J. Plant Biol. (2013) 56:7-12 DOI 10.1007/s12374-012-0333-2
ORIGINAL ARTICLE
Cloning and Characterization of a Putative UDP-Rhamnose Synthase 1 from Populus euramericana Guinier Bong-Gyu Kim†, Woo Dam Jung† and Joong-Hoon Ahn* Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-901, Korea Received: August 30, 2012 / Accepted: October 24, 2012 © Korean Society of Plant Biologists 2012
Abstract L-Rhamnose is a constituent of plant primary cell wall polysaccharides including rhamnogalacturonan-I, rhamnogalacturonan-II, and other natural plant-based compounds. UDP-rhamnose serves as a rhamnose donor whose synthesis is catalyzed by UDP-rhamnose synthase (RHM). A RHM gene, PRHM was cloned from Populus euramericana Guinier. PRHM contains two domains: the NAD dependent epimerase/dehydratase family domain and the RmlD (dTDP-keto-rhamnose-4-keto-reductase) substratebinding domain. Because the recombinant PRHM did not demonstrate any activity during an in vitro assay, complementation with an Escherichia coli mutant was carried out. The rfbD (dTDP-4-dehydrorhamnose reductase), which encodes an enzyme catalyzing the conversion of dTDP-4-keto-rhamnose to TDP-rhamnose, was mutated in E. coli. The mutant strain B-rfbD was transformed with PRHM gene and a flavonoid rhanmosyltransferase gene, AtUGT78D1. The resulting transformant was able to convert quercetin into quercetin 3-O-rhamnoside in a manner similar to that by the wild type E. coli strain harboring AtUGT78D1. This result indicated that PRHM catalyzed the conversion of UDP-glucose into UDP-rhamnose. Key words: Populus euramericana, UDP-rhamnose, UDPrhamnose synthase
Introduction The activated nucleotide sugars (NDP-sugars) serve as sugar donors for biosynthesis of the components of plant cell wall and of diverse secondary metabolite glycones (Gibeaut and †
These two authors are equally contributed.
*Corresponding author; Joong-Hoon Ahn Tel : +82-2-450-3764 E-mail :
[email protected]
Carpita, 1994; Kim et al. 2010). In plants, two nucleotide sugars, UDP-glucose and GDP-mannose, are building blocks for the synthesis of other nucleotides sugars. UDP-glucose is a precursor nucleotide sugar of UDP-glucuronic acid, UDPxylose, UDP-arabinose, UDP-galactose, UDP-apiose, and UDP-rhamnose, whereas GDP-mannose is a precursor nucleotide sugar of GDP-fucose and GDP-galactose (Seifert, 2004). Among the nucleotide sugars, UDP-rhamnose serves as a building block of pectic polysaccharides, rhamnogalacturonan I (RG-1) and rhamnogalacturonan II (RG-II), and is also used a glycosyl donor to supply a rhamnose molecule to aglycone of secondary metabolites such as flavonoids, saponins, triterpenoids, and small phenolic compounds (Ikan, 1999; Ridley et al. 2001). RG-1 is a polymer that consists of over 100 individual units of α (1,4)-linked disaccharide composed of L-rhamnose and D-galacturonic acid units (Ridley et al. 2001). RG-II is a more complex polysaccharide than RG-1, which exists as a dimer that is covalently cross-linked through a borate diester bond in the primary cell wall of the plant (Ridley et al. 2001; O’Neill et al. 1996). Unlike plants, bacteria use dTDP-rhamnose to synthesize the cell surface polysaccharides (Raetz and Whitfield, 2002) and do not synthesize UDP-rhanmnose. In nature, nucleotide diphosphate rhamnose exists in two forms; dTDP-rhamnose and UDP-rhamnose (Jiang et al. 1991; Kamstegg et al. 1978). The biosynthetic pathways of dTDP-rhamnose and UDP-rhamnose are quite distinct. dTDP-rhamnose is synthesized by the catalytic action of three sequential enzymes, rfbB, rfbC, and rfbD (Dong et al. 2003; Jiang et al. 1991). rfbB catalyzes the biosynthesis of dTDP-4-keto-6-deoxy-glucose from dTDP-glucose. dTDP4-keto-6-deoxy-glucose is then converted to dTDP-4-ketorhamnose by the catalytic action of rfbC. Finally, rfbD catalyzes the conversion of dTDP-4-keto-rhamnose to dTDPrhamnose (Fig. 1). UDP-rhamnose is synthesized in plants but not in bacteria (Seifert, 2004). The biosynthesis of UDPrhamnose from UDP-glucose occurs through three-step reaction (Kamsteeg et al. 1978) catalyzed by a single
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analyze the function of the cloned PRHM, we conducted a complementation study with E. coli rfbD mutant, which cannot produce dTDP-rhamnose. To the best of our knowledge, this is the first report characterizing the UDP-rhamnose synthase in woody plants. Our results can be applied in the future for synthesis of the secondary metabolite rhamnoside.
Results and Discussion Cloning and Sequence Analysis of PRHM
Fig. 1. The biosynthetic pathway of the dTDP-rhamnose and UDPrhamnose in bacteria and plant, respectively. In bacteria, dTDPrhamnose is catalytically synthesized from dTDP-glucose by the three enzymes, dTDP-glucose-4,6-dehydratase (RmlB), dTDP-4keto-6-deoxy-glucose-3,5-epimerase (Rmlc), and dTDP-4-ketorhamnose-4-keto-reductase (RmlD). In plants, UDP-rhamnose is synthesized by the catalytic action of UDP-rhamnose synthase (RHM). Specifically, the N-terminus of RHM catalyzes the reaction from UDP-glucose to produce UDP-keto-6-deoxy-glucose, and the C-terminus of RHM is responsible for catalyzing two sequential reactions from UDP-keto-6-deoxy-glucose to produce UDPrhamnose via a UDP-4-keto-rhamnose intermediate.
structural enzyme, UDP-rhamnose synthase (RHM) (Takuji at al. 2007). The N-terminal region of UDP-rhamnose synthase catalyzes the conversion of UDP-glucose to UDP-4-keto-6deoxy-glucose, while the C-terminal region of UDP-rhamnose synthase catalyzes the latter two steps of conversion process, producing UDP-rhamnose from UDP-4-keto-6-deoxy-glucose via a UDP-keto-rhamnose intermediate (Fig. 1). Genes for dTDP-rhamnose biosynthesis have been characterized in several bacteria (Dong et al. 2003; Jiang et al. 1991). However, the RHM gene has only been characterized in Arabidopsis thaliana (Takuji at al. 2007). Poplar is a model woody plant because it has a relatively smaller genome size than other woody plants, and mutants can be generated through Agrobacterium mediated transformation (Tuskan et al. 2006). Additionally, cell wall biosynthesis of poplar is an interesting subject of study. In this study, we aim to investigate nucleotide sugar biosynthesis in poplar, especially UDP-rhamnose biosynthesis. UDPrhamnose is important not only for pectin biosynthesis of plant cell wall but also for the synthesis of secondary metabolites glycone, some of which appears to have therapeutic properties. For example, flavonoid rhamnosides show antipathogenic activities against viruses, bacteria, and fungi (Choi et al. 2009; Choi et al. 2012; Tatsimo et al. 2010; Thapa et al. 2012). The putative RHM gene (PRHM) was cloned from Populus euramericana Guinier. In order to
To clone RHM in poplar, we searched for RHM homologues in the poplar EST database by using the RHM1 and RHM2 gene from A. thaliana. One sequence was found to have high homology with RHM1 and RHM2 and was named PRHM. A full-length cDNA of PRHM was cloned by RT-PCR and sequenced. The blast results for PRHM showed 87% and 85% amino acid identities to RHM1 and RHM2 from A. thaliana, respectively. The open reading frame of PRHM is a 2,013-bp showing 94.2% identity at the amino acid level to the PRHM sequence published in TIGR (TC158686). Comparison of the deduced amino acid sequence of PRHM and TIGR PRHM showed differences in the 38 amino acids. The dissimilarity between the TIGR PRHM sequence and the PRHM sequence obtained in the present study was likely to be due to the use of a different variety of P. euramericana versus the variant P. deltoides used for the genome sequence published in the TIGR. PRHM showed more than 80% amino acid sequence identities with those from Vitis vinifera, Gossypium hirsutum, Glycine max, Hordeum vulgare subsp. Vulgare, Brachypodium distachyon, Zea mays, Oryza sativa, Sorghum bicolor, Ricinus communis, and Selaginella moellendorffii. Phylogenetic analysis of these RHMs showed that RHMs divided into two groups (Fig. 2) although all of them contained two domains which are important for catalysis of UDP-glucose into UDPrhamnose (see below). Because RHMs except those from A. thaliana have been biochemically characterized, it remains uncertain that two phylogenetic groups show different substrate range. PRHM was found to possess two domains; the NADdependent epimerase/dehydratase family domain (Thoden et al. 1997) and the RmlD substrate-binding domain. The NAD-dependent epimerase/dehydratase family domain (Blankenfeldt et al. 2002; Giraud et al. 2000) is located at the N-terminus (amino acids 9-251), and possesses UDP/TDPglucose-4,6-dehydratase activity. The RmlD substrate-binding domain is located at the C-terminus (amino acids, 386-663) and possesses both UDP/TDP-4-keto-6-deoxy-glucose 3,5epimerase and UDP/TDP-4-keto-rhamnose-4-keto-reductase activities. The UDP/TDP rhamnose synthases are part of a
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Fig. 3. Expression and purification of the recombinant PRHM. M, Molecular weight marker; C, uninduced total cells; I, induced total cells; P, purified recombinant PRHM.
Fig. 2. Phylogenetic analysis of RHM proteins possessing multidomain. RHM-like proteins collected from EST database of NCBI were aligned with Clustal X software and analyzed using the MEGA 5.0 software to generate a maximum parsimony tree. Percentages of bootstrap values of 1,000 replicates are presented at each branch point. Abbreviation of each genes and accession numbers are as follows; VvRHM from Vitis vinifera (XP_002285634.1), GhRHM from Gossypium hirsutum (ACJ11756.1), GmRHM from Glycine max (XP_003543185.1), HvRHM from Hordeum vulgare subsp. Vulgare (BAJ97692.1), BdRHM from Brachypodium distachyon (XP_003571828.1), ZmRHM from Zea mays (NP_001151455.1), OsRHM from Oryza sativa (NP_001049724.1), SbRHM from Sorghum bicolor (XP_002468088.1), RcRHM from Ricinus communis (XP_002531238.1), SmRHM from Selaginella moellendorffii (XP_002989472.1), AtRHM2 from A. thaliana (NP_564633.2), AtRHM1 from A. thaliana (AEE36122.1), and AtRHM3 from A. thaliana (AEE75567.1).
subfamily of short chain dehydrogenase/reductase enzymes known for catalyzing the modification of nucleotide sugars. The members of this subfamily possess a putative NAD(P)(H)-binding motif, represented as GXXGXX(G/A) and a conserved catalytic trial, represented as YXXXK (where X represents any amino acid). PRHM was also found to possess two motifs, NAD(P)(H) binding motif between amino acids 392 and 398, and a conserved catalytic trial motif between amino acids 517 and 521. These results suggest that PRHM is likely to be a multifunctional enzyme that catalyzes from UDP/TDP-glucose to UDP/TDPrhamnose via intermediates, the UDP/TDP-4-keto-6-deoxyglucose and UDP/TDP-4-keto-rhamnose. Expression and Characterization of PRHM PRHM was expressed as a GST-tagged fusion protein in E.
coli, and the recombinant protein was purified by using a GST-affinity column. The purified recombinant protein of PRHM was analyzed by SDS-PAGE and the gel was stained with Coomassie Brilliant Blue G-250 solution. The molecular weight of the stained recombinant PRHM was estimated at 101.4 kDa (Fig. 3), corresponding to the sum of the estimated molecular weight of PRHM (~75.4 kDa) calculated from the deduced amino acid sequence plus that of the GST tag (~26 kDa). The enzymatic activity of the recombinant PRHM was assayed using UDP-glucose as a substrate. HPLC analysis of the reaction product provided a chromatogram that was indistinguishable when compared with that of the control reaction, subject to identical conditions except that GST protein is present instead of PRHM recombinant protein (data not shown). This result is likely to be due to the incomplete processing or stability of the PRHM protein. As an alternative method to investigate the function of PRHM, we conducted a complementation study with an E. coli mutant and examined its role in the synthesis of a flavonoid rhamnoside. The E. coli rfbD mutant (B-rfbD), which was unable to generate dTDP-rhamnose, was constructed. The resulting B-rfbD was transformed with AtUGT78D1, known as quercetin 3-O-rhamnosyltransferase. The resulting strain (B-rfbD harboring AtUGT78D1) was used for the biotransformation of quercetin. The wild type E. coli BL21(DE3) harboring AtUGT78D1 was used as a control. The reaction products of wild type E. coli showed two peaks, P1 and P2 (Fig. 4-B). The molecular weight of the first peak, corresponding to an elution time of 10.1 min, was 464-Da (Fig. 4-G; P1), which is likely that a glucose molecule was attached to quercetin. The molecular weight of the second peak, corresponding to an elution time of 11.2 min, was 448Da (Fig. 4-G; P2), which is likely to be quercetin rhamnoside. The structures of the reaction products were determined by comparing the HPLC retention times with authentic quercetin 3-O-glucoside and quercetin 3-O-rhamnoside. On the other hand, B-rfbD harboring AtUGT78D1 only generated quercetin 3-O-glucoside (Fig. 4-D). According to the EcoCyc database
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Fig. 4. HPLC and MS analysis of the reaction products produced by PRHM and AtUGT78D1. Quercetin was used as a substrate in all experiments. A, without AtUGT78D1 in BL21(DE3); B, with AtUGT78D1 in BL21(DE3); C, with PRHM and AtUGT78D1 in BL21(DE3); D, with AtUGT78D1 in B-rfbD; E, with PRHM and AtUGT78D1 in B-rfbD. F, UV-spectra of authentic quercetin (S), reaction product with AtUGT78D1 (P1), reaction product with AtUGT78D1 (P2), reaction product with AtUGT78D1 (P3), reaction product with AtUGT78D1 (P4), and reaction product with AtUGT78D1 (P5); G, MS analysis of reaction product 1 (P1) and reaction product 2 (P2).
(http://biocyc.org/), dTDP-rhamnose, but not UDP-rhamnose is produced in E. coli for O-antigen biosynthesis. This suggests that AtUGT78D1 used dTDP-rhamnose as a sugar donor to produce quercetin 3-O-rhamnoside during the E. coli biotransformation. The B-rfbD strain did not produce dTDP-rhamnose due to the mutation of the gene which catalyzes the final step of dTDP-rhamnose biosynthesis and thereby did not produce quercetin 3-O-rhamnoside. Therefore, in order to investigate the function of the PRHM protein, PRHM gene was transformed into B-rfbD harboring AtUGT78D1. If PRHM were functional, the resulting E. coli transformant would make UDP-rhamnose, which will be a sugar donor of AtUGT78D1 to synthesize quercetin 3-Orhmanoside from quercetin. Analysis of the reaction product displayed one peak with a same retention time as quercetin 3-O-rhamnoside (Fig. 4-E), strongly suggesting that PRHM catalyzed the conversion of dTDP/UDP-glucose into dTDP/ UDP-rhamnose. Therefore, as a result of the stable supply of dTDP/UDP-rhamnose, AtUGT78D1 could specifically catalyze the production of quercetin 3-O-rhamnoside. The disappearance of quercetin 3-O-glucoses could result from a greater supply of nucleotide rhamnose due to overexpression of PRHM compared to that in wild type E. coli.
In the present study, we cloned and characterized PRHM from P. euramericana by using in vivo complementation analysis incorporated with UDP-rhamnose specific glycosyl- transferase, AtUGET78D1. Most plant glycosyltransferases prefer UDP-rhamnose to dTDPrhamnose as a sugar donor (Seifert, 2004). When flavonoid rhamnosides are produced in E. coli or yeast, overexpression of PRHM could increase intrinsic UDP-rhamnose and lead to an increase of flavonoid rhamnoside level. To the best of our knowledge, this is the first report characterizing the UDP-rhamnose synthase in woody plants. Our results will guide future investigation into the biosynthesis of plant primary cell walls and plant secondary metabolite rhamnosides.
Materials and Methods Cloning of PRHM For cloning of the full-length cDNA of PRHM, the cDNA to was synthesized using Omniscript reverse transcriptase (Qiagen, Hilden, Germany) with total RNA isolated from fresh leaves attached stem of Populus euramericana Guinier using a Qiagen plant total RNA isolation kit (Qiagen; Kim et al. 2012b). PRHM was amplified by polymerase chain reaction (PCR) using Hot start Taq DNA polymerase (Qiagen) and primers designed on the basis of the nucleotide sequence published by The Institute for Genomic Research (TIGR, accession number, TC158686). The primers used were 5’-ATgtcgacATGGCTACATATACTCCGAAGA-3’ (forward primer) and 5’-CATgcggccgcTTAGGTTCTCTTGTTGGGT-3’ (reverse primer). To facilitate PRHM
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cloning into E. coli expression vector, the restriction enzyme sites, for SalI and NotI, were inserted in the forward primer and reverse primer, respectively (displayed in the primer sequences above as lowercase letters). The PCR program was as follows: 40 cycles of 1 min denaturation at 94ºC, 1 min annealing at 55ºC, and 2 min amplification at 72ºC. The PCR product was purified using a Bioneer Gel Extraction Kit (Bioneer, Daejeon, Korea) and subcloned into the pGEMT-easy vector (Promega, Madison, WI, USA) and resulting plasmids were sequenced. Expression of PRHM in E. coli The open reading frame of PRHM was subcloned into the E. coli expression vector pGEX 5X-3, and the resulting plasmid was transformed into E. coli BL21 (DE3). The recombinant PRHM protein was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to the final concentration of 0.1 mM. The cells were grown for 20 h with 200 rpm shaking at 25oC. The cell was then harvested by centrifugation at 3,200 rpm for 15 min, washed with the same volume of PBS buffer (10 mM, NaH2PO4, 150 mM NaCl, pH 7.2) and recentrifuged under the same conditions mentioned above. The cells were resuspended in the PBS buffer and lysed by sonication. The sample was centrifuged for 15 min at 4oC with 13,000 rpm to remove cell debris. The supernatant was used to purify the recombinant PRHM protein by using a GST-trap affinity column (GST trap; Amersham Bioscience, Piscataway, NJ) according to the manufacturer’s instructions. The molecular weight and purity of PRHM were estimated by SDS-PAGE analysis. AtUGT78D1, which was cloned previously (Yoon et al, 2012), was subcloned into the EcoRI/NotI site of E. coli expression vector pCDF Duet (Novagen). To determine the PRHM enzyme activity, a reaction mixture containing 50 µg of the purified recombinant PRHM, 3 mM of NAD, 3 mM of NADH, 1 mM of UDP-glucose, and 25 µL of 1M Tris-HCl (pH7.5) in a final volume of 500 µL was prepared. The reaction mixture was incubated at 37oC for 60 min. The reaction was stopped by boiling at 100oC for 5 min. The mixture was centrifuged at 13,000 rpm for 20 min and the supernatant was taken to analyze the reaction product with a high performance liquid chromatography (HPLC, Varian, Walnut Creek, CA, USA) system equipped with a photodiode array detector and a Varian C18 reversed-phase column (4.6×250 mm, 0.45 µm). HPLC analysis was conducted as reported by Kim et al. (2009). Characterization of PRHM by Complementation using E. coli The E. coli rfbD mutant was generated as described in a previous study (Kim et al. 2012a). Briefly, the E. coli rfbD mutant was constructed using the Quick and Easy Conditional Knockout kit (Gene Bridges, Heidelberg, Germany). The forward primer was 5’-TGGCATCATGAGCGAGATGCAAAAATTTGTTAAATTGCCGTAGTCGTAAAAATTAACCCTCACTAAAGGGCG-3’ and the reverse primer was 5’-GGTGCTTATCAGTCGTGGATTGAACAGAACTATGAGGGCCGCCAGTAATGTAATACGACTCACTATAGGGC-TC-3’. An E. coli transformant containing both PRHM and AtUGT78D1 or both PRHM and empty pGEX 5X-1 vector were inoculated into 2 mL of LB medium supplemented with 50 µg/mL ampicillin and 50 µg/mL spectinomycin and cultured overnight at 37oC with 180 rpm shaking. The cultured cells were inoculated into fresh LB medium containing 50 µg/mL ampicillin and 50 µg/mL spectinomycin. The E. coli cells were grown at 37oC until the absorbance reached an OD600 of 0.6. At this point, recombinant protein production was induced by adding IPTG to a final concentration of 0.1 mM. Transformants were grown for an additional 24 h at 25oC with shaking at 180 rpm. The cells were harvested by centrifugation at 13,000 rpm for 1 min, washed once with fresh M9 medium, re-centrifuged, and finally
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resuspended in M9 medium containing 2% glucose, 50 µg/mL ampicillin, and 50 µg/mL spectinomycin. The density of cells was adjusted to an OD600 of 3. The substrate, quercetin was added to a final concentration of 100 µM. The biotransformation mixture was cultured at 30oC with 180 rpm for 2 h. A 500 µL volume of the reaction culture was extracted twice with an equal volume of ethyl acetate. The ethyl acetate layer was dried using a speed vacuum drier and the dried samples were dissolved in 100 µL of dimethyl sulfoxide (DMSO). The samples were analyzed using an HPLC. The molecular weight of the reaction products were determined using mass spectrometry by the method described in Lee et al. (2007).
Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (2012R1A1A2041132), by a grant from Systems and Synthetic AgroBiotech Center through the Next-Generation BioGreen 21 Program (PJ007975), Rural Development Administration, Republic of Korea, and by the Priority Research Centers Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2012-0006686).
References Blankenfeldt W, Kerr ID, Giraud MF, McMiken HJ, Leonard G, Whitfield C, Messner P, Graninger M, Naismith JH (2002) Variation on a theme of SDR. dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) shows a new Mg2+-dependent dimerization mode. Structure (Camb) 10:773−786 Choi HJ, Song JH, Kwon DH (2012) Quercetin 3-rhamnoside exerts antiinfluenza A virus activity in mice. Phytother Res 26:462−464 Choi HJ, Song JH, Park KS, Kwon DH (2009) Inhibitory effects of quercetin 3-rhamnoside on influenza A virus replication. Eur J Pharm Sci 37:329−333 Dong C, Beis K, Giraud MF, Blankenfeldt W, Allard S, Major LL, Kerr ID, Whitfield C, Naismith JH (2003) A structural perspective on the enzymes that convert dTDP-D-glucose into dTDP-Lrhamnose. Biochem Soc Trans 31:532−536 Gibeaut DM, Carpita NC (1994) Biosynthesis of plant cell wall polysaccharides. FASEB J 8:904−915 Giraud MF, Leonard GA, Field RA, Berlind C, Naismith JH (2002) RmlC, the third enzyme of dTDP-L-rhamnose pathway, is a new class of epimerase. Nat Struct Biol 7:398−402 Ikan R (1999) Naturally Occurring Glycosides, Wiley, Chichester, UK Jiang XM, Neal B, Santiago F, Lee SJ, Romana LK, Reeves PR (1991) Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol Microbiol 5:695−713 Kamsteeg J, Van Brederode J, Van Nigtevecht G (1978) The formation of UDP-L-rhamnose from UDP-D-glucose by an enzyme preparation of red campion (Silene Dioica (L) clairv) leaves. FEBS Lett 91:281−284 Kim BG, Kin HJ, Ahn J-H (2012a) Production of bioactive flavonol rhamnosides by expression of plant genes in Escherichia coli. J Agric Food Chem 60:11143-11148 Kim BG, Sung SH, Jung NR, Chong Y, Ahn JH (2010) Biological synthesis of isorhamnetin 3-O-glucoside using engineered glucosyltransferase. J Mol Cat B: Enzym 63:194−199 Kim BG, Lee ER, Ahn JH (2012b) Analysis of flavonoid contents and expression of flavonoid biosynthetic genes in Populus euramericana
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Guinier in response to abiotic stress. J Kor Soc Appl Biol Chem 55:141−145 Kim SK, Kim DH, Kim BG, JeonYM, Hong BS, Ahn JH (2009) Cloning and characterization of the UDP glucose/galactose epimerases of Oryza sativa. J Kor Soc Appl Biol Chem 52:315− 320 Lee YJ, Jeon Y, Lee, JS, Kim BG, Lee CH, Ahn JH (2007) Enzymatic synthesis of phenolic CoAs using 4-coumarate: coenzyme A ligase (4CL) from rice. Bull Kor Chem Soc 28:365−366 O'Neill MA, Warrenfeltz D, Kates K, Pellerin P, Doco T, Darvill AG, Albersheim P (1996) Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester in vitro conditions for the formation and hydrolysis of the dimer. J Biol Chem 271:22923−22930 Raetz C, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71:635−700 Ridley BL, O'Neill MA, Mohnen D (2001) Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry 57:929−967 Schultz J, Copley RR, Doerks T, Ponting CP, Bork P (2000) SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res 28:231−234 Seifert GJ (2004) Nucleotide sugar interconversions and cell wall
J. Plant Biol. (2013) 56:7-12
biosynthesis: how to bring the inside to the outside. Curr Opin Plant Biol 7:277−284 Takufi O, Tadashi N, Yoshifumi J (2007) Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion. J Biol Chem 282:5389−5403 Tatsimo SJN, Tamokou JD, Havyarimana L, Csupor D, Forgo P, Hohmann J, Kuiate J-R, Tane P (2012) Antimicrobial and antioxidant activity of kaempferol rhamnoside derivatives from Bryophyllum pinnatum. BMC Res Notes 5:158 Thapa M, Kim Y, Desper J, Chang K-O, Hua D (2012) Synthesis and antiviral activity of substituted quercetins. Bioorg Med Chem Lett 22:353−356 Thoden JB, Hegeman AD, Wesenberg G, Chapeau MC, Frey PA, Holden HM (1997) Structural analysis of UDP-sugar binding to UDP-galactose 4-epimerase from Escherichia coli. Biochemistry 36:6294−6304 Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, et al (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:1596−1604 Yoon JA, Kim BG, Lee WJ, Lim Y, Chong Y, Ahn JH (2012) Production of a novel quercetin glycoside through metabolic engineering of Escherichia coli. Appl Environ Microbiol 78:4256−4262
