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HCHL expression in hairy roots of Beta vulgaris yields a high accumulation of p-hydroxybenzoic acid (pHBA) glucose ester, and linkage of pHBA into cell walls
Abstract
As part of a study to explore the potential for new or modified bio-product formation, Beta vulgaris (sugar beet) has been genetically modified to express in root-organ culture a bacterial gene of phenylpropanoid catabolism. The HCHL gene, encoding p-hydroxycinnamoyl-CoA hydratase/lyase, was introduced into B. vulgaris under the control of a CaMV 35S promoter, using Agrobacterium rhizogenes LBA 9402. Hairy root clones expressing the HCHL gene, together with non-expressing clones, were analysed and revealed that one expression-positive clone accumulated the glucose ester of p-hydroxybenzoic acid (pHBA) at about 14% on a dry weight basis. This is the best yield achieved in plant systems so far. Determination of cell-wall components liberated by alkaline hydrolysis confirmed that the ratio of pHBA to ferulic acid was considerably higher in the HCHL-expressing clones, whereas only ferulic acid was detected in a non-expressing clone. The change in cell-wall components also resulted in a decrease in tensile strength in the HCHL-expressing clones.
... On the other hand, insertion and expression of this HCHL gene in Datura stramonium hairy roots resulted in the accumulation of glucoside/glucose ester of 4-hydroxybenzoic acid and trace amount of vanillic acid with no vanillin production, which has been interpreted to might have occurred due to lesser availability of feruloyl CoA as its substrate (Mitra et al., 2002). Similarly, on introduction of this gene in Beta vulgaris resulted into the production of p-hydroxy benzaldehyde as the end product and no trace of vanillin was noted (Rahman et al., 2009). The mechanism of action of HCHL was also analysed in Sugarcane (McQualter et al., 2005) and in tobacco plants (Mayer et al., 2001) in which HCHL converts 4-coumaroyl-CoA to 4hydroxybenzaldehyde through retro-aldol cleavage reaction of the phenyl propanoid intermediates. ...
... In the background of these information, the present study was undertaken with the two already established HCHL expressing B. vulgaris hairy root lines (Rahman et al., 2009) to streamline the deciding parameter in converting the naturally occurring phenylpropanoid precursor (ferulic acid) into vanillin (if so occur), through (i) optimization of critical parameters, such as media combination, concentration of carbon source, pH of the media and harvesting time and (ii) enrichment of the availability of precursor by feeding with ferulic acid. In the present paper we are reporting successful production of vanillin from the aforesaid B. vulgaris hairy root clones (Rahman et al., 2009) that reached up to the level of 0.1% of vanillin, which is quite significant being comparable to that of the natural vanillin from Vanilla planifolia pods. ...
... In the background of these information, the present study was undertaken with the two already established HCHL expressing B. vulgaris hairy root lines (Rahman et al., 2009) to streamline the deciding parameter in converting the naturally occurring phenylpropanoid precursor (ferulic acid) into vanillin (if so occur), through (i) optimization of critical parameters, such as media combination, concentration of carbon source, pH of the media and harvesting time and (ii) enrichment of the availability of precursor by feeding with ferulic acid. In the present paper we are reporting successful production of vanillin from the aforesaid B. vulgaris hairy root clones (Rahman et al., 2009) that reached up to the level of 0.1% of vanillin, which is quite significant being comparable to that of the natural vanillin from Vanilla planifolia pods. The present findings indeed facilitated addressing two major concerns: (i) does the HCHL transgene expression in hairy roots really need an intermediate time gap for stabilization of its proper function through long term cultivation as elucidated earlier (Peebles et al., 2008); and (ii) does the availability of precursor and/or other chemical parameters resuscitate the proper function of the HCHL gene? ...
Vanillin is a naturally occurring phenolic compound, which has broad spectrum importance in food, cosmetics, flavouring agent and bioprservative. Aditionally, it also imparts many therapeutic benfits in the form of antimicrobial, antimutagenic and anticarcinogenic agent. Present study demonstrated the production of vanillin from two transgenic hairy root (HR) lines of Beta vulgaris bearing HCHL gene from Pseudomonas fluorescens, encoding p-hydroxycinnamoyl-CoA hydratase/lyase under the control of CaMV 35S promoter. These HCHL expressing HR clones viz. Bv 42 and Bv 18, showed conversion of inherently available phenypropanoid precursor (ferulic acid) into vanillin. Optimization of central parameters, such as media formulation, concentration of sucrose (1-5%), pH of the media (17), culture duration in combination with accessibility to higher amount of precursor by feeding with ferulic acid (2-10 mM), was carried out in detail, which have noticeably influenced the growth as well as vanillin synthesis potential of the roots. The HR clone Bv 42 showed maximum growth (GI = 1426 ± 22.71) in MS medium with 3% sucrose at pH 5.8 within 45 days, which was two times and five times higher than that with pH 7 and pH 3 respectively. Evidently, although the maximum synthesis of vanillin (0.052% ± 0.00013) could be obtained at pH 3 without any additional precursor feeding, its overall yield was better at 5.8 pH, which may be due to retardation in biomass yield as observed at pH 3. Conspicuously, addition of 10 mM of ferulic acid as the precursor enhanced the vanillin yield upto four fold (0.209% ± 0.0021), relative to that in the cultures devoid of any added precursor. The presently established transgenic HR mediated vanillin production technology adds much significance in realising the industrial goal of meeting the global demand of this biomolecule.
... On the other hand, insertion and expression of this HCHL gene in Datura stramonium hairy roots resulted in the accumulation of glucoside/glucose ester of 4-hydroxybenzoic acid and trace amount of vanillic acid with no vanillin production, which has been interpreted to might have occurred due to lesser availability of feruloyl CoA as its substrate (Mitra et al., 2002). Similarly, on introduction of this gene in Beta vulgaris resulted into the production of p-hydroxy benzaldehyde as the end product and no trace of vanillin was noted (Rahman et al., 2009). The mechanism of action of HCHL was also analysed in Sugarcane (McQualter et al., 2005) and in tobacco plants (Mayer et al., 2001) in which HCHL converts 4-coumaroyl-CoA to 4hydroxybenzaldehyde through retro-aldol cleavage reaction of the phenyl propanoid intermediates. ...
... In the background of these information, the present study was undertaken with the two already established HCHL expressing B. vulgaris hairy root lines (Rahman et al., 2009) to streamline the deciding parameter in converting the naturally occurring phenylpropanoid precursor (ferulic acid) into vanillin (if so occur), through (i) optimization of critical parameters, such as media combination, concentration of carbon source, pH of the media and harvesting time and (ii) enrichment of the availability of precursor by feeding with ferulic acid. In the present paper we are reporting successful production of vanillin from the aforesaid B. vulgaris hairy root clones (Rahman et al., 2009) that reached up to the level of 0.1% of vanillin, which is quite significant being comparable to that of the natural vanillin from Vanilla planifolia pods. ...
... In the background of these information, the present study was undertaken with the two already established HCHL expressing B. vulgaris hairy root lines (Rahman et al., 2009) to streamline the deciding parameter in converting the naturally occurring phenylpropanoid precursor (ferulic acid) into vanillin (if so occur), through (i) optimization of critical parameters, such as media combination, concentration of carbon source, pH of the media and harvesting time and (ii) enrichment of the availability of precursor by feeding with ferulic acid. In the present paper we are reporting successful production of vanillin from the aforesaid B. vulgaris hairy root clones (Rahman et al., 2009) that reached up to the level of 0.1% of vanillin, which is quite significant being comparable to that of the natural vanillin from Vanilla planifolia pods. The present findings indeed facilitated addressing two major concerns: (i) does the HCHL transgene expression in hairy roots really need an intermediate time gap for stabilization of its proper function through long term cultivation as elucidated earlier (Peebles et al., 2008); and (ii) does the availability of precursor and/or other chemical parameters resuscitate the proper function of the HCHL gene? ...
Vanillin is a naturally occurring phenolic compound, which has broad spectrum importance in food, cosmetics, flavouring agent and bioprservative. Aditionally, it also imparts many therapeutic benfits in the form of antimicrobial, antimutagenic and anticarcinogenic agent. Present study demonstrated the production of vanillin from two transgenic hairy root (HR) lines of Beta vulgaris bearing HCHL gene from Pseudomonas fluorescens, encoding p-hydroxycinnamoyl-CoA hydratase/lyase under the control of CaMV 35S promoter. These HCHL expressing HR clones viz. Bv 42 and Bv 18, showed conversion of inherently available phenypropanoid precursor (ferulic acid) into vanillin. Optimization of central parameters, such as media formulation, concentration of sucrose (1–5%), pH of the media (17), culture duration in combination with accessibility to higher amount of precursor by feeding with ferulic acid (2–10 mM), was carried out in detail, which have noticeably influenced the growth as well as vanillin synthesis potential of the roots. The HR clone Bv 42 showed maximum growth (GI = 1426 ± 22.71) in MS medium with 3% sucrose at pH 5.8 within 45 days, which was two times and five times higher than that with pH 7 and pH 3 respectively. Evidently, although the maximum synthesis of vanillin (0.052% ± 0.00013) could be obtained at pH 3 without any additional precursor feeding, its overall yield was better at 5.8 pH, which may be due to retardation in biomass yield as observed at pH 3. Conspicuously, addition of 10 mM of ferulic acid as the precursor enhanced the vanillin yield upto four fold (0.209% ± 0.0021), relative to that in the cultures devoid of any added precursor. The presently established transgenic HR mediated vanillin production technology adds much significance in realising the industrial goal of meeting the global demand of this biomolecule.
... On the other hand, insertion and expression of this HCHL gene in Datura stramonium hairy roots resulted in the accumulation of glucoside/glucose ester of 4-hydroxybenzoic acid and trace amount of vanillic acid with no vanillin production, which has been interpreted to might have occurred due to lesser availability of feruloyl CoA as its substrate (Mitra et al., 2002). Similarly, on introduction of this gene in Beta vulgaris resulted into the production of p-hydroxy benzaldehyde as the end product and no trace of vanillin was noted (Rahman et al., 2009). The mechanism of action of HCHL was also analysed in Sugarcane (McQualter et al., 2005) and in tobacco plants (Mayer et al., 2001) in which HCHL converts 4-coumaroyl-CoA to 4hydroxybenzaldehyde through retro-aldol cleavage reaction of the phenyl propanoid intermediates. ...
... In the background of these information, the present study was undertaken with the two already established HCHL expressing B. vulgaris hairy root lines (Rahman et al., 2009) to streamline the deciding parameter in converting the naturally occurring phenylpropanoid precursor (ferulic acid) into vanillin (if so occur), through (i) optimization of critical parameters, such as media combination, concentration of carbon source, pH of the media and harvesting time and (ii) enrichment of the availability of precursor by feeding with ferulic acid. In the present paper we are reporting successful production of vanillin from the aforesaid B. vulgaris hairy root clones (Rahman et al., 2009) that reached up to the level of 0.1% of vanillin, which is quite significant being comparable to that of the natural vanillin from Vanilla planifolia pods. ...
... In the background of these information, the present study was undertaken with the two already established HCHL expressing B. vulgaris hairy root lines (Rahman et al., 2009) to streamline the deciding parameter in converting the naturally occurring phenylpropanoid precursor (ferulic acid) into vanillin (if so occur), through (i) optimization of critical parameters, such as media combination, concentration of carbon source, pH of the media and harvesting time and (ii) enrichment of the availability of precursor by feeding with ferulic acid. In the present paper we are reporting successful production of vanillin from the aforesaid B. vulgaris hairy root clones (Rahman et al., 2009) that reached up to the level of 0.1% of vanillin, which is quite significant being comparable to that of the natural vanillin from Vanilla planifolia pods. The present findings indeed facilitated addressing two major concerns: (i) does the HCHL transgene expression in hairy roots really need an intermediate time gap for stabilization of its proper function through long term cultivation as elucidated earlier (Peebles et al., 2008); and (ii) does the availability of precursor and/or other chemical parameters resuscitate the proper function of the HCHL gene? ...
... Conversely, in most cases, the induction of the efficiency of hypocotyl is higher than the other positions, such as cotyledons. Similarly, Rahman et al. [28] induced the hairy roots of Beta vulgaris by puncture injection and uncovered that the induction efficiency of hypocotyl was higher than that of cotyledons (33% and 8%, respectively). Meng et al. [12] further divided the stem of the hypocotyl into three parts for testing and found that the induction rate was significantly higher in the position closer to the original root of the plant than in the position farther away, which was similar to our results using the injecting method. ...
Rosa roxburghii Tratt. is an emerging fruit endemic to China, which has the reputation of being the “King of Vitamin C” because of its abundance of vitamin C. However, it is also a recalcitrant species that imposes severe limitations on the transformation and whole-plant regeneration processes, restricting the verification of the functional genes. Therefore, developing a feasible and efficient genetic transformation method for R. roxburghii is an urgent requirement. Herein, K599 with eGFP was used as the Agrobacterium strain to optimize the genetic transformation from four factors: bacterial concentration, seedling age, infection site, and method. First, the original roots of 5-day-old seedlings were excised, and then the slant cuts of the remaining hypocotyls with 0.5 cm length were placed in K599 at an OD600 of 0.4. Subsequently, the explants were planted in a moistened sterile vermiculite after the beveled site was stained with a clump of bacteria. The results showed that the transformation efficiency of this cutting method was almost 28% at 30 days post-inoculation, while the transformation efficiency obtained by injecting 5-day-old seedlings 0.5–1.0 cm away from the primary root with K599 at an OD600 of 0.4 was only about 7%. Taken together, the current findings provide evidence that Agrobacterium-mediated transformation is a simple, fast, and efficient approach for generating composite R. roxburghii plants. Thus, this method has a broad application to analyze the gene functions in R. roxburghii and other related plant species.
... A yield of 2.9% 4HBA was obtained; however, the 4HBA was modified and thus isolated as 4HBA glucoside or 4HBA glucose ester from the plant cells. Mitra et al. (1999); McQualter et al. (2005), and Rahman et al. (2009) introduced the P. fluorescens HCHL gene into Datura stramonium, sugarcane, and Beta vulgaris, respectively, to yield a high accumulation of the 4HBA glucose ester. ...
The marine bacterium Microbulbifer sp. A4B-17 produces secondary metabolites such as 4-hydroxybenzoic acid (4HBA) and esters of 4HBA (parabens). 4HBA is a useful material in the synthesis of the liquid crystal. Parabens are man-made compounds that have been extensively used since the 1920s in the cosmetic, pharmaceutical, and food industries for their effective antimicrobial activity. In this study, we completed the sequencing and annotation of the A4B-17 strain genome and found all genes for glucose utilization and 4HBA biosynthesis. Strain A4B-17 uses the Embden-Meyerhof-Parnas (EMP), hexose monophosphate (HMP), and Entner-Doudoroff (ED) pathways to utilize glucose. Other sugars such as fructose, sucrose, xylose, arabinose, galactose, mannitol, and glycerol supported cell growth and 4HBA synthesis. Reverse transcriptional analysis confirmed that the key genes involved in the glucose metabolism were functional. Paraben concentrations were proportionally increased by adding alcohols to the culture medium, indicating that strain A4B-17 synthesizes the 4HBA and the alcohols separately and an esterification reaction between them is responsible for the paraben synthesis. A gene that codes for a carboxylesterase was proposed to catalyze this reaction. The temperature and NaCl concentration for optimal growth were determined to be 35°C and 22.8 g/L.
... The hairy root, induced by a soil bacterium, Agrobacterium rhizogenes, is widely used in biotechnological applications to replace natural sources for the high production of valuable secondary metabolites (Chilton et al., 1982;Mishra and Ranjan, 2008;Sheela and Ramesh, 2011), such as high accumulation of the anticancer compound plumbagin in Plumbago rosea hairy root (Jose et al., 2016) and p-hydroxybenzoic acid (pHBA) in Beta vulgaris hairy root (Rahman et al., 2009). The hairy root system also shows high stability and fast, hormone-independent growth. ...
Scutellarin, a flavonoid 7-O-glucuronide, is an essential bioactive compound of Erigeron breviscapus (Vaniot) Hand.-Mazz. used for the treatment of cerebrovascular diseases. However, due to overexploitation and overuse, E. breviscapus is facing the problems of extinction and habitat degradation. In this study, a correlation analysis between the transcript and metabolite profiles of methyl jasmonate (MeJA)-treated E. breviscapus at different time points indicated that chalcone isomerase (EbCHI) was the primary contributor to scutellarin accumulation during flavonoid biosynthesis. EbCHI was then further characterized as a chalcone isomerase that efficiently converted chalcone to naringenin in vitro. Optimal parameters derived by comparing different culture conditions were successfully used to establish hairy root cultures of E. breviscapus with a maximum transformation rate of 60% in B5 medium. Furthermore, overexpression of EbCHI significantly enhanced scutellarin accumulation in E. breviscapus hairy roots with a maximum content of 2.21 mg g⁻¹ (dw), 10-fold higher than that of natural roots (0.21 mg g⁻¹ dw). This study sheds new light on a method of effective gene-based metabolic engineering by accurate and appropriate strategies and provides a protocol for hairy root cultures that accumulate high levels of scutellarin, providing a promising prospect for relieving the overexploitation and unavailability of E. breviscapus in the future.
... Hairy root lines producing valuable phytochemicals have been developed from various plant species (Dehghan et al. 2012;Cardillo et al. 2013). Recently, successful efforts have been made using hairy root cultures to improve secondary metabolism compounds in Hyoscyamus niger (Zhang et al. 2004) and p-hydroxybenzoic acid (pHBA) glucose ester production in hairy roots of Beta vulgaris (Rahman et al. 2009), express foreign proteins or vaccine in tobacco (Shadwick and Doran 2007), and produce "unnatural" products in C. roseus (Runguphan and O'Connor 2009). Several TIAs' biosynthesis genes have also been overexpressed in C. roseus hairy root cultures (Zhao et al. 2012a, b). ...
Plant cell and tissue culture has been suggested as an alternative means for year-round production of secondary metabolites with an added potential of increasing yields by culture selection and manipulation, genetic transformation, hairy root cultures, and use of bioreactors for mass production. Secondary metabolite pathways and genes involved in those pathways have been identified, and regulation of transcription and transcription factors has been determined by studying functional genomics in conjunction with data- mining tools of bioinformatics. Besides this, advances in metabolic engineering enable researchers to confer new secondary metabolic pathways to crops by transferring three to five, or more, heterologous genes taken from various other species. As an alternative, the metabolic pathways of useful secondary metabolites have been modified to improve their productivity via genetic transformation. However, there is a need to understand metabolic pathways of secondary metabolism at the molecular level. Plant hairy roots offer a novel and sustainable tissue-based system that preserves multiple specialized cell types believed to be important in maintaining a better consistency in synthesis of bioactive secondary molecules. This paper will review stateof-the-art reports on improving production of secondary metabolites in tissue cultures in various plant species.
... Another example is production in hairy roots of p-hydroxybenzoic acid (pHBA) that can be used as polymer feedstock. New conjugates of pHBA were synthesized in transgenic roots of Datura stramonium (Mitra et al. 2002) and sugarbeet (Rahman et al. 2009) by heterologously expressed bacterial p-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) followed by inherent plant dehydrogenase and glucosyltransferases. In sugar beet hairy roots up to 14 % DW pHBA glucose ester were accumulated, that is the best yield achieved in plant systems so far. ...
Metabolite profile of transgenic roots may differ from that of parental plant, but this feature is not necessarily a disadvantage. The review focuses on new substances produced in hairy roots as a result of tumorous root formation by itself or in combination with different treatments and/or genetic engineering manipulations. Examples of new secondary metabolites from hairy root cultures, not known in parental plants, and products of heterologous gene expression are described. In our opinion, transgenic roots may be regarded as a promising origin of natural product biodiversity.
... AaTRI-transformed hairy-root lines were accompanied by a mean 1.87-fold higher level of hyoscyamine and a mean 8-fold higher level of scopolamine compared with control roots, indicating that AaTRI is a promising target for genetic engineering to increase tropane alkaloid in A. acutangulus. Finally, transformed Beta vulgaris (sugar beet) root cultures expressing a bacterial gene for p-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), which involved in phenylpropanoid biosynthesis (Rahman et al., 2009). These results suggest that the availability of HCHL yields a high accumulation of phydroxybenzoic acid (pHBA) glucose ester. ...
Korean Angelica (Angelica gigas) produces pyranocoumarins (decursin and decursinol angelate), which have neuroprotective, anticancer, and anti-androgen receptor-signaling activities. An efficient protocol for the establishment of transgenic Angelica gigas root cultures using Agrobacterium rhizogenes is reported. To further characterise the putative transgenic roots, explant tissues were co-cultivated with A. rhizogenes strain R1601 carrying the pK2GW7-GUS binary vector. Except for the co-cultivation medium, all formulations included 50 mg L -1 kanamycin to select for transformants and 250 mg L -1 cefotaxime to eliminate the Agrobacterium. After 6 to 8 weeks co-cultivation with Agrobacterium rhizogenes, kanamycin-resistant roots appeared on 50% of explants maintained on hormone-free medium. Isolated hairy roots were transferred in liquid medium containing half-strength Schenk and Hildebrandt (SH) salt and 30 mg L -1 sugar. Detection of the neomycin phosphotransferase gene, high levels of β--glucuronidase (GUS) transcripts, and GUS histochemical localisation confirmed the integrative transformation. In the future, these protocols should facilitate the extraction and study of valuable metabolites, such as decursin and decursinol angelate from A. gigas hairy root cultures.
The flavor of vanilla is a complex blend of compounds, with vanillin as the most prominent, along with vanillyl alcohol and 4-hydroxybenzoic acid. Natural vanillin extracted from vanilla beans is expensive, so researchers use heterologous synthesis to produce nature-identical vanillin in plant hosts. Consequently, alternative traditional farming and gathering methods are required to bridge the significant disparity between supply and demand. The current research successfully developed a method to induce hairy root formation from leaves. It integrated the Vanillin synthase (VpVAN) gene into transgenic hairy root lines of Beta vulgaris, synthesizing vanillin-related compounds. The presence of the VpVAN gene in transgenic roots was confirmed using PCR analysis. Additionally, RT-qPCR analysis demonstrated the expression of the VpVAN gene in the transgenic root lines. The transgenic hairy root clones H1, H2, and H5 showed enhanced vanillin production, vanillyl alcohol, and 4-hydroxybenzoic acid. Elicitation with methyl jasmonate (MJ) and salicylic acid (SA) further improved the production of these compounds in B. vulgaris hairy roots. The maximum hairy root biomass was observed after 60 days, with the maximum synthesis of vanillin and 4-hydroxybenzoic acid obtained from hairy root clones H5 and HR2, respectively. Vanillyl alcohol HR2 was obtained on the 45th day of cultivation. Elicitation with wound-associated hormone methyl jasmonate and salicylic acid enhanced the yield of vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid, with a 215-fold increase in vanillin, a 13-fold increase in vanillyl alcohol, and a 21 fold increase in 4-hydroxybenzoic acid. The study results indicate that establishing transgenic hairy root cultures with the VpVAN gene is a promising alternative method for enhancing the production of vanilla flavor compounds such as vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid. A cost-effective protocol has been developed to mass-produce phenolic compounds using a hairy root culture of B. vulgaris. This approach addresses the increasing demand for these substances while reducing the cost of natural vanillin production, making it suitable for industrial-scale applications.
Plant-derived antioxidants are intrinsic components of human diet and factors implicated in tolerance mechanisms against environmental stresses in both plants and humans. They are being used as food preservatives and additives or ingredients of cosmetics. For nearly forty years, Rhizobium rhizogenes-transformed roots (hairy roots) have been studied in respect to their usability as producers of plant specialized metabolites of different, primarily medical applications. Moreover, the hairy root cultures have proven their value as a tool in crop plant improvement and in plant secondary metabolism investigations. Though cultivated plants remain a major source of plant polyphenolics of economic importance, the decline in biodiversity caused by climate changes and overexploitation of natural resources may increase the interest in hairy roots as a productive and renewable source of biologically active compounds. The present review examines hairy roots as efficient producers of simple phenolics, phenylethanoids, and hydroxycinnamates of plant origin and summarizes efforts to maximize the product yield. Attempts to use Rhizobium rhizogenes-mediated genetic transformation for inducing enhanced production of the plant phenolics/polyphenolics in crop plants are also mentioned.
Plants use two distinct isoprenoid biosynthesis routes: methylerythritol phosphate (MEP) and mevalonic acid (MVA) pathways. The rate-limiting enzymes of the MEP pathway are 1-deoxy-D-xylulose5-phosphate synthase (DXS) and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR). 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the rate-limiting step in the MVA pathway. Previously, overexpression of Populus trichocarpa PtDXR was found to upregulate resistance against salt and drought stresses. We showed while PtHMGR overexpressors (OEs) exhibited different MEP- and MVA-related gene expressions than non-transgenic poplars (NT), the PtDXR-OEs revealed upregulated MEP-related and downregulated MVA-related gene expressions. PtDXR and PtHMGR o verexpressions caused changes in MVA-derived trans-zeatin-riboside, isopentenyl adenosine, castasterone, and 6-deoxocastasterone well as MEP-derived carotenoids and gibberellins. In PtHMGR- OEs, the accumulated geranyl diphosphate synthase ( GPS ) and geranyl pyrophosphate synthase ( GPPS) transcript levels in the MEP pathway led to an accumulation of MEP-derived isoprenoids. In contrast, upregulation of farnesyl diphosphate synthase ( FPS ) expression in the MVA pathway contributed to increased levels of MVA-derived isoprenoids. In addition, PtHMGR- OEs increased MEP-related GPS and GPPS transcript levels, expanded MEP-derived isoprenoid levels, changed FPS transcript levels, and affected MVA-derived isoprenoid yields. These results suggest that interaction exists between the MVA- and MEP-pathways.
The root of Angelica gigas Nakai, growing in the high mountains of the Gangwon‐do region in Korea, is known to be a beneficial herbal medicine, which is used in the treatment of various gynecological disorders, chills, anemia, circulatory disorders, and psychological symptoms. The cytotoxic activity of decursin as a potential anticancer drug has been reported along with an extracellular immunostimulating polysaccharide produced by suspension cultures of A. gigas. Angelica gigas root fraction was investigated for the isolation of bergapten, decursinol angelate, decursin, nodakenetin, and nodakenin. The antimicrobial activities of these isolated phytoconstituents were tested against bacterial strains. Angelica dahurica and Angelica pubescentis roots were investigated for the presence of essential oils and their antifungal activities were assessed. Angelica gigas produces decursin and decursinol angelate, demonstrated neuroprotective, anticancer, and anti‐androgen receptor‐signaling activities.
Rational metabolic-flow switching, which we proposed recently, is an effective strategy to produce an exogenous high-value natural product using transformed plant cells; the proof of this concept was demonstrated using bamboo (Phyllostachys nigra; Pn) cells as a model system. Pn cells were transformed to express 4-hydroxycinnamoyl-CoA hydratase/lyase of Pseudomonas putida KT2440 (PpHCHL), which catalyzes the formation of 4-hydroxybenzaldehyde and vanillin from p-coumaroyl-CoA and feruloyl-CoA, respectively. The PpHCHL-transformed cells accumulated glucose conjugates of 4-hydroxybenzoic acid and vanillic acid, indicating that the PpHCHL products (aldehydes) were further metabolized by inherent enzymes in the Pn cells. The production titers of 4-hydroxybenzoic acid glucose ester, vanillic acid glucose ester, and 4-hydroxybenzoic acid glucoside reached 1.7, 0.17, and 0.14 g/L at the maximum, respectively. These results proved the versatility of Pn cells for producing vanillin-related compounds based on rational metabolic-flow switching.
The enormous range of secondary metabolites produced within the plant kingdom includes many of scientific and commercial interest. There are frequently problems associated both with the study of secondary product biosynthesis in planta, and the reliability of agricultural systems for the production of commercially valuable products. In recent decades there has thus been an interest in developing in vitro cell culture systems that produce high levels of selected secondary metabolites. While callus and cell suspension cultures have been widely developed, their secondary metabolite productive capacities can be rather low and unpredictable. A major step forward came with the development of organ cultures, particularly the so-called hairy roots produced by transformation of plants with the bacterium Agrobacterium rhizogenes. The group at the Institute of Food Research, Norwich, was one of the first to exploit the technology, and this article describes their experiences with hairy roots and illustrates the range of approaches that can be taken to maximize their potential. In particular, because hairy root formation already involves a genetic transfer, they are especially good systems in which to study the effects of transgenesis. While some of the techniques described have now been extensively exploited, others have still not reached their full potential, and hopefully this article might serve to throw some light on possible future developments.
The enormous range of secondary metabolites produced within the plant kingdom includes many of scientific and commercial interest. There are frequently problems associated both with the study of secondary product biosynthesis in planta, and the reliability of agricultural systems for the production of commercially valuable products. In recent decades there has thus been an interest in developing in vitro cell culture systems that produce high levels of selected secondary metabolites. While callus and cell suspension cultures have been widely developed, their secondary metabolite productive capacities can be rather low and unpredictable. A major step forward came with the development of organ cultures, particularly the so-called hairy roots produced by transformation of plants with the bacterium Agrobacterium rhizogenes. The group at the Institute of Food Research, Norwich, was one of the first to exploit the technology, and this article describes their experiences with hairy roots and illustrates the range of approaches that can be taken to maximize their potential. In particular, because hairy root formation already involves a genetic transfer, they are especially good systems in which to study the effects of transgenesis. While some of the techniques described have now been extensively exploited, others have still not reached their full potential, and hopefully this article might serve to throw some light on possible future developments.
Molecular biology is arguably the fastest growing fi eld in all biological sciences. New techniques are discovered and they soon fi nd applications. The search for natural products has a long history, as illustrated by the dependence of traditional medicine on botanicals. With the fi nding that microorganisms can produce useful products such as penicillin, scientists took the challenge to explore the microbial world for new natural products. Although the search continues with increasing intensity, fi nding new and more useful products would not have matched with the effort without the support of biotechnology. Although the most powerful approach is genetic manipulation, other techniques such as mutagenesis, breeding and protoplast fusion and the relatively old biotechnology of plant tissue culture are very useful. These also include even more simple approaches such as optimizing culture conditions and design of fermenters. The combination of technologies together with innovative ideas has already increased the production level of already existing natural products and expanded the diversity of products obtainable from biological sources. In addition to harvesting products from living organisms either in the wild or in cultivation, the developments in metagenomics have also paved the way to harness the bioproductforming ability of unculturable microorganisms.
4-Hydroxybenzoate (4HBA) is an important chemical compound used for synthesis of liquid crystal. Production of 4HBA from renewable resources is an effective mean to solve problems such as environmental pollution and petroleum shortage. This review briefly introduces the chemical synthesis of 4HBA from oil compounds, and mainly describes the progress in 4HBA biosynthesis from renewable resources by plants and microorganisms. In most intriguing aspect of plant-based synthesis of 4HBA is the appeal of directly synthesizing a chemical from CO2. However, the glucosylation system in plant cells converting 4HBA to glucose conjugates, causing the post treatment a problem. The recombinant microorganisms produce pure 4HBA, but less efficient. A new strain of Microbulbifer has ability to naturally accumulate 4HBA from glucose. Elucidation of the metabolic pathways and regulation systems would improve 4HBA synthesis efficiency.
To elucidate the function of mevalonate-5-pyrophosphate decarboxylase (MVD) and farnesyl pyrophosphate synthase (FPS) in triterpene biosynthesis, the genes governing the expression of these enzymes were transformed into Panax ginseng hairy roots. All the transgenic lines showed higher expression levels of PgMVD and PgFPS than that by the wild-type control. Among the hairy root lines transformed with PgMVD, M18 showed the highest level of transcription compared to the control (14.5-fold higher). Transcriptions of F11 and F20 transformed with PgFPS showed 11.1 folds higher level compared with control. In triterpene analysis, M25 of PgMVD produced 4.4-fold higher stigmasterol content (138.95 μg/100 mg, dry weight [DW]) than that by the control; F17 of PgFPS showed the highest total ginsenoside (36.42 mg/g DW) content, which was 2.4 folds higher compared with control. Our results indicate that metabolic engineering in P. ginseng was successfully achieved through Agrobacterium rhizogenes-mediated transformation, and that the accumulation of phytosterols and ginsenosides was enhanced by introducing the PgMVD and PgFPS genes into the hairy roots of the plant. Our results suggest that PgMVD and PgFPS play an important role in the triterpene biosynthesis of P. ginseng.
Hydroxycinnamic acids constitute a large class of phenylpropanoid metabolites that are distributed ubiquitously in terrestrial plants. They occur most frequently as esters, amides or glycosides within the cytosol, the particular subcellular compartments such as the vacuole or the cell wall. Hydroxycinnamate conjugates play a vital role in the plant's growth and development and in its defense responses against biotic- and abiotic-stresses. Furthermore, the incorporation of hydroxycinnamate conjugates into the cell wall is a major factor attenuating the wall's biodegradability. Understanding the biosyntheses of hydroxycinnamate conjugates and its molecular regulation may well facilitate the sustainable production of cell wall biomass, and the efficient conversion of lignocellulosic materials. This paper reviews our current molecular and biochemical understandings on the formation of several classes of hydroxycinnamate esters and amides, including the soluble conjugates and the 'wall-bound' phenolics. It also discusses the emerging biotechnological applications in manipulating hydroxycinnamates to improve the degradability of the cell wall biomass and enhance the production of valuable chemicals and biomaterials.
Transgenic hairy root lines from Scutellaria baicalensis overexpressing phenylalanine ammonia-lyase (PAL) (SbPAL1, SbPAL2, and SbAPL3) were established using an Agrobacterium rhizogenes-mediated transformation system. Stable genetic transformation with the SbPAL genes was confirmed by real-time PCR. These transgenic hairy root lines produced higher quantities flavones (i.e., baicalin, baicalein, and wogonin) than the control hairy root line. In particular, the wogonin content was increased by 4-11 times in the SbPAL1-, SbPAL2-, and SbPAL3-overexpressing hairy roots than the S. baicalensis wild-type roots. This research showed the importance of PAL in flavone biosynthesis and demonstrated the efficiency of metabolic engineering in S. baicalensis hairy roots.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the rate-limiting step in the mevalonate pathway. To elucidate the functions of HMGR in triterpene biosynthesis, Platycodon grandiflorum was transformed with a construct expressing Panax ginseng HMGR (PgHMGR). We used PCR analysis to select transformed hairy root lines and selected 6 lines for further investigation. Quantitative real-time PCR showed higher expression levels of HMGR and total platycoside levels (1.5-2.5-fold increase) in transgenic lines than in controls. Phytosterols levels were also 1.1-1.6-fold higher in transgenic lines than in controls. Among these lines, line T7 produced the highest level of total platycosides (1.60 ± 0.2 mg/g D.W.) and α-spinasterol (1.78 ± 0.16 mg/g D.W.). These results suggest that metabolic engineering of P. grandiflorum by Agrobacterium-mediated genetic transformation may enhance production of phytosterols and triterpenoids.
The invention of hairy root induction from various higher plants has provided a new set of hopes for using such organs in vitro as an alternative to cell cultures, chiefly because of their genetic stabilities that impart them high biochemical consistencies. Several research groups characterized different types of red beet hairy root clones, their scaled-up performances as well as their applications for various basic studies. Rather than cell cultures, hairy roots of red beet have attracted the attention of more number of research groups, probably because of their genetic aspects, spectacular colors, various morphologies, clonal stability and for other technological challenges associated with their scale-up and product recovery. The astonishingly variable responses of a cultivar of red beet (Ruby Queen) to different strains of Agrobacterium rhizogenes and the variable spectrum of morpho-physiological responses to nutrient components make red beet an ever interesting material for genetic as well as physiological studies, as discussed in this chapter. The fact that higher plants can transcribe animal genes and perfectly translate them into functional proteins has attracted newer opportunities for obtaining boundless number of important therapeutic proteins from cultured plant cells and organs. Hairy roots are often chosen for such purposes because of their consistent performances under fully manageable in vitro conditions. Many opportunities could also be realized with red beet hairy roots.
Tobacco plants (Nicotiana tabacum cv XHFD 8) were genetically modified to express a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) enzyme which is active with intermediates of the phenylpropanoid pathway. We have previously shown that HCHL expression in tobacco stem resulted in various pleiotropic effects, indicative of a reduction in the carbon flux through the phenylpropanoid pathway, accompanied by an abnormal phenotype. Here, we report that in addition to the reduction in lignin and phenolic biosynthesis, HCHL expression also resulted in several gross morphological changes in poorly lignified tissue, such as abnormal mesophyll and palisade. The effect of HCHL expression was also noted in lignin-free single cells, with suspension cultures displaying an altered shape and different growth patterns. Poorly/non-lignified cell walls also exhibited a greater ease of alkaline extractability of simple phenolics and increased levels of incorporation of vanillin and vanillic acid. However, HCHL expression had no significant effect on the cell wall carbohydrate chemistry of these tissues. Evidence from this study suggests that changes in the transgenic lines result from a reduction in phenolic intermediates which have an essential role in maintaining structural integrity of low-lignin or lignin-deprived cell walls. These results emphasize the importance of the intermediates and products of phenylpropanoid pathway in modulating aspects of normal growth and development of tobacco. Analysis of these transgenic plants also shows the plasticity of the lignification process and reveals the potential to bioengineer plants with reduced phenolics (without deleterious effects) which could enhance the bioconversion of lignocellulose for industrial applications.
An intrinsic improvement is taking place in the methodologies for the development of culture systems with first-rate production of plant-based molecules. The blending of HR (hairy root) cultures with ME (metabolic engineering) approaches offers new insights into, and possibilities for, improving the system productivity for known and/or novel high-value plant-derived active compounds. The introduction and expression of foreign genes in plants results in improvement of cellular activities by manipulating enzymatic, regulatory and transport function of the cell. The rational amendments in the rate-limiting steps of a biosynthetic pathway as well as inactivating the inefficient pathway(s) for by-product formation can be accomplished either through single-step engineering or through the multi-step engineering. The hierarchical control of any metabolic process can lead the engineer to apply the ME ideas and principles to any of the strata, including transcriptional, moving on to translational and enzymatic activity. The HR culture systems offer a remarkable potential for commercial production of a number of low-volume, but high-value, secondary metabolites. Taking HR as a model system, in the present review, we discuss engineering principles and perceptions to exploit secondary-metabolite pathways for the production of important bioactive compounds. We also talk about requisites and possible challenges that occur during ME, with emphasis on examples of various HR systems. Furthermore, it also highlights the utilization of global information obtained from '-omic' platforms in order to explore pathway architecture, structural and functional aspects of important enzymes and genes that can support the design of sets of engineering, resulting in the generation of wide-ranging views of DNA sequence-to-metabolite passageway networking and their control to obtain desired results.
The involvement of genes in flavones biosynthesis was investigated in different organs and suspension cells obtained from Scutellaria baicalensis. Three full-length cDNAs encoding phenylalanine ammonia-lyase isoforms (SbPAL1, SbPAL2, and SbAPL3) and one gene encoding cinnamate 4-hydroxylase (SbC4H) from S. baicalensis were isolated using rapid amplification of cDNA ends (RACE)-PCR. These cDNAs were used together with previously-isolated clones for 4-coumaroyl CoA ligase (4CL) and chalcone synthase (CHS) to show the expression level in different organs of S. baicalensis. These genes were upregulated in suspension cells of S. baicalensis with biotic/abiotic stress factors. The baicalin and baicalein contents in roots were 22 and 107 times higher than those in flowers, respectively. The treatment of suspension cells with methyl jasmonate (MeJa) enhanced the major flavones in S. baicalensis. Cumulatively, the results of this study should advance ability to biosynthesize important and useful medicinal compounds from a variety of plant species.
A number of genes that function in the terpenoid indole alkaloids (TIAs) biosynthesis pathway have been identified in Catharanthus roseus. Except for the geraniol 10-hydroxylase (G10H) gene, which encodes a cytochrome P450 monooxygenase, several of these genes are up-regulated by ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain transcript factor. In this study, the G10H gene was transformed independently, or co-transformed with ORCA3 into C. roseus, using Agrobacterium rhizogenes MSU440. Hairy root clones expressing the G10H gene alone, or both the G10H and ORCA3 genes, were obtained. Alkaloid accumulation level analyses showed that all transgenic clones accumulated more catharanthine, with the highest accumulation level in the transgenic clone OG12 (6.5-fold higher than that of the non-expression clone). Following treatment with ABA, accumulation of catharanthine reached 1.96 mg/g DW in the transgenic clone OG12. The expression levels of TIAs biosynthesis genes in transgenic and non-transgenic clones were also investigated.
Sugar beet (Beta vulgaris L.) is an important industrial crop, being one of only two plant sources from which sucrose (i.e., sugar) can be economically produced. Despite its relatively short period of cultivation (ca. 200 years), its yield and quality parameters have been significantly improved by conventional breeding methods. However, during the last two decades or so, advanced in vitro culture and genetic transformation technologies have been incorporated with classical breeding programs, the main aim being the production of herbicide-and salt-tolerant, disease- and pest-resistant cultivars. Among the many applications of in vitro culture techniques, sugar beet has benefited the most from haploid plant production, protoplast culture, and somaclonal variation and in vitro cell selection. Several genetic transformation technologies have been developed, such as Agrobacterium-meditated, PEG-mediated, particle bombardment, electroporation, sonication and somatic hybridization, the first two being the most successful. Development of herbicide- and salt-tolerant, virus-, pest/nematode-, fungus/Cercospora- and insect-resistant sugar beet has been demonstrated. However, only herbicide-tolerant varieties have been approved for commercialization but not yet available in the marketplace; rhizomania-resistant varieties are being evaluated in field trials. Transgenic plants that convert sucrose into fructan, a polymer of fructose, were also developed. Initial attempts to increase sucrose yields produced promising results, but it still requires additional work. Despite marked progress in improving regeneration and transformation of sugar beet, genotype dependence and low regeneration and transformation frequencies are still serious restrictions for routine application of in vitro culture and, more importantly, transformation technologies. Selected food safety and environmental impact, as well as regulatory and public acceptance issues relating to transgenic sugar beet are also discussed.
Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root-knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest-resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect–host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.
This review addresses recent advances in the understanding of the function and organization of one subfamily of ATP-binding cassette (ABC) transporters, the multidrug resistance-associated proteins (MRPs), from plants and yeast. So named because of the phenotypes conferred by their animal prototypes, many MRPs are glutathione-conjugate or multi-specific organic anion Mg2+-ATPases active in the transport of glutathionated compounds and other bulky amphipathic anions. Acting downstream of the first two phases of toxin detoxification, typified by, but not limited to the reactions catalysed by the cytochromes P450 and glutathione S-transferases, respectively, processes that likely converge and depend on the MRPs from plants and yeast include the detoxification of herbicides and other organic xenobiotics, the alleviation of oxidative damage, the storage of endotoxins, heavy metal sequestration, and the vacuolation of natural pigments. Distinguished by a unique domain organization and the facility to mediate MgATP-energized transport, the MRPs are of profound significance in two respects. First, the fact that the MRPs, like most ABC transporters, are energized directly by MgATP rather than by, for example, a preformed H+ gradient, offers a new, non-chemiosmotic perspective on the molecular basis of energy-dependent solute transport in plants. Second, given the existence of 29 ABC protein genes in the yeast genome alone, characterization of the MRPs - the first class of ABC transporters from a plant source to have been defined at both the molecular and biochemical levels - indicates that many more ABC transporters and ABC transporter-mediated processes remain to be discovered in plants.
A gene encoding a novel enoyl-SCoA hydratase/lyase enzyme for the hydration and nonoxidative cleavage of feruloyl-SCoA to
vanillin and acetyl-SCoA was isolated and characterized from a strain of Pseudomonas fluorescens. Feruloyl-SCoA is the CoASH thioester of ferulic acid (4-hydroxy-3-methoxy-trans-cinnamic acid), an abundant constituent of plant cell walls and a degradation product of lignin. The gene was isolated by
a combination of mutant complementation and biochemical approaches, and its function was demonstrated by heterologous expression
in Escherichia coli under the control of a T7 RNA polymerase promoter. The gene product is a member of the enoyl-SCoA hydratase/isomerase superfamily.
A soil bacterium, designated Pseudomonas fluorescens AN103, was isolated based on its ability to grow on ferulic acid as a sole source of carbon and energy. In addition, this strain was found to metabolize a number of related phenolic substrates which contained a hydroxyl group at the para position of the aromatic ring. During growth on ferulic acid, transient accumulation of vanillic acid and trace amounts of protocatechuic acid were detected in the culture medium. Washed cells grown on ferulic acid readily oxidized vanillin, vanillic acid and protocatechuic acid, the three putative intermediates of the metabolic pathway. The side-chain cleavage of ferulic acid to produce vanillin was demonstrated in vitro for the first time and this enzyme-catalysed reaction was shown to have an essential requirement for CoASH, ATP and MgCl2. This conversion involved a two-step process involving a CoA ligase followed by the side-chain cleavage. The addition of NAD increased the oxidation of vanillin to vanillic acid and had an overall effect of increasing the rate of ferulic acid cleavage. The application of 13C-NMR studies in vitro revealed acetyl-CoA as the C2 side-chain cleavage product. High levels of inducible ferulate-CoA ligase and NAD-linked vanillin dehydrogenase were detected and a novel pathway for ferulic acid metabolism in this organism is proposed.
The gene for a bacterial enoyl-CoA hydratase (crotonase) homolog (HCHL) previously shown to convert 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA to the corresponding hydroxybenzaldehydes in vitro provided an opportunity to subvert the plant phenylpropanoid pathway and channel carbon flux through 4-hydroxybenzaldehyde and the important flavor compound 4-hydroxy-3-methoxybenzaldehyde (vanillin). Expression of the Pseudomonas fluorescens AN103 HCHL gene in two generations of tobacco plants caused the development of phenotypic abnormalities, including stunting, interveinal chlorosis and senescence, curled leaf margins, low pollen production, and male sterility. In second generation progeny, the phenotype segregated with the transgene and transgenic siblings exhibited orange/red coloration of the vascular ring, distorted cells in the xylem and phloem bundles, and lignin modification/reduction. There was depletion of the principal phenolics concomitant with massive accumulation of novel metabolites, including the glucosides and glucose esters of 4-hydroxybenzoic acid and vanillic acid and the glucosides of 4-hydroxybenzyl alcohol and vanillyl alcohol. HCHL plants exhibited increased accumulation of transcripts for phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, and 4-coumarate:CoA ligase, whereas beta-1,3-glucanase was suppressed. This study, exploiting the ability of a bacterial gene to divert plant secondary metabolism, provides insight into how plants modify inappropriately accumulated metabolites and reveals the consequences of depleting the major phenolic pools.
Benzoates are a class of natural products containing compounds of industrial and strategic importance. In plants, the compounds exist in free form and as conjugates to a wide range of other metabolites such as glucose, which can be attached to the carboxyl group or to specific hydroxyl groups on the benzene ring. These glucosylation reactions have been studied for many years, but to date only one gene encoding a benzoate glucosyltransferase has been cloned. A phylogenetic analysis of sequences in the Arabidopsis genome revealed a large multigene family of putative glycosyltransferases containing a consensus sequence typically found in enzymes transferring glucose to small molecular weight compounds such as secondary metabolites. Ninety of these sequences have now been expressed as recombinant proteins in Escherichia coli, and their in vitro catalytic activities toward benzoates have been analyzed. The data show that only 14 proteins display activity toward 2-hydroxybenzoic acid, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid. Of these, only two enzymes are active toward 2-hydroxybenzoic acid, suggesting they are the Arabidopsis salicylic acid glucosyltransferases. All of the enzymes forming glucose esters with the metabolites were located in Group L of the phylogenetic tree, whereas those forming O-glucosides were dispersed among five different groups. Catalytic activities were observed toward glucosylation of the 2-, 3-, or 4-hydroxyl group on the ring. To further explore their regioselectivity, the 14 enzymes were analyzed against benzoic acid, 3-hydroxybenzoic acid, 2,3-, 2,4-, 2,5-, and 2,6-dihydroxybenzoic acid. The data showed that glycosylation of specific sites could be positively or negatively influenced by the presence of additional hydroxyl groups on the ring. This study provides new tools for biotransformation reactions in vitro and a basis for engineering benzoate metabolism in plants.
Three genes from Ralstonia eutropha necessary for poly(3-hydroxybutyrate) (PHB) synthesis were introduced into the hairy roots of sugar beet. Transformation of a vector construct harbouring the PHB genes, each fused to the coding region of the pea ribulose-bisphosphate carboxylase plastid targeting sequence, resulted in 20 transgenic hairy-root clones, producing up to 55 mg high molecular PHB/g dry weight, as identified by gas chromatography, gel permeation chromatography and HPLC. Accumulation of PHB polymer in sugar beet root leucoplasts was confirmed by transmission electron microscopy. Thus, for the first time, plastidic PHB production was demonstrated for roots of a carbohydrate-storing crop plant.
Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.
The gene for a bacterial enoyl-CoA hydratase (crotonase) homolog (HCHL) previously shown to convert 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA to the corresponding hydroxybenzaldehydes in vitro provided an opportunity to subvert the plant phenylpropanoid pathway and channel carbon flux through 4-hydroxybenzaldehyde and the important flavor compound 4-hydroxy-3-methoxybenzaldehyde (vanillin). Expression of the Pseudomonas fluorescens AN103 HCHL gene in two generations of tobacco plants caused the development of phenotypic abnormalities, including stunting, interveinal chlorosis and senescence, curled leaf margins, low pollen production, and male sterility. In second generation progeny, the phenotype segregated with the transgene and transgenic siblings exhibited orange/red coloration of the vascular ring, distorted cells in the xylem and phloem bundles, and lignin modification/reduction. There was depletion of the principal phenolics concomitant with massive accumulation of novel metabolites, including the glucosides and glucose esters of 4-hydroxybenzoic acid and vanillic acid and the glucosides of 4-hydroxybenzyl alcohol and vanillyl alcohol. HCHL plants exhibited increased accumulation of transcripts for phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, and 4-coumarate:CoA ligase, whereas β-1,3-glucanase was suppressed. This study, exploiting the ability of a bacterial gene to divert plant secondary metabolism, provides insight into how plants modify inappropriately accumulated metabolites and reveals the consequences of depleting the major phenolic pools.
An excellent new medium was developed for the production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon. We investigated the effects of all the components of White's medium on the production of these derivatives. Nitrate, phosphate, copper, sulfate and sucrose had especially marked effects. With the new, M-9, medium produced from these studies the yield of shikonin derivatives was 1400 mg/l and the yield for dried cells was about 12%, whereas it was 120 mg/l, or about 2% with White's medium.
A method for quantitative analysis of shikonin derivatives using high pressure liquid chromatography (HPLC) was established. With this method the composition of shikonin derivatives in cultured cells and roots of Lithospermum erythrorhizon (ko-shikon) was compared. The composition of shikonin derivatives produced by cell suspension cultures was similar to that of the ko-shikon, and the composition in cultured cells was found to fluctuate less than that of the ko-shikon.
Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic roots produced by A. rhizogenes infection is characterized by high growth rate and genetic stability. These genetically transformed root cultures can produce higher levels of secondary metabolites or amounts comparable to that of intact plants. Hairy root cultures offer promise for production of valuable secondary metabolites in many plants. The main constraint for commercial exploitation of hairy root cultures is their scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of interconnected tissues unevenly distributed throughout the vessel. Rheological characteristics of heterogeneous system should also be taken into consideration during mass scale culturing of hairy roots. Development of bioreactor models for hairy root cultures is still a recent phenomenon. It is also necessary to develop computer-aided models for different parameters such as oxygen consumption and excretion of product to the medium. Further, transformed roots are able to regenerate genetically stable plants as transgenics or clones. This property of rapid growth and high plantlet regeneration frequency allows clonal propagation of elite plants. In addition, the altered phenotype of hairy root regenerants (hairy root syndrome) is useful in plant breeding programs with plants of ornamental interest. In vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species. The ability to manipulate trees at a cellular and molecular level shows great potential for clonal propagation and genetic improvement. Transgenic root system offers tremendous potential for introducing additional genes along with the Ri T-DNA genes for alteration of metabolic pathways and production of useful metabolites or compounds of interest. This article discusses various applications and perspectives of hairy root cultures and the recent progress achieved with respect to transformation of plants using A. rhizogenes.
Two series of thermotropic copolyesters of p-hydroxybenzoic acid (HBA) were synthesized by direct thermal polycondensation. One comprised aromatic copolyesters from HBA, terephthalic acid, bis(4-hydroxyphenyl) ketone (BHP) and resorcinol. The other comprised semi-aromatic copolyesters from HBA, terephthalic acid, BHP and α,ω-diols with carbon atom number of 4, 6, 8, 10. The properties of the two series were characterized by polarized light microscopy, differential scanning calorimetry and wide angle X-ray diffraction. Most of the resulting copolyesters could form a nematic phase over a wide temperature range above their melting points. The effects of variation in composition and monomer structure on the properties of copolyesters were discussed.
The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. As with all lantibiotics, nisin contains a number of dehydro-residues and thioether amino acids that introduce five lanthionine rings into the target peptide. These atypical amino acids are introduced by post-translational modification of a ribosomally synthesized precursor peptide. In certain cases, the serine residue, at position 33 of nisin, does not undergo dehydration to Dha33. With native nisin this partially processed form represents about 10% of the total peptide, whereas with the engineered variants, [Trp30]nisin A and [Lys27,Lys31]nisin A, the proportion of peptide that escapes full processing was found to be to approximately 50%. This feature of nisin biosynthesis was exploited in an investigation of the role of the NisB protein in pre-nisin maturation. Manipulation of the level of NisB was achieved by cloning and overexpressing the plasmid-encoded nisB gene in a range of different nisin-producing strains. The resulting fourfold increase in the level of NisB significantly increased the efficiency of the dehydration reaction at Ser33. The final secreted product of biosynthesis by these strains was the homogenous form of the fully processed nisin (or nisin variant) molecule. The results presented represent the first experimental evidence for the direct involvement of the NisB protein in the maturation process of nisin.
This paper summarises the occurrence in foods and beverages of the cinnamic acids, their associated conjugates and transformation products. Quantitative data are lacking for some commodities known to contain them, but it is clear that for many people coffee will be the major source. The daily dietary intake of total cinnamates may vary substantially from almost zero to perhaps close to 1 g. The data relating to their absorption and metabolism are presented along with a consideration of their possible in vivo effects. Data for true bioavailability are incomplete: in particular it is not clear whether availability differs markedly with the form of the conjugate, and whether as a consequence some dietary sources may be superior to others.
The highest berberine content of unselected Coptis cells cultured under the best conditions for berberine production (darkness, high aeration, 3% sucrose and White's basal medium) was about 5% on a dry wt basis. Fluoromicroscopy showed that cultured Coptis cells had heterogeneous characters; therefore, selection was used to establish a high berberine-producing culture of Coptis cells. When small cell aggregates were cloned, high berberine-producing cell lines were produced. Repeated cloning, however, was needed to obtain stable cell lines that produced large amounts of berberine. The highest berberine production in a selected cell line was 13.2% on a dry wt basis (1.39 g/l. culture). The average production was 8.2% (0.90 g/l. culture).
Two different methods for sugarbeet (Beta vulgaris L.) transformation were developed, one using Agrobacterium with excised cotyledons, the other, particle bombardment of embryogenic hypocotyl callus. Transformation efficiencies averaged
0.7% for the Agrobacterium method (number of transgenic plants obtained per treated cotyledon) and about 8% for the bombardment method (number of transgenic
plants obtained per plate of embryogenic callus treated). Transgenic sugarbeet plants were produced carrying genes encoding
either pathogen-defense-related proteins or the reporter enzyme β-glucuronidase (GUS) under transcriptional control of stress- or wound-inducible promoters. In addition, two plants were regenerated
carrying a gene associated with enhanced insect resistance, the cytokinin biosynthesis gene, fused to a patatin gene promoter
from potato. Expression of the GUS gene (gusA) under the control of the tobacco osmotin promoter was wound inducible with detectable activity at 8 h and maximal activity
at 72 h post-wounding.
Daucus
carota callus developed red pigments under the influence of indole-3 acetic acid and kinetin. Maximum yield of anthocyanin at the end of 3 weeks was 5.4% on dry weight basis. The callus subjected to phosphate and nitrate stress produced 7.2% and 8.5% anthocyanin respectively. Feeding of sucrose at 7.5% level resulted in production of 15% anthocyanin. Mannitol as osmoticum had positive influence on anthocyanin production.
A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar
beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector
T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of
which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots,
the nematode resistance gene Hs1
pro-1
was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation
from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1
pro-1
-mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture.
Hairy root cultures of red beet (Beta vulgaris) were grown in 3 l bubble column reactor for studying growth and pigment production under the influence of polyamines (PA) and elicitor treatment. Earlier studies with shake flask cultures had shown that combined feeding of spermidine (spd) and putrescine (put) (each 0.75 mM) significantly enhanced betalaine productivity in hairy root cultures of red beet. The present study has been focused on betalaine production in 3 l bubble column bioreactor where the growth pattern and betalaine synthesis under the influence of similar levels of polyamines were followed. A combination of spermidine and putrescine fed to the roots each at levels of 0.75 mM efficiently increased growth and pigment production resulting in 1.23-fold higher biomass (39.2 g FW l−1) and 1.27-fold higher betalaine content (32.9 mg g−1 DW) than control. Treatments with various levels of elicitor-methyl jasmonate (MJ), though progressively retarded biomass, at 40 μM level resulted in a significant increase in betalaine content resulting in 36.13 mg g−1 DW which was 1.4-fold higher than the control. Further higher concentrations of methyl jasmonate treatments supported high as well as rapid accumulation of betalaines, the overall betalaine productivity was hampered mainly because of the inhibitory action on biomass. Pigment release studies with cetyl trimethyl ammonium bromide (CTAB) resulted in optimization of concentration for better efflux of betalaines without showing any inhibitory effect on hairy root viability. These studies on product enhancement and on-line extraction of pigment are useful for developing a bioreactor system for betalaine production using B. vulgaris hairy root cultures. In particular the use of elicitors and efflux studies provide an insight for integrating unit operations and developing a process for continuous operation and higher production of phytochemicals.
The experimental conditions commonly used to detect bound phenolic acids by alkaline hydrolysis result in loss of several phenolic acids, particularly dihydroxy-derivatives (caffeic acid, dihydrocaffeic acid, homogentisic acid). In this study we show that the addition of ascorbic acid, a strong antioxidant, and ethylenediaminetetraacetic acid, a metal chelator, totally prevent the loss of phenolic acids during alkaline hydrolysis. In these conditions, a complete recovery of caffeic acid following hydrolysis of chlorogenic acid (5′-caffeoylquinic acid, an ester of caffeic acid with quinic acid) was found. This procedure has been successfully applied to quantitatively detect bound phenolic acids in coffee brew and apple.
An aqueous sugar-beet pulp extract obtained by pressure-cooking and mainly composed of pectic fragments, was treated with a pectinolytic preparation. A strong, but limited depolymerization occurred fastly, while feruloyl-esterase activities appeared more gradually, pointing to some deficiencies of the enzymic mix. The solubility of ferulic acid in the sugar-rich, acidic hydrolysate proved stable, and no crystallization could be triggered in this complex medium. Comparative batch tests were carried out with preselected, commercially-available adsorbents: activated carbons, polystyrenic divinylbenzene-crosslinked resins, and polyvinylpolypyrrolidone (PVPP). In each case, the ferulic acid uptake is lower at pH values above 4.5, the pKa determined for its carboxylic group. The natural acidity of the hydrolysate, pH 3.4, was chosen as the optimum. The chemically-activated carbon (SA1817) has the highest affinity for ferulic acid, while Amberlite XAD-16 performs best among the polystyrenic resins. PVPP also offers an appreciable adsorption efficiency. Selectivity is ensured, since only the charcoal removes a slight amount of galacturonic acid, which is released on rising the pH to 4. Alkaline dissociation and ethanolic extraction were investigated as desorption strategies, and ethanol was kept as the most suitable common eluent. Dynamic column studies allowed to estimate the maximum specific capacity at about 22, 12 and 8% (w:w ferulic acid/adsorbent) for the chemical granular activated carbon (GAC), XAD-16 and PVPP, respectively. Ferulic acid is quantitatively recovered after ethanolic elution of the three selected adsorbents, and the best purity is achieved with PVPP, followed by GAC.
Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic roots produced by A. rhizogenes infection is characterized by high growth rate and genetic stability. These genetically transformed root cultures can produce higher levels of secondary metabolites or amounts comparable to that of intact plants. Hairy root cultures offer promise for production of valuable secondary metabolites in many plants. The main constraint for commercial exploitation of hairy root cultures is their scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of interconnected tissues unevenly distributed throughout the vessel. Rheological characteristics of heterogeneous system should also be taken into consideration during mass scale culturing of hairy roots. Development of bioreactor models for hairy root cultures is still a recent phenomenon. It is also necessary to develop computer-aided models for different parameters such as oxygen consumption and excretion of product to the medium. Further, transformed roots are able to regenerate genetically stable plants as transgenics or clones. This property of rapid growth and high plantlet regeneration frequency allows clonal propagation of elite plants. In addition, the altered phenotype of hairy root regenerants (hairy root syndrome) is useful in plant breeding programs with plants of ornamental interest. In vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species. The ability to manipulate trees at a cellular and molecular level shows great potential for clonal propagation and genetic improvement. Transgenic root system offers tremendous potential for introducing additional genes along with the Ri T-DNA genes for alteration of metabolic pathways and production of useful metabolites or compounds of interest. This article discusses various applications and perspectives of hairy root cultures and the recent progress achieved with respect to transformation of plants using A. rhizogenes.
In Arabidopsis thaliana, six vacuolar Na(+)/H(+) antiporters (AtNHX1-6) were identified. Among them, AtNHX1, 2 and 5 are functional Na(+)/H(+) antiporters with the most abundant expression levels in seedling shoots and roots. However, the expression of AtNHX3 in Arabidopsis can only be detected by RT-PCR, and its physiological function still remains unclear. In this work, we demonstrate that constitutive expression of AtNHX3 in sugar beet (Beta vulgaris L.) conferred augmented resistance to high salinity on transgenic plants. In the presence of 300 or 500 mm NaCl, transgenic plants showed very high potassium accumulation in the roots and storage roots. Furthermore, the transcripts of sucrose phosphate synthase (SPS), sucrose synthase (SS) and cell wall sucrose invertase (SI) genes were maintained in transgenic plants. The accumulation of soluble sugar in the storage roots of transgenic plants grown under high salt stress condition was also higher. Our results implicate that AtNHX3 is also a functional antiporter responsible for salt tolerance by mediating K(+)/H(+) exchange in higher plants. The salt accumulation in leaves but not in the storage roots, and the increased yield of storage roots with enhanced constituent soluble sugar contents under salt stress condition demonstrate a great potential use of this gene in improving the quality and yield of crop plants.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. As with all lantibiotics, nisin contains a number of dehydro-residues and thioether amino acids that introduce five lanthionine rings into the target peptide. These atypical amino acids are introduced by post-translational modification of a ribosomally synthesized precursor peptide. In certain cases, the serine residue, at position 33 of nisin, does not undergo dehydration to Dha33. With native nisin this partially processed form represents about 10% of the total peptide, whereas with the engineered variants, [Trp30]nisin A and [Lys27,Lys31]nisin A, the proportion of peptide that escapes full processing was found to be to approximately 50%. This feature of nisin biosynthesis was exploited in an investigation of the role of the NisB protein in pre-nisin maturation. Manipulation of the level of NisB was achieved by cloning and overexpressing the plasmid-encoded nisB gene in a range of different nisin-producing strains. The resulting fourfold increase in the level of NisB significantly increased the efficiency of the dehydration reaction at Ser33. The final secreted product of biosynthesis by these strains was the homogenous form of the fully processed nisin (or nisin variant) molecule. The results presented represent the first experimental evidence for the direct involvement of the NisB protein in the maturation process of nisin.
The enzyme 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), which catalyzes a hydration and two-carbon cleavage step in the degradation of 4-hydroxycinnamic acids, has been purified and characterized from Pseudomonas fluorescens strain AN103. The enzyme is a homodimer and is active with three closely related substrates, 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA (Km values: 5.2, 1.6, and 2.4 microM, respectively), but not with cinnamoyl-CoA or with sinapinoyl-CoA. The abundance of the enzyme reflects a low catalytic center activity (2.3 molecules s-1 at 30 degrees C; 4-coumaroyl-CoA as substrate).
Microorganisms able to produce vanillin in excess of 6g/l from ferulic acid have now been isolated. In Pseudomonas strains, the metabolic pathway from eugenol via ferulic acid to vanillin has been characterised at the enzymic and molecular genetic levels. Attempts to introduce vanillin production into other organisms by genetic engineering have begun.
4-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL), a crotonase homologue of phenylpropanoid catabolism from Pseudomonas fluorescens strain AN103, led to the formation of 4-hydroxybenzaldehyde metabolites when expressed in hairy root cultures of Datura stramonium L. established by transformation with Agrobacterium rhizogenes. The principal new compounds observed were the glucoside and glucose ester of 4-hydroxybenzoic acid, together with 4-hydroxybenzyl alcohol- O-beta- D-glucoside. In lines actively expressing HCHL, these together amounted to around 0.5% of tissue fresh mass. No protocatechuic derivatives were found, although a trace of vanillic acid-beta- D-glucoside was detected. There was no accumulation of 4-hydroxybenzaldehydes, whether free or in the form of their glucose conjugates. There was some evidence suggesting a diminished availability of feruloyl-CoA for the production of feruloyl putrescine and coniferyl alcohol. The findings are discussed in the context of a diversion of phenylpropanoid metabolism, and the ability of plants and plant cultures to conjugate phenolic compounds.
We investigated five penicillin derivatives that are popularly used for transformation experiments with Agrobacterium rhizogenes-penicillin G, carbenicillin, ampicillin, amoxicillin and cephalexin-for their effects on the growth and morphology of Beta vulgaris, Capsicum annuum and Glehnia littoralis roots. Attention was given to the relationship between their chemical structures and functions. Ampicillin was found to stimulate root elongation but inhibit root branching, whereas carbenicillin inhibited root elongation but promoted root branching. Root cultures were also exposed to hydrolyzed products of these antibiotics-i.e. phenylmalonic acid (PM), phenylglycine and 6-aminopenicillanic acid (6-APA): PM inhibited root elongation the most, while root elongation was supported best by 6-APA. These results indicate that both the side chains and the major component of penicillin derivatives affect root development and that the nature of the side chains is responsible for the responses. Ampicillin but not carbenicillin was used in subsequent experiments described herein to eliminate bacteria and to support root growth of transformants of the recalcitrant plants.
Feeding of amino acids to hairy roots of the yellow beet (Beta vulgaris var. lutea) usually results in the formation of the respective betaxanthins. One exception is (S)-glutamate whose feeding leads to an increase in the betaxanthin vulgaxanthin I (glutamine as amino-acid moiety) instead of vulgaxanthin II (glutamate as amino-acid moiety). To elucidate this phenomenon, hairy roots were cultivated in modified standard medium and (S)-glutamate was fed. Under most nutrient conditions tested, glutamine and vulgaxanthin I in the tissue dominated over glutamate and vulgaxanthin II. Glutamate, opposed to glutamine, was readily metabolized so that its concentration was lower than that of glutamine. Maximum concentrations of glutamate were reached when the activity of glutamine synthetase was low. Even then, however, vulgaxanthin II stayed on a low level. In contrast, the level of vulgaxanthin I increased with increasing concentrations of glutamine in the tissue. Also 4-aminobutyric acid (GABA) was a major amino acid in the hairy roots. Its concentration reached maximum levels when (S)-glutamate, a GABA precursor, was fed, or when sucrose, the C source of the roots, was replaced by glucose. The respective GABA-betaxanthin, however, was hardly detectable. When both (S)-glutamate and glucose were supplied, the GABA concentration exceeded that of all other amino acids. Only then the GABA-betaxanthin could be characterized in small amounts. Interestingly, the level of the main betaxanthin, miraxanthin V, consisting of betalamic acid and dopamine, was most markedly reduced by a replacement of sucrose with glucose. We conclude that the reaction of betalamic acid with glutamate and GABA was considerably lower than with glutamine and dopamine, irrespective of the concentration of the amino acid in the tissue. Possible reasons will be discussed, also with respect to the occurrence of species-specific patterns of betaxanthins.
To develop an experimental system for studying ginsenoside biosynthesis, we generated thousands of ginseng (Panax ginseng C.A. Meyer) hairy roots, genetically transformed roots induced by Agrobacterium rhizogenes, and analyzed the ginsenosides in the samples. 27 putative ginsenosides were detected in ginseng hairy roots. Quantitative and qualitative variations in the seven major ginsenosides were profiled in 993 ginseng hairy root lines using LC/MS and HPLC-UV. Cluster analysis of metabolic profiling data enabled us to select hairy root lines, which varied significantly in ginsenoside production. We selected hairy root lines producing total ginsenoside contents 4-5 times higher than that of a common hairy root population, as well as lines that varied in the ratio of the protopanaxadiol to protopanaxatriol type ginsenoside. Some of the hairy root lines produce only a single ginsenoside in relatively high amounts. These metabolites represent the end product of gene expression, thus metabolic profiling can give a broad view of the biochemical status or biochemical phenotype of a hairy root line that can be directly linked to gene function.
We report here the accumulation of p-hydroxybenzoic acid in Agrobacterium rhizogenes-induced hairy root cultures of Daucus carota. This phenolic acid finds application in food, pharmaceutical and polymer industries. Metabolic profiling of phenolics by HPLC/ESI-MS from these hairy roots showed a considerable amount of p-hydroxybenzoic acid accumulation both in cytosol and in the cell wall. Analyses of HCl and NaOH treated soluble phenolic fractions resulted in the elution of peaks with same retention time and similar UV-absorption spectra as observed with p-hydroxybenzoic acid standard. This suggests that p-hydroxybenzoic acid is present in the cytosol as free-form (unconjugated). A correlation has been drawn between the accumulation of soluble and wall-bound phenolic acids on a time-course basis. An apparent absence of any p-hydroxybenzoic acid-glucoside supports this observation, which in turn encourages the idea of its incorporation in the cell wall in an alkaline-labile form.
Studies involving transgenic plants with modifications in the lignin pathway reported to date, have received a relatively preliminary characterisation in relation to the impact on vascular integrity, biomechanical properties of tissues and carbon allocation to phenolic pools. Therefore, in this study transgenic tobacco plants (Nicotiana tabacum cv XHFD 8) expressing various levels of a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) gene have been characterised for cell wall and related morphological changes. The HCHL enzyme converts p-coumaroyl-CoA to 4-hydroxybenzaldehyde thereby rerouting the phenylpropanoid pathway. Plants expressing high levels of HCHL activity exhibited reduced lignin deposition, impaired monolignol biosynthesis and vascular integrity. The plants also exhibited reduction in stem toughness concomitant with a massive reduction in both the cell wall esterified and soluble phenolics. A notable result of redirecting the carbon flux was the wall-bound accretion of vanillin and vanillic acid, probably due to the shunt pathway. Intracellular accumulation of novel metabolites such as hydroxybenzoic and vanillic acid derivatives also occurred in the transgenic plants. A line with intermediate levels of HCHL expression conferred correspondingly reduced lignin deposition, toughness and phenolics. This line displayed a normal morphology but distorted vasculature. Coloration of the xylem has been previously attributed to incorporation of alternative phenolics, whereas results from this study indicate that the coloration is likely to be due to the association of low molecular weight phenolics. There was no evidence of increased growth or enhanced cellulose biosynthesis as a result of HCHL expression. Hence, rerouting the phenylpropanoid biosynthetic pathway quantitatively and qualitatively modifies cell wall-bound phenolics and vascular structure.
Sugarcane (Saccharum hybrids) was evaluated as a production platform for p-hydroxybenzoic acid using two different bacterial proteins (a chloroplast-targeted version of Escherichia coli chorismate pyruvate-lyase and 4-hydroxycinnamoyl-CoA hydratase/lyase from Pseudomonas fluorescens) that both provide a one-enzyme pathway from a naturally occurring plant intermediate. The substrates for these enzymes are chorismate (a shikimate pathway intermediate that is synthesized in plastids) and 4-hydroxycinnamoyl-CoA (a cytosolic phenylpropanoid intermediate). Although both proteins have previously been shown to elevate p-hydroxybenzoic acid levels in plants, they have never been evaluated concurrently in the same laboratory. Nor are there any reports on their efficacy in stem tissue. After surveying two large populations of transgenic plants, it was concluded that the hydratase/lyase is the superior catalyst for leaf and stem tissue, and further studies focused on this pathway. p-Hydroxybenzoic acid was quantitatively converted to glucose conjugates by endogenous uridine diphosphate (UDP)-glucosyltransferases and presumably stored in the vacuole. The largest amounts detected in leaf and stem tissue were 7.3% and 1.5% dry weight (DW), respectively, yet there were no discernible phenotypic abnormalities. However, as a result of diverting carbon away from the phenylpropanoid pathway, there was a severe reduction in leaf chlorogenic acid, subtle changes in lignin composition, as revealed by phloroglucinol staining, and an apparent compensatory up-regulation of phenylalanine ammonia-lyase. Although product accumulation in the leaves at the highest level of gene expression obtained in the present study was clearly substrate-limited, additional experiments are necessary before this conclusion can be extended to the stalk.
Morimoto Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon
- Fujita
- Fujita
Cai An improved transformation protocol for studying gene expression in hairy roots of sugar beet
- Kifle
- Kifle
Brumbley Initial evaluation of sugarcane as a production platform for p-hydroxybenzoic acid
- Mcqualter
- Mcqualter