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.

TLC-MS-Bioautographic Identification of Antityrosinase Compounds and Preparation of a Topical Gel Formulation from a Bioactive Fraction of an RSM-Optimized Alcoholic Extract of Rubia Cordifolia L. stem.

BACKGROUND: Rubia cordifolia L., Rubiaceae, is globally reported to treat skin-related problems. The study aimed to assess the antityrosinase potential of Rubia cordifolia (ARC) and the development of gel formulation. METHODS: The AutoDock Vina (version V.1.2.0) program package was used for molecular docking to check for the binding affinity of ligands with protein. Response surface methodology (RSM) software was used to optimize extraction parameters for an alcoholic extract of Rubia cordifolia (ARC). The developed HPTLC method for the quantification of purpurin in ARC was validated as per the International Conference on Harmonization (ICH) guidelines. A bioautographic study for the evaluation of antityrosinase effects was performed; an anthraquinone-enriched fraction (AEF)-loaded gel formulation developed and evaluated physicochemically which could be used to reduce skin pigmentation. RESULTS: Purpurin showed optimum binding affinity (-7.4 kcal/mol) with the molecular target (tyrosinase) when compared to that of standard kojic acid (-5.3 kcal/mol). Quantification of purpurin in ARC, optimized by RSM software, was validated and physiologically significant results were observed for the antityrosinase potential of an AEF, along with TLC-MS-bioautographic identification for antityrosinase compounds: purpurin (m/z 256.21) and ellagic acid (m/z 302.19). Evaluation of an AEF-loaded gel formulation by in vitro and ex vivo permeation studies was performed. CONCLUSION: ARC extraction parameters optimized by RSM, and a bioautographic study helped identify antityrosinase compounds. The development of a gel formulation could be a cost-effective option for the treatment of depigmentation in the future. HIGHLIGHTS: A TLC-MS-Bioautography-based Identification of Antityrosinase Compounds and development of AEF-loaded Topical Gel formulation from a Bioactive Fraction of an RSM-Optimized Alcoholic Extract of Rubia Cordifolia L. stem, which could help with promising results in reducing skin pigmentation and maintaining even tone.

De Novo Transcriptome Analysis Reveals Putative Genes Involved in Anthraquinone Biosynthesis in Rubia yunnanensis.

Rubia yunnanensis Diels (R. yunnanensis), a Chinese perennial plant, is well-known for its medicinal values such as rheumatism, contusion, and anemia. It is rich in bioactive anthraquinones, but the biosynthetic pathways of anthraquinones in R. yunnanensis remain unknown. To investigate genes involved in anthraquinone biosynthesis in R. yunnanensis, we generated a de novo transcriptome of R. yunnanensis using the Illumina HiSeq 2500 sequencing platform. A total of 636,198 transcripts were obtained, in which 140,078 transcripts were successfully annotated. A differential gene expression analysis identified 15 putative genes involved in anthraquinone biosynthesis. Additionally, the hairy roots of R. yunnanensis were treated with 200 µM Methyl Jasmonate (MeJA). The contents of six bioactive anthraquinones and gene expression levels of 15 putative genes were measured using ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. The results showed that the expressions levels for 11 of the 15 genes and the contents of two of six anthraquinones significantly increased by MeJA treatment. Pearson's correlation analyses indicated that the expressions of 4 of the 15 putative genes were positively correlated with the contents of rubiquinone (Q3) and rubiquinone-3-O-ß-d-xylopranosyl-(1→6)-ß-d-glucopyranoside (Q20). This study reported the first de novo transcriptome of R. yunnanensis and shed light on the anthraquinone biosynthesis and genetic information for R. yunnanensis.

Inactivation of the auto-inhibitory domain in Arabidopsis AtCPK1 leads to increased salt, cold and heat tolerance in the AtCPK1-transformed Rubia cordifolia L cell cultures.

Calcium-dependent protein kinases (CDPKs) are essential regulators of plant growth and development, biotic and abiotic stress responses. Inactivation of the auto-inhibitory domain (AID) of CDPKs provides the constitutive activity. This study investigated the effect of overexpressed native and constitutive active (AtCPK1-Ca) forms of the AtCPK1 gene on abiotic stress tolerance and the ROS/redox system in Rubia cordifolia transgenic callus lines. Overexpression of the native AtCPK1 increased tolerance to salinity and cold almost in two times, when AtCPK1-Ca - in three times compare to control culture. A more interesting effect of overexpression of the AtCPK1 and AtCPK1-Ca was observed for heat resistance. The native form of AtCPK1 increased resistance to heating by 45%, while the AtCPK1-Ca increased by 80%. At the same time, another type of mutation of the AID (AtCPK1-Na, not active) did not affect the tolerance of the cell culture to stresses. We suppose, in this process, the ROS/redox system might be involved. Levels of intracellular ROS, ROS-generating enzymes expression and activities (Rbohs, Prx) and ROS-detoxifying enzymes (SOD, Cat, Apx and Prx) changed in a coordinated manner and in strict interconnection, depending of the callus growth phase and correlated with improved stress tolerance caused by AtCPK1. Because overexpression of both the AtCPK1 and AtCPK1-Ca did not significantly change callus growth, we propose that inactivation of AID of the AtCPK1 or its ortholog, might be an interesting instrument for improvement of plant cells resistance to abiotic stress.

Selection and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data.

Real-time quantitative polymerase chain reaction (RT-qPCR) has been widely applied in gene expression and transcription abundance analysis because of its high sensitivity, good repeatability, and strong specificity. Selection of relatively stable reference genes is a precondition in order to obtain the reliable analysis results. However, little is known about evaluation of a set of reference genes through scientific experiments in Rubia plants. Here, 15 candidate reference genes were selected from R. yunnanensis transcriptome database and analyzed under abiotic stresses, hormone treatments, and different tissues. Among these 15 candidate reference genes, heterogeneous nuclear ribonucleoprotein (hnRNP), TATA binding protein (TBP), ribosomal protein L5 (RPL5), malate dehydrogenase (MDH), and elongation factor 1-alpha (EF-1α) were indicated as the five most stable reference genes by four statistical programs (geNorm, NormFinder, BestKeeper, and RefFinder). Ultimately, the validity of reference genes was confirmed by normalizing the expression of o-succinylbenzoate-CoA ligase (OSBL) and isochorismate synthase (ICS) involved in the anthraquinone biosynthesis pathway in different tissues and hormone treatments. Meanwhile, four other putative genes involved in the anthraquinone biosynthesis pathway were also normalized with the selected reference genes, which showed similar expression levels with those given by transcriptome data. This work is the first research that aims at a systematic validation on the stability of reference genes selected from R. yunnanensis transcriptome data and will be conducive to analyze gene expression in R. yunnanensis or other Rubia species.

Effect of salt stress on the genes expression of the vacuolar H+ -pyrophosphatase and Na+/H+ antiporter in Rubia tinctorum.

Salinity which covers vast areas of the world is increasing every year. But some plants like madder can grow in these areas. Madder (Rubia tinctorum) is a perennial plant species from the Rubiaceae family. In this study, madder plants were first treated by different concentration of NaCl (100, 200, 300, and 400 mM). Then gene expression of salinity stress was studied. For gene study, vacuolar H+-pyrophosphatase pump (AVP) and tonoplast Na+/H+ antiporters (NHX) from madder plant were isolated and sequenced. Analyzing protein sequences of these genes demonstrated that the protein sequences have high similarity with the same genes in other plants. Constructing phylogenetic trees based on the protein sequences of the AVP and NHX genes, we found high similarity with Coffea arabica and Capsicum annuum, respectively. Studying gene expression of the AVP and NHX under the condition of salt stress revealed that the genes were up-regulated, which continues up to 400 mM of salt concentration.

Activation of anthraquinone biosynthesis in long-cultured callus culture of Rubia cordifolia transformed with the rolA plant oncogene.

The RolA protein belongs to the RolB class of plant T-DNA oncogenes, and shares structural similarity with the papilloma virus E2 DNA-binding domain. It has potentially as an inducer of plant secondary metabolism, although its role in biotechnology has yet to be realised. In this investigation, a Rubia cordifolia callus culture transformed with the rolA plant oncogene for more than 10 years was analysed. Expression of the rolA gene in the callus line was stable during long-term cultivation, and growth parameters were both elevated and stable, exceeding those of the non-transformed control culture. The rolA-transformed calli not only demonstrated remarkably stable growth, but also the ability to increase the yield of anthraquinones (AQs) in long-term cultivation. After ten years of cultivating rolA callus lines, we observed an activation of AQ biosynthesis from 200 mg/l to 874 mg/l. The increase was mainly due to activation of ruberitrinic acid biosynthesis. The expression of key AQ biosynthesis genes was strongly activated in rolA-transgenic calli. We compared the effects of the rolA gene with those of the rolB gene, which was previously considered the most potent inducer of secondary metabolism, and showed that rolA was more productive under conditions of long-term cultivation.

Accelerated degradation of cFLIPL and sensitization of the TRAIL DISC-mediated apoptotic cascade by pinoresinol, a lignan isolated from Rubia philippinensis.

Plant-derived lignans have numerous biological effects including anti-tumor and anti-inflammatory activities. Screening of purified constituents of Rubia philippinensis from human glioblastoma cells resistant to TNF-related apoptosis-inducing ligand (TRAIL) has suggested that the lignan pinoresinol was a highly active TRAIL sensitizer. Here we show that treatment with nontoxic doses of pinoresinol in combination with TRAIL induced rapid apoptosis and caspase activation in many types of glioblastoma cells, but not in normal astrocytes. Analyses of apoptotic signaling events revealed that pinoresinol enhanced the formation of TRAIL-mediated death-inducing signaling complex (DISC) and complete processing of procaspase-8 within the DISC in glioblastoma cells, in which caspase-8 was inactivated. Mechanistically, pinoresinol downregulated the expression of cellular FLICE-inhibitory protein (cFLIPL) and survivin through proteasome-mediated degradation, without affecting death receptors or downstream intracellular apoptosis-related proteins. Furthermore, the sensitization of TRAIL-mediated apoptosis by pinoresinol strictly depended on the expression level of cFLIPL, which was regulated through de novo protein synthesis, rather than by NF-κB or p53 signaling. Taken together, our results indicate that pinoresinol facilitates DISC-mediated caspase-8 activation by targeting cFLIPL in an early event in apoptotic signaling, which provides a potential therapeutic module for TRAIL-based chemotherapy.

ISSR Characterization and Quantification of Purpurin and Alizarin in Rubia cordifolia L. Populations from India.

Rubia cordifolia L., is an industrially viable medicinal crop and is widely exploited for the therapeutic potential of its bioactive metabolite, purpurin. The present investigation aimed to explore the chemotypic and molecular variability in seven wild populations of R. cordifolia from South Eastern Ghats region of India. Thirty-eight individuals were assessed for molecular fingerprinting (ISSR markers) and densitometric quantification of purpurin and alizarin. The populations of Yelagiri Hills and Shervaroy Hills contained the highest levels of alizarin (0.115 and 0.093%, respectively) while Pachamalai and Kolli Hills revealed the highest purpurin content (0.284 and 0.280%, respectively). Genetic diversity was generally higher in the same populations that produced higher metabolite content, with the exception of Pachamalai, suggesting a highly prioritized conservation concern. The study revealed a Nei's total gene diversity at species level of 0.266 and of 0.187 at population level, with an average population genetic differentiation of 0.28. No clear genetic or chemical structure was retrieved between the studied populations, with individuals from different locations clustering together, and no significant correlation was obtained between metabolites and genetic diversity or between these and the populations' geographic distances.

RA-dimer†B, a New Dimeric RA-series Cyclopeptide Incorporating Two Different Types of Cycloisodityrosine Units, from Rubia cordifolia†L.

RA-dimer B, a new cytotoxic RA-series peptide, was isolated from the roots of Rubia cordifolia L. Its structure was elucidated on the basis of spectroscopic analysis to be a dimeric cyclopeptide composed of deoxybouvardin and allo-RA-V. Those two cyclopeptide units are connected by an ether linkage between the phenolic oxygen atom of deoxybouvardin and the ϵa carbon atom of Tyr-6 of allo-RA-V. RA-dimer B was synthesized by the coupling reaction of deoxybouvardin with the boronic acid derivative of allo-RA-V, and subsequent deprotection, confirming the relative stereochemistry and establishing the absolute configuration of this peptide. RA-dimer B showed cytotoxic activity against human promyelocytic leukaemia HL-60, human colonic carcinoma HCT-116, and human renal cell carcinoma ACHN cells with IC50 values of 0.59, 0.54, and 0.74 µm, respectively.

Increase of anthraquinone content in Rubia cordifolia cells transformed by native and constitutively active forms of the AtCPK1 gene.

KEY MESSAGE: Overexpression of both native and mutant forms of AtCPK1 in Rubia cordifolia cells increased anthraquinone production and transcript abundance of the RcIPPI, RcOSBL, RcOSBS , and RcICS genes to different extents. Calcium-dependent protein kinases (CDPKs) are involved in various cell processes and are regulated by a calcium signal system. CDPKs also function in plant defense against stress factors such as pathogens, temperature, and salinity. In this study, we compared the effect of heterologous expression of two forms of the Arabidopsis AtCPK1 gene, native and constitutively active (Ca(2+)-independent), on anthraquinone production in transgenic Rubia cordifolia cells. Significant qualitative and quantitative differences were found in the content of anthraquinone derivatives in control and AtCPK1-transgenic calli. Expression of the AtCPK1 gene increased anthraquinone production by 3 and 12 times for native and constitutively active forms, respectively, compared with control cells. In addition, we identified and quantified the expression of genes encoding key enzymes of the anthraquinone biosynthesis pathway, including isochorismate synthase (ICS), o-succinylbenzoate synthase (OSBS), o-succinylbenzoate ligase (OSBL), and isopentenyl diphosphate isomerase (IPPi). In all AtCPK1-transgenic cell lines, expression of ICS, OSBS, OSBL, and IPPi increased considerably at 14-15 days of subculture and decreased at the end of cultivation (30 days). The results suggest that both native and constitutively active AtCPK1 forms induced anthraquinone accumulation at the logarithmic growth stage via enhancement of expression of genes involved in the metabolism of anthraquinones or their regulatory mechanisms.

Immobilization of Rubia tinctorum L. Suspension Cultures and Biomass Production.

Plants are natural sources of valuable secondary metabolites used as pharmaceuticals, agrochemicals, flavors, fragrances, colors, biopesticides, and food additives. There is an increasing demand to obtain these metabolites through more productive plant tissue applications and cell culture methods due to the importance of secondary metabolites.Immobilization of plant cells is a method used in plant cell cultures to induce secondary metabolite production. In this method, plant cells are fixed in or on a supporting material or matrix such as agar, agarose, calcium alginate, glass, or polyurethane foam. In the present study, three natural lignocellulosic materials, loofah sponge, and the long fibers of sisal and jute, were used to immobilize suspended R. tinctorum cells.

Toxicity study of dibutyl phthalate of Rubia cordifolia fruits: in vivo and in silico analysis.

Natural toxins from plant sources with wide ranges of biological activities reflect the upswing of drug design in the pharmaceutical industry. Rubia cordifolia L. is one of the most important red dye yielding plants. Most of the former researches have focused on the bioactive compounds from the roots of R. cordifolia, while no attention was paid towards the fruits. For the first time, here we report the presence of dibutyl phthalate in the fruits of R. cordifolia. Structural characterization was carried out using Ultraviolet-Visible spectrophotometer (UV-Vis), Fourier transform infrared (FTIR), gas chromatography-mass spectrophotometer (GC-MS), Nuclear magnetic resonance (NMR). Acute toxicity of the crude ethanolic extracts of the R. cordifolia fruits was examined in Swiss albino mice. No mortality was observed in all treated mice with 100, 500, 1000 mg/kg body weight of crude extract of R. cordifolia fruit and it indicates that the LD50 value is higher than 1000 mg/kg body weight. This study exhibited a significant change in the body weight. Alanine transaminase (ALT), total protein, triglycerides, glucose, and also the histopathological analysis of liver for all treated mice showed difference from the control group. The dibutyl phthalate was further evaluated for the toxicity study through in silico analysis. Together, the results highlighted that the toxic potential of R. cordifolia fruits extracts and also the toxicity profile of the fruit should be essential for the future studies dealing with the long term effect in animals. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1059-1067, 2016.

Natural dye sensitized TiO2 nanorods assembly of broccoli shape based solar cells.

TiO2 nanorods based thin films with rutile phase have been synthesized using template free low temperature hydrothermal method. The scanning electron microscope images showed that the prepared TiO2 samples were made of TiO2 nanorods and the nanorods had arranged by itself to form a broccoli like shape. The X-ray diffraction studies revealed that the prepared TiO2 samples exhibit rutile phase. The grown TiO2 nanorods had been sensitized using the flowers of Sesbania (S) grandiflora, leaves of Camellia (C) sinensis and roots of Rubia (R) tinctorum. Dye sensitized solar cells had been fabricated using the natural dye sensitized TiO2 nanorods based thin film photoelectrode and the open circuit voltage and short circuit current density were found to lie in the range of 0.45-0.6 V and 5.6-6.4 mA/cm(2) respectively. The photovoltaic performance of all the fabricated natural dye sensitized TiO2 solar cells indicate that natural dyes have the potential to be used as effective sensitizer in dye sensitized solar cells.

Quinone derivatives from the genus Rubia and their bioactivities.

The extracts and phytochemicals of the genus Rubia have drawn much attention due to their potent effects; among them, naphthoquinone and cyclopeptide derivatives, with significant biological activities, have great potential to be developed to new drugs. This review updates and compiles a total of 142 quinone derivatives including anthraquinone and naphthoquinone derivatives, occuring in twelve Rubia species. These compounds were listed together with their sources, melting points, bioactivities, as well as 112 corresponding references. Furthermore, the structureactivity relationships of these quinone derivatives were discussed.

Expression profiles of calcium-dependent protein kinase genes (CDPK1-14) in Agrobacterium rhizogenes pRiA4-transformed calli of Rubia cordifolia under temperature- and salt-induced stresses.

Agrobacterium rhizogenes genetically transform plant cells naturally via horizontal gene transfer by the introduction of T-DNA from the Ri plasmid into genomic DNA to create favorable conditions for successful colonization. An intriguing feature of pRiA4-transformed cells is their recently discovered enhanced tolerance to abiotic stress stimuli and activation of antioxidant enzyme expression. The mechanism by which A. rhizogenes modulates the defense responses of transformed cells remains unclear. It has been established that calcium-dependent protein kinase (CDPK) genes mediate crosstalk of signaling pathways in plants, and these genes have been implicated in biotic and abiotic stress signaling. In this study, we identified fourteen CDPK genes from Rubia cordifolia and examined their expression in aerial plant organs as well as in non-transformed and A. rhizogenes A4-transformed calli. Expression of RcCDPK4, RcCDPK5, RcCDPK7, and RcCDPK10 was 1.2- to 3.9-fold higher in pRiA4-transformed cells than in non-transformed cells, whereas expression of RcCDPK1, RcCDPK9, RcCDPK11, and RcCDPK14 was 1.2- to 1.9-fold lower. Agrobacterium transformation substantially modified the transcriptional responses of specific RcCDPK isoforms in pRiA4-transformed cells under conditions of temperature- and salinity-induced stress. On the basis of the results, we suggest that A. rhizogenes T-DNA genes exert their diverse biological functions by altering the expression of various CDPK genes.

Aza-cycloisodityrosine analogue of RA-VII, an antitumor bicyclic hexapeptide.

An aza-cycloisodityrosine analogue of RA-VII, 3, was designed and synthesized. The key aza-cycloisodityrosine unit was prepared by copper(II)-acetate-mediated intramolecular phenylamine/arylboronic acid coupling of dipeptide followed by connection with the tetrapeptide segment to afford a hexapeptide. Subsequent macrocyclization of the hexapeptide with EDC · HCl and HOOBt under dilute conditions gave 3. Analogue 3 showed significant cytotoxic activity against human promyelocytic leukemia HL-60 cells and human colon carcinoma HCT-116 cells, but its activity was weaker than that of parent peptide RA-VII (1).

Effects of lovastatin, clomazone and methyl jasmonate treatment on the accumulation of purpurin and mollugin in cell suspension cultures of Rubia cordifolia.

AIM: To determine the IPP origin of the naphthoquinones (NQs) in Rubia cordifolia, and to evaluate the effects of methyl jasmonate (MeJA) treatment, MEP, and MVA pathway inhibitor treatment on the accumulation of anthraquinones (AQs) and NQs in cell suspension cultures of R. cordifolia. METHODS: Cell suspension cultures of R. cordifolia were established. Specific inhibitors (lovastatin and clomazone) and MeJA were supplied to the media, respectively. Treated cells were sampled every three days. Content determination of purpurin (AQs) and mollugin (NQs) were carried out using RP-HPLC. The yield of the two compounds was compared with the DMSO-supplied group and the possible mechanism was discussed. RESULTS: Lovastatin treatment increased the yield of purpurin and mollugin significantly. Clomazone treatment resulted in a remarkable decrease of both compounds. In the MeJA-treated cells, the purpurin yield increased, meanwhile, the mollugin yield decreased compared with control. CONCLUSION: The IPP origin of mollugin in R. cordifolia cell suspension cultures was likely from the MEP pathway. To explain the different effects of MeJA on AQs and NQs accumulation, studies on the regulation and expression of the genes, especially after prenylation of 1,4-dihydroxy-2-naphthoic acid should be conducted.

Determination of lucidin-specific DNA adducts by liquid chromatography with tandem mass spectrometry in the livers and kidneys of rats given lucidin-3-O-primeveroside.

Lucidin-3-O-primeveroside (LuP) is a component of madder color (MC), a compound which is carcinogenic in the kidney and liver of rats. Since LuP is metabolized to generate genotoxic compounds such as lucidin (Luc) and rubiadin, it is likely that these play key roles in MC carcinogenesis. In fact, after incubation of Luc with calf thymus DNA, Luc-N(2)-dG and N(6)-dA adducts were reportedly formed, possibly via the sulfotransferase metabolic pathway. However, the precise extent of formation in vivo remains uncertain. In the present study, to quantitatively determine Luc-specific DNA adducts in in vivo samples, we developed an online sample purification method using column-switching and an isotope dilution LC-ESI-MS/MS technique. The limits of quantification were 0.2 and 0.04 fmol on column for Luc-N(2)-dG and N(6)-dA adducts, respectively. Using the new analytical method, we attempted to measure adduct levels in the kidneys and livers of rats treated with 0.06, 0.3, and 1.5% LuP in the diet for one week. Luc-N(2)-dG and N(6)-dA adducts in these organs were detected at ranges from 7.97 to 51.67/10(9) dG and from1.83 to 37.10/10(9) dA, respectively. Dose-dependent increases of each adduct were observed in both organs. These quantitative data obtained with our newly developed analytical method might help to improve our understanding of MC carcinogenesis.

The rolB gene suppresses reactive oxygen species in transformed plant cells through the sustained activation of antioxidant defense.

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.