Cadmium induced oxidative stress and changes in soluble and ionically bound cell wall peroxidase activities in roots of seedling and 3–4 leaf stage plants of <Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) czern

Metabolic adaptations to heavy metal toxicity in plants are thought to be related with developmental growth stage and the type of metal by which plant is affected. In the present study, changes in ionically bound CWP, soluble peroxidase activity, H(2)O(2) level and Malonaldehyde content in roots of cadmium and copper stressed seedlings and cadmium stressed 3-4 leaf stage plants of Brassica juncea were investigated. Cadmium inhibits root growth and reduces fresh biomass. The reduction in root growth and fresh biomass is correlated with increased lipid peroxidation and reduced tolerance. Treatment with cadmium resulted in an increase in ionically bound CWP activity in roots of seedlings but no significant change in its activity was found in roots of 3-4 leaf stage plants. Increased level of H(2)O(2) in roots of cadmium and copper treated seedlings, show a direct correlation with increased activity of ionically bound CWP. H(2)O(2) level in 3-4 leaf stage plant roots was found to be very low. Soluble peroxidase activity decreased in cadmium (50 and 100 mu-icroM) treated seedlings but it was ineffective to cause any change in its activity in 3-4 leaf stage plants. Copper treated seedlings showed an increase in ionically bound CWP activity, H(2)O(2) level and MDA content. Ascorbic acid (50 mM) pretreated seedlings shows significant decrease in ionically bound CWP activity when exposed to 50 muM cadmium. Hence, it is concluded that inhibition of root growth in Brassica juncea seedlings by cadmium, is associated with CWP catalyzed H(2)O(2) dependent reactions which are involved in metabolic adaptations to heavy-metal stress.     

Production of reactive oxygen intermediates (O(2)(.-), H(2)O(2), and (.)OH) by maize roots and their role in wall loosening and elongation growth

Cell extension in the growing zone of plant roots typically takes place with a maximum local growth rate of 50% length increase per hour. The biochemical mechanism of this dramatic growth process is still poorly understood. Here we test the hypothesis that the wall-loosening reaction controlling root elongation is effected by the production of reactive oxygen intermediates, initiated by a NAD(P)H oxidase-catalyzed formation of superoxide radicals (O(2)(.-)) at the plasma membrane and culminating in the generation of polysaccharide-cleaving hydroxyl radicals ((.)OH) by cell wall peroxidase. The following results were obtained using primary roots of maize (Zea mays) seedlings as experimental material. (1) Production of O(2)(.-), H(2)O(2), and (.)OH can be demonstrated in the growing zone using specific histochemical assays and electron paramagnetic resonance spectroscopy. (2) Auxin-induced inhibition of growth is accompanied by a reduction of O(2)(.-) production. (3) Experimental generation of (.)OH in the cell walls with the Fenton reaction causes wall loosening (cell wall creep), specifically in the growing zone. Alternatively, wall loosening can be induced by (.)OH produced by endogenous cell wall peroxidase in the presence of NADH and H(2)O(2). (4) Inhibition of endogenous (.)OH formation by O(2)(.-) or (.)OH scavengers, or inhibitors of NAD(P)H oxidase or peroxidase activity, suppress elongation growth. These results show that juvenile root cells transiently express the ability to generate (.)OH, and to respond to (.)OH by wall loosening, in passing through the growing zone. Moreover, inhibitor studies indicate that (.)OH formation is essential for normal root growth.     

Tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> cv. pera) hairy root cultures: Characterization and changes in peroxidase activity under NaCl treatment

Summary Hairy root cultures of Lycopersicon esculentum L. Mill ev. Pera were established by infection of leaf explants with Agrobacterium rhizogenes LBA 9402. The pattern of peroxidase isoenzymes in these tissues was similar to that of roots excised from tomato plants grown in hydroponic cultures. Hairy root cultures may be an appropriate system to analyze the peroxidase involvement in the response of isolated roots to salt stress, avoiding the problem of wounding or changes in hormone levels observed in roots excised from plants. The cultures of hairy roots allowed the evaluation of changes in peroxidase patterns not only in the tissue but also in the culture medium. Hairy roots were subcultured in Murashige and Skoog liquid medium with or without 100 mM NaCl to investigate the evolution of growth, total peroxidase activity of the tissue and culture medium, and changes in the peroxidase isoenzyme patterns under each condition of growth. Control cultures showed a growth index higher than those reported for other hairy root cultures, and it was even higher in the presence of 100 mM NaCl. The total peroxidase activity in the tissue was similar for control and salt-treated roots. Even when the total peroxidase activity of the medium decreased under salt treatment, NaCl induced secretion of a highly basic peroxidase and inhibition of the secretion of some acidic isoenzymes. These changes may explain the physiological role of these enzymes in the response to salt stress that we will possibly establish through a future study of the biochemical properties of those peroxidases.     

Activation of Host Defense Mechanisms by Elevated Production of H2O2 in Transgenic Plants

Active oxygen species have been postulated to perform multiple functions in plant defense, but their exact role in plant resistance to diseases is not fully understood. We have recently demonstrated H2O2-mediated disease resistance in transgenic potato (Solanum tuberosum) plants expressing a foreign gene encoding glucose oxidase. In this study we provide further evidence that the H2O2-mediated disease resistance in potato is effective against a broad range of plant pathogens. We have investigated mechanisms underlying the H2O2-mediated disease resistance in transgenic potato plants. The constitutively elevated levels of H2O2 induced the accumulation of total salicylic acid severalfold in the leaf tissue of transgenic plants, although no significant change was detected in the level of free salicylic acid. The mRNAs of two defense-related genes encoding the anionic peroxidase and acidic chitinase were also induced. In addition, an increased accumulation of several isoforms of extracellular peroxidase, including a newly induced one, was observed. This was accompanied by a significant increase in the lignin content of stem and root tissues of the transgenic plants. The results suggest that constitutively elevated sublethal levels of H2O2 are sufficient to activate an array of host defense mechanisms, and these defense mechanisms may be a major contributing factor to the H2O2-mediated disease resistance in transgenic plants.     

Hairy root cultures of Anethum graveolens (dill): establishment, growth, time-course study of their essential oil and its comparison with parent plant oils

Transformed root cultures of Anethum graveolens were induced by inoculation of aseptically grown seedlings with Agrobacterium rhizogenes carrying plasmid pRi 1855. The main component of the essential oils from the fruits and from the roots of the parent plant was carvone, whereas -phellandrene and apiole were dominant in the oil from, respectively, the aerial parts and the hairy roots. The essential oils from the fruits, aerial parts and roots of the parent plant were at 2%, 0.3% and 0.06% (v/w), respectively, but only 0.02% (v/w) in the hairy root cultures. Growth of the hairy root cultures reached 600 mg dry wt/50 ml medium after 50 days. The essential oil composition did not change significantly during their growth.     

Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots

Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H(+) efflux rate, 1.80 microM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H(+) consumption for O(2)(*-) dismutation to H(2)O(2). Also K(+) influx was strongly depressed by MeJA, even transitorily reverting to K(+) efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H(2)O(2) accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O(2) uptake by roots gave similar results. These and other results for additions of H(2)O(2) or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H(2)O(2) being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN(-)- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O(2)(*-) and H(2)O(2), O(2) uptake, and peroxidase activity by roots.     

Physiological basis of different allelopathic reactions of cucumber and figleaf gourd plants to cinnamic acid

To provide an insight into the mechanism of interspecific interactions mediated by allelochemicals, cucumber and figleaf gourd seedlings were compared on their response to cinnamic acid, an autotoxin from root exudates of cucumber. Reactive oxygen species metabolism and plasma membrane H(+)-ATPase activity were examined in roots upon exposure to cinnamic acid. This exposure resulted in significant increases in activities of NADPH oxidase, superoxide dismutase, guaiacol peroxidase, and catalase, as well as in O(2)(.-) production and H(2)O(2) content, in cucumber roots but not in figleaf gourd roots. Notably, the cucumber roots produced significant amount of reactive oxygen species (ROS) immediately after cinnamic acid treatment, consequently increasing membrane peroxidation, decreasing membrane H(+)-ATPase activity, and losing root viability. By contrast, no such changes were observed in figleaf gourd roots. All these results indicated that there was an interspecies difference in the recognition of allelochemicals, which induced oxidative stress accompanied by root cell death in cucumber, an autotoxic plant, but not in figleaf gourd, a cucumber relative.     

Effect of exogenous H2O2 on antioxidant enzymes of Brassica juncea L. seedlings in relation to 24-epibrassinolide under chilling stress

Hydrogen peroxide is most stable molecule among reactive oxygen species, which play a vital role in growth and development of plant as signaling molecule at low concentration in response to various abiotic and biotic stresses. Exogenous application of H2O2 is known to induce chilling tolerance in plants. Brassinosteroids are plant steroid hormones known for their anti-stress properties. In this study, effect of exogenous H2O2 on antioxidant defense system of Brassica juncea L. seedlings was investigated in 24-epibrassinolide (24-EBL) treated and untreated seedlings under chilling stress. The surface sterilized seeds of B. juncea L. were germinated in petriplates containing different concentrations of H2O2 alone and in combination with 10(-8) M 24-EBL. Chilling treatment (4 degrees C) was given to 10-days old seedlings grown in different treatments for 6 h daily up to 3 days. 24 h recovery period was given to chilling treated seedlings by placing at 25 degrees C + 2 degrees C and harvested for antioxidant enzymes on 14th day after sowing (DAS). Treatment of 24-EBL in combination with H2O2 (15 and 20 mM) helped in reducing the toxicity of seed and seedlings due to H2O2 exposure on their germination rate, shoot and root length respectively. 24-EBL treatment at seed and seedling stage helped in alleviating the toxic effect of H2O2 through antioxidant defense system by increasing the activities of various enzymes involved in antioxidant defense system such as catalase (CAT, E.C. 1.11.1.6), ascorbate peroxidase (APOX, E.C. 1.11.1.11), and superoxide dismutase (SOD, E.C. 1.15.1.1). In conclusion, exogenous pretreatment of H2O2 to seeds of B. juncea L. adapted the seedlings to tolerate chilling stress, which was further ameliorated in combination of H2O2 with 24-EBL.     

Hairy root culture for mass-production of high-value secondary metabolites

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

Agrobacterium tumefaciens-mediated hairy root production from seedlings of Chinese cabbage

Cruciferous hairy roots are often used for improving drought adaptability, peroxidase production, andin vitro subculturing ofPlasmodiophora brassicae. For metabolic engineering,Agrobacterium tumefaciens-mediated systems have previously been developed for hairy root production in other plant species. Here, we used therolABC gene binary construct inA. tumefaciens strain GV3101 to establish cultures of Chinese cabbage hairy roots. On both solid and liquid media, therolABC hairy root lines exhibited a wild-type hairy root syndrome in terms of their growth and morphology. This demonstrates that those three genes are sufficient to induce high-quality hairy roots in Chinese cabbage. Such a system could be useful for the stable production of secondary metabolites in that species.     

Dopamine-derived dopaminochrome promotes H(2)O(2) release at mitochondrial complex I: stimulation by rotenone, control by Ca(2+), and relevance to Parkinson disease

Inhibitors of Complex I of the mitochondrial respiratory chain, such as rotenone, promote Parkinson disease-like symptoms and signs of oxidative stress. Dopamine (DA) oxidation products may be implicated in such a process. We show here that the o-quinone dopaminochrome (DACHR), a relatively stable DA oxidation product, promotes concentration (0.1-0.2 mum)- and respiration-dependent generation of H(2)O(2) at Complex I in brain mitochondria, with further stimulation by low concentrations of rotenone (5-30 nm). The rotenone effect required that contaminating Ca(2+) (8-10 mum) was not removed. DACHR apparently extracts an electron from the constitutively autoxidizable site in Complex I, producing a semiquinone, which then transfers an electron to O(2), generating O(2)(.) and then H(2)O(2). Mitochondrial removal of H(2)O(2) monoamine, formed by either oxidase activity or DACHR, was performed largely by glutathione peroxidase and glutathione reductase, which were negatively regulated by low intramitochondrial Ca(2+) levels. Thus, the H(2)O(2) formed accumulated in the medium if contaminating Ca(2+) was present; in the absence of Ca(2+), H(2)O(2) was completely removed if it originated from monoamine oxidase, but was less completely removed if it originated from DACHR. We propose that the primary action of rotenone is to promote extracellular O(2)(.) release via activation of NADPH oxidase in the microglia. In turn, O(2)(.) oxidizes DA to DACHR extracellularly. (The reaction is favored by the lack of GSH, which would otherwise preferably produce GSH adducts of dopaminoquinone.) Once formed, DACHR (which is resistant to GSH) enters neurons to activate the rotenone-stimulated redox cycle described.     

Isoflavonoid accumulation in soybean hairy roots upon treatment with Fusarium solani.

Hairy roots were initiated from two soybean [Glycine max (L.) Merr.] genotypes with different susceptibility (susceptible 'Spencer' and partially resistant 'PI567.374') to the disease sudden death syndrome (SDS) caused by the soil-borne fungal pathogen Fusarium solani f. sp. glycines (FSG) to study the role of isoflavonoids in the plant response to FSG infection. Hairy root cultures obtained by transformation with Agrobacterium rhizogenes allows normal root growth that can be visually monitored. The principal isoflavones (genistin, daidzin, glycitin and their malonyl conjugates and aglycones) and also isoflavonoid phytoalexins (coumestrol and glyceollin) were measured by HPLC in extracts of the FSG-inoculated and non-inoculated hairy roots. FSG mycelia grew more slowly on inoculated PI567.374 hairy roots than on Spencer hairy roots. The glyceollin content was higher in FSG-inoculated PI567.374 hairy roots than in Spencer hairy roots even though the glyceollin precursor, the isoflavone daidzein, was higher in Spencer. The de novo synthesis of isoflavones and glyceollin was confirmed by [(14)C]Phe incorporation into glyceollin, which was higher both in the FSG-inoculated roots and surrounding medium of the cv. PI567.374 than that of Spencer. Glyceollin was the most inhibitory to FSG growth among eight isoflavonoids tested. The levels of coumestrol, a putative phytoalexin, did not change upon FSG inoculation. The defense response was also elicited by FSG culture filtrates in hairy roots grown in liquid culture. The data obtained indicate that the ability of soybean roots to rapidly produce sufficient amounts of glyceollin in response to FSG infection might be important in providing partial resistance to this fungus.     

Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites

ABSTRACT

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

Hormone independent root organ cultures of rye (Secale cereale)

This work describes the growth of rye root organ cultures which were capable of being repeatedly subcultured in hormone-free medium. They showed morphological characteristics, growth rate, inability to produce shoots, and response to auxins and cytokinins similar to those of the Agrobacterium rhizogenes (Ri plasmid) transformed hairy root cultures of tobacco and red beet which were used for comparison. The root cultures of rye were initiated from callus produced on a medium containing the growth regulators (plant hormones) 2,4-d and kinetin, then transferred to hormone-free medium. However not all rye explants gave rise to callus that would differentiate into stable hairy root cultures and rye seedling root explants did not grow if placed directly on a hormone-free medium. Rice and wheat produced callus and roots on a medium containing hormones but root organ cultures could not be maintained on a hormone-free medium.     

Light-enhanced caffeic acid derivatives biosynthesis in hairy root cultures of <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Light plays an important role in almost all plant developmental processes and provides the fundamental building blocks for growth, development, primary and secondary metabolism. The effects of light on growth rate and caffeic acid derivative (CADs) biosynthesis in hairy root cultures of Echinacea purpurea (Moench) were assessed. Light-grown hairy roots accumulated increased levels of anthocyanins, which became visible in outer cell layer of the cortex as a ring of purple color. The light-grown root cultures also had radially thickened morphology compared with the dark-grown controls. The growth rate and cell viability of the hairy root cultures in light did not show obvious difference in comparison with those in dark. However, biosynthesis of CADs including cichoric acid, caftaric acid, chlorogenic acid and caffeic acid was significantly increased in hairy root cultures grown in the light. The enhanced accumulation of CADs and anthocyanins in E. purpurea hairy root cultures was correlated to an observed light-stimulated activity of phenylalanine ammonium lyase (PAL).     

Effect of copper excess on H2O2 accumulation and peroxidase activities in bean roots

We studied oxidative stress and peroxidase activity resulting from application of excess copper in the nutrient medium on the roots of young bean seedlings. The change in H2O2 content, lipid peroxidation and antioxidant enzymes activities were quantified and located. Excess of copper caused a loss of membrane integrity and the formation of hydrogen peroxide (H2O2) as visualized in the transmission electron microscopy and measured using spectrophotometry. H2O2 accumulated in the intercellular spaces and in the cell wall. The production of H2O2 was accompanied by an increase in the activity of soluble and ionic GPX (guaiacol peroxidase, EC 1.11.17), CAPX (coniferyl alcohol peroxidase) and NADH oxidase.     

Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress

The effect of the arbuscular mycorrhizal (AM) fungus, Glomus versiforme, on growth and reactive oxygen metabolism of trifoliate orange (Poncirus trifoliata) seedlings was studied in potted plants under well-watered (WW) and water stressed (WS) conditions. Water stress significantly decreased root colonization. Shoot dry weight, plant height and stem diameter were higher in AM than in non-AM seedlings regardless of the water status. Inoculation with G. versiforme increased root dry weight and leaf number per plant of WW seedlings. There was less malondialdehyde (MDA) concentration in leaves and roots of AM seedlings, as well as lower hydrogen peroxide (H(2)O(2)) and superoxide anion radical (O(2)(-)) concentrations in AM roots under WW and WS conditions. AM inoculation did not affect the H(2)O(2) and O(2)(-) concentrations of WW leaves. Whether WS or not, AM symbiosis notably increased the guaiacol peroxidase (G-POD) activity of leaves, glutathione reductase (GR) activity of leaves and ascorbate peroxidase (APX) activity of roots. AM infection also markedly increased the APX activity of WS leaves. Soluble proteins and glutathione (GSH) in leaves and roots and ascorbate (ASC) in leaves were higher in WW AM than in WW non-AM seedlings. AM infection also enhanced the ASC and GSH contents of leaves and roots in WS seedlings. Cross-tolerance might occur in AM plants and be enhanced by AM symbiosis. Our results suggest that the increased concentrations of antioxidant enzymes and non-enzymatic antioxidants found in AM plants may serve to protect the organism against oxidative damage, enhancing drought tolerance.     

Propagation of plant viruses in hairy root cultures: a potential method for in vitro production of epitope vaccines and foreign proteins

Hairy roots were used as an in vitro culture system for the propagation of wild-type and transgenic plant viruses. Tobacco mosaic virus (TMV) was added to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by viral infection. Maximum concentrations of TMV in Nicotiana benthamiana hairy roots were 1-2 orders of magnitude greater than in suspended N. benthamiana cells and reached levels of 1-2 mg g(-1) dry weight or 20-28% total soluble protein. Virus accumulated in the roots initially with a constant doubling time of about 1.0 day; subsequent reductions in viral growth rate were correlated with a significant decline in infectivity relative to the inoculum virus. The morphological integrity of the viral particles was maintained during propagation in hairy roots. The contribution to the overall viral titer of passive association of virus with the biomass, for example, by surface adsorption, was negligible compared with active viral replication. N. benthamiana hairy roots were also infected with a TMV-based viral vector developed to express green fluorescent protein (GFP). This vector was about 260-fold less infectious than wild-type TMV and accumulated much more slowly in the roots. Maximum levels of TMV-GFP in the biomass were about 65-fold lower than for TMV. This work demonstrates that hairy root cultures are a feasible means for in vitro propagation of wild-type and transgenic plant viruses under conditions that allow a high degree of environmental containment and control.