Tobacco (Nicotiana tabacum) is an important plant model system that has played a key role in the early development of molecular plant biology. The tobacco genome is large and its characterisation ...challenging because it is an allotetraploid, likely arising from hybridisation between diploid N. sylvestris and N. tomentosiformis ancestors. A draft assembly was recently published for N. tabacum, but because of the aforementioned genome complexities it was of limited utility due to a high level of fragmentation.
Here we report an improved tobacco genome assembly, which, aided by the application of optical mapping, achieves an N
size of 2.17 Mb and enables anchoring of 64% of the genome to pseudomolecules; a significant increase from the previous value of 19%. We use this assembly to identify two homeologous genes that explain the differentiation of the burley tobacco market class, with potential for greater understanding of Nitrogen Utilization Efficiency and Nitrogen Use Efficiency in plants; an important trait for future sustainability of agricultural production.
Development of an improved genome assembly for N. tabacum enables what we believe to be the first successful map-based gene discovery for the species, and demonstrates the value of an improved assembly for future research in this model and commercially-important species.
Backcrossing is often used in cultivar development to transfer one or a few genes to desired genetic backgrounds. The duration necessary to complete such 'trait conversions' is largely dependent upon ...generation times. Constitutive overexpression of the Arabidopsis thaliana gene FT (FLOWERING LOCUS T) induces early-flowering in many plants. Here, we used tobacco (Nicotiana tabacum L.) as a model system to propose and examine aspects of a modified backcross procedure where transgenic FT overexpression is used to reduce generation time and accelerate gene transfer. In this method, the breeder would select for an FT transgene insertion and the trait(s) of interest at each backcross generation except the last. In the final generation, selection would be conducted for the trait(s) of interest, but against FT, to generate the backcross-derived trait conversion. We demonstrate here that constitutive FT overexpression functions to dramatically reduce days-to-flower similarly in diverse tobacco genetic backgrounds. FT-containing plants flowered in an average of 39 days, in comparison with 87-138 days for non-FT plants. Two FT transgene insertions were found to segregate independently of several disease resistance genes often the focus of backcrossing in tobacco. In addition, no undesirable epigenetic effects on flowering time were observed once FT was segregated away. The proposed system would reduce the time required to complete a trait conversion in tobacco by nearly one-half. These features suggest the possible value of this modified backcrossing system for tobacco or other crop species where long generation times or photoperiod sensitivity may impede timely trait conversion.
Germplasm from closely related diploid relatives of tobacco (Nicotiana tabacum L.) could be of value for continued genetic modification of this species and for mapping quantitative trait loci (QTLs). ...We examined near isogenic tobacco lines and hybrids differing for an introgressed genomic region from N. tomentosa Ruiz and Pavon designated as Many Leaves that exhibits a large influence on leaf number and correlated traits. Within a 'Red Russian' genetic background, the region acted in an additive to partially dominant fashion to delay flowering time, and increase leaf number, plant height, and green leaf yield. Evidence of epistasis was observed as the region affected these traits to varying degrees in diverse near isogenic hybrids. Fifteen amplified fragment length polymorphism (AFLP) markers of N. tomentosa origin were mapped within a single linkage group of 34.5 cM using a population of 207 BC₁F₁ individuals segregating for Many Leaves. Composite interval mapping produced 2-LOD confidence intervals for likely QTL positions influencing leaf number (3.1 cM region), plant height (2.9 cM region), and days to flowering (3.3 cM region). These intervals were overlapping. Results demonstrate that genomic regions with large genetic effects can be transferred to tobacco from closely related diploid relatives, and that sufficient recombination within these regions may permit mapping of genes controlling quantitative traits. Materials and results described here may be useful in future research to gain insight on the genetic control of the transition from vegetative to reproductive development in Nicotiana.
A cDNA and genomic clone encoding maize chloroplastic Cu/Zn superoxide dismutase Sod1 were isolated. Southern blot analysis indicated little homology between the chloroplastic (Sod1) and the ...cytosolic (Sod2, Sod4, Sod4A) cDNAs. Sequence analysis of the genomic clone revealed a promoter, transit peptide, and partial coding sequence. The promoter contained several response elements (e.g., for light, cold temperature, xenobiotics) that may be involved in the regulation of the Sod1 gene. Sod1 expression during development and in response to physiological and chemical stressors such as temperature, xenobiotics (paraquat), and light were examined.
Salinity alters general metabolic processes and enzymatic activities, causing increased production of reactive oxygen species (ROS). Expression of antioxidant defense genes would, in turn, be ...triggered to defend the cell against oxidative damage. We report that salt disturbed antioxidant metabolism in maize seedlings, causing detrimental effects on the growth and development of maize plantlets, increased hydrogen peroxide production and altered antioxidant activities and transcripts profiles. Excessive ROS levels were accompanied by increased catalase (CAT) activity in photosynthesizing shoots, along with induction of mRNA accumulation. Increased accumulation of superoxide dismutase (SOD) transcripts was also observed although no significant changes in total SOD enzymatic activity and isozyme profiles were detected. Higher salt concentrations (above 0.25 M NaCl) were highly detrimental to the plants, causing arrested growth and severe wilting, among other effects. Histochemical detection of H
2
O
2
by 3,3-diaminobenzidine (DAB) staining indicated a collapse of the leaf veins, with hydrogen peroxide leaking to neighboring cells. In agreement to these observations, Sod1, Sod2, Sod4, Sod4A, as well as all Cat transcripts were severely inhibited in plants exposed to high salt concentrations.
This study examined the contribution of catalase (CAT) and superoxide dismutase (SOD) in the overall antioxidant response to norflurazon (NF)-induced oxidative stress in leaves, mesocotyls and ...scutella of maize (Zea mays). Maize catalase null mutants were used to provide insights into the role(s) of these isozymes. A substantial increase in Cat1 and Cat2 transcript levels occurred in NF-treated leaves in all maize lines examined. However, these two transcripts did not show a particular pattern of change in NF-treated scutella from 5-day postimbibition (dpi) and 18-day postpollination (dpp) maize. The NF-induced increase in Cat1 appeared to be dependent on excessive light energy caused by a lack of photoprotectant carotenoids, especially in leaves. In NF-treated leaves, the chloroplastic Cu/Zn-SOD-1 isozyme responded strongly compared to the cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD-3 isozymes, suggesting the critical role of SOD-1 as a major component in chloroplastic antioxidant defenses. All SOD isozymes in the NF-treated scutella of various maize lines were consistent in their response to NF. The most significant increase was observed with Sod1 in NF-treated leaves; however, no significant Sod1 changes were observed in similarly treated scutella at 5 dpi and 18 dpp. These results suggest that the response of the Cat and Sod genes to NF is likely developmental and tissue-specific.
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.
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
Yb
1
and
Yb
2
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
Yb
1
and
Yb
2
genes (homologs of
Arabidopsis EGY1
gene) enabled overexpression of the wild-type
Yb
1
allele in
yb
1
yb
1
yb
2
yb
2
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.
Yb
1
overexpression was found to confer an agronomic benefit to
yb
1
yb
1
yb
2
yb
2
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
Yb
1
and
Yb
2
loci. Results indicate that complex changes in the transcriptome of tobacco can be manifested by altered expression of
Yb
1
.
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.
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.
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