Overexpression of the A4-rolB gene from the pRiA4 of Rhizobium rhizogenes modulates hormones homeostasis and leads to an increase of flavonoid accumulation and drought tolerance in Arabidopsis thaliana transgenic plants.

MAIN CONCLUSION: Increased flavonol accumulation and enhanced drought tolerance in A4-rolB-overexpressing plants can be explained by the cooperative action of the SA and ROS signalling pathways. Clarification of function of the A4-rolB plast gene from pRiA4 of Rhizobium rhizogenes will allow a better understanding of the biological principles of the natural transformation process and its use as a tool for plant bioengineering. In the present study, we investigated whether the overexpression of A4-rolB gene could regulate two important processes, flavonoid biosynthesis and drought tolerance. In addition, we investigated some aspects of the possible machinery of the A4-rolB-induced changes in plant physiology, such as crosstalk of the major signalling systems. Based on the data obtained in this work, it can be presumed that constitutive overexpression of A4-rolB leads to the activation of the salicylic acid signalling system. An increase in flavonol accumulation and enhanced drought tolerance can be explained by the cooperative action of SA and ROS pathways.

Biosynthesis and Cytotoxic Properties of Ag, Au, and Bimetallic Nanoparticles Synthesized Using Lithospermum erythrorhizon Callus Culture Extract.

The present study reports a green chemistry approach for the rapid and easy biological synthesis of silver (Ag), gold (Au), and bimetallic Ag/Au nanoparticles using the callus extract of Lithospermum erythrorhizon as a reducing and capping agent. The biosynthesized nanoparticles were characterized with ultraviolet-visible (UV-Vis) spectroscopy, X-ray diffraction (XRD) analysis, and transmission electron microscopy (TEM). Our results showed the formation of crystalline metal nanostructures of both spherical and non-spherical shape. Energy dispersive X-ray (EDX) spectroscopy showed the characteristic peaks in the silver and gold regions, confirming the presence of the corresponding elements in the monometallic particles and both elements in the bimetallic particles. Fourier-transform infrared (FTIR) spectroscopy affirmed the role of polysaccharides and polyphenols of the L. erythrorhizon extract as the major reducing and capping agents for metal ions. In addition, our results showed that the polysaccharide sample and the fraction containing secondary metabolites isolated from L. erythrorhizon were both able to produce large amounts of metallic nanoparticles. The biosynthesized nanoparticles demonstrated cytotoxicity against mouse neuroblastoma and embryonic fibroblast cells, which was considerably higher for Ag nanoparticles and for bimetallic Ag/Au nanoparticles containing a higher molar ratio of silver. However, fibroblast migration was not significantly affected by any of the nanoparticles tested. The obtained results provide a new example of the safe biological production of metallic nanoparticles, but further study is required to uncover the mechanism of their toxicity so that the biomedical potency can be assessed.

Auxin-dependent regulation of growth via rolB-induced modulation of the ROS metabolism in the long-term cultivated pRiA4-transformed Rubiacordifolia L. calli.

Gene transfer from Agrobacterium to plants is the best studied example of horizontal gene transfer (HGT) between prokaryotes and eukaryotes. The rol genes of A. rhizogenes (Rhizobium rhizogenes) provide uncontrolled root growth, or "hairy root" syndrome, the main diagnostic feature. In the present study, we investigated the stable pRiA4-transformed callus culture of Rubia cordifolia L. While untransformed callus cultures need PGRs (plant growth regulators) as an obligatory supplement, pRiA4 calli is able to achieve long-term PGR-free cultivation. For the first time, we described the pRiA4-transformed callus cultures' PGR-dependent ROS status, growth, and specialized metabolism. As we have shown, expression of the rolA and rolB but not the rolC genes is contradictory in a PGR-dependent manner. Moreover, a PGR-free pRiA4 transformed cell line is characterised as more anthraquinone (AQ) productive than an untransformed cell culture. These findings pertain to actual plant biotechnology: it could be the solution to troubles in choosing the best PGR combination for the cultivation of some rare, medicinal, and woody plants; wild-type Ri-plants and tissue cultures may become freed from legal controls on genetically modified organisms in the future. We propose possible PGR-dependent relationships between rolA and rolB as well as ROS signalling targets. The present study highlighted the high importance of the rolA gene in the regulation of combined rol gene effects and the large knowledge gap in rolA action.

Isolation and Characterization of Extracellular Vesicles from Arabidopsis thaliana Cell Culture and Investigation of the Specificities of Their Biogenesis.

Over recent years, extracellular vesicles (EVs), commonly termed exosomes, have gained prominence for their potential as natural nanocarriers. It has now been recognized that plants also secrete EVs. Despite this discovery, knowledge about EV biogenesis in plant cell cultures remains limited. In our study, we have isolated and meticulously characterized EVs from the callus culture of the model plant, Arabidopsis thaliana. Our findings indicate that the abundance of EVs in calli was less than that in the plant's apoplastic fluid. This difference was associated with the transcriptional downregulation of the endosomal sorting complex required for transport (ESCRT) genes in the calli cells. While salicylic acid increased the expression of ESCRT components, it did not enhance EV production. Notably, EVs from calli contained proteins essential for cell wall biogenesis and defense mechanisms, as well as microRNAs consistent with those found in intact plants. This suggests that plant cell cultures could serve as a feasible source of EVs that reflect the characteristics of the parent plant species. However, further research is essential to determine the optimal conditions for efficient EV production in these cultured cells.

Suppression of the HOS1 Gene Affects the Level of ROS Depending on Light and Cold.

The E3 ubiquitin-protein ligase HOS1 is an important integrator of temperature information and developmental processes. HOS1 is a negative regulator of plant cold tolerance, and silencing HOS1 leads to increased cold tolerance. In the present work, we studied ROS levels in hos1Cas9Arabidopsis thaliana plants, in which the HOS1 gene was silenced by disruption of the open reading frame via CRISPR/Cas9 technology. Confocal imaging of intracellular reactive oxygen species (ROS) showed that the hos1 mutation moderately increased levels of ROS under both low and high light (HL) conditions, but wild-type (WT) and hos1Cas9 plants exhibited similar ROS levels in the dark. Visualization of single cells did not reveal differences in the intracellular distribution of ROS between WT and hos1Cas9 plants. The hos1Cas9 plants contained a high basal level of ascorbic acid, maintained a normal balance between reduced and oxidized glutathione (GSH and GSSG), and generated a strong antioxidant defense response against paraquat under HL conditions. Under cold exposure, the hos1 mutation decreased the ROS level and substantially increased the expression of the ascorbate peroxidase genes Apx1 and Apx2. When plants were pre-exposed to cold and further exposed to HL, the expression of the NADPH oxidase genes RbohD and RbohF was increased in the hos1Cas9 plants but not in WT plants. hos1-mediated changes in the level of ROS are cold-dependent and cold-independent, which implies different levels of regulation. Our data indicate that HOS1 is required to maintain ROS homeostasis not only under cold conditions, but also under conditions of both low and high light intensity. It is likely that HOS1 prevents the overinduction of defense mechanisms to balance growth.

Biosynthesis of Functional Silver Nanoparticles Using Callus and Hairy Root Cultures of Aristolochia manshuriensis.

This study delves into the novel utilization of Aristolochia manshuriensis cultured cells for extracellular silver nanoparticles (AgNPs) synthesis without the need for additional substances. The presence of elemental silver has been verified using energy-dispersive X-ray spectroscopy, while distinct surface plasmon resonance peaks were revealed by UV-Vis spectra. Transmission and scanning electron microscopy indicated that the AgNPs, ranging in size from 10 to 40 nm, exhibited a spherical morphology. Fourier-transform infrared analysis validated the abilty of A. manshuriensis extract components to serve as both reducing and capping agents for metal ions. In the context of cytotoxicity on embryonic fibroblast (NIH 3T3) and mouse neuroblastoma (N2A) cells, AgNPs demonstrated varying effects. Specifically, nanoparticles derived from callus cultures exhibited an IC50 of 2.8 µg/mL, effectively inhibiting N2A growth, whereas AgNPs sourced from hairy roots only achieved this only at concentrations of 50 µg/mL and above. Notably, all studied AgNPs' treatment-induced cytotoxicity in fibroblast cells, yielding IC50 values ranging from 7.2 to 36.3 µg/mL. Furthermore, the findings unveiled the efficacy of the synthesized AgNPs against pathogenic microorganisms impacting both plants and animals, including Agrobacterium rhizogenes, A. tumefaciens, Bacillus subtilis, and Escherichia coli. These findings underscore the effectiveness of biotechnological methodologies in offering advanced and enhanced green nanotechnology alternatives for generating nanoparticles with applications in combating cancer and infectious disorders.

The RolB/RolC homolog from sweet potato promotes early flowering and triggers premature leaf senescence in transgenic Arabidopsis thaliana plants.

Expression of the root oncogenic loci (rol) genes from Agrobacterium rhizogenes provokes multiple divergent effects on physiological properties in transgenic plants and cell cultures. Recently, the homolog of the rolB and rolC oncogenes, named Ib-rolB/C, has been identified in the genome of a naturally transgenic food crop, i.e. sweet potato. In this study, we revealed that the Ipomoea batatas genome contains two full-length copies of Ib-rolB/C. The expression level of Ib-rolB/C in leaves of sweet potato showed a clear age-dependent pattern and increased as leaves senesce. Moreover, dark-induced senescence strongly up-regulates transcription of the Ib-rolB/C gene. Though Ib-rolB/C shares homology with its counterparts in A. rhizogenes, this gene was not capable to induce hairy roots or tumors in kalanchoe and tobacco plants. The Ib-rolB/C gene induced early-flowering phenotype, altered leaf morphology, and promoted premature leaf senescence in transgenic Arabidopsis thaliana plants. At the same time, Ib-rolB/C did not affect root morphology and biomass. Our results suggest that Ib-RolB/RolC participates in both age- and dark-triggered leaf senescence programs.

Engineered Fungus Thermothelomyces thermophilus Producing Plant Storage Proteins.

An efficient Agrobacterium-mediated genetic transformation based on the plant binary vector pPZP-RCS2 was carried out for the multiple heterologous protein production in filamentous fungus Thermothelomyces thermophilus F-859 (formerly Myceliophthora thermophila F-859). The engineered fungus Th. thermophilus was able to produce plant storage proteins of Zea mays (α-zein Z19) and Amaranthus hypochondriacus (albumin A1) to enrich fungal biomass by valuable nutritional proteins and improved amino acid content. The mRNA levels of z19 and a1 genes were significantly dependent on their driving promoters: the promoter of tryptophan synthase (PtrpC) was more efficient to express a1, while the promoter of translation elongation factor (Ptef) provided much higher levels of z19 transcript abundance. In general, the total recombinant proteins and amino acid contents were higher in the Ptef-containing clones. This work describes a new strategy to improve mycoprotein nutritive value by overexpression of plant storage proteins.

Biomimetic synthesis of functional silver nanoparticles using hairy roots of Panax ginseng for wheat pathogenic fungi treatment.

Presently, multifunctional silver nanoparticles (AgNPs) show a rapid growth in various commercial applications, leading to increasing demand for new eco-friendly manufacturing technologies. An array of genetic engineering tools can be used to increase the yield in the production of AgNPs using various biological systems. The present study reports a green chemistry approach for the biological synthesis of AgNPs using extracts from non-transformed callus, rolC-transgenic callus and hairy roots of Panax ginseng and an evaluation of their efficacy against crop-damaging fungal pathogens. All types of ginseng cell lines promote the reduction of silver nitrate and formation of spherical AgNPs with an average diameter of 50-90 nm. Notably, hairy root extract possessed the maximal reduction potential among the studied cell lines probably due to higher secondary metabolite content. The biosynthesized nanoparticles were highly toxic against several wheat fungal pathogens including Fusarium graminearum, F. avenaceum, F. poae, and F. sporotrichioides, which are associated with fusarium head blight disease in cereals. Furthermore, the antifungal activity of nanosilver was successfully utilized for surface sterilization of infected wheat kernels without any negative effect on seed germination capability.