Kye-Won Kim

The laccase multigene family in Arabidopsis thaliana: towards addressing the mystery of their gene function(s)

While laccases, multi-copper glycoprotein oxidases, are often able to catalyze oxidation of a broad range of substrates, such as phenols and amines in vitro, their precise physiological/biochemical roles in higher plants remain largely unclear, e.g., Arabidopsis thaliana contains 17 laccases with only 1 having a known physiological function. To begin to explore their roles in planta, spatial and temporal expression patterns of Arabidopsis laccases were compared and contrasted in different tissues at various development stages using RT-PCR and promoter-GUS fusions. Various cell-specific expressions were noted where specific laccases were uniquely expressed, such as LAC4 in interfascicular fibers and seed coat columella, LAC7 in hydathodes and root hairs, LAC8 in pollen grains and phloem, and LAC15 in seed coat cell walls. Such specific cell-type expression patterns provide new leads and/or strategies into determining their precise physiological/biochemical roles. In addition, there was an apparent redundancy of gene expression patterns for several laccases across a wide variety of tissues, lignified and non-lignified, perhaps indicative of overlapping function(s). Preliminary evidence, based on bioinformatics analyses, suggests that most laccases may also be tightly regulated at both transcriptional (antisense transcripts, histone and DNA methylation) and posttranscriptional (microRNAs) levels of gene expression.

Next Generation Sequencing in Predicting Gene Function in Podophyllotoxin Biosynthesis

Background: Biosynthetic pathways to structurally complex plant medicinals are incomplete or unknown. Results: Next generation sequencing/bioinformatics and metabolomics analysis of Podophyllum tissues gave putative unknown genes in podophyllotoxin biosynthesis. Conclusion: Regio-specific methylenedioxy bridge-forming CyP450s were identified catalyzing pluviatolide formation. Significance: Database of several medicinal plant transcriptome assemblies and metabolic profiling are made available for scientific community. Podophyllum species are sources of (−)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (−)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (−)-matairesinol into (−)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (−)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.

Opposite stereoselectivities of dirigent proteins in Arabidopsis and schizandra species.

Background: How vascular plants control phenoxy radical coupling is enigmatic. Results: Two dirigents engendered (−)-pinoresinol formation in Arabidopsis. Coupling stereoselectivity was reversed from (+)- to (−)-pinoresinol through swapping identical regions. Conclusion: Novel insights into stereoselective control over phenoxy radical coupling were obtained. Significance: This is the first report of dirigent-mediated phenoxy radical coupling control leading to opposite stereoselectivities and identification of protein regions involved. How stereoselective monolignol-derived phenoxy radical-radical coupling reactions are differentially biochemically orchestrated in planta, whereby for example they afford (+)- and (−)-pinoresinols, respectively, is both a fascinating mechanistic and evolutionary question. In earlier work, biochemical control of (+)-pinoresinol formation had been established to be engendered by a (+)-pinoresinol-forming dirigent protein in Forsythia intermedia, whereas the presence of a (−)-pinoresinol-forming dirigent protein was indirectly deduced based on the enantiospecificity of downstream pinoresinol reductases (AtPrRs) in Arabidopsis thaliana root tissue. In this study of 16 putative dirigent protein homologs in Arabidopsis, AtDIR6, AtDIR10, and AtDIR13 were established to be root-specific using a β-glucuronidase reporter gene strategy. Of these three, in vitro analyses established that only recombinant AtDIR6 was a (−)-pinoresinol-forming dirigent protein, whose physiological role was further confirmed using overexpression and RNAi strategies in vivo. Interestingly, its closest homolog, AtDIR5, was also established to be a (−)-pinoresinol-forming dirigent protein based on in vitro biochemical analyses. Both of these were compared in terms of properties with a (+)-pinoresinol-forming dirigent protein from Schizandra chinensis. In this context, sequence analyses, site-directed mutagenesis, and region swapping resulted in identification of putative substrate binding sites/regions and candidate residues controlling distinct stereoselectivities of coupling modes.

β-Glucuronidase as Reporter Gene

The beta-glucuronidase (GUS) gene is used extensively in plant biology studies; this analysis summarizes its advantages and limitations. With the advances in genomic sequencing and computational analyses (including bioinformatics), its application in the study of plant gene expression is now an integral component of modern day plant science. This chapter focuses on the detailed challenges of carrying out GUS studies for both qualitative and quantitative analyses, including the increasing employment of GUS from Bacillus strains, rather than E. coli; the Bacillus GUS genes encode proteins with enhanced properties, such as both increased thermostability and stability in the presence of crosslinking fixatives.

Non-host disease resistance response in pea (Pisum sativum) pods: Biochemical function of DRR206 and phytoalexin pathway localization

Continually exposed to potential pathogens, vascular plants have evolved intricate defense mechanisms to recognize encroaching threats and defend themselves. They do so by inducing a set of defense responses that can help defeat and/or limit effects of invading pathogens, of which the non-host disease resistance response is the most common. In this regard, pea (Pisum sativum) pod tissue, when exposed to Fusarium solani f. sp. phaseoli spores, undergoes an inducible transcriptional activation of pathogenesis-related genes, and also produces (+)-pisatin, its major phytoalexin. One of the inducible pathogenesis-related genes is Disease Resistance Response-206 (DRR206), whose role in vivo was unknown. DRR206 is, however, related to the dirigent protein (DP) family. In this study, its biochemical function was investigated in planta, with the metabolite associated with its gene induction being pinoresinol monoglucoside. Interestingly, both pinoresinol monoglucoside and (+)-pisatin were co-localized in pea pod endocarp epidermal cells, as demonstrated using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. In addition, endocarp epidermal cells are also the site for both chalcone synthase and DRR206 gene expression. Taken together, these data indicate that both (+)-pisatin and pinoresinol monoglucoside function in the overall phytoalexin responses.

Trimeric Structure of (+)-Pinoresinol-forming Dirigent Protein at 1.95 Å Resolution with Three Isolated Active Sites

Background: Dirigent protein (DP) discovery gave new paradigm for monolignol-derived coupling in planta. Results: (+)-Pinoresinol-forming DP (PsDRR206) three-dimensional structure was obtained at 1.95 Å resolution. Conclusion: The tightly packed trimeric DP has three putative substrate binding sites spatially far apart, suggesting that each site involves monomer coupling directly. Significance: New insights into monolignol radical-radical coupling in planta. Control over phenoxy radical-radical coupling reactions in vivo in vascular plants was enigmatic until our discovery of dirigent proteins (DPs, from the Latin dirigere, to guide or align). The first three-dimensional structure of a DP ((+)-pinoresinol-forming DP, 1.95 Å resolution, rhombohedral space group H32)) is reported herein. It has a tightly packed trimeric structure with an eight-stranded β-barrel topology for each DP monomer. Each putative substrate binding and orientation coupling site is located on the trimer surface but too far apart for intermolecular coupling between sites. It is proposed that each site enables stereoselective coupling (using either two coniferyl alcohol radicals or a radical and a monolignol). Interestingly, there are six differentially conserved residues in DPs affording either the (+)- or (−)-antipodes in the vicinity of the putative binding site and region known to control stereoselectivity. DPs are involved in lignan biosynthesis, whereas dirigent domains/sites have been implicated in lignin deposition.

1.23 – Lignans (Neolignans) and Allyl/Propenyl Phenols: Biogenesis, Structural Biology, and Biological/Human Health Considerations

This comprehensive, extensively illustrated, critical review chapter addresses the biochemical pathways to two major plant metabolic classes, namely, the rather ubiquitous allyl/propenyl phenols and lignans, which are the subclasses of phenylpropanoids (C6C3). These substances are considered to have diverse physiological/ecological roles within plants, including defense against microbes and insects, as antioxidants, and also as pollinator attractants. Comprehensive analysis of their structural diversity in extant plant groups provides much needed insight into their evolution. In addition, what is known of their biosynthetic pathways is extensively discussed, including the genes/proteins known to be responsible for each biochemical step, their enzymatic kinetic behaviors, three-dimensional structures, and postulated chemical mechanisms. Lastly, many are historically important natural products, and their current and potential human uses in spices, medicines, nutrition, and other fields are broadly reviewed. The text is supported by more than 50 illustrations and schemes including more than 300 compounds, and about 550 key references are presented to the reader.