Roots Of Pisum Sativum Research Articles

Effect of Severe Salt Stress on Respiratory and Biochemical Parameters in Legumes With Differential Nodulation Form

ABSTRACTLegumes are among the most utilised agronomic plant species due to their symbiotic association with N2‐fixing bacteria. Since N2 fixation entails high ATP cost, salt stress disrupts N2 fixation in the symbiont, but increases the production of osmolytes and antioxidant systems in the host plant. This results in competition for C allocation between osmoprotection in the host and continued supply to the symbiont for N acquisition, which may result in different plant responses to salinity. Two‐nodule types of plant species with contrasting carbon requirements for organic N2 fixation can be found within legume species; determinate and indeterminate. In this study, we tested responses of respiratory carbon metabolism, nitrogen assimilation and antioxidant machinery in leaves and roots of Phaseolus vulgaris (determinate nodules) and Pisum sativum (indeterminate nodules) 24 and 72 h after salt treatment (300 mM of NaCl). In P. sativum, we observed that nitrogenase activity was maintained at 24 h, but showed a strong decrease at 72 h together with cytochrome activity. On contrast, in P. vulgaris, respiration rates were maintained by an enhanced antioxidant activity under salinity although at the expense of nodule metabolism. Despite of the severity of the salt stress for N2 fixation, both species showed similar mechanisms to cope with salinity, like the maintenance of alternative respiration and increased antioxidant defence, that are worthy to be tested in the long term under field conditions.

Some biochemical characteristics of the hairy roots of Pisum sativum L. mutants

Two high-protein root cultures of vegetable pea mutants were received [1]. In continuation a PCR analysis of the obtained root cultures genes was carried out according [2] and the amino acid composition of the cultures protein was clarified in a dry product on the AAA 339TM device [3]. Obtained results confirmed the absence of rhizobia contamination of the cultures, which grow steadily on a hormone-free media for 5 years. PCR analysis revealed that fourrolgenesA,B,C,Dwere inserted into the genome of the root culture with genotypeafaftltl, and two —rol Candrol D— in the genome of the root culture with genotypetltl. The protein composition of the obtained cultures was represented by essential and non-essential amino acids and some others. In four inserts culture, the content of essential, ketogenic, proteinogenic and sulfur-containing amino acids prevailed by 1.5–2 times. Two inserts culture has twice as much aspartic acid and proline. Both cultures lacked tryptophan. The number of inserts determines the amino acid composition most likely.

Effect of Endophytic Bacteria Bacillus subtilis on Seedling Growth and Root Lignification of Pisum sativum L. under Normal and Sodium Chloride Salt Conditions

The influence of endophytic bacteria Bacillus subtilis (strain 10-4) was studied on the parameters of growth and tolerance as well as the intensity of lignin deposition in the roots of Pisum sativum L. seedlings under conditions of sodium chloride salinity (1% NaCl). It was found that the impact of salinity reduced the germination energy, viability, length of the roots and shoots of seedlings, their wet and dry weight, and also increased the content of proline and the level of lipid peroxidation (LPO). Pretreatment with strain 10-4 had a stimulating effect on seedlings in normal conditions and a protective effect on salinity, which was reflected in the improvement of germination energy and seed viability, root length, and accumulation of their dry mass under saline conditions; however, in terms of shoot growth under stress, there was no significant difference from the control (nonbacterized) variants. At the same time, strain 10-4 promoted earlier formation of lateral roots as well as a decrease in stress-induced LPO and proline content in seedlings, which indicates that cells are protected from oxidative and osmotic damage under saline conditions. Priority data were obtained on the important role of endophytic B. subtilis strain 10-4 in the process of lignification and strengthening of the barrier properties of the cell walls of the roots, which contributes to the reduction of the toxic effect of sodium chloride salinity and the implementation of the protective effect of these bacteria on pea plants.

Direct introduction MALDI FTICR MS based on dried droplet deposition applied to non-targeted metabolomics on Pisum Sativum root exudates

Non-targeted metabolomic approaches based on direct introduction (DI) through a soft ionization source are nowadays used for large-scale analysis and wide cover-up of metabolites in complex matrices. When coupled with ultra-high-resolution Fourier-Transform ion cyclotron resonance (FTICR MS), DI is generally performed through electrospray (ESI), which, despite the great analytical throughput, can suffer of matrix effects due to residual salts or charge competitors. In alternative, matrix assisted laser desorption ionization (MALDI) coupled with FTICR MS offers relatively high salt tolerance but it is mainly used for imaging of small molecule within biological tissues. In this study, we report a systematic evaluation on the performance of direct introduction ESI and MALDI coupled with FTICR MS applied to the analysis of root exudates (RE), a complex mixture of metabolites released from plant root tips and containing a relatively high salt concentration. Classic dried droplet deposition followed by screening of best matrices and ratio allowed the selection of high ranked conditions for non-targeted metabolomics on RE. Optimization of MALDI parameters led to improved reproducibility and precision. A RE desalted sample was used for comparison on ionization efficiency of the two sources and ion enhancement at high salinity was highlighted in MALDI by spiking desalted solution with inorganic salts. Application of a true lyophilized RE sample exhibited the complementarity of the two sources and the ability of MALDI in the detection of undisclosed metabolites suffering of matrix effects in ESI mode.

Biochemical and physiological characterizations of Rhizobium-Pea (Pisum sativum L.) symbiotic association under abiotic constraints

Pea (Pisum sativum L.) is an important leguminous for the agricultural sector. It is a source of biological nitrogen that efficiently contributes to the soil fertility. In Tunisia, low pea production is due to bad nitrogen management, lack of phosphorus availability and to the abiotic constraints. Thus, in order to improve the pea production ,a new farming technique involving the rhizobia inoculation was applied. The symbiotic, biochemical, physiological characterization and inoculation trials were performed in both the laboratory, greenhouse and open field. Pea Lincoln variety was used as legume species and fifteen Rhizobium strains isolated from the roots of the nodulated pea were collected from different Tunisian areas. Several physiological and biochemical parameters, i.e. pH, temperature, calcium carbonate and salinity were assessed to characterize the strains nodulating pea. All the rhizobia tests were evaluated on Yeast Extract Mannitol Agar medium (YEMA). Pea nodulation and Gallery API test were carried out under controlled conditions. Significant differences (p<0.01) between the nodules number induced by the different bacterial strains and between strains for the dry matter quantities of aerial and root parts were registered. The pH medium test results showed that among 15 strains only 8 strains having a halo diameter greater than 1 cm at basic pH. The most of isolates are able to grow at both low and high temperatures. The limestone test results qualify these rhizobia as calcifuges. Gallery API test results showed a great diversity of rhizobia assimilation of carbohydrates implying genetic diversity. Our results us to select the most efficient solubilizer Rhizobium strains nodulating pea. In order to confirm the previously cited notions on the diversity of Rhizobium strains isolated from Pisum sativum roots in Tunisia, inoculation trial with both selected strains in controlled and open field conditions confirmed the capacity of selected strains to fix atmospheric nitrogen and promote plant growth.

Calcium and Citrate Protect Pisum sativum Roots against Copper Toxicity by Regulating the Cellular Redox Status

Calcium and Citrate Protect Pisum sativum Roots against Copper Toxicity by Regulating the Cellular Redox Status

Metabolic Alterations in Pisum sativum Roots during Plant Growth and Arbuscular Mycorrhiza Development.

Intensive exchange of nutrients is a crucial part of the complex interaction between a host plant and fungi within arbuscular mycorrhizal (AM) symbiosis. For the first time, the present study demonstrates how inoculation with AMF Rhizophagus irregularis affects the pea (Pisum sativum L.) root metabolism at key stages of plant development. These correspond to days 21 (vegetation), 42 (flowering initiation), and 56 (fruiting-green pod). Metabolome profiling was carried out by means of a state-of-the-art GC-MS technique. The content shifts revealed include lipophilic compounds, sugars, carboxylates, and amino acids. The metabolic alterations were principally dependent on the stage of plant development but were also affected by the development of AM fungi, a fact which highlights interaction between symbiotic partners. The comparison of the present data with the results of leaf metabolome profiling earlier obtained did not reveal common signatures of metabolic response to mycorrhization in leaves and roots. We supposed that the feedback for the development and symbiotic interaction on the part of the supraorganismic system (root + AM fungi) was the cause of the difference between the metabolic profile shift in leaf and root cells that our examination revealed. New investigations are required to expand our knowledge of metabolome plasticity of the whole organism and/or system of organisms, and such results might be put to use for the intensification of sustainable agriculture.

Synchrotron Radiation Spectroscopy and Transmission Electron Microscopy Techniques to Evaluate TiO2 NPs Incorporation, Speciation, and Impact on Root Cells Ultrastructure of Pisum sativum L. Plants.

Biosolids (Bs) for use in agriculture are an important way for introducing and transferring TiO2 nanoparticles (NPs) to plants and food chain. Roots of Pisum sativum L. plants grown in Bs-amended soils spiked with TiO2 800 mg/kg as rutile NPs, anatase NPs, mixture of both NPs and submicron particles (SMPs) were investigated by Transmission Electron Microscopy (TEM), synchrotron radiation based micro X-ray Fluorescence and micro X-ray Absorption Near-Edge Structure (µXRF/µXANES) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). TEM analysis showed damages in cells ultrastructure of all treated samples, although a more evident effect was observed with single anatase or rutile NPs treatments. Micro-XRF and TEM evidenced the presence of nano and SMPs mainly in the cortex cells near the rhizodermis. Micro-XRF/micro-XANES analysis revealed anatase, rutile, and ilmenite as the main TiO2 polymorphs in the original soil and Bs, and the preferential anatase uptake by the roots. For all treatments Ti concentration in the roots increased by 38–56%, however plants translocation factor (TF) increased mostly with NPs treatment (261–315%) and less with SMPs (about 85%), with respect to control. In addition, all samples showed a limited transfer of TiO2 to the shoots (very low TF value). These findings evidenced a potential toxicity of TiO2 NPs present in Bs and accumulating in soil, suggesting the necessity of appropriate regulations for the occurrence of NPs in Bs used in agriculture.

The Effect of Cadmium on the Activity of Stress-Related Enzymes and the Ultrastructure of Pea Roots.

The analysis of the effects of cadmium (Cd) on plant cells is crucial to understand defense mechanisms and adaptation strategies of plants against Cd toxicity. In this study, we examined stress-related enzyme activities after one and seven days of Cd application and the ultrastructure of roots of Pisum sativum L. after seven days of Cd treatment (10, 50, 100, and 200 μM CdSO4). Our results showed that phenylalanine ammonia-lyase (PAL) activity and the amount of Cd accumulated in the roots were significantly positively correlated with the Cd concentration used in our experiment. However, Cd caused a decrease of all studied antioxidative enzyme activities (i.e., catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX)). The analysis of the ultrastructure (TEM) showed various responses to Cd, depending on Cd concentrations. In general, lower Cd concentrations (50 and 100 μM CdSO4) mostly resulted in increased amounts of oil bodies, plastolysomes and the accumulation of starch granules in plastids. Meanwhile, roots treated with a higher concentration of Cd (200 μM CdSO4) additionally triggered protective responses such as an increased deposition of suberin lamellae in the endodermal cell walls. This indicates that Cd induces a complex defense response in root tissues.

An integrated approach to highlight biological responses of Pisum sativum root to nano-TiO2 exposure in a biosolid-amended agricultural soil

This study focused on crop plant response to a simultaneous exposure to biosolid and TiO2 at micro- and nano-scale, being biosolid one of the major sink of TiO2 nanoparticles released into the soil environment. We settled an experimental design as much as possible realistic, at microcosm scale, using the crop Pisum sativum. This experimental design supported the hypotheses that the presence of biosolid in the farming soil might influence plant growth and metabolism and that, after TiO2 spiking, the different dimension and crystal forms of TiO2 might be otherwise bioavailable and differently interacting with the plant system. To test these hypotheses, we have considered different aspects of the response elicited by TiO2 and biosolid at cellular and organism level, focusing on the root system, with an integrative approach. In our experimental conditions, the presence of biosolid disturbed plant growth of P. sativum, causing cellular damages at root level, probably through mechanisms not only oxidative stress-dependent but also involving altered signalling processes. These disturbances could depend on non-humified compounds and/or on the presence of toxic elements and of nanoparticles in the biosolid-amended soil. The addition of TiO2 particles in the sludge-amended soil, further altered plant growth and induced oxidative and ultrastructural damages. Although non typical dose-effect response was detected, the most responsiveness treatments were found for the anatase crystal form, alone or mixed with rutile. Based on ultrastructural observations, we could hypothesise that the toxicity level of TiO2 nanoparticles may depend on the cell ability to isolate nanoparticles in subcellular compartments, avoiding their interaction with organelles and/or metabolic processes.The results of the present work suggest reflections on the promising practice of soil amendments and on the use of nanomaterials and their safety for food plants and living organisms.

Progression of Cell Wall Matrix Alterations during Aerenchyma Formation in Pisum sativum Root Cortical Cells

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Cycloheximide-induced phenolic burst in roots of Pisum sativum L.

Chromatography and histochemical analysis of soluble phenolic compounds demonstrated their higher content in the roots of cycloheximide-treated pea plants. These substances accumulated together with lignin in the endodermis and xylem cells of conducting bundles. This finding confirms the antipathogenic cycloheximide effect based on the previous results of proteome analysis.

Humic acid rich vermicompost promotes plant growth by improving microbial community structure of soil as well as root nodulation and mycorrhizal colonization in the roots of Pisum sativum

Humic acid rich vermicompost (HARV) was used to impart plant and soil health as compared to chemical fertilizers (CF), and normal vermicompost (NV) involving pea (Pisum sativum cv. Bonneville) as the host plant. The plant growth parameters of the crop were recorded at the time of harvesting and soil microbial structure was studied by using DGGE (Denaturing Gradient Gel Electrophoresis) on a temporal basis (0day, 12th day, 30th day and 60th day). The growth parameters showed an increase of 34.04%, 50.61% and 33.12% in total height, fresh weight, and dry weight as compared to conventional chemical fertilizers (CF) and 25.53%, 70.13% and 59.49% as compared to control. The DGGE profiles revealed that maximum bacterial and fungal diversity and density occur in soil supplemented with HARV on 12th day (both Shannon’s index and Margalef’s index showed highest bacterial diversity (2.87) and richness (5.52) and highest fungal diversity (2.76) and richness (4.96)). The lowest values for the indices (Margalef’s and Shannon for bacteria 2.51 and 1.78 and Margalef’s and Shannon for fungi 2.12 and 1.43 respectively) were observed in CF applied soils on the 60th day where diversity was even lower than in case of control soil indicating the negative impacts of chemical fertilizers on soil microbial diversities. Amendment of soil with organic fertilizers improved root nodulation and AMF colonization; the increment being higher in HARV. External application of AMF and Rhizobium further improved these parameters suggesting that AMF and Rhizobium act synergistically with HARV in improving plant growth and soil health.

Direct analysis of elemental biodistribution in pea seedlings by LA-ICP-MS, EDX and confocal microscopy: Imaging and quantification

Qualitative and quantitative methods were developed for cadmium, lead, cooper and zinc mapping and quantification in root of pea Pisum sativum L. Plants were cultivated hydroponically in a Hoagland medium with the addition of 50μM of: CdCl2, Pb(NO3)2, CuSO4, or ZnSO4. After 48h, parts of roots were cut and analysed. LA-ICP-MS was used for in vivo quantitative imaging of the distribution of metals in thin sections of plant tissues. Calibration curve was obtained using standard addition method NIST SRM 1515 Apple Leaves spiked with standard and application of sulfur isotope as an internal standard. Energy Dispersive X-ray microanalysis Spectroscopy (EDX) was used in order to confirm results obtained by LA-ICP-MS and identify the exact localization of metals deposits in root tissue. In the electron-microscopic photographs we observed deposits of metals in the cell wall, cell membrane, vacuoles, cytoplasm and organelles, such as: mitochondria and peroxisomes. Formation and localization of O2− and H2O2 in cross-sections of root tips was confirmed by confocal microscopy. The presence of metals induced ROS (reactive oxygen species: O2−, H2O2) production in areas corresponding to patterns of metals accumulation.

Modelling metal accumulation using humic acid as a surrogate for plant roots

Metal accumulation in roots was modelled with WHAM VII using humic acid (HA) as a surrogate for root surface. Metal accumulation was simulated as a function of computed metal binding to HA, with a correction term (EHA) to account for the differences in binding site density between HA and root surface. The approach was able to model metal accumulation in roots to within one order of magnitude for 95% of the data points. Total concentrations of Mn in roots of Vigna unguiculata, total concentrations of Ni, Zn, Cu and Cd in roots of Pisum sativum, as well as internalized concentrations of Cd, Ni, Pb and Zn in roots of Lolium perenne, were significantly correlated to the computed metal binding to HA. The method was less successful at modelling metal accumulation at low concentrations and in soil experiments. Measured concentrations of Cu internalized in L. perenne roots were not related to Cu binding to HA modelled and deviated from the predictions by over one order of magnitude. The results indicate that metal uptake by roots may under certain conditions be influenced by conditional physiological processes that cannot simulated by geochemical equilibrium. Processes occurring in chronic exposure of plants grown in soil to metals at low concentrations complicate the relationship between computed metal binding to HA and measured metal accumulation in roots.

Effect of morphactin (chlorfluorenol IT 3456) on the mitotic activity and cell growth in roots of Pisum sativum L.

Morphactin in a concentration of 100 ppm does not retard pea root growth, however, it reduced the frequency of cell division and shifted the main wave of mitoses in the 24 h period from 1 to 2 mm of root segment. The mean cell cycle duration is prolonged from 14 h in the control to 22 h in morphactin-treated roots. In the presence of morphactin the decrease of <sup>3</sup>H-thymidine incorporation and diminution of labelling index is accompanied by reduction of the nuclear surface area. The described changes are not accompanied by shortening of cell length. The results obtained suggest that morphactin disturbs the mechanisms regulating the initiation of S phase and its regular course. Moreover, it inhibits the endomitotic replication of DNA.

Effect of EDTA washing of metal polluted garden soils. Part II: Can remediated soil be used as a plant substrate?

In a field experiment on metal contaminated and EDTA-remediated soil we studied plant performance, mycorrhizal associations and prospects of potential re-use of remediated soil as a garden substrate. Two experimental plots of 4×1×0.3m were filled, one with remediated and the other with original contaminated soil. Selected cultivars were rotated over the course of 16months. Pb, Zn, Cd and micronutrient plant uptake was measured and their phytoaccessibility was analyzed by the DTPA method. Plant fitness was assessed by chlorophyll fluorescence and gas exchange measurements and evaluation of root colonization were analyzed with mycorrhizal fungi. Remediation reduced Pb and Cd concentrations in roots, green parts and fruits in most of the plants. Phytoaccumulation of Zn was reduced in one half of the cultivars. Some plants suffered from Mn deficiency as total soil Mn was reduced 4-fold and phytoaccessibility of micronutrients Cu, Fe and Mn for 54, 26 and 79%, respectively. Plant biomass was reduced. Photosynthetic parameters of plants grown in original and remediated soil were similar, except for the reduction in Spinacia oleracea. The frequency of mycorrhizal colonization in the roots of Pisum sativum was reduced five-fold and no significant changes were found in Allium cepa roots. Remediation reduced plant uptake of Pb below the concentration stipulated by legislation. Measures to reduce plant accumulation of other toxic metals and to revitalize remediated soil are needed.

Arabidopsis thaliana and Pisum sativum models demonstrate that root colonization is an intrinsic trait of Burkholderia cepacia complex bacteria.

Burkholderia cepacia complex (Bcc) bacteria possess biotechnologically useful properties that contrast with their opportunistic pathogenicity. The rhizosphere fitness of Bcc bacteria is central to their biocontrol and bioremediation activities. However, it is not known whether this differs between species or between environmental and clinical strains. We investigated the ability of 26 Bcc strains representing nine different species to colonize the roots of Arabidopsis thaliana and Pisum sativum (pea). Viable counts, scanning electron microscopy and bioluminescence imaging were used to assess root colonization, with Bcc bacteria achieving mean (±sem) levels of 2.49±0.23×10(6) and 5.16±1.87×10(6) c.f.u. per centimetre of root on the A. thaliana and P. sativum models, respectively. The A. thaliana rhizocompetence model was able to reveal loss of colonization phenotypes in Burkholderia vietnamiensis G4 transposon mutants that had only previously been observed in competition experiments on the P. sativum model. Different Bcc species colonized each plant model at different rates, and no statistical difference in root colonization was observed between isolates of clinical or environmental origin. Loss of the virulence-associated third chromosomal replicon (>1 Mb DNA) did not alter Bcc root colonization on A. thaliana. In summary, Bcc bacteria possess intrinsic root colonization abilities irrespective of their species or source. As Bcc rhizocompetence does not require their third chromosomal replicon, the possibility of using synthetic biology approaches to engineer virulence-attenuated biotechnological strains is tractable.

Effects of Fe Deficiency on Organic Acid Metabolism in Pisum sativum Roots

Iron deficiency induces several responses to iron shortage in plants. Metabolic changes occur to sustain the increased iron uptake capacity of Fe-deficient plants. The aim of this work was to investigate the impact of Fe deficiency on the organic acid metabolism in Pisum sativum roots. For this purpose, seedlings of Pisum sativum (cv. Douce) were grown under controlled conditions, in the presence of iron sufficient (C) or deficient (D) mediums. Our results showed that PEPC activity increased by 290% in root extracts of Fe deficient plants, compared to the control. Citrate concentration increased in Fe deficient Pisum roots (114% of the control). As well, MDH, CS and ICDH activities showed a marked increase in roots subjected to D treatment. However, the extent of stimulation was especially important for MDH and ICDH activities (99% and 150% of the control, respectively). These data suggest that the capacity of Douce cultivar to increase organic acid metabolism enzyme activities under iron deficiency is related to its better Fe-use efficiency, which indicate the tolerance level of this cultivar to iron chlorosis.

Hypoxic stress triggers a programmed cell death pathway to induce vascular cavity formation in Pisum sativum roots.

Flooding at warm temperatures induces hypoxic stress in Pisum sativum seedling roots. In response, some undifferentiated cells in the primary root vascular cylinder start degenerating and form a longitudinal vascular cavity. Changes in cellular morphology and cell wall ultrastructure detected previously in the late stages of cavity formation suggest possible involvement of programmed cell death (PCD). In this study, cytological events occurring in the early stages of cavity formation were investigated. Systematic DNA fragmentation, a feature of many PCD pathways, was detected in the cavity-forming roots after 3 h of flooding in situ by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and in isolated total DNA by gel electrophoresis. High molecular weight DNA fragments of about 20-30 kb were detected by pulse-field gel electrophoresis, but no low-molecular weight internucleosomal DNA fragments were detected by conventional gel electrophoresis. Release of mitochondrial cytochrome c protein into the cytosol, an integral part of mitochondria-dependent PCD pathways, was detected in the cavity-forming roots within 2 h of flooding by fluorescence microscopy of immunolabeled cytochrome c in situ and in isolated mitochondrial and cytosolic protein fractions by western blotting. DNA fragmentation and cytochrome c release remained confined to the undifferentiated cells in center of the root vascular cylinders, even after 24 h of flooding, while outer vascular cylinder cells and cortical cells maintained cellular integrity and normal activity. These findings confirm that hypoxia-induced vascular cavity formation in P. sativum roots involves PCD, and provides a chronological model of cytological events involved in this rare and understudied PCD system.