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The influence of epigenetic factors on plant defense responses and the balance between growth and defense is becoming a central area in plant biology. It is believed that the biosynthesis of secondary metabolites can be regulated by epigenetic factors, but this is not associated with the formation of a "memory" to the previous biosynthetic status. This review shows that some epigenetic effects can result in epigenetic memory, which opens up new areas of research in secondary metabolites, in particular flavonoids. Plant-controlled chromatin modifications can lead to the generation of stress memory, a phenomenon through which information regarding past stress cues is retained, resulting in a modified response to recurring stress. How deeply are the mechanisms of chromatin modification and memory generation involved in the control of flavonoid biosynthesis? This article collects available information from the literature and interactome databases to address this issue. Visualization of the interaction of chromatin-modifying proteins with the flavonoid biosynthetic machinery is presented. Chromatin modifiers and "bookmarks" that may be involved in the regulation of flavonoid biosynthesis through memory have been identified. Through different mechanisms of chromatin modification, plants can harmonize flavonoid metabolism with: stress responses, developmental programs, light-dependent processes, flowering, and longevity programs. The available information points to the possibility of developing chromatin-modifying technologies to control flavonoid biosynthesis.
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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 â 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.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Oncogenes , Plantas Modificadas Genéticamente/genética , ProteómicaRESUMEN
Calcium-dependent protein kinases (CDPKs) are one of the main Ca2+ decoders in plants. Among them, Arabidopsis thaliana AtCPK1 is one of the most studied CDPK genes as a positive regulator of plant responses to biotic and abiotic stress. The mutated form of AtCPK1, in which the autoinhibitory domain is inactivated (AtCPK1-Ca), provides constitutive kinase activity by mimicking a stress-induced increase in the Ca2+ flux. In the present study, we performed a proteomic analysis of Vitis amurensis calli overexpressing the AtCPK1-Ca form using untransformed calli as a control. In our previous studies, we have shown that the overexpression of this mutant form leads to the activation of secondary metabolism in plant cell cultures, including an increase in resveratrol biosynthesis in V. amurensis cell cultures. We analyzed upregulated and downregulated proteins in control and transgenic callus cultures using two-dimensional gel electrophoresis, and Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF). In calli transformed with AtCPK1-Ca, an increased amounts of pathogenesis-related proteins were found. A quantitative real-time PCR analysis confirmed this result.
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Arabidopsis , Vitis , Arabidopsis/genética , Técnicas de Cultivo de Célula , Proteoma/genética , Proteómica , Vitis/genéticaRESUMEN
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.
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Aristolochia , Humanos , Plantas , Medicina Tradicional China , China , Raíces de Plantas/metabolismoRESUMEN
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.
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Arabidopsis , Agrobacterium , Arabidopsis/genética , Sequías , Flavonoides/metabolismo , Flavonoles/metabolismo , Homeostasis , Hormonas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
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Ipomoea batatas , Ipomoea batatas/genética , Ipomoea batatas/metabolismo , Metabolismo Secundario , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Ácido Salicílico/farmacología , Ácido Salicílico/metabolismo , ADN/metabolismo , Técnicas de Cultivo de Célula , Regulación de la Expresión Génica de las PlantasRESUMEN
During our ongoing efforts to investigate biotechnological sources of caffeoylquinic acid (CQA) metabolites, we discovered the plant Scorzonera radiata Fisch. (Asteraceae), which is able to produce callus cultures with high yield and extremely high stability. An actively growing callus line, designated as Sr-L1, retained the ability to produce 11 CQAs during long-term cultivation (more than 20 years). A total of 29 polyphenolic compounds were identified in the leaves and Sr-L1 callus culture of S. radiata, including CQAs, lignol derivatives, flavonoids, and dihydrostilbenes. The composition of CQAs in the Sr-L1 culture was identical to that in the S. radiata leaves. Sr-L1 calli did not produce flavonoids and dihydrostilbenes, but produced lignol derivatives, which were absent in leaves. The HPLC-UV-HRMS determination showed the presence of monoacyl derivatives of CQAs such as 5-CQA, 4-CQA, cis-5-CQA, and 5-O-p-coumaroylquinic acid in the Sr-L1 culture. Among diacyl derivatives, 3,4-diCQA, 3,5-diCQA, cis-3,5-diCQA, 4,5-diCQA, 3-O-p-coumaroyl-5-O-CQA, and 3-O-caffeoyl-5-O-p-coumaroylquinic acid were found. The content of 5-CQA reached 7.54 mg/g dry weight and the content of 3,5-diCQA was as high as 18.52 mg/g dry weight. 3,5-diCQA has been reported to be of high nutritional and pharmacological value, as it alleviates inflammatory pain, reverses memory impairment by preventing neuronal apoptosis, and counteracts excessive adipose tissue expansion, serving as an attractive treatment option for obesity. The high content of 3,5-diCQA and the exceptional stability of biosynthesis make callus cultures of S. radiata a promising source for the development of drugs and nutraceuticals.
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Dihidrostilbenoides , Scorzonera , Ácido Quínico , Ácido Clorogénico , FlavonoidesRESUMEN
The COVID-19 pandemic, as well as the more general global increase in viral diseases, has led researchers to look to the plant kingdom as a potential source for antiviral compounds. Since ancient times, herbal medicines have been extensively applied in the treatment and prevention of various infectious diseases in different traditional systems. The purpose of this review is to highlight the potential antiviral activity of plant compounds as effective and reliable agents against viral infections, especially by viruses from the coronavirus group. Various antiviral mechanisms shown by crude plant extracts and plant-derived bioactive compounds are discussed. The understanding of the action mechanisms of complex plant extract and isolated plant-derived compounds will help pave the way towards the combat of this life-threatening disease. Further, molecular docking studies, in silico analyses of extracted compounds, and future prospects are included. The in vitro production of antiviral chemical compounds from plants using molecular pharming is also considered. Notably, hairy root cultures represent a promising and sustainable way to obtain a range of biologically active compounds that may be applied in the development of novel antiviral agents.
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Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Extractos Vegetales/farmacología , Plantas Medicinales/química , SARS-CoV-2/efectos de los fármacos , Antivirales/química , Antivirales/inmunología , Antivirales/uso terapéutico , Simulación por Computador , Humanos , Agricultura Molecular/métodos , Extractos Vegetales/química , Extractos Vegetales/inmunología , Extractos Vegetales/uso terapéutico , Plantas Medicinales/inmunología , SARS-CoV-2/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
The important regulatory role of brassinosteroids (BRs) in the mechanisms of tolerance to multiple stresses is well known. Growing data indicate that the phenomenon of BR-mediated drought stress tolerance can be explained by the generation of stress memory (the process known as 'priming' or 'acclimation'). In this review, we summarize the data on BR and abscisic acid (ABA) signaling to show the interconnection between the pathways in the stress memory acquisition. Starting from brassinosteroid receptors brassinosteroid insensitive 1 (BRI1) and receptor-like protein kinase BRI1-like 3 (BRL3) and propagating through BR-signaling kinases 1 and 3 (BSK1/3) â BRI1 suppressor 1 (BSU1) -â brassinosteroid insensitive 2 (BIN2) pathway, BR and ABA signaling are linked through BIN2 kinase. Bioinformatics data suggest possible modules by which BRs can affect the memory to drought or cold stresses. These are the BIN2 â SNF1-related protein kinases (SnRK2s) â abscisic acid responsive elements-binding factor 2 (ABF2) module; BRI1-EMS-supressor 1 (BES1) or brassinazole-resistant 1 protein (BZR1)-TOPLESS (TPL)-histone deacetylase 19 (HDA19) repressor complexes, and the BZR1/BES1 â flowering locus C (FLC)/flowering time control protein FCA (FCA) pathway. Acclimation processes can be also regulated by BR signaling associated with stress reactions caused by an accumulation of misfolded proteins in the endoplasmic reticulum.
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Ácido Abscísico/metabolismo , Brasinoesteroides/metabolismo , Plantas/metabolismo , Transducción de Señal , Aclimatación , Reguladores del Crecimiento de las Plantas/metabolismo , Fenómenos Fisiológicos de las Plantas , Proteínas de Plantas/metabolismo , Estrés Fisiológico , Agua/metabolismoRESUMEN
An ability to synthesize extracellular enzymes degrading a wide spectrum of plant and algae polymeric substrates makes many fungi relevant for biotechnology. The terrestrial thermophilic and marine fungal isolates capable of plant and algae degradation have been tested for antibiotic resistance for their possible use in a new genetic transformation system. Plasmids encoding the hygromycin B phosphotransferase (hph) under the control of the cauliflower mosaic virus 35S promoter, the trpC gene promoter of Aspergillus nidulans, and the Aureobasidium pullulans TEF gene promoter were delivered into the fungal cells by electroporation. The effectiveness of different promoters was compared by transformation and growth of Thermothelomyces thermophila (formerly Myceliophthora thermophila) on the selective medium and by real-time PCR analysis. A highly efficient transformation was observed at an electric-pulse of 8.5â¯kV/cm by using 10⯵g of DNA per 1â¯×â¯105 conidia. Although all promoters were capable of hph expression in the Th. thermophila cells, the trpC promoter provided the highest level of hygromycin resistance. We further successfully applied plant binary vector pPZP for co-transformation of hph gene and enhanced green fluorescent protein gene that confirmed this transformation system could be used as an appropriate tool for gene function studies and the expression of heterologous proteins in micromycetes.
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Organismos Acuáticos/genética , Plásmidos/metabolismo , Saccharomycetales/genética , Esporas Fúngicas/genética , Transformación Genética , Organismos Acuáticos/clasificación , Organismos Acuáticos/efectos de los fármacos , Organismos Acuáticos/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Cinamatos/farmacología , Electroporación/métodos , Calor , Higromicina B/análogos & derivados , Higromicina B/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Filogenia , Plásmidos/química , Regiones Promotoras Genéticas , Federación de Rusia , Saccharomycetales/clasificación , Saccharomycetales/efectos de los fármacos , Saccharomycetales/metabolismo , Agua de Mar/microbiología , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/metabolismoRESUMEN
Alkaloids attract great attention due to their valuable therapeutic properties. Stepharine, an aporphine alkaloid of Stephania glabra plants, exhibits anti-aging, anti-hypertensive, and anti-viral effects. The distribution of aporphine alkaloids in cell cultures, as well as whole plants is unknown, which hampers the development of bioengineering strategies toward enhancing their production. The spatial distribution of stepharine in cell culture models, plantlets, and mature micropropagated plants was investigated at the cellular and organ levels. Stepharine biosynthesis was found to be highly spatially and temporally regulated during plant development. We proposed that self-intoxication is the most likely reason for the failure of the induction of alkaloid biosynthesis in cell cultures. During somatic embryo development, the toxic load of alkaloids inside the cells increased. Only specialized cell sites such as vascular tissues with companion cells (VT cells), laticifers, and parenchymal cells with inclusions (PI cells) can tolerate the accumulation of alkaloids, and thus circumvent this restriction. S. glabra plants have adapted to toxic pressure by forming an additional transport secretory (laticifer) system and depository PI cells. Postembryonic growth restricts specialized cell site formation during organ development. Future bioengineering strategies should include cultures enriched in the specific cells identified in this study.
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Alcaloides/metabolismo , Morfogénesis , Stephania/crecimiento & desarrollo , Stephania/metabolismo , Línea Celular , Microdisección , Fenotipo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Stephania/citología , Factores de TiempoRESUMEN
Anthocyanin biosynthesis in Arabidopsis is a convenient and relatively simple model for investigating the basic principles of secondary metabolism regulation. In recent years, many publications have described links between anthocyanin biosynthesis and general defense reactions in plants as well as photomorphogenesis and hormonal signaling. These relationships are complex, and they cannot be understood intuitively. Upon observing the lacuna in the Arabidopsis interactome (an interaction map of the factors involved in the regulation of Arabidopsis secondary metabolism is not available), we attempted to connect various cellular processes that affect anthocyanin biosynthesis. In this review, we revealed the main signaling protein modules that regulate anthocyanin biosynthesis. To our knowledge, this is the first reconstruction of a network of proteins involved in plant secondary metabolism.
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Antocianinas/metabolismo , Metabolismo Secundario , Arabidopsis , Proteínas de Arabidopsis , Regulación de la Expresión Génica de las Plantas , Factores de TranscripciónRESUMEN
The process of silica formation in marine sponges is thought to be mediated by a family of catalytically active structure-directing enzymes called silicateins. It has been demonstrated in biomimicking syntheses that silicateins facilitated the formation of amorphous SiO2. Here, we present evidence that the silicatein LoSiLA1 from the marine sponge Latrunculia oparinae catalyzes the in vitro synthesis of hexa-tetrahedral SiO2 crystals of 200300 nm. This was possible in the presence of the silica precursor tetrakis-(2-hydroxyethyl)-orthosilicate that is completely soluble in water and biocompatible, experiences hydrolysiscondensation at neutral pH and ambient conditions.
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Organismos Acuáticos/enzimología , Catepsinas/química , Nanopartículas/química , Poríferos/enzimología , Dióxido de Silicio/química , Animales , Organismos Acuáticos/genética , Catepsinas/genética , Poríferos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Plant cell cultures are of particular interest in industrial applications as a source of biologically active substances. It is difficult, however, to achieve stable production of secondary metabolites for many plant cell cultures using classical techniques. Novel approaches should be developed for removal of the inhibitor blocks that prevent pathway activation and shift the regulatory balance to the activation of entire biosynthetic pathways. MicroRNAs (miRNAs) are small RNAs that play important regulatory roles in various biological processes. Only recently miRNAs have been demonstrated as active in secondary metabolism regulation. In this work, we summarize recent data on the emerging approaches based on regulation of secondary metabolism by miRNAs.
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MicroARNs/metabolismo , Plantas/genética , ARN de Planta/metabolismo , Metabolismo Secundario , Regulación de la Expresión Génica de las Plantas , MicroARNs/genética , Plantas/metabolismo , ARN de Planta/genéticaRESUMEN
The rolB gene of Agrobacterium rhizogenes renders cells more tolerant of environmental stresses and increases their defense potential. However, these effects, coupled with the developmental abnormalities caused by rolB, have not yet been explained. In rolB-transformed Arabidopsis thaliana cells, we detected a 2.2 to 7-fold increase in the expression of genes encoding core and accessory proteins (DCL1, SE, HYL1, AGO1, TGH, DDL, HEN1, AGO4 and RDR2) of the microRNA processing machinery. However, the rolB gene did not affect the expression of DCL2, DCL3 and HST. The diverse and complex effects of rolB on transformed plant cells may be attributable to changes caused by this gene in particular RNA silencing pathways.
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Agrobacterium/genética , Agrobacterium/metabolismo , Proteínas Bacterianas/metabolismo , Regulación de la Expresión Génica de las Plantas , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , beta-Glucosidasa/metabolismo , Arabidopsis/genética , Proteínas Bacterianas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Glucosidasa/genéticaRESUMEN
The rolB (for rooting locus of Agrobacterium rhizogenes) oncogene has previously been identified as a key player in the formation of hairy roots during the plant-A. rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng, and Arabidopsis (Arabidopsis thaliana) cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione, and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase, and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.
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Agrobacterium/genética , Antioxidantes/metabolismo , Proteínas Bacterianas/metabolismo , Células Vegetales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , beta-Glucosidasa/metabolismo , Arabidopsis/citología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Ascorbato Peroxidasas/genética , Ascorbato Peroxidasas/metabolismo , Proteínas Bacterianas/genética , Muerte Celular , Supervivencia Celular , Medios de Cultivo/metabolismo , Glutatión/metabolismo , Luz , Estrés Oxidativo , Panax/citología , Panax/efectos de los fármacos , Panax/genética , Panax/metabolismo , Paraquat/farmacología , Células Vegetales/efectos de los fármacos , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Rubia/efectos de los fármacos , Rubia/genética , Rubia/metabolismo , Plantas Tolerantes a la Sal/citología , Plantas Tolerantes a la Sal/efectos de los fármacos , Plantas Tolerantes a la Sal/genética , Plantas Tolerantes a la Sal/metabolismo , Cloruro de Sodio/farmacología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Vitamina K 3/farmacología , beta-Glucosidasa/genéticaRESUMEN
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.
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Botánica , Técnicas de Cultivo de Célula , Rubia , Rubia/química , Rubia/metabolismo , Antraquinonas/metabolismo , Células Vegetales , Especies Reactivas de Oxígeno/metabolismo , Ácidos Indolacéticos/farmacología , Reguladores del Crecimiento de las Plantas/metabolismo , Botánica/métodos , Técnicas de Cultivo de Célula/métodos , Transformación GenéticaRESUMEN
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.
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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.
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Rosmarinic acid (RA) is one of the first secondary metabolites produced in plant cell cultures in extremely high yields, up to 19% of the cell dry weight. More complex derivatives of RA, such as rabdosiin and lithospermic acid B, later were also obtained in cell cultures at high yields. RA and its derivatives possess promising biological activities, such as improvement of cognitive performance, prevention of the development of Alzheimer's disease, cardioprotective effects, reduction of the severity of kidney diseases and cancer chemoprevention. The TNF-α-induced NF-κB signaling pathway has emerged as a central target for RA. Despite these impressive activities and high yields, the biotechnological production of these metabolites on an industrial scale has not progressed. We summarized data suggesting that external stimuli, the Ca(2+)-dependent NADPH oxidase pathway and processes of protein phosphorylation/dephosphorylation are involved in the regulation of biosynthesis of these substances in cultured plant cells. In spite of growing information about pathways regulating biosynthesis of RA and its derivatives in cultured plant cells, the exact mechanism of regulation remains unknown. We suggest that further progress in the biotechnology of RA and its derivatives can be achieved by using new high-throughput techniques.