Four glucosyltransferases from rice: cDNA cloning, expression, and characterization

Four UDP-dependent glucosyltransferase (UGT) genes, UGT706C1, UGT706D1, UGT707A3, and UGT709A4 were cloned from rice, expressed in Escherichia coli, and purified to homogeneity. In order to find out whether these enzymes could use flavonoids as glucose acceptors, apigenin, daidzein, genistein, kaempferol, luteolin, naringenin, and quercetin were used as potential glucose acceptors. UGT706C1 and UGT707A3 could use kaempferol and quercetin as glucose acceptors and the major glycosylation position was the hydroxyl group of carbon 3 based on the comparison of HPLC retention times, UV spectra, and NMR spectra with those of corresponding authentic flavonoid 3-O-glucosides. On the other hand, UGT709A4 only used the isoflavonoids genistein and daidzein and transferred glucose onto 7-hydroxyl group. In addition, UGT706D1 used a broad range of flavonoids including flavone, flavanone, flavonol, and isoflavone, and produced at least two products with glycosylation at different hydroxyl groups. Based on their substrate preferences and the flavonoids present in rice, the in vivo function of UGT706C1, UGT706D1, and UGT707A3 is most likely the biosynthesis of kaempferol and quercetin glucosides.     

Molecular cloning, expression and characterization of a glycosyltransferase from rice

Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40–42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4′ flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized.     

Synthesis and bioactivity of C-2 and C-3 methyl ether derivatives of brassinolide

The following six novel methyl ether derivatives of brassinolide were prepared and their brassinosteroid activity was measured by means of the rice leaf lamina inclination bioassay: 2-O-methylbrassinolide, 3-O-methylbrassinolide, 2,22,23-tri-O-methylbrassinolide, 3,22,23-tri-O-methylbrassinolide, 2-O-methyl-25-methoxybrassinolide and 3-O-methyl-25-methoxybrassinolide. Brassinolide was used as a standard for comparison. All six compounds were also tested in the presence of 1000 ng of indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids. The 2-O-methyl- and 3-O-methylbrassinolide derivatives showed weak activity at high doses, which was enhanced by IAA, especially in the case of the 3-O-methyl derivative. Similarly, the 2,22,23-tri-O-methyl- and 3,22,23-tri-O-methyl derivatives displayed weak bioactivity on their own, but significantly stronger activity when applied with IAA. The 3-O-methyl and 3,22,23-tri-O-methyl analogues plus IAA were comparable in bioacivity to brassinolide alone, but were less active than brassinolide plus IAA. In each case, O-methylation at C-2 resulted in a greater loss of activity than O-methylation at C-3 under the same conditions. The relatively strong activity of 3,22,23-tri-O-methylbrassinolide in the presence of IAA is especially noteworthy as it indicates that free hydroxyl groups at C-3, C-22, and C-23 are not essential for bioactivity. Finally, 2-O-methyl- and 3-O-methyl-25-methoxybrassinolide were essentially inactive alone, and showed only a modest increase in bioactivity when coapplied with IAA.     

cDNA cloning and gene expression of the major prolamins of rice

A full-length cDNA (pS 18) encoding the 16 kDa rice prolamin composed of 158 amino acids was sequenced. Analysis of N-terminal amino acid sequence of a major rice prolamin indicated that an 18 amino acid signal peptide was removed from 16 kDa precursor prolamin to form the 14 kDa prolamin during seed development. Synthesis of the 16 kDa precursor prolamin began around 8 days after flowering (DAF), increased remarkably at 8–11 DAF and gradually reached maximum levels with the maturation of rice seeds.     

Cation dependent O-methyltransferases from rice

Two lower molecular mass OMT genes (ROMT-15 and -17) were cloned from rice and expressed in Escherichia coli as glutathione S-transferase fusion proteins. ROMT-15 and -17 metabolized caffeoyl-CoA, flavones and flavonols containing two vicinal hydroxyl groups, although they exhibited different substrate specificities. The position of methylation in both luteolin and quercetin was determined to be the 3′ hydroxyl group and myricetin and tricetin were methylated not only at 3′ but also at 5′ hydroxyl groups. ROMT-15 and -17 are cation-dependent and mutation of the predicted metal binding sites resulted in the loss of the enzyme activity, indicating that the metal ion has a critical role in the enzymatic methylation.     

Chemical components of callus tissues of rice

Chemical components of callus and other tissues (leaf, root, and seed) of rice were investigated to find the variation in callus metabolism. The isolated compounds were squalene, three sterols, three triterpenes and a fatty alcohol; the last being present only in the callus. Cylindrin, isoarborinol and chlorophyll were not found in the callus. Fatty acids were detected in all tissues in varying amounts, the callus containing lauric acid in greater quantity than the leaves, roots, and seeds.     

Antibacterial diterpenes and their fatty acid conjugates from rice leaves

Six structurally oryzalide-related compounds, oryzadione (1), 2, 3, 4, 5 and 6, were isolated from a neutral fraction of the extract of healthy leaves using a bacterial leaf blight-resistant cultivar of a rice plant, "Norin-27", as a group of antimicrobial substances. Their structures were determined by spectroscopic studies to be kaurane analogues and kaurane analogues conjugated with fatty acids, i.e., 1: ent-15,16-epoxy-kauran-2,3-dione (enol form: ent-15,16-epoxy-2-hydroxy-kauran-1-en-3-one), 2: ent-15,16-epoxy-3beta-hydroxy-kauran-2-one, 3: ent-15,16-epoxy-3-oxa-kauran-2-one, 4: ent-15,16-epoxy-3beta-myristoyloxy-kauran-2-one, 5: ent-15,16-epoxy-3alpha-palmitoyloxy-kauran-2-one, and 6: ent-15,16-epoxy-2beta-palmitoyloxy-kauran-2-one.     

Detection and characterization of anS-adenosyl-l-methionine: Flavonoid 7-O-methyltransferase from leaves ofPrunus x yedoensis matsum. with special reference to prunetrin formation

An enzyme,S-adenosyl-l-methionine: flavonoid 7-O-methyltransferase (F7OMT), catalyzing the transfer of the methyl group fromS-adenosyl-l-methionine (SAM) to the 7 position of sophoricoside (5, 7, 4′-trihydroxyisoflavone 4′-O-glucoside) and some of the other flavonoids, was detected in extracts from leaves ofPrunus x yedoensis, and it was partially purified (about 203-fold) by a combination of gel filtration and ion-exchange column chromatographies. F7OMT was isolated as a soluble enzyme with a pH optimun of 7.5 in K-phosphate buffer. The molecular mass of F7OMT, which had an isoelectric point at pH 4.1, was estimated by elution from a column of Sephadex G-100 to be about 36 kDa. The activity of F7OMT was stimulated by 14 mM 2-Co²⁺ and reagents that react with sulfhydryl groups. The apparentKm values for sophoricoside, its aglycone genistein (5, 7, 4′-trihydroxyisoflavone) and quercetin were 1.49, 2.19 and 1.89 μM, respectively. The apparentKm value for SAM as methyl donor was 2.08 mM. The specificity of F7OMT for methyl acceptors was not strict; flavonols, flavanones and flavanonols in addition to isoflavones served as methyl acceptor. An examination ofP. x yedoensis leaves during spring and autumn showed variations in the activities of F7OMT and UDP-glucose: isoflavone 4′-O-glucosyltransferase (I4′ GT). The activities of F7OMT and I4′GT increased in enlarging leaf tissues and then markedly declined when the leaves approached maturation. In autumn leaves F7OMT activity was scarcely detected, but a small peak of I4′GT activity was observed during autumnal reddening.     

Rice functional genomics research: progress and implications for crop genetic improvement

Rice is a staple food crop and has become a reference of monocot plant for functional genomic research. With the availability of high quality rice genome sequence, there has been rapid accumulation of functional genomic resources, including: large mutant libraries by T-DNA insertion, transposon tagging, and chemical mutagenesis; global expression profiles of the genes in the entire life cycle of rice growth and development; full-length cDNAs for both indica and japonica rice; sequences from resequencing large numbers of diverse germplasm accessions. Such resource development has greatly accelerated gene cloning. By the end of 2010, over 600 genes had been cloned using various methods. Many of the genes control agriculturally useful traits such as yield, grain quality, resistances to biotic and abiotic stresses, and nutrient-use efficiency, thus have potential utility in crop genetic improvement. This review was aimed to provide a comprehensive summary of such progress. We also presented our perspective for future studies.     

Anthocyanin 3-galactosides from Cornus alba 'Sibirica' with glucosidation of the B-ring

The three anthocyanins, delphinidin 3-O-beta-galactopyranoside-3',5'-di-O-beta-glucopyranoside (1), delphinidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (2) and cyanidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (3), and the 3-O-beta-galactopyranosides of delphinidin (4) and cyanidin (5) were isolated from the bluish white berries and compound umbel of Siberian dogwood, Cornus alba 'Sibirica'. The ornamental autumn leaves and the characteristic purplish red bark of this variety were found to contain only pigment 5.     

Synthesis and bioactivity of C-29 brassinosteroid analogues with different functional groups at C-6

In this paper, we report the synthesis and bioactivity of four synthetic analogues of 28-homobrassinosteroids, in order to evaluate the influence in bioactivity when the C-6 keto group is replaced by different functional groups. The synthetic analogues are 6-deoxo-28-homocastasterone [(22R,23R)-stigmasta-2alpha,3alpha,22,23-tetraol], 6alpha-hydroxy-28-homocastasterone [(22R,23R)-stigmasta-2alpha,3alpha,6alpha,22,23-pentaol], 6beta-hydroxy-28-homocastasterone [(22R,23R)-stigmasta-2alpha,3alpha,6beta,22,23-pentaol], and [(22R,23R)-6alpha-fluorostigmasta-2alpha,3alpha,22,23-tetraol]. Results indicate that replacement of the 6-keto moiety by an beta or alpha hydroxyl group led to a decrease in activity, whereas the 6-deoxo analogue showed a very low activity, confirming the importance of an electronegative moiety at C-6 to observe hormonal potency. The 6alpha-fluorinated analogue elicited a low activity, similar to that of the 6-deoxo analogue.     

Geranyl diphosphate synthase from Abies grandis: cDNA isolation,functional expression,and characterization

Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library. When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1). Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.     

Molecular characterization of a cDNA encoding caffeoyl-coenzyme A 3-O-methyltransferase ofStellaria longipes

A cDNA clone encodingS-adenosyl-L-methionine:trans-caffeoyl-CoA 3-O-methyl-transferase (EC 2.1.1.104; CCoAOMT) fromStellana longipes Goldie (long-stalked chick-weed) was isolated and studied. Structural analysis of both the nucleotide sequence and the predicted amino acid sequence suggests that our cloned sequence encoded a CCoAOMT enzyme ofStellaria longipes, which shared overall structural similarity with other plant CCoAOMTs but exhibited certain distinct characteristics. Southern blot hybridization and cloning analyses indicating a small CCoAOMT gene family in theStellana longipes genome and the absence of introns in the coding region of the cDNA-corresponding gene. Sequence variations in the coding region were found among three genotypes from geographically isolated populations. Higher levels of CCoAOMT mRNA were detected in stems and leaves than in roots. The cDNA-encoded protein expressed inEschendia coli was shown to utilize caffeoyl-CoA, but not caffeic acid or 5-hydroxy ferulic acid, as its substrate.     

Heavy metal-regulated new microRNAs from rice

MicroRNAs (miRNAs) are a novel class of short, endogenous non-coding small RNAs that have the ability to base pair with their target mRNAs to repress their translation or induce their degradation in both plants and animals. To identify heavy metal stress-regulated novel miRNAs, we constructed a library of small RNAs from rice seedlings that were exposed to toxic levels of cadmium (Cd²⁺). Sequencing of the library and subsequent analysis revealed 19 new miRNAs representing six families. These cloned new rice miRNAs have sequence conservation neither in Arabidopsis nor in any other species. Most of the new rice miRNAs were up- or down-regulated in response to the metal exposure. On the base of sequence complementarity, a total of 34 miRNA targets were predicted, of which 23 targets are functionally annotated and the other 11 records belong to unknown proteins. Some predicted targets of miRNAs are associated with the regulation of the response to heavy metal-induced stresses. In addition to the new miRNAs, we detected nine previously reported miRNAs and 56 other novel endogenous small RNAs in rice. These findings suggest that the number of new miRNAs in rice is unsaturated and some of them may play critical roles in plant responses to environmental stresses.     

Molecular cloning and functional expression of chitinase-encoding cDNA from the cabbage moth, Mamestra brassicae

Chitinase is a rate-limiting and endo-splitting enzyme involved in the bio-degradation of chitin, an important component of the cuticular exoskeleton and peritrophic matrix in insects. We isolated a cDNA-encoding chitinase from the last larval integument of the cabbage moth, Mamestra brassicae (Lepidoptera; Noctuidae), cloned the ORF cDNA into E. coli to confirm its functionality, and analyzed the deduced amino acid sequence in comparison with previously described lepidopteran chitinases. M. brassicae chitinase expressed in the transformed E. coli cells with the chitinase-encoding cDNA enhanced cell proliferation to about 1.6 times of the untransformed wild type strain in a colloidal chitin-including medium with only a very limited amount of other nutrients. Compared with the wild type strain, the intracellular levels of chitin degradation derivatives, glucosamine and N-acetylglucosamine were about 7.2 and 2.3 times higher, respectively, while the extracellular chitinase activity was about 2.2 times higher in the transformed strain. The ORF of M. brassicae chitinaseencoding cDNA consisted of 1686 nucleotides (562 amino acid residues) except for the stop codon, and its deduced amino acid composition revealed a calculated molecular weight of 62.7 and theoretical pI of 5.3. The ORF was composed of N-terminal leading signal peptide (AA 1-20), catalytic domain (AA 21-392), linker region (AA 393-498), and C-terminal chitin-binding domain (AA 499-562) showing its characteristic structure as a molting fluid chitinase. In phylogenetic analysis, the enzymes from 6 noctuid species were grouped together, separately from a group of 3 bombycid and 1 tortricid enzymes, corresponding to their taxonomic relationships at both the family and genus levels.