Changes of lignin biosynthesis in tobacco leaves during maturation.

Lignin is one of the most valuable renewable industrial materials. To elucidate the mechanism via which lignin is synthesised, we compared the lignin content, leaf hardness, cell wall thickness of palisade tissue, and gene expression patterns of lignin biosynthetic enzymes in three tobacco (Nicotiana tabacum L.) varieties during maturation. The results consistently showed that during maturation, the accumulation of lignin gradually increased in tobacco leaves, reaching a peak at full maturity (45 days after topping), and then gradually decreased. Similarly, the transcript level analysis revealed that the gene expression pattern of NtPAL, NtC4H, NtCCoAOMT and NtCOMT were relatively high, and consistent with the lignin content changes. Thus, the four genes may play regulatory roles in the synthesis of tobacco lignin. Analysis of tissue expression patterns of the lignin synthesis-related gene showed that the NtPAL, NtC4H, Nt4CL, NtHCT, NtCCoAOMT, NtCOMT, NtCCR, NtCAD, and NtPAO were all expressed in stems, roots, and leaves. NtC3H and NtF5H were specifically expressed in stems and roots, and not in leaves. Consistently, the NtC3H promoter induced high GUS expression in stems and petioles, marginal in roots, and no GUS activity in leaves. These results provide insights into molecular regulation of lignin biosynthesis in tobacco.

Plant-derived compounds: A potential source of drugs against Tobacco mosaic virus.

Tobacco mosaic virus (TMV) is an important plant virus that led to significant losses in the crops worldwide. In this study, the antiviral activities of Ursolic Acid (UA) and 4-methoxycoumarin against TMV and their underlying mechanisms were initially investigated for the first time. The results demonstrated that the antiviral effects of UA and 4-methoxycoumarin were as effective as those of the commercial agent lentinan, in either the protective effect, inactivation effect or curative effect. In addition, both plant-derived compounds could induce the resistance responses of tobacco plants against TMV, showing increased antioxidant enzyme activities (SOD and POD) and H2O2 accumulation in tobacco leaves after treatment with UA or 4-methoxycoumarin, along with highly expressed regulatory and defence genes in the salicylic acid signaling pathway. Meanwhile, electrolyte leakage and malondialdehyde experiments indicated that these effects did not result in phytotoxicity or damage to the leaf plasma membrane of tobacco plants. Collectively, the results demonstrate that UA and 4-methoxycoumarin have potential as eco-friendly and safe strategies to control TMV in the future.

Comparative transcriptomic analysis reveals that multiple hormone signal transduction and carbohydrate metabolic pathways are affected by Bacillus cereus in Nicotiana tabacum.

Bacillus cereus is thought to be a beneficial bacterium for plants in several aspects, such as promoting plant growth and inducing plant disease resistance. However, there is no detailed report on the effect of Bacillus cereus acting on Nicotiana tabacum. In the present study, RNA-based sequencing (RNA-seq) was used to identify the molecular mechanisms of the interaction between B. cereus CGMCC 5977 and N. tabacum. A total of 7345 and 5604 differentially expressed genes (DEGs) were identified from leaves inoculated with Bacillus cereus at 6 and 24 hpi, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the most DEGs could be significantly enriched in hormone signal transduction, the MAPK signaling pathway, photosynthesis, oxidative stress, and amino sugar, and nucleotide sugar metabolism. Furthermore, glycolysis/gluconeogenesis was severely affected by inoculation with Bacillus cereus. In the hormone signal pathway, multiple DEGs were involved in plant defense-related major hormones, including activation of jasmonic acid (JA), salicylic acid (SA), and ethylene (Eth). Further analyses showed that other hormone-related genes involved in abscisic acid (ABA), gibberellin (GA), auxin (AUX), and cytokinin (CK) also showed changes. Notably, a large number of genes associated with glycolysis/gluconeogenesis, catabolism of starch and oxidative stress were induced. In addition, the majority of DEGs related to nucleic acid sugar metabolism were also significantly upregulated. Biochemical assays showed that the starch content of B. cereus-treated leaves was reduced to 2.51 mg/g and 2.38 mg/g at 6 and 24 hpi, respectively, while that of the control sample was 5.42 mg/g. Overall, our results demonstrated that multiple hormone signal transduction and carbohydrate metabolic pathways are involved in the interaction of tobacco and B. cereus.

Foliar exposure of Fe3O4 nanoparticles on Nicotiana benthamiana: Evidence for nanoparticles uptake, plant growth promoter and defense response elicitor against plant virus.

Nanoparticles are recently employed as a new strategy to directly kill pathogens (e.g., bacteria and fungus) and acted as nanofertilizers. However, the influences of this foliar deposition of nanoparticles on plant physiology particularly plant immunity are poorly understood. The uptake and physiological effects of Fe3O4 nanoparticles (Fe3O4NPs), and plant resistance response against Tobacco mosaic virus (TMV) after foliar spraying were studied. Specifically, Fe3O4NPs entered leaf cells and were transported and accumulated throughout the whole Nicotiana benthamiana plant, and increased plant dry and fresh weights, activated plant antioxidants, and upregulated SA synthesis and the expression of SA-responsive PR genes (i.e., PR1 and PR2), thereby enhancing plant resistance against TMV. Conversely, the viral infection was not inhibited in the NahG transgenic plants treated by Fe3O4NPs, suggesting the involvement of salicylic acid (SA) induced by Fe3O4NPs in the production of plant resistance. Moreover, no inhibition was observed of the infection after inoculating with the pretreated TMV mixtures. Thus, the deposition of Fe3O4NPs induced the accumulation of endogenous SA, which was correlated with the plant resistance against TMV infection. Such information is vital for valuing the risk of Fe3O4NPs products and broadens the researching and applying nanoparticles in the fight against plant diseases meantime.

The cold shock family gene cspD3 is involved in the pathogenicity of Ralstonia solanacearum CQPS-1 to tobacco.

Cold shock proteins (Csps) are small and highly conserved proteins that have target RNA- and DNA-binding activities. Csps play roles in different cellular processes and show functional redundancy. Ralstonia solanacearum, the agent of bacterial wilt, has 4 or 5 Csps based on genome analysis. However, the functions of all Csps in R. solanacearum remain unclear. According to phylogenetic analysis, the Csps from R. solanacearum are clustered into a group with CspD from E. coli. Here, we studied the role of CspD3, which was closer to CspD of E. coli in the phylogenetic tree. A cspD3 deletion strain was constructed to assess its effect on the phenotype of R. solanacearum, including growth, biofilm formation, motility, and virulence. The results showed that cspD3 of R. solanacearum was not necessary for normal growth, cold-shock adaptation, or biofilm formation. However, deletion of cspD3 in R. solanacearum CQPS-1 led to increased swimming motility, and the mean diameters of swimming haloes produced by the ΔcspD3 mutant were 1.3-fold larger than those produced by wild-type strain and 1.2-fold larger than those produced by the complemented strain. More importantly, the virulence of the cspD3 deletion mutant on susceptible tobacco plants was significantly attenuated compared to the wild-type strain. At 20 days after inoculation, the disease index of the ΔcspD3 mutant was 2.27, which was reduced by 1.6-fold relative to the wild-type strain. To assess the molecular response influenced by cspD3, the expressions of the main motility-associated genes and virulence-associated genes including flgM, fliA, pehS, pehR, hrpG, xpsR, and prhI in R. solanacearum were measured. The results showed that the expressions of hrpG, xpsR, and prhI were significantly decreased in cspD3 deletion mutant. Collectively, our findings showed that Csps are involved in the regulation of motility and virulence in R. solanacearum.

Dual-Action Pesticide Carrier That Continuously Induces Plant Resistance, Enhances Plant Anti-Tobacco Mosaic Virus Activity, and Promotes Plant Growth.

Improving plant resistance against systemic diseases remains a challenging research topic. In this study, we developed a dual-action pesticide-loaded hydrogel with the capacity to significantly induce plant resistance against tobacco mosaic virus (TMV) infection and promote plant growth. We produced an alginate-lentinan-amino-oligosaccharide hydrogel (ALA-hydrogel) by coating the surface of an alginate-lentinan drug-loaded hydrogel (AL-hydrogel) with amino-oligosaccharide using electrostatic action. We determined the formation of the amino-oligosaccharide film using various approaches, including Fourier transform infrared spectrometry, the ζ potential test, scanning electron microscopy, and elemental analysis. It was found that the ALA-hydrogel exhibited stable sustained-release activity, and the release time was significantly longer than that of the AL-hydrogel. In addition, we demonstrated that the ALA-hydrogel was able to continuously and strongly induce plant resistance against TMV and increase the release of calcium ions to promote Nicotiana benthamiana growth. Meanwhile, the ALA-hydrogel maintained an extremely high safety to organisms. Our findings provide an alternative to the traditional approach of applying pesticide for controlling plant viral diseases. In the future, this hydrogel with the simple synthesis method, green synthetic materials, and its efficiency in the induction of plant resistance will attract increasing attention and have good potential to be employed in plant protection and agricultural production.

Genome-wide identification and characterization of Hsp70 gene family in Nicotiana tabacum.

Heat shock proteins 70 (Hsp70) constitute a highly conserved protein family of cellular chaperones widely distributed in plants, where they play a fundamental role in response to biotic and abiotic stress. Until now, genome-wide analyses of the Hsp70 gene family have been conducted for some species. However, reports about Hsp70 genes in Nicotiana tabacum are scarce. In this study, we systematically conducted genome-wide identification and expression analysis of the Hsp70 gene family in tobacco, including gene structure, classification, evolutionary relationships, promoters, and transcript levels in response to abiotic stress treatments. In all, 61 Hsp70 members were identified and classified into six groups that were mapped onto 18 chromosomes, where most were distributed on both ends of the chromosome. The conserved structures and motifs of NtHsp70 proteins in the same subfamily were highly consistent. At least 15 pairs of NtHsp70 genes underwent gene duplication by segment and tandem duplications. Most NtHsp70 proteins contained N-terminal hexokinase conserved motifs. Phylogenetic analysis showed that most species expanded according to their own species-specific approach during the evolution of Hsp70s. Tissue-specific expression analysis indicated that all NtHsp70 genes were involved in at least one or more abiotic stress responses, highlighting the wide participation of NtHsp70 genes in environmental adaptation. This is the first genome-wide analysis of Hsp70 in N. tabacum. These results indicate that each NtHsp70 member fulfilled distinct functions in response to various abiotic stresses.

Green Synthesis of an Alginate-Coated Silver Nanoparticle Shows High Antifungal Activity by Enhancing Its Cell Membrane Penetrating Ability.

As a kind of promising nanopesticide, in contrast to traditional synthesis strategies, the application of a polysaccharide in silver nanoparticle preparation can improve its stability and avoid the usage of harmful substances. In this work, a two-step strategy for synthesizing silver nanoparticles (A-AgNPs) from aldehyde-modified sodium alginate (ASA) was introduced. The size of the A-AgNPs synthesized can be controlled from 6 to 40 nm with a high dispersibility in water. Furthermore, compared to naked AgNPs (n-AgNPs), the A-AgNPs showed improved broad-spectrum antimicroorganism activity. We found that the A-AgNPs mainly exerted their antifungal activity through the changing of cell membrane permeability and affecting the soluble protein synthesis, destruction of DNA structure, and inhibition of DNA replication. Meanwhile, the A-AgNPs showed no inhibition of rice and N. benthamiana seed germination. Considering its high biocompatibility and the highly efficient antimicroorganism activity, A-AgNPs can be potentially applied in plant protection science research.

Molecular identification and functional characterization of GhAMT1.3 in ammonium transport with a high affinity from cotton (Gossypium hirsutum L.).

Ammonium (NH4 + ) represents a primary nitrogen source for many plants, its effective transport into and between tissues and further assimilation in cells determine greatly plant nitrogen use efficiency. However, biological components involved in NH4 + movement in woody plants are unclear. Here, we report kinetic evidence for cotton NH4 + uptake and molecular identification of certain NH4 + transporters (AMTs) from cotton (Gossypium hirustum). A substrate-influx assay using 15 N-isotope revealed that cotton possessed a high-affinity transport system with a Km of 58 µM for NH4 + . Sequence analysis showed that GhAMT1.1-1.3 encoded respectively a membrane protein containing 485, 509 or 499 amino acids. Heterologous functionality test demonstrated that GhAMT1.1-1.3 expression mediated NH4 + permeation across the plasma membrane (PM) of yeast and/or Arabidopsis qko-mutant cells, allowing a growth restoration of both mutants on NH4 + . Quantitative PCR measurement showed that GhAMT1.3 was expressed in roots and leaves and markedly up-regulated by N-starvation, repressed by NH4 + resupply and regulated diurnally and age-dependently, suggesting that GhAMT1.3 should be a N-responsive gene. Importantly, GhAMT1.3 expression in Arabidopsis improved plant growth on NH4 + and enhanced total nitrogen accumulation (∼50% more), conforming with the observation of 2-fold more NH4 + absorption by GhAMT1.3-transformed qko plant roots during a 1-h root influx period. Together with its targeting to the PM and saturated transport kinetics with a Km of 72 µM for NH4 + , GhAMT1.3 is suggested to be a high-affinity NH4 + permease that may play a significant role in cotton NH4 + acquisition and utilization, adding a new member in the plant AMT family.

NtMYB12 Positively Regulates Flavonol Biosynthesis and Enhances Tolerance to Low Pi Stress in Nicotiana tabacum.

Phosphorus (P) is an essential macronutrient for plant growth and development. The concentration of flavonol, a natural plant antioxidant, is closely related to phosphorus nutritional status. However, the regulatory networks of flavonol biosynthesis under low Pi stress are still unclear. In this study, we identified a PFG-type MYB gene, NtMYB12, whose expression was significantly up-regulated under low Pi conditions. Overexpression of NtMYB12 dramatically increased flavonol concentration and the expression of certain flavonol biosynthetic genes (NtCHS, NtCHI, and NtFLS) in transgenic tobacco. Moreover, overexpression of NtMYB12 also increased the total P concentration and enhanced tobacco tolerance of low Pi stress by increasing the expression of Pht1-family genes (NtPT1 and NtPT2). We further demonstrated that NtCHS-overexpressing plants and NtPT2-overexpressing plants also had increased flavonol and P accumulation and higher tolerance to low Pi stress, showing a similar phenotype to NtMYB12-overexpressing transgenic tobacco under low Pi stress. These results suggested that tobacco NtMYB12 acts as a phosphorus starvation response enhancement factor and regulates NtCHS and NtPT2 expression, which results in increased flavonol and P accumulation and enhances tolerance to low Pi stress.