Search Results (19)

Rhizopus oryzae lipase (ROL) is a key enzyme involved in olive oil spoilage and acts as a virulence factor in fungal infections. Natural lipophilic lipase inhibitors are crucial for mitigating economic losses resulting from lipid degradation in stored or decaying olive fruits. This study evaluated a series of enzymatically synthesized piperate esters with varying alkyl chain lengths (butyryl, C4; octyl, C8; dodecyl, C12) for their inhibitory effects on ROL activity. Octyl piperate (C8) demonstrated the highest potency, with IC₅₀ values of 0.05 mg/mL using methods B and C or 0.25 mg/mL using method A. Molecular docking indicated that C8 achieved the most favorable predicted binding energy (Gscore: –11.134 kcal/mol), primarily through hydrophobic interactions (Val329, Ala212, Phe209) and hydrogen bonds with oxyanion hole residues (Ser268, Thr206, Gln241). Molecular dynamics simulations confirmed that C8 maintained stable binding and stabilized the catalytic residues. In comparison, C4 exhibited weaker interactions, and the longer C12 chain induced conformational instability and steric hindrance. These results establish a parabolic structure–activity relationship, identifying the octyl chain (C8) as optimal for ROL inhibition among the tested derivatives. The rational design of lipophilic, biodegradable lipase inhibitors thus positions octyl piperate as a promising candidate for extending olive storage and shelf life, and as a scaffold for developing natural antifungal agents targeting virulent R. oryzae strains. Full article

Sweet potato (Ipomoea batatas) is a globally important crop and one of a growing number of plants recognized as naturally transgenic, harboring Agrobacterium-derived T-DNA genes whose functions remain largely uncharacterized. In this proof-of-concept study, we applied CRISPR/Cas9 technology to generate targeted knockouts of the Ib-rolB/C and Ib-rolD-like genes located within the sweet potato cellular T-DNA2 (IbT-DNA2) region. Mutations were introduced into sweet potato callus cultures using an optimized genome editing protocol, with most edits consisting of single-nucleotide insertions. Knockout of Ib-rolB/C did not affect callus growth but significantly reduced levels of chlorogenic acid derivatives. Validation in planta using transient expression in I. batatas leaves confirmed the suppressive effect of Ib-rolB/C disruption on polyphenol content. In contrast, Ib-rolD-like knockout lines showed reduced biomass accumulation and downregulation of cell cycle–related genes, but did not display significant changes in metabolite content in either callus cultures or leaf tissues. These findings suggest that Ib-rolB/C and Ib-rolD-like may differentially contribute to growth and secondary metabolism in sweet potato. Full article

We investigated the effect of Agrobacterium rhizogenes-mediated transformation mof rolB on adventitious root development and endogenous hormones in ‘duli’ (Pyrus betulaefolia) via transcriptomic analysis of wild-type (WT) and rolB-transformed plants. The formation of root primordia occurred earlier in transgenic ‘duli’ than in the WT plants. At seven days, 57% of the transgenic seedlings had formed root primordia, whereas root primordia first appeared at seven days in WT ‘duli’. The rooting rate of transgenic ‘duli’ and WT plants was 90% and 77.14%, respectively. rolB significantly promoted the formation of secondary roots. Within 20 days, auxin (IAA), gibberellic acid (GA₃), and zeatin riboside (ZR) were higher and abscisic acid (ABA) was lower in transgenic ‘duli’ than in WT plants. Gene Ontology analysis revealed high enrichment in signaling pathways and ADP binding, and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that several differentially expressed genes were enriched in flavonoid and carotenoid-related pathways and plant hormone signal transduction. rolB induced changes in the expression patterns of several genes involved in hormone biosynthesis, metabolism, and signal transduction pathways in ‘duli’. Weighted gene co-expression network analysis identified the DEGs associated with endogenous hormone levels and indicated that the central genes of modules most strongly correlated with ABA, ZR, IAA, and GA₃ regulate protein synthesis, signaling, and root tissue meristem activity. Protein–protein interaction analysis yielded a co-expression network of physiological and transcriptomic data during rooting and identified key genes at the network core. These findings provide valuable insights into the regulatory mechanisms of rolB and its influence on root development in ‘duli’. Full article

Aluminum alloys, serving as critical structural materials in the aviation and aerospace industry, frequently endure variable amplitude loading under complex service conditions. The resulting non-steady-state crack propagation behavior directly impacts structural safety. This study considers the engineering application requirements of the 7055-T7751 aluminum alloy and conducts fatigue crack growth experiments on compact tensile specimens subjected to constant amplitude loading and periodic variable amplitude overloading conditions. The findings indicate that the 7055 aluminum alloy exhibits an instantaneous acceleration period under tensile overload, which is important in the comprehensive analysis of crack growth life. The experimental findings show no significant correlation between post-overload minimum crack growth rate deviation and thickness or crack size at overload, where the values are 50.3% and 94.8% at 1.4 and 1.7 R_OL, respectively. An analytical model for the crack growth increment a_ii during this period was developed. Additionally, the delay distance influenced by overloads a_d and the number of delay cycles N_d are identified as effective parameters for evaluating the retardation effects induced by overloading. Our comparative analysis of crack growth experimental data under varying overload ratios R_OL and specimen thicknesses B revealed that existing plastic zone models inadequately assess a_d, prompting the establishment of a corresponding evaluation model. By incorporating the parameters a_ii and a_d into the Wheeler model, a method for calculating the delay cycles N_d was constructed, which effectively captured the variation trend. Finally, an analysis of fractography revealed numerous secondary cracks within the overload damage zone, and the ductile fracture characteristics in this region were significantly weaker compared to areas subjected to fatigue loading. Full article

In Arctic regions, ship structures face low temperatures, overloads, thickness effects, and fluctuating stress ratios, which significantly influence the fatigue crack growth (FCG) behavior of marine steels. This study investigates the FCG behaviors of DH36 steel by a series of experiments under the combined effects of low temperatures, overload ratios R_ol, specimen thickness B, and stress ratios R. Experiment results show that the yield strength, ultimate tensile strength, and elastic modulus of DH36 steel exhibit negative correlations with temperature varying within the Arctic temperature range. A reduction in fatigue crack growth rate (FCGR) is observed under the combined effects of low temperature and overload, and the magnitude of decrease shows a positive correlation with R_ol. Notably, low temperatures weaken the FCG retardation effect induced by overload, and this attenuation becomes more pronounced as temperature decreases. Under low temperatures, while maintaining constant peak load, increasing R significantly reduces both initial and terminal stress intensity factor ranges ΔK₀ and ΔK_e, resulting in diminished effective crack driving force and thereby substantially extending FCG life. Although increased B enhances FCGR at low temperatures, thinner plates demonstrate shorter FCG life due to their higher ΔK₀ values. Full article

This study reports the first successful establishment of Perovskia atriplicifolia hairy root cultures using Rhizobium rhizogenes and evaluates their potential for bioactive phenolic acid production, particularly rosmarinic acid (RA). Hairy roots were induced using two R. rhizogenes strains, A4 and ATCC 15834; transformation was confirmed by PCR analysis targeting the rol and aux genes. The A4 strain exhibited higher transformation efficiency (41.3%) than ATCC 15834 (30.2%). Eight transgenic root clones (C1–C8) were established and confirmed as transformed. The clones exhibited significant variation in biomass accumulation and phenolic acid production. RA production was most strongly correlated with PAL, RAS, and CYP98A14 expression. Hierarchical clustering clustered the clones into three groups based on growth, metabolite content, and gene expression. Lines C1 and C2 exhibiting the highest RA, total polyphenol content, and the highest productivity were selected for further experiments. McCown Woody Plant (WP) and Schenk and Hildebrandt (SH) media demonstrated the greatest biomass accumulation, with growth indexes exceeding 13. Conversely, Gamborg (B5) medium enhanced RA content, achieving 38.3 and 40.8 mg/g dry weight (DW) for clones C1 and C2, respectively, representing a fourfold increase compared to the least favorable Murashige and Skoog (MS) medium. These findings establish P. atriplicifolia hairy roots as efficient systems for RA biosynthesis and can provide a basis for metabolic engineering and scale-up production of phenolic acids in medicinal plants. Full article

Dittrichia viscosa is a ruderal plant species growing along roadsides and well adapting to extreme environmental conditions. D. viscosa plant tissues, especially leaves, are known to be a rich source of bioactive metabolites which have antioxidant, cytotoxic, antiproliferative and anticancer properties. Hairy root cultures are a suitable biotechnological system for investigating plant metabolic pathways and producing specialized metabolites in in vitro conditions. In this study, D. viscosa hairy root transformed lines induced by Agrobacterium rhizogenes ATCC15834 were obtained using leaf explants, and the integration of rolB and rolC genes in the genomes of transformed hairy roots were confirmed by PCR analysis. Three hairy root D. viscosa lines (DvHrT1, DvHrT4 and DvHrT5) having different phenotypic features were characterized in terms of total phenolics, flavonoids and antioxidant activity. Correlated with antioxidant activity, phenolic and flavonoid content of DvHrT1 was significantly higher than control roots and the other DvHrT lines. Our results suggest that D. viscosa hairy roots can be a valuable tool for producing various bioactive compounds having antioxidant activity and are to be further investigated to produce other specific molecules that could find application in agricultural or pharmaceutical fields. Full article

Kalanchoë gastonis-bonnieri Raym.-Hamet & Perrier is a plant used for medicinal purposes in the treatment of several ailments. The aim of this study was to analyze the chemical profile of extracts from K. gastonis-bonnieri embryogenic calli, generated from genetically transformed roots by Agrobacterium rhizogenes. Putative transformants were verified by PCR. Hydroalcoholic extracts were obtained and the chemical profile was analyzed by LC-ESI-MS/MS. Root formation was obtained from 80% of infected seedlings. Fifteen root lines were isolated, and two lines showed prominent longitudinal growth and profuse branching in the B5 semi-solid medium. In all lines, the formation of nodules and later embryogenic callus was observed. Putative transgenic root lines were cultivated in free-plant growth regulators B5 medium. In the two selected lines, the PCR amplification of rolA, rolB, rolC, rolD, and aux1 genes was detected. The extract of embryogenic calli showed 60 chemical compounds tentatively identified, such as ferulic acid, quinic acid, neobaisoflavone, and malic acid, among others, and the chemical profile was different in comparison to wild-type extracts. This is the first study reporting the analysis of the chemical profile of hairy root extracts derived from Kalanchoë gastonis-bonnieri. This work displays the great potential for obtaining chemical compounds of pharmacological importance from hairy roots and facilitates the identification of new useful drugs against human chronic-degenerative diseases. Full article

Leishmaniasis is a complex disease caused by infection with different Leishmania parasites. The number of medications used for its treatment is still limited and the discovery of new drugs is a valuable approach. In this context, here we describe the in vitro leishmanicidal activity and the in silico interaction between trypanothione reductase (TryR) and (-)-5-demethoxygrandisin B from the leaves of Virola surinamensis (Rol.) Warb. The compound (-)-5-demethoxygrandisin B was isolated from V. surinamensis leaves, a plant found in the Brazilian Amazon, and it was characterized as (7R,8S,7′R,8′S)-3,4,5,3′,4′-pentamethoxy-7,7′-epoxylignan. In vitro antileishmanial activity was examined against Leishmania amazonensis, covering both promastigote and intracellular amastigote phases. Cytotoxicity and nitrite production were gauged using BALB/c peritoneal macrophages. Moreover, transmission electron microscopy was applied to probe ultrastructural alterations, and flow cytometry assessed the shifts in the mitochondrial membrane potential. In silico methods such as molecular docking and molecular dynamics assessed the interaction between the most stable configuration of (-)-5-demethoxygrandisin B and TryR from L. infantum (PDB ID 2JK6). As a result, the (-)-5-demethoxygrandisin B was active against promastigote (IC₅₀ 7.0 µM) and intracellular amastigote (IC₅₀ 26.04 µM) forms of L. amazonensis, with acceptable selectivity indexes. (-)-5-demethoxygrandisin B caused ultrastructural changes in promastigotes, including mitochondrial swelling, altered kDNA patterns, vacuoles, vesicular structures, autophagosomes, and enlarged flagellar pockets. It reduced the mitochondria membrane potential and formed bonds with important residues in the TryR enzyme. The molecular dynamics simulations showed stability and favorable interaction with TryR. The compound targets L. amazonensis mitochondria via TryR enzyme inhibition. Full article

Aristolochia manshuriensis is a relic liana, which is widely used in traditional Chinese herbal medicine and is endemic to the Manchurian floristic region. Since this plant is rare and slow-growing, alternative sources of its valuable compounds could be explored. Herein, we established hairy root cultures of A. manshuriensis transformed with Agrobacterium rhizogenes root oncogenic loci (rol)B and rolC genes. The accumulation of nitrogenous secondary metabolites significantly improved in transgenic cell cultures. Specifically, the production of magnoflorine reached up to 5.72 mg/g of dry weight, which is 5.8 times higher than the control calli and 1.7 times higher than in wild-growing liana. Simultaneously, the amounts of aristolochic acids I and II, responsible for the toxicity of Aristolochia species, decreased by more than 10 fold. Consequently, the hairy root extracts demonstrated pronounced cytotoxicity against human glioblastoma cells (U-87 MG), cervical cancer cells (HeLa CCL-2), and colon carcinoma (RKO) cells. However, they did not exhibit significant activity against triple-negative breast cancer cells (MDA-MB-231). Our findings suggest that hairy root cultures of A. manshuriensis could be considered for the rational production of valuable A. manshuriensis compounds by the modification of secondary metabolism. Full article

Various strategies have been used to increase the efficiency of secondary metabolite production in Salvia plants. This report is the first to examine the spontaneous development of Salvia bulleyana shoots transformed by Agrobacterium rhizogenes on hairy roots and the influence of light conditions on the phytochemical profile of this shoot culture. The transformed shoots were cultivated on solid MS medium with 0.1 mg/L of IAA (indole-3-acetic acid) and 1 mg/L of m-Top (meta-topolin), and their transgenic characteristic was confirmed by PCR-based detection of the rolB and rolC genes in the target plant genome. This study assessed the phytochemical, morphological, and physiological responses of the shoot culture under stimulation by light-emitting diodes (LEDs) with different wavelengths (white, WL; blue, B; red, RL; and red/blue, ML) and under fluorescent lamps (FL, control). Eleven polyphenols identified as phenolic acids and their derivatives were detected via ultrahigh-performance liquid chromatography with diode-array detection coupled to electrospray ionization tandem mass spectrometry (UPLC-DAD/ESI-MS) in the plant material, and their content was determined using high-performance liquid chromatography (HPLC). Rosmarinic acid was the predominant compound in the analyzed extracts. The mixed red and blue LEDs gave the highest levels of polyphenol and rosmarinic acid accumulation (respectively, 24.3 mg/g of DW and 20.0 mg/g of DW), reaching two times greater concentrations of polyphenols and three times greater rosmarinic acid levels compared to the aerial parts of two-year-old intact plants. Similar to WL, ML also stimulated regeneration ability and biomass accumulation effectively. However, the highest total photosynthetic pigment production (1.13 mg/g of DW for total chlorophyll and 0.231 mg/g of DW for carotenoids) was found in the shoots cultivated under RL followed by BL, while the culture exposed to BL was characterized as having the highest antioxidant enzyme activities. Full article

A variety of plant species found in nature contain agrobacterial T-DNAs in their genomes which they transmit in a series of sexual generations. Such T-DNAs are called cellular T-DNAs (cT-DNAs). cT-DNAs have been discovered in dozens of plant genera, and are suggested to be used in phylogenetic studies, since they are well-defined and unrelated to other plant sequences. Their integration into a particular chromosomal site indicates a founder event and a clear start of a new clade. cT-DNA inserts do not disseminate in the genome after insertion. They can be large and old enough to generate a range of variants, thereby allowing the construction of detailed trees. Unusual cT-DNAs (containing the rolB/C-like gene) were found in our previous study in the genome data of two Vaccinium L. species. Here, we present a deeper study of these sequences in Vaccinium L. Molecular-genetic and bioinformatics methods were applied for sequencing, assembly, and analysis of the rolB/C-like gene. The rolB/C-like gene was discovered in 26 new Vaccinium species and Agapetes serpens (Wight) Sleumer. Most samples were found to contain full-size genes. It allowed us to develop approaches for the phasing of cT-DNA alleles and reconstruct a Vaccinium phylogenetic relationship. Intra- and interspecific polymorphism found in cT-DNA makes it possible to use it for phylogenetic and phylogeographic studies of the Vaccinium genus. Full article

The flavonoids in Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) have high medicinal value. However, because of slow growth and harsh ecological environments, T. hemsleyanum is currently an endangered species. In light of this, we present a detailed hairy root induction procedure as a promising alternative to true roots with medicinal value. The percentage of explants induced by Agrobacterium rhizogenes (A. rhizogenes) to produce hairy roots out of the total number of explants infected (induction rate 1) was 95.83 ± 7.22%, and the proportion of hairy roots that contained Rol B fragments among all the hairy roots with or without Rol B fragments (positive rate) was 96.57 ± 1.72%. The transformation was further confirmed by the expression of the GUS protein. A high-productive hairy root line was screened for the comparative profiling of six flavonoids with true roots using high-performance liquid chromatography (HPLC). The contents of (+)-catechin, (−)-epicatechin, neochlorogenic acid, luteolin-6-C-glucoside, and orientin were 692.63 ± 127.24, 163.34 ± 31.86, 45.95 ± 3.46, 209.68 ± 6.03, and 56.82 ± 4.75 μg/g dry weight (DW) of 30-day-old hairy roots, respectively, which were higher than those of 3-year-old true roots. Hairy roots have stronger antioxidant activity than true roots. Overall, the hairy roots of T. hemsleyanum could serve as promising alternative sources for the production of flavonoids with medicinal uses. Full article

During Agrobacterium rhizogenes–plant interaction, the rolB gene is transferred into the plant genome and is stably inherited in the plant’s offspring. Among the numerous effects of rolB on plant metabolism, including the activation of secondary metabolism, its effect on plant defense systems has not been sufficiently studied. In this work, we performed a proteomic analysis of rolB-expressing Arabidopsis thaliana plants with particular focus on defense proteins. We found a total of 77 overexpressed proteins and 64 underexpressed proteins in rolB-transformed plants using two-dimensional gel electrophoresis and MALDI mass spectrometry. In the rolB-transformed plants, we found a reduced amount of scaffold proteins RACK1A, RACK1B, and RACK1C, which are known as receptors for activated C-kinase 1. The proteomic analysis showed that rolB could suppress the plant immune system by suppressing the RNA-binding proteins GRP7, CP29B, and CP31B, which action are similar to the action of type-III bacterial effectors. At the same time, rolB plants induce the massive biosynthesis of protective proteins VSP1 and VSP2, as well as pathogenesis-related protein PR-4, which are markers of the activated jasmonate pathway. The increased contents of glutathione-S-transferases F6, F2, F10, U19, and DHAR1 and the osmotin-like defense protein OSM34 were found. The defense-associated protein PCaP1, which is required for oligogalacturonide-induced priming and immunity, was upregulated. Moreover, rolB-transformed plants showed the activation of all components of the PYK10 defense complex that is involved in the metabolism of glucosinolates. We hypothesized that various defense systems activated by rolB protect the host plant from competing phytopathogens and created an effective ecological niche for A. rhizogenes. A RolB → RACK1A signaling module was proposed that might exert most of the rolB-mediated effects on plant physiology. Our proteomics data are available via ProteomeXchange with identifier PXD037959. Full article

Ipomoea batatas is a vital root crop and a source of caffeoylquinic acid derivatives (CQAs) with potential health-promoting benefits. As a naturally transgenic plant, I. batatas contains cellular T-DNA (cT-DNA) sequence homologs of the Agrobacterium rhizogenes open reading frame (ORF)14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n of unknown function. This study aimed to evaluate the effect of abiotic stresses (temperature, ultraviolet, and light) and chemical elicitors (methyl jasmonate, salicylic acid, and sodium nitroprusside) on the biosynthesis of CQAs and cT-DNA gene expression in I. batatas cell culture as a model system. Among all the applied treatments, ultraviolet irradiation, methyl jasmonate, and salicylic acid caused the maximal accumulation of secondary compounds. We also discovered that I. batatas cT-DNA genes were not expressed in cell culture, and the studied conditions weakly affected their transcriptional levels. However, the Ib-rolB/C gene expressed under the strong 35S CaMV promoter increased the CQAs content by 1.5–1.9-fold. Overall, our results show that cT-DNA-encoded transgenes are not involved in stress- and chemical elicitor-induced CQAs accumulation in cell cultures of I. batatas. Nevertheless, overaccumulation of RolB/RolC transcripts potentiates the secondary metabolism of sweet potatoes through a currently unknown mechanism. Our study provides new insights into the molecular mechanisms linked with CQAs biosynthesis in cell culture of naturally transgenic food crops, i.e., sweet potato. Full article