Pretreatment Of Roots Research Articles

CLSM-Guided Imaging for Quantifying Endodontic Disinfection.

Elimination of microbes in the root canal system is crucial for achieving long-term success in endodontic treatment. Further efforts in study design and standardization are needed in order to improve the validity and comparability of in vitro results on endodontic disinfection procedures, in turn improving clinical outcomes. This study optimizes two models at all steps: tooth selection, pretreatment, inoculation method (by growth or centrifugation), and confocal laser scanning microscopy (CLSM)-guided imaging of LIVE/DEAD-stained specimens. Individual anatomical conditions lead to substantial differences in penetration depth. Sclerosis grading (SCG), a classification system introduced in this study, provides information about the sclerosis status of the dentine and is helpful for careful, specific, and comparable tooth selection in in vitro studies. Sonically activated EDTA for the pretreatment of roots, inoculation of Enterococcus faecalis in an overflow model, 3-4 weeks of incubation, as well as polishing of dentine slices before staining, led to advances in the visualization of bacterial penetration and irrigation depths. In contrast, NaOCl pretreatment negatively affected performance reproducibility and should be avoided in any pretreatment. Nonsclerotized teeth (SCG0) can be used for microbial semilunar-shaped inoculation by centrifugation as a "quick-and-dirty" model for initial orientation. In conclusion, CLSM-guided imaging for quantifying endodontic infection/disinfection is a very powerful method after the fine-tuning of materials and methods.

Salicylic Acid Improves Cold Resistance of Solanum tuberosum Regenerants via Regulation of the Antioxidant System

Pretreatment of roots with salicylic acid (SA) and its role in control over morphophysiological parameters characterizing cold resistance of plant regenerants were examined in potato (Solanum tuberosum L.). It was found that potato plants exposed to low above-zero temperatures (4°C, 3 days) failed to restore initial growth parameters (plant height, leaf surface area, number of stolons, and weight of the organs) and oxidative status (intensity of lipid peroxidation) of the roots and leaves after transfer to favorable conditions (22°C, 10 days). A short-term (4-h-long) pretreatment of the roots with 0.1 μM SA minimized the adverse effect of deferred chilling. SA treatment promoted restoration of the initial plant phenotype after hypothermia, which caused a rise in growth parameters (number and surface area of the leaves and number of stolons) as compared with plants exposed to chilling without SA treatment. It was shown that pretreatment with SA improves cold resistance of potato plants owing to changes in the activity of antioxidant enzymes (superoxide dismutase and guaiacol-dependent peroxidase) and in the level of nonenzymatic antioxidants (ascorbic acid, anthocyans, total flavonoids and phenolic compounds). Depending on conditions, SA alters the ratio between individual flavonoids in the leaf. An ambiguous response to chilling and SA treatment was observed in two lines of potato regenerants produced by means of in vitro microcloning from the apical and middle part of the shoot, which is probably associated with their differing hormonal status.

Enhancing potato resistance against root-knot nematodes using a plant-defence elicitor delivered by bacteria

The root-knot nematode Meloidogyne chitwoodi is a pest that affects potato production in the Pacific Northwest of the United States. Here, to develop new strategies against M. chitwoodi infection of potato, we engineered Bacillus subtilis to secrete the plant-defence elicitor peptide StPep1. Pre-treatment of potato roots with the bacteria secreting StPep1 substantially reduced root galling, indicating that a bacterial secretion of a plant elicitor is an effective strategy for plant protection.

Development of novel processes for the aqueous extraction of natural rubber from Taraxacum kok‐saghyz (TK)

AbstractBACKGROUNDTaraxacum kok‐saghyz (TK) is a rubber‐producing plant being developed as a commercial alternative source of natural rubber (NR). The objective of this study was to investigate processes to extract high quality NR at high yield and purity from TK. Rubber is formed in the roots of TK and exists in mostly solid form. Previously used mechanical processes extract NR from TK roots at low purity (< 90%) that does not meet industry standards (≥ 99.8%). To improve this, the effects of root chopping, enzymatic hydrolysis, pebble milling and alkaline pretreatment and purification on NR yield, purity and quality were investigated.RESULTSEnzymatic digestion (with and without alkaline pretreatment), yielded rubber at the highest yield (∼80%) and purity (99.4%). Alkaline pretreatment of roots improved NR yield and did not adversely affect quality. More rubber was extractable from chopped roots than whole roots, but the rubber was less pure. Alkaline purification of the extracted crude rubber further increased rubber purity but reduced rubber molecular weight.CONCLUSIONEnzymatic digestion with or without alkaline pretreatment forms the basis of aqueous processes that could be used for large‐scale, efficient extraction of TK rubber at high yield, purity and quality to meet industry standards for NR. © 2019 Society of Chemical Industry

Formaldehyde assimilation through coordination of the glyoxylate pathway and the tricarboxylic acid cycle in broad bean roots

Formaldehyde (HCHO) assimilation in broad bean (Vicia faba L. cv. YD) roots was investigated using 13C-labeled HCHO followed by 13C-NMR analysis. Results revealed that H13CHO was first oxidized to H13COOH in the roots treated with 2 mM H13CHO in a time-dependent manner. Subsequently, a massive signal peak of [2, 4–13C]citrate (Cit) and a signal peak of [2, 3–13C]succinate (Su) were observed in accompany with an enhancement in the signal intensity of [3–13C]Cit. The data suggested that the glyoxylate pathway and the tricarboxylic acid (TCA) cycle functioned simultaneously in the subsequent assimilation of H13COOH. The yield of [2, 4–13C]Cit accounted for more than 80% of the total metabolites. The activity of isocitrate lyase (ICL), a key enzyme in the glyoxylate pathway, was stimulated by HCHO in a dosage-dependent manner. As a result, [2, 4–13C]Cit production was increased significantly in YD roots treated with high concentrations (4 and 6 mM) of H13CHO. Moreover, induction of the ICL activity by methanol resulted in a simultaneous elevation in the production of [2, 4–13C]Cit and [3–13C]Cit in methanol-pretreated roots under 2 mM H13CHO stress. Pretreatment of roots with cyclosporin A, which hinders the transport of 13C-enriched compounds into mitochondria, caused a notable decline in the signal peak and yield of [2, 4–13C]Cit and consequently induced a notable accumulation of [2, 3–13C]Su and an increase in the HCO3− production (generated from H13COOH oxidation) in H13CHO-treated roots. These results suggested that the glyoxylate pathway and the TCA cycle function coordinately in HCHO assimilation in broad bean roots.

DETERMINACIÓN DEL CARIOTIPO EN HELICONIAS NATIVAS DE MÉXICO

Heliconia es un género que agrupa a especies tropicales y subtropicales; se distribuye de manera natural desde Brasil hasta México; la vistosidad de su inflorescencia permite su uso con fines ornamentales. Estudios cariológicos en Heliconia reportan especies con 2n = 2x = 22, 2n = 2x = 24 y 2n = 3x = 36 cromosomas. Debido a que en las especies de México se desconoce el número y morfología cromosómica, el presente estudio tuvo por objetivo determinar el cariotipo de Heliconia uxpanapensis, endémica de México, H. latispatha y H. stricta cv. Iris. El pretratamiento de las raíces se realizó con un medio físico (4 ± 1 ºC). El genoma haploide de H. uxpanapensis mide 22.21 μm, el de H. latispatha 26.56 μm y el de H. stricta 25.37 μm. Las tres especies tienen 2n = 2x = 24 cromosomas, la fórmula cariotípica de H. uxpanapensis está representada por 2n = 8m + 10sm (2sat) + 6st, el de H. latispatha 2n = 4m + 14sm + 6st (2sat) y el de H. stricta 2n = 16m + 8sm. El primero y segundo par de cromosomas de H. latispatha y H. uxpanapensis, respectivamente, presentan satélites, mientras que en H. stricta la presencia del satélite se observó en un solo cromosoma del genoma. Este es el primer estudio en el género Heliconia que reporta la presencia de cromosomas subtelocéntricos.

Hydrogen peroxide pretreatment of roots protected tomato cells against copper induced oxidative damage and genotoxicity

Hydrogen peroxide pretreatment of roots protected tomato cells against copper induced oxidative damage and genotoxicity

Depletion of extracellular calcium increases cadmium toxicity in barley root tip via enhanced Cd uptake-mediated superoxide generation and cell death

Whereas severe Cd stress (150 µM Cd) causes root growth arrest as a consequence of marked superoxide generation leading to extensive cell death in the root tips, mild Cd stress (15 µM Cd) evokes morphogenic responses, such as reduced root elongation and radial root expansion, resulting in shorter and thicker roots. Similar to the low Cd concentration-caused mild stress, treatment of roots with either Ba to remove exchangeable or EDTA to remove both exchangeable and tightly bound cations, including Ca and Mg, from the apoplast, induced root growth inhibition and swelling. However, pre-treatment of roots with Ba had a synergistic effect on the development of these mild Cd stress-induced morphogenic responses, but without the development of any other symptoms in the root tips. In turn, EDTA pre-treatment markedly increased the toxicity of Cd in barley root tips via enhanced Cd uptake-mediated superoxide generation, which evoked extensive cell death in the transition zone of root tips identically to the high Cd concentration-induced severe stress. While the mild stress-induced responses were alleviated by the inhibition of auxin signalling pathway, the severe stress-induced symptoms were prevented by Ca, but not Mg, supplementation or by the inhibition of Cd uptake into the root symplasm. Therefore, the appropriate concentration of Ca in the apoplast is crucial to prevent the rapid accumulation of Cd in the symplasm, which above a certain threshold level leads to the huge superoxide generation and cell death.

Roles of sodium hydrosulfide and sodium nitroprusside as priming molecules during drought acclimation in citrus plants.

Emerging evidence suggests that the gaseous molecules hydrogen sulfide (H2S) and nitric oxide (NO) enhances plant acclimation to stress; however, the underlying mechanism remains unclear. In this work, we explored if pretreatment of citrus roots with NaHS (a H2S donor) or sodium nitroprusside (SNP, a NO donor) for 2days (d) could elicit long-lasting priming effects to subsequent exposure to PEG-associated drought stress for 21d following a 5d acclimation period. Detailed physiological study documented that both pretreatments primed plants against drought stress. Analysis of the level of nitrite, NOx, S-nitrosoglutahione reductase, Tyr-nitration and S-nitrosylation along with the expression of genes involved in NO-generation suggested that the nitrosative status of leaves and roots was altered by NaHS and SNP. Using a proteomic approach we characterized S-nitrosylated proteins in citrus leaves exposed to chemical treatments, including well known and novel S-nitrosylated targets. Mass spectrometry analysis also enabled the identification of 42 differentially expressed proteins in PEG alone-treated plants. Several PEG-responsive proteins were down-regulated, especially photosynthetic proteins. Finally, the identification of specific proteins that were regulated by NaHS and SNP under PEG conditions provides novel insight into long-term drought priming in plants and in a fruit crop such as citrus in particular.

Control of tomato bacterial wilt and root-knot diseases by Bacillus thuringiensis CR-371 and Streptomyces avermectinius NBRC14893

Ralstonia solanacearum and Meloidogyne incognita are two soilborne pathogens that cause serious damage and great losses in the production of tomato. For this purpose, a bacterial isolate, Bacillus thuringiensis CR-371, and an actinomyces isolate, Streptomyces avermectinius NBRC14893, were examined for their ability to protect tomato from root-knot nematode and bacterial wilt diseases under glasshouse conditions. Treatment of tomato roots with B. thuringiensis CR-371 and S. avermectinius NBRC14893 followed by challenge inoculation with R. solanacearum and M. incognita significantly decreased disease severity of bacterial wilt alone, root-knot nematode alone, or mixed infection by both pathogens compared to the control. Furthermore, pretreatment of tomato roots with B. thuringiensis CR-371 and S. avermectinius NBRC14893 significantly reduced bacterial proliferation of R. solanacearum both in pathogen alone inoculated plants and in plants co-inoculated with R. solanacearum and M. incognita. In conclusion, our results suggest that the treatment of tomato roots with B. thuringiensis CR-371 and S. avermectinius NBRC14893 simultaneously suppresses bacterial wilt and root-knot nematode diseases. Therefore, B. thuringiensis CR-371 and S. avermectinius NBRC14893 could provide new options for integrated pest management strategies against plant diseases, especially against bacterial-nematode disease complexes that cause synergistic yield losses.

Influence of abscisic acid and fluridone on the content of phytohormones and polyamines and the level of oxidative stress in plants of Mesembryanthemum crystallinum L. under salinity

The effect of abscisic acid (ABA) and fluridone on the content of endogenous phytohormones and free polyamines and the intensity of oxidative stress was studied in plants of Mesembryanthemum crystallinum L. under salinity. It was shown that the pretreatment of plant roots with 1 μM ABA, followed by the action of 300 mM NaCl, caused a protective effect and improved the physiological state of the plants, which was manifested in increased biomass and content of available cytokinins and reduced values of the indicators of oxidative stress. It was noted that the inhibitor fluridone reduced the effect of ABA and acted as a pro-oxidant.

Ralstonia solanacearum Requires PopS, an Ancient AvrE-Family Effector, for Virulence and To Overcome Salicylic Acid-Mediated Defenses during Tomato Pathogenesis

ABSTRACTDuring bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression of popS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in the R. solanacearum species complex. The phylogeny of popS mirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. A popS mutant of R. solanacearum UW551 had reduced virulence on agriculturally important Solanum spp., including potato and tomato plants. However, the popS mutant had wild-type virulence on a weed host, Solanum dulcamara, suggesting that some species can avoid the effects of PopS. The popS mutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, the popS mutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated the popS mutant virulence defect. Finally, the popS mutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which are R. solanacearum’s natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed in Nicotiana leaf tissue, unlike the AvrE homolog DspEPcc from the necrotroph Pectobacterium carotovorum subsp. carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophic R. solanacearum.

Nitrogen-Dependent Regulation of De Novo Cytokinin Biosynthesis in Rice: The Role of Glutamine Metabolism as an Additional Signal

Cytokinin activity in plants is closely related to nitrogen availability, and an Arabidopsis gene for adenosine phosphate-isopentenyltransferase (IPT), IPT3, is regulated by inorganic nitrogen sources in a nitrate-specific manner. In this study, we have identified another regulatory system of cytokinin de novo biosynthesis in response to nitrogen status. In rice, OsIPT4, OsIPT5, OsIPT7 and OsIPT8 were up-regulated in response to exogenously applied nitrate and ammonium, with accompanying accumulation of cytokinins. Pre-treatment of roots with l-methionine sulfoximine, a potent inhibitor of glutamine synthetase, abolished the nitrate- and ammonium-dependent induction of OsIPT4 and OsIPT5, while glutamine application induced their expression. Thus, neither nitrate nor ammonium, but glutamine or a related metabolite, is essential for the induction of these IPT genes in rice. On the other hand, glutamine-dependent induction of IPT3 occurs in Arabidopsis, at least to some extent. In transgenic lines repressing the expression of OsIPT4, which is the dominant IPT in rice roots, the nitrogen-dependent increase of cytokinin in the xylem sap was significantly reduced, and seedling shoot growth was retarded despite sufficient nitrogen. We conclude that plants possess multiple regulation systems for nitrogen-dependent cytokinin biosynthesis to modulate growth in response to nitrogen availability.

The Mutualistic Fungus Piriformospora indica Protects Barley Roots from a Loss of Antioxidant Capacity Caused by the Necrotrophic Pathogen Fusarium culmorum

Fusarium culmorum causes root rot in barley (Hordeum vulgare), resulting in severely reduced plant growth and yield. Pretreatment of roots with chlamydospores of the mutualistic root-colonizing basidiomycete Piriformospora indica (subdivision Agaricomycotina) prevented necrotization of root tissues and plant growth retardation commonly associated with Fusarium root rot. Quantification of Fusarium infections with a real-time polymerase chain reaction assay revealed a correlation between root rot symptoms and the relative amount of fungal DNA. Fusarium-infected roots showed reduced levels of ascorbate and glutathione (GSH), along with reduced activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, GSH reductase, dehydroascorbate reductase, and monodehydroascorbate reductase. Consistent with this, Fusarium-infected roots showed elevated levels of lipid hydroperoxides and decreased ratios of reduced to oxidized forms of ascorbate and GSH. In clear contrast, roots treated with P. indica prior to inoculation with F. culmorum showed levels of ascorbate and GSH that were similar to controls. Likewise, lipid peroxidation and the overall reduction in antioxidant enzyme activities were largely attenuated by P. indica in roots challenged by F. culmorum. These results suggest that P. indica protects roots from necrotrophic pathogens, at least partly, through activating the plant's antioxidant capacity.

Hydrogen sulfide induces systemic tolerance to salinity and non-ionic osmotic stress in strawberry plants through modification of reactive species biosynthesis and transcriptional regulation of multiple defence pathways

Hydrogen sulfide (H2S) has been recently found to act as a potent priming agent. This study explored the hypothesis that hydroponic pretreatment of strawberry (Fragaria × ananassa cv. Camarosa) roots with a H2S donor, sodium hydrosulfide (NaHS; 100 μM for 48h), could induce long-lasting priming effects and tolerance to subsequent exposure to 100mM NaCI or 10% (w/v) PEG-6000 for 7 d. Hydrogen sulfide pretreatment of roots resulted in increased leaf chlorophyll fluorescence, stomatal conductance and leaf relative water content as well as lower lipid peroxidation levels in comparison with plants directly subjected to salt and non-ionic osmotic stress, thus suggesting a systemic mitigating effect of H2S pretreatment to cellular damage derived from abiotic stress factors. In addition, root pretreatment with NaHS resulted in the minimization of oxidative and nitrosative stress in strawberry plants, manifested via lower levels of synthesis of NO and H2O2 in leaves and the maintenance of high ascorbate and glutathione redox states, following subsequent salt and non-ionic osmotic stresses. Quantitative real-time RT-PCR gene expression analysis of key antioxidant (cAPX, CAT, MnSOD, GR), ascorbate and glutathione biosynthesis (GCS, GDH, GS), transcription factor (DREB), and salt overly sensitive (SOS) pathway (SOS2-like, SOS3-like, SOS4) genes suggests that H2S plays a pivotal role in the coordinated regulation of multiple transcriptional pathways. The ameliorative effects of H2S were more pronounced in strawberry plants subjected to both stress conditions immediately after NaHS root pretreatment, rather than in plants subjected to stress conditions 3 d after root pretreatment. Overall, H2S-pretreated plants managed to overcome the deleterious effects of salt and non-ionic osmotic stress by controlling oxidative and nitrosative cellular damage through increased performance of antioxidant mechanisms and the coordinated regulation of the SOS pathway, thus proposing a novel role for H2S in plant priming, and in particular in a fruit crop such as strawberry.

Hydrogen peroxide pretreatment of roots enhanced oxidative stress response of tomato under cold stress

In the view of physiological role of H2O2, we investigated whether exogenous H2O2 application would affect short-term cold response of tomato and induce acclimation. Pretreatments were performed by immersing roots into 1 mM H2O2 solution for 1 h when transferring seedlings from seedling substrate to soil (acclimated group). Cold stress (3 °C for 16 h) caused significant reduction in relative water content (RWC) of control and non-acclimated (distilled water treated) groups when compared with unstressed plants. H2O2 promoted maintenance of relatively higher RWC under stress. Anthocyanin level in leaves of acclimated plants under cold stress was significantly higher than that of unstressed control and non-acclimated plants. Malondialdehyde (MDA) levels demonstrated low temperature induced oxidative damage to control and non-acclimated plants. MDA remained around unstressed conditions in acclimated plants, which demonstrate that H2O2 acclimation protected tissues against cold induced lipid peroxidation. H2O2 acclimation caused proline accumulation in roots under cold stress. Ascorbate peroxidase (APX) activity in roots of cold stressed and unstressed H2O2 acclimated plants increased when compared with control and non-acclimated plants, with highest increase in roots of acclimated plants under cold stress. CAT levels in roots of acclimated plants also increased, whereas levels remained unchanged in unstressed plants. Endogenous H2O2 levels significantly increased in roots of control and non-acclimated plants under cold stress. On the other hand, H2O2 content in roots of acclimated plants was significantly lower than control and non-acclimated plants under cold stress. The results presented here demonstrated that H2O2 significantly enhanced oxidative stress response by elevating the antioxidant status of tomato.

Interaction of Extracellular Proteinases of Phytophthora cinnamomi with Proteinase Inhibitors Produced by Avocado Root

The interaction avocado plant/Phytophtora cinnamomi has not been explored at the level of defense responses induced by this pathogen. Here, we show that the infection of avocado plants by Phytophthora cinnamomi induced proteinase inhibitor, which had inhibitor activity against extracellular proteases of P. cinnamomi. Extracellular proteinase activity was not increased with the addition of the avocado root cell wall to liquid culture but decreased in the presence of root of healthy avocado plants. The pretreatment of avocado roots with the avocado inhibitor before the infection by P. cinnamomi inhibited root colonization by the pathogen. Avocado inhibitor had no effect on in vitro growth of the oomycete, suggesting that the inhibition of root colonization could be due to its interaction with pathogen proteinases.

Early signalling pathways in rice roots under vanadate stress

Vanadate is beneficial to plant growth at low concentration. However, plant exposure to high concentrations of vanadate has been shown to arrest cell growth and lead to cell death. We are interested in understanding the signalling pathways of rice roots in response to vanadate stress. In this study, we demonstrated that vanadate induced rice root cell death and suppressed root growth. In addition, we found that vanadate induced ROS accumulation, increased lipid peroxidation and elicited a remarkable increase of MAPKs and CDPKs activities in rice roots. In contrast, pre-treatment of rice roots with ROS scavenger (sodium benzoate), serine/threonine protein phosphatase inhibitor (endothall), and CDPK antagonist (W7), reduced the vanadate-induced MAPKs activation. Furthermore, the expression of a MAPK gene (OsMPK3) and four tyrosine phosphatase genes (OsDSP3, OsDSP5, OsDSP6, and OsDSP10) were regulated by vanadate in rice roots. Collectively, these results strongly suggest that ROS, protein phosphatase, and CDPK may function in the vanadate-triggered MAPK signalling pathway cause cell death and retarded growth in rice roots.

Impact of Pseudomonads and Ethylene on the Cadmium and Nickel Rhizofiltration of Sunflower, Squash, and Indian Mustard

Three hydroponic experiments were set up to study the rhizofiltration of cadmium (Cd) or nickel (Ni) from artificially contaminated nutrient solution with sunflower, squash, or Indian mustard. After 48 h of exposure with 2 mg L−1 Cd‐contaminated water, 460, 415, or 1092 µg Cd g−1 (dry weight) was detected in roots of 33‐day‐old sunflower and squash or in 50‐day‐old Indian mustard, respectively. As calculated, 1 g of root dry matter of the tested crop species removed 5.7–12.4% of total Cd content present in the nutrient solution. It was supposed that pseudomonads (soil rhizoplane bacteria) and the plant growth hormone ethylene can enhance the specific surface of roots and hence roots' metal adsorption capacity. As a trend, pretreatment of Indian mustard with Pseudomonas fluorescens bacteria enhanced slightly the Cd (from 1793 to 2346 µg g−1) or Ni (from 1088 to 1192 µg g−1) concentration of roots. Cadmium concentration in roots was also enhanced from 2694 to 3273 µg g−1 when the roots of Indian mustard were pretreated with Cd‐tolerant rather than Cd‐sensitive Pseudomonas cepacia. In spite of the occurrence of new root hairs, the pretreatment of roots with ethylene proved to be ineffective in enhancement of the Cd rhizofiltration capacity of Indian mustard.

ROS and CDPK-like kinase-mediated activation of MAP kinase in rice roots exposed to lead

Lead (Pb2+) is a cytotoxic metal ion in plants, the mechanism of which is not yet established. The aim of this study is to investigate the signalling pathways that are activated by elevated concentrations of Pb2+ in rice roots. Root growth was stunted and cell death was accelerated when exposed to different dosages of Pb2+ during extended time periods. Using ROS-sensitive dye and Ca2+ indicator, we demonstrated that Pb2+ induced ROS production and Ca2+ accumulation, respectively. In addition, Pb2+ elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot and immunoprecipitation analysis, 40- and 42-kDa MBP kinases that were activated by Pb2+ were identified to be mitogen-activated protein (MAP) kinases. Pre-treatment of rice roots with an antioxidant and a NADPH oxidase inhibitor, glutathione (GSH) and diphenylene iodonium (DPI), effectively reduced Pb2+-induced cell death and MAP kinase activation. Moreover, calcium-dependent protein kinase (CDPK) antagonist, W7, attenuated Pb2+-induced cell death and MAP kinase activation. These results suggested that the ROS and CDPK may function in the Pb2+-triggered cell death and MAP kinase signalling pathway in rice roots.