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The bHLH transcription factors play pivotal roles in plant growth and development, production of secondary metabolites and responses to various environmental stresses. Although the bHLH genes have been well studied in model plant species, a comprehensive investigation of the bHLH genes is required for tobacco with newly obtained high-quality genome. In the present study, a total of 309 NtbHLH genes were identified and can be divided into 23 subfamilies. The conserved amino acids which are essential for their function were predicted for the NtbHLH proteins. Moreover, the NtbHLH genes were conserved during evolution through analyzing the gene structures and conserved motifs. A total of 265 NtbHLH genes were localized in the 24 tobacco chromosomes while the remained 44 NtbHLH genes were mapped to the scaffolds due to the complexity of tobacco genome. Moreover, transcripts of NtbHLH genes were obviously tissue-specific expressed from the gene-chip data from 23 tobacco tissues, and expressions of 20 random selected NtbHLH genes were further confirmed by quantitative real-time PCR, indicating their potential functions in the plant growth and development. Importantly, overexpressed NtbHLH86 gene confers improve drought tolerance in tobacco indicating that it might be involved in the regulation of drought stress. Therefore, our findings here provide a valuable information on the characterization of NtbHLH genes and further investigation of their functions in tobacco.

Cytoplasmic male sterile (CMS) lines are valuable resources in plant breeding for heterosis utilization, guaranteeing quality of hybrid seeds, and genetic integrity. Nta(sua)S K326, which contains the nuclei of Nicotiana tabacum cv. K326 and cytoplasm of N. suaveolens, is currently the dominant CMS source used in tobacco leaf production in Yunnan, China. Therefore, the alternative CMS lines are required. Here, the Nta(gla)S K326 with cytoplasm of N. glauca has been generated from asymmetric somatic hybridization between protoplasts of K326 and N. glauca followed by backcrossing with K326. Flow cytometry analysis revealed that Nta(gla)S has almost identical ploidy level as that in K326. Moreover, Nta(gla)S, Nta(sua)S, and K326 plants showed similar relative growth rate, yield, and contents of several chemical components in the field. Further scaning electron microscopic observation of floral organs revealed that the Nta(gla)S has shortened filaments and shrivelled stamens, whereas the Nta(sua)S has severe degenerated and carpel-liked stamens, compared with fertile K326 plants. In addition, the expression patterns of several mitochondrial genes in Nta(gla)S, Nta(sua)S, and K326 plants were compared. Furthermore, the cytoplasm source of Nta(gla)S and Nta(sua)S could be distinguished by cleaved amplified polymorphic sequence (CAPS) analysis with chloroplast-specific universal primers. Taken together, these results showed the development of a Nicotiana CMS hybrid via asymmetric somatic hybridization, suggesting that flow cytometry analysis at early stage could facilitate and accelerate the selection process of generating CMS lines. Importantly, the Nta(gla)S provides a valuable germplasm as an alternative CMS line instead of Nta(sua)S in Nicotiana hybrid breeding. The Nicotiana CMS hybrid Nta(gla)S has been generated by asymmetric protoplast hybridization. The Nta(gla)S provides a valuable germplasm as an alternative CMS line instead of Nta(sua)S in Nicotiana hybrid breeding.

Most mutualisms are exploited by parasites, which must strike an evolutionary balance between virulence and long‐term persistence. Fig‐associated nematodes, living inside figs and dispersed by fig wasps, are thought to be exploiters of the fig–fig wasp mutualism. The life history of nematodes is synchronized with the fig development and adapted to particular developmental characteristics of figs. We expect host breeding systems (monoecious vs. gynodioecious figs) and seasonality to be central to this adaptation. However, the details of the adaptation are largely unknown. Here, we conducted the first field surveys on the prevalence of nematodes from monoecious Ficus microcarpa L.f. (Moraceae), gynodioecious Ficus hispida L.f., and their pollinating fig wasps in two seasons and two developmental stages of figs in Xishuangbanna, China. We followed this up by quantifying the effects of nematodes on fitness‐related traits on fig wasps (e.g., egg loads, pollen grains, and longevity) and fig trees (seed production) in gynodioecious F. hispida. The magnitude of nematode infection was compared between pre‐ and post‐dispersal pollinators to quantify the probability of nematodes being transported to new hosts. Our results showed that Ficophagus microcarpus (Nematoda: Aphelenchoididae) was the only nematode in F. microcarpa. In F. hispida, Martininema guangzhouensis (Nematoda: Aphelenchoididae) was the dominant nematode species, whereas Ficophagus centerae was rare. For both species of Ficus, rainy season and inter‐floral figs had higher rates of nematode infection than the dry‐hot season and receptive figs. Nematodes did not affect the number of pollen grains or egg loads of female wasps. We did not detect a correlation between seed production and nematode infection. However, carrying nematodes reduced the lifespan and dispersal ability of pollinator wasps, indicating higher rates of post‐emergence mortality in infected fig wasps. Severely infected fig wasps were likely ‘filtered out’, preventing the overexploitation of figs by wasps and stabilizing the interaction over evolutionary time.

Theory identifies factors that can undermine the evolutionary stability of mutualisms. However, theory's relevance to mutualism stability in nature is controversial. Detailed comparative studies of parasitic species that are embedded within otherwise mutualistic taxa (e.g., fig pollinator wasps) can identify factors that potentially promote or undermine mutualism stability. We describe results from behavioral, morphological, phylogenetic, and experimental studies of two functionally distinct, but closely related, Eupristina wasp species associated with the monoecious host fig, Ficus microcarpa, in Yunnan Province, China. One (Eupristina verticillata) is a competent pollinator exhibiting morphologies and behaviors consistent with observed seed production. The other (Eupristina sp.) lacks these traits, and dramatically reduces both female and male reproductive success of its host. Furthermore, observations and experiments indicate that individuals of this parasitic species exhibit greater relative fitness than the pollinators, in both indirect competition (individual wasps in separate fig inflorescences) and direct competition (wasps of both species within the same fig). Moreover, phylogenetic analyses suggest that these two Eupristina species are sister taxa. By the strictest definition, the nonpollinating species represents a "cheater" that has descended from a beneficial pollinating mutualist. In sharp contrast to all 15 existing studies of actively pollinated figs and their wasps, the local F. microcarpa exhibit no evidence for host sanctions that effectively reduce the relative fitness of wasps that do not pollinate. We suggest that the lack of sanctions in the local hosts promotes the loss of specialized morphologies and behaviors crucial for pollination and, thereby, the evolution of cheating.

Nicotine is one of the most important secondary metabolites in tobacco, and its biosynthesis can be induced by topping and jasmonic acid treatment. NtMYC2s play pivotal roles in the regulation of nicotine. The mediator server as a bridge betwen the transcription factors and RNA polymerase in order to facilitates transcription and functions in plants. However, the role of mediator in the regulation of nicotine biosynthesis remains unknown. In this study, we firstly identify the NtMED25 through homologous analysis. NtMED25 interacts with NtMYC2s through the MD region. Interestingly, the nicotine content is decreased in the the knock-down transgenic lines of NtMED25, and the expression levels of two nicotine biosynthesis genes, NtQPT2 and NtPMT2, are also reduced when compared with that in the wild-type plants. Furthermore, NtMED25 enhances the binding of NtMYC2a/ NtMYC2b to the promoter of NtPMT2 and NtQPT2, and then facilitates the nicotine biosynthesis. Therefore, our study revealed the function of mediator in the regulation of nicotine, and provide the insight role on the transcriptional regulation of plant secondary metabolites.

Background Abscisic acid (ABA) is an important phytohormone for plant growth, development and responding to stresses such as drought, salinity, and pathogen infection. Pyrabactin Resistance 1 (PYR1)/PYR1-Like (PYL)/Regulatory Component of ABA Receptor (RCAR) (hereafter referred to as PYLs) has been identified as the ABA receptors. The PYL family members have been well studied in many plants. However, the members of PYL family have not been systematically identified at genome level in cultivated tobacco (Nicotiana tabacum) and its two ancestors. In this study, the phylogenic relationships, chromosomal distribution, gene structures, conserved motifs/regions, and expression profiles of NtPYLs were analyzed. Results We identified 29, 11, 16 PYLs in the genomes of allotetraploid N. tabacum, and its two diploid ancestors N. tomentosiformis and N. sylvestris, respectively. The phylogenetic analysis revealed that NtPYLs can be divided into three subfamilies, and each NtPYL has one counterpart in N. sylvestris or N. tomentosiformis. Based on microarray analysis of NtPYL transcripts, four NtPYLs (from subfamily II, III), and five NtPYLs (from subfamily I) are highlighted as potential candidates for further functional characterization in N. tabacum seed development, response to ABA, and germination, and resistance to abiotic stresses, respectively. Interestingly, the expression profiles of members in the same NtPYL subfamily showed somehow similar patterns in tissues at different developmental stages and in leaves of seedlings under drought stress, suggesting particular NtPYLs might have multiple functions in both plant development and drought stress response. Conclusions NtPYLs are highlighted for important functions in seed development, germination and response to ABA, and particular in drought tolerance. This work will not only shed light on the PYL family in tobacco, but also provides some valuable information for functional characterization of ABA receptors in N. tabacum. Electronic supplementary material The online version of this article (10.1186/s12864-019-5839-2) contains supplementary material, which is available to authorized users.

1. Species interactions in tightly bound ecological mutualisms often feature highly specialised species' roles in which competitive exclusion may preclude multi‐species coexistence. Among the 800 fig (Ficus) species, it was originally considered that each was pollinated by their own wasp (Agaonidae). However, recent investigations show that this ‘one‐to‐one’ rule often breaks down, as fig species regularly host multiple agaonids but in ways suggesting that competitive processes still mediate biodiversity outcomes. 2. A phenological survey was conducted of the fig–fig wasp pair, Ficus microcarpa and its associated pollinating wasp, alongside its sister species, the cheating wasp, in Xishuangbanna, China. 3. Reproductive output underwent extreme seasonal variation. Seed and pollinator production fell markedly during cooler, drier months, although high levels of fig production continued. However, this resource was predominantly utilised by the cheater species, which offers no pollination services. Pollinators and cheaters rarely co‐occur, suggesting that temporal coexistence is constrained by competition for access to figs. 4. The overall findings indicate periodic rearrangements of mutualism dynamics, probably resulting from a strongly seasonal environment. Sympatric co‐occurrence may result from a window of opportunity for a functionally divergent agaonid, potentially due to constraints on the main pollinator in adapting to variable year‐round conditions that prevent competitive exclusion.

The biosynthesis of flavonols and anthocyanins is precisely regulated by different transcription factors in plants. WRKY11 promotes the biosynthesis of flavonoids in apple, but the molecular mechanism of WRKY11 regulating flavonols biosynthesis, and whether WRKY11 plays the same roles in other plants species remains to be further studied. Here, we cloned four NtWRKY11 genes from tobacco, which all contained the conserved WRKYGQK heptapeptide and a zinc-finger motif. The NtWRKY11b showed higher expression levels than the other NtWRKY11 genes in all the tobacco tissues examined, especially in tobacco leaves. Silencing of NtWRKY11b in tobacco leaves reduced the content of flavonols to 45.2 %-69.8 % of that in the WT plants, but overexpression of NtWRKY11b increased the flavonols content by 37.8 %-80.7 %. Transcriptome analysis revealed 8 flavonoids related differentially expressed genes (DEGs) between NtWRKY11b-OE and WT plants, among which the transcription of NtMYB12, NtFLS, NtGT5, and NtUFGT was significantly induced by posttranslational activation of NtWRKY11b with the presence of protein synthesis inhibitor, indicating a putative direct promotion of NtWRKY11b on the transcription of these flavonoids related genes. Chromatin immunoprecipitation assays further demonstrated that NtWRKY11b could bind to the promoter regions of NtMYB12, NtFLS, NtGT5, and NtUFGT to activate the transcription of these genes. Moreover, ectopic expression of NtWRKY11b also promoted the expression levels of NtCML38, NtCTL1, NtWRKY44, and NtCML37 genes, which have been shown to enhance plant resistance to various stresses. Our findings revealed the molecular mechanism of NtWRKY11b regulating flavonols biosynthesis, and provided a promising target for increasing flavonols content in tobacco.

MADS-box genes play a pivotal role in various processes, including floral and seed development, controlling flowering time, regulation of fruits ripening, and respond to abiotic and biotic stressors in planta. Tobacco (Nicotiana tabacum) has been widely used as a model plant for analyzing the gene function, however, there has been less information on the regulation of flowering, and the associated genes. In the present study, a total of 168 NtMADS-box genes were identified from tobacco, and their phylogenetic relationship, chromosome locations, and gene structures were further analyzed. NtMADS-box genes can be clustered into four sub-families of M&alpha, M&gamma, MIKC*, and MIKCC. A total of 111 NtMADS-box genes were distributed on 20 chromosomes, and 57 NtMADS-box genes were located on the unanchored scaffolds due to the complex and incomplete assembly of the tobacco genome. Expression profiles of NtMADS-box genes by microarray from 23 different tissues indicated that members in different NtMADS-box gene subfamilies might play specific roles in the growth and flower development, and the transcript levels of 24 NtMADS-box genes were confirmed by quantitative real-time PCR. Importantly, overexpressed NtSOC1/NtMADS133 could promote early flowering and dwarfism in transgenic tobacco plants. Therefore, our findings provide insights on the characterization of NtMADS-box genes to further study their functions in plant development.

An effective method for Agrobacterium tumefaciens inhibition using silica nanoparticles (NPs) as the carrier and melittin as the antibacterial drug is developed. The diameters of the inhibition zones of A. tumefaciens treated with different concentrations of melittin-conjugated silica NPs, pure silica NPs, gentamicin and free melittin are determined. The results indicate that the pure silica NPs have no impact on A. tumefaciens. The melittin-conjugated silica NPs have strong antibacterial activity against A. tumefaciens. In addition, the antibacterial activity of the melittin-conjugated silica NPs is dose dependent. The diameter of the inhibition zone caused by the melittin-conjugated silica NPs is almost the same as the positive control. Compared with the free drug molecules, silica NPs as an ideal carrier can gather a large number of drug molecules on their surface so as to greatly enhance the antibacterial activity.

To investigate the protective effects and the possible molecular mechanisms of chlorogenic acid on H2O2-induced dysfunction and apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were divided into three groups: H2O2groups (0, 5, 10, 20, 50mM), chlorogenic acid (100nmol/L) groups and atorvastatin (10μmol/L) groups. The eNOS mRNA expression was measured by RT-PCR. The content of NO in the supernatant was detected by the nitrate reductase assay. Cells activity was evaluated by the MTT assay. The level of reactive oxygen species (ROS) in cells was measured using H2DCF-DA as a fluorescence probe. Results demonstrated that H2O2caused a significant down-regulation of eNOS mRNA expression and decreased the secretion of NO in a dose-dependent manner compared with the control group. Furthermore, H2O2 induced cell apoptosis, increased ROS accumulation. Nevertheless, these effects of H2O2could be blocked by chlorogenic acid in chlorogenic acid pretreatment groups. CONCLUSION:chlorogenic acid can inhibite H2O2-induced dysfunction and apoptosis in HUVECs, which may be related to its effects on suppressing oxidative stress, up-regulating eNOS pathway. These results suggest the usefulness of chlorogenic acid particularly in the treatment of cardiovascular disease.

Plants have varying abilities to tolerate chilling (low but not freezing temperatures), and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance., Author Summary Compared to cold acclimation (enhancement of freezing tolerance by a prior exposure to low non-freezing temperature), the tolerance mechanism to non-freezing chilling temperatures is not well understood. Here, we performed a genome-wide mutant screen for chilling sensitive phenotype and identified 49 candidate genes important for chilling tolerance in Arabidopsis. Among the proteins encoded by these 49 genes, 16 are annotated as having chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. We further studied RBD1, one of the four RNA-binding proteins localized to chloroplast. RBD1 is only expressed in the green photosynthetic tissues and is localized to nucleoid of chloroplasts. Furthermore, RBD1 is found to be a regulator of 23S rRNA processing likely through direct binding to the precursor of 23S rRNA in a temperature dependent manner. Our study thus reveals the importance of chloroplast function especially protein translation in chilling tolerance at genome-wide scale and suggests an adaptive mechanism involving low temperature enhanced activities from proteins such as RBD1 in chilling tolerance.

Microbial-derived volatiles are ubiquitous in the environment and actively engaged in bio-communication with other organisms. Recently, some volatile-producing endophytes (VPEs), cryptic fungal symbionts persisting in healthy plant tissues, have attracted great attention due to their strong antibiotic activity or production of carbon chains that are identical to many of those found in petroleum, while other fragrant volatiles can be used in the flavoring industries. From an application-oriented and biotechnological point of view, these findings show significant promise for sustainable development of agriculture, forestry, and industry, especially in the control of fruit postharvest diseases, soil-borne pathogen management, and bio-fuel production. In comparison, the ecological importance of VPEs has only rarely been addressed and warrants further exploration. In this review, we summarize the current knowledge and future directions in this fascinating research field, and also highlight the constraints and progresses towards commercialization of VPEs products.

Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress.

graphical a bstract abstract Fig trees (Ficus spp.) are popular ornamental trees that are entirely dependent on a mutalistic association with host-specific pollinating fig wasps for reproduction. They can become naturalized and invasive in countries where the associated pollinator is also established. Figs. (syconia) are also also utilized by a diverse community of organisms that are potentially detrimental to the pollinators or seed production. Ficus benjamina is a widely-planted fig tree with the ability to establish outside its native range. We examined the impact of an undescribed gall midge species associated with F. benjamina within the plant's natural range in Xishuangbanna, south-western China. Observations on the levels of abundance of the midge together with fig abortion and seed germination rates showed that the gall midge had a strong negative effect on reproduction. The gall midge reduced pollinator survival and at high densities elimi- nated all pollinators, due primarily to premature abortion of figs. Seed numbers were only reduced at high gall midge densities, but seed quality, as measured by germination success and root growth rates, was greatly reduced whenever the gall midge was present. Within its presumed natural range the gall midge appears to be host specific, and given its dramatic impact on host reproductive success, is a poten- tial candidate for the biological control of F. benjamina.

The plant hormone abscisic acid (ABA) plays important roles in regulating plant growth, development, and responses to environmental stresses. Proteins in the PYR/PYL/RCAR family (hereafter referred to as PYLs) are known as ABA receptors. Since most studies thus far have focused on Arabidopsis PYLs, little is known about PYL homologs in crop plants. We report here the characterization of 21 PYL homologs (GmPYLs) in soybean. Twenty-three putative GmPYLs can be found from soybean genome sequence and categorized into three subgroups. GmPYLs interact with AtABI1 and two GmPP2Cs in diverse manners. A lot of the subgroup I GmPYLs interact with PP2Cs in an ABA-dependent manner, whereas most of the subgroup II and III GmPYLs bind to PP2Cs in an ABA-independent manner. The subgroup III GmPYL23, which cannot interact with any of the tested PP2Cs, differs from other GmPYLs. The CL2/gate domain is crucial for GmPYLs-PP2Cs interaction, and a mutation in the conserved proline (P109S) abolishes the interaction between GmPYL1 and AtABI1. Furthermore, the ABA dependence of GmPYLs-PP2Cs interactions are partially correlated with two amino acid residues preceding the CL2/gate domain of GmPYLs. We also show that GmPYL1 interacts with AtABI1 in an ABA-dependent manner in plant cells. Three GmPYLs differentially inhibit AtABI1 and GmPP2C1 in an ABA-dependent or -enhanced manner in vitro. In addition, ectopically expressing GmPYL1 partially restores ABA sensitivity of the Arabidopsis triple mutant pyr1/pyl1/pyl4. Taken together, our results suggest that soybean GmPYLs are ABA receptors that function by interacting and inhibiting PP2Cs.

Through bioassay-guided fractionation, the EtOAc extract of a culture broth of the endophytic fungus Phoma species ZJWCF006 in Arisaema erubescens afforded a new α-tetralone derivative, (3S)-3,6,7-trihydroxy-α-tetralone (1), together with cercosporamide (2), β-sitosterol (3), and trichodermin (4). The structures of compounds were established on the basis of spectroscopic analyses. Compounds 1, 2, and 3 were obtained from Phoma species for the first time. Additionally, the compounds were subjected to bioactivity assays, including antimicrobial activity, against four plant pathogenic fungi (Fusarium oxysporium, Rhizoctonia solani, Colletotrichum gloeosporioides, and Magnaporthe oryzae) and two plant pathogenic bacteria (Xanthomonas campestris and Xanthomonas oryzae), as well as in vitro antitumor activities against HT-29, SMMC-772, MCF-7, HL-60, MGC80-3, and P388 cell lines. Compound 1 showed growth inhibition against F. oxysporium and R. solani with EC50 values of 413.22 and 48.5 μg/mL, respectively. Additionally, compound 1 showed no cytotoxicity, whereas compound 2 exhibited cytotoxic activity against the six tumor cell lines tested, with IC50 values of 9.3 ± 2.8, 27.87 ± 1.78, 48.79 ± 2.56, 37.57 ± 1.65, 27.83 ± 0.48, and 30.37 ± 0.28 μM, respectively. We conclude that endophytic Phoma are promising sources of natural bioactive and novel metabolites.