Effects of paraquat on lipid peroxidation and antioxidant enzymes in aquatic fern <Emphasis Type="Italic">Azolla microphylla</Emphasis>

Paraquat is most extensively used methyl viologen herbicide to control weeds in the rice-Azolla ecosystem. The effects of different paraquat (PQ) dosages on growth, lipid peroxidation, and activity of antioxidant enzymes of Azolla microphylla Kaul. were investigated. The results indicated that Azolla fronds survived only at the concentrations of 2–6 μM PQ. Frond fragmentation and browning occurred after 24 h at 8 μM PQ. At 24 h, the amount of proteins decreased by 48.7 % in Azolla fronds exposed to 10 μM PQ than that in control fronds. The supplementation of 10 μM PQ increased the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX) by 2,4-, 1,8-, 3,0-, and 2,2-fold, respectively, as compared with control. The content of PQ and activities of SOD, CAT, GPX, and APX were found to be positively correlated. Our study showed that PQ (2–6 μM) caused ROS overproduction in Azolla fronds, which were scavenged by induced activities of antioxidant enzymes.     

Effects of Barium Stress in <i>Brassica juncea</i> and <i>Cakile maritima</i>: The Indicator Role of Some Antioxidant Enzymes and Secondary Metabolites

Soil contamination by toxic trace metal elements, like barium (Ba), may stimulate various undesirable changes in the metabolic activity of plants. The plant responses are fast and with, direct or indirect, generation of reactive oxygen species (ROS). To cope with the stress imposed by the ROS production, plants developed a dual cellular system composed of enzymatic and non-enzymatic players that convert ROS, and their by-products, into stable nontoxic molecules. To assess the Ba stress response of two Brassicaceae species (Brassica juncea, a glycophyte, and Cakile maritime, a halophyte), plants were exposure to different Ba concentrations (0, 100, 200, 300 and 500 μM). The plants response was evaluated through their morphology and development, the determination of plant leaves antioxidant enzymatic activities and by the production of plants secondary metabolites. Results indicated that the two Brassicaceae species have the ability to survive in an environment containing Ba (even at 500 μM). The biomass production of C. maritima was slightly affected whereas an increase in biomass B. juncea was noticed. The stress imposed by Ba activated the antioxidant defense system in the two species, noticed by the changes in the leaves activity of catalase (CAT), ascorbate peroxidase (APX) and guaicol peroxidase (GPX), and of the secondary metabolites, through the production of total phenols and flavonoids. The enzymatic response was not similar within the two plant species: CAT and APX seem to have a more important role against the oxidative stress in C. maritima while in B. juncea is GPX. Overall, total phenols and flavonoids production was more significant in the plants aerial part than in the roots, of the both species. Although the two Brassicaceae species response was different, in both plants catalytic and non-catalytic transformation of ROS occurs, and both were able to overcome the Ba toxicity and prevent the cell damage.     

Bio indices for 2,4-D sensitivity between two plant species: <Emphasis Type="Italic">Azolla pinnata</Emphasis> R.Br. and <Emphasis Type="Italic">Vernonia cinerea</Emphasis> L. with their cellular responses

In the present experiment a pteridophytic species Azolla and an angiospermic species Vernonia were evaluated on the basis of cellular reactivity for herbicidal action through ongoing concentrations. Initially, both the species recorded a significant activity of IAA-oxidase as mark of IAA metabolism with herbicidal sensitivity. Still, Vernonia species were more affected on 2,4-D mediated auxin catabolism. The loss of auxin concentrations on the tissues by 2,4-D reaction was also reflected on growth parameters including relative growth rate and chlorophyll biosynthesis. In a dose dependent manner Vernonia plants were more affected with loss of chlorophyll content and decline in relative growth rate. On the other hand, both those parameters were adjusted significantly with 2,4-D accumulation in Azolla. The stability of cellular metabolism was documented by significant down regulation of protein and lipid peroxidation with concomitant moderation to superoxide and hydrogen peroxide accumulation. The later two were more vulnerable to damage in the Vernonia plant with profuse accumulation of protein and lipid peroxidation products. Similarly, tissue specific reaction to superoxide and hydrogen peroxide accumulation were distinctly demarcated in two species significantly. As a whole, the cellular responses and metabolite distribution to 2,4-D sensitization are the features to describe bio-indices for aquatic fern species Azolla with comparison to angiospermic species Vernonia.     

Effect of Water Stress Induced by Polyethylene Glycol on Growth,Proline Accumulation in <i>Agave americana</i> L.

The effect of water deficit was determined on both in vitro and soil seedling as well as in cells in suspension of Agave americana L. In order to do the establishment of cells, the formation of callus was induced; for it two auxins were evaluated: 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-mino-3,5,6-trichloropicolinic acid (picloram) at three concentrations (0.25, 0.5 and 0.75 mg L⁻¹) in three explants (leaf, root and meristems) cultured in MS semisolid medium. The callogenesis response was related to the type and section of the explant, as well as the regulator used, and a cell suspension was established using 0.5 mg L⁻¹ naphthaleneacetic acid (NAA) + 0.5 mg L⁻¹ Benzylaminopurine (BAP). Seedlings were exposed to polyethyleneglycol (15% and 30% w/v) with a water potential of −0.87 and −2.67 MPa, respectively, under soil conditions. Water stress was applied through restricted irrigation. Fresh weight, root system growth, and chlorophyll concentration were some of the parameters that were affected by the effect of water deficit on A. americana L. Chlorophyll concentration values were significantly decreased by 15 at 30% PEG (19.6 SPAD units) compared to the control treatment. In in vitro plants, the highest concentration of proline was found in the roots, being the treatment with 30% polyethylene glycol where the highest concentration of this osmoregulator was obtained (62.5 mg g⁻¹ DW). Under restricted irrigation conditions, an increase in proline concentration was observed both in the aerial part (2.2 µg 100 g⁻¹ DW) and in the root system (1.8 µg 100 g⁻¹ DW). However, the concentrations found were approximately ten times greater, less than those found under in vitro conditions. Therefore, the accumulation of proline can be considered an indicator of stress in Agave Americana L. growth in vitro.     

Silicon and Nitric Oxide-Mediated Regulation of Growth Attributes,Metabolites and Antioxidant Defense System of Radish (<i>Raphanus sativus</i> L.) under Arsenic Stress

Arsenic (As) contaminated food chains have emerged as a serious public concern for humans and animals and are known to affect the cultivation of edible crops throughout the world. Therefore, the present study was designed to investigate the individual as well as the combined effects of exogenous silicon (Si) and sodium nitroprusside (SNP), a nitric oxide (NO) donor, on plant growth, metabolites, and antioxidant defense systems of radish (Raphanus sativus L.) plants under three different concentrations of As stress, i.e., 0.3, 0.5, and 0.7 mM in a pot experiment. The results showed that As stress reduced the growth parameters of radish plants by increasing the level of oxidative stress markers, i.e., malondialdehyde and hydrogen peroxide. However, foliar application of Si (2 mM) and pretreatment with SNP (100 µM) alone as well as in combination with Si improved the plant growth parameters, i.e., root length, fresh and dry weight of plants under As stress. Furthermore, As stress also reduced protein, and metabolites contents (flavonoids, phenolic and anthocyanin). Activities of antioxidative enzymes such as catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD), and polyphenol oxidase (PPO), as well as the content of non-enzymatic antioxidants (glutathione and ascorbic acid) decreased under As stress. In most of the parameters in radish, As III concentration showed maximum reduction, as compared to As I and II concentrations. However, the individual and combined application of Si and NO significantly alleviated the As-mediated oxidative stress in radish plants by increasing the protein, and metabolites content. Enhancement in the activities of CAT, APX, POD and PPO enzymes were recorded. Contents of glutathione and ascorbic acid were also enhanced in response to co-application of Si and NO under As stress. Results obtained were more pronounced when Si and NO were applied in combination under As stress, as compared to their individual application. In short, the current study highlights that Si and NO synergistically regulate plant growth through lowering the As-mediated oxidative stress by upregulating the metabolites content, activity of antioxidative enzymes and non-enzymatic antioxidants in radish plants.

     

Morphometric and Biochemical Changes in <i>Agave americana</i> L. Plantlets Induced By Ethyl Methanesulfonate

A. americana L. is a crop with very little genetic variability. In order to evaluate the effect of ethyl methanesulfonate (EMS) to induce variability in in vitro plantlets of A. americana, different explants (meristems, leaves and roots) were evaluated for the production of callus. MS medium supplemented with ANA (2.68 μM) and BAP (2.68 μM) was used. Callus obtained from apical meristem were treated with 15 mM EMS for two hours after which shoot formation was induced using 2,4-D (0.11 μM) and BAP (44 μM). The EMS induced variations in the morphometric and morphological parameters of the plantlets obtained, with 60% of the plantlets presenting differences such as dwarfism and different leaf forms, without the presence of spines, as well as an increase in fructan content of 30% with respect to the control plantlets. PAL was increased and this activity is related with higher anthocyanins concentration in A. americana L. plantlets.     

Modulation of physiological responses with TiO<Subscript>2</Subscript> nano-particle in <Emphasis Type="Italic">Azolla pinnata</Emphasis> R.Br. under 2,4-D toxicity

The present work is emphasised with the herbicidal tolerance of Azolla pinnata R.Br. and its modulation with TiO₂ nano-particle. Both carbohydrate and nitrogen metabolism were effected with 2,4-D as herbicide and in few cases TiO₂-NP had recovered few detrimental effects. From the nutrient status in Azolla it recorded the recovery of nitrogen as well as potassium by TiO₂-NP but not in case of phosphorus. However, a conversion of nitrate to ammonium was more induced by TiO₂-NP under herbicidal toxicity. Similar results were obtained for inter-conversion of amino acid–nitrate pool, but no changes with glutamine synthase activity with TiO₂-NP. Initially, the effects of 2,4-D was monitored with changes of chlorophyll content but had not been recovered with nanoparticle. Photosynthetic reserves expressed as both total and reducing sugar were insensitive to TiO₂-NP interference but activity of soluble and wall bound invertase was in reverse trend as compared to control. The 2,4-D mediated changes of redox and its oxidative stress was ameliorated in plants with over expressed ADH activity. As a whole the Azolla bio system with TiO₂ supplementation may be useful in sustenance against 2,4-D toxicity through recovery of nitrogen metabolism. Thus, Azolla-TiO₂-NP bio system would be realised to monitor the herbicidal toxicity in soil and its possible bioremediation.     

Organ-specific effects of the auxin herbicide 2,4-D on the oxidative stress and senescence-related parameters of the stems of pea plants

Oxidative stress and senescence have been shown to participate in the toxicity mechanism of auxin herbicides in the leaves and roots of sensitive plants. However, their role in stem toxicity has not been studied yet. In this work, we report the effect of foliar or root applications of the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the parameters of oxidative stress and senescence of stems of pea (Pisum sativum L.) plants. Contrary to their effect on the pea leaves, in the stems 2,4-D applications did not cause oxidative stress, as shown by the parameters of lipid peroxidation, protein carbonyls, and total and protein thiols. Moreover, they inhibited the superoxide radical (O₂^.−)-producing xanthine oxidase (XOD) activity and stimulated the antioxidant activities of catalase (CAT), guaiacol peroxidase (GPOX), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione S-transferase (GST) and Krebs cycle NAD⁺-isocitrate dehydrogenase (IDH). Applications of 2,4-D also did not induce senescence in the pea stems, as shown by the increase of proteins, the lack of stimulation of proteolytic activity, and the inhibition of senescence-related isocitrate lyase (ICL) activity. However, they stimulated the H₂O₂-producing acyl-CoA oxidase (ACOX) activity of fatty acid beta oxidation. Results suggest that oxidative stress and senescence are not involved in the mechanism of toxicity of 2,4-D in the stems of pea plants, and that these phenomena are not whole-plant toxicity mechanisms for auxin herbicides in susceptible plants. Results also suggest that the effect of 2,4-D on the oxidative metabolism of pea plants might be organ-specific.     

Stress Ameliorative Effects of Indole Acetic Acid on <i>Hordeum vulgare</i> L. Seedlings Subjected to Zinc Toxicity

The heavy metals present in the environment accumulate in the plants and affect their productivity and yield. By entering the food chain, metals cause several serious health problems in human beings as well as in other organisms. Indole acetic acid (IAA) is known to act as a signaling molecule between symbiotic association of metal accumulating plants and plant growth promoting rhizobacteria (PGPR). Present study demonstrated a protective role of IAA against surplus Zinc (Zn)-induced toxicity to Hordeum vulgare seedlings. Elevated Zn concentrations suppressed the plant growth, caused a reduction in leaf relative water contents (RWC) and elevated free proline and non-protein thiols (NPT) accumulation. Zinc treatment also led to enhanced lipid peroxidation (MDA contents) as well as the activity of ascorbate peroxidase (APX), showing the involvement of antioxidative defense mechanism to reduce Zn induced toxicity. IAA oxidase activity was also observed to increase due to Zn treatment. IAA pretreatment of H. vulgare caryopsis could partly revert the Zn-induced toxicity in seedlings.     

Biological Control of Root-Knot Nematode <i>Meloidogyne incognita</i> in <i>Psoralea corylifolia</i> Plant by Enhancing the Biocontrol Efficacy of <i>Trichoderma harzianum</i> Using Press Mud

Meloidogyne incognita is a plant pathogen causing root-knot disease and loss of crop yield. The present study aimed to use Trichoderma harzianum as a biocontrol agent against plant-parasitic nematodes and used press mud, which is a solid waste by-product of sugarcane, as a biocontrol agent and biofertilizer. Therefore, the combined application of T. harzianum and press mud may enhance nematode control and plant growth. Elemental analysis of press mud using scanning electron microscopy (SEM) integrated with an Energy Dispersive X-ray (EDX) analyzer revealed the presence of different elements such as C, O, Mg, Si, P, K, Ca, Cu and Zn. In addition, a greenhouse study was conducted to investigate the combined effects of press mud and T. harzianum on M. incognita reproduction and growth and the biochemical features of Psoralea corylifolia. The results showed that plant length, dry biomass, leaf area, the number of seeds per plant, chlorophyll a, chl b, carotenoid content, nitrate reductase, carbonic anhydrase, and nitrogen content were significantly increased (P ≤ 0.05) in the T2 plants (plants were treated with 100 g of press mud + 50 mL T. harzianum before one week of M. incognita inoculation), over inoculated plants (IC). Antioxidant enzyme activity of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in the foliage of P. corylifolia was significantly increased when plants were treated with press mud + T. harzianum. A significant reduction in the number of egg masses, nematode population, and root-knot index (RKI) was found in plants with T2 plants. These results suggest that the combined application of T. harzianum and press mud has the potential to control the M. incognita infection and can be used as an environmentally safe alternative to chemical nematicides and also help in the removal of sugarcane waste that causes environmental pollution.     

Inhibitory Effect of <i>N</i>, <i>N</i>-Dimethylhexadecylamine on the Growth of White-Rot Fungus <i>Trametes versicolor</i> (L.) in Wood

Wood is an organic material that is a source of carbon of organisms called Wood-decay fungi, and to preserve the wood, various toxic compounds to man and the environment have been used. To analyze the effect of N,N-Dimethylhexadecylamine (DMHDA) on wood attacked by the rotting fungus Trametes versicolor L. We used an in vitro system to expose the fungus T. versicolor to different concentrations of the DMHDA (50, 150 and 450 μM). We quantified the diameter of mycelial growth and laccase activity, also, under these experimental conditions we studied morphological details of the organisms using different scanning equipment including scanning electron microscopy. The growth of T. versicolor exposed to DMHDA for 60 days, showed a concentration-dependent dose behavior, also, the electron microscopy analysis revealed that the morphology and mycelial density was affected by the DMHDA, showing a formation of atypical morphological and thickener folds. Finally, the pieces of wood treated with DMHDA and exposed to the fungus had a lower mass loss, after a period of 60 days of exposure, the values obtained were 0.7, 1.0 and 0.5 g of mass lost for the control, LoC and LoDMHDA treatments respectively. Wood-rot fungi have represented economic losses worldwide, the strategies used have been supported by toxic compounds for the environment. The DMHDA both in the Petri dish system and as a wood preservative was shown to significantly inhibit the growth of T. versicolor.     

Differential responses of hydrogen peroxide, lipid peroxidation and antioxidant enzymes in Azolla microphylla exposed to paraquat and nitric oxide

The present investigation was carried out to decipher the interplay between paraquat (PQ) and exogenously applied nitric oxide (NO) in Azolla microphylla. The addition of PQ (8 ??M) increased the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) by 1.7, 2.7, 3.9 and 1.9 folds respectively than that control in the fronds of Azolla. The amount of H₂O₂ was also enhanced by 2.7 times in the PQ treated plants than that of control. The supplementation of sodium nitroprusside (SNP) from 8?C100 ??M along with PQ, suppressed the activities of antioxidative enzymes and the amount of H₂O₂ compared to PQ alone. The drop in the activity of antioxidative enzymes ?? SOD, GPX, CAT and APX was highest (39.9%, 48.4%, 41.6% and 41.3% respectively) on the supplementation of 100 ??M SNP with PQ treated fronds compared to PQ alone. The addition of NO scavengers along with NO donor in PQ treated fronds neutralized the effect of exogenously supplied NO. This indicates that NO can effectively protect Azolla against PQ toxicity by quenching reactive oxygen species. However, 200 ??M of SNP reversed the protective effect of lower concentration of NO donor against herbicide toxicity. Our study clearly suggests that (i) SNP released NO can work both as cytoprotective and cytotoxic in concentration dependent manner and (ii) involvement of NO in protecting Azolla against PQ toxicity.     

Short-term effect of elevated CO2 concentration and high irradiance on the antioxidant enzymes in bean plants

The effect of short-term exposure to elevated CO₂ concentration and high irradiance on the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidases (GPX) and catalase (CAT), and on the extent of the lipid peroxidation was studied in bean (Phaseolus vulgaris L.) plants. Plants were exposed for 4 d (8 h a day) to irradiance of 100 (LI) or 1000 (HI) μmol m⁻² s⁻¹ at ambient (CA, 350 μmol mol⁻¹) or elevated (CE, 1300 μmol mol⁻¹) CO₂ concentration. Four-day exposure to CE increased the leaf dry mass in HI plants and RuBPC activity and chlorophyll content in LI plants. Total soluble protein content, leaf dry matter and RuBPC activity were higher in HI than in LI plants, although the HI and CE increased the contents of malonyldialdehyde and H₂O₂. Under CA, exposure to HI increased the activity of APX and decreased the total SOD activity. Under CE, HI treatment also activated APX and led to reduction of both, SOD and GPX, enzymes activities. CE considerably reduced the CAT activity at both irradiances, possibly due to suppressed rate of photorespiration under CE conditions.     

The Physiological Mechanisms Underlying N₂-Fixing Common Bean (<i>Phaseolus vulgaris</i> L.) Tolerance to Iron Deficiency

Iron is an essential element for plants as well as all living organisms, functioning in various physiological and biochemical processes such as photosynthesis, respiration, DNA synthesis, and N₂ fixation. In the soil, Fe bioavailability is extremely low, especially under aerobic conditions and at high pH ranges. In contrast, plants with nodules on their roots that fix atmospheric nitrogen need much more iron. To highlight the physiological traits underlying the tolerance of N₂-fixing common bean to iron deficiency, two genotypes were hydroponically cultivated in a greenhouse: Coco nain (CN) and Coco blanc (CB). Plants were inoculated with an efficient strain of Rhizobium tropici, CIAT899, and received a nutrient solution added with 0 µM Fe (severe Fe deficiency, SFeD), 5 µM Fe (moderate Fe deficiency, MFeD) or 45 µM Fe (control, C). Several physiological parameters related to photosynthesis and symbiotic nitrogen fixation were then analyzed. Iron deficiency significantly reduced whole plant and nodule growth, chlorophyll biosynthesis, photosynthesis, leghemoglobin (LgHb), nitrogenase (N₂ase) activity, nitrogen, and Fe nutrition, with some genotypic differences. As compared to CB, CN maintained better Fe allocation to shoots and nodules, allowing it to preserve the integrity of its photosynthetic and symbiotic apparatus, thus maintaining the key functional traits of the plant metabolism (chlorophyll biosynthesis and photosynthesis in shoots, leghemoglobin accumulation, and nitrogenase activity in root nodules). Plant growth depends on photosynthesis, which needs to be supplied with sufficient iron and nitrogen. Fe deficiency stress index (FeD-SI) and Fe use efficiency (FeUE) are two physiological traits of tolerance that discriminated the studied genotypes.     

<i>In Vitro</i> Propagation of <i>Agave guiengola</i> Gentry Using Semisolid Medium and Temporary Immersion Bioreactors

Agave guiengolaGentry is an endemic plant from a very small locality in Oaxaca, Mexico. Its conservation status is fragile and can rapidly worsen. Because of its scarcity, this agave has been used solely for ornamental purposes, but it could have other uses if more plants were available. In vitro propagation by enhanced axillary sprouting from stem segments was attained using Murashige and Skoog Basal Medium (MS) as well as basal medium supplemented with cytokinins 6-Benzylaminopurine (BA) or 6-(γ,γ-Dimethylallylamino)purine (2iP). The best treatment for shoot induction in semisolid medium consisted in MS supplemented with 2 mg l^–1 BA, obtaining a mean of 3.7 shoots per explant. Other interesting responses were observed, such as nodular callus induction using combinations of BA and 2,4-Dichlorophenoxyacetic acid (2,4-D); root induction without Plant Growth Regulators (PGR); and generation of shoot clusters. These clusters constituted an excellent explant for micropropagation in temporary immersion bioreactors, obtaining a propagation rate of 43 shoots per explant with 1 min immersion and 6 h immersion frequencies. All new plants rooted and survived the transfer to soil. This study developed an in vitro propagation scheme to produce individuals that can be used either for reforestation, economical purposes, or to carry out studies in this species to assess its full potential, avoiding exploitation from wild plants.     

The effect of 2,4-dichlorophenol and pentachlorophenol on antioxidant system in the leaves of Phalaris arudinacea

The purpose of this work was to evaluate the effect of 2,4-dichlorophenol (2,4-DCP) and pentachlorophenol (PCP) on the activity of antioxidative system and lipid peroxidation in the leaves of reed canary grass (Phalaris arudinacea). The activity of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR) and glutathione S-transferase (GST) as well as the content of glutathione, ascorbate and phenolic compounds were determined. An induced-increase in the APX, CAT, GPX and GR activities was stronger for PCP, while a significant increase in the GST activity was noted only for 2,4-DCP. Both compounds increased the content of phenolic compounds, oxidized and reduced glutathione as well as the content of ascorbic acid. PCP induced stronger increase in lipid peroxidation than 2,4-DCP. The observed changes revealed that chlorophenols induce oxidative stress and oxidative damage in the leaves of reed canary grass.     

Identification of Resistance to Pathogenesis Related Protein <i>GmPR1L</i> in Tobacco <i>Botrytis cinerea</i> Infection

Soybean (Glycine max (Linn.) Merr.) annual leguminous crop is cultivated all over the world. The occurrence of diseases has a great impact on the yield and quality of soybean. In this study, based on the RNA-seq of soybean variety M18, a complete CDS (Coding sequence) GmPR1L of the pathogenesis-related protein 1 family was obtained, which has the ability to resist fungal diseases. The overexpression vector and interference expression vector were transferred into tobacco NC89, and the resistance of transgenic tobacco (Nicotiana tabacum L.) to Botrytis cinerea infection was identified. The results show that: Compared with the control, the activities of related defense enzymes SOD (Superoxide dismutase), POD (Peroxidase), PAL (L-phenylalanine ammonia-lyase) and PPO (Polyphenol oxidase) in the over-expressed transgenic tobacco OEA1 and OEA2 increased to different degrees, and increased significantly at different infection time points. The activities of defense enzymes in the interfering strains IEA1 and IEA2 were significantly lower than those in the control strains. The results of resistance level identification showed that the disease spot rate of OEA1 was significantly lower than that of the control line, and the disease spot rate of OEA2 was significantly lower than that of the control line. The plaque rate of the interfering expression line IEA1-IEA2 was significantly higher than that of the control line. It is preliminarily believed that the process related protein GmPR1L can improve the resistance of tobacco to B. cinerea.     

Induction of metal binding compounds and antioxidative defence in callus cultures of two black poplar (<Emphasis Type="Italic">P. nigra</Emphasis>) clones with different tolerance to cadmium

Callus cultures of two parental clones of Populus nigra L., Poli and 58-861, originating from contrasting environments, were exposed to different cadmium concentrations (0, 150 and 250 μM CdSO₄). Clones showed different growth responses to cadmium, evaluated by the tolerance index (Ti), with Poli being more tolerant to the metal at both concentrations. The cadmium concentration at the end of the treatment was very similar between clones at 150 μM CdSO₄, while a higher value in 58-861 compared to Poli was detected at 250 μM CdSO₄. The bioconcentration factor evidenced the lowest value in Poli at 250 μM CdSO₄. Unlike 58-861, cadmium provoked a strong induction of thiols and phytochelatins in clone Poli. In both clones, organic acid concentration differed notably in untreated calli and cadmium treatment induced a general lowering of these compounds. A notably higher antioxidant enzyme activity (ascorbate peroxidase, APX; catalase, CAT; guaiacol peroxidase, GPX) was measured in control calli of clone Poli compared to 58-861. Cadmium induced a remarkable enhancement of APX and CAT, but not GPX, activity at 150 μM CdSO₄ in Poli. Conversely, in 58-861 at 150 μM CdSO₄, and in both clones at 250 μM CdSO₄, a decrease in the antioxidant activity occurred. This investigation provided evidence that these two contrasting genotypes of P. nigra are characterised by a different response to cadmium in callus cultures. In particular, in Poli, the higher tolerance to cadmium is associated with a higher activity of antioxidative enzymes and the ability to strongly increase thiol and PC concentration in response to metal exposure.     

Novel avenues for passion fruit in vitro regeneration from endosperm culture,and morpho-agronomic and physiological traits of triploid Passiflora cincinnata Mast. emblings

The present study describes a new regeneration system based on somatic embryogenesis from mature endosperm Passiflora cincinnata Mast. cultures. Moreover, the morpho-agronomic and phenological traits, as well as enzymatic activity of regenerated triploid emblings are compared to those of diploids. Mature endosperms were cultured on Murashige and Skoog medium supplemented with various concentrations (4.5–45.2 µM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 μM 6-benzylaminopurine (BA). No plant growth regulators were included in the control group. Embryogenic calli were observed only in treatments supplemented with 13.6 and 18.1 µM 2,4-D?+?4.5 µM BA, with the highest number of somatic embryos per explant and regenerated plants (emblings) obtained with 18.1 µM 2,4-D. Most emblings (70%) were triploid (2n?=?3x?=?27), with a DNA amount (4.38 pg) similar to that of endosperm and 1.5 times greater than in diploid P. cincinnata seedlings (2n?=?2x?=?18), that contained 2.98 pg of DNA. While the number of organs and/or structures was akin to that in diploids, triploid emblings generally exhibited larger and longer vegetative and floral structures. The flower lifespan was also slightly altered by triploidy, nectar concentration was 27% higher, and the activity of plant defense enzymes β-1,3-glucanase and polyphenol oxidase was 29.8% and 22.1% higher. This study describes a new regeneration system for the production of phenotypic variants of this ornamental passion fruit species, opening new perspectives for future studies on genetic passion fruit breeding.

     

Regulation of ascorbate peroxidase activity in dark-grown radish cotyledons by a catalase inhibitor, 3-Amino-1,2,4-Triazole

The present study was performed to see the physiological role of cytosolic ascorbate peroxidase (APX) and its relationship to other enzymes involved in the H₂O₂ scavenging metabolism, and also to elucidate the regulation of APX expression in dark-grown radish (Raphanus sativus L. cv Taiwang) cotyledons. To do so, 3-amino-l,2,4-triazole (aminotriazole), a known specific inhibitor of catalase, was used to simulate a catalase-deficient phenomenon in cotyledons. Aminotriazole, in very low concetration (10^-4 M), inhibited remarkably the development of catalase activity in cotyledons during dark germination. This inhibition of catalase by aminotriazole, however, did not result in any significant changes in the growth response and the H₂O₂ level of developing cotyledons. In addition, the development of guaiacol peroxidase (GPX) activity was also not significantly affected. Unlike GPX, cytosolic APX activity was induced rapidly and reached a 1.7-fold increase in aminotriazole treated cotyledons at day 7 after germination. However,in vitro incubation of cytosolic APX preparation from cotyledons with aminotriazole did not result in any significant change in activity. One cytosolic APX isozyme (APXa) band involved in this APX activation was predominantly intensified in a native polyacrylamide gel by activity staining assay. This means that this APXa isozyme seems to play a key role in the expression of cytosolic APX activity. On the other hand, 2-day-old control seedlings treated with exogenous 1 mM H₂O₂ for 1 h showed a significant increase of cytosolic APX acitivity even in the absence of aminotriazole. Also, 2 μM cycloheximide treatment substantially inhibited the increase of APX activity due to aminotriazole. Based on these results, we suggest that a radish cytosolic APX could probably be substituted for catalase in H₂O₂ removal and that the expression of APX seems to be regulated by a change of endogenous H₂O₂ level which couples to APX protein synthesis in a translation stage in cotyledons.