Endophytic fungi from rhizomes of <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

About 63 fungal endophytic isolates were separated from rhizomes of Paris polyphylla var. yunnanensis, which is a traditional medicinal plant mainly distributed in China. The isolates were characterized and grouped based on the culture characteristics and the morphology of colony growth and conidia. Eleven representative ones were selected for further taxonomical identification. Five genera namely Fusarium, Gliocladiopsis, Gliomastix, Aspergillus and Cylindrocarpon were identified on the basis of their morphological characterizations. Of them, the most frequent genus was Fusarium (i.e. Ppf1, Ppf3 and Ppf14). Their ITS-rDNA sequences were compared with those available in the GeneBank databases to obtain the closest related species by BLAST analysis as well as to analyze their phylogenetic affiliation. The isolates were identified as Gliocladiopsis irregularis (Ppf2), Plectosphaerella cucumerina (Ppf4), Padospora sp. (Ppf6), Gliomastix murorum var. murorum (Ppf7), Aspergillus fumigatus (Ppf9), Pichia guilliermondii (Ppf10), Neonectria radicicola (anamorph: Cylindrocarpon) (Ppf12) and one uncultured mycorrhizal ascomycete (Ppf13) separately based on their morphological and molecular features. The molecular characters of the endophytic fungi were basically coincident with their morphology. The broad diversity and taxonomic spectrum were exhibited by the endophytic fungi from P. polyphylla var. yunnanensis.     

Stimulation of saponin production in <Emphasis Type="Italic">Panax ginseng</Emphasis> hairy roots by two oligosaccharides from <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5–30 mg l⁻¹. HS and OS at 30 mg l⁻¹, fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70% to ∼20 g l⁻¹ from 13 g l⁻¹ and the total saponin content of roots by more than 1-fold to ∼3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides may have plant growth-regulatory activity in plant tissue cultures.     

Premature cytokinesis in pollen mother cells of transgenic tobacco plants (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.)

For the majority of dicotyledonous plants, cytokinesis in PMC is staged only once, i.e., after the completion of two cycles of caryokinesis. In the article, a cytological picture and the frequency characteristics of anomalies are shown, in which the cytokinesis in the PMCs of transgenic tobacco plants was already initiated after the first meiotic division. It is shown that, in such cells, the basic processes of cytoskeletal reorganization, which is typical for the simultaneous type of cytokinesis, remained unmodified. However, in most cases, premature division of cytoplasm took place with abnormalities, e.g., with the formation of a membranous “tunnel” or “gash.” It has been detected that the initialization of an additional round of cytokinesis is not an obstacle to the performance of the nuclear cycle or cytokinesis after the second meiotic division. Thus, in the presence of this anomaly, there is a change in the type of cytoplasm division, i.e., of simultaneous to successive.     

Whole genome resequencing of tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.) genotypes and high-throughput SNP discovery

Genome-wide single-nucleotide polymorphisms (SNPs) are highly useful in unraveling genetic insights and are essential to accelerate selections for genetic improvement in tobacco. The discovery of genome-wide SNPs in tobacco is very complex due to its high level of repetitive genome and polyploidy. At present, publicly available genomic data on SNPs are very limited, which warrants the need for high-throughput SNPs for application in tobacco breeding. In this research paper, we describe our efforts on SNP discovery by whole genome resequencing of 18 flue-cured Virginia (FCV) tobacco genotypes and annotation of SNPs in the tobacco genome. A large amount of data of about 225 GB per genotype was generated, with an average read depth of 50× using paired-end next-generation sequencing (NGS) with the HiSeq 2500 platform. The discovery of a large number of SNPs and indels was attempted to assist mapping and, thus, the selection processes to develop superior tobacco breeding lines. Discovered SNPs, their functional annotation, mapping to the reference genome, and their relative positioning in the linkage group are discussed in this paper.     

Stable Expression of Adalimumab in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Production of monoclonal antibodies and pharmaceutical proteins in transgenic plants has been the focus of many research efforts for close to 30 years. Use of plants as bioreactors reduces large-scale production costs and minimizes risk for human pathogens contamination. Stable nuclear transformation of the plant genome offers a clear advantage in agricultural protein production platforms, limited only by the number of hectares that can be cultivated. We report here, for the first time, successful and stable expression of adalimumab in transgenic Nicotiana tabacum plants. The plant-derived adalimumab proved fully active and was shown to rescue L929 cells from the in vitro lethal effect of rhTNFα just as effectively as commercially available CHO-derived adalimumab (Humira). These results indicate that agricultural biopharming is an efficient alternative to mammalian cell-based expression platforms for the large-scale production of recombinant antibodies.     

Photosynthesis in leaves of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. infected with tobacco mosaic virus

In tobacco leaves inoculated with tobacco mosaic virus (TMV), changes in chlorophyll (Chl) and carotenoid contents, parameters of slow Chl fluorescence kinetics, i.e. the maximum quantum yield of photosystem (PS2) photochemistry F_v/F_m, the effective quantum yield of photochemical energy conversion in PS2 Φ₂, ratio of quantum yields of photochemical and concurrent non-photochemical processes in PS2 F_v/F₀, non-photochemical quenching (NPQ), and photochemical activities of isolated chloroplasts from systemically infected tobacco leaves were investigated. We compared two successive stages of infection, the first in the stage of vein clearing at 9^th day post inoculation (dpi) and the second at 22^nd dpi when two different regions, i.e. light- (LGI) or dark-green (DGI) islands in the infected leaf were apparent and symptoms were fully developed. These two different regions were measured separately. The Chl and carotenoid contents in infected leaves decreased with a progression of infection and were lowest in LGI in the second stage. Also the ratio of Chl a/b declined in similar manner. The maximum quantum yield of PS2 photochemistry F_v/F_m, was decreased in the following order: first stage, DGI, and LGI. The same is true for the ratio F_v/F₀. The decrease of Φ₂ in infected leaves declined as compared to their controls. On the contrary, NPQ increased in infected leaves, the highest value was found in the first infection stage. Photochemical activities of the whole electron transport chain in isolated chloroplasts dramatically declined with the progression of symptoms, the lowest value was in LGI. Similarly, but to a lesser extent, the activity of PS2 in isolated chloroplasts decreased in infected leaves. Generally, the most marked impairment of the photosynthetic apparatus was manifested in the LGI of infected leaves.     

Phenolic compounds induced by <Emphasis Type="Italic">Bemisia tabaci</Emphasis> and <Emphasis Type="Italic">Trialeurodes vaporariorum</Emphasis> in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. and their relationship with the salicylic acid signaling pathway

Changes in the levels of secondary compounds can trigger plant defenses. To identify phenolic compounds induced by Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) in tobacco (Nicotiana tobacco L.), the content changes of 11 phenolic compounds in plants infested by B. tabaci MEAM1 or Trialeurodes vaporariorum were compared. The chlorogenic acid, catechin, caffeic acid, p-coumaric acid, rutin, and ferulic acid contents in B. tabaci MEAM1-infested tobacco plants increased significantly, having temporal and spatial effects, compared with uninfested control and T. vaporariorum infested plants. The contents were 4.10, 2.84, 2.25, 3.81, 1.46, and 1.91 times higher, respectively, than those in the control. However, a T. vaporariorum nymphal infestation just caused smaller chlorogenic acid, catechin, caffeic acid, and rutin contents increase, which were 2.33, 2.13, 1.59, and 3.19 times higher, respectively, than those in the control. In B. tabaci MEAM1 third-instar nymph-infested plants, chlorogenic acid, catechin, caffeic acid, and rutin increased more significantly in systemic than in local leaves. Salicylate-deficient plants inhibited the induction of the content of 10 phenolic compounds, but not caffeic acid, after a B. tabaci MEAM1 nymphal infestation. Thus, the elevated levels of phenolic compounds induced by B. tabaci MEAM1 were correlated with the salicylic acid signaling pathway and induced the responses of defense-related phenolic compounds.     

Random amplified DNA polymorphism of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. cultivars

The polymorphism, similarities and relationships among Nicotiana tabacum L. cultivars were assessed with RAPD analyses. One hundred and forty-nine bands were detected, of which 94 were polymorphic (63.1 %). A primer distinguishing all of the tested cultivars was found. High similarity between cultivars was revealed, and cultivar relationships were estimated through cluster analysis (UPGMA) based on RAPD data.The experiments in this study were carried out at the South Center Tobacco Breeding Research of China; the expense was provided by Yunnan Tobacco Company.     

Expression of HPV-11 L1 protein in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

Background  

We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine.     

Effect of seed-specific expression of the <Emphasis Type="Italic">ipt</Emphasis> Gene on <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. seed composition

A transgenic approach to manipulation of endosperm development has been investigated. Nicotiana tabacum cv. Xanthi, an endosperm-containing dicotyledon, has been used as a model plant and the 2.6 kb wheat high molecular weight (HMW) glutenin subunit 12 promoter has been used fused either to the gus reporter gene (HMWgus construct)—to study promoter characteristics—or to the Agrobacterium ipt gene—to study the effect of cytokinin (CK) over-expression on assimilate accumulation in the seed. In transgenic tobacco the promoter:gus fusion showed that HMW is an endosperm-specific promoter with maximum expression 20 days after anthesis (DAA), corresponding to the mid to late stages of seed development. Transgenic plants containing the HMWipt construct showed no morphological abnormalities but they had an average increase in seed weight and total ethanol-insoluble carbohydrates and protein content of 8.1%, 7.0% and 8.3%, respectively. SDS PAGE analysis demonstrated that the effect on protein accumulation was non-specific. The highest values of the parameters analysed correlated with moderate increases in the levels of biologically active CKs. These results suggest that ectopic expression of small amounts of CKs can be used to increase storage assimilate accumulation without a detrimental effect on development.     

Association of diamine oxidase and <Emphasis Type="Italic">S</Emphasis>-adenosylhomocysteine hydrolase in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> extracts

The oxidative deamination of methylated putrescine by a diamine oxidase activity (DAO) is an important step in the biosynthesis of nicotine in tobacco and tropane alkaloids in several Solanaceous plants. A polyclonal rabbit antiserum was previously developed to a purported purified DAO enzyme from Nicotiana tabacum. The antiserum bound to a single 53 kDa protein and immunoprecipitated 80 of DAO activity from tobacco root extracts. In an effort to obtain DAO cDNAs, this antiserum was used to screen a tobacco cDNA expression library and three distinct immunoreactive cDNA clones were isolated. These cDNAs encoded predicted proteins that were either identical or nearly identical to predicted S-adenosylhomocysteine hydrolase (SAHH) from two Nicotiana species. Thus, the rabbit antiserum was not specific to DAO, even though it immunodepleted the majority of DAO activity from root extracts. Alternative hypotheses to explain the DAO immunodepletion results (such as poisoning of DAO activity or that SAHH is a bifunctional enzyme) were tested and ruled out. Therefore, we hypothesize that SAHH associates with DAO as part of a larger multienzyme complex that may function in planta as a nicotine metabolic channel.     

Changes in cytokinin levels and metabolism in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.) explants during in vitro shoot organogenesis induced by <Emphasis Type="Italic">trans</Emphasis>-zeatin and dihydrozeatin

The uptake and metabolism of trans-zeatin and/or dihydrozeatin, in correlation with cytokinin oxidase/dehydrogenase (CKX) and β-glucosidase activity, were studied in leaf segments derived from wild-type (WT) and transgenic (T) tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) during in vitro induction of shoot organogenesis. T explants harbored the maize gene Zm-p60.1β-glucosidase. Higher levels of shoot regeneration were observed on T explants in the early stages of cultivation. In WT explants, the content of cytokinin (CK)-O- and N-glucosides increased. In T explants, a higher content of Z-9-riboside and Z-9-riboside-5′-monophosphate and higher CKX activity during the early stage of cultures were found. A positive correlation was obtained for bioactive CK content and the organogenic response in T explants. Our results indicate a connection between the organogenic capacity of tobacco explants, metabolism of endogenous CKs and uptake of exogenous CKs from the cultivation medium.     

Induced expression of <Emphasis Type="Italic">Serratia marcescens</Emphasis> ribonuclease III gene in transgenic <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. cv. SR1 tobacco plants

Transgenic Nicotiana tabacum L. cv. SR1 plants, characterized by an increase in the level of dsRNA-specific hydrolytic activity after induction by wounding, were obtained. The Solanum lycopersicum anionic peroxidase gene promoter (new for plant genetic engineering) was for the first time used for the induced expression of the target Serratia marcescens RNase III gene. Upon infection with the tobacco mosaic virus (TMV), the transgenic plants of the obtained lines did not differ significantly from the control group in the level of TMV capsid protein accumulation. In general, no delay in the development of the infection symptoms was observed in transgenic plants as compared with the control group. The obtained transgenic plants represent a new model for the study of the biological role of endoribonucleases from the RNase III family, including in molecular mechanisms of resistance to pathogens.     

Responses of transgenic <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> seedlings expressing a <Emphasis Type="Italic">Cucurbita pepo</Emphasis> antisense <Emphasis Type="Italic">PHYA</Emphasis> RNA to far-red radiation

The Nicotiana tabacum transgenic plants expressing a Cucurbita pepo antisense PHYA RNA were obtained. The seedlings of transgenic tobacco with reduced phytochrome A (PHYA) content displayed decreased sensitivity to continuous broad-band far-red radiation (λ > 680 nm). Under far-red irradiance transgenic seedlings showed less elongation of the hypocotyls, more rapid plastid development, more chlorophyll accumulation, less repression of lightdependent NADPH:protochlorophyllide oxidoreductase than wild-type plants that was in accordance with PHYA control of plant development. Dynamics of the far-red radiation dependent changes in low temperature chlorophyll fluorescence spectra for the transgenic and wild-type seedlings were consistent with the more rapid formation of photosynthetic apparatus in the seedlings with reduced PHYA.     

Biochemical Characterization of Cytokinin Oxidases/Dehydrogenases from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Expressed in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Transgenic tobacco plants overexpressing single Arabidopsis thaliana cytokinin dehydrogenase (CKX, EC 1.5.99.12) genes AtCKX1, AtCKX2, AtCKX3, AtCKX4, AtCKX5, AtCKX6, and AtCKX7 under the control of a constitutive 35S promoter were tested for CKX-enzymatic activity with varying pH, electron acceptors, and substrates. This comparative analysis showed that out of these, only AtCKX2 and AtCKX4 were highly active enzymes in reaction with isoprenoid cytokinins (N ⁶ -(2-isopentenyl)adenine (iP), zeatin (Z)) and their ribosides using the artificial electron acceptors 2,6-dichlorophenol indophenol (DCPIP) or 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q₀). Turnover rates of these cytokinins by four other AtCKX isoforms (AtCKX1, AtCKX3, AtCKX5, and AtCKX7) were substantially lower, whereas activity of AtCKX6 was almost undetectable. The isoenzymes AtCKX1 and AtCKX7 showed significant preference for cytokinin glycosides, especially N ⁶ -(2-isopentenyl)adenine 9-glucoside, under weakly acidic conditions. All enzymes preferentially cleave isoprenoid cytokinins in the presence of an electron acceptor, but aromatic cytokinins are not resistant and are degraded with lower reaction rates as well. Cytokinin nucleotides, considered as resistant to CKX attack until now, were found to be potent substrates for some of the CKX isoforms. Substrate specificity of AtCKXs is discussed in this study with respect to the structure of the CKX active site. Further biochemical characterization of the AtCKX1, AtCKX2, AtCKX4 and AtCKX7 enzymes showed pH-dependent activity profiles.     

Cloning of new rubisco promoters from <Emphasis Type="Italic">Brassica rapa</Emphasis> and determination of their activity in stably transformed <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> plants

The aim of our study was to identify the highest expressing rubisco small subunit (RbcS) promoters (pRbcS) from the cotyledons of germinating seedlings of Brassica rapa var. oleifera to drive high-level and preferably stage-specific transgenic protein expression in Brassicaceae plants. We cloned four new pRbcS promoters using several approaches, including the construction of a cDNA library and use of genome walking technique. Real-time PCR analysis of RbcS mRNA expression clearly showed that two of these promoters exhibited the highest activity on the germination stage of plant development. We used gusA expression as a reporter of promoter activity in Brassica napus and Nicotiana tabacum plants that were transformed with the constructs using an Agrobacterium-mediated transformation strategy. The mRNA level of RbcS and of gusA was quantified in transformed plants. The data obtained demonstrate that the promoter most active in seedlings under native conditions was also most active in transgenic constructs at the same stage of plant development. The fine structure of the promoters is discussed herein.