Effect of Glycine Betaine Nanocomposite Coated with Chitosan and Moderate Salinity Stress on In vitro Microtuberization of Potato (Solanum tuberosum L.) cv. Agria

Introduction Potato (Solanum tuberosum L.) is an important food and cash crop having the first rank in the world from non grain crops to ensure food security. The tubers produced through the conventional propagation are characterized by low multiplication rate and susceptibility to pathogens. Microtubers are an ideal propagating material for producing high quality seed potatoes. Nowadays, the production, application, and biological risk assessment of nano-scaled products have attracted global concerns in various fields such as agricultural, biotechnological, medicinal, and plant sciences. Chitosan, a biocompatible polymer, has been widely utilized to improve the production of nano-chemicals, thereby improving crop growth, productivity, and immunity. Nanotechnology plays an important role in modern agriculture to address global challenges such as climate change, severity of plant diseases and the limited availability of important plant nutrients. Polymer-based nano-formulations have recently received the greatest attention with the key objectives of developing less harmful, plant growth promoting and protective agents of biodegradable and natural origin. Use of chitosan-based nanoparticles in agriculture field is still in a budding phase. Significant outcomes have been reported in in vitro and a few in vivo studies in plant growth and protection by chitosan-based nanomaterials.   Materials and Methods MS medium containing 80 g of sucrose, containing glycine betaine at concentrations of 20 and 40 mg/l, chitosan at a concentration of 240 mg/l, glycine betaine 20 and chitosan 240 mg/l, glycine betaine 40 and chitosan 240 mg/l, 120 mg/l, glycine betaine nanocomposite coated with chitosan 120 and 240 mg/l and control treatment, as well as nonsalinity treatments with the same compounds and concentrations mentioned for salinity were cultured. The study was performed in randomized complete block design with three replications and sixteen treatment. Glycine betaine nanocomposite coated with chitosan were prepared at Maragheh University as follows. Chitosan, glycine betaine, and triphosphate are major consumables. First, chitosan was dissolved well with acetic acid under the influence of temperature. Then, a certain amount of glycine betaine was dissolved in distilled water and added to the chitosan. Tri-polyphosphate (TPP) was dissolved in distilled water at a certain volume ratio and added dropwise to the mixture of chitosan and glycine betaine. The precipitate obtained under the freeze-drying process lead to the preparation of its powder.     Results and Discussion According to the comparison of the means, treatment of glycine betaine coated with chitosan nanocomposite at a concentration of 120 mg/l produced the highest number of microtuber during the experiment from the first month to the final month. The nanocomposite was more capable of improving growth and biomass than the bare ZnONPs in pepper. The application of the nanocomposite increased the concentration of chlorophylls (51%), carotenoids (70%), proline (2-fold), and proteins (about 2- fold). The supplementation of culture medium with the nanomaterials upregulated enzymatic antioxidant biomarkers (catalase and peroxidase) (Asgari-targhi et al., 2021). The highest mean microtuber weight was related to the nanocomposite treatment of 120 mg/l with a weight of 29 mg. In the microtuber diameter, this treatment had the highest value. The results of analysis of variance in Table 1 indicate that the effect of moderate salinity and the interaction of moderate salinity and nanocomposite treatments are not significant and the effect of experimental treatments is significant at the level of 5% probability. Due to the fact that the most important trait in the potato microtuberization is microtuber yield, so in the experiment, the highest microtuber yield was earned nanocomposite treatment with 131 mg and the lowest in control treatment with 87 mg. It seems that by using stress-reducing compounds such as glycine betaine and chitosan and nanocomposites, these compounds increase the amount of genes responsible for the formation of microtuber, and as a result, increasing the proteins involved in stresses induce more microtuberization. Also, nanocomposite materials, more assimilated materials may be transferred from the roots to microtuberization processes.   Conclusion  The microtuber produced in the glycine betaine coated with chitosan nanocomposite treatment produced the highest number of microtubers in the first, second, third, and final months. In treatments with moderate salinity in the first, second, third and final months, the number of microtuber and eyes and sprouted microtubers had the highest amount compared to the treatment without moderate salinity. Also, plants treated with glycine betaine voated with chitosan nanocomposite in the microtuber trate showed a greater effect than chitosan and glycine betaine with chitosan. According to the findings of this study, it seems that the use of nanocomposite materials in increasing the microtuber and reducing the vegetative growth of potato shoots has been made in Agria cultivar

High-salinity growth conditions promote tat-independent secretion of tat substrates in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways

Environmental Salinity Determines the Specificity and Need for Tat-Dependent Secretion of the YwbN Protein in Bacillus subtilis

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described “minimal Tat translocase” consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate

Transport of Folded Proteins by the Tat System

The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of Escherichia coli and the two-component TatAC systems of Bacillus subtilis as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations

The acute effect of inhaled NaCl 5%, per CF TRUST protocol, on spirometry indices in patients over six years with cystic fibrosis

Background: Our aim was to investigate the acute effect of inhaled NaCl 5% on spirometry indices in patients aged over six years with cystic fibrosis (CF). Methods: 44 children over 6 years of age with CF took part in this study. Spirometry indices were measured. After receiving two puffs of salbutamol spray, the children were administered with 5 ml NaCl 5% by a medical compressed air system using a nebulizer kit and immediately a spirometric test was taken again. Spirometry results were recorded and the data were analyzed by SPSS v.18. Results: The percentages of the annual decline of forced expiratory volume in one second (FEV1) were 10.12±31.28, 7.26±17.10, and 13.8±21.7 in children aged 6 - 8 years, 9 - 12 years, and over 13 years, respectively. There were significant differences in FVC, FEV1/FVC, MEF50, MEF75, MMEF25/27 before and after hypertonic NaCl 5% inhalation (P < 0.05). A significant decline in FEV1 was observed only in 2% of the children. Conclusions: Patients with CF exhibited a suitable response to acute inhalation of hypertonic NaCl 5%. © 2018, Archives of Pediatric Infectious Diseases