Clive Lo

Cinnamon Bark Proanthocyanidins as Reactive Carbonyl Scavengers To Prevent the Formation of Advanced Glycation Endproducts

Cinnamon bark has been reported to be effective in the alleviation of diabetes through its antioxidant and insulin-potentiating activities. In this study, the inhibitory effect of cinnamon bark on the formation of advanced glycation endproducts (AGEs) was investigated in a bovine serum albumin (BSA)-glucose model. Several phenolic compounds, such as catechin, epicatechin, and procyanidin B2, and phenol polymers were identified from the subfractions of aqueous cinnamon extract. These compounds showed significant inhibitory effects on the formation of AGEs. Their antiglycation activities were not only brought about by their antioxidant activities but also related to their trapping abilities of reactive carbonyl species such as methylglyoxal (MGO), an intermediate reactive carbonyl of AGE formation. Preliminary study on the reaction between MGO and procyanidin B2 revealed that MGO-procyanidin B2 adducts are primary products which are supposed to be stereoisomers. This is the first report that proanthocyanidins can effectively scavenge reactive carbonyl species and thus inhibit the formation of AGEs. As proanthocyanidins behave in a similar fashion as aminoguanidine (AG), the first AGE inhibitor explored in clinical trials, they show great potential to be developed as agents to alleviate diabetic complications.

Gene discovery and gene function assignment in filamentous fungi

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.

A Stilbene Synthase Gene (SbSTS1) Is Involved in Host and Nonhost Defense Responses in Sorghum

A chalcone synthase (CHS)-like gene, SbCHS8, with high expressed sequence tag abundance in a pathogen-induced cDNA library, was identified previously in sorghum (Sorghum bicolor). Genomic Southern analysis revealed that SbCHS8 represents a single-copy gene. SbCHS8 expression was induced in sorghum mesocotyls following inoculation with Cochliobolus heterotrophus and Colletotrichum sublineolum, corresponding to nonhost and host defense responses, respectively. However, the induction was delayed by approximately 24 h when compared to the expression of at least one of the other SbCHS genes. In addition, SbCHS8 expression was not induced by light and did not occur in a tissue-specific manner. SbCHS8, together with SbCHS2, was overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) tt4 (transparent testa) mutants defective in CHS activities. SbCHS2 rescued the ability of these mutants to accumulate flavonoids in seed coats and seedlings. In contrast, SbCHS8 failed to complement the mutation, suggesting that the encoded enzyme does not function as a CHS. To elucidate their biochemical functions, recombinant proteins were assayed with different phenylpropanoid-Coenzyme A esters. Flavanones and stilbenes were detected in the reaction products of SbCHS2 and SbCHS8, respectively. Taken together, our data demonstrated that SbCHS2 encodes a typical CHS that synthesizes naringenin chalcone, which is necessary for the formation of different flavonoid metabolites. On the other hand, SbCHS8, now retermed SbSTS1, encodes an enzyme with stilbene synthase activity, suggesting that sorghum accumulates stilbene-derived defense metabolites in addition to the well-characterized 3-deoxyanthocyanidin phytoalexins.

Identification of flavone phytoalexins and a pathogen-inducible flavone synthase II gene (SbFNSII) in sorghum

Following inoculation with the anthracnose pathogen Colletotrichum sublineolum, seedlings of the sorghum resistant cultivar SC748-5 showed more rapid and elevated accumulation of luteolin than the susceptible cultivar BTx623. On the other hand, apigenin was the major flavone detected in infected BTx623 seedlings. Luteolin was demonstrated to show stronger inhibition of spore germination of C. sublineolum than apigenin. Because of their pathogen-inducible and antifungal nature, both flavone aglycones are considered sorghum phytoalexins. The key enzyme responsible for flavone biosynthesis has not been characterized in monocots. A sorghum pathogen-inducible gene encoding a cytochrome P450 protein (CYP93G3) in the uncharacterized CYP93G subfamily was identified. Transgenic expression of the P450 gene in Arabidopsis demonstrated that the encoded protein is a functional flavone synthase (FNS) II in planta. The sorghum gene was then termed SbFNSII. It is a single-copy gene located on chromosome 2 and the first FNSII gene characterized in a monocot. Metabolite analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in precursor ion scan mode revealed the accumulation of 2-hydroxynaringenin and 2-hydroxyeriodictyol hexosides in the transgenic Arabidopsis plants. Hence, SbFNSII appears to share a similar catalytic mechanism with the licorice and Medicago truncatula FNSIIs (CYP93B subfamily) by converting flavanones to flavone through the formation of 2-hydroxyflavanones.

An anther-specific dihydroflavonol 4-reductase-like gene (DRL1) is essential for male fertility in Arabidopsis.

Arabidopsis contains only one functional dihydroflavonol 4-reductase (DFR) gene, but several DFR-like genes encoding proteins with the conserved NAD(P)H binding domain. At4g35420, named DRL1 (Dihydroflavonol 4-reductase-like1), is a closely related homolog of the rice anther-specific gene OsDFR2 reported previously. Two T-DNA mutants (drl1-1 and drl1-2) were found to have impaired pollen formation and seed production. Histological analysis revealed defective microspore development after tetrad release in both mutants. Microspore walls were found to rupture, releasing the protoplasts which eventually degenerated. The DRL1 promoter is anther-specific in closed flower buds. Promoter-GUS analysis in transgenic Arabidopsis revealed expression in tapetum, tetrads, and developing microspores, but not in mature anthers. Enhanced yellow fluorescent protein (EYFP)-localization analysis demonstrated that DRL1 is a soluble cytosolic protein that may also be localized in the nucleus. Restoration of male fertility and seed formation was only achieved by a native promoter-DRL1 construct, but not by a 35S-DRL1 construct, demonstrating the importance of spatial and temporal specificities of DRL1 expression. DRL1 may be involved in a novel metabolic pathway essential for pollen wall development. DRL1 homologs were identified as anther- and floral-specific expressed sequence tags from different species, suggesting that DRL1 may have a conserved functional role in male fertility in flowering plants.

Development of online high-/low-pH reversed-phase-reversed-phase two-dimensional liquid chromatography for shotgun proteomics: A reversed-phase-strong cation exchange-reversed-phase approach

Previously, we described an online high-/low-pH RP-RP LC system exhibiting high-throughput, automatability, and performance comparable with that of SCX-RP. Herein, we report a variant of the RP-RP platform, RP-SCX-RP, featuring an additional SCX trap column between the two LC dimensions. The SCX column in combination with the second-dimension RP can be used as an SCX-RP biphasic column for trapping peptides in the eluent from the first RP column. We evaluated the performance of the new platform through proteomic analysis of Arabidopsis thaliana chloroplast samples and mouse embryonic mouse fibroblast STO cell lysate at low-microgram levels. In general, RP-SCX-RP enhanced protein identification by allowing the detection of a larger number of hydrophilic peptides. Furthermore, the platform was useful for the quantitative analyses of crude chloroplast samples for iTRAQ applications at low-microgram levels. In addition, it allowed the online removal of sodium dodecyl sulfate and other chemicals used in excess in iTRAQ reactions, avoiding the need for time-consuming offline SCX clean-up prior to RP-RP separation. Relative to the RP-RP system, our newly developed RP-SCX-RP platform allowed the detection of a larger number of differentially expressed proteins in a crude iTRAQ-labeled chloroplast protein sample.

Accumulation of isoflavone genistin in transgenic tomato plants overexpressing a soybean isoflavone synthase gene

Isoflavones are legume-specific flavonoids best known for their potential cancer preventive and phytoestrogenic properties. In this study, we attempted to engineer the isoflavone pathway in the popular fruit crop tomato (Solanum lycopersicum L). Tomato plants were transformed with a soybean (Glycine max L) isoflavone synthase (GmIFS2) cDNA under the control of the cauliflower mosaic virus 35S promoter. LC-MS/MS analysis demonstrated the presence of genistin (genistein 7-O-glucoside) as the major isoflavone metabolite in the transgenic plants. Substantial amounts of genistin (up to 90 nmol/g FW) were found in leaves, while the levels were marginally detectable (less than 0.5 nmol/g FW) in fruit peels. In either case, no drastic variations in endogenous phenolic contents were observed. Fruit peels were found to accumulate high levels of naringenin chalcone, implicating the limitation of naringenin substrates for isoflavone synthesis. Our results suggested that tomato plants could be engineered to produce isoflavones without comprising the levels of endogenous flavonols, which are also health-beneficial, but it may be necessary to enhance the expression levels of chalcone isomerase simultaneously to achieve significant yields in edible tissues such as fruit peels.

Trapping of Phenylacetaldehyde as a Key Mechanism Responsible for Naringenin’s Inhibitory Activity in Mutagenic 2-Amino-1-methyl-6-phenylimidazo [4,5-b]Pyridine Formation

Chemical model reactions were carried out to investigate the mechanism of inhibition by a citrus flavonoid, naringenin, on the formation of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), the most abundant mutagenic heterocyclic amine found in foods. GC-MS showed that naringenin dose dependently reduced the level of phenylacetaldehyde, a key intermediate on the pathway to the formation of PhIP. Subsequent LC-MS analyses of samples from a wide range of model systems consisting of PhIP precursors, including phenylalanine, glucose, and creatinine, suggested that naringenin scavenged phenylacetaldehyde via adduct formation. An isotope-labeling study showed that the postulated adducts contain fragment(s) of phenylalanine origin. Direct reaction employing phenylacetaldehyde and naringenin further confirmed the capability of naringenin to form adducts with phenylacetaldehyde, thus reducing its availability for PhIP formation. Two of the adducts were subsequently isolated and purified. Their structure was elucidated by one- and two-dimensional NMR spectroscopy as 8- C-( E-phenylethenyl)naringenin (1) and 6- C-( E-phenylethenyl)naringenin (2), respectively, suggesting that C-6 and C-8 are two of the active sites of naringenin in adduct formation. These two adducts were also identified from thermally processed beef models, highlighting phenylacetaldehyde trapping as a key mechanism of naringenin to inhibit PhIP formation.

Molecular dissection of the pathogen-inducible 3-deoxyanthocyanidin biosynthesis pathway in sorghum.

3-Deoxyanthocyanidins are the unique phytoalexins synthesized by sorghum in response to fungal inoculation. They are structurally related to anthocyanins but the final steps of their pathogen-inducible biosynthesis are not fully understood. We have identified new flavonoid structural genes from the recently completed sorghum BTx623 genome sequence. The biochemical functions of the different expressed sorghum genes were established in planta by complementation in the appropriate Arabidopsis transparent testa mutants. There is a family of nine chalcone synthase genes which are all inducible by fungal inoculation in sorghum seedlings. Specific dihydroflavonol 4-reductase (DFR) genes responsive to conditions which stimulated anthocyanin accumulation (SbDFR1) or 3-deoxyanthocyanidin production (SbDFR3) were identified. Recombinant SbDFR1 and SbDFR3 were found to function as typical DFRs by accepting dihydroflavonol substrates. On the other hand, both DFRs showed substantially lower but detectable NADPH-dependent activities toward flavanones. Reduction of flavanones to flavan-4-ols is a reaction step required for 3-deoxyanthocyanidin production. Flavanone 3-hydroxylase (F3H) converts flavanones to dihydroflavonols for anthocyanin biosynthesis. In sorghum seedlings, expression of two F3H genes was either absent or strongly suppressed during the accumulation of 3-deoxyanthocyanidins. Under such conditions, most flavanones are expected to be reduced by the pathogen-induced SbDFR3 for the formation of flavan-4-ols. Our work also revealed that 3-deoxyanthocyanidin accumulation and SbDFR3 expression were induced by methyl jasmonate treatment in sorghum roots but the stimulation effects were antagonized by salicylic acid.

OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1: implication for a highly conserved plant cGTPase essential for chloroplast function

*The bacterial protein YqeH is a circularly permuted GTPase with homologs encoded by plant nuclear genomes. The rice homolog OsNOA1/RIF1 is encoded by the single-copy gene Os02g01440. OsNOA1/RIF1 is expressed in different tissues and is light-inducible. The OsNOA1/RIF1-EYFP fusion protein was targeted to chloroplasts in transgenic Arabidopsis plants. In addition, the rice homolog was able to rescue most of the growth phenotypes in an Arabidopsis rif1 mutant. *Rice (Oryza sativa) OsNOA1/RIF1 RNAi mutant seedlings were chlorotic with reduced pigment contents and lower photosystem II (PSII) efficiency. However, the expressions of the chloroplast-encoded genes rbcL, atpB, psaA and psbA were not affected. By contrast, reduced abundance of the chloroplast 16S rRNA was observed in the mutant. *Quantitative iTRAQ-LC-MS/MS proteomics investigations revealed proteome changes in the rice mutant consistent with the expected functional role of OsNOA1/RIF1 in chloroplast translation. The RNAi mutant showed significantly decreased expression levels of chloroplast-encoded proteins as well as nuclear-encoded components of chloroplast enzyme complexes. Conversely, upregulation of some classes of nonchloroplastic proteins, such as glycolytic and phenylpropanoid pathway enzymes, was detected. *Our work provides independent indications that a highly conserved nuclear-encoded cGTPase of likely prokaryotic origin is essential for proper chloroplast ribosome assembly and/or translation in plants.