Pisum Sativum Seedlings Research Articles

Caspase-like activity in the seedlings of Pisum sativum eliminates weaker shoots during early vegetative development by induction of cell death.

Activation of aspartate-specific cysteine proteases (caspases) plays a crucial role in programmed cell death (PCD) in animals. Although to date caspases have not been identified in plants, caspase-like activity was described in tobacco during a hypersensitive response to pathogens and in Arabidopsis and tomato cell cultures during chemical-induced PCD. Caspase-like activity was also detected in the course of plant development during petal senescence and endosperm PCD. It is shown here that caspase-like proteases play a crucial role in the developmental cell death of secondary shoots of pea seedlings that emerge after removal of the epicotyl. Caspase-like activity was induced in senescing secondary shoots, but not in dominant growing shoots, in contrast to the papain-like cysteine protease activity that was stronger in the dominant shoot. Revitalization of the senescing shoot by cutting of the dominant shoot reduced the caspase-like activity. Injection of caspase or cysteine protease inhibitors into the remaining epicotyl tissue suppressed the death of the secondary shoots, producing seedlings with two equal shoots. These results suggest that shoot selection in pea seedlings is controlled by PCD, through the activation of caspase-like proteases.

Nucleoside diphosphate kinase is a possible component of the ethylene signal transduction pathway.

In etiolated seedlings of Pisum sativum and leaves of Arabidopsis thaliana, in vivo ethylene treatment resulted in an increase in in vitro phosphorylation of 17 kD (P. sativum) or 16 and 17 kD (A. thaliana) polypeptides. These polypeptides were identified as nucleoside diphosphate kinase (NDPK) based on both biochemical properties and interaction with antibodies against NDPK from P. sativum. Using the receptor-directed antagonist of ethylene action 2,5-norbornadiene and the ethylene-insensitive mutants of A. thaliana etr1-1 and eti5, ethylene specificity and receptor dependence of NDPK phosphorylation have been demonstrated. In pea epicotyls, ethylene treatment also led to increase in nucleoside transferase activity unlike in A. thaliana leaves. The increases in nucleoside transferase activity and NDPK phosphorylation were very rapid and transient. The results suggest a role for NDPK as a possible component of the ethylene signal transduction chain.

Activities of amine oxidase, peroxidase and catalase in seedlings of Pisum sativum L. under different light conditions

The activities of amine oxidase, peroxidase and catalase were studied in 12 cultivars of field pea (Pisum sativum L.) and one accession of wild pea (Pisum sativum subsp. transcaucasicum). The influence of different light conditions on the enzyme activities was studied in extracts of 8-d-old seedlings. Substantially higher amine oxidase activity was detected in etiolated pea seedlings than in plants growing under controlled light conditions (12h photoperiod). Higher peroxidase and catalase activities indicate more intensive production of toxic hydrogen peroxide evolved by reactions of different type in green plants in comparison with etiolated ones. Significantly lower activity of peroxidases in etiolated plants could be related to a lower degree of lignification. Marked differences in enzyme activities between etiolated field pea and P. sativum subsp. transcaucasicum were observed for all studied enzymes. A very interesting result was the exceptionally low activity of amine oxidase in etiolated plants that was hardly detectable in green plants of Malton cultivar. Two bands with amine oxidase activity were found by the method of native PAGE in extracts of 8-d-old plants. A different relationship of these isoenzymes was detected in field pea and wild pea. Two isoenzymes were present in pea shoots but only one isoenzyme was detected in pea roots. Amine oxidase isoenzymes were studied in the roots and shoots of cv. Smaragd for three weeks. The profile of isoenzymes was opposite in 8- and 36-d-old stems of pea.

Application of capillary electrophoresis for DNA-protein binding tests

The combination of affinity chromatography and capillary electrophoresis (CE-SDS) has been found to be a useful tool to analyse populations of proteins which specifically bind to ssor dsDNA. Proteins were extracted from tissue, cytosol or nuclei of meristems of Pisum sativum seedlings and separated on cellulose column functionalized with ss-, dsDNA (calf thymus) and ssDNA (P. sativum) at 2M concentration of sodium chloride. Electropherograms of the crude protein extracts show two fractions of proteins specific for dsDNA (calf thymus) and three fractions specific for ssDNA (calf thymus). Four and five fractions of proteins specific for ssDNA (P. sativum) were identified in the material isolated from cytosolic and nuclear extracts, respectively. Both ds- and ssDNA (calf thymus) form complexes with ca. 4.0 % of the total amount of proteins, while ssDNA (P. sativum) binds to ca. 11.0 % of cytosolic and 5.0 % of nuclear proteins.

Sulphate uptake and xylem loading of young pea ( Pisum sativum L.) seedlings

Sulphate uptake and xylem loading was analysed in young pea (Pisum sativum) seedlings. The rate of sulphate uptake into intact 8-days-old pea seedlings (determined by a 1 h exposure to radiolabelled sulphate in the nutrient solution) was 585 nmol sulphate g−1 root fresh weight h−1. When the cotyledons were removed on day 6 the 8-days-old seedlings took up only 7% of the controls. Interruption of the phloem transport by steam girdling of the stem or the root (1 h before incubation with radiolabelled sulphate) diminished sulphate uptake by approximately 50%. The addition of sucrose to the nutrient solution during incubation did not restore sulphate uptake rates indicating that the decrease was not due to a lack of energy. Apparently, a signal from the shoot and/or the cotyledons is necessary to stimulate sulphate uptake into the roots of pea seedlings. Glutathione fed to the roots for 3 h prior to incubation with radiolabelled sulphate diminished sulphate uptake by approximately 50%. The relative proportion of the sulphate taken up that was loaded into the xylem remained unchanged (between 7 and 9% of total uptake), even when the stem was girdled above the cotyledons or when the seedlings were pre-exposed to glutathione. Only removal of the cotyledons or girdling of the root below the cotyledons increased the proportion of sulphate loaded into the xylem to 13–15% of total uptake upon exposure to glutathione. Apparently, a signal from the cotyledons represses xylem loading to some extent.

Influence of different natural physical fields on biological processes.

In space flight conditions gravity, magnetic, and electrical fields as well as ionizing radiation change both in size, and in direction. This causes disruptions in the conduct of some physical processes, chemical reactions, and metabolism in living organisms. In these conditions organisms of different phylogenetic level change their metabolic reactions undergo changes such as disturbances in ionic exchange both in lower and in higher plants, changes in cell morphology for example, gyrosity in Proteus (Proteus vulgaris), spatial disorientation in coleoptiles of Wheat (Triticum aestivum) and Pea (Pisum sativum) seedlings, mutational changes in Crepis (Crepis capillaris) and Arabidopsis (Arabidopsis thaliana) seedling. It has been found that even in the absence of gravity, gravireceptors determining spatial orientation in higher plants under terrestrial conditions are formed in the course of ontogenesis. Under weightlessness this system does not function and spatial orientation is determined by the light flux gradient or by the action of some other factors. Peculiarities of the formation of the gravireceptor apparatus in higher plants, amphibians, fish, and birds under space flight conditions have been observed. It has been found that the system in which responses were accompanied by phase transition have proven to be gravity-sensitive under microgravity conditions. Such reactions include also the process of photosynthesis which is the main energy production process in plants. In view of the established effects of microgravity and different natural physical fields on biological processes, it has been shown that these processes change due to the absence of initially rigid determination. The established biological effect of physical fields influence on biological processes in organisms is the starting point for elucidating the role of gravity and evolutionary development of various organisms on Earth.

Lithium-induced changes in gravicurvature, statocyte ultrastructure and calcium balance of pea roots.

Calcium signaling has been implicated in plant graviperception. In order to investigate the role of intracellular calcium in the process, I used lithium ions (LiCl), which suppress inositol 1,4,5-trisphosphate (IP3) cycling and signaling by inhibiting inositol-1-phosphatase. After 4 h of gravistimulation, no curvature was observed in 81% of the roots of 5-day Pisum sativum seedlings pretreated with 5 mM LiCl. Structural features of statocyte ultrastructure in these roots were the following: loss of a cellular polarity, appearance of amyloplast clusters, condensed mitochondria, local dilations in a perinuclear space, increases in a relative volume of vacuoles. The intensity of a cytochemical reaction (pyroantimonate staining which detected Ca2+ ions) was moderate: the Ca2+ pyroantimonate deposits were observed in all organelles. There were few granules of this precipitate in a hyaloplasm of the statocytes. Mitochondria and vacuoles were found to contain more granules of the precipitate compared with the controls. Additionally, Ca(2+)-ATPase activity in the statocytes of pea roots pretreated with LiCl was approximately the same as in control roots. Data obtained by using inhibitor of inositol signaling suggest that the observed effects of LiCl on root gravicurvature and ultrastructure of root statocytes were due to effects on Ca2+ homeostasis, particularly on IP3-mediated release of intracellular Ca2+ which can be inhibited by inositol depletion. The work demonstrates the key role played by second messengers (Ca2+ and IP3) in a gravity perception and response.

Gibberellin-enhanced Growth and Sugar Accumulation in Growing Subhooks of Etiolated Pisum sativum Seedlings: Effects of Actinomycin D on Invertase Activity, Soluble Sugars and Stem Elongation

Summary The possible involvement of soluble acid invertase in the action of gibberellic acid (GA) in stimulating sugar accumulation in relation to osmoregulation and growth was studied in growing subhooks of etiolated Alaska pea (Pisum sativum L. cv. Alaska). GA enhanced elongation growth, soluble sugar accumulation and invertase activity, which appears to be involved in phloem unloading, in the growing subhook. Actinomycin 0 inhibited the effects ofGA on invertase activity, accumulation of soluble sugars and elongation growth. Positive correlations were found between invertase activity, the amount of soluble sugars and elongation growth. These results suggest that GA-induced synthesis of mRNA seems to be involved in the enhancement of invertase activity which is responsible for sugar translocation from the phloem to the growing cells of the stem.

Cu I-semiquinone radical species in plant copper-amine oxidases

The intermediate Cu I-semiquinone radical species in the catalytic mechanism of copper-amine oxidase from Lens esculenta and Pisum sativum seedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped-flow and temperature-jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.

Photosynthetic activities of Pisum sativum seedlings grown in presence of cadmium

One-month-old pea seedlings ( Pisum sativum L. cv. Bonneville) raised in sand culture, were provided with a nutrient solution containing 0, 2.5, 5, 7.5 and 10 mM cadmium and the effect on various aspects of photosynthesis were investigated after 6 and 12 d of the treatment. The rate of photosynthesis, chlorophyll content, activities of photosystem I (PS I) and II (PS II) and a few selected photosynthetic enzymes viz. ribulose-1,5-bisphosphate carboxylase (EC 4.1.3.9), NADP-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13), fructose-1,6-bisphosphatase (EC 3.1.3.11) and NADP-malate dehydrogenase (EC 1.1.1.82) declined progressively with increasing concentration of applied cadmium. As compared to the other parameters such as per dm 2 leaf area, per mg fresh weight (FW) and per mg chlorophyll (chl), the rate of photosynthesis showed maximum decline on per plant basis. On extending the period of exposure to 12 d, the rate of photosynthesis and activities of enzymes showed a further decline. Cadmium had a more pronounced effect on the activity of PS II during the initial stages; however, on prolongation of the exposure for 12 d to the heavy metal, the functioning of PS I was also equally affected. Addition of cadmium to the chloroplasts isolated from untreated plants impaired the functioning of PS II without any discernible effect on that of PS I. In presence of 0.1 mM cadmium in the reaction mixture, activity of PS II was inhibited by 46 % whereas that of PS I remained unaffected. Compared to the effect on the rate of photosynthesis (per mg chl basis) and on photosystems, activities of photosynthetic enzymes showed far greater inhibition 12 d after the treatment.

Enhancement of polar auxin transport by cycloheximide in etiolated pea seedlings

Pretreatment of tissue with cycloheximide resulted in enhancement of [5- 3H]IAA transport in isolated stem segments of etiolated Pisum sativum seedlings. The cycloheximide effect was diminished if the antibiotic treatment was delayed after the segments were excised from the seedlings. Elimination of cycloheximide-exposed cut surfaces by having the segments shortened from both ends after cycloheximide pretreatment also abolished the enhancement of auxin transport by the protein synthesis inhibitor. The promotive effect of cycloheximide on auxin transport was fully manifested following pretreatment for 30 min, and this was maintained with pretreatment periods up to 4 h. Partial inhibition of auxin transport by non-saturating concentrations of NPA and TIBA was alleviated by cycloheximide pretreatment. Increases in [5- 3H]IAA net-uptake by NPA and TIBA in tissue slices were diminished by cycloheximide pretreatment. Implications for a possible wound inducible proteineous inhibitor of auxin transport are discussed.

Metabolic routes of β-(isoxazolin-5-on-2-yl)-l-alanine (bia), the precursor of the neurotoxin odap ( β-n-oxalyl-l- α, β,-diaminopropionic acid), in different legume seedlings

BIA, β-(isoxazolin-5-on-2-yl)-alanine, is formed in high concentrations in the seedling stage of many plants belonging to the tribe Vicieae. The metabolism of this compound in different legume seedlings has been studied by radioisotope feeding. In Lathyrus sativus, BIA is metabolised to the neurotoxin ODAP ( β- N-oxalyl- α, β-diaminopropionic acid) and the γ-glutamyl derivative of BIA. In L. odoratus seedlings BIA is metabolised to the decarboxylation product of the glutamyl derivative, and the glutamyl derivative is proposed as an intermediate. In Pisumsativum seedlings, the main part of BIA is not further metabolised. While in Lensculinaris seedlings, besides the formation of the glutamyl derivative, another four unknown amino acids were found. Approaches for the detoxification of Lathyrus sativus by genetic transformation are suggested.

Inhibitors of plant copper amine oxidases.

In this review, inhibitors of plant copper amine oxidases from Lens esculenta seedlings, Pisum sativum seedlings, and Euphorbia characias latex are described. Reversible competitive inhibitors and non-competitive inhibitors, irreversible active-site directed inhibitors and mechanism-based inactivators are reviewed in regard to their mechanisms of action.

Induction and ionic basis of slow wave potentials in seedlings of Pisum sativum L.

Slow wave potentials (SWPs) are transient depolarizations which propagate substantial distances from their point of origin. They were induced in the epidermal cells of pea epicotyls by injurious methods such as root excision and heat treatment, as well as by externally applied defined steps in xylem pressure (Px) in the absence of wounding. The common principle of induction was a rapid increase in Px. Such a stimulus appeared under natural conditions after (i) bending of the epicotyl, (ii) wounding of the epidermis, (iii) rewatering of dehydrated roots, and (iv) embolism. The induced depolarization was not associated with a change in cell input resistance. This result and the ineffectiveness of ion channel blockers point to H(+)-pumps rather than ion channels as the ionic basis of the SWP. Stimuli such as excision, heat treatment and pressure steps, which generate SWPs, caused a transient increase in the fluorescence intensity of epicotyls loaded with the pH-indicator DM-NERF, a 2',7'-dimethyl derivative of rhodol, but not of those loaded with the pH indicator 2',7'bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Matching kinetics of depolarization and pH response identify a transient inactivation of proton pumps in the plasma membrane as the causal mechanism of the SWP. Feeding pump inhibitors to the cut surface of excised epicotyls failed to chemically simulate a SWP; cyanide, azide and 2,4-dinitrophenol caused sustained, local depolarizations which did not propagate. Of all tested substances, only sodium cholate caused a transient and propagating depolarization whose arrival in the growing region of the epicotyl coincided with a transient growth rate reduction.

Control of Chenopodium album by soil application of Ascochyta caulina under greenhouse conditions

Summary.Effects of soil application of Ascochyta caulina spores on seedlings of Chenopodium album and five cultivated plant species were investigated under greenhouse conditions as a part of a study on biological control of C. album.Application of A. caulina spores to soils resulted in disease development on C. album and to a lesser degree on Spinacia oleracea seedlings, but not on Beta vulgaris subspecies vulgaris, Zea mays, Triticum aestivum and Pisum sativum seedlings. Affected C. album seedlings had an abnormal olive‐green colour or necrotic spots on cotyledons and hypocotyls, and were stunted or died. Affected S. oleracea seedlings were pale in colour or had necrotic spots on the cotyledons, but did not die. Time courses of disease incidence and of mortality of C. album could be described by a monomolecular model. Effects of spore density, sowing depth, soil water content, soil type and time of sowing on disease development were examined. Disease incidence and mortality were influenced by spore density, soil water content and soil type, but not by sowing depth. Spores in a moist soil maintained infectivity at least 2 wk. Spore densities of 109 to 1010 spores m‐2 were required for 50% mortality of emerged C. album plants.Aspects of the development of A. caulina into a soil‐applied mycoherbicide are discussed.

Gibberellin induces endopeptidase activity in detached cotyledons of Pisum sativum

Endopeptidase activity in cotyledons of 5-day seedlings of Pisum sativum increased rapidly during germination. However, the increase of the activity in detached cotyledons was depressed. We examined whether a growth regulator can be substituted for the embryonic axis on the development of endopeptidase activity. As monitored by an assay with azoalbumin, the development of endopeptidase activity from crude extracts of detached cotyledons appeared to be slightly accelerated by incubation with 10−5M GA3. However, the pattern after gelatin-polyacrylamide gel suggested that the activity induced in detached cotyledons during a 5-d incubation at 10−7M GA3 was the same as that in attached ones during germination for 5 days and an even greater increase in activity was obtained with 10−5M GA3. These results suggest that GA3 from the embryonic axis induces endopeptidase activity in attached cotyledons at the first stage of germination.

The influence of herbicides on the growth and proline content of primary roots and on the ultrastructure of root caps

The root tip of a germinating seed is usually the first organ of a plant to contact possible herbicide residues in the soil. Therefore, we examined electron microscopically the responses of the root caps of very young seedlings of Pisum sativum, Phaseolus vulgaris and Vicia faba to low concentrations of three frequently used herbicides, chlorsulfuron, norflurazon and triallate. Additionally, herbicide-induced increase of proline content and growth response of the roots were determined. All three herbicides caused changes in root cap ultrastructure; the outermost cell layer of the secretory tissue was destroyed, and the sloughing of cells from the root cap periphery was inhibited. Moreover, chlorsulfuron damaged amyloplasts in statocytes. Root growth was reduced by all three herbicides, but mostly by chlorsulfuron. The increase in proline content was highest in Pisum and Vicia after chlorsulfuron treatment, whereas triallate and norflurazon were less effective. Our results indicate that the root caps of germinating bean and pea seeds are highly sensitive to low concentrations of herbicides. The herbicide-induced growth reduction may be partially based on the injuries of the root caps described.

Pyrimidine metabolism and secondary product formation; biogenesis of albizziine, 4-hydroxyhomoarginine and 2,3-diaminopropanoic acid

In relation to the biogenesis of pyrimidine-derived secondary products, the relative activities and substrate specificities of the enzymes of pyrimidine metabolism were determined in extracts of seedlings from a number of leguminous species. Carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase) were 4 to 6 × more active, and 5′-nucleotidase and uridine hydrolase were 2 to 3 × more active in the secondary metabolite producing species examined. As it was also confirmed that UMP is the most effective nucleotide inhibitor of ATCase activity, these relatively high rates of hydrolysis of UMP to uracil would lessen effective negative feedback control of pyrimidine synthesis in the secondary metabolite producers and further increase the availability of uracil for secondary metabolism. The key enzyme of pyrimidine catabolism, NADPH-dependent dihydrouracil dehydrogenase was shown to use uracil, thymine, lathyrine and 5-aminouracil as substrates and its activity was 2 to 3 × higher in those species not yielding pyrimidine secondary products. This observation is consistent with the diversion, in the secondary product-forming plants examined, of significant amounts of uracil away from catabolism and into secondary product synthesis. The dihydrouracil dehydrogenase of Albizia julibrissin exhibited higher activity with 5-aminouracil as substrate than with uracil or thymine. Dihydropyrimidinase, the next enzyme in the catabolic pathway, was extracted from the various species and shown to use 5,6-dihydrouracil 5,6-dihydrothymine and 5-amino-5,6-dihydrouracil as substrates. Using a NADPH-regenerating system, the combined action of the two enzymes, dihydrouracil dehydrogenase and dihydropyrimidinase, was shown to produce 4-hydroxyhomoarginine from lathyrine. This was identified by co-chromatography and electrophoresis with authentic samples of 4-hydroxy-homoarginine. Preparations of β-ureidopropionase, the third enzyme in the catabolic pathway, were obtained from seedlings of Pisum sativum and shown to hydrolyse N-carbamoyl- β-alanine to 3-aminopropanoic acid, and N-carbamoyl-2-methyl-3-aminopropanoic acid to 3-amino-2-methylpropanoic acid. Additionally, the preparations from P. sativum and A. julibrissin hydrolysed albizziine to 2,3-diaminopropanoic acid, identified by co-chromatography and electrophoresís with authentic samples. It is concluded that there is a greater production and diversion of uracil into secondary product formation in the plants producing these compounds than in those that do not. In the non-producing plants, there is a more active catabolism of uracil. With A. julibrissin, degradation of 5-aminouracil by the pyrimidine catabolic process was shown to yield the non-protein amino acids albizziine and 2,3-diamino-propanoic acid. In Lathyrus tingitanus, this process demonstrably produces 4-hydroxyhomoarginine from lathyrine. The observations described are consistent with the view that production of pyrimidine-derived secondary products is attributable to the operation of detoxication processes for bioactive pyrimidines.

A Soluble High Affinity Auxin-Binding Protein from Pea Apex

Summary A soluble auxin-binding protein (ABP 44 ) from etiolated seedlings of Pisum sativum L. has been purified. It was detected in the apex but not in the basal, non-dividing parts of the pea epicotyls. Different set-ups of affinity chromatography were found to be powerful tools for the purification and characterization of ABP 44 . The determination of the binding kinetics was done using equilibrium dialysis binding tests. The modifications and improvements of the equilibrium binding assay, described previously (Reinard and Jacobsen, 1989), allowed the assignment of auxin binding activity to a purified soluble protein (ABP 44 ) with a dissociation constant of K D = 7.5 nM for the naturally occurring IAA. The data presented indicate that ABP 44 binds active auxins both with a high affinity and great specificity.

Quantitative analysis of gibberellins by isotope dilution mass spectrometry: A comparison of the use of calibration curves, an isotope dilution fit program and arithmetical correction of isotope ratios

AbstractThe quantitative analysis of gibberellin A1 (GA1) in apices of 19‐day‐old seedlings of Pisum sativum L. cultivar Alaska is described using isotope dilution mass spectrometry. Three methods are compared for determining the dilution of isotope in the internal standards, [17‐2H2]GA1 and [2β‐2H1]GA1. Similar results were obtained with each method. The use of calibration curves gave the most precise determination with [2H2]GA1 as internal standard irrespective of the amount added. The use of an isotope dilution fit program was more precise using [2H1]GA1 as internal standard. A simpler arithmetical correction procedure gave consistently higher estimates with both internal standards. The relative merits of the methods are discussed and the requirements for reliable quantification by isotope dilution gas chromatography‐mass spectrometry are presented.