Plenary Lecture
Highlights of My Long Journey in Plant Biochemistry
Ragai K. IbrahimPlant Biochemistry Laboratory, and Centre for Structural
&Functional Genomics, Concordia University, MontrÉal, QuÉbec, Canada H4B 1R6
Plants are endowed by their ability to synthesize and accumulate an enormous variety of organic molecules, collectively known as secondary metabolites. These include the alkaloids, phenolics and terpenoids, among others. Of the phenolic compounds, flavonoids constitute one of the most important group of compounds; since they form an important part of human diet and active principles of many medicinal plants. This lecture highlights some of my original contributions to the field of plant biochemistry, particularly flavonoid compounds, during mtfollowing studies focused mainly on some novel aspects of the enzymes involved in later steps of flavonoid biosynthesis, with emphasis on O-methyltransferases (OMTs), glucosyltransferases (GTs), a flavonol 6-hydroxylase (a novel oxoglutarate-dependent dioxygenase), sulfotransferases (STs) and prenyltransferases (PTs), as well as cloning, and inferences about their phylogenetic relationships, of the genes encoding some of these enzymes. We started with the OMTs, and demonstrated the enzymatic methylation of almost all hydroxyl groups on the flavonoid ring system, including the rare 8-position of flavonols and 5-position of isoflavones, all of which involved single methylation steps. Our search for a system that catalyzes multiple steps of methylations was realized in a unique semi-aquatic weed, Chrysosplenium americanum (Saxifragaceae) which served as the ‘treasure-chest’ of our laboratory for close to two decades, in studies of the enzymology and compartmentation of
polymethylated flavonol glucosides
(PMFGs) of this plant, and cloning of the first cDNA encoding a flavonol-specific OMT. We also demonstrated, for the first time, that (a) the stepwise methylation of quercetin (Q) to its pentamethyl derivative is catalyzed by five distinct enzymes, (b) with an orderly sequence of methyl transfers from: Q→ 3-MeQ → 3,7-MeQ → 3,7,4'- MeQ → 3,6,7,4'-MeQ → 3,6,7,2',4'-/
3,6,7,4',5'-MeQ, (c) four of these methylation steps take place at the aglycone level, whereas the last one occurs at the glucoside level, and (d) glucosylation of the 2'- and 5'-hydroxyls is also catalyzed by two distinct GTs; thus providing a strong evidence for the substrate- and regio-specificities of both groups of enzymes. These studies allowed us to develop a compartmentation model for the ten enzymes involved in PMFG synthesis, and secretion of the end metabolites
to the external leaf surface by the use of ultrastructural, immunofluorescence and immunocyto-chemical techniques. One of the important hallmarks of our research was the introduction of
‘
flavonoid sulfonation
’ to the plant biochemistry literature (1985-1992), as a result of the pioneering contributions of Denis Barron to the phytochemistry, organic synthesis, chemical detection and spectroscopic analysis of sulfated flavonoids, and of Luc Varin to the enzymology and molecular biology of flavonoid sulfonation. The availability of an extensive synthetic depository of sulfated flavonoids, the use of affinity chromatography ligands and the development of a reliable sulfotransferase (ST) enzyme assay, allowed the purification and characterization of four, substrate- and stereospecific STs involved in the biosynthesis of flavonol tetrasulfates in Flaveria spp.(Asteraceae), and establish the sequence of their sulfonation from quercetin (Q) → Q-3S → Q-3,3'/3,4'S → Q-3,7,3'/374' → Q-3,7,3',4'S.
Furthermore, the molecular cloning of flavonol 3-ST gene facilitated its application to the metabolic engineering of the ‘undesirable’
glucosinolate (GI) metabolism in Brassica napus (canola). This was achieved by creating in canola a new pathway for flavonol sulfate (FS) synthesis, to replace GI biosynthesis, since both groups of compounds derive their sulphur from a common sulfate donor, 3'-phosphodenosine-5'-phosphosulfate (PAPS). This strategy resulted in the creation of transgenic canola plants with significantly low GI content and expressing a novel set of metabolites, flavonol sulfates!
We recently isolated and characterized a cDNA clone (TaOMT2) from a cold-acclimated wheat(Triticum vulgare) library, whose gene product catalyzes the stepwise methylation of tricetin (5,7,3',4',5'- pentahydroxyflavone) to its 3'-monomethyl- (selgin), 3',5'-dimethyl-(tricin) and 3',4',5'-trmethyl derivatives. The fact that this novel gene forms a small OMT-gene family amongs members of the Gramineae, prompted us to investigate the structural basis for sequential methylation. The generous collaboration and expertise of Professor Yoongho Lim made it possible to construct a 3-D molecular model for TaOMT2 based on its 63% protein sequence identity with the Medicago sativa caffeic/5-hydroxyferulic acid OMT (MsCOMT) as a template. Time permitting, I shall propose a catalytic mechanism for the sequential methylation of tricetin by this novel protein. Finally, I wish to acknowledge the valuable contributions of all the men and women –graduate students, research associates and guest researchers - who made my long trip such a great pleasure, with many pleasant and unforgettable memories. I also wish to thank the Natural Sciences and Engineering Research Council (NSERC) of Canada, and the QuÉbec Department of Higher Education for their generous financial support.
KSABC Research Award
Structure, Function and Biotechnology of Oat
β-Glucosidase (Avenacosidase) Multimer
In-Soo Kim
School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, South Korea
β-Glucosidases (EC 3.2.1.21) are widely distributed in prokaryotes and eukaryotes and have scientific, medical and economic implications.
An oat β-glucosidase (avenacosidase) hydrolyzes avenacosides to C26-desgluco-avenacosides which have anti-fungal activity. The enzyme exists in a superamolecular assembly of long fibrillar multilers in oat plastid. It has two isomeric forms of type I and type II. Type I isozyme is a homomultimer of As-Glu 1 subunit and type II is a heteromultimer of As-Glu 1 and As-Glu 2 subunits in 1:1 stoichiometry. The cDNAs encodes a plastid-directing transit peptide of 57 amino acid residues and mature proteins of 510 amino acid residues, and their amino acid sequences are highly homologous each other. The cDNA of the As-Glu 1 and As-Glu 2 were cloned and expressed in E. coli into soluble and active enzymes. The As-Glu 1 assembled to fibrillar homomultimers. The As-Glu 2 formed mainly dimmer, but co-expression of the As-Glu 1 and As-Glu 2 assembled to fibrillar heteromultimers. Type I multimer is more stable than type II multimer but lower in catalytic activity. The assembly of the long fibrillar structure of type I enzyme has been elucidated by cryo-electron microscopy. Type I multimer has a novel quaternary structure that is assembled by a linear stacking of hollow trimeric units and the resulting fibril has a long central tunnel connecting to the outer medium via regularly distributed side fenestrations. The enzyme kinetics and chemical modification of type I indicate that the enzyme active site is localized within the central tunnel of the long fibrillar assembly.
This multimer assembly increased enzyme affinity to the in vivo substrate, avenacosides. Molecular swapping of cDNA indicates that the C-terminal segment of AsGlu1 is critical for the fibrillar multimer formation. In fact, a single substitution of glutamic acid-495 of AsGlu2 in the C-terminal region with lysine assembled the As-Glu2 into fibrillar homomultimers. In comparison to the As-Glu2 dimer, the mutant
AsGlu2 homomultimer increases enzyme affinity to substrates. Their kinetic data suggest that the side fenestrations of the fibrillar assembly may have a regulatory role of the substrate entry to the active sites which may function to discriminate the in vivo substrate, avenacosides, from many other kinds of β-glucosides in oat seedlings. Oat β -glucosidase is a good resource for enzyme biotechnology, as it is exceptionally stable in vitro. In attempt to enhance catalytic activity toward flavonoid-β-glucosides, the polarity and size of aglycone binding site of As-Glu 1 was modified. All mutants tested assembled into a stable multimers. A mutant of S186V that was modified the hydrophobicity of the binding site, and two mutants of H197F and F371W that were modified the binding site size efficiently hydrolyzed isoflavonoid-β-glucosides. The S186V mutant increased catalytic efficiency toward the isoflavones by 28-62 times. In particular, the S186V mutant hydrolyzed naringenin-β-glucoside that is relatively resistant to β-glucosidases. These results imply that oat β-glucosidase could be a good resource for enzyme biotechnology for the production of biologically active aglycones.
References
1. Su-Nam Kwak, Sang-Yeob Kim and In-Soo Kim (2008) Assembly and function of AsGlu2 fibrillar multimer of oat β-glucosidase. Biochim. Biophy. Acta (submitted)
2. S.-Y. Kim, Y.-W. Kim, R. Hegerl, M. Cyrklaff, I.-S. Kim (2005) Novel type of enzyme multimerization enhances substrate affinity of oat β-glucosidase, J. Struct. Biol., 150, 1-10
3. Y.-W Kim, K.-S. Kang S.-Y Kim and I.-S Kim (2000) Formation of fibrillar multimers of oat β- glucosidase isoenzymes is mediated by the As-Glu 1 monomer, J. Mol.
Biol., 303, 831-842
4. Y.-W and I.-S. Kim (1998) Subunit composition and oligomer stability of oat β-glucosidase isozymes. Biochim.
Biophy. Acta, 1388, 457-464
5. Y.-W Kim, P.-S. Song and I.-S. Kim (1996) Purification and characterization of isoenzymes of β- glucosidase from etiolated oat seedlings. Mol. Cells, 6, 773-779
6. Su-Nam Kwak, Sang-Yeob Kim and In-Soo Kim (2008) Modification of aglycone binding site of oat β-glucosidase to hydrolyze isoflavonoid-β-glucosides (in preparation)
KOFST Award
Relationship between Cell Cycle and Growth of Plants
Myeong-Hyeon Wang
Department of Molecular and Medical Biotechnology, College of Bioscience and Biotechnology, Kangwon National University
Plants offer a unique possibility to study the integration ofcell division, growth, and development of multicellular organisms.During the mitotic cell cycle genomic DNA of the organism is replicated and segregated equally to the two daughter cells formed upon cytokinesis. Cell division is controlled by the activity of cyclin dependent kinases (CDKs). CDKs play important roles in the plant cell cycle, a highly coordinated process in plant growth and development. CDKs are regulated by the presence of cyclins. CDKs associate with specific cyclins for activation and the timing of CDK activation depends on the kinetics and localization of cyclin expression.
Numerous cyclins were identified in plants and classified into several groups. Different groups of cyclins possess different characteristics of expression. Both A- and B-type cyclins are expressed during mitosis and are known as mitotic cyclins, however, D-type cyclins have a prominent role in the G1-to-S transition and are referred to as G-specific
cyclins. Cyclin D1 has been considered to be an oncogene, and it is believed that it has an important role in the neoplastic transformation of some tumors. The existence of GTcyc was confirmed in genetic tumors by Southern blot analysis and it is also observed that the GTcyc level of expression was significantly higher in the genetic tumor than in the normal leaves of the hybrid and in the parents. A cyclin D1 interacting protein (p22ack1) was isolated by two-hybrid screening system and overexpression of p22ack1 in transgenic Arabidopsis resulted in growth retardation due to a strong inhibition of cell division in the leaf primordial and meristematic tissue. Plant CDK inhibitors such as ICK1 and ICK2 show distinct distributions in different tissues and have specific roles in development. Overexpression of ACK1 in transgenic Arabidopsis significantly inhibited growth, leading to effects such as serration of leaves, as a result of strong inhibition of cell division in the leaf meristem. The relationship extends to the molecular cell cycle regulatory mechanism comes from a comparison of 18 Arabidopsis ecotypes, where a positive correlation between overall cell production rates and cyclin-dependent kinase (CDK) activity was observed. Similarly, local rates of cell division and CDK activity covaried in corn leaves growing under control. Some findings provide a direct link between cell cycle regulation and variation in whole-organ growth rate.
Invited Lectures
Topic A: Biochemistry and Molecular Biology
IL-A1 IL-A1
Regulation of Plant Growth by Immunomodulational Approach
Yoshihito SuzukiUniversity of Tokyo, Japan
Since Hiatt et al. showed that functional antibodies (Abs) could be produced in plants, antibody production in planta has been used to modulate plant functions by capturing target antigens, which could be endogenous molecules, and exogenous substances or organisms.
This methodological notion is called ‘immunomodulation’.
Immunomodulational studies in plants have seen a steady increase in the number of antibodies produced, including those against phytohormones to repress phenomena regulated by phytohormones such as abscisic and jasmonic acid, metabolic enzymes to modulate pigment metabolism and starch synthesis, plant viral coat proteins to confer virus resistance, herbicides to confer herbicide resistance, and against small heat shock proteins that prevent the normal heat stress response. In this study, we applied immunomodulational method in Arabidopsis to repress the function of gibberellin, a class of phytohormones responsible for various plant growth and development, i.e. shoot elongation, seed germination, fruit development, flower induction, etc. There are two possible modes of immunomodulation for GA function. One is inhibition of GA signaling by capture of bioactive GAs by using Abs against bioactive GAs, and the other is inhibition of GA biosynthesis by using Abs against biosynthetic precursors of bioactive GAs. These two types of immunomodulational studies of GA function are described below.
1. Inhibition of GA signaling
Two different antibodies against bioactive GA4 were produced in Arabidopsis as single-chain Fv (scFv) fused to GFP with four different subcellular localizations: endoplasmic reticulum (ER), cytosol, apoplastic space, or the outer surface of the plasma membrane. When targeting scFv-GFP to ER, plants showed highest accumulation of scFv-GFP with binding activity, strong GFP fluorescence in ER-derived compartments and mild but clear GA-deficient phenotypes, including reduced leaf size, delayed bolting, reduced inflorescence length and decreased germination. Plants expressing scFv-GFP in ER responded to exogenous GA4 and contained 15 to 40 times higher endogenous GA4 than wild-type plants. They also showed increased gene expression for GA3ox1, GA20ox1 and GA20ox2, but decreased expression for GA2ox1, which are respectively feedback- and feedforward-regulated by GA signaling. These results suggest that the level of free functional GA4 decreased by being trapped in the ER with scFv to the extent that mild GA-deficient phenotypes were created. A dramatic increase in the total sum of GA4(free plus scFv-GFP bound) was detected due to the up-regulation of GA biosynthesis (feedback regulated), and a reduction in GA4 catabolism due to protection by scFv-GFP binding.
2. Inhibition of GA biosynthesis
A scFv against GA24, a precursor GA, was utilized to repress the biosynthesis of bioactive gibberellins. Stable accumulation of the scFv in ER was achieved by being produced as a fusion with GFP.
The transgenic plants showed GFP fluorescence in the reticulate cortical ER network in epidermal cells. The GFP-scFv fusion produced in plants maintained its binding activity. The transgenic plants showed GA-deficient phenotypes, including reduced rosette leaf development, delayed flower induction and reduced stem elongation of the main culm, especially in the early stage of inflorescence growth. Contrarily, stem elongation of the main culm at a later stage, or that of lateral
shoots was much less affected by scFv production. These phenotypes were different from anti-bioactive GA scFv-producing lines, whose stem elongation was continuously repressed throughout the inflorescence development. The GA-deficient phenotypes were recovered by treatment with GA24 and bioactive GA4, the latter being more effective. The transgenic lines contained conspicuously higher endogenous GA24 and clearly less GA4 than wild-type plants. The expression of GA 20-oxidase and GA 3-oxidase genes were up-regulated in those plants. These results demonstrate that the scFv trapped GA24 in ER and inhibited metabolism of GA24 to bioactive GA4.
IL-A2 IL-A2
Bioinformatics Application of Chemical Transformation Rules in Metabolic Reactions
Susumu Goto, Masahiro Hattori, Yugo Shimizu, Masaaki Kotera, Toshiaki Tokimatsu, Yuki Moriya,
Zen-ichi Nakagawa, Minoru Kanehisa
Bioinformatics Center, Institute for Chemical Research, Kyoto University
The progress in omics technologies such as genome sequencing, transcriptome and proteome profiling, and metabolome analysis has produced an increasing amount of data. These are the basis for understanding biological systems in molecular and cellular levels. We have been developing KEGG, Kyoto Encyclopedia of Genes and Genomes, to bridge the information and current knowledge in molecular biology and biochemistry [1]. The current knowledge is accumulated in the KEGG PATHWAY and KEGG BRITE databases in the form of metabolic and other network diagrams and functional hierarchies of proteins and chemical compounds, respectively.
In contrast to the omics data that is produced basically for all information available in a genome or cell, the information in the PATHWAY database are manually annotated based on published articles and do not cover the information as a whole yet. For example, there are many missing parts in plant secondary metabolism and bacterial biodegradation compared with the intermediary metabolisms that is relatively well elucidated. In some missing pathways, we know the initial substrate and the final product, but do not know the intermediate compounds, and want to know them by predicting the corresponding enzyme reactions and genes in a given genome.
A simple way to predict or compute the missing pathway is to use reaction database by searching a list of reactions whose substrate and product match to those in question. However, it is not the usual case, and chemical transformation rules (reaction patterns) will be useful in that case. We have developed RPAIR database that stores substrate and product pairs of reactions in the KEGG REACTION database, and extracted a chemical transformation rule from each pair by aligning its two compounds. We defined reaction center, different part and matched part for each alignment and use them as the transformation rule [2].
If some part of a query substrate can be aligned to the substrate part of a transformation rule, we can infer the product by applying the rule. This will, however, produce a large number of possible products, because we took only adjacent atoms to the reaction center and there are many possible transformation rules that can be aligned to the query substrate. To reduce such possibility, we have classified the rules according to how the original reactions are used in terms of functional classification such as those defined for the PATHWAY and BRITE database. We have applied this method to predict biodegradation pathways [3] and currently try to predict plant secondary metabolisms. For the current and future work, we still need to develop an efficient and automatic pathway computation system using the chemical transformation rules and linking the rules to the genomic information for genome annotation.
[1] Kanehisa, M. et al., KEGG for linking genomes to life and the environment. Nucleic Acids Res. 36, D480-D484 (2008).
[2] Kotera, M. et al., Computational assignment of the EC numbers of genomic-scale analysis of enzymatic reactions.
J. Am. Chem. Soc. 126, 16487-16498 (2004).
[3] Oh, M. et al., Systematic analysis of enzyme-catalyzed reaction patterns and prediction of microbial biodegradation pathways. J. Chem. Inf. Model. 47, 1702-1712 (2007).
IL-A3 IL-A3
Plant Hormones and Rice Growth
Dongsu ChoiDepartment of Biology, Kunsan National Universit
Deepwater rice is cultivated as a subsistence crop in many flood-prone areas of Southeast Asia because of its remarkable capability of stem elongation under submergence. Some deepwater rice cultivars can grow up to 25 cm a day when partially submerged. Rapid internodal growth of deepwater rice is induced by an environmental signal (submergence) and is mediated by the interaction of at least three plant hormones: ethylene, abscisic acid (ABA), and gibberellin (GA). Upon submergence, the gas atmosphere and hormone ratio in deepwater rice internodes changes dramatically. Submergence increases the ethylene level, which leads to a rapid reduction in ABA content. Since ABA is a strong antagonist of GA action in rice internodes, the reduced level of ABA renders the internodal tissue more responsive to GA. Ethylene and submergence enhance stem elongation in deepwater rice, at least in part, by reducing in the internode the endogenous abscisic acid (ABA) content and increasing the level of gibberellin A1 (GA1). We observed the expression of the OsGA20ox2 and OsGA20ox4 genes, which encode GA-20 oxidase, and of the OsGA3ox2 gene, which encodes the enzyme that converts GA20 to GA1, was upregulated by ethylene treatment. We cloned and characterized the CYP707A5 which encode ABA 8'hydroxylase, the enzyme that oxidizes ABA.
Expression of CYP707A5 was upregulated significantly by ethylene treatment indicating that CYP707A5 may play a role in ABA catabolism during submergence- or ethylene-induced stem elongation in deepwater rice. All the evidence indicates GA is the ultimate hormone that promotes elongation of deepwater rice internodes. We are currently identifying GA-responsive genes that are differentially regulated in GA-induced stem growth of deepwater rice.
Among GA-responsive genes, OsGRF1 (Oryza sativa Growth-Regulating Factor1) is a novel plant gene encoding a transcription regulator whose expression is regulated by GA.
Expansin genes are another GA-responsive genes encoding primary cell wall-loosening factors that mediate stress relaxation of cell walls in a pH-dependent manner. A close correlation between growth, expression of expansin genes, and the activity of expansin proteins has been found in a number of plant species.
To further understand the mechanism by which GA promotes internodal elongation in deepwater rice, we performed microarray experiment. Using the 60K Rice Whole Oligomeric DNA Microarray system, we found 605 genes were upregulated and 623 genes were downregulated specifically by GA treatment.
Genes encoding expansins, enzymes for modification of cell wall polymers and factors regulating DNA replication were upregulated. With this study, we will be able to build a model for the molecular events in the early stage of stem growth. In addition, The results from the study will contribute to elucidating mechanisms of rice vegetative growth, and to introducing elongation capacity into high yielding rice cultivars that are usually susceptible to flooding.
IL-A4 IL-A4
Energy, Carbon, and Nitrogen Metabolism in Hydrogen Bacteria
Masaharu Ishii1, Masafumi Kameya1, Akane Miura1, Ki-Seok Yoon2, Hiroyuki Arai1, Hirofumi Nishihara2,Yasuo Igarashi1
1Department of Biotechnology, The University of Tokyo, 2Faculty of Agriculture, Ibaraki University
Hydrogen bacteria utilize the energy liberated from the oxidation of molecular hydrogen for the fixation of carbon dioxide. Most of them are facultative autotrophs, while a few are known as obligate autotrophs. In my talk, I will select one facultatively autotrophic strain (Ralstonia eutropha) and one obligately autotrophic strain (Hydrogenobacter thermophilus). Because the prominent characteristic of hydrogen bacteria is their ability to utilize molecular hydrogen as an energy source, I will introduce the enzymatic system for metabolizing molecular hydrogen, hydrogenase. Also, recent our achievements for the utilization of hydrogenase are presented. In this section of the talk, Ralstonia eutropha will be selected. Autotrophic mode of living is quite interesting in terms of biochemistry. It should be much more interesting if the strain has a deep branching point in 16S rDNA analysis. In fact, Hydrogenobacter is the very example.
In the talk, I will introduce the brief history of unraveling the metabosism of H. thermophilus with special attention toward the carbon dioxide fixation system of the strain (Reductive TCA cycle). Then, I will talk our recent experimental results.
(1) Purification and characterization of fumarate reduatase (2) GS-GOGAT pathway
(3) Ferredoxin-related energy metabolism
(4) (If possible) Aminotransferase system I will finish my talk by presenting the future of hydrogen bacteria.
IL-A5 IL-A5
Structural Genomics of Photosynthetic, Nitrogen-Fixing,
Bradyhizobiumand Functional Genomics of
Bradyrhizobium japonicumMichael Sadowsky
Department of Soil, water, and Climate; and BioTechnology Institute, University of Minnesota, St. Paul, MN(USA)
Members of the genus Bradyrhizobium refer to slow-growing, nitrogen-fixing microsymbionts of legumes in the family Rhizobiaceae.
Photosynthetic Bradyrhizobium, like other members of the genus, form nitrogen-fixing nodules on the roots of legumes. These strains, however, also form nodules on the stems of some aquatic legumes belonging to the genus Aeschynomene. Collectively, these bacteria have been referred to as the rhizobia. The photosynthetic bradyrhizobia comprise two host-specificity groups: group II strains are able to nodulate all stem-nodulating species of Aeschynomene, whereas group III strains only nodulate some species of this legume. Interestingly, while the common nod genes, nodABC, have been found in group I strains, and all other rhizobia, they have never been identified among the strains belonging to group III. Here I report on a joint France-U.S.
structural genomics project, initiated in 2005, to sequence the genomes of two photosynthetic Bradyrhizobium strains, ORS278 and Btai1, both in group III. Our objectives were to enhance our understanding of the genomics of photosynthetic rhizobia with regards to their carbon and nitrogen–fixing capacities and to use comparative genomics to understand the evolution of symbiotic and photosynthetic systems in the rhizobia. This project was done in collaboration with GÉnoscope in France, the DOE Microbial Genome Program, the LSTM, the Microbial Ecology Laboratory at the University of Minnesota, the National Center for Soybean Technology at the University of Missouri, and the laboratory of BioÉnergÉtique CÉllulaire (CEA) in France.
The manual annotation of the sequences of these 2 genomes (7.4 Mb for ORS278 and 8.2 Mb for Btai1) has been completed. Here I present the most relevant characteristics found in these unique bacteria relative to those found in other rhizobia, and discuss the possible evolution of these unique, autotrophic, photosynthetic, nitrogen-fixing symbiotic bacteria.
Aside from structural genomic studies, we have also been examining the functional genomics of Bradyrhizobium japonicum USDA110, the nitrogen-fixing microsymbiont of temperate legumes This genome for this bacterium, which forms nodules on the roots of soybean, was sequenced in 2002 by the Kasuza Institute in Japan. The strain USDA 110 has a genome size of 9,105,828 nucleotides, and encodes for approximately 8,317 CDS. The functional genomic studies are being done in collaboration with the University of Minnesota in St.
Paul, MN (USA) and the University of Missouri, Columbia, MO (USA). Here I report on functional genomic studies of this bacterium done using 70-mer microarrays. Our laboratory has focused on studying the regulation of genes involved in soil survival. Drought and desiccation can significantly inhibit survival and nodulation efficiency of rhizobium species. Nonetheless, despite the environmental significance of desiccation, the mechanisms specifically involved in desiccation resistance and regulation in soil bacteria and more specifically in rhizobia are still poorly understood. The objective of this study was to acquire a comprehensive understanding of genetic mechanisms potentially involved in drought tolerance in rhizobia in order to enhance the desiccation tolerance capacity of future rhizobium strains. Bradyrhizobium japonicum cells were incubated for 4, 24 and 72 hours under severe drought (27% RH) and hydrated conditions.
The vitality of desiccated cells decreased by more than 50% in the first 24 hours but remained relatively constant for the remaining 48 hours thereafter. Desiccation tolerant genes and gene pathways potentially upregulated as a result of desiccation were assessed using genome wide transcriptional profiling of desiccated vs. fully hydrated cells complemented by additional biochemical analyses. Two hundred and five genes of 8317 total CDS’s in the B. japonicum genome showed induced (>3 times) transcriptional expression levels in the desiccated cells vs. the hydrated ones. These included several gene groups encoding for proteins potentially involved in the desiccation resistance response; most notably, trehalose synthesis genes, reactive oxygen species (ROS) scavengers, DNA modification and repair, alternative sigma factors, heat shock proteins, proteins involved in EPS formation and proteins form the pentose-phosphate pathway.
A substantial fraction of the upregulated genes were of unknown function indicating the presence of additional uncharacterized mechanisms potentially involved in desiccation tolerance. Here I present a discussion of the regulation of the genes most relevant for survival of this bacterium to soil desiccation, salt, and osmotic stresses and discuss how these and other genes are involved in the symbiotic interaction with soybean.
IL-A6 IL-A6
Glucose & Energy Signaling Networks in Plants
Sang-Dong Yoo2, Young-Hee Cho1, Jen Sheen11Molecular Biology/Genetics, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114, USA,
2Department of Biological Science, Sungkyunkwan University, Suwon, Korea
Plant life relies and revolves around sugar production, utilization, storage and mobilization. In contrast to conventional concepts limiting sugar’s roles in supplying energy, metabolites and polymers, glucose and energy signaling play central regulatory roles in orchestrating diverse plant processes from embryogenesis to senescence. To elucidate the fundamental and complex plant glucose and energy signaling networks, we have taken a combination of cellular, genetic,
genomic, and proteomic approaches. Using Arabidopsis as a genetic model, we have isolated and characterized glucose insensitive (gin) and glucose oversensitive (glo) mutants. Our studies of gin and glo mutants have revealed extensive and intimate molecular connections between glucose and plant hormone signaling pathways. We have also provided compelling evidence for the uncoupling of glucose signaling from glucose metabolism in controlling gene expression and developmental processes. The evolutionarily conserved glucose sensor hexokinase and energy sensor protein kinases KIN10/11 control the energy budget and resource utilization through novel nuclear signaling complexes and numerous plant metabolic pathways in anabolism and catabolism. The flexible and reversible responses to low and high glucose signals and energy status in plant growth promotion and inhibition depend on cell types, developmental state, multiple nutrient status, and environmental conditions. The plasticity of plant developmental programs could be attributed to versatile sugar and energy signaling activities in the plant signal transduction networks.
Key References
1. Baena-Gonzalez, E.*, Rolland, F.*, Thevelein, J., and Sheen, J. 2007. A central integrator of transcription networks in plant stress and energy signalling. Nature, 448: 938-942.
2. Cho, Y.H.*, Yoo, S.D.*, and Sheen, J. 2006. Regulatory functions of nuclear hexokinase1 complex in glucose signaling. Cell, 127: 579-589.
3. Rolland, F., Baena-Gonzalez, E., and Sheen, J. 2006. Sugar sensing and signaling in plants. Ann. Rev. Plant Biol. 57:
675-709.
4. Moore, B.*, L., Zhou, L.*, Rolland, F., Hall, Q., Cheng, W.-H., Liu, Y.-X., Jones, T. L., and Sheen, J. 2003. Role of the Arabidopsis glucose sensor HXK1 in nutrient, light and hormonal signaling. Science, 300: 332-336.
IL-A7 IL-A7
A Capped Small Nucleolar RNA Is a Splicing Regulator in Arabidopsis thaliana
Jun Yong Lee, Seung Kook Choi, Sang Hyon Kim,
Division of Biosciences and Bioinformatics, Myongji University, Yongin, Korea
Small nucleolar RNAs (snoRNAs) are nucleolar RNAs involved in biogenesis of ribosomal RNA (rRNA). A portion of snoRNAs are involved in cleavage reactions from long primary rRNA precursor in eukaryotes and archaebacteria, to generate mature 18S, 5.8S and 25S rRNAs. However, the majority are guide RNAs to determine the precise sites of 2’-O-ribose methylation and pseudouridylation of rRNA. Two major structurally different families of snoRNAs are well categorized: box C/D snoRNAs responsible for 2’-O-ribose methylation and box H/ACA snoRNAs responsible for pseudouridylation.
SnoRNAs have a number of different modes of expression. Some genes are monocistronic being transcribed from their own promoters, while others are intronic (encoded within an intron of a pre-mRNA) or polycistronic. Yeast contains five polycistronic clusters and many intron-encoded snoRNAs and the vast majority of metazoan snoRNAs are intronic. Intronic snoRNAs are expressed as part of the precursor mRNA (pre-mRNA) of the host gene and require splicing for production of the snoRNA. Plant snoRNAs have all three types of gene organisation but are unique in that the majority are organised into polycistronic gene clusters and processing of primary transcripts to produce mature snoRNAs is not splicing-dependent. Plant snoRNA gene clusters usually contain around 2 to 8 genes but exceptionally long clusters, made up of repeated smaller clusters have been found in rice. Plant polycistronic clusters are transcribed as polycistronic precursor snoRNAs (pre-snoRNAs) which are then processed via
endonucleolytic activity followed by exonucleolytic trimming to produce the mature snoRNA.
To identify further Arabidopsis snoRNAs, we carried out a comprehensive analysis of short RNAs from RNA of purified Arabidopsis nucleoli by constructing cDNA libraries from size-selected(89-400 nt), capped and uncapped RNAs. The uncapped library contained full-length sequences of over 100 different small nucleolar RNAs (snoRNAs). These snoRNAs derive from gene clusters, would be processed from pre-snoRNA transcripts and are therefore not capped. The capped library contained spliceosomal small nuclear RNAs (snRNAs) and known capped snoRNAs (U3 and MRP) as expected, and three capped box C/D snoRNAs and a novel capped snoRNA, snoR113.
The three capped box C/D snoRNAs unusually have the box C sequence 20 nt from the 5’ end and this 20 nt region is exactly conserved in all three genes. Examination of the genomic sequence of the three genes found promoter elements of snRNAs –namely an Upstream Sequence Element (USE) at ~90 and a TATA box at ~25 bp upstream of the transcription start site. These are well-characterised plant promoter elements found upstream of tri-methyl-guanosine capped spliceosomal snRNAs (U1, U2, U4 and U5) and U3 and MRP snoRNAs with essential functions in splicing and in the cleavage of pre-rRNA to produce ribosomal RNA. Expression from such promoters is assumed to be constitutive, making it all the more surprising that three uncharacterised snoRNAs also rely on this mode of expression.
In addition, the novel snoRNAs do not have complementarity to rRNA or snRNA but instead initial BLAST searches identified significant complementarity to a range of Arabidopsis mRNAs. The cloning of the novel snoRNAs from the capped library, the presence of snRNA promoter elements, the novel structure of the snoRNAs, the wide-spread occurrence of such snoRNAs in different species and their complementarity to mRNAs suggest an important function in regulating gene expression of the target mRNAs. We have demonstrated expression of all three capped snoRNAs by RT-PCR and northerns.
We have begun to characterize one of these snoRNAs, snoR113, in detail and show the RNA to be enriched in the nucleolus by RT-PCR and in situ hybridisation. In addition, we have identified a knock-out mutant in snoR113 which has a transposable element insertion between the promoter and snoRNA coding sequence and snoR113 transcripts are undetectable by RT-PCR. The snoR113 mutant has a phenotype with delayed flowering, and first indications suggest that alternative splicing of one putative target is altered in the knock-out. Some extensive complementarity was found including a 20 nt exact complementarity between snoR113 and the 45 nt exon of a kinase of unknown function. Interestingly, a small RNA corresponding exactly to this region of snoR113 is present in the Arabidopsis small RNA project (ASRP) database and is potentially derived from the RNA duplex formed between the snoRNA and pre-mRNA or mRNA template.
A key question is whether snoRNAs, as nucleolar RNAs, can interact with mRNAs or pre-mRNAs. Firstly, the human HBII-52 snoRNA interacts with a pre-mRNA to affect alternative splicing, presumably in the nucleus. Secondly, the maturation pathway of snRNAs involves the export of pre-snRNAs to the cytoplasm where the cap is converted to a tri-methyl-G cap and core Sm proteins are assembled, prior to re-import into the nucleus. A similar pathway has recently been demonstrated for U3 snoRNA and therefore the novel capped snoRNAs are likely to also localize to the cytoplasm during maturation providing an opportunity to interact with mRNAs.
Finally, although, the nucleolus is the site of rRNA transcription and processing and of ribosome subunit assembly, it is clear that the nucleolus is multi-functional and is involved in many aspects of RNA processing and RNP metabolism and some mammalian mRNAs have been found in the nucleolus. More importantly, we recently showed that exon junction complex (EJC) proteins were present in the nucleolus
by proteomics and GFP-fusion protein localisation. The EJC is deposited on mRNAs following splicing of introns and is therefore associated with mRNAs. We have now demonstrated that the plant nucleolus contains fully spliced mRNAs and aberrant mRNAs providing the opportunity for the snoRNAs to interact with mRNAs.
Here we provide an evidence that a capped snoRNA is involved in splicing of a bunch of protein-coding mRNAs gene whereby the RNA is supposed to regulate aging.
IL-A8 IL-A8
RiceArrayNet: A Database for Correlating Gene Expression from Transcriptome Profiling with a 60k Gene Rice Microarray
Yeon-Ki Kim1, Tae-Ho Lee2, Baek-hie Nahm21GreenGene Biotech Inc. and Division of BioScience and Bioinformatics, Yongin, 449-728, 2
Division of BioScience and Bioinformatics,, Myongji University, Yongin, Korea
Transcriptome profiling with microarrays has been widely used to reveal individual or groups of gene expression patterns on a genomic scale. The data accumulated thus far have made it possible to understand the relationships between genes involved in various biological contexts of an organism, given the complete spectrum of experimental conditions and materials included in a database. However, there have been no reports to date regarding a database for rice (Oryza sativa), a model plant for monocotyledons. Here, we built a database, called RiceArrayNet (RAN), that provides co-expression information between genes in terms of correlation coefficients. Given the correlation of a gene pair, the degrees of closeness between genes are calculated using a ‘neighbor-joining’ me-thod and are visualized in a relational tree. In addition, RAN supports scatter plots of log ratios between genes and links them to pathway maps, while providing a common cis-element list of promoter regions that are involved. As such, RAN allows users to grasp the meaningful biological context of clustered or co-expressed genes.
We applied RAN database to study co-expression pattern in Rice.
In an example of 8 membered-L7Ae ribosomal protein family on rice genome, Os01g0276000 and Os01g0276000 show that the 589 and 519 genes are co-expressed, respectively, under the criteria of r=.5 and depth=1 and many of them are ribosomal proteins implying these proteins are expressed in stoichiometric ratios for efficient translation as suggested in arabidopsis. In contrast, Os05g0490100, Os07g0150200, and Os08g0326400 in the same family have about 30 or more genes. These data show that the genes comprizing a family are differently controlled and expressed depending on cellular processes such as development and stresses. We retrieved the same group of genes from TAIR arabidopsis genome v.8 and performed similar analysis in ACT (http://www.arabidopsis.leeds.ac.uk/act/) which provides functions for the analysis of co-expressed genes in arabidopsis. We observed the similar partitioning of members of a gene family according to co-expression pattern in stressed related genes sush as a DREB2, a trehalose-6-phosphate synthase, and a small heat shock protein genes. A phylogenetic and statistical analysis suggested that a functional gene according to a co-expression pattern in a species is identifiable in the other species and vice versa. The functional equivalents in model organisms could be identifiable and information could be applicable to the other organisms to identify gene functions.
* Corresponding Author: [email protected], 017-225-5460 Acknowledgements : Sponsored by BioGreen 21 program and Crop Functional Genomics Center
Topic B: Functional Materials IL-B1
IL-B1
Brassica Vegetables and Cancer Prevention
Elizabeth H. JefferyDepartment of Food Science and Human Nutrition and the Division of Nutritional Science, University of Illinois, 445 Bevier Hall, 905 S. Goodwin Ave., Urbana, IL., 61801, USA.
Epidemiological studies suggest that 3 –5 servings per week of broccoli (Brassica oleracea L.), and/or other vegetables from the Brassicaceae family such as cabbage or radish, can decrease the risk for a number of cancers, including colon, prostate, breast and kidney cancer [1, 2]. Animal studies have confirmed cancer prevention in several experimental cancer models [2]. The Brassicaceae contain a group of plant secondary compounds called glucosinolates. These thioglucosides have no biological activity. However, hydrolytic removal of the glucose, by the plant thiohydrolase enzyme myrosinase, produces an unstable intermediate that rearranges to form one of several products, including the isothiocyanates that have been identified as anticarcinogenic [3]. When myrosinase is destroyed by heating, as in cooking, then hydrolysis of ingested glucosinolates occurs in the lower gut, catalyzed by gut microflora [4]. Understanding glucosinolate metabolism and bioavailability can aid in determining how to overcome variability of response and optimize the health benefits.
Most brassica contain a mixture of glucosinolates, including both indolyl and aliphatic glucosinolates. The prototype aliphatic glucosinolate hydrolysis product is sulforaphane, the most studied isothiocyanate, although several other isothiocyanates also have strong cancer- preventive properties [5]. Aliphatic isothiocyanates upregulate detoxification enzymes and other antioxidant enzymes that play a role in defending man against carcinogens and other toxic chemicals, and it is this property that led to the idea that brassica inhibit the initiation of cancer. However, isothiocyanates also inhibit proliferation of cancer cells, disrupt histone silencing of tumor suppressor genes and promote apoptosis [6]. These changes suggest that isothiocyanates are able to prevent later stages of cancer. A study of prevention of dimethyl benzanthracene- induced papillomas suggests an even greater role for isothiocyanates in preventing tumor growth than in preventing initiation [7]. The prototype indolyl glucosinolate is glucobrassicin, which upon hydrolysis yields indole-3-carbinol (I3C). This upregulates cytochrome P450 1A1, altering estrogen metabolism and decreasing risk for estrogen-dependent breast and ovarian cancers [8]. Recent work suggests that both aliphatic isothiocyanates and I3C may play a role in preventing inflammation [9]. This work was supported in part by USDA, in part by NIH.
References
[1] van Poppel G, Verhoeven DT, Verhagen H, Goldbohm RA.
Brassica vegetables and cancer prevention. Adv Exp Med Biol. 1999; 472:159-68.
[2] Jeffery EH, Keck A-S Translating knowledge generated by epidemiological and in vitro studies into dietary cancer prevention. Mol Nutr Food Res 2008; 52; S7-S17.
[3] Matusheski NV, Jeffery EH. Comparison of the bioactivity of two glucoraphanin hydrolysis products found in broccoli, sulforaphane and sulforaphane nitrile. J Agric Food Chem.
2001; 49:5743-9.
[4] Bheemreddy RM, Jeffery EH. The metabolic fate of purified glucoraphanin in F344 rats. J Agric Food Chem. 2007 Apr 18;55(8):2861-6
[5] Munday R, Munday CM. Induction of phase II detoxification enzymes in rats by plant-derived isothiocyanates:
comparison of allyl isothiocyanate with sulforaphane and related compounds. J Agric Food Chem. 2004; 52: 1867-71 [6] Juge N, Mithen RF, Traka M. Molecular basis for
chemoprevention by sulforaphane: a comprehensive Cell Mol Life Sci. 2007;64:1105-27
[7] Gills JJ, Jeffery EH, Matusheski NV, Moon RC, Lantvit DD, Pezzuto JM. Sulforaphane prevents mouse skin tumorigenesis during the stage of promotion. Cancer Lett.
2006; 236:72-9
[8] Wong GY, Bradlow L, Sepkovic D, Mehl S, Mailman J, Osborne MP. Dose-ranging study of indole-3-carbinol for breast cancer prevention. J Cell Biochem Suppl.
1997;28-29:111-6.
[9] Araya M, Jeffery, EH. Physiological effects of broccoli consumption. Phytochem. Rev. 2008, in press.
IL-B2 IL-B2
Lycopene, Tomatoes and Prostate Cancer
John W. Erdman Jr.Department of Food Science and Human Nutrition and the Division of Nutritional Science, University of Illinois, 445 Bevier Hall, 905 S. Goodwin Ave., Urbana, IL., 61801, USA.
Numerous epidemiological trials have associated the intake of high carotenoid-containing foods or blood carotenoid concentrations with reduced incidence of a number of chronic diseases such as cardiovascular disease and some forms of cancer. Epidemiological and animal studies suggest that consumption of tomato products reduces the risk of prostate cancer. Prostate cancer is the most common malignancy in American men and dietary approaches that reduce its risk or delay its progression could have profound impact on public health. Most of the research interest on tomato and prostate cancer has focused upon the single, red tomato carotenoid, lycopene. Lycopene is only found in significant concentrations in a very select number of foods (tomato, watermelon, guava, pink grapefruit), with about 85% of lycopene intake in the U.S. coming from fresh and processed tomato products. The parent molecule, lycopene, may indeed provide some of the cancer protection associated with tomato intake but it is not the only bioactive compound in the tomato. In fact, the tomatoes contain significant quantities of vitamins C and E, folate, polyphenols and other carotenoids such as phytoene and phytofluene. It is possible that dietary intake of lycopene, or blood or tissue lycopene simply serves as a marker of tomato intake.
Our laboratory, in conjunction with other scientists from the University of Illinois and The Ohio State University, have carried out a series of in vitro and in vivo trials to evaluate the impact of tomato and tomato bioactives upon development and progression of prostate cells in culture or tumors in animals. We have determined that feeding whole, freeze-dried tomato powder lowers prostate cancer tumor growth while lycopene by itself results in a small, non-significant reduction [1, 2]. Clearly, other bioactives in tomatoes are important to prostate cancer reduction in addition to lycopene We have also found that the addition of freeze-dried broccoli powder to tomato powder results in reduced growth of Dunning transplantable tumors in Copenhagen rats more than either powder alone [2]. Of all the carotenoids tested, lycopene has been demonstrated to be the most potent in vitro antioxidant, primarily acting as a singlet oxygen quencher. However, it is not clear whether lycopene is an important antioxidant in vivo. Our laboratory has postulated that metabolic products of lycopene, lycopenoids [3], may be more biologically active than the parent molecule. We propose that lycopene is cleaved by the eccentric carotenoid monooxygenase cleavage enzyme, CMO-II, to initially produce lycopenals which could then be oxidized to lycopenoic acids,
chain-shortened, or further metabolized. Lycopenoids, due to structural and polarity similarities to retinoids, may act as agonists or antagonists for a number of nuclear receptors such as RARs, RXRs, PPARs, LXRs, etc., or may activate response elements such as the ARE. Kiefer and coworkers [4] demonstrated in E. coli engineered to produce lycopene and expressing CMO-II exhibited a color shift while E. coli engineered to produce β-carotene and expressing CMO-II did not. Hessel et al [5] showed in mice lacking the central cleavage enzyme, CMO-I, that β-carotene was not converted to vitamin A, an observation that we have recently confirmed [6]. Moreover, we found in CMO-I KO mice fed lycopene that there was an altered tissue lycopene biodistribution and isomer pattern compared with wild-type mice. In a pilot study with CMO-II KO mice fed lycopene, many tissues had enhanced lycopene accumulation but unaltered isomer distribution [unpublished data]. Thus, there is emerging in vitro and in vivo evidence to suggest that lycopene is metabolized in mammalian tissues to produce retinoid-like compounds. Thus, lycopenoids, along with metabolites of other tomato carotenoids, may contribute to the reduced prostate cancer risk associated with tomato consumption. (Supported in part by PHS R03 CA112649 A &PHS 1 R01 CA125384 A)
References
[1] T.W. Boileau, Z. Liao, S. Kim, S. Lemeshow, J.W. Erdman Jr., S.K Clinton. J. Natl. Cancer Inst.
95
, 1578-1586 (2003) [2] K. Canene-Adams, B.L. Lindshield, S. Wang, E.J. Jeffery,S.K. Clinton, J.W. Erdman Jr.
67
, 836-843 (2007) [3] B.L. Lindshield, K. Canene-Adams and J.W. Erdman Jr.,Arch Biochem. Biophy.
458
, 136-140 (2008).[4] C. Kiefer, S. Hessel, J.M. Lampert, K. Vogt, M.O. Lederer, D.E. Breithaupt and J. von Lintig. J. Biol. Chem.
276
, 14110-14116 (2001).[5] S. Hessel, A. Eichinger, A. Isken, J. Amengual, S.
Hunzelmann, U.Hoeller, V. Elste, W. Hunziker, R.
Goralczyk, V. Oberhauser, J. von Lintig and A. Wyss.
J.Biol Chem.
228
, 33553-33561 (2007)[6] B.L. Lindshield, J.L. King, A. Wyss, R. Goralczyk, C-H Lu, N.A. Ford and J.W. Erdman Jr.. (in submission).
IL-B3 IL-B3
Inhibition of Colon Cancer Cell Growth by the Dietary Bioactive Compound Fisetin
Do Y Lim, Xianghua Lu, Jung H Y Park
Department of Food Science and Nutrition, Hallym University, Korea
Colon cancer is one of the leading causes of cancer death in Western countries, and modifications of life style and diet offer measures for reducing the risk of developing colon cancer.
Epidemiological studies have shown that a diet containing abundant vegetables and fruits may reduce the risk of colon cancer.
Flavonoids are a group of naturally occurring polyphenolic compounds present in vegetables, fruits, and other edible plants.
Some of these flavonoids are reported to possess cancer chemopreventive properties, and many studies have recently been conducted to evaluate the mechanisms by which flavonoids prevent cancer, including induction of apoptosis, cell cycle arrest, and antiproliferation of cancer cells.
Fisetin is a natural flavonol found in apple, kiwi fruit, strawberry, and persimmon and has been reported to exert anticarcinogenic effects. We examined the effect of fisetin on induction of apoptosis and the cell cycle progression of the human colon cancer cells. Fisetin inhibited cell growth and induced apoptosis in HCT-116 and HT-29 cells in dose-dependent manners.
Western blot analysis of total cell lysates revealed that fisetin increased cleavage of caspase-8, -9, -7, -3, and poly (ADP-ribose) polymerase. In addition, fisetin increased the translocation of cytochrome c and Smac/Diablo from the mitochondria to the cytosol and induced depolarization of mitochondrial membranes.
Furthermore, fisetin decreased the protein levels of Bcl-xL and Bcl-2 but increased levels of Bim, Bak, Bik, truncated Bid and mitochondrial Bax. Fisetin increased the levels of membrane-bound Fas ligand but had no effect on Fas levels. Treating cells with an inhibitor of caspase-9 or caspase-8 led to a suppression of fisetin-induced apoptosis, caspase-3 activation, and cleavage of PARP. Fisetin increased the expression of p53 tumor suppressor protein and inhibition of p53 expression by RNA interference resulted decreases in fisetin-induced apoptosis and Bax translocation to mitochondria. These results indicate that the induction of apoptosis by fisetin is mediated through activation of cell surface death receptors and changes in mitochondrial membrane permeability, and the subsequent activation of the caspase pathways.
Perturbed cell cycle progression from the G1 to S phase was observed at 8 h with 60 µmol/L fisetin treatment, whereas a G2/M phase arrest was observed after 24 h. The phosphorylation state of the retinoblastoma proteins shifted from hyperphosphorylated to hypophosphorylated, in cells treated with 40 mmol/L fisetin. Fisetin decreased the activities of cyclin-dependent kinase (CDK)2 and CDK4, which were probably attributable to the decreases in the levels of cyclin E and D1 and increase in p21CIP1/WAF1 levels.
However, fisetin also inhibited the CDK4 activity in a cell-free system, indicating it may directly inhibit the CDK4 activity. The protein levels of cell division cycle (CDC)2 and CDC25C and activity of CDC2 were also decreased in fisetin-treated cells. These results indicate that inhibition of cell cycle progression in HT-29 cells after treatment with fisetin can be explained, at least in part, by modification of CDK activities. The present results indicate that fisetin inhibits colon cancer cell growth by delaying cell cycle progression and inducing apoptosis. We provide some of the molecular basis for using fisetin as a potential anti-tumorigenic agent. Further in vivo evaluations of the potential of fisetin as an anti-tumorigenic agent are clearly warranted.
IL-B4 IL-B4
Molecular Targets for Cancer Chemoprevention
Zigang DongHormel Institute, University of Minnesota, Austin, MN 55912 USA Consumption of plant-derived food, especially fruits and vegetables, has been linked to decreased risk of cancer. Laboratory studies with animals and cells in culture have shown cancer preventive activity of phytonutrients isolated from soy, tea, rice, noni and many green, yellow and orange fruits and vegetables. We have used cell culture, transgenic mice and knockout mice models to examine the anti-cancer effects of these dietary factors at the molecular level. We have identified several high-affinity binding proteins with epigallocatechin gallate (EGCG), resveratrol and other chemopreventive agents. By knockdown or knockout of these targeting proteins, we have shown that these proteins are critical in cell growth and cell transformation. We show EGCG in green tea and theaflavins, the major active components in black tea, inhibit epidermal growth factor (EGF)- or 12-O-tetradecanoylphorbol- 13-acetate (TPA)-induced AP-1 and NFkB-dependent transcriptional activation. EGCG binds and inhibits the activity of IGF-1R, GRP78, and Fyn. Active compound from rice and other grains, inhibited TPA- or EGF-induced transformation and signal transduction through its effects on PI3 kinase. Resveratrol inhibited cell transformation through the induction of apoptosis, mediated through JNK pathways. Phenethyl isothiocyante (PEITC) inhibited
cell transformation, correlated with the induction of apoptosis. An elevation of p53 is required for PEITC-induced apoptosis.
Chemicals in ginger and hot pepper showed inhibition of AP-1 and cell transformation and apoptotic pathways in cells. Our studies indicated that the chemopreventive effect of these phytonutrients may target different signal transduction pathways.
[Supported by The Hormel Foundation and NIH Grants CA27502, CA77646, CA81064, CA88961, CA120388, and CA111536]
IL-B5 IL-B5
Bioactive Secondary Metabolites from Fungi and Mushrooms for Health Care
Jong-Pyung Kim
Functional Metabolite Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Korea
It is generally accepted that oxidative stress plays a crucial role in the promotion of aging process, For these reasons, much attention has been paid to antioxidants in the prevention of aging and aging-related disease for the human health care. Skin aging is a complex process that renders several morphological and chemical changes to human skin. Particularly, the photo-aging is induced by the UV irradiation of skin. The conspicuous clinical signs accompanied by the photo-aging are optically hyper-pigmentation and wrinkles of skin. In this aging process, free radicals and related reactive oxygen species plays a crucial role to injure the DNA and extracellular matrix in the skin cells. Therefore, development of natural antioxidants is essential in cosmetics and health care products. Fungi and mushrooms produce diverse secondary metabolites and exhibit various biological effects. Many bioactive compounds were discovered form the metabolites and developed to drugs and beneficial health care products. We have isolated and identified several promising antioxidative, anti-wrinkle, and anti-obesity compounds from the fermented fungal and mushroom metabolites. Here, I will present the isolation and identification of the bioactive secondary metabolites and their biological activities for the application of human health care.
IL-B6 IL-B6
Development of Biological Active Substances and Oriental Treatment of Non-insulin Dependent Diabetes Mellitus from the Recorded Herbs on the Donguibogam
Byoung Seob Ko
Korea Institute of Oriental Medicine, korea
Donguibogam by Heo Jun (AD1610) is the greatest masterpiece, and in Korea it is even today considered a bible of Korean traditional medicines. We have previously investigated a study on the screening for anti-diabeticactivities of traditional herbs and formulas selected in Donguibogam. The symptoms of sogal (消渴) are classified into sangso (上消), joongso (中消), and haso (下消) according to samcho (三焦) part in Oriental Medicine. The characteristic of sangso is that the body becomes thirsty making it drink lots of water (渴而多飮).
Joongso's symptoms are a voracious appetite and getting hungry easily (消穀善飢). Haso's symptoms are drinking lots of water because of thirst, and frequent urinating (渴而尿數). The symptoms are similar to those of diabetes, which are thirst due to hyperglycemia, weight loss, and frequent urinating. Traditional medicines have been used for patients with diabetes, and case reports have indicated improvements in their quality of life as well as prolongation of life in people with type 2 diabetes. However, it is not clear how the bio-activity of these medicines contributes towards retarding the progression of type 2 diabetes.
1. Effects of Okchun-San, a herbal formulation on Type 2 diabetes The effects of herbal medicine on type 2 diabetic animal model were investigated. Herbal medicine were composed with the addition of Coicis Semen into Okchun-san (OCS), Commelinae Herba into Gangsim-tang (GST), Scrophulariae Radix into Hyunsamsunki-san (HSK), and Erythrinae Cortex into Yukmijihuang-hwan (YMH). We evaluated anti-hyperglycemic and body weight reduction activity in diabetic db/db mice. On day 14, OCS-treated db/db mice had significantly lower fasting blood glucose levels compared to control group (296±25.9 versus 593±16.4 mg/dl, p<0.001). The water extracts of these new remedies were treated in 3T3-L1 fibroblasts and adipocytes in order to investigate insulin-like substances and insulin sensitizers, respectively. The treatment of OCS with 1 ng/mL insulin increased glucose uptake much more than only insulin 1 ng/mL treatment.
These result suggest that OCS could be effective on insulin-independent type 2 diabetes.
2. Insulin sensitizing and α-glucoamylase inhibitory action of sennosides, rheins and rhaponticin in Rhei Rhizoma
The fractions fractionated from Rhei Rhizoma (RR) extracts by XAD-4 column revealed that 60%, 80% and 100% methanol fractions enhanced insulin sensitivity and inhibited α-glucoamylase activity. The major compounds of these fraction were sennosides, rheins and rhaponticin. Rheins and rhaponticin enhanced insulin-stimulated glucose uptake in 3T3-L1 adipocytes.
Rhaponticin increased adipocytes with a differentiating effect similar to pioglitazone, but rhein and sennosid B decreased triglyceride accumulation. Sennoside A and B inhibited α -glucoamylase activity as much as acabose. In conclusion, crude extracts of Rhei Rhizoma improves glucose intolerance by enhancing insulin-stimulated glucose uptake and decreasing carbohydrate digestion via inhibiting α-glucoamylase activity. Rhein and rhaponticin are potential candidates for hypoglycemic agents.
3. Insulin sensitizing and insulinotropic action of berberine from Cortidis Rhizoma
We fractionated 70% ethanol Cortidis Rhizoma extracts (CR) extract by XAD-1 column and which fractions had insulin sensitizing and insulinotropic action. Insulin sensitizing action and fat accumulation capacity was investigated in 3T3-L1 adipocytes and insulinotropic action was in Min6 cells. The 20, 40 and 60%
MeOH fractions from XAD-1 column had the most insulin sensitizing activities. The common major peak in theses fractions was berberine. A significant insulin sensitizing activity was observed in 3T3-L1 adipocytes, giving 50 M berberine with 1 ng/mL insulin to reach glucose uptake level increased by 50 ng/mL of insulin alone. This was associated with increased GLUT4 translocation into plasma membrane via enhancing insulin signaling pathway, IRS1-PI3 kinase-Akt. Berberine also increased insulin secretion and enhanced IGF-1 signaling cascade in Min6 cells.
4. Hypoglycemic effects of Schizandrae Fructus fractions To determine hypoglycemic effect of Schizandrae Fructus (SF) extract containing in Okchun-san, one of the Chinese diabetic medicine, we measured insulin-like action, insulin sensitizing action and a-glucoamylase suppressing action of SF extract. SF fractions had a remarkable insulin sensitizing effects, meaning that SF extracts with 1 ng/mL insulin increased glucose uptake more than 50 ng/mL insulin treatment in 3T3-L1 adipocytes. Especially, the treatment of 1 ng/mL insulin plus Fr. 4 (60% methanol) or Fr. 5 (80% methanol) from 70% ethanol SF extracts increased glucose uptake more than 7 folds compared to 1 ng/mL insulin treatment alone. Increased glucose uptake by Fr. 4 and 5 was resulted from increased GLUT4 contents in the cell membrane due to enhanced insulin signaling. In conclusions, SF extracts contains very effective insulin sensitizing compounds to enhance insulin signaling.
IL-B7 IL-B7
Regulation of Terpene Secondary Metabolism by Multiple Gene in Gymnosperms
Sang-Min Kim2, Yeon-Bok Kim2, Jin Hee Kim2, Tomohisa Kuzuyama3, Soo-Un Kim1
1Program in Applied Life Chemistry, School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea,
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. It is now well established that plant plastids, most bacteria except archebacteria, and apicomplexan parasites harbor MEP pathway for isoprenoid building blocks, whereas cytosol of most eukaryotes carry MVA pathway. 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. 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. 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 analyzed by real-time quantitative PCR. 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.
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. Light and methyl jasmonate (MeJA) were applied to the ginkgo embryos and P. densiflora 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. These results thus imply that class 1 genes are involved in the primary metabolism, while class 2 genes in the secondary metabolism.
IL-B8 IL-B8
Metabolic Engineering of Benzophenanthridine Alkaloid Biosynthesis in Poppy
Sang Un Park
Division of Plant Science and Resources, Chungnam National University, 220 Gung-Dong, Yuseong-Gu, Daejeon, 305-754, Korea
This work presents a study on the plant regeneration, genetic transformation, and metabolic engineering in the Papaveraceae. For metabolic engineering in the Papaveraceae, We had to establish the optimized protocols of plant regeneration and transformation as follows: 1) somatic embryogenesis and plant regeneration from seed-derived embryogenic callus and cell suspension cultures of California poppy (Eschscholzia californica Cham.) 2) Agrobacterium-mediated stable genetic transformation of E.
californica via somatic embryogenesis 3) Agrobacterium-mediated protocol for the stable genetic transformation of intact opium poppy, Papaver somniferum L., plants via shoot organogenesis 4) Establishment of transgenic opium poppy and California poppy root
cultures using Agrobacterium rhizogenes.
California poppy cell and hairy root cultures produce several benzophenanthridine alkaloids, with potent pharmacological activity.
Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3'-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures.
Transformed cell lines expressing antisense-BBE or antisense-CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared to control cultures transformed with a β- glucuronidase gene.
Sense and antisense constructs of genes encoding the BBE were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a β -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways.
Topic C: Bioenergy IL-C1
IL-C1
Transgenic Sweetpotato for Industrial Materials in Marginal Lands
Sang-Soo Kwak
Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea
The dramatic increase in population accompanied by rapid industrialization in developing countries has caused imbalances in the supply of food and energy. To cope with these global crises over food and energy supplies as well as environmental problems, it is urgently required to develop new crop varieties to be grown in marginal lands for sustainable agriculture. Sweetpotato (Ipomoea batatas (L.) Lam.) ranks seventh in annual production among food crops in the world. It is also an alternative source of bio-energy and industrial materials such as natural antioxidants. Moreover, it does not require large amounts of fertilizers and pesticides and is rather tolerant to some environmental stresses. The nonprofit Center for Science in the Public Interest (CSPI) designated sweetpotato as one of ten super foods for better health. Taken together, the sweetpotato can be developed to produce valuable industrial materials including bio-energy in marginal lands by molecular breeding. Recently we have established transformation systems of sweetpotato by particle bombardment and Agrobacterium-mediated method. In this respect, we focus on the metabolic engineering for sweetpotato as an industrial plant in the marginal lands to contribute in solving global environment, energy and food problems. For this purpose, we are developing the gene expression platform technology for industrial sweetpotato as follows.
1. Stress-inducible gene expression system
We previously isolated a strong oxidative stress-inducible peroxidase (SWPA2) promoter from the cultured cells of
