Engineering Sclareol Production on the Leaf Surface of Nicotiana tabacum.

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.

A comparison of three automated root-knot nematode egg counting approaches using machine learning, image analysis, and a hybrid model.

Meloidogyne spp. (root-knot nematodes, RKN) are a major threat to a wide range of agricultural crops worldwide. Breeding crops for RKN resistance is an effective management strategy, yet assaying large numbers of breeding lines requires laborious bioassays that are time-consuming and require experienced researchers. In these bioassays, quantifying nematode eggs through manual counting is considered the current standard for quantifying establishing resistance in plant genotypes. Counting RKN eggs is highly laborious, and even experienced researchers are subject to fatigue or misclassification, leading to potential errors in phenotyping. Here, we present three automated egg counting models that rely on machine learning and image analysis to quantify RKN eggs extracted from tobacco and sweetpotato plants. The first method relied on convolutional neural networks trained using annotated images to identify eggs (M. enterolobii R2 = 0.899, M. incognita R2 = 0.927, M. javanica R2 = 0.886), while a second contour-based approach used image analysis to identify eggs from their morphological characteristics and did not rely on neural networks (M. enterolobii R2 = 0.977, M. incognita R2 = 0.990, M. javanica R2 = 0.924). A third hybrid model combined these approaches and was able to detect and count eggs nearly as well as human raters (M. enterolobii R2 = 0.985, M. incognita R2 = 0.992, M. javanica R2 = 0.983). These automated counting protocols have the potential to provide significant time and resource savings annually for breeders and nematologists, and may be broadly applicable to other nematode species.

Modified physiology of burley tobacco plants genetically engineered to express Yb1, a functional EGY enzyme.

MAIN CONCLUSION: Transgenic overexpression of a NtEGY2 gene restores normal green color of burley tobacco plants, but does not increase nitrogen utilization efficiency beyond that exhibited by wild-type individuals. Nitrogen physiology is important in tobacco because of its role in generation of leaf yield and accumulation of nitrogen-containing alkaloids that can react with nitrosating agents in the formation of carcinogenic tobacco-specific nitrosamines. Cultivars of the burley tobacco market class are homozygous for deleterious mutant alleles at the duplicate Yb1 and Yb2 loci which have previously been associated with decreased nitrogen use and utilization efficiency; increased leaf nitrate, total nitrogen, and alkaloid levels; and reduced yields. How mutant alleles at these two loci affect these traits is not well understood. Recent characterization of the Yb1 and Yb2 genes (homologs of Arabidopsis EGY1 gene) enabled overexpression of the wild-type Yb1 allele in yb1yb1yb2yb2 plants to determine if observed unfavorable effects were due to linkage or pleiotropy, and to determine if overexpression could lead to beneficial modifications in any of these traits in transgenic plants relative to naturally-occurring wild-type genotypes. Yb1 overexpression was found to confer an agronomic benefit to yb1yb1yb2yb2 genotypes but no advantage to wild-type genotypes. RNA-Seq was used to carry out a comparative transcriptome analysis of genetically engineered and wild-type nearly isogenic lines (NILs) to gain insight on metabolic pathways affecting carbon and nitrogen metabolism that might be altered as the result of genetic variability at the Yb1 and Yb2 loci. Results indicate that complex changes in the transcriptome of tobacco can be manifested by altered expression of Yb1.

Combined reduced expression of two gene families lowers nicotine content to ultra-low levels in cultivated tobacco.

KEY MESSAGE: Reduced expression of two gene families results in ultra-low nicotine accumulation in Nicotiana tabacum. The potential for mandated lowering of tobacco cigarette filler nicotine levels to below 0.4 mg g-1 is currently being discussed by regulatory and public health organizations. Commercial tobacco cultivars that would routinely meet this proposed standard do not currently exist. Inactivation or silencing of gene families corresponding to single enzymatic steps in the nicotine biosynthetic pathways have not resulted in tobacco genotypes that would meet this standard under conventional agronomic management. Here, we produced and evaluated under field conditions tobacco genotypes expressing an RNAi construct designed to reduce expression of the Methyl Putrescine Oxidase (MPO) gene family associated with nicotine biosynthesis. In a standard flue-cured genetic background, cured leaf nicotine levels were reduced to only 1.08 to 1.65 mg g-1. When MPO RNAi was combined with reduced Berberine Bridge Like (BBL) activity conferred by induced mutations, genotypes producing cured leaf nicotine levels slightly lower than 0.4 mg g-1 were generated. Past research has suggested that MPO activity may contribute to the biosynthesis of nornicotine in a route that does not involve nicotine. However, nornicotine was not reduced to zero in MPO-silenced plants that were also homozygous for induced mutations in known Nicotine Demethylase genes that are responsible for the vast majority of nornicotine accumulation.

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

Nitrate accumulation in tobacco (Nicotiana tabacum L.) leaf, particularly in the burley (BU) type, is a reservoir for the generation of nitrosating agents responsible for the formation of tobacco-specific nitrosamines (TSNAs). TSNAs are mainly produced via the nitrosation of alkaloids occurring during the curing of tobacco leaves. Additional formation of TSNAs may also occur during tobacco storage, leaf processing and in some circumstances via pyrosynthesis during combustion. Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are found in the tobacco products and have been documented to be animal carcinogens. A previous study showed that decreasing the accumulation of nitrate in tobacco leaf via the overexpression of a deregulated form of nitrate reductase is efficient to reduce the production of TSNAs. We pursue in finding another molecular genetic target to lower nitrate in BU tobacco. Suppressing expression or knocking-out CLCNt2 has a direct impact on leaf nitrate and TSNA reduction in cured leaves without altering biomass. This study provides now a straight path toward the development of new commercial tobacco varieties with reduced TSNA levels by breeding of variants deficient in active CLCNt2 copies.

Editing of A622 genes results in ultra-low nicotine whole tobacco plants at the expense of dramatically reduced growth and development.

Due to potential regulations that could affect nicotine levels in some tobacco products, there is interest in using genetic modification to reduce levels of this pyridine alkaloid in tobacco leaves. Enzymes coded by A622 genes have previously been indicated to be involved in one of the latter steps of tobacco alkaloid biosynthesis. Whole tobacco plants with reduced A622 activity have never been evaluated, however. We utilized CRISPR/Cas9-based editing to introduce deleterious mutations into the two A622 genes present in the Nicotiana tabacum genome. Double homozygous A622 mutant genotypes established in four recipient genotypes varying for the presence/absence of mutations in other alkaloid biosynthetic genes exhibited severely reduced nicotine accumulation in field and greenhouse experiments. A622 knockout lines exhibited lower nicotine levels than previously created genotypes with deleterious mutations in BBL genes also associated with one of the latter steps in tobacco alkaloid biosynthesis. Reduced A622 activity resulted in plants with drastically reduced growth and development, however. A622 mutant lines were later flowering and produced green leaf yields that were 60.6% lower, on average, than those for non-A622-mutated control lines. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01293-w.

Analyses of diverse low alkaloid tobacco germplasm identify naturally occurring nucleotide variability contributing to reduced leaf nicotine accumulation.

Recent suggestions for mandated lowering of nicotine content in cigarettes have prompted tobacco breeders to search for N. tabacum germplasm with allelic variability contributing to low alkaloid accumulation. In this research, we phenotyped a series of 81 selected diverse tobacco introductions (TIs) to identify a sub-group with authentic low alkaloid phenotypes. We also genotyped these materials for sequences associated with the Nic1 and Nic2 loci previously reported to influence tobacco alkaloid biosynthesis. Only five low alkaloid TIs possessed previously described deletions of Ethylene Response Factor (ERF) genes at the Nic2 locus that contribute to lower alkaloid accumulation. Eleven TIs possessed an apparent deletion of ERF199, a gene recently reported to underlie the effect at the Nic1 locus. Quantitative trait locus (QTL) mapping was performed using populations derived from three selected low alkaloid TIs to possibly identify new genomic regions affecting alkaloid accumulation. A major QTL was identified on linkage group 7 in all three populations that aligned with the Nic1 locus. A newly discovered 5 bp deletion in the gene MYC2a on linkage group 5 was found to likely partially underlie the ultra-low alkaloid phenotype of TI 313. This new information is useful for tobacco breeders attempting to assemble novel genetic combinations with the potential for meeting future levels of tolerance for nicotine concentration in cigarette tobacco. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01274-5.

Homoeologous chromosome exchange explains the creation of a QTL affecting soil-borne pathogen resistance in tobacco.

Crop plant partial resistance to plant pathogens controlled by quantitative trait loci (QTL) is desirable in cultivar development programmes because of its increased durability. Mechanisms underlying such resistance are difficult to study. We performed RNA-seq analyses for tobacco (Nicotiana tabacum) nearly isogenic lines (NILs) with and without favourable allele(s) at Phn7.1, a major QTL influencing partial resistance to the soil-borne pathogens Phytophthora nicotianae and Ralstonia solanacearum. Based upon combined analyses of transcriptome-based sequence variation and gene expression profiles, we concluded that allelic variability at the Phn7.1 locus was likely generated from homoeologous exchange, which led to deletion of low-expressing members of the SAR8.2 gene family and duplication of high-expressing SAR8.2 genes from a different subgenome of allotetraploid tobacco. The high expression of endogenous Phn7.1-associated SAR8.2 genes was correlated with observed resistance to P. nicotianae. Our findings suggest a role for genomic rearrangements in the generation of favourable genetic variability affecting resistance to pathogens in plants.

Adaptation of Phytophthora nicotianae to Multiple Sources of Partial Resistance in Tobacco.

Host resistance is an important tool in the management of black shank disease of tobacco. Race development leads to rapid loss of single-gene resistance, but the adaptation by Phytophthora nicotianae to sources of partial resistance from Beinhart 1000, Florida 301, and the Wz gene region introgressed from Nicotiana rustica is poorly characterized. In greenhouse environments, host genotypes with quantitative trait loci (QTLs) conferring resistance from multiple sources were initially inoculated with an aggressive isolate of race 0 or race 1 of P. nicotianae. The most aggressive isolate was selected after each of six host generations to inoculate the next generation of plants. The race 0 isolate demonstrated a continuous gradual increase in disease severity and percentage root rot on all sources of resistance except the genotype K 326 Wz/-, where a large increase in both was observed between generations 2 and 3. Adaptation by the race 0 isolate on Beinhart 1000 represents the first report of adaptation to this genotype by P. nicotianae. The race 1 isolate did not exhibit significant increases in aggressiveness over generations but exhibited a large increase in aggressiveness on K 326 Wz/- between generations 3 and 4. Molecular characterization of isolates recovered during selection was completed via double digest restriction-site associated DNA sequencing, but no polymorphisms were associated with the observed changes in aggressiveness. The rapid adaptation to Wz resistance and the gradual adaptation to other QTLs highlights the need to study the nature of Wz resistance and to conduct field studies on the efficacy of resistance gene rotation for disease management.

QTL analysis of seed germination traits in tobacco (Nicotiana tabacum L.).

Genetic mapping of seed germination traits has been performed with many plant species. In tobacco, however, investigations are rare. In the present study, a bi-parental mapping population consisting of 118 doubled haploid lines and derived from a cross between 'Beinhart-1000' and 'Hicks' was investigated. Four germination-related traits, total germination (TG), normal germination (NG), time to reach 50% of total germination (T50), and the area under the curve after 200 h of germination (AUC) were considered by examining seeds either untreated or after a moderate controlled deterioration (CD). Quantitative trait loci were found for all traits distributed on 11 out of the 24 linkage groups. It was demonstrated that, as in many other species, germination-related traits are very complex and under polygenic control.

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

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 F1 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.

Identification and editing of a hybrid lethality gene expands the range of interspecific hybridization potential in Nicotiana.

KEY MESSAGE: Identification and inactivation of hybrid lethality genes can be used to expand the available gene pool for improvement of a cultivated crop species. Hybrid lethality is one genetic mechanism that contributes to reproductive isolation in plants and serves as a barrier to use of diverse germplasm for improvement of cultivated species. A classic example is the seedling lethality exhibited by progeny from the Nicotiana tabacum × N. africana interspecific cross. In order to increase the body of knowledge on mechanisms of hybrid lethality in plants, and to potentially develop tools to circumvent them, we utilized a transposon tagging strategy to identify a candidate gene involved in the control of this reaction. N. tabacum gene Nt6549g30 was identified to code for a class of coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) proteins, the largest class of plant defense proteins. Gene editing, along with other experiments, was used to verify that Nt6549g30 is the gene at the N. tabacum Hybrid Lethality 1 (NtHL1) locus controlling the hybrid lethality reaction in crosses with N. africana. Gene editing of Nt6549g30 was also used to reverse interspecific seedling lethality in crosses between N. tabacum and eight of nine additional tested species from section Suaveolentes. Results further implicate the role of disease resistance-like genes in the evolution of plant species and demonstrate the possibility of expanding the gene pool for a crop species through gene editing.

Genetic and Agronomic Analysis of Tobacco Genotypes Exhibiting Reduced Nicotine Accumulation Due to Induced Mutations in Berberine Bridge Like (BBL) Genes.

Genetic methodologies for reducing nicotine accumulation in the tobacco plant (Nicotiana tabacum L.) are of interest because of potential future regulations that could mandate lowering of this alkaloid in conventional cigarettes. Inactivation of tobacco genes such as the Berberine Bridge Like (BBL) gene family believed to encode for enzymes involved in one of the latter steps of nicotine biosynthesis could be a viable strategy for producing new tobacco cultivars with ultra-low leaf nicotine accumulation. We introduced deleterious mutations generated via ethyl methanesulfonate treatment of seed or gene editing into six known members of the BBL gene family and assembled them in different combinations to assess their relative contribution to nicotine accumulation. Significant reductions (up to 17-fold) in percent leaf nicotine were observed in genotypes homozygous for combined mutations in BBL-a, BBL-b, and BBL-c. The addition of mutations in BBL-d1, BBL-d2, and BBL-e had no additional significant effect on lowering of nicotine levels in the genetic background studied. Reduced nicotine levels were associated with reductions in cured leaf yields (up to 29%) and cured leaf quality (up to 15%), evidence of physiological complexities within the tobacco plant related to the nicotine biosynthetic pathway. Further nicotine reductions were observed for a BBL mutant line cultivated under a modified production regime in which apical inflorescences were not removed, but at the expense of further yield reductions. Plants in which BBL mutations were combined with naturally occurring recessive alleles at the Nic1 and Nic2 loci exhibited further reductions in percent nicotine, but no plant produced immeasurable levels of this alkaloid. Findings may suggest the existence of a minor, alternative pathway for nicotine biosynthesis in N. tabacum. The described genetic materials may be of value for the manufacture of cigarettes with reduced nicotine levels and for future studies to better understand the molecular biology of alkaloid accumulation in tobacco.

Characterization of Phn15.1, a Newly Identified Phytophthora nicotianae Resistance QTL in Nicotiana tabacum.

Phytophthora nicotianae is an oomycete that causes black shank, one of the most economically important diseases affecting tobacco production worldwide. Identification and introgression of novel genetic variability affecting partial genetic resistance to this pathogen is important because of the increased durability of partial resistance over time as compared with genes conferring immunity. A previous mapping study identified a quantitative trait locus (QTL), hereafter designated as Phn15.1, with a major effect on P. nicotianae resistance in tobacco. In this research, we describe significantly improved resistance of nearly isogenic lines (NILs) of flue-cured tobacco carrying the introgressed Phn15.1 region derived from highly resistant cigar tobacco cultivar Beinhart 1000. The Phn15.1 region appeared to act in an additive or partially dominant manner to positively affect resistance. To more finely resolve the position of the gene or genes underlying the Phn15.1 effect, the QTL was mapped with an increased number of molecular markers (single-nucleotide polymorphisms) identified to reside within the region. Development and evaluation of subNILs containing varying amounts of Beinhart 1000-derived Phn15.1-associated genetic material permitted the localization of the QTL to a genetic interval of approximately 2.7 centimorgans. Importantly, we were able to disassociate the Beinhart 1000 Phn15.1 resistance alleles from a functional NtCPS2 allele(s) which contributes to the accumulation of a diterpene leaf surface exudate considered undesirable for flue-cured and burley tobacco. Information from this research should be of value for marker-assisted introgression of Beinhart 1000-derived partial black shank resistance into flue-cured and burley tobacco breeding programs.

Increasing Photosynthesis: Unlikely Solution For World Food Problem.

Increasing the photosynthesis rate of plants has been recently revitalized as an approach for increasing grain crop yields and solving world food crises. The idea that photosynthesis is the key to increasing grain crop yields is not new. Considerable research in the 1970s and 1980s showed that carbon input was not limiting for crop growth and yield. Instead, the availability and uptake of water and nutrients were found to be critical for increasing grain yield, and that conclusion still applies today. In this Opinion article, nitrogen limitation is given particular attention because of its quantitative linkage with vegetative and reproductive growth and its essential role as a quantitative component of seeds.

Genetic Control of Facultative Parthenocarpy in Nicotiana tabacum L.

Investigation of parthenocarpy, the production of fruit without fertilization, in multiple plant species could result in development of technologies for conferring seedless fruits and increased stability of fruit formation in economically important plants. We studied parthenocarpy in the model species Nicotiana tabacum L., and observed variability for expression of the trait among diverse genetic materials. Parthenocarpy was found to be partially dominant, and a single major quantitative trait locus on linkage group 22 was found to control the trait in a doubled haploid mapping population derived from a cross between parthenocarpic cigar tobacco cultivar "Beinhart 1000" and nonparthenocarpic flue-cured tobacco cultivar, "Hicks." The same genomic region was found to be involved with control of the trait in the important flue-cured tobacco cultivar, "K326." We also investigated the potential for the production of maternal haploids due to parthenogenesis in parthenocarpic tobacco seed capsules. Maternal haploids were not observed in parthenocarpic capsules, suggesting a requirement of fertilization for maternal haploid production due to parthenogenesis in N. tabacum.

Potential Mandated Lowering of Nicotine Levels in Cigarettes: A Plant Perspective.

The Food and Drug Administration has announced the potential for mandated lowering of nicotine levels in combustible cigarettes. The World Health Organization has recommended a lowering of cigarette filler nicotine levels to below 0.4 mg/g. To devise appropriate nicotine control strategies, regulators must consider technical feasibility, timelines for compliance, and potential impediments to implementation. Outlined here is previously unsummarized information on genetic approaches that might be used to reduce nicotine levels in cured tobacco leaves. For the benefit of regulators, altered alkaloid or toxicant profiles that might result by implementation of some of these methodologies are discussed. Also mentioned are potential licensing or regulatory impediments to use of some of the technologies per se. Implications: An understanding of technical feasibility of plant-based nicotine reduction technologies, along with the potential for corresponding alterations in alkaloid or toxicant profiles, is needed by regulators to develop effective nicotine control strategies with minimal impediments or undesirable consequences.

Filter paper-based spin column method for cost-efficient DNA or RNA purification.

We describe herein a method of recharging used commercial spin columns or assembling homemade spin columns using filter paper as binding material for cost-effective, low throughput nucleic acid purification. The efficiency of filter paper-based spin columns was evaluated for purification of nucleic acids from various sources. Following protocols of commercial kits, we found filter paper to be a useful binding material for purification of nucleic acids, including plant genomic DNA, plant total RNA, PCR products, and DNA from agarose gels. However, filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Protocols for the use of filter paper recharged spin columns or homemade spin columns for low throughput purification of plant genomic DNA and total RNA with unused commercial kit buffers or less expensive homemade buffers are presented.