About

Ralph Dewey is the Philip Morris Professor and Associate Department Head in the Department of Crop and Soil Sciences at NC State University. His research focuses on the application of molecular biology techniques toward crop improvement. Current projects include reducing carcinogenic compounds in tobacco products, developing ultra-low nicotine tobaccos to assist smokers in quitting, and utilizing genome editing tools to modify the mitochondrial genome in plants. These efforts aim to develop novel hybrid seed production systems and create seedless fruit phenotypes. Dewey's work contributes to advancements in crop biotechnology with a particular emphasis on tobacco and related plant systems.

Research topics

• Biology
• Botany
• Computer Science
• Biochemistry
• Horticulture
• Pathology
• Computational biology
• Food science
• Cell biology
• Medicine
• Organic chemistry
• Chemistry
• Genetics
• Bioinformatics

Selected publications

• Involvement of NtInvVR1 in the increase of reducing sugars during the curing of virginia tobacco

  Scientific Reports · 2026-04-22

  articleOpen access

  Flue-cured leaves of the Virginia tobacco (Nicotiana tabacum L.) market type are characterized by their high concentrations of sugars, namely sucrose (Suc), glucose (Glc), and fructose (Fru). During leaf curing, the so-called yellowing phase is associated with a dark-senescence process generating Suc and the main reducing sugars (RS), Glc and Fru. Invertase enzymes are reported to convert Suc into Glc and Fru throughout leaf senescence, but also fruit maturation. In this study, we actualize the complete family of genes encoding invertases (Invs) in N. tabacum. Two genes coding for vacuolar Invs, NtInvVR1_S and NtInvVR1_T, originating from the tobacco ancestors N. sylvestris and N. tomentosiformis, were identified to be the highest expressed Inv genes during the first 48 h of flue-curing (yellowing phase). Silencing both genes in the Virginia tobacco significantly reduced the accumulation of Glc and Fru by more than 90%, while Suc levels increased approximately 140% in a greenhouse environment. These data suggested that NtInvVR1_S and NtInvVR1_T play a key role in the hydrolysis of Suc, contributing to the accumulation of RS in cured leaves. NtInvVR1-RNAi plants were also cultivated in the field, showing a significant reduction in Glc and Fru of about 40% at the expense of Suc.

  PublisherOA PDFDOI

• Optimization of Adventitious Shoot Regeneration Protocols for Six Vaccinium corymbosum Cultivars

  HortScience · 2025-06-13 · 1 citations

  articleOpen access

  Limitations in tissue culture and regeneration techniques present significant barriers to the development of improved blueberry ( Vaccinium spp.) cultivars, restricting the adoption of tissue culture in commercial production. This challenge emphasizes the urgent need for a standardized regeneration protocol. Addressing this, we developed enhanced in vitro regeneration protocols for six highbush blueberry ( Vaccinium corymbosum L.) cultivars—Emerald, NC5288, Rowan (NC3104), Legacy, Pinnacle, and Jewel—each with varying levels of tissue culture recalcitrance. We evaluated the effects of auxins, specifically α-Naphthaleneacetic Acid, cytokinins [including trans-Zeatin (ZT), 1-(2-Chloro-4-pyridyl)-3-phenylurea 98.0+%, TCI America™ (CPPU), and 6-γ-γ-[Dimethylallylamino]-purine (2iP)], and Thidiazuron (TDZ), which exhibits both auxin- and cytokinin-like activities, in various plant species cultured in vitro, on callus induction and regeneration from leaf explants. Media supplemented with ZT + 2iP (PZ9, PZ10) consistently outperformed all other treatments, enhancing regeneration significantly across all cultivars with high genotype-independent reproducibility, particularly in treatment PZ10. In contrast, TDZ-based treatments yielded highly variable results and were ineffective in recalcitrant cultivars such as Jewel and Pinnacle. ZT + CPPU treatments performed moderately across all metrics and may offer utility for cultivars with intermediate responsiveness. These findings provide critical insights into refining tissue culture techniques, offering a foundation for improving blueberry cultivar development and efficient genome editing. This work represents the first published report of in vitro regeneration for the highbush blueberry cultivars Rowan (NC3104), NC5288, and Pinnacle, expanding the scope of possibilities for advancing blueberry crop improvement.

  PublisherOA PDFDOI

• BBL enzymes exhibit enantiospecific preferences in the biosynthesis of pyridine alkaloids in Nicotiana tabacum L.

  Phytochemistry · 2024-12-16 · 4 citations

  article
  PublisherDOI

• Nitrate assimilation pathway is impacted in young tobacco plants overexpressing a constitutively active nitrate reductase or displaying a defective CLCNt2

  BMC Plant Biology · 2024-11-27 · 1 citations

  articleOpen access

  BACKGROUND: We have previously shown that the expression of a constitutively active nitrate reductase variant and the suppression of CLCNt2 gene function (belonging to the chloride channel (CLC) gene family) in field-grown tobacco reduces tobacco-specific nitrosamines (TSNA) accumulation in cured leaves and cigarette smoke. In both cases, TSNA reductions resulted from a strong diminution of free nitrate in the leaf, as nitrate is a precursor of the TSNA-producing nitrosating agents formed during tobacco curing and smoking. These nitrosating agents modify tobacco alkaloids to produce TSNAs, the most problematic of which are NNN (N-nitrosonornicotine) and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). The expression of a deregulated nitrate reductase enzyme (DNR) that is no longer responsive to light regulation is believed to diminish free nitrate pools by immediately channeling incoming nitrate into the nitrate assimilation pathway. The reduction in nitrate observed when the two tobacco gene copies encoding the vacuolar nitrate transporter CLCNt2 were down-regulated by RNAi-mediated suppression or knocked out using the CRISPR-Cas technology was mechanistically distinct; likely attributable to the inability of the tobacco cell to efficiently sequester nitrate into the vacuole where this metabolite is protected from further assimilation. In this study, we used transcriptomic and metabolomic analyses to compare the nitrate assimilation response in tobacco plants either expressing DNR or lacking CLCNt2 function. RESULTS: When grown in a controlled environment, both DNR and CLCNt2-KO (CLCKO) plants exhibited (1) reduced nitrate content in the leaf; (2) increased N-assimilation into the amino acids Gln and Asn; and (3) a similar pattern of differential regulation of several genes controlling stress responses, including water stress, and cell wall metabolism in comparison to wild-type plants. Differences in gene regulation were also observed between DNR and CLCKO plants, including genes encoding nitrite reductase and asparagine synthetase. CONCLUSIONS: Our data suggest that even though both DNR and CLCKO plants display common characteristics with respect to nitrate assimilation, cellular responses, water stress, and cell wall remodeling, notable differences in gene regulatory patterns between the two low nitrate plants are also observed. These findings open new avenues in using plants fixing more nitrogen into amino acids for plant improvement or nutrition perspectives.

  PublisherOA PDFDOI

• Engineering Sclareol Production on the Leaf Surface of <i>Nicotiana tabacum</i>

  Journal of Agricultural and Food Chemistry · 2024-06-06 · 10 citations

  article

  Sclareol, a diterpene alcohol, is the most common starting material for the synthesis of ambrox, which serves as a sustainable substitute for ambergris, a valuable fragrance secreted by sperm whales. Sclareol has also been proposed to possess antibacterial, antifungal, and anticancer activities. However, in nature, sclareol is only produced by a few plant species, including Cistus creticus, Cleome spinosa, Nicotiana glutinosa, and Salvia sclarea, which limits its commercial application. In this study, we cloned the two genes responsible for sclareol biosynthesis in S. sclarea, labda-13-en-8-ol diphosphate synthase (LPPS) and sclareol synthase (SS), and overexpressed them in tobacco (Nicotiana tabacum L.). The best transgenic tobacco lines accumulated 4.1 μg/cm2 of sclareol, which is comparable to the sclareol production of N. glutinosa, a natural sclareol producer. Thus, sclareol synthesis in tobacco represents a potential alternative means for the production of this high-value compound.

  PublisherDOI

• Cytoplasmic Male Sterility and Abortive Seed Traits Generated through Mitochondrial Genome Editing Coupled with Allotopic Expression of <i>atp1</i> in Tobacco

  bioRxiv (Cold Spring Harbor Laboratory) · 2023-06-12 · 1 citations

  preprintOpen access1st authorCorresponding

  ABSTRACT Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δ atp1 ) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δ atp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δ atp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δ atp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1 -targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

  PublisherOA PDFDOI

• Cytoplasmic male sterility and abortive seed traits generated through mitochondrial genome editing coupled with allotopic expression of atp1 in tobacco

  Frontiers in Plant Science · 2023-09-15 · 6 citations

  articleOpen access1st authorCorresponding

  Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δ atp1 ) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δ atp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δ atp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δ atp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1 -targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

  PublisherOA PDFDOI

• Knockout of a key gene of the nicotine biosynthetic pathway severely affects tobacco growth under field, but not greenhouse conditions

  BMC Research Notes · 2022-09-06 · 8 citations

  articleOpen accessSenior author

  OBJECTIVE: There is great interest in developing tobacco plants containing minimal amounts of the addictive compound nicotine. Quinolate phosphoribosyltransferase (QPT) is an important enzyme both for primary (NAD production) and secondary (pyridine alkaloid biosynthesis) metabolism in tobacco. The duplication of an ancestral QPT gene in Nicotiana species has resulted in two closely related QPT gene paralogs: QPT1 which is expressed at modest levels throughout the plant, and QPT2 which is coordinately regulated with genes dedicated to alkaloid biosynthesis. This study evaluated the utility of knocking out QPT2 function as a means for producing low alkaloid tobacco plants. RESULTS: CRISPR/Cas9 vectors were developed to specifically mutate the tobacco QPT2 genes associated with alkaloid production. Greenhouse-grown qpt2 plants accumulated dramatically less nicotine than controls, while displaying only modest growth differences. In contrast, when qpt2 lines were transplanted to a field environment, plant growth and development was severely inhibited. Two conclusions can be inferred from this work: (1) QPT1 gene function alone appears to be inadequate for meeting the QPT demands of the plant for primary metabolism when grown in a field environment; and (2) the complete knockout of QPT2 function is not a viable strategy for producing agronomically useful, low nicotine tobaccos.

  PublisherOA PDFDOI

• CLCNt2 Mediates Nitrate Content in Tobacco Leaf, Impacting the Production of Tobacco-Specific Nitrosamines in Cured Leaves

  Frontiers in Plant Science · 2022 · 13 citations

  • Chemistry
  • Food science
  • Horticulture

  copies.

  PublisherOA PDFDOI

• Constitutive activation of nitrate reductase in tobacco alters flowering time and plant biomass

  Scientific Reports · 2021-02-19 · 13 citations

  articleOpen accessSenior author

  Abstract Pyridine alkaloids produced in tobacco can react with nitrosating agents such as nitrite to form tobacco-specific nitrosamines (TSNA), which are among the most notable toxicants present in tobacco smoke. The market type known as burley tobacco is particularly susceptible to TSNA formation because its corresponding cultivars exhibit a nitrogen-use-deficiency phenotype which results in high accumulation of nitrate, which, in turn, is converted to nitrite by leaf surface microbes. We have previously shown that expression of a constitutively activated nitrate reductase (NR) enzyme dramatically decreases leaf nitrate levels in burley tobacco, resulting in substantial TSNA reductions without altering the alkaloid profile. Here, we show that plants expressing a constitutively active NR construct, designated 35S:S523D-NR, display an early-flowering phenotype that is also associated with a substantial reduction in plant biomass. We hypothesized that crossing 35S:S523D-NR tobaccos with burley cultivars that flower later than normal would help mitigate the undesirable early-flowering/reduced-biomass traits while maintaining the desirable low-nitrate/TSNA phenotype. To test this, 35S:S523D-NR plants were crossed with two late-flowering cultivars, NC 775 and NC 645WZ. In both cases, the plant biomass at harvest was restored to levels similar to those in the original cultivar used for transformation while the low-nitrate/TSNA trait was maintained. Interestingly, the mechanism by which yield was restored differed markedly between the two crosses. Biomass restoration in F 1 hybrids using NC 645WZ as a parent was associated with delayed flowering, as originally hypothesized. Unexpectedly, however, crosses with NC 775 displayed enhanced biomass despite maintaining the early-flowering trait of the 35S:S523D-NR parent.

  PublisherOA PDFDOI

Frequent coauthors

• Joseph W. Burton

  Royal London Hospital

  13 shared

• Ramsey S. Lewis

  North Carolina State University

  11 shared

• Balazs Siminszky

  University of Kentucky

  10 shared

• C. S. Levings

  10 shared

• Richard F. Wilson

  9 shared

• D. H. Timothy

  University of Utah

  8 shared

• William P. Novitzky

  8 shared

• Andrea J. Cardinal

  Syngenta (United States)

  8 shared

Labs

• Dewey LabPI

Education

• PhD, Crop Science

  North Carolina State University

  1987

• MS, Crop Science

  North Carolina State University

  1985

• BS, Biology

  Utah State University

  1982