-15:00 Comparative Analysis of ABA Signaling between Moss and Higher Plants

(Dr. Yo-ichi Sakata, Tokyo University) 15:00-15:30 Regulation of seed germination by light (Dr. Giltsu Choi, Korea Advanced

Institute of Science and Technology (KAIST) � Coffee break (15:30-16:00) � Session 2: Plant Secondary Metabolism (16:00-18:00) Chair: Dr. Yi Lee (Chungbuk National University) 16:00-16:30 Metabolic Engineering in Benzylisoquinoline Alkaloid Biosynthesis (Dr.

Fumihiko Sato, Kyoto University) 16:30-17:00 Aromatic substrate prenyltransferase involved in plant secondary

metabolism. (Dr. Kazufumi Yazaki, Kyoto University) 17:00-17:30 Regulation of Terpene Secondary Metabolism by Multiple Gene in

Gymnosperms (Dr. Soo Un Kim, Seoul National University) Chair: Dr. Ray Bressan (Purdue University) 17:30-18:00 Enzymolgy and molecular biology of aurone biosynthesis (Dr. Toru

Nakayama, Tohoku University) 18:00-18:30 Molecular genetics of nicotine biosynthesis and transport (Dr. Takashi

Hashimoto, Nara Institue of Science and Technology) � Reception (18:30-20:00) < November 10, Sat>

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� Session 3: Metabolomics and future technologies (9:30-11:00) Chair: Dr. Dae-Jin Yun (Gyeongsang National University) 9:30-10:00 NMR and LC/MS as a tool for metabolomics analysis (Dr. Nam In Baek,

Kyunghee University) 10:00-10:30 Biosynthesis of Isoflavonoids and Triterpenoids in a Model Legume, Lotus

japonicus (Dr. Shin-ich Ayabe, Nihon University) 10:30-11:00 Camptothecin Biosynthetic System: Pathway Elucidation, Gene Discovery

and Self Resistance (Dr. Kazuki Saito, Chiba University) � Coffee break (11:00-11:15) � Session 4: Metabolism and Signal Transduction of Plant Hormones 2

(11:15-12:30) Chair: Dr. Woo Taek Kim (Yonsei University) 11:15-11:45 Brassinosteroid biosynthesis and its effects on other hormones (Dr.

Sunghwa Choe, Seoul National University) 11:45-12:15 Beta-glucosidase homologs play critical roles in homeostasis of ABA (Dr.

Inhwan Hwang, Pohang University of Science and Technology (POSTECH) � Concluding ceremony (12:15-12:30)

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Biosynthesis of isoflavonoids and triterpenoids in a model

legume, Lotus japonicus

Shin-ichi Ayabe Department of Applied Biological Sciences, Nihon University, 252-8510 Kanagawa,

Japan. E-mail: [email protected]

Isoflavonoids and triterpenoids are typical products of leguminous plants with significant ecological and physiological functions. New findings on the biosynthesis of these metabolites, e.g., involvement of enzymes apparently recruited from primary metabolism (carboxylesterase-like dehydratase) and another secondary metabolism (lignan pathway) in isoflavonoid synthesis and discovery of plant lanosterol synthase, an oxidosqualene cyclase believed to be restricted to animals and fungi, are presented. Organization of biosynthetic genes in Lotus japonicus as extensive clusters of paralogs implicated local gene duplication as a major molecular mechanism of evolution of phytochemicals. 5-Deoxyisoflavonoids are characteristic components of the Leguminosae that function in symbiotic and defensive relationship with environmental organisms. Also, dietary isoflavonoid phytoestrogens are known as disease-preventing and health-promoting agents. Triterpenoids of leguminous plants, such as glycyrrhizin of licorice (rhizome of Glycyrrhiza spp.), are pharmacologically important as the crude drug constituents. Understanding of the biosynthetic mechanism and utilization of these compounds in the future agriculture and human life can be achieved through studies with model legumes, the representatives of which are Lotus japonicus (Regel) K. Larsen and Medicago truncatula Gaertn. Comparative genomic studies incorporating the information from Arabidopsis thaliana and other plant genomes can further bring about evolutionary insight into the plant secondary metabolism. Recent advances in the characterization of the enzymes using L. japonicus and other legumes and genome-wide analysis of the structural genes of the pathways in L. japonicus are summarized. New enzymes of isoflavonoid biosynthesis

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L. japonicus produces an isoflavan phytoalexin, (-)-vestitol, a 4’-methoxy-5-deoxy-isoflavonoid (Fig. 1). The first committed intermediate of 5-deoxyisoflavonoid biosynthesis is 2,7,4’-trihydroxyisoflavanone produced by a P450 isoflavonoid synthase (IFS) from a 5-deoxyflavanone, liquiritigenin. In L. japonicus, Medicago spp. (including alfalfa) and licorice (G. echinata), 2,7,4’-trihydroxyisoflavanone is methylated by SAM: 2-hydroxyisoflavanone 4’-O-methyltransferase (HI4’OMT) and dehydrated [non-enzymatically or by 2-hydroxyisoflavanone dehydratase (HID)] to yield formononetin. In soybean and other legumes that produce 4’-hydroxylated isoflavonoids, 2,7,4’-trihydroxyisoflavanone is directly dehydrated to give daidzein. Through screening of repeatedly fractionated E. coli expressing a licorice cDNA library, a HID cDNA was isolated, and another HID cDNA was cloned from soybean based on the sequence information in its EST library (Akashi et al. 2005). Kinetic studies with recombinant proteins revealed that licorice HID is specific to 2,7-dihydroxy-4’-methoxyisoflavanone, while soybean HID has broader specificity to both 4’-hydroxylated and 4’-methoxylated 2-hydroxyisoflavanones, reflecting the structures of isoflavones contained in each plant species. Strikingly, the kcat of soybean HID was 105 times greater than the rate constant of spontaneous dehydration of 2-hydroxyisoflavanone, strongly indicating the involvement of this enzyme in isoflavone biosynthesis (also see the section In planta characterization of genes of 5-deoxyisoflavonoid biosynthesis of this presentation). HID proteins were found to be members of carboxylesterase family, of which plant proteins form a monophyletic group and some are assigned defensive functions. The finding represents an example of recruitment of enzymes of primary metabolism during the molecular evolution of plant secondary metabolism.

OHO

O

OHH OHO

O

OH

OMe

H

OHO

O

OH

OH

H

IFS(CYP93C) HI4'OMT4'

(2S)-Liquiritigenin((2S)-Flavanone)

2,7,4'-Trihydroxyisoflavanone(2-Hydroxyisoflavanone)

2

4'

277

55

O

OHOMe

HOH

(-)-Medicarpin

6a

11a

3OHO

OMeHO

(-)-Vestitol

HOHO

OOMe

HID 4'

FormononetinPTR

Fig. 1. Isoflavonoid biosynthesis in L. japonicus.

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The steps from isoflavones to pterocarpans have already been biochemically and/or molecular biologically characterized, and isoflavans and pterocarpans have been supposed to be biosynthetically connected. A search of the EST library of L. japonicus for homologs of phenylcoumaran benzylic ether reductase catalyzing the reductive cleavage of dihydrofurans in the lignan pathway yielded cDNAs encoding pterocarpan reductase (PTR) that catalyzes the cleavage of the dihydrofuran of a pterocarpan medicarpin to yield an isoflavan (-)-vestitol (Akashi et al. 2006). Two PTRs displayed enantiospecificity to (-)-medicarpin, representing genuine L. japonicus PTRs, while the other two lacked enantiospecificity and were presumed to be evolutionarily primitive types. Analyses of the gene families of 5-deoxyisoflavonoid biosynthesis As a cooperative project with Kazusa DNA Research Institute, we analyzed the genes encoding enzymes involved in the biosynthesis of the legume-specific 5-deoxyisoflavonoid of L. japonicus (Shimada et al. 2007). The paralogous biosynthetic genes were assigned as comprehensively as possible by biochemical experiments, similarity searches, comparison of the gene structures, and phylogenetic analyses. Among the 10 biosynthetic genes investigated, seven (chalcone synthase, chalcone polyketide reductase, chalcone isomerase, IFS, isoflavone reductase, vestitone reductase, PTR) comprise multigene families, and in many cases they form gene clusters in the chromosomes. HI4’OMT, HID and isoflavone 2’-hydroxylase were found as single copies. Semi-quantitative RT-PCR analyses showed coordinate up-regulation of most of the genes during phytoalexin induction and complex accumulation patterns of the transcripts in different organs. Some paralogous genes exhibited similar expression specificities, suggesting their genetic redundancy. The results provide reliable annotations of the genes and genetic markers for comparative and functional genomics of leguminous plants. In planta characterization of genes of 5-deoxyisoflavonoid biosynthesis -- toward the metabolic engineering of (iso)flavonoid pathway Hairy root cultures of Lotus japonicus were established to characterize two heterologous cDNAs encoding enzymes in the isoflavone biosynthesis, i.e., licorice IFS and soybean HID catalyzing sequential reactions to yield isoflavones (Shimamura et al, 2007). While the control and the IFS overexpressor did not accumulate detectable isoflavones, the HID overexpressors did accumulate daidzein and genistein, showing

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that HID is a critical determinant of isoflavone productivity. Production of coumestrol in all the genotypes and isoliquiritigenin/liquiritigenin in IFS+HID overexpressing lines was also noted. The branching point of the 5-deoxyisoflavonoid pathway characteristic to leguminous plants is the formation of the deoxy-type chalcone (isoliquiritigenin), which is catalyzed by the coaction of chalcone synthase and polyketide reductase (PKR). A putative cDNA for PKR (PKR1) of L. japonicus was overexpressed in a red-flowered cultivar of petunia, “Polo Red Target”, which reduced anthocyanin accumulation and caused the formation of isoliquiritigenin and its putative derivatives (Shimada et al. 2006). These results suggested that PKR1 encodes a PKR that functions in planta. Functional and structural analysis of genes encoding oxidosqualene cyclases of L. japonicus The structures of cyclic triterpenoids of higher plants, whose skeletons are constructed by oxidosqualene cyclases (OSCs), are remarkably diverse in contrast to the limited variation in sterol/triterpenoid structures of other organisms. We comprehensively analyzed the functions and structures of OSC genes of L. japonicus, and compared them with those of A. thaliana in order to elucidate the diversification process of plant cyclic triterpenoids (Sawai et al. 2005). Eight OSC genes (OSC1–OSC8) formed two sets of clusters in chromosomes 2 and 3 of L. japonicus. OSC1, OSC3 and OSC5 were identified as β-amyrin synthase, lupeol synthase and cycloartenol synthase, respectively, by heterologous expression of the cDNAs in an OSC-disrupted yeast mutant (Fig. 2). A phylogenetic tree based on deduced amino acid sequences categorized eudicot OSCs into four groups and suggested an extensive diversification of the group IV OSCs.

Isoprenoidpathway

HO H

H

H

cycloartenol

Fig. 2. Triterpenoid biosynthesis in L. japonicus.

lanosterolβ-amyrin

OSC4,8

HO H

H

HH

lupeol

OSC3

OSC52,3-Oxidosqualene

OSC7 HO

H

H

H

?

HO H

H

OSC1

HO

H

H

H

α-amyrin

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Sterols are biosynthesized through either cyclic triterpenes, lanosterol (fungi and animals) or cycloartenol (plants). The cDNA for OSC7 of L. japonicus was shown to encode lanosterol synthase (LAS) by the complementation of a LAS-deficient mutant yeast and structural identification of the accumulated lanosterol (Sawai et al. 2006). A double site-directed mutant of OSC7, in which amino acid residues crucial for the reaction specificity were changed to the cycloartenol synthase (CAS) type, produced parkeol and cycloartenol. The multiple amino acid sequence alignment of a conserved region suggests that the LAS of different eukaryotic lineages emerged from the ancestral CAS by convergent evolution. Akashi, T., Aoki, T. and Ayabe, S. (2005) Molecular and biochemical characterization of

2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis. Plant Physiol. 137: 882-891.

Akashi, T., Koshimizu, S., Aoki, T. and Ayabe, S. (2006) Identification of cDNAs encoding pterocarpan reductase involved in isoflavan phytoalexin biosynthesis in Lotus japonicus by EST mining. FEBS Lett. 580: 5666-5670.

Sawai, S., Shindo, T., Sato, S., Kaneko, T., Tabata, S., Ayabe, S and Aoki, T. (2006) Functional and structural analysis of genes encoding oxidosqualene cyclases of Lotus japonicus. Plant Sci. 170: 247–257.

Sawai, S., Akashi, T., Sakurai, N., Suzuki, H., Shibata, D., Ayabe, S. and Aoki, T. (2006) Plant lanosterol synthase: divergence of the sterol and triterpene biosynthetic pathways in eukaryotes. Plant Cell Physiol. 47: 673-677.

Shimada, N., Nakatsuka, T., Nishihara, M., Yamamura, S., Ayabe, S. and Aoki, T. (2006) Isolation and characterization of a cDNA encoding polyketide reductase in Lotus japonicus. Plant Biotechnol. 23: 509–513.

Shimada, N., Sato, S., Akashi, T., Nakamura, Y., Tabata, S., Ayabe, S. and Aoki, T. (2007) Genome-wide analyses of the structural gene families involved in the legume-specific 5-deoxyisoflavonoid biosynthesis of Lotus japonicus. DNA Research 14: 25-36.

Shimamura, M., Akashi, T., Sakurai, N., Suzuki, H., Saito, K., Shibata, D., Ayabe, S and Aoki, T. (2007) 2-Hydroxyisoflavanone dehydratase is a critical determinant of isoflavone productivity in hairy root cultures of Lotus japonicus. Plant Cell Physiol. in press.

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Molecular Genetics of Nicotine Biosynthesis and Transport Takashi Hashimoto Graduate School of Biological Sciences, Nara Institute of Science and Technology, 630-0192 Nara, Japan. E-mail: [email protected] Nicotine is most familiar to us as a principal pharmacologically active component of cigarettes. This alkaloid is synthesized in the root in response to insect damage to the leaf, and then transported to the aerial parts of tobacco plants. We have been characterizing genes involved in nicotine biosynthesis and transport, utilizing the tobacco NIC regulatory mutants. Our recent strategy to tap on natural variations in alkaloid accumulation patterns in wild Nicotiana species will be introduced. Putrescine, a symmetrical diamine, is formed from basic amino acids, ornithine and/or arginine, and is metabolized to higher polyamines in all organisms and to particular alkaloids in restricted plant species (Hashimoto and Yamada 1994). Putrescine is metabolized to nicotine in tobacco and other Nicotiana and related species, and to pharmacologically active tropane alkaloids, such as hyoscyamine and scopolamine, in some medical solanaceous plants. The pyridine moiety of nicotine is derived from nicotinic acid or its metabolite, which is synthesized from aspartate via a salvage pathway of NAD synthesis (Kato et al. 2006). Since nicotine and tropane

alkaloids are expected to share the same evolutionary origin during the diversification of the Solanaceae, basic principles and molecular components revealed in the nicotine regulation may well be applied to tropane alkaloid biosynthesis.

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Results and Discussion 1. Site of nicotine formation and transport Putrescine N-methyltransferase (PMT) is the first committed enzyme in the biosynthetic pathways of nicotine and tropane alkaloids. PMT and A622 oxidoreductase genes are specifically expressed in the root of tobacco plants (Hibi et al. 1994). The A622 gene is coordinately regulated with the PMT gene, is required for accumulation of tobacco alkaloids (our unpublished results), and thus is postulated to encode an enzyme in nicotine pathway. Analysis by immunohistochemistry and promoter::GUS fusion reporters showed that both enzymes are localized in the same cell types in the root (Shoji et al. 2000; Shoji et al. 2002). High expression was observed at epidermis and cortex cells in the root tips, whereas in the differentiated region of the root, the outermost layer of the cortex and parenchyma cells surrounding xylem in vascular bundle were stained. These expression patters indicate that nicotine is synthesized in

sly and might be trapped inside the

enetic pproaches, we hope to identify the gene(s) responsible for the alkaloid transport.

root cells that are suitable for transport via xylem. Nicotine translocated to the leaf and other aerial tissues finally accumulates in the vacuole. We recently identified tobacco genes for tonoplast-localized tobacco transporters (NtMATE1 and NtMATE2) of the Multidrug and Toxic Compound Extrusion family that are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by Nic regulatory loci, and induction by methyljasmonate (our unpublished results). Nicotine might pass through tonoplast membrane spontaneouvacuole after forming ion-pairs with organic acids. Wild Nicotiana plants accumulate nicotine-related alkaloids in distinct amounts and patterns. Diploid tobacco species Nicotiana langsdorffii synthesize mainly nicotine in the root and transport it efficiently to the leaf, while closely related diploid species N. alata, known as an ornamental tobacco, synthesize nicotine, nornicotine, and anatabine in the root but does not transport these alkaloids to the aerial parts. To explore genetic loci controlling this transporting difference, we crossed these two species and analyzing alkaloid contents in their F1 and F2 progenies. Through molecular ga 2. Intracellular localization of biosynthetic enzymes The first three enzymes in the NAD pathway (aspartate oxidase, quinolinate synthase, and quinolinic acid phosphoribosyl transferase) are all localized in the plastid. Interestingly, we found that A622 oxidoreductase is targeted to the plastid, despite its apparent lack of a signal peptide (our unpublished results). Furthermore, a novel

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tobacco oxidase which is required for synthesis of tobacco alkaloids was localized in the vacuole of the root (our unpublished results). These results indicate that nicotine

iosynthesis occurs in several subcellular compartments.

Ha id biogenesis: Molecular aspects. Ann Rev Plant

Hi Y (1994) Gene expression in tobacco

Kaidopsis start with aspartate and occur in the plastid. Plant Physiol.

Sherase genes in the root of Nicotiana sylvestris. Plant Cell Physiol

Sheir possible roles in

secondary metabolism in tobacco. Plant Mol Biol 50:427-440

b

shimoto T, Yamada Y (1994) AlkaloPhysiol Plant Mol Biol 45:257-285 bi N, Higashiguchi S, Hashimoto T, Yamada low-nicotine mutants. Plant Cell 6:723-735 toh A, Uenohara K, Akita M, Hashimoto T (2006) Early steps in the biosynthesis of NAD in Arab141:851-857 oji T, Yamada Y, Hashimoto T (2000) Jasmonate induction of putrescine N-methyltransf41:831-839 oji T, Winz R, Iwasa T, Nakajima K, Yamada Y, Hashimoto T (2002) Expression patterns of two tobacco isoflavone reductase-like genes and th

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Dual biosynthetic pathways of phytosterol in plant Toshiya Muranaka KIHARA Institute for Biological Research, Yokohama City University, 641-12, Maioka-cho, Totsuka-ku, Yokohama, Kanagawa, 244-0813 Japan. E-mail:[email protected] Sterols are important as structural components of plasma membranes and precursors of steroidal hormones in both animals and plants. The differences in the biosynthesis of sterols between plants and animals are believed to begin at the step of cyclization of oxidosqualene, which is cyclized to lanosterol in animals and to cycloartenol in plants. Last year, three laboratories independently identified lanosterol synthase genes from dicotyledonous plant species including Arabidopsis, Panax, and Lotus. It is important to understand whether biosynthetic pathway of phytosterols via lanosterol generally exists or not. We show here the direct evidence that the biosynthetic pathway of phytosterol via lanosterol exists in plant cells. Sterols are 6,6,6,5-tetra cyclic triterpene alcohols, which are formed by the cyclization

of 2,3-oxidosqualene. This cyclization, which is catalyzed by oxidosqualene cyclases

(OSCs), is one of the most complicated and fascinating reactions found in nature. While

OSCs in vertebrates and fungi convert 2,3-oxidosqualene only to lanosterol, a sterol

precursor, OSCs in plants convert 2,3-oxidosqualene to cycloartenol and some other

cyclic triterpenes. For several decades, cycloartenol was thought to possibly replace

lanosterol as the first cyclic triterpene during phytosterol biosynthesis. The first cloned

CAS gene was isolated from Arabidopsis in 1993 using a chromatographic screen of a

heterologous expression system (Corey et al. 1993). This was the second OSC gene to

be cloned, following the LAS gene from Candida albicans. Following the cloning of

CAS1 from Arabidopsis, many cycloartenol synthase genes were isolated from

dicotyledonous plants.

Results and Discussion

1. Lanosterol synthase in plants

Despite the consensus that cycloartenol is the plant sterol precursor and the

identification of CAS genes from many plant species, lanosterol has also been identified

in some plants (Giner and Djerassi 1995, Itoh et al. 1977). As plants lack the ability to

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convert cycloartenol to lanosterol, lanosterol must be synthesized directly from

oxidosqualene. In other words, LAS must be present in the plant kingdom. Very recently

three laboratories independently identified LAS genes, including LAS1 (also called as

LSS) from Arabidopsis (Kolesnikova et al. 2006, Suzuki et al 2006), PNZ from Panax

ginseng (Suzuki et al 2006), and OSC7 from Lotus japonica (Sawai et al. 2006).

Arabidopsis At3g45130 (LAS1) was previously postulated to be a CAS, based on its

77% similarity in amino acid sequence to CAS1. LAS1 has been shown to complement

LAS-deficient yeast and to function as a LAS in plant cells (Suzuki et al 2006). LAS1 is

the first plant OSC whose function has been verified in planta. CAS1 mutant analyses

demonstrated that Tyr410, His477, and Ile481 are important for the CAS product

specificity. With just two amino acid substitutions, the CAS activity of CAS1

(His477Asn and ILe481Val) is converted to LAS activity (Lodeiro et al. 2005). It is

interesting to note that the amino acid residues of LAS1, PNZ, and OSC7, which

correspond to His477 and ILe481 of CAS1 are Asn and Val, respectively. This is

consistent with LAS1, PNZ, and OSC7 having LAS rather than CAS activity.

Phylogenetic analyses showed that LAS1, PNZ, and OSC7 belong to a branch

that is most closely related to but distinct from the plant CAS branch. The plant

LAS-type and CAS branches are different from other plant triterpenoid synthase

branches. It has been proposed that other plant triterpenoid synthases, such as lupeol

synthase and β-amyrin synthase, evolved in that order from ancestral CAS. Although

CAS and LAS cyclize 2,3-oxidosqualene through chair–boat–chair conformations, other

triterpenoid synthases cyclize 2,3-oxidosqualene through chair–chair–chair

conformations. The differences in the primary structure of plant OSCs may reflect

differences in their reaction mechanisms. Some other plant OSCs, such as TRV from

Taraxacum officinale, CPR from Cucurbita pepo, LcOSC2 from Luffa cylindrical, and

OSC6 from Lotus japonica belong to the plant LAS-type branch. However, no LAS

activity for these four OSCs has been identified. TRV, CPR, LcOSC2, and OSC6 may

represent intermediates in the evolution of plant LAS to other triterpenoid synthases.

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2. Biosynthetic pathway of phytosterols via lanosterol exists in plant

Plant CAS and LAS genes have been cloned from many plant species, and their activities have been characterized in heterologous expression systems. Yet little biological function of these genes has been determined. Only Arabidopsis LAS1 has been characterized in planta. It is important to understand whether biosynthetic pathway of phytosterols via lanosterol generally exists or not. To answer this question, we designed a tracer experiment using [6-13C2H3]mevalonate. By the elucidation of deuterium on C-19 behavior of phytosterol, it is possible to clarify that phytosterol is biosynthesized via either cycloartenol or lanosterol; two deuterium retained and three deuterium retained phytosterol are biosynthesized via cycloartenol and lanosterol, respectively. The number of deuterium can be analyzed by 13C-NMR; carbon spectrum connected a deuterium will be theoretically shifted to approximately 0.3 ppm up-field by isotope effect. According to this stratagy, [6-13C2H3]mevalonate was synthesized and fed to the seedlings of wild type and LAS1 overexpression plant of Arabidopsis. To enhance the incorporation ratio of the labeled mevalonate, lovastatin (inhibitor of HMG-CoA reductase) was applied at the same time. After incubation,

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extracted sitosterols were analyzed by 1H, 2H-double decoupled 13C-NMR. Although main peak was two deuterium-retained peak, three deuteriums retained peak was observed as minor peak. The peak intensity of sitosterol in LAS1 overexpression was approximately three times as strong as that in WT. This is the first direct evidence that the biosynthetic pathway of phytosterol via lanosterol existed in plant cells.

Corey, E.J., Matsuda, S.P.T., and Bartel, B. (1993) Isolation of an Arabidopsis thaliana

gene encoding cycloartenol synthase by functional expression in a yeast mutant

lacking lanosterol synthase by the use of a chromatographic screen, Proc. Natl. Acad.

Sci. USA 90, 11628-11632.

Giner, J.-L., and Djerassi, C. (1995) A Reinvestigation of the Biosynthesis of Lanosterol

in Euphorbia lathyris, Phytochemistry 39, 333-335.

Itoh, T., Jeong, M.T., Hirano, Y., Tamura, T., and Matsumoto, T. (1977) Occurrence of

Lanosterol and Lanostenol in Seeds of Red Pepper (Capsicum annuum), Steroids 29,

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569-577.

Kolesnikova, M.D., Xiong, Q., Lodeiro, S., Hua, L., and Matsuda, S.P.T. (2006)

Lanosterol biosynthesis in plants, Arch. Biochem. Biophys. 447, 87-95.

Lodeiro, S., Schulz-Gasch, T., and Matsuda, S.P.T. (2005) Enzyme Redesign: Two

Mutations Cooperate to Convert Cycloartenol Synthase into an Accurate Lanosterol

Synthase, J. Am. Chem. Soc. 127, 14132-14133.

Suzuki, M., Xiang, T., Ohyama, K., Seki, H., Saito, K., Muranaka, T., Hayashi, H.,

Katsube, Y., Kushiro, T., Shibuya, M., and Ebizuka, Y. (2006) Lanosterol Synthase in

Dicotyledonous Plants, Plant Cell Physiol. 47, 565-571.

Sawai, S., Akashi, T., Sakurai, N., Suzuki, H., Shibata, D., Ayabe, S., and Aoki, T.

(2006) Plant Lanosterol Synthase: Divergence of the Sterol and Triterpene

Biosynthetic Pathway in Eukaryotes, Plant Cell Physiol. 47, 673-677.

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Enzymology and molecular biology of aurone biosynthesis

Toru Nakayama Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-11, Aoba-ku, Sendai, Miyagi 980-8579, Japan. E-mail: [email protected]

Abstract. Aurones belong to a class of plant flavonoids that provide the bright yellow color of some important ornamental flowers, such as snapdragon (Antirrhinum majus). Although the aurone biosynthetic genes have been attractive tools to engineer novel yellow flowers, biochemical and genetic details of aurone biosynthesis were not established until recently. During the past several years, enzymes and genes involved in aurone biosynthesis in yellow snapdragon flowers have been identified. Aureusidin synthase, one of key enzymes in aurone biosysthesis, is a chalcone-specific vacuolar homolog of plant polyphenol oxidase (PPO), providing new insights into the role in plant secondary metabolism and subcellular localization of plant PPOs. The finding presented here will open the way to engineer yellow flowers for ornamental species lacking this color variant. Aurones confer yellow color to flowers of a variety of popular ornamental plants such as snapdragon, cosmos, and dahlia [1,2]. It has been suggested that aurones are closely related to chalcones in their biosynthesis [1]. The aurone biosynthetic genes have been attractive tools to engineer novel yellow flowers by genetic engineering approaches. However, details of the biosynthetic pathway of aurones have remained unknown since the discovery of aurones nearly 50 years ago. In 2000, aurones biosynthetic pathway in yellow snapdragon (Antirrhinum majus) flowers, which contain aurones (such as aureusidin and bracteatin 6-glucosides) in abundance, was clarified (Scheme I), and a key enzyme of the pathway, aureusidin synthase, and its cDNA (AmAS1) were isolated [1,2].

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Here I describe the enzymological and molecular biological aspects of aurone biosynthesis in yellow snapdragon flowers and the application of aurone biosynthetic genes in molecular breeding of yellow flowers.

Results and Discussion 1. Aureusidin synthase is a homolog of plant polyphenol oxidase

Aureusidin synthase is a binuclear copper enzyme with sugar chain(s) and specifically acted on chalcones with a 4-monohydroxy or 3,4-dihydroxy B-ring to produce aurones, for whose production the oxidative cyclization of chalcones must be preceded by 3-oxygenation [3]. Primary structure analysis of this enzyme revealed that it is a homolog of plant polyphenol oxidase (PPO), providing the first example of the biological role of plant PPO for flower coloration [2]. A mechanism of aurone synthesis by aureusidin synthase was consistently proposed on the basis of known PPO-catalyzed reactions [3], leading to the conclusion that the enzyme is a chalcone-specific PPO specialized for aurone biosynthesis in yellow snapdragon flowers. 2. Subcellular localization studies of aureusidin synthase

Despite many attempts, transgenic flowers that heterologously overexpress AmAS1 gene failed to produce aurones. To find a clue to overcome these circumstances, the subcellular localization of this enzyme in petal cells of the yellow snapdragon was investigated. Primary structural characteristics and some molecular properties of aureusidin synthase contradicted the enzyme’s localization in plastids and cytoplasm, despite the fact that known plant PPOs are all localized in plastids and flavonoid biosynthesis generally takes place in cytoplasm (or on the cytoplasmic surface of ER). Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER [4]. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme is localized within vacuole lumen. It was also found that the necessary information for the vacuolar targeting of the PPO is encoded within the 53-residue N-terminal sequence (NTPP), but not in C-terminal sequence of the precursor [4]. The NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera fused with a dominant negative mutant of Sar1 GTPase of Arabidopsis. A sequence-specific vacuolar sorting determinant in the NTPP of the precursor was also identified. These findings provide the first example of the biosynthesis of a flavonoid skeleton in vacuoles (Fig. 1). This metabolic compartmentation should serve as a

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strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower.

Figure 1. The proposed intracellular compartmentation of aurone biosynthetic pathway in petal cells of

snapdragon flowers. Synthesis, glycosylation, and isomerization of chalcones may take place on the

cytoplasmic surface of the ER. We previously showed that aureusidin can be produced from either THC

or PHC, whereas bracteatin arises solely from PHC. Moreover, the 4’-O-glucosides of these chalcones

serve as very good substrates for enzymatic aurone synthesis [1,2]. AS, aureusidin synthase; CHI,

chalcone isomerase; C4’GT, chalcone 4’-O-glucosyltransferase; and Glc, β-D-glucopyranosyl.

3. Engineering of yellow flowers by molecular breeding

These considerations, in turn, imply the importance of intracellular translocation of substrate chalcones from cytoplasm to vacuoles for aurone synthesis (Fig. 1), and this process should most likely be facilitated by the 4’-O-glucosylation of the substrate. Thus, a cDNA coding for a chalcone 4’-O-glucosyltransferase from yellow snapdragon flowers was isolated [5]. This enzyme, AmC4’GT, was a member of UGT88-related plant secondary metabolite glycosyltransferases with a regiospecificity of glucosyl

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transfer to 4’-position of chalcones. As expected, co-expression of the AmC4’GT and AmAS1 genes was sufficient for the accumulation of aureusidine 6-O-glucoside in transgenic flowers (Torenia hybrida). Moreover, their co-expression, combined with down-regulation of anthocyanin biosynthesis by RNA interference (RNAi), resulted in yellow flowers. An AmC4’GT-GFP chimeric protein localized in the cytoplasm, whereas the AmAS1(N1-60)-RFP chimeric protein was localized to the vacuole. It was therefore concluded that chalcones are 4’-O-glucosylated in the cytoplasm, then transported to the vacuoles, and therein oxidatively converted to aurone 6-O-glucosides [5]. These findings not only demonstrate the biochemical basis of aurone biosynthesis but also open the way to engineer yellow flowers for major ornamental species lacking this color variant.

Acknowledgements This work has been carried out in collaboration of Tohoku University with Suntory Ltd, Kobe Gakuin University, Minami-Kyushu University, Kyoto University, and Osaka Medical College. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by grants from The Asahi Glass Foundation, Shorai Foundation, and Novartis Foundation. Literatures Cited [1] Sato T, Nakayama T, Kikuchi S, Fukui Y, Yonekura-Sakakibara K, Ueda T, Nishino

T, Tanaka Y, Kusumi T (2001) Enzymatic formation of aurones in the extracts of yellow snapdragon flowers. Plant Sci 160: 229-236

[2] Nakayama T, Yonekura-Sakakibara K, Sato T, Kikuchi S, Fukui Y,

Fukuchi-Mizutani M, Ueda T, Nakao M, Tanaka Y, Kusumi T, Nishino T (2000) Aureusidin synthase: a polyphenol oxidase homolog responsible for flower coloration. Science 290: 1163-1166

[3] Nakayama T, Sato T, Fukui Y, Yonekura-Sakakibara K, Hayashi H, Tanaka Y,

Kusumi T, Nishino T (2001) Specificity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase homolog responsible for flower coloration. FEBS Lett 499: 107-111

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[4] Ono E, Hatayama M, Isono Y, Sato T, Watanabe R, Yonekura-Sakakibara K,

Fukuchi-Mizutani M, Tanaka Y, Kusumi T, Nishino T, Nakayama T (2006) Localization of a flavonoid biosynthetic polyphenol oxidase in vacuoles. Plant J 45: 133-143

[5] Ono E, Fukuchi-Mizutani M, Nakamura N, Fukui Y, Yonekura-Sakakibara K,

Yamaguchi M, Nakayama T, Tanaka T, Kusumi T, Tanaka Y. (2006) Yellow flowers generated by expression of the aurone biosynthetic pathway. Proc. Natl. Acad. Sci. USA, 103: 11075-11080

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Camptothecin Biosynthetic System – Pathway Elucidation,

Gene Discovery and Self-Resistance

Kazuki Saito1,2, Supaart Sirikantaramas1, Hiroshi Sudo1, Takashi Asano1,3, Mami Yamazaki1,3

1Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522,

Japan; 2RIKEN Plant Science Center, Yokohama 230-0045, Japan; 3CREST, Japan

Science and Technology Agency, Saitama 332-0012, Japan. E-mail:

[email protected]

Camptothecin, a plant-originated alkaloid, exhibits an antitumor activity due to its inhibitory action to DNA topoisomerase I (1). At present, semisynthetic water-soluble camptothecin analogues, topotecan and irinotecan, are prescribed as clinical antitumor drugs throughout the world. Despite its quinoline structure, camptothecin belongs biogenetically to a family of modified monoterpenoid indole alkaloids. However, the information about genes and pathway after strictosidine is limited. In the last several years we have been investigating the camptothecin biosynthetic systems for pathway elucidation, gene discovery and self-resistance.

Production system by cell culture

Initially we have established a hairy root culture of Ophiorrhiza pumila (Rubiaceae) transformed by Agrobacterium rhizogenes strain 15834. This hairy root culture grew well, increasing by 16-fold during 5 weeks in liquid culture, and it produced camptothecin as a main alkaloid up to 0.1% per dry weight of the cells. Interestingly, not only the hairy root cells contained camptothecin, but the culture medium also accumulated substantial amounts. Camptothecin content in the medium was increased by the presence of a polystyrene resin (Diaion HP-20) that absorbed camptothecin. Camptothecin was easily recovered from the resin. This hairy root culture is a feasible

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system not only for practical production of camptothecin and excretion of camptothecin, but this system is ideal also for pathway elucidation and gene discovery (2-4). Regeneration method of O. pumila plantlets from hairy roots was also established (5).

Pathway elucidation and gene expression studies

We investigated the biosynthetic pathway of camptothecin from [1-13C]glucose by in silico and in vivo studies. The in silico study measured the incorporation of glucose into alkaloids using the Atomic Reconstruction of Metabolism software and predicted the labeling patterns of successive metabolites from [1-13C] glucose. The in vivo study followed incorporation of [1-13C] glucose into camptothecin with hairy roots of O. pumila by 13C nuclear magnetic resonance spectroscopy. The 13C-labeling pattern of camptothecin isolated from the hairy roots clearly showed that the monoterpene-secologanin moiety was synthesized via the 2C-methyl-D-erythritol 4-phosphate pathway, not via the mevalonate pathway. This conclusion was supported by differential inhibition of camptothecin accumulation by the pathway-specific inhibitors (fosmidomycin and lovastatin). The quinoline moiety from tryptophan was also labeled as predicted by the Atomic Reconstruction of Metabolism program via the shikimate pathway. These results indicate that camptothecin is formed by the combination of the 2C-methyl-D-erythritol 4-phosphate pathway and the shikimate pathway (6).

We have cloned and characterized the cDNAs encoding strictosidine synthase (OpSTR; EC 4.3.3.2) and tryptophan decarboxylase (OpTDC; EC 4.1.1.28), two key enzymes in the biosynthesis of monoterpenoid indole alkaloids from hairy roots of O. pumila. We also isolated the cDNA coding for NADPH: cytochrome P450 reductase (OpCPR; EC 1.6.2.4) that is presumed to be indirectly involved in camptothecin synthesis. The recombinant OpSTR and OpTDC proteins exhibit STR and TDC activities, respectively, when expressed in Escherichia coli. The tissue-specific and stress-inducible expression patterns of OpSTR and OpTDC were quite similar, unlike those of OpCPR. The high expression of OpSTR and OpTDC observed in hairy roots, roots and stems were closely correlated with STR protein accumulation as observed by immunoblot analysis. Plant stress compounds like salicylic acid repressed expression of OpSTR and OpTDC, suggesting coordinate regulation of these genes for camptothecin biosynthesis (7).

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Comprehensive gene discovery

From this hairy root culture, we have established the non-differentiated cell-suspension culture that does not produce these secondary products. Thus, the comparison of these hairy root and cell suspension cultures is a desirable experimental system for research of molecular biology and biochemistry of camptothecin biosynthesis. We have conducted PCR-select cDNA subtraction for those two cultures to isolate cDNA fragments which are specifically expressed in camptothecin-producing tissues. In addition, the full-length cDNA clones have been sequenced from the both sides. Functional identification of those cDNAs that are presumed to be involved in biosynthesis of camptothecin is now undertaken by RNAi strategy in transformed roots and analysis of recombinant proteins.

Transport of camptothecin

We have investigated the subcellular accumulation and transport of camptothecin in hairy roots of O. pumila. When the hairy roots were exposed to UV radiation, autofluorescence emitted from CPT showed subcellular localization of CPT in the vacuole. Treatment with several inhibitors suggested that camptothecin excretion is a transporter-independent passive transport controlled by the concentration gradient of the compound. Interestingly, the hairy roots treated with brefeldin A, a vesicle transport inhibitor, showed increased camptothecin excretion. This could be explained by an increased transport rate of camptothecin from the endoplasmic reticulum (ER) to the cytoplasm when transport of camptothecin to the vacuole is blocked. The much higher concentration of camptothecin in the cytoplasm resulted in the increased excretion rate. This result indicates that camptothecin is biosynthesized at the ER and transported to accumulate in the vacuole by the same machinery that is used for vacuolar protein sorting (8).

Self-resistance mechanism of camptothecin-producing plants

Camptothecin exhibits eukaryotic topoisomerase I (TOP1) poisoning activity, resulting in cell death. Because of its toxicity to a house-keeping enzyme of cells, we addressed the question how the camptothecin-producing plant cells survive in the presence of camptothecin. We hypothesized that these plants might possess camptothecin-resistant type topoisomerase I. In fact, the recombinant OpTOPI from O. pumila expressed in yeast was resistant to camptothecin. Amino acid sequence analysis of OpTOP1 revealed

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that the highly conserved residue next to the catalytic site has been mutated. Coincidently, the identical mutation has been observed with the camptothecin-resistant human topoisomerase I. To confirm that the mutation is concomitant with and presumably caused by the presence of camptothecin, we compared the amino acid sequence of topoisomerase I from O. japonica, which is closely related to O. pumila but does not produce camptothecin. As expected, no mutation was confirmed in this non-producing species. Our findings suggest the possibility of adaptive co-evolution of topoisomerase I with camptothecin biosynthetic pathway in camptothecin-producing plants.

1. Sirikantaramas S, Asano T, Sudo H, Yamazaki M, Saito K: Camptothecin Therapeutic

potential and biotechnology. Curr. Pharm. Biotech., 8, 196-202 (2007) 2. Saito S, Sudo H, Yamazaki M, Koseki-Nakamura M, Kitajima M, Takayama H, Aimi N:

Feasible production of camptothecin by hairy root culture of Ophiorrhiza pumila. Plant

Cell Rep., 20, 267-271 (2001) 3. Sudo H, Yamakawa T, Yamazaki M, Aimi N, Saito K: Bioreactor production of

camptothecin by hairy root cultures of Ophiorrhiza pumila. Biotech. Lett., 24, 359-363 (2002)

4. Yamazaki Y, Urano A, Sudo H, Kitajima M, Takayama H, Yamazaki M, Aimi N. Saito K: Metabolite profiling of alkaloids and strictosidine synthase activity in camptothecin

producing plants. Phytochemistry, 62, 461-470 (2003) 5. Watase I, Sudo H, Yamazaki M, Saito K: Regeneration of transformed Ophiorrhiza

pumila plants producing camptothecin. Plant Biotech., 21, 337-342 (2004) 6. Yamazaki Y, Kitajima M, Arita M, Takayama H, Sudo H, Yamazaki M, Aimi N, Saito K:

Biosynthesis of camptothecin. In silico and in vivo tracer study from [1-13C]glucose. Plant

Physiol., 134, 161-170 (2004) 7. Yamazaki Y, Sudo H, Yamazaki M, Aimi N, Saito K: Camptothecin biosynthetic genes in

hairy roots of Ophiorrhiza pumila: Cloning, characterization and differential expression in

tissues and by stress compounds. Plant Cell Physiol., 44, 395-403 (2003) 8. Sirikantaramas S, Sudo H, Asano T, Yamazaki M, Saito K: Transport of camptothecin in

hairy roots of Ophiorrhiza pumila. Phytochemistry, in press (2007)

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Comparative analysis of ABA signaling between moss and

higher plants Yoichi Sakata Department of BioSciences, Tokyo Univ. of Agriculture, Tokyo 156-8502, Japan E-mail: [email protected] Introduction The phytohormone abscisic acid (ABA) not only regulates processes occurring during seed development, but also controls processes associated with responses to drought during vegetative development of seed plants. ABA is also found in most land plants (Finkelstein et al., 2002) and has demonstrated physiological and molecular responses to exogenous ABA in non-seed plants such as mosses (Werner et al., 1991; Goode et al., 1993; Minami et al., 2003) that are considered as basal land plants in the evolutional tree of plants. With sequenced genome and advanced tools for studying gene function such as RNA interference, inducible promoters and gene targeting, the moss Physcomitrella patens is emerging as a model plant for comparative and functional genomics (Quatrano et al., 2007). To gain insights into the evolution of ABA functions and the signaling pathway in land plants, we have decided to take a comparative genomic approach using P. patens. ABI3 A genetic approach using a model plant Arabidopsis thaliana has contributed to the identification of several important factors involved in ABA signal transduction pathway. ABA-insensitive (abi) mutants were identified by screening seedlings that were able to germinate in the presence of ABA (Koornneef et al., 1984). Subsequent cloning of the responsible genes revealed that ABI1 and ABI2 encoded homologous type 2C protein phosphatases (PP2Cs) (Leung et al., 1994; Meyer et al., 1994; Leung et al., 1997) and ABI3, ABI4 and ABI5 encoded different types of transcription factors (Giraudat et al., 1992; Finkelstein et al., 1998; Finkelstein and Lynch, 2000). Among these factors, ABI3 functions mainly in seeds and is believed as a regulatory factor of seed maturation. However, we have identified three ABI3-like genes named PpABI3A, PpABI3B and PpABI3C from P. patens. These three genes were expressed through the life cycle of P. patens and were activated by exogenous ABA. We tested their activity in both P. patens

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and barley aleurone cells using promoters of ABA-responsive late embryogenesis abundant (LEA) protein genes from P. patens and wheat (Em). As with ABI3/VP1, PpABI3A enhanced ABA-induced PpLea1-GUS and Em-GUS expression in protonemal tissue and in barley aleurone cells. Furthermore, it was demonstrated that PpABI3A driven by the Arabidopsis ABI3 promoter partially complemented the phenotypes of the Arabidopsis abi3-6 mutant. The partial molecular complementation of abi3-6 might be due in part to a failure of PpABI3A to interact with the bZIP transcription factor ABI5, which is required for proper expression of the ABI3-regulated genes in Arabidopsis. To investigate further the function of PpABI3, we established the transgenic P. patens plants that overexpress PpABI3A (PpABI3A OEs), and triple knockout plants of PpABI3 genes (PpABI3 KOs). Protonemata of P. patens are known to enhance the freezing tolerance in response to exogenous ABA (Minami et al., 2005). The ABA-induced freezing tolerance was enhanced in PpABI3 OEs, and was reduced in PpABI3 KOs. These results demonstrated that ABI3 evolved before the separation of bryophytes and vascular plants to control the ABA responses in vegetative tissue. It is possible that ABI3 continued to evolve in angiosperms to regulate the ABA signaling specifically during seed development. ABI1-related PP2C Among ABI genes, ABI1 and ABI2 are distinct from others in respect that they encode protein phosphatases which would be involved in phospholylation signaling and, in addition, that the functions extend though seed maturation and germination to vegetative growth. Previously we reported that abi1-1 that is a dominant allele of ABI1 blocked the ABA-induced activation of Em-GUS in P. patens transient assay (Marella et al., 2006). The result indicated that bryophytes also possess the PP2C-regulated ABA signaling pathway. We searched the EST database PHYSCObase (Nishiyama et al., 2003) as well as the genome database (http://genome.jgi-psf.org/Phypa1_1/Phypa1_1.home.html) for genes encoding PP2Cs. As the result, we have identified 51 genes encoding the highly conserved catalytic domain of PP2C. We generated a phylogenetic tree of the 51 putative PP2Cs with Arabidopsis 76 PP2Cs, and identified a clade that contains Arabidopsis 9 ABI1-related PP2Cs and closely-related two PP2Cs from P. patens. The expressions of the two P. patens genes, termed PpABI1A and PpABI1B, were observed through the life cycle of P. patens, although PpABI1A was predominant. Interestingly, the expression of PpABI1A but not PpABI1B was activated by cold treatment. Single disruption of PpABI1A resulted in the enhanced responses to exogenous ABA. ABA-induced expressions of

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LEA protein genes were increased in PpABI1A KO plants compared to the wild type. The most prominent phenotype of PpABI1A KO plants was observed in the freezing experiment. The freezing tolerance at -5°C of PpABI1A KO plants in the absence of ABA was slightly higher than that of wild type. A marked increase of the survival rate was observed when PpABI1A KO plants were subjected to freezing stress after treatment with extremely low concentration of ABA (0.01µM), whereas that of wild type was barely affected with this treatment. These results demonstrated that PpABI1A is a negative regulator of ABA signaling and regulates the expression of stress responsive genes and the physiological response to freezing stress. We showed ABI1-related PP2Cs also evolved before the separation of bryophytes and vascular plants, suggesting that PP2C-mediated regulation of ABA signaling is an ancient mechanism to control the response of land plants to environmental stresses. Unlike ABI3, ABI1-related PP2Cs increased in number during the evolution of land plants. It is possible that the increase in number of the ABI1-related PP2Cs enabled the fine-tuning of ABA signaling in complex tissues/organs of angiosperms. ABA catabolism To understand the regulation of ABA level in P. patens, we examined protonemata using LC/MS/MS system for the presence of known ABA catabolites, ABA glucosyl ester (ABA-GE), phaseic acid (PA), dihydrophaseic acid (DPA), 7′-hydroxy ABA (7′-OH ABA), 8′-hydroxy ABA (8′-OH ABA) and neophaseic acid (neoPA). Interestingly, ABA-GE, PA and DPA, the major ABA catabolites in seed plants (Nambara and Marion-Poll, 2005), were detected neither in cellular extracts nor in media extracts. Instead, considerable amount of ABA was recovered from media extracts. We also detected neoPA, a 9′-hydroxylated catabolite (Zhou et al., 2004) from cellular extracts and media extracts. These results indicated that P. patens has the 9′-hydroxylation pathway and also the system for exracellular export of ABA and neoPA to control cellular ABA level. To see the effect of manipulation of ABA catabolism in P. patens, we generated transgenic P. patens lines that overexpress Arabidopsis CYP707A3 gene encoding ABA 8′-hydroxylase (Kushiro et al., 2004) under the control of the rice actin promoter. Because ABA 8′-hydroxylase catalyzes the first step of ABA catabolic pathway in Arabidopsis, CYP707A3 plants were expected to accumulate less ABA. We found that CYP707A3 plants resulted in the spontaneous appearance of macrochloroplasts in protonemal cells. The macrochloroplast phenotype of CYP707A3 plants was relieved

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by exogenously applied ABA, and application of an ABA biosynthesis inhibitor Abamine SG to wild type caused the macrochloroplasts phenotype. Inversely, application of ABA to wild type increased the number of chloroplasts in protonemal cells. These results suggest that ABA is involved in the chloroplast division. Conclusions Our comparative analysis suggested that the fundamental regulatory network of ABA signaling was already established when the first drought-tolerant land plants appeared. Genomic- and full-length EST resources coupled with efficient gene targeting and experimentally tractable system will make P. patens the model system to dissect the ancient ABA regulatory network as well as to provide new findings in ABA functions and the regulation of ABA metabolism. . Finkelstein, R.R., and Lynch, T.J. (2000). The Arabidopsis abscisic acid response gene ABI5 encodes a

basic leucine zipper transcription factor. Plant Cell 12, 599-609.

Finkelstein, R.R., Gampala, S.S., and Rock, C.D. (2002). Abscisic acid signaling in seeds and seedlings.

Plant Cell 14 Suppl, S15-45.

Finkelstein, R.R., Wang, M.L., Lynch, T.J., Rao, S., and Goodman, H.M. (1998). The Arabidopsis

abscisic acid response locus ABI4 encodes an APETALA 2 domain protein. Plant Cell 10,

1043-1054.

Giraudat, J., Hauge, B.M., Valon, C., Smalle, J., Parcy, F., and Goodman, H.M. (1992). Isolation of

the Arabidopsis ABI3 gene by positional cloning. Plant Cell 4, 1251-1261.

Goode, J.A., Stead, A.D., and Duckett, J.G. (1993). Redifferentiation of moss protonemata: an

experimental and immunofluorescence study of brood cell formation. Can. J. Bot. 71,

1510-1519.

Koornneef, M., Reuling, G., and Karssen, C.M. (1984). The isolation and characterization of abscisic

acid-insensitive mutants of Arabidopsis thaliana. Physiol. Plant. 61, 377-383.

Kushiro, T., Okamoto, M., Nakabayashi, K., Yamagishi, K., Kitamura, S., Asami, T., Hirai, N.,

Koshiba, T., Kamiya, Y., and Nambara, E. (2004). The Arabidopsis cytochrome P450

CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism. EMBO J. 23,

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Leung, J., Merlot, S., and Giraudat, J. (1997). The Arabidopsis ABSCISIC ACID-INSENSITIVE2

(ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid

signal transduction. Plant Cell 9, 759-771.

Leung, J., Bouvier-Durand, M., Morris, P.C., Guerrier, D., Chefdor, F., and Giraudat, J. (1994).

Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase.

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Science 264, 1448-1452.

Marella, H.H., Sakata, Y., and Quatrano, R.S. (2006). Characterization and functional analysis of

ABSCISIC ACID INSENSITIVE3-like genes from Physcomitrella patens. Plant J. 46, 1032-1044.

Meyer, K., Leube, M.P., and Grill, E. (1994). A protein phosphatase 2C involved in ABA signal

transduction in Arabidopsis thaliana. Science 264, 1452-1455.

Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., and Takezawa, D. (2003). Abscisic acid-induced

freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of

stress-related genes. J. Plant. Physiol. 160, 475-483.

Minami, A., Nagao, M., Ikegami, K., Koshiba, T., Arakawa, K., Fujikawa, S., and Takezawa, D.

(2005). Cold acclimation in bryophytes: low-temperature-induced freezing tolerance in

Physcomitrella patens is associated with increases in expression levels of stress-related genes but

not with increase in level of endogenous abscisic acid. Planta 220, 414-423.

Nambara, E., and Marion-Poll, A. (2005). Abscisic acid biosynthesis and catabolism. Annu. Rev. Plant

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Nishiyama, T., Fujita, T., Shin, I.T., Seki, M., Nishide, H., Uchiyama, I., Kamiya, A., Carninci, P.,

Hayashizaki, Y., Shinozaki, K., Kohara, Y., and Hasebe, M. (2003). Comparative genomics of

Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: implication for

land plant evolution. Proc. Natl. Acad. Sci. USA 100, 8007-8012.

Quatrano, R.S., McDaniel, S.F., Khandelwal, A., Perroud, P.-F., and Cove, D.J. (2007).

Physcomitrella patens: mosses enter the genomic age. Current Opinion in Plant Biology 10, 182.

Werner, O., Ros Espín, R.M., Bopp, M., and Atzorn, R. (1991). Abscisic-acid-induced drought

tolerance in Funaria hygrometrica Hedw. Planta 186, 99.

Zhou, R., Cutler, A.J., Ambrose, S.J., Galka, M.M., Nelson, K.M., Squires, T.M., Loewen, M.K.,

Jadhav, A.S., Ross, A.R.S., Taylor, D.C., and Abrams, S.R. (2004). A New Abscisic Acid

Catabolic Pathway. Plant Physiol. 134, 361-369.

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Metabolic Engineering in Benzylisoquinoline Alkaloid

Biosynthesis Fumihiko Sato Department of Plant Gene and Totipotency, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan. E-mail: [email protected] Higher plants produce diverse classes of metabolites. Metabolic engineering offers tremendous potential to improve the production and quality of these chemicals. This report summarizes the possibility of using metabolic engineering in benzylisoquinoline alkaloid biosynthesis. First, the overexpression of a rate-limiting enzyme in an early pathway to increase the overall alkaloid yield is discussed. Second, the possibility of accumulating a pathway intermediate by the knock-down of a key step is examined. Finally, the introduction of a new branch into the pathway has been shown to produce novel metabolites. Further metabolic modification is also discussed, since the latter two modifications may lead to the production of novel compound(s) from an accumulated intermediate through metabolic activation. Introduction Isoquinoline alkaloids are a large and diverse group of alkaloids with ~2500 defined structures. They include the analgesic morphine from Papaver somniferum L.; the antigout colchicine from Colchicum autumnale L.; the emetic and antiamoebic emetine from Cephaelis ipecacuanha (Brot.) A. Rich.; the skeletal muscle relaxant tubocurarine from Chondodendron tomentosum; and the antimicrobial compounds berberine and sanguinarine from diverse plant species including Berberis spp., Sanguinaria spp., and Coptis spp., many of which are used as pharmaceuticals [1,2]. Isoquinoline alkaloid biosynthesis begins with the conversion of tyrosine to both dopamine and 4-hydroxyphenylacetaldehyde (4HPAA) by decarboxylation, ortho-hydroxylation, and deamination (Fig. (1)) [2,3]. Dopamine and 4HPAA are condensed by norcoclaurine synthase (NCS) to yield (S)-norcoclaurine, which is the central precursor to all isoquinoline alkaloids. [4].

(S)-Norcoclaurine is sequentially converted to coclaurine by S-adenosyl methionine (SAM)-dependent norcoclaurine 6-O-methyltransferase (6OMT) [5], to N-methylcoclaurine by coclaurine N-methyltransferase [6], to 3'-hydroxy-N-methyl

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coclaurine by P450 hydroxylase [7], and then to (S)-reticuline by 3'-hydroxy N-methylcoclaurine 4'-O-methyltransferase (4'OMT) [5]. cDNAs for these reactions have been isolated from several plant species and their enzymological properties for several recombinant proteins have been characterized. Whereas detailed biochemical studies using recombinant enzymes have been limited, available data suggest that these enzymes show strict reaction specificities and distinct properties, and regulate biosynthesis sequentially and in a coordinated manner.

Many isoquinoline alkaloids are produced from the central intermediate reticuline, while P450-dependent oxidase (berbamunine synthase, CYP80A1) [8] produces dimeric bisbenzylisoquinoline alkaloids, such as berbamunine and tubocurarine, from the intermediates of the (S)-reticuline pathway. Berberine bridge enzyme (BBE) plays a role in the first committed step in the biosynthesis of benzophenanthridine (e.g. sanguinarine and marcarpine), and protoberberine alkaloid (e.g. berberine and palmatine), which converts the N-methyl group of (S)-reticuline into the methylene bridge moiety of (S)-scoulerine [9]. In benzophenanthridine alkaloid biosynthesis, (S)-scoulerine can be converted to (S)-stylopine by two P450-dependent oxidases, (S)-chelanthifoline synthase and (S)-stylopine synthase, which result in the formation of two methylenedioxy groups (see refs. [2, 10]). On the other hand, in protoberberine biosynthesis, (S)-scoulerine is converted to (S)-tetrahydrocolumbamine by the SAM-dependent scoulerine 9-O-methyltransferase (SMT) [11], and then to tetrahydroberberine (canadine) by a P450-dependent canadine synthase (CDS or CYP719A1) [12], and to berberine by tetrahydroberberine oxidase [13, Minami et al. unpublished data]. Metabolic engineering in isoquinoline alkaloid biosynthesis While breeding for secondary metabolite productivity in medicinal plants is not currently popular, this approach may offer considerable potential. Classical breeding is, however, limited by the need for time-consuming processes to establish stable lines and high gene redundancy, which hinders isolation of the mutant phenotype. Thus, metabolic engineering in isoquinoline alkaloid biosynthesis with transgenic technology is needed, especially now, when major pathways have been characterized at enzymological and molecular levels; i.e., many biosynthetic genes are available and technological tools for the transformation and analysis of metabolites have been developed [2]. 1) Strategy to increase yield A practical approach to increasing the yield is to find the most critical

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rate-limiting step in the pathway. An overall analysis of enzyme activities in the target pathway should help to identify the rate-limiting step. For example, the clear difference in enzyme activities in the early steps of berberine biosynthesis between high- and low-producing C. japonica cells [14] indicates the importance of early steps in high productivity. The over-expression of enzyme gene in host cells can provide direct evidence for examining the rate-limiting step. In California poppy (Eschscholzia californica) cells, the ectopic expression of two O-methyltransferase (Cj6OMT and Cj4’OMT) cDNAs isolated from Coptis japonica cells clearly illustrated the usefulness of this strategy. Ectopic expression of Cj6OMT markedly increased the production of benzophenanthridine alkaloid biosynthesis, whereas that of 4’OMT gave only a marginal increase in alkaloid production [15]. Since the early step in isoquinoline biosynthesis from tyrosine to reticuline is common to the biosynthesis of many isoquinoline alkaloids, our results may provide the general basis for improving isoquinoline alkaloid production.

Overall gene expression with a master transcriptional factor would be a more effective way to increase metabolite production. Our current efforts to isolate master transcription factors in alkaloid biosynthesis are still in the primary stage, while our recent successful isolation of a WRKY gene may lead to the transcriptional regulation of isoquinoline alkaloid biosynthesis [16,17]. 2) Inhibition of metabolic pathway to accumulate the intermediates.

Important approach to modify the alkaloid profiles is the inhibition of pathway. As shown in the top1 mutant of poppy [18], the alkaloid profile can be converted from morphine to thebaine and oripavine. Similarly, knock-down of the last step in morphine biosynthesis by RNAi of COR [19] induced the accumulation of reticuline and its methylated products although we could not explain the mechanism of this accumulation. These data suggest that pathway trimming is an efficient way to increase chemical diversity as in the introduction of a new pathway. On the other hand, we also found that when we carefully inhibit a pathway, especially at a point where branching pathways are limited, we can accumulate the intermediate of that pathway. Thus, direct gene silencing of BBE in a pathway with RNAi enabled the accumulation of reticuline in transgenic California poppy cells [20], whereas transgenic California poppy cells with antisense BBE RNA did not accumulate the intermediate [21]. While RNAi technology still needs further improvement before it can be widely applied in metabolic engineering, RNAi is clearly a very powerful tool for gene silencing. 3) Introduction of a new pathway to increase the metabolite-profile diversity

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Full-length cDNA resources of biosynthetic genes now provide more opportunities to modify metabolic pathways in plant cells, although there are still few published results. Molecular engineering in isoquinoline alkaloids was first performed in California poppy with the over-expression of C. japonica scoulerine O-methyltransferase (SMT) cDNA [22]. Complete modification of the alkaloid profile with the introduction of CjSMT cDNA into California poppy cells was observed; i.e., the alkaloid profile changed from sanguinarine (benzophenanthridine-type) to columbamine (berberine-type).

Interestingly, a newly introduced pathway can provide the substrate for further enzymatic conversion and produce novel compounds which are not detected in wild-type cells. Whereas new compounds are still being identified, more than 10 new peaks, which were not found in non-transformed cells, have been detected [22, Takemura et al. unpublished data]. LC-MS/LC-NMR analyses suggested that transgenic California poppy cells with CjSMT accumulated a novel protopine-type alkaloid and also modified benzophenanthrizine alkaloids, which suggests that pre-existing enzymes would function as newly accumulated metabolites to produce a more diverse array of chemicals.

Future perspectives While the above two strategies, i.e., the introduction of a new branch pathway and the knock-down of a pathway, enhanced metabolic diversity through pathway modification, cultured cells offer greater opportunity to increase metabolic diversity. The chemical diversity in transgenic California poppy cells is quite high. While metabolically modified transgenic California poppy cells with CjSMT overproduction or RNAi of BBE clearly showed marked differences from wild type cells in metabolite profiles, these transgenic cells lines also showed marked heterogeneity within the same transformants with identical constructs. This observed divergence may be due to the activation of a silent pathway, which was inactive when metabolism was unmodified and the substrate level was low. Another possibility is that cells might respond to the new metabolite levels and induce responsive genes. Furthermore, additional somaclonal variation might occur after modification of the biosynthetic pathway. While the molecular mechanism of how secondary metabolism evolved is not clear, our results suggest that plant cells have high potential to adapt to new chemical conditions and produce a greater diversity of chemicals.

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Acknowledgements This work was supported in part by the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS- RFTF00L01606) and by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to F.S.), [1] Croteau, R., Kutchan, T.M. and Lewis, N.G. (2000) Natural products (Secondary

metabolites), in Biochemistry & Molecular Biology of Plants, (Buchanan, B.B.; Gruissem, W. and Jones, R.L. Ed.), Am. Soc. Plant Physiol., Maryland, pp. 1250-1318.

[2] Sato F., Inui T. and Takemura T. (2007) Curr. Pharmaceut. Biotech., 8, 211-218. [3] Facchini, P. (2001) Annu. Rev. Plant Physiol. Plant Mol. Biol., 52, 29-66. [4] Minami, H., Dubouzet, E., Iwasa, K. and Sato, F. (2007) J. Biol. Chem., 282,

6274-6282. [5] Morishige, T., Tsujita, T., Yamada, Y. and Sato, F. (2000) J. Biol. Chem., 275,

23398-23405. [6] Choi, K.B. Morishige, T., Shitan, N., Yazaki, K. and Sato, F. (2002) J. Biol. Chem.,

277, 830-835. [7] Pauli, H.H. and Kutchan, T.M. (1998) Plant J., 13, 793-801. [8] Kraus, P.F.X. and Kutchan, T.M. (1995) Proc. Natl. Acad. Sci. USA, 92 (6),

2071-2075. [9] Dittrich, H. and Kutchan, T.M. (1991) Proc. Natl. Acad. Sci. USA, 88, 9969-9973. [10] Ikezawa, N., Iwasa, K. and Sato, F. (2007) FEBS J., 274(4),1019-1035. [11] Takeshita, N., Fujiwara, H., Mimura, H., Fitchen, J.H., Yamada, Y. and Sato, F.

(1995) Plant Cell Physiol., 36, 29-36. [12] Ikezawa, N., Tanaka, M., Nagayoshi, M., Shinkyo, R., Sakaki, T., Inouye, K. and

Sato, F. (2003) J. Biol. Chem., 278, 38557-38565. [13] Yamada, Y. and Okada, N. (1985) Phytochemistry, 24, 63-65. [14] Sato, F., Takeshita, N., Fujiwara, H., Katagiri, Y., Huan, L. and Yamada, Y. (1994)

Plant Cell, Tissue Organ Culture, 38, 249-256. [15] Inui, T., Tamura, K., Fujii, N., Morishige, T. and Sato, F. (2007) Plant Cell Physiol. 48(2), 252-62. [16] Dubouzet, J.G., Morishige, T., Fujii, N., An, C.-I., Fukusaki, E., Ifuku, K., and

Sato, F. (2005) Biosci., Biotech., Biochem., 69, 63-70. [17] Kato, N., Dubouzet, E., Kokabu, Y., Yoshida, S., Taniguchi, Y., Dubouzet, J.G.,

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Yazaki, K. and Sato, F. (2007) Plant Cell Physiol. 48(1), 8-18 [18] Millgate, A.G., Pogson, B.J., Wilson, I.W., Kutchan, T.M., Zenk, M.H., Gerlach,

W.L., Fist, A.J. and Larkin, P.J. (2004) Nature, 431, 413-414. [19] Allen, R.S., Millgate, A.G., Chitty, J.A., Thisleton, J., Miller, J.A., Fist, A.J.,

Gerlach, W.L. and Larkin, P.J. (2004) Nat. Biotechnol. 22, 1559-1566. [20] Fujii, N., Inui, T., Iwasa, K., Morishige, T. and Sato, F. (2007) Transgenic Res. 16,

363-375. [21] Park,S.U., Yu, M. and Facchini, P.J. (2002) Plant Physiol., 128, 696-706. [22] Sato, F., Hashimoto, H., Hachiya, A., Tamura, K., Choi, K.B., Morishige, T.,

Fujimoto, H. and Yamada, Y. (2001) Proc. Natl. Acad. Sci. USA, 98, 367-372.

Unbroken arrows indicate single enzymatic conversions and broken arrows indicate

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multiple enzymatic steps. Enzymes for which the corresponding genes have been cloned are indicated in bold. tyrosine/dopa decarboxylase, TYDC; norcoclaurine synthase, NCS; norcoclaurine 6-O-methyltransferase, 6OMT; coclaurine N-methyltransferase, CNMT; berbamunine synthase, CYP80A1; N-methylcoclaurine 3'-hydroxylase, CYP80B1; 3' hydroxy N-methylcoclaurine 4'-O-methyltransferase, 4'OMT; berberine bridge enzyme, BBE; reticuline 7-O-methyltransferase, 7OMT; canadine synthase (methylene dioxy bridge-forming enzyme), CYP719; scoulerine 9-O-methyltransferase, SMT; salutaridine synthase, SAS; salutaridine reductase, SAR; acetylcoenzyme A:salutaridinol-7-O-acetyltransferase, SAT; codeinone reductase, COR; berbamunine synthase, CYP80A1; stylopine synthase, CYP719A2/3.

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Aromatic substrate prenyltransferase involved in plant

secondary metabolism.

Kazufumi Yazaki Laboratory of Plant Gene Expression, Research Institute for Sustainable

Humanosphere, Kyoto University, Gokasho, Uji 611-0011, Japan. E-mail:

[email protected]

Prenylation of an aromatic compound is a critical step to diversify the chemical structures and biological activities of secondary metabolites, and this reaction step is also involved in the biosynthesis of important endogenous quinone compounds like coenzyme Q and plastoquinone. We are interested in the cloning of new prenyltransferase genes responsible for the modification of aromatic natural products in plants, and also those involved in the formation of quinone compounds, which would be benefitial for human health. In the meeting I will introduce some aromatic substrate prenyltransfearses and compare their enzymatic properties each other. Finally an attempt to increase of a valuable natural compound by use of such a gene via metabolic enginieering is also introduced.

Prenyltransferase for diversification of secondary metabolites The prenylation of aromatic compounds is a major biosynthetic reaction step contributing to the diversification of plant secondary metabolites due to differences in prenylation position on the aromatic ring, various lengths of prenyl chain, and further modifications of the prenyl moiety, e.g. cyclization and hydroxylation, resulting in the occurrence of more than 1,000 prenylated compounds in plants (Tahara and Ibrahim 1995, Barron and Ibrahim, 1996). Prenylated flavonoids (Welle and Griesebach, 1991), coumarins (Hammerski et al., 1990), cannabinoid (Fellermaier and Zenk, 1998) and phenylpropanoids often occur in some particular plant families, such as Moraceae, Guttiferae, Leguminosae, Umberiferae, etc. The substitution of aromatic proton with prenyl moiety is also involved in biosynthetic reactions of many quinone compounds, and the prenylation is regarded as a rate-limiting reaction step. One of such example is geranylation of p-hydroxybenzoic acid for the biosynthesis of red naphthoquinone derivative shikonin and its isomer, alkannin, in boraginaceous plants (Yazaki et al., 2002). The enzyme responsible for this key reaction belongs to the membrane-bound protein family involved in ubiquinone (coenzyme Q) biosynthesis (Kawamukai 2002).

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PPT family as membrane-bound enzymes Prenyltransferase accepting p-hydroxybenzoic acid as a substrate is one of the most well-characterized subfamily (abbreviated as PPT) in the aromatic substrate prenyltransferase family. PPT members, which have been studied mostly for the importance in the coenzyme Q biosynthesis (Ohara et al., 2006), occur from bacteria to human. All PPT members characterized so far recognize prenyl diphosphate of different chain length from diprenyl diphosphate (GPP) to decaprenyl diphosphate, which are localized at mitochondrial membrane in eukaryotes, whereas one exceptional member of them responsible for a particular secondary metabolism, i.e. red naphthoquinone pigment formation, LePGT1 (Lithospermum erythrorhizon p-hydroxybenzoate geranyltransferase 1) is localized to endoplasmic reticulum membrane, which accepts only GPP as its prenyl substrate. The final lipophilic secondary product shikonin is secreted out of the cells (Yazaki et al., 2002), while the final product by other PPTs, coenzyme Q, is retained mainly at the mitochondrial membrane. Even forcibly localized to mitochondrial membrane, LePGT1 did not complement the function of other PPTs for coenzyme Q biosynthesis. The possible molecular evolution of this enzyme from PPT is also discussed

Recognition of substrate by LePGT LePGT1 was further utilized as a model enzyme of aromatic substrate prenyltransferase of membrane-type to clarify the molecular basis of this enzyme family. Site-directed mutagenesis studies of LePGT1 with yeast expression system indicated that three out of six conserved aspartates play the critical role for its enzymatic activity. By detailed kinetic studies of mutant enzymes, amino acid residues responsible for substrate binding were also identified. In vitro and in vivo analyses of chimera suggested that determinant region of this prenyl substrate specificity was included in 155 amino acids of N-terminus. A molecular modeling study demonstrated the reasonable structure of substrate binding domain, which will be also presented in the meeting.

Metabolic engineering using membrane-bound prenyltransferase Using the gene coding for the yeast polyprenyltransferase coq2, an attempt to increase quinone compounds was performed. Overexpression of coq2 gene in tobacco resulted in 2-fold increase in coenzyme Q accumulation compared to the control, whereas the modified coq2, whose gene product was designed to target to endoplasmic reticulum, gave higher increment in the coenzyme Q production (Ohara et al., 2004). The possible mechanism of the effect of the non-native localization of biosynthetic enzyme will be discussed. I will also discuss about

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prenyltransfease genes of other aromatic secondary metabolites.

Acknowledgement This work was supported in part by a Grant-in-Aid for Scientific Research (No. 17310126 to K. Y.) from the Ministry of Education, Science, Sports and Culture of Japan.

Barron D, Ibrahim RK (1996) Isoprenylated flavonoids- A survey. Phytochemistry 43: 921-982

Fellermeier M, Zenk MH (1998) Prenylation of olivetolate by a hemp transferase yields cannabigerolic acid, the precursor of tetrahydrocannabinol. FEBS Lett 427: 283-285

Kawamukai M (2002) Biosynthesis, bioproduction and novel roles of ubiquinone. J Biosci Bioeng 94: 511-517

Ohara K, Yamamoto K, Hamamoto M, Sasaki K, Yazaki K (2006) Functional characterization of OsPPT1, which encodes p-hydroxybenzoate polyprenyltransferase

involved in ubiquinone biosynthesis in Oryza sativa. Plant Cell Physiol 47: 581-590 Ohara K, Kokado Y, Yamamoto H, Sato F, Yazaki K (2004) Engineering of ubiquinone

biosynthesis using the yeast coq2 gene confers oxidative stress tolerance in transgenic

tobacco. Plant J 40: 734-743 Tahara S, Ibrahim RK (1995) Prenylated isoflavonoids-An update. Phytochemistry 38:

1073-1094

Welle R, Grisebach H (1991) Properties and solubilization of the prenyltransferase of isoflavonoid phytoalexin biosynthesis in soybean. Phytochemistry 30: 479-484

Yazaki K, Kunihisa M, Fujisaki T, Sato F (2002) Geranyl diphosphate:4-hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon. Cloning and characterization of a key

enzyme in shikonin biosynthesis. J Biol Chem 277: 6240-6246

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NMR and LC/MS as a tool for metabolomics study

Nam-In Baek Graduate School of Biotechnology & Plant Metabolism Research Center, KyungHee University Suwon 449-701, Korea. E-mail : [email protected]

The metabolite occurred in a living cell is classified to primary and secondary metabolite. While the former one is commonly present in all of the cells, the latter one is differently present according to the kind of cells and the circumstance to lie in. The role of the former one in cell is known well, but that of the latter one is yet generally unknown. Many interests have been concentrated on the secondary metabolites because of their versatile biological activities. Though the plant cell theoretically has about some thousands of metabolites, less than fifty metabolites have been actually isolated. Metabolomics study means analysis of metabolites occurred in the living cells, which comprises target analysis, metabolite profiling and metabolomics, and interpretation of their roles in the cell or systematic networks among them. Target analysis indicates the analysis of a few metabolites, metabolite profiling does some selected metabolites, and metabolomics does more than two hundreds metabolites. LC/MS and NMR have been mainly exploited for this purpose. Metabolite profiling of red pepper (Capsicum annuum L.) for natural product screening using LC-time of flight mass spectrometry

Unbiased analysis of natural products from plant products needs to comprise accurate annotations of known compounds as well as the ability to screen for potentially novel chemicals that are present in a given matrix. We here exemplify the use case of Korean red pepper, an important plant product produced and exported in excess of 17,000 Mt yearly. Red pepper is an example of a plant product that is known for its health effects, similar to ginger, which are ascribed for the complement of natural products (secondary metabolites). Although some active constituents in red pepper are well known such as capsaicin, which has a preventive role in the etiology of human cancers and other human diseases, usually the exact role and contribution of the known secondary metabolites cannot be unraveled by classical quantitative analytical chemistry. It is a valid hypothesis that beneficial health effects cannot be explained by additive actions but rather by synergistic contributions of the complement of all ingredients, the

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‘metabolome’. We therefore present an approach using LC-TOF mass spectrometry to utilize and quantify such metabolome profiles, following the aim of elucidating additional, yet unknown compounds and establishing a baseline of natural products in red pepper that may later be utilized for quality monitoring in food products.

Metabolomics analysis for red pepper was carried out using LC/MS. ODS column with 2.1 mm diameter was used for separation, and ESI for ionization and Tof for mass analysis. Because of broad spectrum of the metabolites of red pepper extracts in polarity, polar, non-polar and moderate polar metabolites were separately analyzed. Finally, the pseudo-molecular ions of more than three hundreds of metabolites were detected. Each pseudo-molecular ions were compared for the exact value (less than 50 ppm in error value) and isotopic patterns with those of the metabolites isolated from the red pepper so far. Mere than seventy metabolites were identified

Polar

components Nonpolar

components

Moderately polar

components

Figure 1. Analytical ion chromatogram of red pepper under general solvent gradient conditions (GSG).

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OHO

O

OH

Capsanthin-3,6-epoxide

Figure 2. Extracted ion trace chromatogram of red pepper under non polar solvent gradient conditions (NPSG), displaying some known secondary plant compounds in this food product.

Quantitative Analysis of Paeoniflorin from Paeonia lactiflora Using 1H-NMR The methods for analysis of metabolites from living cells include thin layer

chromatography (TLC), high performance liquid chromatography (HPLC), capillary electrophoresis (CE), but up to now HPLC has been used more often for metabolite quantification. However, HPLC analysis wasted to equilibration time and organic solvents and more preprocessing. Therefore, an alternative method for the analysis of metabolites would be highly desirable. Conventionally, nuclear magnetic resonance spectrometry (NMR) has been used to elucidate the molecular structure of purified compounds as an analytical tool. But, NMR is possible to quantitative spectroscopic tool, because the intensity of a resonance line is directly proportional to the number of resonant nuclei (spins). Quantitative NMR (qNMR) has particularly due to specific advantages like, (i) the possibility to determine structures, (ii) no need for intensity calibrations in case of determination of ratios, (iii) relatively short measuring time, (iv) its non-destructive character, (v) no prior isolation of the analyte in a mixture, (vi) the possibility of a simultaneous determination of more than one analyte in a mixture.

Paeoniflorin, the major component of the root of Paeonia lactiflora, was quantitatively analyzed using 1H-NMR spectrometry. The quantity of paeoniflorin was

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calculated by the ratio of the intensity of the signals (H-9, H-10, H-2", 6") to the aldehyde peak of the known amount of internal standard, 2,4,6-trihydroxybenzaldehyde. These results were compared with the conventional HPLC method. The advantages of quantitative 1H-NMR analysis are that can be analyzed to identify and quantify, and no reference compounds required for calibration curves. Besides, it allows rapid and simple quantification for paeoniflorin with an analysis time for only 20 min without any preprocessing.

O O

O

CHOO

OH

Glc

paeoniflorin

Figure 3. 1H-NMR Spectra of (A) paeoniflorin, (B) the D2O extract of the root of Paeonia lactiflora in D2O in the range of δ 0.0-12.0 after 128 scans. Table Comparison of the concentration (%, w/w) of paeoniflorin in the D2O extract as determined by integration of H-9, H-10 and H-2", 6" in the 1H-NMR spectrum and by peak area in the HPLC

Analysis method 1H-NMR HPLC Peak H-9 H-10 H-2", 6"

Paeoniflorin content 2.60 ± 0.07 2.44 ± 0.09 2.77 ± 0.12 2.46 ± 0.16

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Brassinosteroid biosynthesis and its effects on other hormones Bo Kyung Kim1, Shozo Fujioka2, Suguru Takatsuto3, Masafumi Tsujimoto2, and Sunghwa Choe1 1 Department of Biological Sciences, College of Natural Sciences, Seoul National

University, Seoul 151-747, Korea. E-mail: [email protected] 2 RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama

351-0198, Japan 3 Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata

943-8512, Japan Brassinosteroids (BRs) collectively refer to steroidal plant hormones that are essential for proper growth and development of plants. It has been proposed that BRs are synthesized via multiple grid pathways, the early and the late C-22 and C-6 oxidation pathways according to the order of C-22 and C-6 oxidation status. One is the early C-6 oxidation pathway, in which oxidation at C-6 occurs before the introduction of a vicinal hydroxyl group at C-22 and C-23 of the side chain. The other is the late C-6 oxidation pathway in which C-6 is oxidized after the introduction of the hydroxyl group on the side chain. The C-6 oxidation of BR intermediates is catalyzed by the enzymes encoded by Cytochrome P450 85 (CYP85) genes. Arabidopsis CYP85 enzymes have been shown to catalyze C-6 oxidation of 6-deoxo intermediates. Interestingly, Arabidopsis CYP85A2 was also found to mediate the ultimate step which is Baeyer-Villiger type oxidation of C-6. However, the functions of rice CYP85 protein as a BL synthase are still unknown. Therefore, we aimed to understand the function through feeding experiments with a yeast strain that is heterologously expressing the rice CYP85 gene. Feeding tests following GC-MS based analyses revealed that both Arabidopsis CYP85A2 and rice CYP85 metabolize 6-deoxo-BRs into 6-oxo-BRs. However, unlike Arabidopsis CYP85A2, rice CYP85 does not metabolize Castasterone into brassinolide. This result and previously reported profiles of endogenous brassinosteroids in rice plants suggests that rice has Castasterone as its end product due to lack of brassinolide synthase activity in the rice CYP85 enzyme.

Brassinosteroids (BRs) are widely distributed among the species of plant

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kingdom. Currently more than 50 compounds have been discovered (Bajguz and Tretyn, 2003). Genetic evidence suggested that BRs are responsible for a flurry of physiological processes including organ elongation, stress biology, flowering, vascular system development, photomorphogenesis, and skotomorphogenesis in Arabidopsis (Kwon and Choe, 2005). Similar to the effects on organ elongation in Arabidopsis, rice plants defective in BR biosynthesis or signal transduction pathways also display dwarf phenotype, small crinkled lamia, and sterility (Hong et al., 2003; Mori et al., 2002).

Brassinolide (BL) is known to be the most bioactive among the BRs examined to date. The biosynthetic pathways leading to BL starts from Campesterol (CR). CR goes through two alternative pathways depending on the order of the two reactions: C-22 hydroxylation and C-5 reduction. The C-22 hydroxylation reactions are mediated by the Arabidopsis DWARF4 enzyme which belongs to Cytochrome P450 monooxygenase family (CYP90B1) (Choe et al., 1998; Choe et al., 2001, Fujita et al., 2006, Ohnishi et al., 2006). The C-22 hydroxylated compounds are further hydroxylated at C-23 position by another group of Cytochrome P450 enzymes classified as CYP90C1 and CYP90D1 (Ohnishi et al., 2006). Once the C-5 reducion takes place, C-3 epimerization, and C-2 hydroxylation reactions follow, and finally the C-6 position is oxidized to generate Castasterone (CS) and BL.

BL is biosynthesized by catalytic activities of Arabidopsis CYP85A2 and Tomato CYP85A3 enzymes using 6-DeoxoCS as a substrate. These enzymes mediate three consecutive steps including C-6 hydroxylation, C-6 dehydrogenation, and Baeyer-Villiger type oxidation to create lactone at the ring B in the steroidal backbone (Kim et al., 2005; Nomura et al., 2005). Arabidopsis genome possesses two copies of CYP85 genes. Due to functional redundancy in these two genes, a single loss-of-function mutation for each gene does not display any conventional BR dwarf phenotype. However, when the function of the two genes was simultaneously disrupted, they exhibited severe growth retardation phenotypes (Kwon et al., 2005).

In contrast to the Arabidopsis and tomato genomes, recently completed rice genome revealed that it contains only one copy of the CYP85 gene. In addition, different groups of scientists examined the endogenous level of BRs in rice plants, and found that there is no BL detected even in the mutant plants that known to greatly accumulate BRs (Yamamuro et al., 2000). To examine if the rice CYP85A1 enzyme has only the function of CS synthase than BL and this is the reason that rice contains only up to CS, we expressed rice CYP85A1 gene in yeast and carried out metabolic conversion analysis.

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Results and Discussion Arabidopsis genome possesses more than 270 genes classified as Cytochrome

P450 proteins. Still functions of the genes remain mostly unknown due to lack of genetic and biochemical analysis. To systemically express the genes in different cellular system such as yeast, insect, plant protoplasts, bacteria, or human cells, we chose to employ a Gateway cloning system. Coding sequences for rice OsCYP85A1 as well as Arabidopsis CYP85A1 and CYP85A2 were cloned into pENTR/SD/D-TOPO vector after amplification of the genes using gene specific primers. The cDNA clones that contain the expected cDNA in a right direction for expression were transferred to a destination vector pYES_DEST52 which is designed for inducible expression by Galactose. The yeast expression clones were transformed into the strain WAT11 whose nascent NADH:Cytochrome P450 reductase (CPR) has been replaced with the one in Arabidopsis for functional optimization of plant P450 proteins (Pompon et al., 1996). BR biosynthetic intermediates such as 6-deoxocastasterone (6-DeoxoCS) and CS were supplied to the yeast strains that heterologously expressing the P450 genes.

When 6-DeoxoCS was tested, Arabidopsis successfully converted the compound into CS as well as BL, which is consistent with the previous report. Furthermore, Arabidopsis CYP85A2 could transform CS to BL efficiently. Rice CYP85 also converted 6-DeoxoCS into CS. However, it failed to convert CS into BL at repeated experiments. Based on these results, it is likely that rice plants have CS as the end product in the BR biosynthetic pathways. Implication of undetectable level of brassinolide in rice plants could be explained by multiple ways. First, rice plants indeed are devoid of BL due to lack of the BL synthase function rather than simple technical problem of detection limit. Evolutionarily, CYP85A1 might have failed to duplicate in the rice genome. Second, rice plants are typical of cultivars generated after intensive breeding program. The BL biosynthetic genes may have segregated out of current cultivars due to its detrimental effects on the breeding lines. In fact application of BL to rice plants induces the lamina being bent away from the main axis of the stem. This is considered an undesirable trait of cultivated rice, which favors erect stature to better harvest sun light for maximum photosynthesis. Third, there had been a gene that was responsible for the BL synthase function in rice sometime during evolutionary times. However, due to independent evolution of this function, their amino acid sequences are quite diverged from CYP85 proteins, they may still wait for being discovered.

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Bajguz A, Tretyn A (2003) The chemical characteristic and distribution of brassinosteroids in plants. Phytochemistry 62: 1027-1046

Choe S, Dilkes BP, Fujioka S, Takatsuto S, Sakurai A, Feldmann KA (1998) The DWF4 gene of Arabidopsis encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps in brassinosteroid biosynthesis. Plant Cell 10: 231-243

Choe S, Fujioka S, Noguchi T, Takatsuto S, Yoshida S, Feldmann KA (2001) Overexpression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis. Plant J 26: 573-582

Fujita S, Ohnishi T, Watanabe B, Yokota T, Takatsuto S, Fujioka S, Yoshida S, Sakata K, Mizutani M (2006) Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols. Plant J 45: 765-774

Hong Z, Ueguchi-Tanaka M, Shimizu-Sato S, Inukai Y, Fujioka S, Shimada Y, Takatsuto S, Agetsuma M, Yoshida S, Watanabe Y, Uozu S, Kitano H, Ashikari M, Matsuoka M (2002) Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6 oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem. Plant J 32: 495-508

Kim TW, Hwang JY, Kim YS, Joo SH, Chang SC, Lee JS, Takatsuto S, Kim SK (2005) Arabidopsis CYP85A2, a cytochrome P450, mediates the Baeyer-Villiger oxidation of castasterone to brassinolide in brassinosteroid biosynthesis. Plant Cell 17: 2397-2412

Kwon M, Choe S (2005) Brassinosteroid biosynthesis and dwarf mutants. J Plant Biol 48: 1-15

Kwon M, Fujioka S, Jeon JH, Kim HB, Takatsuto S, Yoshida S, An CS, Choe S (2005) A double mutant for the CYP85A1 and CYP85A2 genes of Arabidopsis exhibits a brassinosteroid dwarf phenotype. J Plant Biol 48: 237-244

Mori M, Nomura T, Ooka H, Ishizaka M, Yokota T, Sugimoto K, Okabe K, Kajiwara H, Satoh K, Yamamoto K, Hirochika H, Kikuchi S (2002) Isolation and characterization of a rice dwarf mutant with a defect in brassinosteroid biosynthesis. Plant Physiol 130: 1152-1161

Nomura T, Kushiro T, Yokota T, Kamiya Y, Bishop GJ, Yamaguchi S (2005) The last reaction producing brassinolide is catalyzed by cytochrome P-450s, CYP85A3 in tomato and CYP85A2 in Arabidopsis. J Biol Chem 280: 17873-17879

Ohnishi T, Watanabe B, Sakata K, Mizutani M (2006) CYP724B2 and CYP90B3

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function in the early C-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato. Biosci Biotechnol Biochem 70: 2071-2080

Yamamuro C, Ihara Y, Wu X, Noguchi T, Fujioka S, Takatsuto S, Ashikari M, Kitano H, Matsuoka M (2000) Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint. Plant Cell 12: 1591-1606

HO

HO O HO HO

OH

HO

O

HO

OH

HO

OH

O

HO

OH

OH

HO

OH

OH

OOH

OH

O

OH

OH

O

O

OH

OH

HO

OH

OH

HOO

OH

OH

HO

HO

OH

OH

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HO

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6α-Hydroxycastasterone6-Deoxocastasterone

6-Deoxotyphasterol

3-Dehydro-6-deoxoteasterone

6-Deoxoteasterone

6-Deoxocathasterone

Castasterone(CS)

Typhasterol(TY)

3-Dehydroteasterone(3-DT)

Teasterone (TE)

Cathasterone (CT)

6-Oxocampestanol(6-OxoCN)

6α-Hydroxycampestanol (6-OHCN)

Campestanol(CN)

Campesterol (CR)

24-Methylenecholesterol

DWF4CYP90B1

DET2 (DWF6)

C-24 Reductase

Brassinolide(BL)

C-22α Hydroxylase

C-23α Hydroxylase

C-3 Dehydrogenase

C-3 Reductase

C-2α Hydroxylase

H

HHH

HH

HH

H H

HH

HHHHOHO

OH

OHO

CYP85A1CYP85A2

Early C22 Oxidation Pathway

Late C22 Oxidation Pathway

DDWF1 DDWF1

HO

DWF4OH OH OH OH

(22S)-22-Hydroxycampesterol (22-OH CR)

Δ5-Δ4 Isomerase 3-Dehydrogenase 5α-reductase3-Dehydrogenase

(22S, 24R)-22-hydroxy-ergost-4-en-3-one

(22S, 24R)-22-hydroxy-5α-ergostan-3-one

(22S, 24R)-22-hydroxy-ergost-4-en-3β-ol

HOHO5-Dehydroepisterol Episterol

Δ7 Sterol C-7 reductase

H

Δ7 Sterol C-5 desaturase

CYP85A1CYP85A2

CYP85A2

DWF1

DWF7 DWF5

DWF4CYP90B1

CYP90C1CYP90D1CYP90C1

CYP90D1

CYP90C1CYP90D1

Figure 1. Brassinosteroid biosynthetic pathways.

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Phytochromes negatively regulate their interacting bHLH

transcription factors in Arabidopsis Giltsu Choi Department of Biological Sciences, KAIST, Daejeon 305-701, Korea. E-mail:

[email protected]

Although light is ubiquitous, the light in a given locale may vary in terms of its wavelength, irradiance, direction, and periodicity. Since plants acquire energy solely from light, it is not surprising to find that plants are equipped with various photoreceptor systems to detect local light conditions and adjust their growth and development according to the conditions. Years of research show that higher plants contain at least four independent photoreceptor systems that sense either blue light or red light spectrum. Blue light is perceived by three different photoreceptors called cryptochromes, phototropins, and zeitlupes, whereas red light is perceived by a photoreceptor called phytochromes. Three blue light photoreceptors, though recognize the same blue spectra, have shared, but distinct functions. Cryptochromes, encoded by two genes, CRY1 and CRY2 in Arabidopsis, regulate seedling photomorphogenesis, flowering, and circadian clock in Arabidopsis. Unlike cyrptochromes, phototropins, encoded also by two genes, PHOT1 and PHOT2 in Arabidopsis, regulate phototropism and chloroplast movement. Zeitlupes are newly identified blue light photoreceptors consisting of three proteins called ZTL, FKF1, and LKP2 in Arabidopsis. Zeitlupes are F-box proteins having a chromophore-binding LOV domain and kelch repeats and regulate circadian clock and flowering. Red and far-red light is perceived by plant photoreceptors called phytochromes. In Arabidopsis, five different phytochromes (PHYA-E) perceive red and far-red light and regulate various aspect of plant development including seed germination, seedling photomorphogenesis, shade avoidance, circadian clock. Phytochromes convert the information contained in external red and far-red light into biological signals. The conversion process starts with the perception of red light, which occurs through

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photoisomerization of a chromophore located within the phytochrome, leading to structural changes needed for interactions with other proteins. Several studies have shown that light information represented by the concentration of the Pfr form is converted into biological signals through modulating activity of phytochrome-interacting proteins. Total 20 phytochrome-interacting proteins have been identified up to now. Among them, a set of bHLH transcription factors including PIF3, PIF4, PIL5, and PIL6 bind to the Pfr form of phytochromes and play key roles in understanding phytochrome-mediated light signaling. These transcription factors have transcriptional activation activities when assayed in the transient expression system or in transgenic plants. Functionally, they negatively regulate various light responses such as seed germination (PIL5), hypocotyl elongation (PIF3, PIF4, PIL6), and chloroplast development (PIF3, PIL5). Upon exposure of red light, these bHLH transcription factors are rapidly degraded in phytochrome-dependent manner, suggesting that phytochromes induce these light responses by removing negative bHLH transcription factors. In the symposium, I will discuss how phytochromes regulate various responses by negatively modulating activities of these bHLH transcription factors in Arabidopsis.

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Regulation of Terpene Secondary Metabolism by Multiple

Gene in Gymnosperms Sang-Min Kim1, 2, Yeon-Bok Kim1, 2, Tomohisa Kuzuyama 3, Soo-Un Kim 1, 2

1Program in Applied Life Chemistry, School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea. E-mail: [email protected]

2Plant Metabolism Research Center, Kyung Hee University, Yongin 449-701, Korea 3Laboratory of Cell Biotechnology, Biotechnology Research Center, University of Tokyo, Tokyo 113-8657, Japan Isoprenoids, characterized by extreme structural diversity, are a group of natural products that play important roles in all living organisms. Isoprenoids function primarily in photosynthesis, growth regulation, cell division, signal transduction, and respiration. They also are prominently involved in the exchange of chemical signals between plants and their environment or in defense of plants against pathogens. One of the prominent examples is complex terpenoid resin excreted from conifer resin duct. The resin contains diterpenoid resin acid that defends the plant from pathogens and herbivores. Terpenoids also play important role in human health. For example, the diterpenoid ginkgolides produced by Ginkgo biloba are important ingredients in now widely sold ginkgo extract, and the ginkgolides are potent platelet-activating factor antagonists. Despite structural and functional complexities of isoprenoids, they are biosynthesized

from two simple five-carbon building units, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Most terpenoids are made through the head-to-tail or head-to-head condensation of these units. In the 1950s, mevalonic acid (MVA) pathway was discovered in the liver cytosolic fraction, and, at that time, the pathway had been thought to be the single source of the five-carbon units. However, accumulating experimental data not compatible with MVA pathway in plants and microorganisms led to the discovery of a novel 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. It is now well established that plant plastids, most bacteria except archebacteria, and apicomplexan parasites harbor MEP pathway, whereas cytosol of most eukaryotes carry MVA pathway. Furthermore, though not strict, both pathways have separate roles in that MVA pathway ultimately leads to sesquiterpenoids and triterpenoids, and MEP pathway to monoterpenoids, diterpenoids, and tetraterpenoids.

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Results and discussion All MEP pathway genes of G. biloba were isolated and characterized using concensus PCR methods. In G. biloba, two DXS isogenes (GbDXS1, GbDXS2) were found as expected. However, CMEK was found as two-copy genes (GbCMEK1, GbCMEK2) and IDS as three isogenes (GbIDS1, GbIDS2, GbIDS2-1) in G. biloba. Until now, there have been no reports on multi-type genes besides DXS among genes involved in plant MEP pathway. On the other hand, one CMEK gene and two isogenes of IDS were assembled from Pinus taeda EST database. The multiple MEP pathway genes of G. biloba in this research were thus classified into two classes according to their transcription pattern: class 1 containing GbDXS1, GbCMEK1, and GbIDS1; class 2 containing GbDXS2, GbCMEK2, GbIDS2, and GbIDS2-1. To identify the functional activities of the isolated MEP pathway proteins, E. coli

disruptants of each MEP pathway gene were used for the complementation assay. Each MEP pathway proteins expressed in E. coli could complement each mutant, except GbHDS, GbIDS2-1, an indication that most proteins had in vivo functional activities.

All MEP pathway protein sequences deduced from the isolated cDNAs possessed conserved motifs or residues needed for the functional activities as revealed by alignment with the known plant sequences. Phylogenetic tree showed that these MEP pathway proteins belong to the plant group of MEP pathway proteins. Interestingly, phylogenetic tree showed that multiplication of plant DXS protein occurred before the separation of angiosperm from gymnosperm, while other novel multiple enzymes, CMEK and IDS in G. biloba, multiplied after the separation of the angiosperm. In addition, multiple IDSs were discovered in two other gymnosperm families, Pinophyta (P. taeda and Picea sitchensis) and Cycadophyta (Cycas revoluta), alluding that multiple IDS is a signature of gymnosperms. Recent study, however, reported multiple DXR isogenes in Hevea brasiliensis, a angiosperm.

Arabidopsis protoplast system was employed to determine the subcellular location of MEP pathway proteins using smGFP protein as a reporter. As expected, green fluorescence of most MEP pathway proteins were detected in the chloroplast. Green fluorescence of two pine IDSs were also found in the chloroplast. However, in the cases of GbCMEK1 and GbIDS1, GFPs were found in the cytosol and nucleus, in addition to the expected site, chloroplast, implying the flow of the MEP pathway metabolites among plastid, cytoplasm, and nucleus is possible. Transcript profiles of G. biloba MEP pathway genes from 4-week-old culture were

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analyzed by real-time quantitative PCR. There was a distinct difference in the transcription patterns between single- and multi-copy genes. In general, the expression levels of single-copy genes were similar in both roots and leaves. Multi-copy genes, however, displayed different transcription levels between roots and leaves. Class 1 genes were discovered in roots and leaves at similar levels, while class 2 genes were distinctively abundant in roots, where ginkgolide biosynthesis takes place. GbLPS, used as a reference gene, was also detected almost exclusively in roots, since LPS catalyzes the committed cyclization step in ginkgolide biosynthesis route. Furthermore, transcripts of PtDXS1 and PtIDS1 were detected evenly in all organs of P. taeda, whereas PtDXS2 and PtIDS2 showed 4-5 folds higher transcript levels in the wood, where diterpene resin acid is known to be biosynthesized. Accumulation of the major secondary metabolites, ginkgolides (ginkgolides A and B) and their degradation product bilobalides, were determined from the ginkgo embryos grown in a hormone-free MS medium for 4 weeks. The most abundant product in roots and leaves was bilobalide. When roots and leaves were compared, leaves contained about three times more ginkgolides. These facts further supported that ginkgolides were biosynthesized in the root and transported to the leaves. Light and methyl jasmonate (MeJA) were applied to the ginkgo embryos to investigate

the transcription patterns of multi-type genes in the primary and secondary metabolisms. Generally, class 1 genes were induced to a higher level upon illumination than class 2 genes. In particular, transcripts of GbIDS1 increased about 18 folds upon illumination. However, MeJA elicited the reversed trends. Transcription of class 2 genes were distinctively induced by methyl jasmonate, whereas that of class 1 genes decreased by 1/3 to 1/5. These results thus imply that class 1 genes are involved in the primary metabolism, while class 2 genes in the secondary metabolism.

Kim SM, Kuzuyama T, Chang YJ, Kim SU (2005) Functional identification of Ginkgo biloba 1-deoxy-D-xylulose 5-phosphate synthase (DXS) gene by using Escherichia coli disruptants defective in DXS gene. Agr Chem Biotechnol 48:101-104

Kim SM, Kuzuyama T, Chang YJ, Kim SU (2006a) Cloning and characterization of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECS) gene from Ginkgo biloba. Plant Cell Rep 25:829-835

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Kim SM, Kuzuyama T, Chang YJ, Kwon HJ, Kim SU (2006b) Cloning and functional

characterization of 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (GbMECT) gene from Ginkgo biloba. Phytochemistry 67:1435-1441

Kim SM, Kuzuyama T, Chang YJ, Song KS, Kim SU (2006c) Identification of class 2

1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase genes from Ginkgo biloba and their transcription in embryo culture with respect to ginkgolide biosynthesis. Planta Med 72:234-240

Kuzuyama T and Seto H (2003) Diversity of the biosynthesis of the isoprene units. Nat

Prod Rep 20:171-183 Walter MH, Hans J, Strack D (2002) Two distantly related genes encoding

1-deoxy-d-xylulose 5-phosphate synthases: differential regulation in shoots and apocarotenoid-accumulating mycorrhizal roots. Plant J 31:243-254

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Cooperation and Functional Diversification of Two Closely

Related Galactolipase Genes for Jasmonate Biosynthesis

Youbong Hyuna, Sang-Jip Namb, Ju-Young Parkc, Young Sam Seod Woo Taek Kimd, Yong-Hwan Leec, Heonjoong Kangb, and Ilha Lee a,e,1 a National Research Laboratory of Plant Developmental Genetics, Department of

Biological Sciences, Seoul National University, Seoul 151-742, Korea. E-mail: [email protected]

b Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University, NS80, Seoul 151-747, Korea.

c Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea.

d Department of Biology, College of Science, Yonsei University, Seoul 120-749, Korea e Global Research Laboratory for Flowering at SNU and UW, Seoul 151-742, Korea. Jasmonic acid (JA) plays pivotal roles in diverse plant biological processes including wound response. Chloroplast lipid hydrolysis is a critical step for JA biosynthesis, but the mechanism of this process remains elusive. We report here that DONGLE (DGL), a homolog of DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), encodes a chloroplast-targeted lipase having both phospholipase A1 and galactolipase activity. DGL is expressed in the leaves and have a specific role in maintaining basal JA content under normal conditions, which regulates vegetative tissue growth and is required for a rapid JA burst after wounding. During wounding, DGL and DAD1 have partially redundant function for JA production but show different induction kinetics, indicating temporally separated roles – DGL for the early phase and DAD1 for the late phase of JA production. While DGL and DAD1 are necessary and sufficient for JA production, phospholipase D seems to induce wound response through the transcriptional activation of them. Introduction Jasmonic acid (JA) and its derivatives, collectively referred to as the jasmonates, are lipid-derived plant hormones that are ubiquitous throughout the plant kingdom. These compounds play pivotal roles during plant developmental processes, such as seed

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maturation, viable pollen production, root growth, and tendril coiling, and also function as important signaling molecules in plant defense responses to biotic and abiotic stress. JA is reported to be biosynthesized from α-linolenic acid, which is released from membrane lipids, via the so-called octadecanoid pathway that involves enzymes located in two different subcellular compartments – the chloroplast and the peroxisome.

A proportional response to wounding is critical for plants to cope with their biotic/abiotic environments. Various responses related with defense and wound healing are induced by mechanical wounding and insect herbivory, and global changes in gene expression profiles are required to evoke such a wide variety of responses. It has been suggested that wound-induced JA and its cyclopentenone precursor, OPDA, are major components regulating such changes. To date, however, the precise mechanism by which JA is produced as a response to wounding is still unclear.

The Arabidopsis DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) protein has been reported to be a chloroplastic phospholipase that catalyzes the production of α-linolenic acid from chloroplast membrane lipid. The mutant dad1 is defective in terms of anther dehiscence, pollen maturation, and flower opening, but the overall morphology of the dad1 mutant is almost the same as that of the wild type. However, wounding causes a rapid induction of DAD1 expression in the leaves, and the kinetics of this expression correlates well with the kinetics of JA accumulation. Based on these studies, it has been proposed that DAD1 contributes to the wound induction of JA biosynthesis. However, the levels of JA accumulation following wounding in dad1 mutants and the wild type were similar, thereby leaving open the question of which components provide α-linolenic acid for JA production after wounding.

In the present study, we identified a new component of the chloroplast-targeted lipase required for JA biosynthesis, DONGLE (DGL). DGL is a member of the AtPLA1-I family and is expressed in vegetative tissues in the absence of wounding. We show that DGL has the enzymatic activity of both PLA1 and galactolipase. In addition, DGL is required for the production of basal levels of endogenous JA, which regulates the growth of seedlings and vegetative organs, and that this endogenous JA is involved in the rapid induction of DAD1 following wounding. We also show that DGL and DAD1 are the only genes participating in JA biosynthesis among the AtPLA1-I family and that wound-induced JA production is dependent on the function of DGL and DAD1. Finally, we show that PLDα1 may provide a secondary signaling molecule for the wound-inducible expression of DGL and DAD1. Results and Discussion Overexpression of DONGLE causes the dwarf phenotype

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Mutant plant lines showing developmental and growth defects were obtained by activation-tagging mutagenesis on a Columbia-0 (Col-0) ecotype of Arabidopsis thaliana. One mutant (among 40,000 T1 transformants) exhibiting a dominantly inherited dwarf phenotype was isolated and designated as dongle-D on the basis of its round-leaf morphology (dgl-D; ‘dongle’ means ‘round-shaped’ in Korean). The dgl-D mutant was smaller than the wild type and showed a defect in apical dominance, short anthers. Thermal asymmetric interlaced (TAIL)-PCR analysis and the recapitulation analsysis identified the DGL gene as At1G05800. DGL, a chloroplast-localized phospho-/galactolipase A, mediates an initial step of JA biosynthesis

The cellular localization of DGL was determined by means of a protoplast transient assay using constructs encoding DGL:GFP (Green Fluorescent Protein). The DGL was shown to be localized in the chloroplast. The biochemical analysis showed that the DGL has both phospholipase A1 and galactolipase activity. The genetic analysis showed that DGL acts upstream of OPR3 in the JA biosynthetic pathway and is capable of replacing DAD1 activity. Because overexpression of JA causes enhanced resistance to pathogens, dgl-D plants were assayed for resistance to necrotrophic and biotrophic pathogens. As expected, dgl-D was resistant to both A. brassicicola (a necrotrophic pathogen) and P. syringae DC3000 (a biotrophic pathogen).

The levels of JA and methyl jasmonate (MeJA) in the dgl-D mutant and wild type were measured by gas chromatography/mass spectrometry (GC/MS). In triplicate analyses, the dgl-D mutant exhibited much higher amounts than the wild type. Taken together, our results suggest that DGL is a chloroplast-localized phospho-/galacto-lipase A participating in an initial step of JA biosynthesis.

Among the AtPLA1-I family, only DGL and DAD1 participate in JA biosynthesis Because JA production is strongly induced by wounding, we measured the JA level in dgl-i and dad1 mutants after wounding. At 1 hr after wounding, dad1 showed wild type levels of JA induction as previously reported whereas dgl-i showed weak induction. However, dgl-i recovered to almost wild type level of JA at 4 hrs after wounding, suggesting that only the early phase of JA production is defective in dgl-i. When checked, the JA level in dgl-i dad1 double mutant was below the detection limit of our experimental instruments, irrespective of wound treatment. It indicates that only DGL and DAD1 among the 7 members of the AtPLA1-I family have roles for JA biosynthesis, although they are closely related in amino acid sequences.

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To examine if other members among the AtPLA1-I family genes participate JA biosynthesis, we analyzed the expression patterns after wounding and the phenotypes of transgenic plants overexpressing each family member. In contrast to DGL and DAD1, AtPLA1-Iα2 and AtPLA1-Iβ2 expressions were not detected even at 4 h after wounding. The remaining members, AtPLA1-Iγ1, AtPLA1-Iγ2, and AtPLA1-Iγ3, which belong to a separate sub-class, were constitutively expressed at a high level and did not show any significant induction by wounding. Consistent with this, the transgenic plants overexpressing AtPLA1-Iα2, AtPLA1-Iβ2, AtPLA1-Iγ1, AtPLA1-Iγ2, or AtPLA1-Iγ3, did not exhibit the JA overproduction phenotype observed in 35S::DGL. Therefore, these results confirm that only DGL and DAD1 participate in JA biosynthesis.

Roles of DGL and DAD1 in wound-inducible JA biosynthesis are temporally separated

In addition to the difference in biochemical activity, we found differences in the wound response between DGL and DAD1. In the wild type, both DGL and DAD1 exhibited peak expression 1 h after wounding. However, while the expression of DGL decreased rapidly after 1 h, that of DAD1 decreased slowly and remained at a relatively high level until 4 h after wounding. To assess whether the higher expression level of DAD1 in the late phase has any function, we compared the expression of VSP1 in wild-type and dad1 plants. The expression of VSP1 in both plant lines showed a similar increase up to 1–2 h after wounding; thereafter, it started to decrease in dad1 plants but showed a continuous increase in the wild type. This result suggests that a relatively longer duration of DAD1 expression after wounding is required for the continuous increase of VSP1 in the late phase. In contrast, VSP1 expression in dgl-i plants decreased in the early phase, indicating that DGL is necessary for the early induction of VSP1 expression.

To confirm such temporally diversified functions of DGL and DAD1 in wound-inducible JA production, we compared the JA induction kinetics between dgl-i and dad1 mutants. At 1 h after wounding, the level of JA was very low in the dgl-i mutant, but JA accumulation was normal in the dad1, indicating that the early phase of wound-induced JA production requires DGL. Conversely, the level of JA fell rapidly in the dad1 mutant at 2 h after wounding compared to the wild type, thereby showing that DAD1 is necessary for the accumulation of JA during the late phase. The JA level in dgl-i was recovered to the wild type level 4 hrs after wounding.

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Conclusion Through activation tagging mutagenesis, we have isolated a dwarf mutant, dgl-D, that overexpresses a gene encoding a member of AtPLA1-I. Similar to DAD1, which catalyzes a critical step in the JA biosynthetic pathway, DGL has a function for JA production. Here, we show that DGL has strong but DAD1 has weak galactolipase activity for DGDG, and the two closely related lipases are necessary and sufficient for wound-inducible JA production. We also show that DGL and DAD1 have temporally and spatially separate functions. Finally, our results indicate that PLD induces wound response through the transcriptional activation of DGL and DAD1 and does not provide the JA precursor molecules.

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Beta-glucosidase homologs play critical roles in homeostasis of

ABA Kwanghee Lee, Zhengyi Zu, Hongju Byeon, Hyangju Kang, Junho Lee, Inhwan Hwang Department of Life Science, POSTECH, Pohang, 790-784, Korea. E-mail: [email protected] The phytohormone, abscisic acid (ABA), is critical for plant growth and development, as well as for adaptive responses. To elicit proper physiological responses, ABA levels have to be constantly adjusted to changing physiological and environmental conditions. To date, the mechanisms for fine-tuning ABA levels remain elusive. In this presentation, I will present evidence that AtBG1 and AtBG2, two �-glucosidase homologs localized to the ER and the vacuole, respectively, hydrolyze glucose-conjugated ABA (ABA-GE) to produce ABA. Loss-of-function mutants, atbg1 and atbg2, displayed early germination, abiotic stress-sensitive phenotypes, and lower ABA levels, whereas transgenic plants overexpressing AtBG1 or AtBG2 accumulated higher ABA levels and displayed enhanced tolerance to abiotic stress. Under dehydration stress, AtBG1 undergoes rapid polymerization into a high molecular weight forms, resulting in a 4-fold increase in its enzymatic activity and AtBG2 was protected from degradation, resulting in higher protein levels. Furthermore, diurnal increases in ABA levels were attributable to polymerization-mediated AtBG1 activation. We propose that plants accomplish rapid increases in ABA levels in response to environmental cues by hydrolysis of ABA-GE using organelle-specific AtBG isoforms whose activity is modulated by mechanisms involving transcriptional and post-translational modulation.

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<<뒷표지>> (사) 한국식물생명공학회 (305-701) 대전시 유성구 구성동 373-1 한국과학기술원 생물과학과 2201호 http://www.kspbt.org Phone: 042-862-0986 Fax: 042-860-4608 [email protected] / [email protected]

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