Carolyn Lee-Parsons | Northeastern University (original) (raw)

Papers by Carolyn Lee-Parsons

Plant cell reports, May 14, 2024

Key message A GLK homologue was identified and functionally characterized in Catharanthus roseus.... more Key message A GLK homologue was identified and functionally characterized in Catharanthus roseus. Silencing CrGLK with VIGS or the chloroplast retrograde signaling inducer lincomycin increased terpenoid indole alkaloid biosynthesis. Abstract Catharanthus roseus is the sole source of the chemotherapeutic terpenoid indole alkaloids (TIAs) vinblastine and vincristine. TIA pathway genes, particularly genes in the vindoline pathway, are expressed at higher levels in immature versus mature leaves, but the molecular mechanisms responsible for this developmental regulation are unknown. We investigated the role of GOLDEN2-LIKE (GLK) transcription factors in contributing to this ontogenetic regulation since GLKs are active in seedlings upon light exposure and in the leaf's early development, but their activity is repressed as leaves age and senesce. We identified a GLK homologue in C. roseus and functionally characterized its role in regulating TIA biosynthesis, with a focus on the vindoline pathway, by transiently reducing its expression through two separate methods: virus-induced gene silencing (VIGS) and application of chloroplast retrograde signaling inducers, norflurazon and lincomycin. Reducing CrGLK levels with each method reduced chlorophyll accumulation and the expression of the light harvesting complex subunit (LHCB2.2), confirming its functional homology with GLKs in other plant species. In contrast, reducing CrGLK via VIGS or lincomycin increased TIA accumulation and TIA pathway gene expression, suggesting that CrGLK may repress TIA biosynthesis. However, norflurazon had no effect on TIA gene expression, indicating that reducing CrGLK alone is not sufficient to induce TIA biosynthesis. Future work is needed to clarify the specific molecular mechanisms leading to increased TIA biosynthesis with CrGLK silencing. This is the first identification and characterization of GLK in C. roseus and the first investigation of how chloroplast retrograde signaling might regulate TIA biosynthesis.

Journal of Biological Chemistry, 2023

한국생물공학회 학술대회, Apr 1, 2007

bioRxiv (Cold Spring Harbor Laboratory), Apr 4, 2020

The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with... more The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by stably transforming a Cas9 expression construct into the plant genome. The efficiency with which mutations are obtained in genes of interest can vary considerably depending on specific features of the constructs, including the source and nature of the promoters and terminators used for expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency with which mutations could be obtained in target genes in Arabidopsis thaliana with the Cas9 nuclease, we have investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLS) and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on Cas9 activity and that two NLSs work better than one. However, the most important impact on the efficiency of the constructs was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies of the constructs; none of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of primary transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

Transgenic hairy root cultures of the medicinal plant, Catharanthus roseus, were established with... more Transgenic hairy root cultures of the medicinal plant, Catharanthus roseus, were established with green fluorescent protein (GFP) expression under the control of an estrogen-inducible system. The Agrobacterium-mediated transformation process was optimized with an overall efficiency of 33%. Furthermore, GFP expression was tightly regulated and highly inducible using the estrogen-inducible system as demonstrated in hairy roots. This paper describes the optimized transformation method using the estrogen-inducible system for engineering the production of pharmaceutically important alkaloid compounds from C. roseus hairy root cultures.

Plant direct, Dec 1, 2019

Due to their sessile existence, plants have developed rapid and effective responses to environmen... more Due to their sessile existence, plants have developed rapid and effective responses to environmental stresses, herbivory, and pathogens. When stressed, plants can shift primary metabolic resources to produce specialized metabolites that act as defense compounds. For instance, the Madagascar periwinkle, Catharanthus roseus, produces numerous monoterpenoid indole alkaloids (MIA) in defense against pathogens and herbivory (Luijendijk, van der Meijden, & Verpoorte, 1996; Dugé de Bernonville et al., 2017). Many of these MIAs are pharmaceutically valuable, including the antihypertensive ajmalicine, the sedative serpentine, and most notably the anticancer compounds vincristine and vinblastine (Van der Heijden, Jacobs,

Biochemical Engineering Journal, Nov 1, 2004

The potential of strategies for increasing secondary metabolite accumulation from plant cell cult... more The potential of strategies for increasing secondary metabolite accumulation from plant cell cultures may still be partially limited by unfavorable conditions for biosynthesis (i.e. feedback inhibition, unfavorable equilibrium state) or product metabolism. In this paper, the rate of product removal was altered and used as a tool for probing the impact of these limiting conditions on product accumulation, using the production of ajmalicine and serpentine from Catharanthus roseus suspensions as a model system. The method for altering the in situ product removal rate was simple; Amberlite ® XAD-7HP resin was enclosed in either a porous or less porous material, and experiments confirmed that the product removal rate was higher with resin enclosed in the porous Miracloth versus the less porous nylon. When resin was added to suspensions, higher ajmalicine production and recovery were associated with Miracloth-enclosed resin which had higher product removal rates. This simple experiment illustrated that further improvements in production and recovery were possible, indicating that conditions unfavorable for biosynthesis and/or product metabolism existed in cultures with nylon-enclosed resin. These results also suggest the importance of optimizing the product removal rate for improving the overall production and recovery of secondary metabolites from plant cell cultures.

Plant Cell Reports, Aug 11, 2005

Cytosolic Ca 2+ and jasmonate mediate signals that induce defense responses in plants. In this st... more Cytosolic Ca 2+ and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca 2+ and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl 2 (3, 23, and 43 mM) and MJ (0, 10, and 100 µM) on day 6 of growth. Increased Ca 2+ influx through the addition of extracellular CaCl 2 suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 µM). In the presence of 3 mM CaCl 2 in the medium, the addition of Ca 2+ chelator EGTA (1, 2.5, and 5 mM) or Ca 2+ channel blocker verapamil (1, 10, and 50 µM) to MJ-induced (100 µM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca 2+ inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca 2+ concentration and a low extracellular Ca 2+ concentration (3 mM) enhanced MJ-induced ajmalicine production.

Plant Cell Reports, Jan 24, 2006

Jasmonates enhance the expression of various genes involved in terpenoid indole alkaloid (TIA) bi... more Jasmonates enhance the expression of various genes involved in terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus. We applied precursor feeding to our C. roseus suspensions to determine how methyl jasmonate (MJ) alters the precursor availability for TIA biosynthesis. C. roseus suspensions were induced with MJ (100 µM) on day 6 and fed loganin (0.30 mM), tryptamine (0.15 mM), loganin plus tryptamine, or geraniol (0.1-1.0 mM) on day 7. While MJ increased ajmalicine production by 3-fold, induced cultures were still limited by terpenoid precursors. However, both induced and non-induced cultures became tryptamine-limited with excess loganin. Geraniol feeding also increased ajmalicine production in non-induced cultures. But MJ appeared to increase geraniol availability in induced cultures, due presumably to the increased expression of Dxs with MJ addition.

PLOS ONE, Oct 17, 2012

The abundant flavonoid aglycone, naringenin, which is responsible for the bitter taste in grapefr... more The abundant flavonoid aglycone, naringenin, which is responsible for the bitter taste in grapefruits, has been shown to possess hypolipidemic and anti-inflammatory effects both in vitro and in vivo. Recently, our group demonstrated that naringenin inhibits hepatitis C virus (HCV) production, while others demonstrated its potential in the treatment of hyperlipidemia and diabetes. However, naringenin suffers from low oral bioavailability critically limiting its clinical potential. In this study, we demonstrate that the solubility of naringenin is enhanced by complexation with b-cyclodextrin, an FDA approved excipient. Hydroxypropoyl-b-cyclodextrin (HPbCD), specifically, increased the solubility of naringenin by over 400fold, and its transport across a Caco-2 model of the gut epithelium by 11-fold. Complexation of naringenin with HPbCD increased its plasma concentrations when fed to rats, with AUC values increasing by 7.4-fold and C max increasing 14.6-fold. Moreover, when the complex was administered just prior to a meal it decreased VLDL levels by 42% and increased the rate of glucose clearance by 64% compared to naringenin alone. These effects correlated with increased expression of the PPAR co-activator, PGC1a in both liver and skeletal muscle. Histology and blood chemistry analysis indicated this route of administration was not associated with damage to the intestine, kidney, or liver. These results suggest that the complexation of naringenin with HPbCD is a viable option for the oral delivery of naringenin as a therapeutic entity with applications in the treatment of dyslipidemia, diabetes, and HCV infection.

Analytical Methods, 2012

Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for ob... more Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for obtaining natural products of medical relevance. Cultures can be optimized for high yields of desired metabolites using rapid profiling assays such as HPLC. We describe an approach to establishing a rapid assay for profiling cell culture expression systems using a novel microscale LC-UV-MS-NMR platform, designed to acquire both MS and NMR each at their optimal sensitivity, by using nanosplitter MS from 4 mm analytical HPLC columns, and offline microdroplet NMR. The approach is demonstrated in the analysis of elicited Eschscholzia californica cell cultures induced with purified yeast extract to produce benzophenanthridine alkaloids. Preliminary HPLC-UV provides an overview of the changes in the production of alkaloids with time after elicitation. At the time point corresponding to the production of the most alkaloids, the integrated LC-MS-microcoil NMR platform is used for structural identification of extracted alkaloids. Eight benzophenanthridine alkaloids were identified at the sub-microgram level. This paper demonstrates the utility of the nanosplitter LC-MS/microdroplet NMR platform when establishing cell culture expression systems. † Electronic supplementary information (ESI) available. See

Biotechnology Progress, Aug 31, 2009

The production of pharmaceutically important terpenoid indole alkaloids (TIAs) from Catharanthus ... more The production of pharmaceutically important terpenoid indole alkaloids (TIAs) from Catharanthus roseus is partly regulated at the transcriptional level. In this study, limitations in TIA biosynthesis from C. roseus hairy root cultures were assessed through gene expression profiling and precursor feeding. The transcript levels of key TIA pathway genes (G10h, Tdc, Str, and Sgd) and metabolite levels associated with the TIA pathway (tryptamine, loganin, secologanin, strictosidine, ajmalicine, serpentine, and tabersonine) were monitored using quantitative RT-PCR and HPLC, respectively. In cultures elicited with methyl jasmonate (250 lM MeJA on day 21), G10h, Tdc, Str, and Sgd expression increased by 9.1, 3.1, 6.7, and 8.3-fold, respectively, after 24 h. Up-regulation of gene expression was followed by a 160, 440, and 420% increase in strictosidine, ajmalicine, and tabersonine levels, respectively, after 5 days. Precursors loganin, tryptamine, or their combination were fed to noninduced and MeJA-induced cultures to complement the above studies. TIA production was not significantly enhanced in either noninduced or MeJA-induced cultures with precursor feeding. In noninduced cells, steps downstream of loganin and tryptamine were limiting (SLS, STR, or SGD) because either loganin or tryptamine accumulated in the cells with precursor feeding. These bottlenecks were partly overcome in MeJA-induced cultures as the expression of Str and Sgd genes and TIA production increased. However, secologanin accumulated in MeJA-induced cultures with precursor feeding, suggesting that STR was likely limiting under MeJA-induced conditions. V

Biotechnology Letters, Oct 1, 2004

Biotechnology Letters, Jul 13, 2007

Biotechnology Progress, Sep 19, 2013

The effects of methyl jasmonate (MJ) dosage on terpenoid indole alkaloid (TIA) biosynthesis in Ca... more The effects of methyl jasmonate (MJ) dosage on terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus are correlated with the relative levels of specific MJ-responsive transcription factors. In this study, the expression of transcription factors (Orca, Zct, Gbf, Myc2, At-hook, and Wrky1), TIA pathway genes (G10h, Tdc, Str, and Sgd), and TIA metabolites (secologanin, strictosidine, and tabersonine) were investigated in C. roseus hairy root cultures elicited with a range of MJ dosages (0-1,000 mM) during mid-exponential growth. The highest production of TIA metabolites occurs at 250 lM MJ, increasing by 150-370% compared with untreated controls. At this MJ dosage, the expression of the transcriptional activators (Orca) is dramatically increased (29-40 fold) while the levels of the transcriptional repressors (Zct) remain low (2-7 fold). Simultaneously, the expression of genes coding for key enzymes involved in TIA biosynthesis increases by 8-15 fold. In contrast, high MJ dosages (1,000 mM) inhibit the production of TIA metabolites. This dosage is correlated with elevated expression levels of Zct (up to 40-fold) relative to Orca (13-19-fold) and minimal induction of the TIA biosynthetic genes (0-6 fold). The significant changes in the expression of Orca and Zct with MJ dosage do not correspond to changes in the expression of the early-response transcription factors (AT-hook, Myc2, and Wrky1) believed to regulate Orca and Zct. In summary, these observations suggest that the dependence of alkaloid production on MJ dosage in C. roseus may be partly mediated through the relative levels of Orca and Zct family transcription factors.

Journal of Biotechnology, May 20, 2008

To develop an optimal bioprocess for secondary metabolite production and explain the bioprocess a... more To develop an optimal bioprocess for secondary metabolite production and explain the bioprocess at the molecular level, we examine the synergistic effects of sequential treatment with methyl jasmonate (MJ), salicylic acid (SA) and yeast extract (YE) on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures. Serial treatment of MJ, SA and YE at 24h intervals enhanced the accumulation of dihydrosanguinarine (2.5 times) and sanguinarine (5.5 times). This sequential treatment using different signal elicitors was more effective than single elicitor or simultaneous treatment of the elicitors; it induced benzophenanthridine alkaloid accumulation to 917.7+/-42.0mg/L. Also, (S)-methylcoclaurine-3'-hydroxylase (CYP80B1) and 3'-hydroxy-(S)-N-methylcoclaurine-4'-O-methyltransferase (4'OMT) expressions among enzymes in sanguinarine biosynthetic pathway explained the synergistic effects by sequential treatment of the elicitors. The sequential treatment strategy using elicitors related to different signal transduction pathways can be used to design better processes to increase accumulation of secondary metabolites in plant cell culture. Analysis of protein expression provides the detailed information about metabolite accumulation through the correlated results.

Chemical engineering education, Jul 1, 2005

Enzyme and microbial technology, Sep 1, 2005

Despite their low solubility in aqueous medium, dissolved gases play important roles in the culti... more Despite their low solubility in aqueous medium, dissolved gases play important roles in the cultivation and successful scale-up of plant cell cultures. In this paper, the effects of O 2 and CO 2 on growth and secondary metabolism were investigated using the production of ajmalicine from Catharanthus roseus cultures. The effects of gas composition were investigated using shear-protected alginate-immobilized cells (diameter < 2 mm) cultured in bubble columns sparged with premixed gases, including nine combinations of O 2 and CO 2. A wider range of concentrations (10-95% O 2 , 0.03-10% CO 2 by mole) was studied to explore potential benefits or drawbacks. Sparge gas composition significantly altered growth and ajmalicine production. Low and high O 2 concentrations (10, 90, 95% O 2) were either inhibitory or toxic to growth and ajmalicine production. The effects of CO 2 depended on O 2 concentration. At lower O 2 concentrations (21% O 2), increasing the CO 2 concentration decreased both growth and specific ajmalicine production. At higher O 2 concentrations (78.4% O 2), increasing the CO 2 concentration decreased growth while specific ajmalicine production was not affected. In these studies, extracellular ajmalicine concentration was maximized with a gas mixture of 50% O 2 + 0.03% CO 2 .

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Algal Research-Biomass Biofuels and Bioproducts, Apr 1, 2016

The authors would like to make the following corrections in the paper: Fig. 4. Sequence homology ... more The authors would like to make the following corrections in the paper: Fig. 4. Sequence homology comparison of CvuDGAT1 with DGAT1 protein sequences from green algae, diatoms, and higher plants. The CvuDGAT1 sequence was aligned against characterized DGAT1 amino acid sequences from green algae, plants, and diatoms with MEGA 6 [54]. Green algae: Chlorella variabilis (CvaDGAT1, EFN50697.1), Chlamydomonas reinhardtii (CrDGAT1) [34], and Coccomyxa subellipsoidea (CsDGAT1, EIE20990.1); Oil-producing or model plants: Arabidopsis thaliana (AtDGAT1; NP179535.1), Glycine max (GmDGAT1, AAS78662.1), Olea europaea (OeDGAT1, AAS01606.1), Ricinus communis (RcDGAT1, XP002514132.1), Vernicia fordii (VfDGAT1, ABC94471.1), Zea mays (ZmDGAT1, ABV91586.1); diatoms: Phaeodactylum tricornutum (PtDGAT1, ADY76581.1) and Thalassiosira pseudonana CCMP1335 (TpDGAT1, XP_002287215.1). Conserved motifs and putative signatures are numbered from I to V (see text for details). We identified the acyl-CoA binding motif (I), the putative active site of DGAT1 (II, black box), the highly conserved fatty acid protein signature (III), containing a putative tyrosine phosphorylation site (•), the DAG/phorbol ester binding motif (IV), and the putative retrieval motif (V). Within the predicted active site (II) the amino acids arginine (R; denoted by asterisk) and Algal Research 15 (2016) 224

Plant cell reports, May 14, 2024

Key message A GLK homologue was identified and functionally characterized in Catharanthus roseus.... more Key message A GLK homologue was identified and functionally characterized in Catharanthus roseus. Silencing CrGLK with VIGS or the chloroplast retrograde signaling inducer lincomycin increased terpenoid indole alkaloid biosynthesis. Abstract Catharanthus roseus is the sole source of the chemotherapeutic terpenoid indole alkaloids (TIAs) vinblastine and vincristine. TIA pathway genes, particularly genes in the vindoline pathway, are expressed at higher levels in immature versus mature leaves, but the molecular mechanisms responsible for this developmental regulation are unknown. We investigated the role of GOLDEN2-LIKE (GLK) transcription factors in contributing to this ontogenetic regulation since GLKs are active in seedlings upon light exposure and in the leaf's early development, but their activity is repressed as leaves age and senesce. We identified a GLK homologue in C. roseus and functionally characterized its role in regulating TIA biosynthesis, with a focus on the vindoline pathway, by transiently reducing its expression through two separate methods: virus-induced gene silencing (VIGS) and application of chloroplast retrograde signaling inducers, norflurazon and lincomycin. Reducing CrGLK levels with each method reduced chlorophyll accumulation and the expression of the light harvesting complex subunit (LHCB2.2), confirming its functional homology with GLKs in other plant species. In contrast, reducing CrGLK via VIGS or lincomycin increased TIA accumulation and TIA pathway gene expression, suggesting that CrGLK may repress TIA biosynthesis. However, norflurazon had no effect on TIA gene expression, indicating that reducing CrGLK alone is not sufficient to induce TIA biosynthesis. Future work is needed to clarify the specific molecular mechanisms leading to increased TIA biosynthesis with CrGLK silencing. This is the first identification and characterization of GLK in C. roseus and the first investigation of how chloroplast retrograde signaling might regulate TIA biosynthesis.

Journal of Biological Chemistry, 2023

한국생물공학회 학술대회, Apr 1, 2007

bioRxiv (Cold Spring Harbor Laboratory), Apr 4, 2020

The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with... more The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by stably transforming a Cas9 expression construct into the plant genome. The efficiency with which mutations are obtained in genes of interest can vary considerably depending on specific features of the constructs, including the source and nature of the promoters and terminators used for expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency with which mutations could be obtained in target genes in Arabidopsis thaliana with the Cas9 nuclease, we have investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLS) and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on Cas9 activity and that two NLSs work better than one. However, the most important impact on the efficiency of the constructs was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies of the constructs; none of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of primary transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

Transgenic hairy root cultures of the medicinal plant, Catharanthus roseus, were established with... more Transgenic hairy root cultures of the medicinal plant, Catharanthus roseus, were established with green fluorescent protein (GFP) expression under the control of an estrogen-inducible system. The Agrobacterium-mediated transformation process was optimized with an overall efficiency of 33%. Furthermore, GFP expression was tightly regulated and highly inducible using the estrogen-inducible system as demonstrated in hairy roots. This paper describes the optimized transformation method using the estrogen-inducible system for engineering the production of pharmaceutically important alkaloid compounds from C. roseus hairy root cultures.

Plant direct, Dec 1, 2019

Due to their sessile existence, plants have developed rapid and effective responses to environmen... more Due to their sessile existence, plants have developed rapid and effective responses to environmental stresses, herbivory, and pathogens. When stressed, plants can shift primary metabolic resources to produce specialized metabolites that act as defense compounds. For instance, the Madagascar periwinkle, Catharanthus roseus, produces numerous monoterpenoid indole alkaloids (MIA) in defense against pathogens and herbivory (Luijendijk, van der Meijden, & Verpoorte, 1996; Dugé de Bernonville et al., 2017). Many of these MIAs are pharmaceutically valuable, including the antihypertensive ajmalicine, the sedative serpentine, and most notably the anticancer compounds vincristine and vinblastine (Van der Heijden, Jacobs,

Biochemical Engineering Journal, Nov 1, 2004

The potential of strategies for increasing secondary metabolite accumulation from plant cell cult... more The potential of strategies for increasing secondary metabolite accumulation from plant cell cultures may still be partially limited by unfavorable conditions for biosynthesis (i.e. feedback inhibition, unfavorable equilibrium state) or product metabolism. In this paper, the rate of product removal was altered and used as a tool for probing the impact of these limiting conditions on product accumulation, using the production of ajmalicine and serpentine from Catharanthus roseus suspensions as a model system. The method for altering the in situ product removal rate was simple; Amberlite ® XAD-7HP resin was enclosed in either a porous or less porous material, and experiments confirmed that the product removal rate was higher with resin enclosed in the porous Miracloth versus the less porous nylon. When resin was added to suspensions, higher ajmalicine production and recovery were associated with Miracloth-enclosed resin which had higher product removal rates. This simple experiment illustrated that further improvements in production and recovery were possible, indicating that conditions unfavorable for biosynthesis and/or product metabolism existed in cultures with nylon-enclosed resin. These results also suggest the importance of optimizing the product removal rate for improving the overall production and recovery of secondary metabolites from plant cell cultures.

Plant Cell Reports, Aug 11, 2005

Cytosolic Ca 2+ and jasmonate mediate signals that induce defense responses in plants. In this st... more Cytosolic Ca 2+ and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca 2+ and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl 2 (3, 23, and 43 mM) and MJ (0, 10, and 100 µM) on day 6 of growth. Increased Ca 2+ influx through the addition of extracellular CaCl 2 suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 µM). In the presence of 3 mM CaCl 2 in the medium, the addition of Ca 2+ chelator EGTA (1, 2.5, and 5 mM) or Ca 2+ channel blocker verapamil (1, 10, and 50 µM) to MJ-induced (100 µM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca 2+ inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca 2+ concentration and a low extracellular Ca 2+ concentration (3 mM) enhanced MJ-induced ajmalicine production.

Plant Cell Reports, Jan 24, 2006

Jasmonates enhance the expression of various genes involved in terpenoid indole alkaloid (TIA) bi... more Jasmonates enhance the expression of various genes involved in terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus. We applied precursor feeding to our C. roseus suspensions to determine how methyl jasmonate (MJ) alters the precursor availability for TIA biosynthesis. C. roseus suspensions were induced with MJ (100 µM) on day 6 and fed loganin (0.30 mM), tryptamine (0.15 mM), loganin plus tryptamine, or geraniol (0.1-1.0 mM) on day 7. While MJ increased ajmalicine production by 3-fold, induced cultures were still limited by terpenoid precursors. However, both induced and non-induced cultures became tryptamine-limited with excess loganin. Geraniol feeding also increased ajmalicine production in non-induced cultures. But MJ appeared to increase geraniol availability in induced cultures, due presumably to the increased expression of Dxs with MJ addition.

PLOS ONE, Oct 17, 2012

The abundant flavonoid aglycone, naringenin, which is responsible for the bitter taste in grapefr... more The abundant flavonoid aglycone, naringenin, which is responsible for the bitter taste in grapefruits, has been shown to possess hypolipidemic and anti-inflammatory effects both in vitro and in vivo. Recently, our group demonstrated that naringenin inhibits hepatitis C virus (HCV) production, while others demonstrated its potential in the treatment of hyperlipidemia and diabetes. However, naringenin suffers from low oral bioavailability critically limiting its clinical potential. In this study, we demonstrate that the solubility of naringenin is enhanced by complexation with b-cyclodextrin, an FDA approved excipient. Hydroxypropoyl-b-cyclodextrin (HPbCD), specifically, increased the solubility of naringenin by over 400fold, and its transport across a Caco-2 model of the gut epithelium by 11-fold. Complexation of naringenin with HPbCD increased its plasma concentrations when fed to rats, with AUC values increasing by 7.4-fold and C max increasing 14.6-fold. Moreover, when the complex was administered just prior to a meal it decreased VLDL levels by 42% and increased the rate of glucose clearance by 64% compared to naringenin alone. These effects correlated with increased expression of the PPAR co-activator, PGC1a in both liver and skeletal muscle. Histology and blood chemistry analysis indicated this route of administration was not associated with damage to the intestine, kidney, or liver. These results suggest that the complexation of naringenin with HPbCD is a viable option for the oral delivery of naringenin as a therapeutic entity with applications in the treatment of dyslipidemia, diabetes, and HCV infection.

Analytical Methods, 2012

Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for ob... more Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for obtaining natural products of medical relevance. Cultures can be optimized for high yields of desired metabolites using rapid profiling assays such as HPLC. We describe an approach to establishing a rapid assay for profiling cell culture expression systems using a novel microscale LC-UV-MS-NMR platform, designed to acquire both MS and NMR each at their optimal sensitivity, by using nanosplitter MS from 4 mm analytical HPLC columns, and offline microdroplet NMR. The approach is demonstrated in the analysis of elicited Eschscholzia californica cell cultures induced with purified yeast extract to produce benzophenanthridine alkaloids. Preliminary HPLC-UV provides an overview of the changes in the production of alkaloids with time after elicitation. At the time point corresponding to the production of the most alkaloids, the integrated LC-MS-microcoil NMR platform is used for structural identification of extracted alkaloids. Eight benzophenanthridine alkaloids were identified at the sub-microgram level. This paper demonstrates the utility of the nanosplitter LC-MS/microdroplet NMR platform when establishing cell culture expression systems. † Electronic supplementary information (ESI) available. See

Biotechnology Progress, Aug 31, 2009

The production of pharmaceutically important terpenoid indole alkaloids (TIAs) from Catharanthus ... more The production of pharmaceutically important terpenoid indole alkaloids (TIAs) from Catharanthus roseus is partly regulated at the transcriptional level. In this study, limitations in TIA biosynthesis from C. roseus hairy root cultures were assessed through gene expression profiling and precursor feeding. The transcript levels of key TIA pathway genes (G10h, Tdc, Str, and Sgd) and metabolite levels associated with the TIA pathway (tryptamine, loganin, secologanin, strictosidine, ajmalicine, serpentine, and tabersonine) were monitored using quantitative RT-PCR and HPLC, respectively. In cultures elicited with methyl jasmonate (250 lM MeJA on day 21), G10h, Tdc, Str, and Sgd expression increased by 9.1, 3.1, 6.7, and 8.3-fold, respectively, after 24 h. Up-regulation of gene expression was followed by a 160, 440, and 420% increase in strictosidine, ajmalicine, and tabersonine levels, respectively, after 5 days. Precursors loganin, tryptamine, or their combination were fed to noninduced and MeJA-induced cultures to complement the above studies. TIA production was not significantly enhanced in either noninduced or MeJA-induced cultures with precursor feeding. In noninduced cells, steps downstream of loganin and tryptamine were limiting (SLS, STR, or SGD) because either loganin or tryptamine accumulated in the cells with precursor feeding. These bottlenecks were partly overcome in MeJA-induced cultures as the expression of Str and Sgd genes and TIA production increased. However, secologanin accumulated in MeJA-induced cultures with precursor feeding, suggesting that STR was likely limiting under MeJA-induced conditions. V

Biotechnology Letters, Oct 1, 2004

Biotechnology Letters, Jul 13, 2007

Biotechnology Progress, Sep 19, 2013

The effects of methyl jasmonate (MJ) dosage on terpenoid indole alkaloid (TIA) biosynthesis in Ca... more The effects of methyl jasmonate (MJ) dosage on terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus are correlated with the relative levels of specific MJ-responsive transcription factors. In this study, the expression of transcription factors (Orca, Zct, Gbf, Myc2, At-hook, and Wrky1), TIA pathway genes (G10h, Tdc, Str, and Sgd), and TIA metabolites (secologanin, strictosidine, and tabersonine) were investigated in C. roseus hairy root cultures elicited with a range of MJ dosages (0-1,000 mM) during mid-exponential growth. The highest production of TIA metabolites occurs at 250 lM MJ, increasing by 150-370% compared with untreated controls. At this MJ dosage, the expression of the transcriptional activators (Orca) is dramatically increased (29-40 fold) while the levels of the transcriptional repressors (Zct) remain low (2-7 fold). Simultaneously, the expression of genes coding for key enzymes involved in TIA biosynthesis increases by 8-15 fold. In contrast, high MJ dosages (1,000 mM) inhibit the production of TIA metabolites. This dosage is correlated with elevated expression levels of Zct (up to 40-fold) relative to Orca (13-19-fold) and minimal induction of the TIA biosynthetic genes (0-6 fold). The significant changes in the expression of Orca and Zct with MJ dosage do not correspond to changes in the expression of the early-response transcription factors (AT-hook, Myc2, and Wrky1) believed to regulate Orca and Zct. In summary, these observations suggest that the dependence of alkaloid production on MJ dosage in C. roseus may be partly mediated through the relative levels of Orca and Zct family transcription factors.

Journal of Biotechnology, May 20, 2008

To develop an optimal bioprocess for secondary metabolite production and explain the bioprocess a... more To develop an optimal bioprocess for secondary metabolite production and explain the bioprocess at the molecular level, we examine the synergistic effects of sequential treatment with methyl jasmonate (MJ), salicylic acid (SA) and yeast extract (YE) on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures. Serial treatment of MJ, SA and YE at 24h intervals enhanced the accumulation of dihydrosanguinarine (2.5 times) and sanguinarine (5.5 times). This sequential treatment using different signal elicitors was more effective than single elicitor or simultaneous treatment of the elicitors; it induced benzophenanthridine alkaloid accumulation to 917.7+/-42.0mg/L. Also, (S)-methylcoclaurine-3&amp;amp;amp;amp;amp;amp;amp;amp;#39;-hydroxylase (CYP80B1) and 3&amp;amp;amp;amp;amp;amp;amp;amp;#39;-hydroxy-(S)-N-methylcoclaurine-4&amp;amp;amp;amp;amp;amp;amp;amp;#39;-O-methyltransferase (4&amp;amp;amp;amp;amp;amp;amp;amp;#39;OMT) expressions among enzymes in sanguinarine biosynthetic pathway explained the synergistic effects by sequential treatment of the elicitors. The sequential treatment strategy using elicitors related to different signal transduction pathways can be used to design better processes to increase accumulation of secondary metabolites in plant cell culture. Analysis of protein expression provides the detailed information about metabolite accumulation through the correlated results.

Chemical engineering education, Jul 1, 2005

Enzyme and microbial technology, Sep 1, 2005

Despite their low solubility in aqueous medium, dissolved gases play important roles in the culti... more Despite their low solubility in aqueous medium, dissolved gases play important roles in the cultivation and successful scale-up of plant cell cultures. In this paper, the effects of O 2 and CO 2 on growth and secondary metabolism were investigated using the production of ajmalicine from Catharanthus roseus cultures. The effects of gas composition were investigated using shear-protected alginate-immobilized cells (diameter < 2 mm) cultured in bubble columns sparged with premixed gases, including nine combinations of O 2 and CO 2. A wider range of concentrations (10-95% O 2 , 0.03-10% CO 2 by mole) was studied to explore potential benefits or drawbacks. Sparge gas composition significantly altered growth and ajmalicine production. Low and high O 2 concentrations (10, 90, 95% O 2) were either inhibitory or toxic to growth and ajmalicine production. The effects of CO 2 depended on O 2 concentration. At lower O 2 concentrations (21% O 2), increasing the CO 2 concentration decreased both growth and specific ajmalicine production. At higher O 2 concentrations (78.4% O 2), increasing the CO 2 concentration decreased growth while specific ajmalicine production was not affected. In these studies, extracellular ajmalicine concentration was maximized with a gas mixture of 50% O 2 + 0.03% CO 2 .

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Algal Research-Biomass Biofuels and Bioproducts, Apr 1, 2016

The authors would like to make the following corrections in the paper: Fig. 4. Sequence homology ... more The authors would like to make the following corrections in the paper: Fig. 4. Sequence homology comparison of CvuDGAT1 with DGAT1 protein sequences from green algae, diatoms, and higher plants. The CvuDGAT1 sequence was aligned against characterized DGAT1 amino acid sequences from green algae, plants, and diatoms with MEGA 6 [54]. Green algae: Chlorella variabilis (CvaDGAT1, EFN50697.1), Chlamydomonas reinhardtii (CrDGAT1) [34], and Coccomyxa subellipsoidea (CsDGAT1, EIE20990.1); Oil-producing or model plants: Arabidopsis thaliana (AtDGAT1; NP179535.1), Glycine max (GmDGAT1, AAS78662.1), Olea europaea (OeDGAT1, AAS01606.1), Ricinus communis (RcDGAT1, XP002514132.1), Vernicia fordii (VfDGAT1, ABC94471.1), Zea mays (ZmDGAT1, ABV91586.1); diatoms: Phaeodactylum tricornutum (PtDGAT1, ADY76581.1) and Thalassiosira pseudonana CCMP1335 (TpDGAT1, XP_002287215.1). Conserved motifs and putative signatures are numbered from I to V (see text for details). We identified the acyl-CoA binding motif (I), the putative active site of DGAT1 (II, black box), the highly conserved fatty acid protein signature (III), containing a putative tyrosine phosphorylation site (•), the DAG/phorbol ester binding motif (IV), and the putative retrieval motif (V). Within the predicted active site (II) the amino acids arginine (R; denoted by asterisk) and Algal Research 15 (2016) 224