PRISM :: Browsing by Author "Ro, Dae-Kyun"

Browsing by Author "Ro, Dae-Kyun"

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Open Access
A chromosome level reference genome of Diviner’s sage (Salvia divinorum) provides insight into salvinorin A biosynthesis
(2024-10-01) Ford, Scott A.; Ness, Rob W.; Kwon, Moonhyuk; Ro, Dae-Kyun; Phillips, Michael A.
Abstract Background Diviner’s sage (Salvia divinorum; Lamiaceae) is the source of the powerful hallucinogen salvinorin A (SalA). This neoclerodane diterpenoid is an agonist of the human Κ-opioid receptor with potential medical applications in the treatment of chronic pain, addiction, and post-traumatic stress disorder. Only two steps of the approximately twelve step biosynthetic sequence leading to SalA have been resolved to date. Results To facilitate pathway elucidation in this ethnomedicinal plant species, here we report a chromosome level genome assembly. A high-quality genome sequence was assembled with an N50 value of 41.4 Mb and a BUSCO completeness score of 98.4%. The diploid (2n = 22) genome of ~ 541 Mb is comparable in size and ploidy to most other members of this genus. Two diterpene biosynthetic gene clusters were identified and are highly enriched in previously unidentified cytochrome P450s as well as crotonolide G synthase, which forms the dihydrofuran ring early in the SalA pathway. Coding sequences for other enzyme classes with likely involvement in downstream steps of the SalA pathway (BAHD acyl transferases, alcohol dehydrogenases, and O-methyl transferases) were scattered throughout the genome with no clear indication of clustering. Differential gene expression analysis suggests that most of these genes are not inducible by methyl jasmonate treatment. Conclusions This genome sequence and associated gene annotation are among the highest resolution in Salvia, a genus well known for the medicinal properties of its members. Here we have identified the cohort of genes responsible for the remaining steps in the SalA pathway. This genome sequence and associated candidate genes will facilitate the elucidation of SalA biosynthesis and enable an exploration of its full clinical potential.
Open Access
Adapting RNA interactome capture to explore plant RNA-binding proteins and their RNA targets
(2022-09-23) Atugala, Dilini M; Muench, Douglas; Chua, Gordon; Ro, Dae-Kyun
RNA-binding proteins (RBPs) function in every step of RNA metabolism, from synthesis todecay. Thus, the global identification of RBPs and their RNA targets provide important insight intopost transcriptional gene regulation during organismal development and in response toenvironmental cues. To better understand the repertoire of RBPs in Arabidopsis, an RNAinteractome capture (RIC) approach was applied to root and leaf tissues, and to suspensionculture cells. RIC identified 416, 611 and 110 proteins comprising the root, leaf and cell culturemRNA-binding proteomes (RBPomes), respectively. Similar to other RNA interactome studiesthere were a large number of ‘moonlighting’ RBPs (up to 46%) that had activities not related toRNA biology, but rather to processes that included intermediate metabolic pathways,photosynthesis, cytoskeleton, and membrane transporters. These results highlight the overlapbetween interactome studies and reports RBPs that have not been identified in other Arabidopsisstudies. Additionally, the root RIC results provide insight into the RBPome of a tissue that has notbeen reported previously.To understand the responses of cellular RBPs to an abiotic stress, we applied RIC coupledwith stable isotope dimethyl labeling-mass spectrometry to Arabidopsis roots treated withnaphthenic acid fraction compounds (NAFCs) isolated from oil sands tailings pond water. NAFCtreatment resulted in a remodeling of the cellular RBPome, identifying 205 RBPs that increasedin their association with RNA. In addition to RBPsrelated to RNA biology processes, classical stressresponsive proteins were detected and included metabolic enzymes involved in carbohydratemetabolism, suggesting that these RBPs may govern metabolic changes during stress responses.Lastly, an approach was developed and tested for the identification of in vivo target RNAsof RBPs under stringent, denaturing extraction conditions. This immunoprecipitation approachidentified over 300 mRNA targets of a specific RBP (AtGRP7). This approach has the potential tobe used as a simplified option to identify RNA targets over other more advanced techniques suchas crosslinking and immunoprecipitation (CLIP).
Open Access
An Exploration of Cross-dehydrogenative Coupling Methodology and The Synthesis of Transient Receptor Potential Melastatin Member 8 (TRPM8) Channel Modulators
(2017) LeGay, Christina; Derksen, Darren; Back, Thomas; Sutherland, Todd; Ro, Dae-Kyun
Structure-activity relationship (SAR) studies of natural product analogues that modulate transient receptor potential (TRP) channels have been a useful tool for the development of potent and selective channel modulators. TRP channels play numerous physiological roles, including temperature and pain sensation, and have emerged as viable therapeutic targets. Recently, the Derksen group produced a library of TRP melastatin member 8 (TRPM8) channel modulators based on the natural product menthol. The first library of menthol analogues was prepared via an optimized and cost-effective synthetic route from commercially available starting materials. The biological evaluation of the first library revealed three novel TRPM8 channel modulators: an agonist with an EC50 of 11 ± 1 μM, an antagonist with an IC50 of 2 ± 1 μM, and an allosteric modulator that boosted the channel response towards consecutive applications of menthol. This thesis describes the results of the first library, and the tailoring of established synthetic methods to prepare a second generation library of natural product analogues based on the TRPM8 antagonist from the first library. Also described is the exploration of the organo-mediated cross-dehydrogenative coupling (CDC) of silyl enol ether and ketone substrates with the goal of preparing aldol adducts, which are key moieties in bioactive compounds including TRP channel modulators. CDC was employed for the direct functionalization of carbon-hydrogen bonds to prepare aldol adducts in an effort to overcome the issues of controlling reactivity and selectivity in aldol addition reactions. This thesis describes the variety of substrates and reaction conditions screened, and the measurements and calculations conducted in an attempt to optimally produce aldol like adducts directly from silyl protected alcohols. The highest yield achieved was an 18% GC-MS yield despite considerable effort and exploration of the reaction mechanism.
Open Access
Assessment and Characterization of Various Gene-editing Platforms for Brassica napus (Canola) using TRANSPARENT TESTA 8 (TT8) as the Target Gene
(2023-12-21) Coates, Ethan Robin; Samuel, Marcus; Muench, Douglas; Ro, Dae-Kyun; Chua, Gordon
Climate change poses a significant threat to agriculture by causing more frequent and extreme weather events, altering temperature and precipitation patterns, and increasing the prevalence of pests and diseases. These changes can lead to reduced crop yields and jeopardize food security on a global scale. CRISPR-Cas9, a revolutionary gene editing tool, offers a promising solution to address these challenges by enabling the development of crops with enhanced resilience to environmental stressors. Through targeted genetic modifications, it is possible to create crop varieties that are more drought-tolerant, heat-resistant, and better equipped to adapt to changing conditions, ultimately ensuring a more sustainable and secure food supply in the face of climate change. However, a multitude of different CRISPR systems with varying success, contingent on factors such as the plant species, sgRNA expression, and the choice of endonuclease present unique considerations. For instance, the choice between Cas9 versus an alternative, and the selection between a polycistronic or single guide complex system, can impact outcomes. In this thesis, I aimed to assess efficiency of several CRISPR systems in Brassica napus, a globally important oilseed crop also known as canola, in order to identify the most efficient editing system for canola. By investigating sgRNA expression and endonuclease efficiency using a hairy root system, the findings revealed that the 35S:Cas9:m35S:tRNA guide system was the most effective for editing the target gene, the transcription factor TT8 (Transparent Testa 8), in B. napus hairy roots. The choice of the 35S promoter driving the Cas9 and sgRNA expression in the 35S:Cas9:m35S:tRNA guide system appeared to contribute to its success, allowing for simultaneous and stoichiometric availability of Cas9 protein and its target guide RNA. This system can be efficiently utilized in future stable of hairy root systems to target candidate genes.
Open Access
Biochemical and evolutionary studies of sesquiterpene lactone metabolism in the sunflower (Asteraceae) family
(2013-05-06) Nguyen, Trinh Don; Ro, Dae-Kyun
Plants have evolved the capacity to synthesize a myriad of specialized metabolites which enhance their fitness in specific living conditions. These compounds are also widely utilized for human purposes. Elucidating the enzymes in plant specialized metabolism has been one of the main forces driving plant biochemistry. The more intriguing question, however, is how these enzymes evolved to acquire their existent functions. The Asteraceae, the largest flowering plant family, is well-known for its enormously diverse and lineage-characteristic contents of sesquiterpene lactones (STLs). Thousands of compounds in this subclass of specialized metabolites have been studied extensively for their structures and valuable bioactivities. However, the details of their metabolism are poorly understood. Studying STLs in the Asteraceae thus improves our knowledge of the biosynthesis of these compounds. Furthermore, the tight links between STLs and the Asteraceae family provide an excellent model to explore enzyme adaptive evolution. My thesis aims to advance our understanding of STL metabolism by focusing on elucidating the enzyme that is responsible for the oxidation of sesquiterpene to sesquiterpene carboxylic acid in the general STL biosynthetic route of the Asteraceae. In lettuce, two cytochrome P450-dependent monooxygenase (P450) isoforms responsible for oxidizing the three consecutive oxidations of germacrene A to germacrene A carboxylic acid in the biosynthesis of costunolide were characterized. This was achieved using a combination of genomic and biochemical approaches, and the aid of a metabolically-engineered yeast system. Furthermore, this germacrene A oxidase (GAO) activity was demonstrated to be highly conserved throughout the Asteraceae, even in the phylogenetically basal subfamily Barnadesioideae, which split from the rest of the family at least 50 million years go. Previous studies have characterized an Artemisia annua-specific sesquiterpene oxidase, amorphadiene oxidase (AMO), which is considered to have diverged from an ancestral GAO. The substrate specificity/promiscuity of AMO and GAOs towards each other’s natural substrates and seven other non-natural substrates was investigated to test the general hypothesis of enzyme evolution from ancestral promiscuity. The results from these combinatorial biochemistry studies and phylogenetic relations of AMO and GAOs provided deep insights into the evolution of these P450s in the context of the chemical diversity of the Asteraceae.
Open Access
Biochemical Studies of Terpenoid Metabolism in Aztec Sweet Herb
(2016) Hurd, Matthew; Ro, Dae-Kyun; Zaremberg, Vanina; Samuel, Marcus
Analysis of ethnobotanical literature led scientists to discover the Aztec sweet herb (Lippia dulcis), a highly aromatic plant containing the potent sesquiterpene ketone sweetener, hernandulcin. Despite the intense sweetness of L. dulcis, the main volatile constituent is the bitter-tasting and toxic monoterpenoid, camphor. To date, the biosynthesis of hernandulcin and camphor have yet to be fully elucidated in L. dulcis. Identification of key genes involved in both pathways could be used to enhance the safety and quality of hernandulcin-based alternative sweetener product. Two terpene synthases (TPSs) were identified from L. dulcis transcriptomics, one of which exhibited bornyl diphosphate synthase (LdBPPS) activity, catalyzing the first step of camphor biosynthesis in L. dulcis. Interestingly, the N-terminal region of LdBPPS contains a duplicated RRX8W motif that was found to be inhibitory in vitro, suggesting that it may be cleaved with the chloroplast transit peptide. Phylogenetic analysis demonstrated that LdBPPS evolved independently from the homologous enzyme in sage, despite both performing the same biochemical reaction. A cytochrome P450 monooxygenase (P450) was hypothesized to convert (+)-epi-α-bisabolol to hernandulcin. The induction of hernandulcin biosynthesis by methyl jasmonate was investigated to generate an elicitor-based transcriptome library but was found to be ineffective. Therefore, a homology-based cloning was used to identify ten putative P450s and their expression and activity were tested in yeast. A total of seven P450s were expressed in yeast, determined by immunoblot analyses, in either the native form or after N-terminal replacement. Six P450s showed no unique product formation after GC-MS analysis while one P450 (LdO10) showed minor affinity to (+)-epi-α-bisabolol substrate and produced bisabolol oxide B. Future efforts should be used to generate better bioinformatic resources to facilitate the mining for genes involved in hernandulcin biosynthesis.
Open Access
Biophysical studies of bacterial proteins; can we address antibiotic resistance?
(2017) Paul, Subrata; Vogel, Hans; Ro, Dae-Kyun; Zaremberg, Vanina
It is forecast that the single biggest challenge to medical care in the twenty-first century will be the control of antibiotic resistant bacteria, including multidrug resistant strains. Although numerous factors are associated with the rapid emergence of pathogen resistance, the limited number of core structures of currently used antibiotics is one of the reasons for this development. Unlike other conventional antibiotics, the recently discovered class of lipopeptide antibiotics consists of a cyclic peptide head group with an acyl chain that are synthesized by a non-ribosomal peptide synthetase in association with an acyl carrier protein (ACP). This dissertation presents the solution structure of an ACP, LipD that is involved in the acylation of the lipopeptide antibiotic friulimicin. Enhanced stability of holo-LipD (LipD with the phosphopantetheine group attached) was observed through biophysical investigations. This may originate from a subtle change in the helix angles, required for the attachment of the acyl chain. Moreover, interaction studies of ACPs with antimicrobial peptides (AMPs) revealed that bacterial ACPs can be novel targets for AMPs and that this may perturb the fatty acid synthesis to abrogate the pathogenesis of recalcitrant pathogens such as Pseudomonas aeruginosa. We also investigated the ligand binding properties of two selected periplasmic binding proteins (PBP). Understanding the mechanisms by which PBPs can bind and release various ligands has implications for designing biosensors and developing therapeutic compounds that will perturb the pathways mediated by PBPs. Detailed biophysical studies of the Escherichia.coli ferric citrate binding PBP, FecB, revealed that it could form complexes with a wide variety of different tricarboxylic acid-iron complexes. Moreover unexpectedly FecB was found to bind apo-citrate and other iron-free tricarboxylic acids with great avidity. Information regarding the plasticity of substrate binding by FecB will allow us to design new strategies to overcome bacterial resistance. On the other hand, we demonstrated that E. coli HisJ interacts with L-histidine with nM affinity and can also bind 3-methyl-L-histidine, which has been identified as a biomarker for muscle wasting. Therefore, HisJ can potentially be used to design a reagentless protein-based biosensor for the early detection of this muscle disease.
Open Access
Comparative transcriptomics and proanthocyanidin metabolism in pea (Pisum sativum) seed coat
(2014-02-07) Ferraro, Kiva; Ro, Dae-Kyun
Plants produce a vast array of specialized compounds known as secondary metabolites, which were originally thought to be non-essential for plant survival. However, we now know that secondary metabolites play integral roles in plant defense, signalling, reproduction and more. Proanthocyanidins (PAs) are a class of flavonoid polymers derived from the phenylpropanoid pathway. PAs accumulate in the seed coat, bark, and leaves of many plants and are believed to play a role in plant defense. Recent evidence of health benefits associated with PA consumption has spurred new research interests in PA biosynthesis. Many of the studies of seed coat PA biosynthesis have been conducted in non-crop species that produce a limited variety of PAs. Pea (Pisum sativum) offers a number of unique advantages for PA research. Peas produce large seed coats, which are easy to isolate. Dry peas are an important source of nutrition for both humans and livestock, enabling research integration into the human diet and commercial agriculture. Finally, centuries of breeding have produced a wide variety of pea cultivars, making pea a valuable phenotypic and genetic resource for plant research. Despite these advantages, PA biosynthesis in pea has not been well characterized. This work presents the seed coat PA chemical profile of three pea cultivars and the biochemical characterization of two PA branch point enzymes, anthocyanidin reductase and leucoanthocyanidin reductase, from pea. In addition, the seed coat transcriptomes of these three varieties were compared to those of two varieties lacking PAs in an effort to elucidate novel genetic mechanisms relating to PA biosynthesis. This comparative transcriptomic analysis was expanded to study general seed phenotypic differences between pea cultivars. Two target genes were identified; one related to seed weight and another to PAs, the latter of which was further characterized.
Open Access
Developing base-editing for the improvement of agronomic traits in field peas (Pisum sativum) using glyphosate resistance as a target
(2024-07-04) Roth, Susan Alexandra; Ro, Dae-Kyun; Samuel, Marcus; Chua, Gordon
The world’s population is estimated to surpass 10 billion by 2059, and alongside the increasing challenges posed by climate change, current global food systems are not sustainable. With these challenges has come an increased push towards more plant-based diets as plant agriculture has lower greenhouse gas emissions (GHGs) and land use requirements than animal agriculture. Field peas (Pisum sativum), an important Canadian crop, lend themselves well to this shift due to their high protein content, which can be used to replace animal-derived protein. Expanding the gene-editing toolkit for field peas will allow for more agronomic traits to be targeted and increase the ease with which field pea crops can be grown. Canonical CRISPR/Cas systems have revolutionized the field of crop improvement; however, their application has been limited to imprecise knockout mutations through non-homologous end joining (NHEJ) of double-stranded breaks (DSBs). Cytosine base-editing (CBE) is a new CRISPR-based technology that increases the precision of gene editing to the single base pair resolution in plants and opens the doors to producing gain of function mutations. This technology has yet to be used in peas and will increase the number of agronomic traits that can be targeted. This study used CBE to target 5- enolypyruvulshikimate-3-phosphate (EPSP) synthase in yellow field pea hairy roots to induce resistance to the herbicide glyphosate (commercially known as RoundUp). The base-editing window that predicts where editing is most likely to occur with CBEs was found to be expanded when using the PmCDA1 cytidine deaminase. Novel mutations R105C and P106F were obtained and characterized to show a complete knockout of EPSP synthase function with an R105C mutation and a 100-fold increase in glyphosate resistance with a P106F mutation. A P106S mutation, which has previously been characterized in other species, was also obtained and presented an 18-fold increase in glyphosate resistance, consistent with previous works.
Open Access
Engineering Streptavidin with Switchable Ligand Binding Affinity Using Disulfide Bonds at the Biotin Entry Gateway
(2020-07-22) Marangoni, Jesse M; Wong, Sui-Lam; Ng, Kenneth; Evans, Stephen; Schriemer, David; Ro, Dae-Kyun; Gedamu, Lashitew
Streptavidin is widely used in biotechnological applications for its specific, high-affinity interaction with its natural ligand biotin, a small vitamin that can be easily conjugated to biomolecules of interest. However, its utility is often limited since many applications require reversibility in binding whereas others require higher affinity than that offered by wild-type streptavidin. For some applications, it would even be beneficial to allow extremely tight binding and subsequent ligand release within a single protocol. To combine both extremely tight and reversible binding into a single protein, two streptavidin muteins, designated M88 and M112, were engineered to each contain a distinct disulfide on opposite sides of a flexible loop critical for ligand binding. Each disulfide bond has markedly different effects on protein structure and binding kinetics. While the disulfide in M112 caused a detrimental conformational change which decreased biotin binding affinity, oxidized M88 showed a ~250-fold decrease in off-rate constant at 21°C and increased thermostability when compared to wild-type streptavidin. Furthermore, reduction of the disulfide bond increased the off-rate constant ~19,000-fold compared to the oxidized form, reducing the half-life for dissociation from 50 years to 1 day. Increasing the temperature to 50°C allows ligand release from the reduced form with a half-life of 9 minutes. M88 thus displays redox and temperature dependent ligand binding, both of which can be used to switch between high- and low-affinity states. M88 coupled to a matrix can be used to capture and release biotinylated biomolecules. For applications where increased temperature is not viable, further engineering of M88 has been used to reduce ligand binding affinity. The relative ease of controlling protein disulfide bond redox state with mild chemical agents allows switchable affinity of M88 towards biotin.
Open Access
Genetics and evolution of ultraviolet reflectance in flowers
(2018-01-25) Liu, Yan; Vamosi, Jana; Samuel, Marcus; Ro, Dae-Kyun; Melin, Amanda; Caruso, Christina
Many flowers have ultraviolet (UV) reflectance patterns, which are invisible to humans but visible to pollinators, such as bumblebees and hummingbirds. In bees and hummingbirds, photoreceptors are sensitive to UV wavelengths, and it is therefore necessary to incorporate this variable to model pollinators’ perception and assess floral UV evolution. In this thesis, I explore micro- and macroevolutionary patterns in floral UV patterns, specifically concentrating on the effect of this phenotype on pollinators. I first explore the ways in which UV patterns can be measured and characterized, as well as explore the underlying basis of UV patterning in flowers. By gathering UV reflectance data (between 300 to 400nm) on 150 species, I found evidence that the phylogenetic distribution of UV trait disparity is consistent with a stabilizing selection model of evolution, but the magnitude of stabilizing selection varies with geography and pollinator syndrome. Mimulus species have become key model species for investigating the genetics of floral adaptations, in part because it is tremendous diversity in floral phenotypes. I firstly estimate genetic diversity in six populations in Alberta and British Columbia. Historical contingency (via geographic and bioclimatic events) provides the evidence of restricted gene flow. Variance in phenotypes depends not only on allelic interactions but also on environmental factors. Variation and heritability of the floral UV reflectance are further explored with experimental interspecific crosses between Mimulus guttatus and Mimulus luteus. By recoding 12 floral traits throughout the parental to F4 generations, I find that phenotypic covariance is strongest between UV reflectance and other floral traits, lending evidence to the idea that UV reflectance in flowers evolves along with other floral traits as a response to selection from pollinators. My research has implications for forecasting plant adaptation through hybridization and polyploidization, which may occur in concert with the evolution of plant-pollinator relationships.
Open Access
Identification and characterization of diterpene synthases in the salvinorin A biosynthetic pathway
(2012-08-31) Mitchell, Rod; Ro, Dae-Kyun
Salvia divinorum is a hallucinogenic plant that is used for divination and spiritual communion by the Mazatecs of Mexico. The active component of the plant is salvinorin A, a neoclerodane diterpenoid that selectively acts as a potent κ opioid receptor agonist. Salvinorin A's novel receptor binding profile makes derivatives of the compound potentially useful in the treatment of various psychiatric and mood disorders. In this work, next generation sequencing (Roche 454) was used to generate a S. divinorum transcript database. Five distinct putative diterpene synthase cDNAs (two type II and three type I) were identified in the database. We report here a recombinant type II diterpene synthase capable of catalyzing the rearrangement of GGPP into terpentedienyl diphosphate (a neoclerodane diphosphate), and a recombinant type I diterpene synthase that renders terpentedienyl diphosphate into kolavenol. These enzymatic products are potential intermediates in the salvinorin A biosynthetic pathway.
Open Access
In-vitro Characterizing of Thebaine Inhibition on Salutaridinol-7-O-acetyltransferase in Morphine Biosynthesis
(2024-12-13) Kapasi, Moiz Shabbir; Facchini, Peter James; Ng, Kenneth; Ro, Dae-Kyun; Kapoor, Manju
Opiates, such as morphine, have been used for medicinal purposes throughout human history. These bioactive compounds are produced in the opium poppy (Papaver somniferum) through complex biosynthetic pathways. This study aimed to characterize the in-vitro enzyme kinetics and inhibition of salutaridine reductase (SalR) and the coupled enzymes salutaridinol-7O-acetyltransferase (SalAT) and thebaine synthase (THS2). It was found that SalR exhibits substrate inhibition to salutaridine, while the coupled system of SalAT/THS2 demonstrated mixed model inhibition by the product thebaine. This inhibition was characterized using deuterated thebaine-d3. These findings could significantly enhance the potential for synthetic engineering of the alkaloid pathway and remove bottlenecks in production, offering a more adaptable approach to medicinal production via bacterial or yeast fermentation. This research provides valuable insights into the regulatory mechanisms of the morphine biosynthetic pathway and opens new avenues for the synthetic production of medicinal opiates.
Open Access
Increasing Reproductive Output of Brassica napus (canola) Through Manipulation of Shoot Architecture
(2019-11) Stanic, Matija; Samuel, Marcus A.; Ro, Dae-Kyun; Muench, Douglas G.
Food security and yield increases of important crops is of paramount concern to an ever-increasing human population. A key determinant of yield in plants is shoot architecture which is controlled through a complex interplay between endogenous and exogenous signals and exhibits high plasticity throughout a plant’s lifetime. Strigolactones (SL) are a class of terpenoid lactone plant hormones which have recently been characterized as key regulators of shoot development. Mutants in this pathway exhibit various developmental deviations, most notably a dwarfed and highly branched shoot architecture. Canola transgenic lines suppressed in key SL biosynthetic and signalling components were generated to assess effects on architecture. Lines suppressed in the SL receptor, BnD14, displayed reductions in overall shoot height as well as an increase in lateral branching. This was accompanied by an increase in total flowers per plant. Consistent with this, BnD14 knockout lines generated through a CRISPR/Cas9-mediated system also exhibited a dwarf, highly branched phenotype with significant increases in total flowers per plant. This study provides evidence of the potential use of the SL pathway in future crop modifications to promote increased crop yield.
Open Access
Investigation of the Role of Kinase-Associated Protein Phosphatase (KAPP) in Pistil-Pollen Interactions in Brassica napus (canola)
(2021-06-25) Ribano, Alynne Kris B.; Samuel, Marcus A.; Ro, Dae-Kyun; Moorhead, Gregory
Climate change and our increasing population threatens the availability of sustainable food sources, creating greater pressure to find solutions. Increasing crop yield can mitigate these problems. Given that seeds are the most important agronomic trait, a key component of this reproductive success is pollination. The pollination pathway, both compatible and self-incompatible (SI), is comprised of complex signalling mechanisms which govern pollen-pistil communications. Even with significant contributions to our understanding of the pollination response through the identification of key players and its signalling mechanism, there remains a gap in our understanding of the SI pathway.Although kinase-associated protein phosphatase (KAPP) can interact in vitro with various receptor kinases, for decades, its specific role in SI remained unknown. In this study, biochemical approaches determined that KAPP was highly expressed in mature stigmas with poor expression in pollen. While compatible pollen did not affect KAPP levels, following SI pollination, KAPP levels were significantly increased. Through transgenic approaches, overexpressing only the phosphatase type 2C (PP2C) domain of KAPP sufficiently reduced pollen acceptance, leading to shorter pods and reduced seed production. These observations indicate that KAPP could be a positive regulator in the incompatibility response where it acts through its PP2C domain.When a PP2C domain mutated in the mitogen-activated protein kinase (MAPK) docking site (KAPP?290M) was overexpressed, a more compatible phenotype relative to PP2C overexpression was observed in the KAPP?290M transgenic lines. However, biochemical analysis revealed that this phenotype was not due to the inability of the KAPP?290M to dephosphorylate MPK4 as the mutated version KAPP?290M was able to effectively dephosphorylate MPK4 in vitro. Confocal imaging of KAPP?290M-RFP expression indicated that, lack of these key MAPK docking residues resulted in localization of KAPP?290M-RFP from the cytosol to the plasma membrane. Collectively, in this study, I have identified KAPP as a positive regulator of SI acting through its PP2C domain and provided evidence for the requirement of the MAPK docking site for precise intracellular localization. Therefore, this study bridges the knowledge gap in SI and provides another protein which can be manipulated to create novel hybrids to improve crop yield.
Embargo
Let There be Morphine: Structural Insights into Functional and Evolutionary Relationships of Morphine Biosynthesis in Opium Poppy
(2023-11-30) Carr, Samuel Clyde; Facchini, Peter; Ng, Kenneth; Ro, Dae-Kyun; Moorhead, Gregory; Noskov, Sergei
Benzylisoquinoline alkaloids are a large and diverse class of plant specialized metabolites known for their pharmaceutical properties, including the tumor suppressant noscapine, vasodilator papaverine, antimicrobial sanguinarine, and the important analgesics codeine, morphine, and their semisynthetic derivatives. The latter group of analgesics are known as morphinan alkaloids or opiates and are produced solely in select members of the genus Papaver, most importantly Papaver somniferum commonly known as opium poppy. This thesis highlights the use of structural biology in multidisciplinary approaches to understand the biosynthesis of morphinan alkaloids in opium poppy and related species. Dehydroreticuline reductase and codeinone reductase are closely related enzymes from the aldo-keto reductase superfamily catalyzing the second and second-last steps, respectively, in morphine biosynthesis in opium poppy. The elucidation of the crystal structure of codeinone reductase reveals novel structural features allowing for the in-depth analysis of substrate binding and catalysis leading to the engineering of substrate specificity. This structure also provides the means for the homology modeling of dehydroreticuline reductase giving insight into its novel catalytic mechanism. Transcriptomic analysis of representative Papaver species reveals putative morphinan biosynthetic enzyme orthologues and the ubiquitous distribution of dehydroreticuline reductase and codeinone reductase, among other morphinan biosynthetic enzymes. Structural analysis and functional/kinetic characterization of aldo-keto reductases demonstrates the ubiquitous conservation of dehydroreticuline reductase and codeinone reductase activity in the genus Papaver. These results challenge the current evolutionary narrative of morphinan biosynthesis suggesting: [1] a more ancient neofunctionalization of early enzymatic steps of morphinan biosynthesis than previously believed and [2] that morphinan biosynthesis evolved via the patchwork evolutionary model. Enzymatic latency and ligand binding affinity both provide a platform for the neofunctionalization of novel enzymatic activities. The alkaloid binding capabilities of pathogenesis related 10 proteins provides a means to understand the neofunctionalization of the morphine biosynthetic enzymes thebaine synthase and neopinone isomerase, alongside a biological role in alkaloid storage for abundant non-catalytic pathogenesis related 10 proteins. Their binding to alkaloids is predicted to promote the formation of protein-alkaloid aggregates based on binding-induced conformational changes observed through X-ray crystallography and dramatic changes in sucrose gradient fractionation.
Open Access
Molecular characterization of cis-prenyltransferase complexes in guayule (Parthenium argentatum)
(2017-12-17) Lakusta, Adam; Ro, Dae-Kyun; Muench, Douglas; Moorhead, Gregory; Wasmuth, James
Natural Rubber (NR) is a biopolymer with an irreplaceable role in the fields of medicine, manufacturing, and transport. Despite its necessity, the Brazilian Rubber Tree (Hevea brasiliensis) remains virtually the sole source of NR. As the majority of NR plantations are in Southeast Asia, its production is threatened by climate and disease. While NR supply remains insecure, biochemical studies have identified two essential proteins: cis-prenyltransferases (CPTs) and cis-prenyltransferase Binding Proteins (CBPs). With recent studies elucidating the biochemical properties of plant CPTs and CBPs, a potential mechanism for NR biosynthesis has been proposed. As the bulk of NR research has so far focused on two phylogenetically close relatives, lettuce and dandelion, further evidence is required from a more genetically distant plant species. This research is focused on guayule (Parthenium argentatum, Asteraceae family) – an alternative NR-producing plant long-diverged from lettuce and dandelion. CBP and CPT homologs, PaCBP and PaCPT1-3, were identified through publicly available transcriptomics databases and various biochemical assays were undertaken. In vivo split-ubiquitin yeast two hybrid and co-immunoprecipitation assays have indicated strong levels of interaction between the PaCPTs and PaCBP. Interestingly, our findings also show that guayule CPTs, together with guayule’s CBP, are responsible for the production of dolichol-like polymers in vitro rather than NR. This was shown by using harvested microsomes from yeast that expressed heterologous guayule CBP and CPTs in a biochemical assay. The biochemical assay was performed using [14C]-radiolabeled isopentenyl pyrophosphate as substrate. The polymer sizes were determined through reverse-phase thin layer chromatography. Another method used to determine the function of CPT and CBP was to rescue the lethal phenotype of dolichol synthase-deficient yeast by co-expressing CPT and CBP. Furthermore, phylogenetic and expression analyses of PaCPT1-3 revealed PaCPT3 as both likely involved in NR biosynthesis and highly expressed throughout the plant. PaCPT3 may therefore prove to be suitable for future NR studies and biotechnological applications. Taken together, our data provide further evidence of a NR biosynthetic mechanism that is conserved across the Asteraceae family, while also increasing guayule’s viability as both a production alternative and model for the study of NR.
Open Access
Molecular cloning and characterization of (+)-epi-a-bisabolol synthase, the entry point enzyme involved in the hernadulcin biosynthetic pathway in lippia dulcis
(2012) Attia, Mohamed; Ro, Dae-Kyun
Hemandulcin, a C 15 sesquiterpene ketone, is a natural sweetener isolated from the leaves of Lippia dulcis, an indigenous plant to the Central America. Hernandulcin is a promising sugar substitute due to its safety and low caloric potential. However, its biosynthesis in L. dulcis remains unknown. The first biochemical step in hemandulcin biosynthetic pathway is the synthesis of (+)-epi-a-bisabolol from famesyl diphosphate, which is presumed to be catalyzed by a unique sesquiterpene synthase in L. dulcis. To identify(+)epi-a-bisabolol synthase gene in L. dulcis, deep transcript sequencings ( 454 and Illumina) were performed and five potential candidates were identified by bioinformatic analysis. One of these candidates was successfully identified as a-bisabolol synthase by in vivo functional characterization in metabolically engineered yeast. Deep structural analysis of the produced a-bisabolol confirmed its configuration to be ( + )-epi-a-bisabolol, the core skeleton of hemandulcin. Bisabolol yield from the engineered yeast was quantified and kinetic constants of the identified ( + )-epi-a-bisabolol synthase were determined.
Open Access
Molecular cloning and characterization of sesquiterpene synthases from valeriana officinalis
(2012-07-19) Pyle, Bryan Wilkinson; Ro, Dae-Kyun
Valeriana officinalis (valerian) is a popular medicinal plant in North America and Europe. Its root extract is commonly used as a mild sedative and anxiolytic. Valerenic acid, a C15 sesquiterpenoid, has been suggested as the active ingredient responsible for the sedative effect. Recently, medical uses of valerenic acid as anti-depressant and anti-inflammatory drugs were suggested due to its affinity for the γ-aminobutyric acid type A (GABAA) receptor as an agonist and its inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, respectively. Despite its importance, biochemistry of valerenic acid in valerian remains unknown. To identify the first committed enzymatic step in valerenic acid biosynthesis, next-generation sequencing (Roche 454 titanium) was used to generate ~1 million transcript reads from valerian root. Subsequently, three cDNAs for sesquiterpene synthases (VoTPS1/2/3) were identified and their corresponding recombinant enzymes were purified. Three recombinant enzymes efficiently catalyze the synthesis of valerena-4,7(11)-diene, germacrene C/D, and drimenol, respectively, based on the spectral match in the mass spectrometry library. Additional structural analyses using GC-MS and 13C-NMR spectrometry in comparison to a semi-synthesized standard confirmed the chemical identity of valerena-4,7(11)-diene. This is the first report of valerena-4,7(11)-diene and drimenol synthases, and the biosynthetic mechanisms of these two products from the substrate, farnesyl diphosphate, were proposed.
Open Access
Molecular genetic and biochemical studies of cis-prenyltransferase and small rubber particle protein for natural rubber biosynthesis in lettuce (Lactuca saliva)
(2013-09-24) Qu, Yang; Ro, Dae-Kyun
Plant derived metabolites have been extensively implicated in medication and industry. Natural rubber (cis-1,4-polyisoprene) has become one of the most important raw materials since the rise of automotive industry due to its elastic characteristics. Despite the invention of synthetic elastomers, the demand and production of natural rubber has been increasing because it has higher polymer properties such as elasticity, resilience, abrasion and heat dispersion. Natural rubber belongs to the metabolites family termed isoprenoid that is synthesized from the same precursor isopentenyl pyrophosphate (IPP). Natural rubber is classified in a subgroup of linear molecules condensed from various IPP in cis-stereochemistry called cis- polyisoprenoids. The responsible enzyme is termed cis-prenyltransferase (CPT). Bacterial CPTs have been extensively studied in both biochemistry and 3D structure. Eukaryotic CPTs are not studied in such detail. They are not enzymatically active when purified, and the activity requires biomembrane and/or other undetermined membrane associated proteins. Based on natural rubber structure, CPT is suggested in the biosynthesis of this extremely long-chain cis-polyisoprenoid. However, there had not been convincing experimental evidence at the beginning of this study. Using proteomics, next-generation-sequencing, RNAi-silencing, and protein-protein interaction study, we identified two key proteins directly involved in the natural rubber biosynthesis in rubber producing plant lettuce (Lactuca sativa). RNAi silencing of CPT-like gene CPT Scaffold protein 2 (CSF2) resulted in robust decrease of natural rubber in planta. CSF2 interacts with a regular CPT (CPT3), and this interaction mutually influences the subcellular localization of both proteins. In agreement with the recently reported human dolichol synthase comprising both CSF and CPT partners, our data suggests the previously unknown heterodimeric synthase infrastructure may be conserved in eukaryotic long-chain cis-polyisoprenoid synthesis. Rubber Elongation Factor (REF) and its closely related homolog Small Rubber Particle Protein (SRPP) have also been implicated in natural rubber synthesis, despite the lack of convincing evidence. RNAi-silencing of their orthologs (lettuce SRPPs) in planta instead suggests that they are not crucial in the natural rubber synthesis. For the first time, CPT-like gene CSF is shown indispensible in natural rubber synthesis. The discovery of the interaction between CSF and CPT has built the molecular base for future improvement of natural rubber production and the eventual reconstitution of natural rubber synthesis in other organism at industrial scale.