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Background: Leaf morphology plays a crucial role in photosynthetic efficiency and yield potential in crops. Cigar tobacco plants, which are derived from common tobacco (Nicotiana tabacum L.), possess special leaf characteristics including thin and delicate leaves with few visible veins, making it a good system for studying the genetic basis of leaf morphological characters., Results: In this study, GWAS and QTL mapping were simultaneously performed using a natural population containing 185 accessions collected worldwide and an F 2 population consisting of 240 individuals, respectively. A total of 26 QTLs related to leaf morphological traits were mapped in the F 2 population at three different developmental stages, and some QTL intervals were repeatedly detected for different traits and at different developmental stages. Among the 206 significant SNPs identified in the natural population using GWAS, several associated with the leaf thickness phenotype were co-mapped via QTL mapping. By analyzing linkage disequilibrium and transcriptome data from different tissues combined with gene functional annotations, 7 candidate genes from the co-mapped region were identified as the potential causative genes associated with leaf thickness., Conclusions: These results presented a valuable cigar tobacco resource showing the genetic diversity regarding its leaf morphological traits at different developmental stages. It also provides valuable information for novel genes and molecular markers that will be useful for further functional verification and for molecular breeding of leaf morphological traits in crops in the future., (© 2024. The Author(s).)

Fluctuating light is a typical light condition in nature and can cause selective photodamage to photosystem I (PSI). The sensitivity of PSI to fluctuating light is influenced by the amplitude of low/high light intensity. Tobacco mature leaves are tended to be horizontal to maximize the light absorption and photosynthesis, but young leaves are usually vertical to diminish the light absorption. Therefore, we tested the hypothesis that such regulation of the leaf angle in young leaves might protect PSI against photoinhibition under fluctuating light. We found that, upon a sudden increase in illumination, PSI was over-reduced in extreme young leaves but was oxidized in mature leaves. After fluctuating light treatment, such PSI over-reduction aggravated PSI photoinhibition in young leaves. Furthermore, the leaf angle was tightly correlated to the extent of PSI photoinhibition induced by fluctuating light. Therefore, vertical young leaves are more susceptible to PSI photoinhibition than horizontal mature leaves when exposed to the same fluctuating light. In young leaves, the vertical leaf angle decreased the light absorption and thus lowered the amplitude of low/high light intensity. Therefore, the regulation of the leaf angle was found for the first time as an important strategy used by young leaves to protect PSI against photoinhibition under fluctuating light. To our knowledge, we show here new insight into the photoprotection for PSI under fluctuating light in nature.

Delayed pollination is widely used to overcome pre-fertilization incompatibility, but its regulatory mechanisms are unclear. When Nicotiana tabacum was cross-pollinated with pollen of N. alata, the incompatibility occurring in the basal 1/4 region of the style (pollinated at anthesis: 0-day-old pistil) was overcome by delayed pollination (of 6-day-old pistil), and the morphological changes and corresponding physiological basis are explored here. The structure and ultrastructure of the pistil were observed under fluorescence microscopy and transmission electron microscopy. Differentially expressed proteins were screened with a monoclonal antibody chip for Nicotiana, and protein expression and distribution were analysed by immunofluorescence. Cellulase and pectinase activities were tested using enzyme-linked immunosorbent assay kits. The style of Nicotiana is solid in the basal region and pollen tubes grow in the extracellular spaces (ECM) of the transmitting tissue (TTS) cells. Seven of the 22 identified proteins were cell wall-associated proteins and were expressed at higher levels during pistil senescence. Cellulase and pectinase activities increased. The TTS cells in the basal 1/4 region of the 0-day-old style were polygonal and tightly arranged, with narrow ECM, but these were oval or partially dissolved in the 6-day-old pistil, leading to wider ECM and richer secretions. The increased expression of cell wall proteins and enhanced enzyme activity during pistil senescence might partially be responsible for the cells becoming oval and the ECM enlarged, providing the morphological basis for delayed pollination overcoming the pre-fertilization incompatibility between N. tabacum and N. alata., (© 2020 German Society for Plant Sciences, Royal Botanical Society of the Netherlands.)

Scanning electron microscopy (SEM) is widely used to investigate the surface morphology, and physiological state of plant leaves. Conventionally used methods for sample preparation are invasive, irreversible, require skill and expensive equipment, and are time and labor consuming. This study demonstrates a method to obtain in vivo surface information of plant leaves by imaging replicas with SEM that is rapid and non-invasive. Dental putty was applied to the leaves for 5 minutes and then removed. Replicas were then imaged with SEM and compared to fresh leaves, and leaves that were processed conventionally by chemical fixation, dehydration and critical point drying. The surface structure of leaves was well preserved on the replicas. The outline of epidermal as well as guard cells could be clearly distinguished enabling determination of stomatal density. Comparison of the dimensions of guard cells revealed that replicas did not differ from fresh leaves, while conventional sample preparation induced strong shrinkage (-40% in length and -38% in width) of the cells when compared to guard cells on fresh leaves. Tilting the replicas enabled clear measurement of stomatal aperture dimensions. Summing up, the major advantages of this method are that it is inexpensive, non-toxic, simple to apply, can be performed in the field, and that results on stomatal density and in vivo stomatal dimensions in 3D can be obtained in a few minutes., Competing Interests: The authors have declared that no competing interests exist.

Tobacco (Nicotiana tabacum) is extensively cultivated all over the world for its economic value. During curing and storage, senescence occurs, which is associated with physiological and biochemical changes in postharvest plant organs. However, the molecular mechanisms involved in accelerated senescence due to high temperatures in tobacco leaves during curing need further elaboration. We studied molecular mechanisms of senescence in tobacco leaves exposed to high temperature during curing (Fresh, 38 °C and 42 °C), revealed by isobaric tags for relative and absolute quantification (iTRAQ) for the proteomic profiles of cultivar Bi'na1. In total, 8903 proteins were identified, and 2034 (1150 up-regulated and 1074 down-regulated) differentially abundant proteins (DAPs) were obtained from tobacco leaf samples. These DAPs were mainly involved in posttranslational modification, protein turnover, energy production and conversion. Sugar- and energy-related metabolic biological processes and pathways might be critical regulators of tobacco leaves exposed to high temperature during senescence. High-temperature stress accelerated tobacco leaf senescence mainly by down-regulating photosynthesis-related pathways and degrading cellular constituents to maintain cell viability and nutrient recycling. Our findings provide a valuable inventory of novel proteins involved in senescence physiology and elucidate the protein regulatory network in postharvest organs exposed to high temperatures during flue-curing., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

Because carotenoids act as accessory pigments in photosynthesis, play a key photoprotective role and are of major nutritional importance, carotenogenesis has been a target for crop improvement. Although carotenoids are important precursors of phytohormones, previous genetic manipulations reported little if any effects on biomass production and plant development, but resulted in specific modifications in carotenoid content. Unexpectedly, the expression of the carrot lycopene β-cyclase (DcLCYB1) in Nicotiana tabacum cv. Xanthi not only resulted in increased carotenoid accumulation, but also in altered plant architecture characterized by longer internodes, faster plant growth, early flowering and increased biomass. Here, we have challenged these transformants with a range of growth conditions to determine the robustness of their phenotype and analyze the underlying mechanisms. Transgenic DcLCYB1 lines showed increased transcript levels of key genes involved in carotenoid, chlorophyll, gibberellin (GA) and abscisic acid (ABA) biosynthesis, but also in photosynthesis-related genes. Accordingly, their carotenoid, chlorophyll, ABA and GA contents were increased. Hormone application and inhibitor experiments confirmed the key role of altered GA/ABA contents in the growth phenotype. Because the longer internodes reduce shading of mature leaves, induction of leaf senescence was delayed, and mature leaves maintained a high photosynthetic capacity. This increased total plant assimilation, as reflected in higher plant yields under both fully controlled constant and fluctuating light, and in non-controlled conditions. Furthermore, our data are a warning that engineering of isoprenoid metabolism can cause complex changes in phytohormone homeostasis and therefore plant development, which have not been sufficiently considered in previous studies., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Jatropha curcas is an important bioenergy oil plant, and often planted on barren land to save the area of arable land. It is significant to improve the adaptability of J. curcas to various abiotic stresses. In the present study, we transferred a J. curcas gene, encoding a CBF2 transcription factor, into Nicotiana benthamiana. Under drought treatment, the JcCBF2 transgenic lines showed improved survival rate, leaf water retention and active oxygen scavenging capacity, but reduced photosynthesis and transpiration rate, suggesting that JcCBF2 played an important role in improving plant drought tolerance. Overexpressing JcCBF2 decreased leaf area and increased leaf thickness. To explore the possible mechanisms for the change of leaf anatomical structure, the leaves of wild-type and overexpression lines under drought stress were RNA sequenced. Genes involved in the plant hormones signal transduction were found to be enriched. Cytokinin and indole-3-acetic acid were the major plant hormones whose abundance increased. Quantitative RT-PCR analysis showed expression of NbMYB21, NbMYB86 and NbMYB44 and both abscisic acid (ABA) and jasmonic acid (JA) related genes in the overexpression lines were increased under drought stress. These results indicated that JcCBF2 was able to positively regulate plant drought response by changing the leaf anatomical structure and possibly through JA and ABA signalling pathways. Our work may help us to understand the drought tolerant mechanism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Developing automated methods to efficiently process large volumes of point cloud data remains a challenge for three-dimensional (3D) plant phenotyping applications. Here, we describe the development of machine learning methods to tackle three primary challenges in plant phenotyping: lamina/stem classification, lamina counting, and stem skeletonization. For classification, we assessed and validated the accuracy of our methods on a dataset of 54 3D shoot architectures, representing multiple growth conditions and developmental time points for two Solanaceous species, tomato ( Solanum lycopersicum cv 75 m82D ) and Nicotiana benthamiana Using deep learning, we classified lamina versus stems with 97.8% accuracy. Critically, we also demonstrated the robustness of our method to growth conditions and species that have not been trained on, which is important in practical applications but is often untested. For lamina counting, we developed an enhanced region-growing algorithm to reduce oversegmentation; this method achieved 86.6% accuracy, outperforming prior methods developed for this problem. Finally, for stem skeletonization, we developed an enhanced tip detection technique, which ran an order of magnitude faster and generated more precise skeleton architectures than prior methods. Overall, our improvements enable higher throughput and accurate extraction of phenotypic properties from 3D point cloud data., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Reproductive isolation, including prezygotic and postzygotic barriers, is a mechanism that separates species. Many species in the Nicotiana section Suaveolentes exhibit reproductive isolation in crosses with Nicotiana tabacum. In this study, we investigated whether the chromosome numbers and ploidy levels of eight Nicotiana suaveolens accessions are related to the reproductive isolation after crosses with N. tabacum by flow cytometry and chromosome analyses. Additionally, the internal transcribed spacer (ITS) regions of the eight N. suaveolens accessions were sequenced and compared with the previously reported sequences of 22 Suaveolentes species to elucidate the phylogenetic relationships in the section Suaveolentes. We revealed that four N. suaveolens accessions comprised 64 chromosomes, while the other four accessions carried 32 chromosomes. Depending on the ploidy levels of N. suaveolens, several types of reproductive isolation were observed after crosses with N. tabacum, including decreases in the number of capsules and the germination rates of hybrid seeds, as well as hybrid lethality and abscission of enlarged ovaries at 12-17 days after pollination. A phylogenetic analysis involving ITS sequences divided the eight N. suaveolens accessions into three distinct clades. Based on the results, we confirmed that N. suaveolens accessions vary regarding ploidy levels and reproductive isolation mechanisms in crosses with N. tabacum. These accessions will be very useful for revealing and characterizing the reproductive isolation mechanisms in interspecific crosses and their relationships with ploidy levels.

Litchi (Litchi chinesis Sonn.) is the most economically significant member of Sapindaceae family, especially in sub-tropical regions. However, its tall tree body often brings many inconveniences to production management. In order to modify the tree size or growth for productivity optimization and simplifying management, it is urgent to reveal the dwarf mechanism of litchi for dwarfing rootstocks or cultivar breeding. However, to date, the mechanisms on litchi dwarfism is still poor known. In the present study, transcriptome profiling were performed on L. chinensis cv. 'Feizixiao' (FZX, vigorous cultivar) and 'Ziniangxi' (ZNX, dwarf cultivar). A total of 55,810 unigenes were obtained, and 9,190 unigenes were differentially expressed between vigorous and dwarf litchi samples. Gene functional enrichment analysis indicated that the differentially expressed unigenes (DEGs) were related to phytohormone metabolism and signal transduction, and energy metabolism pathways. In particular, GA2ox were only up-regulated in ZNX samples, indicating GA might play an important role in regulating huge difference between vigorous and dwarf litchi cultivars. In addition, the 35S::LcGA2ox transgenic tobacco plants were dwarf and had smaller leaves or branches than wild type plants. Our study provided a series of candidate genes to reveal the mechanism of litchi dwarf., Competing Interests: The authors have declared that no competing interests exist.

Anthropogenic radioactivity generated by nuclear or chemical events results in the liberation of quadrillions of Becquerel and tons of materials to the environment. These events include nuclear accidents, nuclear weapon experiments, and high levels of generated radioactive and chemical waste. 210 Po is a high-energy α emitter that presents in the environment at extremely low concentration. It is considered as one of the highly toxic elements and is estimated to contribute about 7% of the total effective dose equivalent to humans from ingested natural internal radiation. The assessment of 210 Po activity/concentration in the environment could be used as an indicator of the level of anthropogenic radioactivity. The 210 Po concentration present in the most frequently sold cigarette produced in Lebanon was assayed using α spectrometry after a radiochemical separation and spontaneous deposition of Po on a copper disk. Although the geographical nature of Lebanese land is an extension of Syrian territory, the polonium activity concentration obtained is 8.8 times higher and attributable to the excessive use of phosphate fertilizers in agriculture. The individual committed effective dose was estimated to be equal to 219 ± 17 μSv/year of cigarette smoking.

The LONGIFOLIA1 (LNG1) gene of Arabidopsis regulates leaf shape by polar cell elongation independent of ROTUNDAFOLIA3 (ROT3). To expand our knowledge on the function of this gens in plant systems, Arabidopsis LNG1 (AtLNG1) was introduced both sense and antisense orientation under the control of 35S CaMV promoter into tobacco plants that lack AtLNG1 homolog. Resulting transgenic tobacco plants were analyzed by their phenotype, anatomy and transcript levels. AtLNG1-overexpressing tobacco lines showed increase in the leaf petiole and leaf blade compared with wild type tobacco line. The overexpressors also showed elongated palisade cells as well as epidermal cells in the leaf length direction, but no increase in cell number. Ectopic expression of AtLNG1 in tobacco plants also increased the expression of cell wall modification-related genes, such as NT_XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE9 (NT_XTH9), NT_XTH15 and NT_XTH33, indicating that these genes appear to be target of AtLNG1. As results of molecular and cellular examination, AtLNG1 seemed to have a conserved functional role in shaping leaf morphology in both Arabidopsis and tobacco.

Main Conclusion: Downregulation in the expression of the signal recognition particle 43 (SRP43) gene in tobacco conferred a truncated photosynthetic light-harvesting antenna (TLA property), and resulted in plants with a greater leaf-to-stem ratio, improved photosynthetic productivity and canopy biomass accumulation under high-density cultivation conditions. Evolution of sizable arrays of light-harvesting antennae in all photosynthetic systems confers a survival advantage for the organism in the wild, where sunlight is often the growth-limiting factor. In crop monocultures, however, this property is strongly counterproductive, when growth takes place under direct and excess sunlight. The large arrays of light-harvesting antennae in crop plants cause the surface of the canopies to over-absorb solar irradiance, far in excess of what is needed to saturate photosynthesis and forcing them to engage in wasteful dissipation of the excess energy. Evidence in this work showed that downregulation by RNA-interference approaches of the Nicotiana tabacum signal recognition particle 43 (SRP43), a nuclear gene encoding a chloroplast-localized component of the photosynthetic light-harvesting assembly pathway, caused a decrease in the light-harvesting antenna size of the photosystems, a corresponding increase in the photosynthetic productivity of chlorophyll in the leaves, and improved tobacco plant canopy biomass accumulation under high-density cultivation conditions. Importantly, the resulting TLA transgenic plants had a substantially greater leaf-to-stem biomass ratio, compared to those of the wild type, grown under identical agronomic conditions. The results are discussed in terms of the potential benefit that could accrue to agriculture upon application of the TLA-technology to crop plants, entailing higher density planting with plants having a greater biomass and leaf-to-stem ratio, translating into greater crop yields per plant with canopies in a novel agronomic configuration.

Main Conclusion: The software FlowerMorphology is designed for automatic morphometry of actinomorphic flowers. The novel complex parameters of flowers calculated by FlowerMorphology allowed us to quantitatively characterize a polyploid series of tobacco. Morphological differences of plants representing closely related lineages or mutants are mostly quantitative. Very often, there are only very fine variations in plant morphology. Therefore, accurate and high-throughput methods are needed for their quantification. In addition, new characteristics are necessary for reliable detection of subtle changes in morphology. FlowerMorphology is an all-in-one software package to automatically image and analyze five-petal actinomorphic flowers of the dicotyledonous plants. Sixteen directly measured parameters and ten calculated complex parameters of a flower allow us to characterize variations with high accuracy. The program was developed for the needs of automatic characterization of Nicotiana tabacum flowers, but is applicable to many other plants with five-petal actinomorphic flowers and can be adopted for flowers of other merosity. A genetically similar polyploid series of N. tabacum plants was used to investigate differences in flower morphology. For the first time, we could quantify the dependence between ploidy and size and form of the tobacco flowers. We found that the radius of inner petal incisions shows a persistent positive correlation with the chromosome number. In contrast, a commonly used parameter-radius of outer corolla-does not discriminate 2n and 4n plants. Other parameters show that polyploidy leads to significant aberrations in flower symmetry and are also positively correlated with chromosome number. Executables of FlowerMorphology, source code, documentation, and examples are available at the program website: https://github.com/Deyneko/FlowerMorphology .

Nicotiana otophora contains Agrobacterium-derived T-DNA sequences introduced by horizontal gene transfer (Chen et al., 2014). Sixty-nine contigs were assembled into four different cellular T-DNAs (cT-DNAs) totalling 83 kb. TC and TE result from two successive transformation events, each followed by duplication, yielding two TC and two TE inserts. TC is also found in other Nicotiana species, whereas TE is unique to N. otophora. Both cT-DNA regions are partially duplicated inverted repeats. Analysis of the cT-DNA divergence patterns allowed reconstruction of the evolution of the TC and TE regions. TC and TE carry 10 intact open reading frames. Three of these are TE-6b genes, derived from a single 6b gene carried by the Agrobacterium strain which inserted TE in the N. otophora ancestor. 6b genes have so far only been found in Agrobacterium tumefaciens or Agrobacterium vitis T-DNAs and strongly modify plant growth (Chen and Otten, 2016). The TE-6b genes were expressed in Nicotiana tabacum under the constitutive 2 × 35S promoter. TE-1-6b-R and TE-2-6b led to shorter plants, dark-green leaves, a strong increase in leaf vein development and modified petiole wings. TE-1-6b-L expression led to a similar phenotype, but in addition leaves show outgrowths at the margins, flowers were modified and plants became viviparous, i.e. embryos germinated in the capsules at an early stage of their development. Embryos could be rescued by culture in vitro. The TE-6b phenotypes are very different from the earlier described 6b phenotypes and could provide new insight into the mode of action of the 6b genes., (© 2018 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

Currently, capsule cigarettes have been introduced by tobacco industries and their sales have increased exponentially. However, the capsule flavoring ingredients and their safety are still unknown. Although the contents of menthol and other ingredients directly added to cigarettes have been determined extensively, no analogous study exists for menthol-containing cigarette capsules. Basic physical properties of capsules in all 31 different capsule cigarettes commercially available in Korea were investigated, and their menthol contents were determined in this study. Other ingredients in these capsules were also qualitatively analyzed; methyl octanoate, menthone and isopulegol as well as menthol were detected in all the capsules. There is considerable variability in basic physical properties and menthol contents of cigarette capsules depending on their brand styles. The menthol contents of capsules and whole cigarettes are similar or higher than those previously reported for conventional menthol cigarettes. This is the first report on the physical properties of capsules and the fact that a wide range of menthol contents in capsule cigarettes, regardless of flavor types, allows smokers to select menthol content to their liking., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Transport networks serve critical functions in biological and engineered systems, and yet their design requires trade-offs between competing objectives. Due to their sessile lifestyle, plants need to optimize their architecture to efficiently acquire and distribute resources while also minimizing costs in building infrastructure. To understand how plants resolve this design trade-off, we used high-precision three-dimensional laser scanning to map the architectures of tomato, tobacco, or sorghum plants grown in several environmental conditions and through multiple developmental time points, scanning in total 505 architectures from 37 plants. Using a graph-theoretic algorithm that we developed to evaluate design strategies, we find that plant architectures lie along the Pareto front between two simple length-based objectives-minimizing total branch length and minimizing nutrient transport distance-thereby conferring a selective fitness advantage for plant transport processes. The location along the Pareto front can distinguish among species and conditions, suggesting that during evolution, natural selection may employ common network design principles despite different optimization trade-offs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The anatomy and ultrastructure of the short glandular trichomes occurring on young expanding leaves of Nicotiana tabacum were investigated using light and transmission electron microscopy. The objective of the present research was to characterize the cellular changes that occur during morphogenesis of short glandular trichomes, from initiation to senescence. Ultrastructural analysis of their secretory cells revealed characteristics common to gland cells: numerous mitochondria in highly organized cytoplasm, large nuclei, and an elaborate network of endoplasmic reticulum. Initial changes in nuclear and plastidial organization were observed at a more advanced secretory stage, marking the onset of senescence. During trichome senescence, gradual reduction of the cytoplasm density occurred along with structural changes of the plastids and the tonoplast. As a result of inward blebbing of the cytoplasm into the vacuole, membrane bound vesicular structures appeared in the vacuolar space. At the late secretory stage, marked by an increase in vacuolation and extraplasmic space, degenerative changes included further fragmentation of the cytoplasm and deterioration of the tonoplast. Multimembrane myelin bodies observed in the vacuolar space were indicative of membrane digestion although plasma membrane did not appear massively degraded., (© 2017 Wiley Periodicals, Inc.)

Production of recombinant proteins in plants through Agrobacterium-mediated transient expression is a promising method of producing human therapeutic proteins, vaccines, and commercial enzymes. This process has been shown to be viable at a large scale and involves growing large quantities of wild-type plants and infiltrating the leaf tissue with a suspension of Agrobacterium tumefaciens bearing the genes of interest. This study examined one of the steps in this process that had not yet been optimized: the scale-up of Agrobacterium production to sufficient volumes for large-scale plant infiltration. Production of Agrobacterium strain C58C1 pTFS40 was scaled up from shake flasks (50-100 mL) to benchtop (5 L) scale with three types of media: Lysogeny broth (LB), yeast extract peptone (YEP) media, and a sucrose-based defined media. The maximum specific growth rate (μ max ) of the strain in the three types of media was 0.46 ± 0.04 h -1 in LB media, 0.43 ± 0.03 h -1 in YEP media, and 0.27 ± 0.01 h -1 in defined media. The maximum biomass concentration reached at this scale was 2.0 ± 0.1, 2.8 ± 0.1, and 2.6 ± 0.1 g dry cell weight (DCW)/L for the three media types. Production was successfully scaled up to a 100-L working volume reactor with YEP media, using k L a as the scale-up parameter.

Objective: Our purpose was to quantify 36 inorganic chemical elements in aerosols from disposable electronic cigarettes (ECs) and electronic hookahs (EHs), examine the effect of puffing topography on elements in aerosols, and identify the source of the elements., Methods: Thirty-six inorganic chemical elements and their concentrations in EC/EH aerosols were determined using inductively coupled plasma optical emission spectroscopy, and their source was identified by analyzing disassembled atomizers using scanning electron microscopy and energy dispersive X-ray spectroscopy., Results: Of 36 elements screened, 35 were detected in EC/EH aerosols, while only 15 were detected in conventional tobacco smoke. Some elements/metals were present in significantly higher concentrations in EC/EH aerosol than in cigarette smoke. Concentrations of particular elements/metals within EC/EH brands were sometimes variable. Aerosols generated at low and high air-flow rates produced the same pattern of elements, although the total element concentration decreased at the higher air flow rate. The relative amount of elements in the first and last 60 puffs was generally different. Silicon was the dominant element in aerosols from all EC/EH brands and in cigarette smoke. The elements appeared to come from the filament (nickel, chromium), thick wire (copper coated with silver), brass clamp (copper, zinc), solder joints (tin, lead), and wick and sheath (silicon, oxygen, calcium, magnesium, aluminum). Lead was identified in the solder and aerosol of two brands of EHs (up to 0.165 μg/10 puffs)., Conclusion: These data show that EC/EH aerosols contain a mixture of elements, including heavy metals, with concentrations often significantly higher than in conventional cigarette smoke. While the health effects of inhaling mixtures of heated metals is currently not known, these data will be valuable in future risk assessments involving EC/EH elements/metals.

Key Message: Functional characterization of two tobacco genes, one involved in xylan synthesis and the other, a positive regulator of secondary cell wall formation, is reported. Lignocellulosic secondary cell walls (SCW) provide essential plant materials for the production of second-generation bioethanol. Therefore, thorough understanding of the process of SCW formation in plants is beneficial for efficient bioethanol production. Recently, we provided the first proof-of-concept for using virus-induced gene silencing (VIGS) approach for rapid functional characterization of nine genes involved in cellulose, hemicellulose and lignin synthesis during SCW formation. Here, we report VIGS-mediated functional characterization of two tobacco genes involved in SCW formation. Stems of VIGS plants silenced for both selected genes showed increased amount of xylem formation but thinner cell walls than controls. These results were further confirmed by production of stable transgenic tobacco plants manipulated in expression of these genes. Stems of stable transgenic tobacco plants silenced for these two genes showed increased xylem proliferation with thinner walls, whereas transgenic tobacco plants overexpressing these two genes showed increased fiber cell wall thickness but no change in xylem proliferation. These two selected genes were later identified as possible members of DUF579 family involved in xylan synthesis and KNAT7 transcription factor family involved in positive regulation of SCW formation, respectively. Glycome analyses of cell walls showed increased polysaccharide extractability in 1 M KOH extracts of both VIGS-NbDUF579 and VIGS-NbKNAT7 lines suggestive of cell wall loosening. Also, VIGS-NbDUF579 and VIGS-NbKNAT7 lines showed increased saccharification rates (74.5 and 40 % higher than controls, respectively). All these properties are highly desirable for producing higher quantities of bioethanol from lignocellulosic materials of bioenergy plants.

Polyploidy is an important driving force in angiosperm evolution, and much research has focused on genetic, epigenetic and transcriptomic responses to allopolyploidy. Nicotiana is an excellent system in which to study allopolyploidy because half of the species are allotetraploids of different ages, allowing us to examine the trajectory of floral evolution over time. Here, we study the effects of allopolyploidy on floral morphology in Nicotiana, using corolla tube measurements and geometric morphometrics to quantify petal shape. We show that polyploid morphological divergence from the intermediate phenotype expected (based on progenitor morphology) increases with time for floral limb shape and tube length, and that most polyploids are distinct or transgressive in at least one trait. In addition, we show that polyploids tend to evolve shorter and wider corolla tubes, suggesting that allopolyploidy could provide an escape from specialist pollination via reversion to more generalist pollination strategies.

Nicotiana tabacum (Solanaceae) is the only species whose leaves can be legally marketed as tobacco according to the Japanese Tobacco Business Act. Nicotine, a major alkaloid produced by N. tabacum leaves, is regulated in pharmaceuticals by the Japanese Pharmaceutical Affairs Law. However, the use of N. tabacum stems as an excipient in pharmaceuticals is permitted, because these contained only a small amount of nicotine. Recently, several reports showed that a substantial amount of nicotine was detected in an OTC pharmaceutical product, in which N. tabacum stems were used as an excipient. Therefore, products containing N. tabacum stems could be contaminated with the leaf material. In the present study, we established a method to detect contamination of N. tabacum stem materials with its leaves, using microscopy to obtain standard reference microphotographs for identification. Cultivated N. tabacum stems and leaves, commercial cigarette leaves, and N. tabacum tissue imported as excipient material were used for preparing the microphotographs. The characteristic N. tabacum leaf structures found in the powdered fragments included: epidermal cells with sinuous anticlinal cell walls, hairs, mesophyll parenchyma with crystalized calcium oxalate (calciphytoliths), and branching vascular bundles derived from reticulate net-veins. A comparison of the microscopic characteristics of an OTC powder with those from the standard reference microphotographs was an effective method for N. tabacum stem and leaf identification. Thus, we evaluated the powdered pharmaceutical product containing N. tabacum stem tissue and Hydrangea serrata (Hydrangeaceae) leaf tissue as excipients, and confirmed the presence of N. tabacum leaf material.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG1) catalyzes the formation of mevalonic acid, the key intermediate of the cytosolic isoprenoid synthesis pathway. The parameters of stem and leaf growth were studied in the transgenic tobacco plants that express the HMG1 gene in both sense and antisense orientations towards the constitutive promoter. The transgenic plant height did not significantly differ from that of the control plants, though the plants carrying the sense copy of the HMG1 gene were considerably taller than plants that carried the antisense gene copy. Plants carrying an extra copy of the HMG1 gene were also characterized by increased leaf area. The number of mesophyll cells calculated per square unit of transgenic plants leaves was smaller than in the control plant leaves, though their volume was not considerably changed in any of the variants, suggesting changes in the cell packing density in leaves.

Cost efficient foraging is of especial importance for animals like hawkmoths or hummingbirds that are feeding 'on the wing', making their foraging energetically demanding. The economic decisions made by these animals have a strong influence on the plants they pollinate and floral volatiles are often guiding these decisions. Here we show that the hawkmoth Manduca sexta exhibits an innate preference for volatiles of those Nicotiana flowers, which match the length of the moth's proboscis. This preference becomes apparent already at the initial inflight encounter, with the odour plume. Free-flight respiration analyses combined with nectar calorimetry revealed a significant caloric gain per invested flight energy only for preferred-matching-flowers. Our data therefore support Darwin's initial hypothesis on the coevolution of flower length and moth proboscis. We demonstrate that this interaction is mediated by an adaptive and hardwired olfactory preference of the moth for flowers offering the highest net-energy reward.

Key Message: Transgenic Nicotiana benthamiana lines with constitutive expression of an Arabidopsis lectin receptor kinase gene (LecRK - I.9 or LecRK - IX.1) show enhanced resistance to Phytophthora pathogens, demonstrating conserved gene functionality after interfamily transfer. In plants, cell surface receptors mediate the first layer of innate immunity against pathogenic microbes. In Arabidopsis several L-type lectin receptor kinases (LecRKs) were previously found to function as Phytophthora resistance components. In this study, we determined the functionality of Arabidopsis LecRK-I.9 or LecRK-IX.1 in Phytophthora resistance when transferred into the Solanaceous plant Nicotiana benthamiana. Multiple transgenic lines were generated for each LecRK gene and molecular analyses revealed variation in transgene copy number, transgene expression levels and LecRK protein accumulation. Infection assays showed that transgenic N. benthamiana plants expressing either Arabidopsis LecRK-I.9 or LecRK-IX.1 are more resistant to Phytophthora capsici and to Phytophthora infestans. These results demonstrate that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their Phytophthora resistance function when transferred into N. benthamiana. Therefore, these LecRKs have the potential to function as a complementary Phytophthora resistance resource in distantly related plant species next to the canonical Phytophthora resistance genes encoding nucleotide-binding leucine-rich repeat proteins.

After germination, plants progress through juvenile and adult phases of vegetative development before entering the reproductive phase. The character and timing of these phases vary significantly between different plant species, which makes it difficult to know whether temporal variations in various vegetative traits represent the same, or different, developmental processes. miR156 has been shown to be the master regulator of vegetative development in plants. Overexpression of miR156 prolongs the juvenile phase of development, whereas knocking-down the level of miR156 promotes the adult phase of development. Therefore, artificial modulation of miR156 expression is expected to cause corresponding changes in vegetative-specific traits in different plant species, particularly in those showing no substantial difference in morphology during vegetative development. To identify specific traits associated with the juvenile-to-adult transition in tobacco, we examined the phenotype of transgenic tobacco plants with elevated or reduced levels of miR156. We found that leaf shape, the density of abaxial trichomes, the number of leaf veins, the number of stomata, the size and density of epidermal cells, patterns of epidermal cell staining, the content of chlorophyll and the rate of photosynthesis, are all affected by miR156. These newly identified miR156-regulated traits therefore can be used to distinguish between juvenile and adult phases of development in tobacco, and provide a starting point for future studies of vegetative phase change in the family Solanaceae., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Main Conclusion: The T-DNA 6b oncogene induces complex and partly unprecedented phenotypic changes in tobacco stems and leaves, which result from hypertrophy and hyperplasia with ectopic spot-like, ridge-like and sheet-like meristems. The Agrobacterium T-DNA oncogene 6b causes complex growth changes in tobacco including enations; this unusual phenotype has been called "6b enation syndrome". A detailed morphological and anatomical analysis of the aerial part of Nicotiana tabacum plants transformed with a dexamethasone-inducible dex-T-6b gene revealed several striking growth phenomena. Among these were: uniform growth of ectopic photosynthetic cells on the abaxial leaf side, gutter-like petioles with multiple parallel secondary veins, ectopic leaf primordia emerging behind large glandular trichomes, corniculate structures emerging from distal ends of secondary veins, pin-like structures with remarkable branching patterns, ectopic vascular strands in midveins and petioles extending down along the stem, epiascidia and hypoascidia, double enations and complete inhibition of leaf outgrowth. Ectopic stipule-like leaves and inverted leaves were found at the base of the petioles. Epinastic and hyponastic growth of petioles and midveins yielded complex but predictable leaf folding patterns. Detailed anatomical analysis of over sixty different 6b-induced morphological changes showed that the different modifications are derived from hypertrophy and abaxial hyperplasia, with ectopic photosynthetic cells forming spot-like, ridge-like and sheet-like meristems and ectopic vascular strands forming regular patterns in midveins, petioles and stems. Part of the enation syndrome is due to an unknown phloem-mobile enation factor. Graft experiments showed that the 6b mRNA is mobile and could be the enation factor. Our work provides a better insight in the basic effects of the 6b oncogene.

Plant growth is a dynamic process, and the precise course of events during early plant development is of major interest for plant research. In this work, we investigate the growth of rosette plants by processing time-lapse videos of growing plants, where we use Nicotiana tabacum (tobacco) as a model plant. In each frame of the video sequences, potential leaves are detected using a leaf-shape model. These detections are prone to errors due to the complex shape of plants and their changing appearance in the image, depending on leaf movement, leaf growth, and illumination conditions. To cope with this problem, we employ a novel graph-based tracking algorithm which can bridge gaps in the sequence by linking leaf detections across a range of neighboring frames. We use the overlap of fitted leaf models as a pairwise similarity measure, and forbid graph edges that would link leaf detections within a single frame. We tested the method on a set of tobacco-plant growth sequences, and could track the first leaves of the plant, including partially or temporarily occluded ones, along complete sequences, demonstrating the applicability of the method to automatic plant growth analysis. All seedlings displayed approximately the same growth behavior, and a characteristic growth signature was found.

Cigarette smoke is a complex mixture of more than 8000 smoke constituents. The quantification of selected mainstream smoke constituent yields is one of the methods to evaluating and comparing the performance of different products. Numerous regulatory and scientific advisory bodies have used cigarette smoke constituent yield data for reporting and product comparison purposes. For more than a decade limitations of the indiscriminate application of traditional statistical methods such as the t-test for differences in comparative smoke constituent yield assessments lacking a specific study design, have been highlighted. In the present study, the variability of smoke constituent yields is demonstrated with data obtained under the ISO smoking regime for the Kentucky reference cigarette 3R4F and one commercial brand, analyzed on several occasions between 2007 and 2014. Specifically it is shown that statistically significant differences in the yields of selected smoke constituents do not readily translate to differences between products, and that tolerances need to be defined. To this end, two approaches have been proposed in the literature--minimal detectable differences, and the statistical equivalence. It is illustrated how both approaches provide more meaningful comparison outcomes than the statistical t-test for differences. The present study provides considerations relevant for comparative tobacco product assessments both in the scientific and regulatory contexts., (Copyright © 2015 Philip Morris Products S.A. Published by Elsevier Inc. All rights reserved.)

Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.

Chimeric genes resulting from the rearrangement of a mitochondrial genome were generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). In the study, earlier we reported that identifying a 47 bp deletion at 3'- flanking of atp9 that was linked to male sterile cytoplasm in kenaf. The truncated fragment was fused with atp9, a mitochondrial transit signal (MTS) and/or GFP, comprised two chimeric genes MTS-HM184-GFP and MTS-HM184. The plant expression vector pBI121 containing chimeric genes were then introduced to tobacco plants by Agrobacterium-mediated T-DNA transformation. The result showed that certain transgenic plants were male sterility or semi-sterility, while some were not. The expression analysis further demonstrated that higher level of expression were showed in the sterility plants, while no expression or less expression in fertility plants, the levels of expression of semi-sterility were in between. And the sterile plant (containing MTS-HM184-GFP) had abnormal anther produced malformed/shriveled pollen grains stained negative that failed to germinate (0%), the corresponding fruits was shrunken, the semi-sterile plants having normal anther shape produced about 10-50% normal pollen grains, the corresponding fruits were not full, and the germination rate was 58%. Meanwhile these transgenic plants which altered on fertility were further analyzed in phenotype. As a result, the metamorphosis leaves were observed in the seedling stage, the plant height of transgenic plants was shorter than wild type. The growth duration of transgenic tobacco was delayed 30-45 days compared to the wild type. The copy numbers of target genes of transgenic tobacco were analyzed using the real-time quantitative method. The results showed that these transgenic plants targeting-expression in mitochondrial containing MTS-HM184-GFP had 1 copy and 2 copies, the other two plants containing MTS-HM184 both had 3 copies, but 0 copy in wild type. In summary, the two manual chimeric genes might be related to male sterility in kenaf.

Nicotiana tabacum 'Bel-W3' is widely used as an ozone bioindicator species, showing typical necrosis preceded by microscopic markers of oxidative stress. This study aimed to follow the development of symptoms in tobacco exposed in São Paulo highlighting the temporal dynamics of the cellular events. Leaves with and without necrosis were processed according to standard techniques for anatomical analyses. Leaves from the site with higher SUM00 presented thinner palisade parenchyma, fewer layers of spongy parenchyma, higher stomatal density, clusters of vessel elements in the midrib, erosion of cuticular waxes and stomatal damage. The sequence of microscopic events from the third day of exposure were condensation of the cytoplasm in parenchyma tissue, sinuosity of anticlinal walls, pectinaceous cell wall protrusions, chromatin condensation and changes in chlorophyll autofluorescence. On the 14th day of exposure, these events finally led to cell death in the palisade parenchyma and necrosis on the leaf. The markers observed indicated oxidative stress caused by ozone.

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Main Conclusion: Modifications in leaf anatomy of tobacco plants induced greater leaf water transport capacity, meeting greater transpirational demands and acclimating to warmer temperatures with a higher vapor pressure deficit. Temperature is one of the most important environmental factors affecting photosynthesis and growth of plants. However, it is not clear how it may alter leaf hydraulic architecture. We grew plants of tobacco (Nicotiana tabacum) 'k326' in separate glasshouse rooms set to different day/night temperature conditions: low (LT 24/18 °C), medium (MT 28/22 °C), or high (HT 32/26 °C). After 40 days of such treatment, their leaf anatomies, leaf hydraulics, photosynthetic rates, and instantaneous water-use efficiency (WUEi) were measured. Compared with those under LT, plants exposed to HT or MT conditions had significantly higher values for minor vein density (MVD), stomatal density (SD), leaf area, leaf hydraulic conductance (K leaf), and light-saturated photosynthetic rate (A sat), but lower values for leaf water potential (ψ l) and WUEi. However, those parameters did not differ significantly between HT and MT conditions. Correlation analyses demonstrated that SD and K leaf increased in parallel with MVD. Moreover, greater SD and K leaf were partially associated with accelerated stomatal conductance. And then stomatal conductance was positively correlated with A sat. Therefore, under well-watered, fertilized conditions, when relative humidity was optimal, changes in leaf anatomy seemed to facilitate the hydraulic acclimation to higher temperatures, meeting greater transpirational demands and contributing to the maintenance of great photosynthetic rates. Because transpiration rate increased more with temperature than photosynthetic rate, WUEi reduced under warmer temperatures. Our results indicate that the modifications of leaf hydraulic architecture are important anatomical and physiological strategies for tobacco plants acclimating to warmer temperatures under a higher vapor pressure deficit.

Cell movement is the central mechanism for animal morphogenesis. Plant cell development rather relies on flexible alignment of cell axis adjusting cellular differentiation to directional cues. As central input, vectorial fields of mechanical stress and gradients of the phytohormone auxin have been discussed. In tissue contexts, mechanical and chemical signals will always act in concert; experimentally it is difficult to dissect their individual roles. We have designed a novel approach, based on cells, where directionality has been eliminated by removal of the cell wall. We impose a new axis using a microfluidic set-up to generate auxin gradients. Rectangular microvessels are integrated orthogonally with the gradient. Cells in these microvessels align their new axis with microvessel geometry before touching the wall. Auxin efflux is necessary for this touch-independent geometry exploration and we suggest a model, where auxin gradients can be used to align cell axis in tissues with minimized mechanical tensions.

Angiosperms developed floral nectaries that reward pollinating insects. Although nectar function and composition have been characterized, the mechanism of nectar secretion has remained unclear. Here we identify SWEET9 as a nectary-specific sugar transporter in three eudicot species: Arabidopsis thaliana, Brassica rapa (extrastaminal nectaries) and Nicotiana attenuata (gynoecial nectaries). We show that SWEET9 is essential for nectar production and can function as an efflux transporter. We also show that sucrose phosphate synthase genes, encoding key enzymes for sucrose biosynthesis, are highly expressed in nectaries and that their expression is also essential for nectar secretion. Together these data are consistent with a model in which sucrose is synthesized in the nectary parenchyma and subsequently secreted into the extracellular space via SWEET9, where sucrose is hydrolysed by an apoplasmic invertase to produce a mixture of sucrose, glucose and fructose. The recruitment of SWEET9 for sucrose export may have been a key innovation, and could have coincided with the evolution of core eudicots and contributed to the evolution of nectar secretion to reward pollinators.

Scanning electron microscopy (SEM) is a powerful technique that can image exposed surfaces in 3D. Modern scanning electron microscopes, with field emission electron sources and in-lens specimen chambers, achieve resolutions of better than 0.5 nm and thus offer views of ultrastructural details of subcellular structures or even macromolecular complexes. Obtaining a reliable image is, however, dependent on sample preparation methods that robustly but accurately preserve biological structures. In plants, exposing the object of interest may be difficult due to the existence of a cell wall. This protocol shows how to isolate plant nuclei for SEM imaging of the nuclear envelope and associated structures from both sides of the nuclear envelope in cultured cells as well as in leaf or root cells. Further, it provides a method for uncovering membrane-associated cytoskeletal structures.

Background: The Gibberellic Acid (GA) signal is governed by the GAI (Gibberellic Acid Insensitive) repressor, which is characterized by a highly conserved N-terminal DELLA domain. Deletion of the DELLA domain results in constitutive suppression of GA signaling. As the GAI transcript is transportable in phloem elements, a Δ-DELLA GAI (gai) transgenic stock plant can reduce the stature of a scion through transport of gai mRNA from the stock. However, little is known about the characteristics of a scion on a gai stock., Results: Arabidopsis Δ-DELLA GAI (gai) was fused with a T7 epitope tag and expressed under the control of a companion cell-specific expression promoter, Commelina yellow mottle virus promoter (CoYMVp), to enhance transport in the phloem. The CoYMVp:Atgai-T7 (CgT) transgenic Nicotiana benthamiana exhibited a dwarf phenotype and lower sensitivity to GA enhancement of shoot stature. A wild-type (WT) scion on a CgT stock contained both Atgai-T7 mRNA and the translated product. Microarray analysis to clarify the effect of the CgT stock on the gene expression pattern in the scion clearly revealed that the WT scions on CgT stocks had fewer genes whose expression was altered in response to GA treatment. An apple rootstock variety, Malus prunifolia, integrating CoYMVp:Atgai moderately reduced the tree height of the apple cultivar scion., Conclusions: Our results demonstrate that Atgai mRNA can move from companion cells to sieve tubes and that the translated product remains at the sites to which it is transported, resulting in attenuation of GA responses by reducing the expression of many genes. The induction of semi-dwarfism in an apple cultivar on root stock harbouring Atgai suggests that long-distance transport of mRNA from grafts would be applicable to horticulture crops.

Nucleus-encoded ribonucleases and RNA-binding proteins influence chloroplast gene expression through their roles in RNA maturation and stability. One mechanism for mRNA 5' end maturation posits that sequence-specific pentatricopeptide repeat (PPR) proteins define termini by blocking the 5'→3' exonucleolytic activity of ribonuclease J (RNase J). To test this hypothesis in vivo, virus-induced gene silencing was used to reduce the expression of three PPR proteins and RNase J, both individually and jointly, in Nicotiana benthamiana. In accordance with the stability-conferring function of the PPR proteins PPR10, HCF152 and MRL1, accumulation of the cognate RNA species atpH, petB and rbcL was reduced when the PPR-encoding genes were silenced. In contrast, RNase J reduction alone or combined with PPR deficiency resulted in reduced abundance of polycistronic precursor transcripts and mature counterparts, which were replaced by intermediately sized species with heterogeneous 5' ends. We conclude that RNase J deficiency can partially mask the absence of PPR proteins, and that RNase J is capable of processing chloroplast mRNAs up to PPR protein-binding sites. These findings support the hypothesis that RNase J is the major ribonuclease responsible for maturing chloroplast mRNA 5' termini, with RNA-binding proteins acting as barriers to its activity.

The cell wall plays crucial roles in establishing the morphology of the plant cell, defence response to biotic and abiotic stresses, and mechanical properties of organs. The COBRA gene encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein that possesses the ability to modulate cellulose deposition and orient cell expansion in plant cell. We reported here the functional characterization of ClCOBL1, a conifer COBRA-like gene from the differentiating xylem of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook). ClCOBL1 belonged to a woody plant-specific clade of the COBRA protein family with several conserved motifs. Expression pattern demonstrated that ClCOBL1 was constitutively expressed but with high level in cambium region. ClCOBL1 protein was mainly located in the cell wall and plasma membrane. Overexpression of ClCOBL1 in tobacco plants yielded altered leaf adaxial-abaxial patterning and short, swollen corolla tubes. The changed leaf architecture in the ClCOBL1 overexpressors was associated with the differential expression of leaf adaxial-abaxial identity genes. Our results indicated that ClCOBL1 was involved in the determination of leaf dorsoventrality and anisotropic expansion possibly by affecting the expression of adaxial and abaxial identity genes., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Leafy gall is a plant hyperplasia induced upon Rhodococcus fascians infection. Previously, by genomic and transcriptomic analysis, it has been reported that, at the early stage of symptom development, both primary and secondary metabolisms are modified. The present study is based on the hypothesis that fully developed leafy gall, could represent a potential source of new bioactive compounds. Therefore, non-targeted metabolomic analysis of aqueous and chloroform extracts of leafy gall and non-infected tobacco was carried out by 1H-NMR coupled to principal component analysis (PCA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA). Polar metabolite profiling reflects modifications mainly in the primary metabolites and in some polyphenolics. In contrast, main modifications occurring in non-polar metabolites concern secondary metabolites, and gas chromatography and mass spectrometry (GC-MS) evidenced alterations in diterpenoids family. Analysis of crude extracts of leafy galls and non-infected tobacco leaves exhibited a distinct antiproliferative activity against all four tested human cancer cell lines. A bio-guided fractionation of chloroformic crude extract yield to semi-purified fractions, which inhibited proliferation of glioblastoma U373 cells with IC50 between 14.0 and 2.4 µg/mL. Discussion is focused on the consequence of these metabolic changes, with respect to plant defense mechanisms following infection. Considering the promising role of diterpenoid family as bioactive compounds, leafy gall may rather be a propitious source for drug discovery.

Transgenic tobacco plants that overexpress the ARGOS-LIKE (ARL) gene of Arabidopsis thaliana have been developed. The transgenic plants possessed increased dimensions of leaves and stem, whereas the magnitude of flowers was modified to a lesser degree. The increase in the organ dimensions was a result of stimulation of cell expansion; the cell quantity in the organ was even decreased. Ectopic expression of the ARL gene was promoted in order to increase in the level of mRNA of tobacco expansine NtEXPA5. It has been shown that the ARL gene of A. thaliana can be used to obtain transgenic plants with increased sizes of the leaves and stem.

Transgenic tobacco plants expressing the AINTEGUMENTA gene of rape under control of the 35S promoter and the promoter of dahlia mosaic virus were obtained. The transgenic plants were characterized by increase in the length of the leaves, flower sizes, stem height, and weight of seeds; at the same time, the degree of increase was greater in the case of use of the dahlia mosaic virus promoter as a regulator of transcription. Ectopic expression of the AINTEGUMENTA gene promoted prolongation of leaf growth, while sizes of epidermal cells of the leaves remained unchanged.

The mechanism underlying pollen tube growth involves diverse genes and molecular pathways. Alterations in the regulatory genes or pathways cause phenotypic changes reflected by cellular morphology, which can be captured using fluorescence microscopy. Determining and classifying pollen tube morphological phenotypes in such microscopic images is key to our understanding the involvement of genes and pathways. In this context, we propose a computational method to extract quantitative morphological features, and demonstrate that these features reflect morphological differences relevant to distinguish different defects of pollen tube growth. The corresponding software tool furthermore includes a novel semi-automated image segmentation approach, allowing to highly accurately identify the boundary of a pollen tube in a microscopic image., (© 2012 Institute of Botany, Chinese Academy of Sciences.)

KEY MESSAGE : We characterized a white flower mutant of allotetraploid N. tabacum as a DFR-deficient mutant; one copy of DFR has a cultivar-specific frameshift, while the other was deleted by heavy-ion irradiation. In most plants, white-flowered mutants have some kind of deficiency or defect in their anthocyanin biosynthetic pathway. Nicotiana tabacum normally has pink petals, in which cyanidin is the main colored anthocyanidin. When a relevant gene in the cyanidin biosynthetic pathway is mutated, the petals show a white color. Previously, we generated white-flowered mutants of N. tabacum by heavy-ion irradiation, which is accepted as an effective mutagen. In this study, we determined which gene was responsible for the white-flowered phenotype of one of these mutants, cv. Xanthi white flower 1 (xwf1). Southern blot analysis using a DNA fragment of the dihydroflavonol 4-reductase (DFR) gene as a probe showed that the xwf1 mutant lacked signals that were present in wild-type genomic DNAs. Sequence analysis demonstrated that one copy of the DFR gene (NtDFR2) was absent from the genome of the xwf1 mutant. The other copy of the DFR gene (NtDFR1) contained a single-base deletion resulting in a frameshift mutation, which is a spontaneous mutation in cv. Xanthi. Introduction of NtDFR2 cDNA into the petal limbs of xwf1 by particle bombardment resulted in production of the pink-colored cells, whereas introduction of NtDFR1 cDNA did not. These results indicate that xwf1 is a DFR-deficient mutant. One copy of NtDFR1 harbors a spontaneous frameshift mutation, while the other copy of NtDFR2 was deleted by heavy-ion beam irradiation.

In some species, a crucial role has been demonstrated for the seed endosperm during germination. The endosperm has been shown to integrate environmental cues with hormonal networks that underpin dormancy and seed germination, a process that involves the action of cell wall remodeling enzymes (CWREs). Here, we examine the cell wall architectures of the endosperms of two related Brassicaceae, Arabidopsis (Arabidopsis thaliana) and the close relative Lepidium (Lepidium sativum), and that of the Solanaceous species, tobacco (Nicotiana tabacum). The Brassicaceae species have a similar cell wall architecture that is rich in pectic homogalacturonan, arabinan, and xyloglucan. Distinctive features of the tobacco endosperm that are absent in the Brassicaceae representatives are major tissue asymmetries in cell wall structural components that reflect the future site of radicle emergence and abundant heteromannan. Cell wall architecture of the micropylar endosperm of tobacco seeds has structural components similar to those seen in Arabidopsis and Lepidium endosperms. In situ and biomechanical analyses were used to study changes in endosperms during seed germination and suggest a role for mannan degradation in tobacco. In the case of the Brassicaceae representatives, the structurally homogeneous cell walls of the endosperm can be acted on by spatially regulated CWRE expression. Genetic manipulations of cell wall components present in the Arabidopsis seed endosperm demonstrate the impact of cell wall architectural changes on germination kinetics.

The C-function, according to the ABC model of floral organ identity, is required for stamen and carpel development and to provide floral meristem determinacy. Members of the AG lineage of the large MADS box gene family specify the C-function in a broadly conserved manner in angiosperms. In core eudicots, two sub-lineages co-exist, euAG and PLE, which have been extensively characterized in Antirrhinum majus and Arabidopsis thaliana, where strong sub-functionalization has led to highly divergent contributions of the respective paralogs to the C-function. Various scenarios have been proposed to reconstruct the evolutionary history of the euAG and PLE lineages in eudicots, but detailed functional analyses of the roles of these genes in additional representative species to validate evolutionary hypotheses are scarce. Here, we report functional characterization of euAG- and PLE-like genes in Nicotiana benthamiana through expression analyses and phenotypic characterization of the defects caused by their specific down-regulation. We show that both paralogs redundantly contribute to the C-function in this species, providing insights on the likely evolution of these gene lineages following divergence of the major groups within the eudicots (rosids and asterids). Moreover, we have demonstrated a conserved role for the PLE-like genes in controlling fruit dehiscence, which strongly supports the ancestral role of PLE-like genes in late fruit development and suggests a common evolutionary origin of late developmental processes in dry (dehiscent) and fleshy (ripening) fruits., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Interfering with small RNA production is a common strategy of plant viruses. A unique class of small RNAs that require microRNA and short interfering (siRNA) biogenesis for their production is termed trans-acting short interfering RNAs (ta-siRNAs). Tomato (Solanum lycopersicum) wiry mutants represent a class of phenotype that mimics viral infection symptoms, including shoestring leaves that lack leaf blade expansion. Here, we show that four WIRY genes are involved in siRNA biogenesis, and in their corresponding mutants, levels of ta-siRNAs that regulate AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 are reduced, while levels of their target ARFs are elevated. Reducing activity of both ARF3 and ARF4 can rescue the wiry leaf lamina, and increased activity of either can phenocopy wiry leaves. Thus, a failure to negatively regulate these ARFs underlies tomato shoestring leaves. Overexpression of these ARFs in Arabidopsis thaliana, tobacco (Nicotiana tabacum), and potato (Solanum tuberosum) failed to produce wiry leaves, suggesting that the dramatic response in tomato is exceptional. As negative regulation of orthologs of these ARFs by ta-siRNA is common to land plants, we propose that ta-siRNA levels serve as universal sensors for interference with small RNA biogenesis, and changes in their levels direct species-specific responses.

The plastid (chloroplast) genomes of seed plants typically encode 30 tRNAs. Employing wobble and superwobble mechanisms, most codon boxes are read by only one or two tRNA species. The reduced set of plastid tRNAs follows the evolutionary trend of organellar genomes to shrink in size and coding capacity. A notable exception is the AUN codon box specifying methionine and isoleucine, which is decoded by four tRNA species in nearly all seed plants. However, three of these four tRNA genes were lost from the genomes of some parasitic plastid-containing lineages, possibly suggesting that less than four tRNA species could be sufficient to decode the triplets in the AUN box. To test this hypothesis, we have performed knockout experiments for the four AUN-decoding tRNAs in tobacco (Nicotiana tabacum) plastids. We find that all four tRNA genes are essential under both autotrophic and heterotrophic growth conditions, possibly suggesting tRNA import into plastids of parasitic plastid-bearing species. Phylogenetic analysis of the four plastid tRNA genes reveals striking conservation of all those bacterial features that are involved in discrimination between the different tRNA species containing CAU anticodons.