High Yield of Bioactive Abietane Diterpenes in Salvia sclarea Hairy Roots by Overexpressing Cyanobacterial DXS or DXR Genes

Planta Medica ◽  
2019 ◽  
Vol 85 (11/12) ◽  
pp. 973-980 ◽  
Author(s):  
Mariacarmela Vaccaro ◽  
Vivian Ocampo Bernal ◽  
Nicola Malafronte ◽  
Nunziatina De Tommasi ◽  
Antonietta Leone
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Biological Activities ◽  
Ectopic Expression ◽  
Fold Increase ◽  
Carnosic Acid ◽  
High Yield ◽  
Salvia Sclarea ◽  
Cyanobacterial Genes ◽  
Phosphate Synthase

AbstractAbietane diterpenoids, containing a quinone moiety, are synthesized in the roots of several Salvia species. Promising cytotoxicity and antiproliferative activities have been reported for these compounds in various cell and animal models. We have recently shown that aethiopinone, an o-naphto-quinone diterpene, produced in the roots of different Salvia species, is selectively cytotoxic against the A375 melanoma cell line. To enhance the synthesis of this abietane diterpenoid, we have engineered the plastidial 2-C-methyl-D-erythritol 4-phosphate-derived isoprenoid pathway in Salvia sclarea hairy roots by ectopic expression and plastid targeting of cyanobacterial genes encoding the 1-deoxy-D-xylulose 5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene, the first two enzymatic steps of the plastidial MEP pathway, from which plant diterpenes primarily derive. Plastid-targeted expression of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase proteins significantly enhanced the yield of aethiopinone by a 3-fold and about 6-fold increase, respectively. The accumulation of other abietane-type diterpenes (ferruginol, salvipisone, and carnosic acid), with interesting antiproliferative activity, was also increased. Compared to our previous data obtained by overexpressing the plant orthologous 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase genes in S. sclarea hairy roots, the results presented here confirm that the bacterial 1-deoxy-D-xylulose-5-phosphate reductoisomerase enzyme plays a major role than the DXS enzyme in the biosynthetic pathway of this class of compounds and that its ectopic expression does not conflict with active hairy root growth, resulting in a balanced trade-off between the transgenic hairy root final biomass and the increased content of o-naphto-quinone diterpenes, with interesting biological activities.

scholarly journals Ectopic Expression of Production of Anthocyanin Pigment 1 (PAP1) Improves the Antioxidant and Anti-Melanogenic Properties of Ginseng (Panax ginseng C.A. Meyer) Hairy Roots

Antioxidants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 922
Author(s):  
Sora Jin ◽  
Tae Kyung Hyun
Keyword(s):  
Panax Ginseng ◽  
Hairy Roots ◽  
Biological Activities ◽  
Radical Scavenging Activity ◽  
Differentially Expressed Gene ◽  
Ectopic Expression ◽  
Radical Scavenging ◽  
Ginseng Root ◽  
Anthocyanin Pigment ◽  
Anthocyanin Production

The development of genetically engineered cell cultures has been suggested as a potential approach for the production of target compounds from medicinal plants. In this study, we generated PAP1 (production of anthocyanin pigment 1)-overexpressing ginseng (Panax ginseng C.A. Meyer) hairy roots to improve the production of anthocyanins, as well as the bioactivity (e.g., antioxidant and whitening activities) of ginseng. Based on differentially expressed gene analysis, we found that ectopic expression of PAP1 induced the expression of genes involved in the ‘phenylpropanoid biosynthesis’ (24 genes), and ‘flavonoid biosynthesis’ (17 genes) pathways, resulting in 191- to 341-fold increases in anthocyanin production compared to transgenic control (TC) hairy roots. Additionally, PAP1-overexpressing ginseng hairy roots exhibited an approximately seven-fold higher DPPH-free radical scavenging activity and 10-fold higher ORAC value compared to the TC. In α-melanocyte-stimulating hormone-stimulated B16F10 cells, PAP1-overexpressing ginseng hairy roots strongly inhibited the accumulation of melanin by 50 to 59% compared to mock-control. Furthermore, results obtained by quantitative real-time PCR, western blot, and tyrosinase inhibition assay suggested that the anti-melanogenic activity of PAP1-overexpressing ginseng hairy roots is mediated by tyrosinase activity inhibition. Taken together, our results suggested that the ectopic expression of PAP1 is an effective strategy for the enhancement of anthocyanin production, which improves the biological activities of ginseng root cultures.

scholarly journals Induction of Platycodon grandiflorum hairy roots through the mediation of four Agrobacterium rhizogenes strains

2016 ◽  
Vol 19 (4) ◽  
pp. 64-75
Author(s):  
Phuong Dong Tra ◽  
Phuong Thi Bach Vu ◽  
Phuong Ngo Diem Quach
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Biological Activities ◽  
Root Induction ◽  
Infection Frequency ◽  
Platycodon Grandiflorum ◽  
Metabolites Production ◽  
Valuable Compounds ◽  
Agrobacterium Rhizogenes Strains

Balloon flower (Platycodon grandiflorum (Jacq.) A. DC.), the only species in Platycodon genus (Campanulaceae), is mainly distributed in East Asia. The rhizomes of P. grandiflorum, a traditional herbal medicine, have been widely used for the treatment of cough, sore throat, asthma, tuberculosis and other diseases. Recently, pharmacological researches identified important biological activities compounds in the rhizomes. Thus, to study and extract valuable compounds, a hairy root induced technique was achieved on P. grandiflorum for stable material with fast growth rates (in hormone-free media) and metabolites production. To achieve this, the “natural genetic tool” Agrobacterium rhizogenes, which can transfer DNA segments into genome of plant, was exploited. The results suggested two (A. rhizogenes ATCC 15834 and C34) of four A. rhizogenes strains could induce hairy roots. RolB and rolC genes, which are responsible for the induction of hairy roots, were inserted into the genome of hairy roots. Leaves had the highest infection frequency of hairy root induction 100 %. The optimization of protocol, including time of immersion and co-culture, had the best results with 10 and 15 mins (10 mins for A. rhizogenes ATCC 15834 and 15 mins for A. rhizogenes C34) and 72 hours, respectively. In the future, this protocol, which was described in this paper, should be useful for studying and isolating valuable compounds from P. grandiflorum hairy root cultures.

scholarly journals Antioxidant compounds in Salvia officinalis L. shoot and hairy root cultures in the nutrient sprinkle bioreactor

2011 ◽  
Vol 79 (1) ◽  
pp. 7-10 ◽  
Author(s):  
Izabela Grzegorczyk ◽  
Halina Wysokińska
Keyword(s):  
Hairy Root ◽  
Shoot Culture ◽  
Growth Period ◽  
Biomass Accumulation ◽  
Fold Increase ◽  
Operating Conditions ◽  
Salvia Officinalis ◽  
Carnosic Acid ◽  
Antioxidant Compounds ◽  
Shoot Cultures

The study focused on the production of compounds with antioxidant activity in hairy root and shoot cultures of <em>Salvia officinalis</em> grown in laboratory-scale sprinkle nutrient bioreactors. HPLC analysis showed that production of rosmarinic acid in transformed roots (34.65 ±1.07 mg l<sup>-1</sup>) was higher that in shoot culture (26.24 ±0.48 mg l<sup>-1</sup>). In the latter diterpenoids: carnosic acid (1.74 ±0.02 mg l<sup>-1</sup>) and carnosol (1.34 ±0.01 mg l<sup>-1</sup>) were also found. Biomass accumulation after a growth period in the bioreactor was also studied. An 18-fold increase in hairy root biomass was recorded after 40 days of culture. In sage shoot culture, biomass increased 43 times after 21 days of bioreactor run. The current operating conditions of the bioreactor were not suitable for the propagation of <em>Salvia officinalis</em> mainly due to the hyperhydricity problem of leaves and stems.

scholarly journals Biological activities and hairy roots induction of Abutilon indicum (L.)

2016 ◽  
Vol 19 (4) ◽  
pp. 95-104
Author(s):  
Phuong Thi Bach Vu ◽  
Minh Thi Thanh Hoang ◽  
Hong Thi Anh Pham ◽  
Phuong Ngo Diem Quach
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Biological Activities ◽  
Reducing Power ◽  
Artemia Salina ◽  
Root Production ◽  
Pcr Analysis ◽  
Root Extract ◽  
Root Induction ◽  
Abutilon Indicum

Abutilon indicum L. belonging to Malvaceae is used as traditional medicinal herbs to treat syphilis, hypoglycemia, hepatic disorders, and malaria... Because of the medicinal value of A. indicum, the aims of this study are the evaluation of bioactivities of A. indicum and production of transformed hairy root for pharmaceutical production. In this study, the reducing power of ethanol root extract and stem extract are higher than that of leave. The root extract exhibited potent brine shrimp (Artemia salina) lethality with LC50 37.04 μg/mL. The α-glucosidase and acetylcholinesterase inhibitory activities of the root extract are the highest. These results showed that roots are higher bioactivities than stems and leaves. This study induced successfully hairy roots of A. indicum L. via Agrobacterium rhizogenes ATCC 15834 in the plant cells. The frequency of hairy root and number of hairy root induction from the wounded sites of leaves are the highest (88.66 % and 8.66 roots). The stable introduction of the rolB and rolC genes of A. rhizogenes strain 15834 into A. indicum plants was confirmed by PCR analysis. Besides, the absence of the virD gene confirmed hairy roots as a bacteria-free. Subsequently, these results demonstrated that A. indicum, particularly the roots, have great potential as pharmacological values and hairy root production maybe used for pharmaceutical sources.

Mitragyna speciosa: Hairy Root Culture for Triterpenoid Production and High Yield of Mitragynine by Regenerated Plants

2008 ◽  
Vol 63 (9-10) ◽  
pp. 691-698 ◽  
Author(s):  
Siriwan Phongprueksapattana ◽  
Waraporn Putalun ◽  
Niwat Keawpradub ◽  
Juraithip Wungsintaweekul
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
High Yield ◽  
Naphthaleneacetic Acid ◽  
Hairy Root Cultures ◽  
Root Cultures ◽  
Mitragyna Speciosa ◽  
In Vitro Plantlets ◽  
Regenerated Plants

Hairy root cultures of Mitragyna speciosa were established by infection of Agrobacterium rhizogenes ATCC 15834 and maintained in McCown woody plant medium (WPM) supplemented with 0.5 mg/l naphthaleneacetic acid. The hairy roots were identified for the rooting genes loci of rolA and rolB by polymerase chain reaction. For studying the secondary metabolite production, the n-hexane extract of the hairy roots was prepared and the compounds were isolated by silica gel column chromatography, affording triterpenoids (ursolic acid and oleanolic acid) and phytosterols (β-sitosterol and stigmasterol). The shoots from the hairy root cultures were regenerated and differentiated to the plantlets. For micropropagation, shoot multiplication was successfully induced from the axillary buds of the regenerated plantlets in WPM supplemented with 0.1 mg/l thidiazuron. The mitragynine contents of 5-monthold regenerated plants and in vitro plantlets (germinated from seeds) were determined using the TLC-densitometric method. The regenerated plants contained (14.25 ± 0.25) mg/g dry wt mitragynine, whereas the in vitro plantlets contained (4.45 ± 0.09) mg/g dry wt.

scholarly journals Carrot hairy roots: factories for secondary metabolite production

2020 ◽  
Vol 71 (22) ◽  
pp. 6861-6864
Author(s):  
María A Pedreño ◽  
Lorena Almagro
Keyword(s):  
Phenolic Compounds ◽  
Secondary Metabolite ◽  
Hairy Root ◽  
Hairy Roots ◽  
Hairy Root Cultures ◽  
Root Cultures ◽  
Black Carrot ◽  
Metabolite Production ◽  
Experimental Botany ◽  
Induced Changes

This article comments on: Barba-Espín G, Chen S-T, Agnolet S, Hegelund JN, Stanstrup J, Christensen JH, Müller R, Lütken H. 2020. Ethephon-induced changes in antioxidants and phenolic compounds in anthocyanin-producing black carrot hairy root cultures. Journal of Experimental Botany 71, 7030–7045.

scholarly journals Resveratrol Biosynthesis in Hairy Root Cultures of Tan and Purple Seed Coat Peanuts

Agronomy ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 975
Author(s):  
Ye-Eun Park ◽  
Chang-Ha Park ◽  
Hyeon-Ji Yeo ◽  
Yong-Suk Chung ◽  
Sang-Un Park
Keyword(s):  
Arachis Hypogaea ◽  
Hairy Root ◽  
Hairy Roots ◽  
Seed Coat ◽  
Biological Properties ◽  
Fresh Weight ◽  
Good Source ◽  
Plant Parts ◽  
Seedling Roots

Peanut (Arachis hypogaea) is a crop that can produce resveratrol, a compound with various biological properties, such as those that exert antioxidant, anticancer, and anti-inflammatory effects. In this study, trans-resveratrol was detected in the roots, leaves, and stems of tan and purple seed coat peanuts (Arachis hypogaea) cultivated in a growth chamber. Both cultivars showed higher levels of resveratrol in the roots than the other plant parts. Thus, both cultivars were inoculated with Agrobacterium rhizogenes, in vitro, to promote hairy root development, thereby producing enhanced levels of t-resveratrol. After 1 month of culture, hairy roots from the two cultivars showed higher levels of fresh weight than those of seedling roots. Furthermore, both cultivars contained higher t-resveratrol levels than those of their seedling roots (6.88 ± 0.21 mg/g and 28.07 ± 0.46 mg/g, respectively); however, purple seed coat peanut hairy roots contained higher t-resveratrol levels than those of tan seed coat peanut hairy roots, ranging from 70.16 to 166.76 mg/g and from 46.61 to 54.31 mg/g, respectively. The findings of this study indicate that peanut hairy roots could be a good source for t-resveratrol production due to their rapid growth, high biomass, and substantial amount of resveratrol.

A reliable and efficient protocol for induction of hairy roots in Agastache foeniculum

Biologia ◽  
2014 ◽  
Vol 69 (7) ◽  
Author(s):  
Elnaz Nourozi ◽  
Bahman Hosseini ◽  
Abbas Hassani
Keyword(s):  
Salicylic Acid ◽  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
High Performance ◽  
Root Culture ◽  
Hairy Root Culture ◽  
Biochemical Properties ◽  
Separate Experiment ◽  
Transformed Roots

AbstractHairy root culture system is a valuable tool to study the characteristics of gene expression, gene function, root biology, biochemical properties and biosynthesis pathways of secondary metabolites. In the present study, hairy roots were established in Anise hyssop (Agastache foeniculum) via Agrobacterium rhizogenes. Three strains of Agrobacterium rhizogenes (A4, A7 and 9435), were used for induction of hairy roots in four various explants (hypocotyl, cotyledon, one-month-old leaf and five-month-old leaf) of Anise hyssop. The highest frequency of transformation was achieved using A4 strain in one-month-old leaves (51.1%). The transgenic states of hairy root lines were confirmed by PCR (Polymerase chain reaction) method. High performance liquid chromatography analysis revealed that the production of rosmarinic acid (RA) in transformed roots of A. foeniculum was almost 4-fold higher than that of the non-transformed roots. In a separate experiment, hairy roots obtained from one-month-old leaves inoculated with A4 strain, were grown in liquid medium and the effects of different concentrations of salicylic acid (0.0, 0.01, 0.1 and 1 mM) and chitosan (0, 50, 100 and 150 mg L−1) (as elicitor) and sucrose (20, 30, 40 and 50 g L−1) on the growth of hairy roots were evaluated. The results showed that, 30 g L−1 sucrose and 100 mg L−1 chitosan increased the biomass of hairy root cultures and application of salicylic acid reduced the growth of hairy roots compared with control roots.

HAIRY ROOT TRANSFORMATION SYSTEM IN LARGE-SEEDED GRAIN LEGUMES

1995 ◽  
Vol 43 (1) ◽  
pp. 1-5 ◽  
Author(s):  
H.J. Siefkes-Boer ◽  
M.J. Noonan ◽  
D.W. Bullock ◽  
A.J. Conner
Keyword(s):  
Vicia Faba ◽  
Hairy Root ◽  
Hairy Roots ◽  
Faba Bean ◽  
Grain Legumes ◽  
Transformation System ◽  
Root Transformation ◽  
Specific Strain ◽  
Size And Morphology ◽  
Root Size

Hairy roots were produced on faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.) plants by inoculation with Agrobacterium root-inducing strains. Examination of 14 plant genotypes and eight Agrobacterium strains in all possible combinations revealed specific strain/genotype interactions. Hairy root size and morphology differed substantially between faba bean and chickpea hairy roots. Sixty percent of chickpea hairy roots were 10–15 mm in length and forty percent, 15–25 mm. All were <1.0 mm in thickness. Sixty-three percent of faba bean hairy roots were 15–25 mm long and thirty-seven percent, 25–40 mm. All faba bean hairy roots were between 1.0 and 1.5 mm in thickness.

scholarly journals Subcellular Targeting of p33ING1b by Phosphorylation-Dependent 14-3-3 Binding Regulates p21WAF1 Expression

2006 ◽  
Vol 26 (8) ◽  
pp. 2947-2954 ◽  
Author(s):  
Wei Gong ◽  
Michael Russell ◽  
Keiko Suzuki ◽  
Karl Riabowol
Keyword(s):  
Kinase Inhibitor ◽  
Biological Activities ◽  
Ectopic Expression ◽  
Growth Stress ◽  
Nuclear Localization Sequence ◽  
Binding Motif ◽  
Subcellular Targeting ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Cargo Proteins ◽  
Localization Sequence

ABSTRACT ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33ING1b splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33ING1b protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33ING1b increased levels of the p21Waf1 cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21Waf1 by p33ING1b, consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33ING1b by directing its subcellular localization.

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