Selection of Nicotiana tabacum Haploids of High Photosynthetic Efficiency

Anthers of Nicotiana tabacum cultivar Wisconsin 38 were treated with the mutagen ethyl methane sulfonate and cultured to obtain populations of haploid plants of high genetic variability. The selection of these populations by their photosynthetic efficiency was carried out in a hydroponic culture chamber with a CO₂ atmosphere concentration close to the compensation point. Plants that survived 45 days of treatment were grown in pots in a greenhouse and their performance was compared to a population of unselected haploids. The growth characteristics, net photosynthesis, and chlorophyll content were measured and the haploid character verified. Selected plants were propagated by in vitro culture of buds and then diploidized to obtain seeds. Growth and other characteristics of the plants obtained were compared with those of the parental variety (Wisconsin 38) in a field assay. Growth, dry weight, leaf area, and net photosynthesis of selected plants were higher than in control plants.     

Phenotypic, genetic and genomic consequences of natural and synthetic polyploidization of Nicotiana attenuata and Nicotiana obtusifolia

Background and Methods

Polyploidy results in genetic turmoil, much of which is associated with new phenotypes that result in speciation. Five independent lines of synthetic allotetraploid N. × obtusiata (N × o) were created from crosses between the diploid N. attenuata (Na) (♂) and N. obtusifolia (No) (♀) and the autotetraploids of Na (NaT) and No (NoT) were synthesized. Their genetic, genomic and phenotypic changes were then compared with those of the parental diploid species (Na and No) as well as to the natural allotetraploids, N. quadrivalvis (Nq) and N. clevelandii (Nc), which formed 1 million years ago from crosses between ancient Na and No.

Key Results

DNA fingerprinting profiles (by UP-PCR) revealed that the five N × o lines shared similar but not identical profiles. Both synthetic and natural polyploidy showed a dosage effect on genome size (as measured in seeds); however, only Nq was associated with a genome upsizing. Phenotypic analysis revealed that at the cellular level, N × o lines had phenotypes intermediate of the parental phenotypes. Both allo- and autotetraploidization had a dosage effect on seed and dry biomass (except for NaT), but not on stalk height at first flower. Nc showed paternal (Na) cellular phenotypes but inherited maternal (No) biomass and seed mass, whereas Nq showed maternal (No) cellular phenotypes but inherited paternal (Na) biomass and seed mass patterns. Principal component analysis grouped Nq with N × o lines, due to similar seed mass, stalk height and genome size. These traits separated Nc, No and Na from Nq and N × o lines, whereas biomass distinguished Na from N × o and Nq lines, and NaT clustered closer to Nq and N × o lines than to Na.

Conclusions

Both allo- and autotetraploidy induce considerable morphological, genetic and genomic changes, many of which are retained by at least one of the natural polyploids. It is proposed that both natural and synthetic polyploids are well suited for studying the evolution of adaptive responses.Key words: Nicotiana, polyploidy, genome size, DNA fingerprinting, phenotypic variation, Solanaceae     

Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata

After herbivore attack, plants launch a suite of direct and indirect defense responses that must be coordinated if plants are to realize a fitness benefit from these responses. Here we characterize the volatile emissions in the native tobacco plant, Nicotiana attenuata Torr. ex Wats., that are elicited by tobacco hornworm (Manduca sexta L.) attack and are known to function as attractants for parasitoids. To provide the first ecophysiological comparison of examples of both types of defense in the same species, we characterize the elicitation and signaling mechanisms, the resources required, and the potential costs and benefits of the volatile release and compare these traits with those of the well-described induced direct defense in this species, nicotine production. The release of (E)-β-ocimene, cis-α-bergamotene and linalool is dramatically induced within 24 h by application of methyl jasmonate (MeJA), caterpillar feeding, and the treatment of mechanical wounds with larval oral secretions (OS), but not by mechanical damage alone. Plants from different geographic locations produce volatile blends that differ in composition. The most consistently released component from all genotypes, cis-α-berga-motene, is positively related to the amount of MeJA and the level of wounding if OS are applied to the wounds. The volatile release is strongly light dependent, dropping to undetectable quantities during dark periods, even when temperatures are elevated to match those of the light period. Inhibitors of wound-induced jasmonate accumulation (salicylates and auxins), which are known to inhibit wound-induced nicotine production, do not inhibit the release of volatiles. By individually inducing different leaf positions with OS and, on other plants, excising them after induction, we demonstrate that the emission is largely a systemic, whole-plant response, which is maximally triggered when the second fully expanded leaf is induced. We conclude that while both are whole-plant, systemic responses that utilize recently acquired resources for their production and are activated by the jasmonate cascade, the elicitation of the volatile release exhibits greater tissue sensitivity and utilizes additional signaling components than does nicotine production. In contrast to the large investment of fitness-limiting resources required for induced nicotine production or the resources used in benzyl acetone release from flowers for pollinator attraction, the resource requirements for the volatile release are minor. Hence the argument that the volatile release incurs comparatively large physiological costs cannot be supported in this system. Received: 4 November 1999 / Accepted: 1 March 2000     

Induced defense in Nicotiana attenuata (Solanaceae) fruit and flowers

Plants protect themselves against herbivory using a continuum of strategies, ranging from constitutive defenses to intermittent induced responses. Induced defenses may not provide immediate and maximum protection, but could be advantageous when continuous defense is either energetically or ecologically costly. As such, induced defenses in flowers could help defend relatively valuable tissue while keeping reproductive structures accessible and attractive to pollinators. Thus far, no one has demonstrated the efficacy of induced defenses against floral herbivores (florivores) in the field. Here we show that mechanical leaf damage in wild tobacco, Nicotiana attenuata (Solanaceae), reduced both flower and fruit herbivory in the field and that exogenous application of methyl jasmonate, a potent elicitor of induced responses, reduced both leaf and floral damage in natural populations. This result is consistent with a survey of damage in the field, which showed a negative relationship between leaf damage and flower and fruit damage. Although optimal defense theory predicts that induced defenses should be rare in reproductive tissues, owing to their high fitness value, our results suggest otherwise. Induced defenses in leaves and reproductive tissues may allow plants to respond effectively to the concomitant pressures of defending against herbivory and attracting pollinators.     

Solar ultraviolet-B radiation and insect herbivory trigger partially overlapping phenolic responses in Nicotiana attenuata and Nicotiana longiflora

BACKGROUND AND AIMS: Plants exposed to solar ultraviolet-B radiation (UV-B, 280-315 nm) frequently suffer less insect herbivory than do plants that receive attenuated levels of UV-B. This anti-herbivore effect of solar UV-B exposure, which has been documented in several ecosystems, is in part mediated by changes in plant tissue quality. Exposure to UV-B can modify the abundance of a number of secondary metabolites, including phenolic compounds with potential impacts on insect herbivores. The aim of this study is to assess the potential anti-herbivore role of UV-B-induced phenolic compounds by comparing the phenolic profiles induced by UV-B and simulated insect herbivory in two wild species of the genus Nicotiana. METHODS: Plants grown under field and glasshouse conditions were exposed to contrasting levels of UV-B. Half of the plants of the attenuated UV-B treatment were given a simulated herbivory treatment, where leaves were mechanically damaged and immediately treated with oral secretions of Manduca sexta caterpillars. This treatment is known to mimic the impact of real herbivory on the expression of plant defences in Nicotiana. Phenolic profiles induced by UV-B and simulated herbivory were characterized using high-performance liquid chromatography-mass spectrometry (HPLC-MS). KEY RESULTS: UV-B induced the accumulation of several UV-absorbing phenolic compounds that are known to play a significant role in UV-B screening. Interestingly, there was a significant convergence in the phenolic profiles induced by UV-B and simulated herbivory: chlorogenic acid and dicaffeoylspermidine isomers, in particular, displayed a similar pattern of response to these stimuli. In contrast, rutin, the only flavonoid that accumulated in significant quantities in the experiments, was only induced by UV-B. CONCLUSIONS: The results suggest that the anti-herbivory effect induced by UV-B may be mediated at least in part by the accumulation of phenylpropanoid derivatives that are similar to those induced by the plant in response to insect herbivory.     

Fitness consequences of altering floral circadian oscillations for Nicotiana attenuata

Ecological interactions between flowers and pollinators are all about timing.Flower opening/closing and scent emissions are largely synchronized with pollinator activity,and a circadian clock regulates these rhythms.However,whether the circadian clock increases a plant's reproductive success by regulating these floral rhythms remains untested.Flowers of Nicotiana attenuata,a wild tobacco,diurnally and rhythmically open,emit scent and move vertically through a 140° arc to interact with nocturnal hawkmoths.We tethered flowers to evaluate the importance of flower positions for Manduca sexta-mediated pollinations;flower position dramatically influenced pollination.We examined the pollination success of phase-shifted flowers,silenced in circadian clock genes,NaZTL,NaLHY,and NaTOCi,by RNAi.Circadian rhythms in N.attenuata flowers are responsible for altered seed set from outcrossed pollen.     

Heritability of a Quantitative and Qualitative Protease Inhibitor Polymorphism in Nicotiana attenuata

Abstract: Many plant species contain chemical defenses that protect them against herbivores. Despite the benefit of these chemical defenses, not all individuals contain high levels of these compounds. In the native tobacco Nicotiana attenuata we found that plants from three natural populations differed considerably in their ability to produce trypsin protease inhibitors (PIs), which are defensive proteins that reduce herbivore damage to plants. Plants from a Utah (U) population produced high levels, whereas plants from Arizona (A) contained no detectable PI levels. Californian (C) plants had intermediate levels. The PI-producing U and C plants thus differ quantitatively from each other, whereas they both differ qualitatively from PI-deficient A plants. Here we analyze how PI production is inherited in N. attenuata with the ultimate goal of better understanding how the quantitative and qualitative differences between the three populations have evolved. Using a series of classical crossing designs, we determined that the ability to produce PIs is inherited as a dominant Mendelian trait. PI-deficient plants contain two non-functional recessive alleles, whereas heterozygous plants or homozygous dominant plants both are able to produce PIs. Similarly, the level of constitutive PIs may be determined by its genotype, either by an interaction between a functional and a non-functional allele in heterozygotes, or by a factor on the PI allele itself in homozygous C plants. Based on these data and on previous studies with A and U plants we postulate that the PI-deficient A plants may have originated from a mutant that lost its ability to produce PIs. The fitness loss due to reduced herbivore resistance may be offset by the fitness gain associated with increased competitive ability, a trade-off which may maintain this mutation in the Arizona population.     

Unpredictability of nectar nicotine promotes outcrossing by hummingbirds in Nicotiana attenuata

Many plants use sophisticated strategies to maximize their reproductive success via outcrossing. Nicotiana attenuata flowers produce nectar with nicotine at concentrations that are repellent to hummingbirds, increasing the number of flowers visited per plant. In choice tests using native hummingbirds, we show that these important pollinators learn to tolerate high‐nicotine nectar but prefer low‐nicotine nectar, and show no signs of nicotine addiction. Nectar nicotine concentrations, unlike those of other vegetative tissues, are unpredictably variable among flowers, not only among populations, but also within populations, and even among flowers within an inflorescence. To evaluate whether variations in nectar nicotine concentrations increase outcrossing, polymorphic microsatellite markers, optimized to evaluate paternity in native N. attenuata populations, were used to compare outcrossing in plants silenced for expression of a biosynthetic gene for nicotine production (Napmt1/2) and in control empty vector plants, which were antherectomized and transplanted into native populations. When only exposed to hummingbird pollinators, seeds produced by flowers with nicotine in their nectar had a greater number of genetically different sires, compared to seeds from nicotine‐free flowers. As the variation in nectar nicotine levels among flowers in an inflorescence decreased in N. attenuata plants silenced in various combinations of three Dicer‐like (DCL) proteins, small RNAs are probably involved in the unpredictable variation in nectar nicotine levels within a plant.     

渐狭叶烟草高效基因编辑体系的建立

渐狭叶烟草(Nicotiana attenuata)是烟草属植物中研究植物与昆虫、植物与病原菌互作的模式植物。本研究以八氢番茄红素脱氢酶基因(PDS)为靶标基因,建立一套以pHSE401为基因编辑载体,以潮霉素为抗性筛选标记的渐狭叶烟草高效基因编辑体系。利用该体系,获得PDS基因约80%的基因编辑效率,远远超过目前在渐狭叶烟草中报道的约30%的基因编辑效率。进一步使用WRKY70基因为靶标,对该体系对进行编辑效率验证,经测序发现WRKY70基因编辑材料中的基因编辑效率为83%,其中发生大片段缺失突变的频率为50%。因此,本研究成功建立了渐狭叶烟草高效基因编辑体系,为以后渐狭叶烟草的基因功能研究奠定基础。     

Localized micronutrient patches induce lateral root foraging and chemotropism in Nicotiana attenuata

Nutrients are distributed unevenly in the soil.Phenotypic plasticity in root growth and proliferation may enable plants to cope with this variation and effectively forage for essential nutrients. However, how micronutrients shape root architecture of plants in their natural environments is poorly understood. We used a combination of field and laboratory-based assays to determine the capacity of Nicotiana attenuata to direct root growth towards localized nutrient patches in its native environment. Plants growing in nature displayed a particular root phenotype consisting of a single primary root and a few long, shallow lateral roots. Analysis of bulk soil surrounding the lateral roots revealed a strong positive correlation between lateral root placement and micronutrient gradients, including copper, iron and zinc. In laboratory assays, the application of localized micronutrient salts close to lateral root tips led to roots bending in the direction of copper and iron. This form of chemotropism was absent in ethylene and jasmonic acid deficient lines,suggesting that it is controlled in part by these two hormones. This work demonstrates that directed root growth underlies foraging behavior, and suggests that chemotropism and micronutrient-guided root placement are important factors that shape root architecture in nature.     

渐狭叶烟草组培苗生根条件优化及在基因转化中的应用

针对渐狭叶烟草(Nicotiana attenuata)组培苗生根困难的问题，本研究测试了不同浓度萘乙酸(NAA)、吲哚丁酸(IBA)、吲哚乙酸(IAA)、嘧啶醇、矮壮素、生根粉及活性炭等外源添加物对组培苗生根的影响。结果表明，对照处理的组培苗在90 d内基本不生根；不同浓度矮壮素处理也不能诱导组培苗生根；而合适浓度的NAA、IBA或IAA处理均能较好地诱导组培苗生根，其中0.8 mg·L^-1 IBA不仅具有较好的诱导生根效果，生根率达72.73%，且根及地上部分均生长良好。进一步测试发现0.8 mg·L^-1 IBA处理对转基因组培苗具有良好的生根效果，20 d后生根率达60%以上，而对照组未生根；经伤口诱导和0.8 mg·L^-1 IBA处理，40 d内总生根率可达90%以上。该研究为高效获得渐狭叶烟草转基因生根苗提供了解决方案，也为渐狭叶烟草作为烟草属模式植物的研究提供了便利。     

Allocation of nitrogen to an inducible defense and seed production in Nicotiana attenuata

Resource-based tradeoffs in the allocation of a limiting resource are commonly invoked to explain negative correlations between growth and defense in plants, but critical examinations of these tradeoffs are lacking. To rigorously quantify tradeoffs in a common currency, we grew Nicotiana attenuata plants in individual hydroponic chambers, induced nicotine production by treating roots with methyl jasmonate (MJ) and standardized leaf puncturing, and used ¹⁵N to determine whether nitrogen-based tradeoffs among nicotine production, growth, and seed production could be detected. Plants were treated with a range of MJ quantities (5, 45 or 250 μg plant^?1) to effect a physiologically realistic range of changes in endogenous jasmonic acid levels and increases in nicotine production and accumulation; MJ treatments were applied to the roots to target JA-induced nicotine production, since nicotine biosynthesis is restricted to the roots. Leaf puncturing and 5 μg MJ treatments increased de novo nicotine synthesis and whole-plant (WP) nicotine pools by 93 and 66%, while 250 μg MJ treatments increased these values 3.1 and 2.5-fold. At these high rates of nicotine production, plants incorporated 5.7% of current nitrogen uptake and 6.0% of their WP nitrogen pools into nicotine. The ¹⁵N-labeled nicotine pools were stable or increased for the duration of vegetative growth, indicating that the N-nicotine was not metabolized and re-used for growth. Plants with elevated nicotine production grew more slowly and the differences in plant biomass gain between MJ-treated plants and controls were linearly related to the differences in nicotine accumulation. Despite the reductions in rosette-stage growth associated with nicotine production, estimates of lifetime fitness (cumulative lifetime seed production, mass/seed, seed viability) were not affected by any treatment. Only two treatments (leaf puncturing and 250 μg MJ) increased the allocations of ¹⁵N acquired at the time of induction to seed production. On average, plants used only 14.9% of their WP nitrogen pool for seed production, indicating that either the nitrogen requirements for seed production or the reproductive effort of these hydroponically-grown plants are low. To determine if seed production is strongly influenced by the amount of vegetative biomass attained before reproduction, the experiment was repeated with plants that had 44% of their leaf area (or 29% of their WP biomass) removed before MJ treatments with a removal technique that minimized the nicotine response. MJ treatments of these plants dramatically increased nicotine production and accumulation, but these plants also suffered no measurable fitness consequences from either the leaf removal or MJ treatments. We conclude that when N. attenuata plants are grown in these individual hydroponic chambers, their allocation to reproduction is sufficiently buffered to obscure the large increases in nitrogen allocations to an inducible defense. To determine whether soil-grown plants are similarly buffered, we grew two genotypes of plants in the high-nutrient soil from a 1-year-old burn in a piñyon-juniper forest (the plants' natural habitat) and in low-nutrient soil from an adjacent unburned area, and induced nicotine production in half of the plants with a 500 μg root MJ treatment. Plants grown in burned soils had an estimated lifetime fitness that was on average 2.8-fold greater than that of plants grown in unburned soils. MJ treatment reduced fitness estimates by 43% and 71% in the burned and unburned soils, respectively. We conclude that while hydroponic culture allows one to rigorously quantitate nitrogen allocation to growth, reproduction and defense, the allocation patterns of plants grown in hydroponic culture differ from those of plants grown in soil. Under hydroponic conditions, plants have low reproductive allocations and reproductive-defense tradeoffs are not detected. Reproductive-defense tradeoffs are readily discernible in soil-grown plants, but under these growing conditions, the nitrogen-basis for the tradeoff is difficult to quantify.