Studies upon the copper fungicides: VII. The solution of copper from dressings on the pea seed

During the process of swelling pea seeds yielded substances to water which were capable of dissolving the insoluble copper dressings normally applied to the seed to prevent 'pre-emergence damping off'. This occurred under conditions in which soil factors were eliminated and irrespective of whether the dressing was on the surface of the seed or treated separately. Copper was readily taken into solution from dried 4:4:50 Bordeaux Mixture by water in which peas had been soaked, a circumstance which proved that the formation of soluble complex forms of copper was involved.
A chemical investigation on the solution obtained by soaking peas in water showed that volatile acids or their salts were present together with a protein-like material in colloidal solution. The occurrence of amino derivatives and other nitrogenous materials as well as carbohydrates was indicated and evidence of the presence of asparagine and citrate was also obtained.
The suggestion is made that both the fungicidal and phytocidal actions of insoluble copper dressings on pea seed depend largely on the effect of the pea seed exudates, notably colloidal protein-like material and certain amino derivatives, in bringing copper into solution.     

Leakage during Seed Imbibition

When pea embryos are placed in water solutes leak from themat a rate which declines rapidly at first and then more slowly.Embryos can be dried down over calcium chloride and will thenleak as before when returned to water. Similar results wereobtained with Ricinus seeds after removal of the testa. Pea embryos that have first been allowed to imbibe some waterthrough a small part of their surface (by placing them on dampfilter-paper) leak relatively slowly when subsequently immersedin water; the greater the initial imbibition the slower thesubsequent leakage. Likewise, embryos taken from peas that wereharvested when succulent and tender show only slow leakage It is proposed that as seeds dry out in the course of developmentcell membranes lose their integrity. When such dry seeds areallowed to imbibe water there is a short period, before membraneintegrity is re-established, during which solutes can leak outof the cells.     

An Analysis of Seed Development in Pisum sativum: VI. ABSCISIC ACID ACCUMULATION

The abscisic acid (ABA) content of wrinkled (rr) pea seed tissueshas been quantified during development using multiple-ion-monitoringcombined gas chromatography-mass spectrometry and a deuteratedinternal standard. The level of ABA in the embryo generallyincreased with increasing cotyledon fresh weight while thatin the testa showed a distinct maximum at the time of maximumendosperm volume and the slowing in the growth of the testa.Pods contained relatively little ABA on a fresh weight basis.The total seed ABA content showed a biphasic distribution, thefirst maximum following the maximum growth rate of the testaand the second that of the embryo. The biphasic distributionof ABA in the pea seed was confirmed using a second pea genotype,near-isogenic to the first except for the r locus, and by theanalysis of individual seeds using a radioimmunoassay for ABA.The first maximum was composed mainly of a testa component andthe second mainly of an embryo component. When plants were grownin different environments, wrinkled seeds were found to containslightly more ABA than round (RR) but this was only significantlate in development. Immature seeds were capable of metabolizing17'-deoxy ABA to ABA, as determined by incorporation of either³H or ²H, and the metabolite was present mainly in the testa.The production of ABA in pea seeds is discussed in relationto the development of the different seed tissues. Key words: Abscisic acid, peas, seed development     

Transport of copper and manganese to the xylem exudate of sunflower

Abstract Absorption of copper and manganese by sunflower roots from solution cultures of varying composition was followed by measuring the concentrations of the metals appearing in whole roots, root cell sap and xylem exudate. Total copper in the fibrous roots was linearly related to the concentration of copper in the external solution but the concentration of copper released to the xylem exudate was buffered somewhat against the changes made externally. No such buffering was observed for managenese. A copper-sensitive electrode, responsive only to free cupric ions was used in conjunction with total copper analysis by atomic absorption spectrophotometry to show that little of the copper (usually < 1%) existed as a free ion in any phase of the system. Copper in the xylem exudate may be strongly complexed. An electron paramagnetic resonance spectrum of the xylem exudate indicated that manganese probably was a free divalent ion. Calculation of the electrochemical potential gradient for free cupric ions showed that no special metabolically-linked mechanism need be postulated to account for absorption of copper (or manganese) other than that necessary to maintain the transmembrane potential.     

The Importance of Genetically Determined Seed Coat Characteristics to Seed Quality in Grain Legumes

Comparisons of five pairs of isogenk lines of peas, differingonly in the A gene for seed coat colour showed that white seeds(genotype aa) imbibed more rapidly than coloured seeds (AA),suffered greater imbibition damage revealed by dead tissue onthe cotyledons, and higher solute leakage. Seed-coat pigmentationwas closely associated with slow water uptake, since when expressionof the A gene was suppressed by the recessive pollens gene,the resulting white seeds {palpal AA) imbibed rapidly. The slowwater uptake by coloured seeds was not due to the restrictionof water entry by the seed coat since the differences in imbibitionrate were maintained when a portion of the seed coat was removedand seeds were imbibed with the exposed cotyledon in contactwith moist filter paper. Imbibition of similarly treated seedsby immersion in polyethylene glycol solutions (1–4%) whichincreased the seed/solution wettability, had little effect onthe water uptake of coloured seeds compared to imbibition inwater whereas that of white seeds increased in the first 10mins imbibition. Poor wettability of the inner surface of colouredseed coats did not therefore explain the slow imbibition ofthese seeds. The white seed coats loosened rapidly during imbibitionwhilst the coloured seed coats remained closely associated withthe cotyledons suggesting that the adherence of the seed coatto the cotyledons and therefore the ease of access of waterbetween the testa and cotyledons determines the rate of imbibition.The rapid water uptake by white-coated seeds and the subsequentimbibition damage may explain the high incidence of infectionof these seeds by the soil-bome fungus Pythhan after 2 d insoil. Improved seed quality and emergence may therefore be achievedby breeding for seed coat characteristics leading to reducedrates of imbibition Pisum sativum, isogenic lines, A gene, seed coat colour, imbibition, imbibition damage, wettability, pollens gene, seed quality, grain legumes     

The Damaging Effect of Water on Dry Pea Embryos During Imbibition

When pea seeds were imbibed in water without their seed coats,vital staining revealed that cells on the abaxial surface ofthe cotyledons were dead. No damage occurred on the surfaceof cotyledons when the seeds were imbibed intact, or beneaththe testa when only half of the testa was removed. Cell deathoccurred as a result of rapid water uptake within the first2 min of imbibition, since reducing the rate of imbibition insolutions of Carbowax 4000 lessened the damage. Cell death wasrestricted to the outer layers of the cotyledons; inner tissuesremained alive. These observations supported the hypothesisthat rapid early leakage during imbibition of dry embryos resultedfrom the death of cells caused by the physical disruption ofmembranes. Imbibition damage resulted in reduced respirationand germination, a decline in the rate of food reserve transferfrom the cotyledons to the growing axis, and a lower growthrate in the seedlings produced. Greater sensitivity of embryosto imbibition damage at low temperature, and similarities betweenfeatures of imbibition damage and chilling injury led to thesuggestion that so-called chilling injury is the result of imbibitiondamage rather than the effects of low temperature.     

Evaluation of copper speciation and water quality factors that affect aqueous copper tasting response

This study determined taste thresholds for copper as its speciation was varied among free cupric ion, complexed cupric ion, and precipitated cupric particles. The impact of copper chemistry on taste is important as copper is added to many beverages and can be present in drinking water as a natural mineral or due to corrosion of copper plumbing. A one-of-five test was used to define thresholds with solutions containing 0.025-8 mg/l Cu (from copper sulfate) in distilled or mineralized water of varying pH. The mineralized water was designed to mimic the composition of a typical tap water. Group thresholds for copper in either distilled-deionized water or mineralized water were not significantly different and ranged from 0.4 to 0.8 mg/l Cu. A difference from control test was used to assess the impact of soluble and particulate copper on taste. Soluble copper species, including free cupric ion and complexed copper species, were readily tasted, while particulate copper was poorly tasted.     

Oxidative Processes in Soybean and Pea Seeds: Effect of Light, Temperature, and Water Content

Oxidative processes are probable determinants of longevity of seeds in storage. Measurements of actual oxygen uptake rates were made for soybean and pea seeds as a comparison of short and long lived seeds when light, temperature, and moisture contents were varied. In both peas and soybeans, the oxygen uptake was depressed at low temperatures (<16°C) and low water contents (<0.25 gram H₂O per gram dry weight). Apparent activation energies under these conditions are very high, while apparent activation energies of seeds at higher water contents and at temperatures greater than 22°C are much less. Light enhances the level of oxygen uptake in pea, but reduces the level of oxygen uptake in soybean. The complexities of the interactions of oxygen uptake with environmental conditions in soybean compared to pea suggest that oxidative processes occur in soybean at low water contents, but are essentially absent in pea. It is suggested that the additional oxidative processes in soybean with moisture contents between 0.10 and 0.24 gram per gram may contribute to the poorer longevity of soybean seed compared to pea seed.     

Bijdragen tot de kennis van enkele Hollandsche groente-erwten. I.

Summary Several crosses werer made between some races of peas frequently grown in Holland for culinary purposes. Of these Blauwpeul is a large brown seeded pea with indent seeds. It has pigment in its testa, but does not possess the factor for spots.Krombek, a pea with curved pods, yellow cotyledons and pigmentless testa possesses this factor for spots; thus the cross Krombek x Blauwpeul gives spotted seeds in the F₁.Kroonerwt, a green pea with big globular seeds has also the spto factor, but American Wonder, green with wrinkled seeds, has not. Low Cape Capucin has small spotted seeds of indent shape. Crossed with American Wonder it gives rise to all kinds of peas, but a remarkable fact in all crosses mentioned is, that where pigment is present in the testa, the form of the seeds always is indent. Pure wrinkled and globular seeds have colourless testa.The factor for indent is hyperstatic over globular.     

Copper toxicity: evidence for the conversion of cupric to cuprous copper in vivo under anaerobic conditions.

We have determined the toxicity to cells of Escherichia coli B of cupric copper applied under aerobic and anaerobic conditions in two ways. The growth of cells in liquid medium incorporating cupric copper shows differential inhibition, comparing aerobic and anaerobic conditions--toxicity being greater under anoxia. The growth of colonies upon agar plates incorporating cupric copper does not show such a differential effect. We conclude that colonies on plates are largely anoxic even when incubated aerobically. EPR spectra of cells obtained at various times after application of cupric copper under anoxic conditions indicate the conversion of a considerable proportion of the Cu(II) to a non-paramagnetic species, probably Cu(I). We demonstrate that Cu(I) is more toxic than Cu(II) to cells when applied under anoxic conditions and conclude that the difference in toxicity of Cu(II) applied to cells under aerobic and anaerobic conditions results from the greater extent of reduction of Cu(II) to Cu(I) under anaerobic conditions.     

Leaf surface wax and plant morphology of peas influence insect density

Insect predators and parasitoids adhere better, forage more effectively, and take more aphid prey on pea plants (Pisum sativum L.) (Leguminosae) with mutations that reduce the crystalline wax bloom on the plant surface. To assess the agronomic potential of this trait for pest management, abundance of pea aphids (Acyrthosiphon pisum L.) (Homoptera: Aphididae) and coccinellid predators, and percent parasitism of the aphids were evaluated on pea lines differing in wax bloom and plant architecture over two field seasons. Three pairs of pea lines were evaluated, each pair with a different architecture and differing within the pair in the amount of surface wax bloom (reduced or normal). The trials included plots treated with a narrow spectrum insecticide (pymetrozine) to reduce aphid populations and untreated controls. Reduced wax peas had significantly fewer aphids per plant in 2002 but not in 2003. Total natural enemy abundance was greater on reduced wax than on normal wax pea lines in both years of the study. Pymetrozine reduced aphid densities significantly in both years. Among the four pea lines evaluated for yield, seed yield per plant was affected by plant morphology and insecticide treatment. Yield was greatest on semileafless plants and on pymetrozine sprayed plots in both years. Yield of the reduced wax line in the semileafless background was similar to or exceeded yield in its normal wax sister line, suggesting that this morphological type was best for an agronomically viable reduced wax phenotype. Pea weevil (Bruchus pisorum L.) (Coleoptera: Bruchidae) damage to seed was overall more frequent on seeds from reduced wax varieties than from normal wax varieties. The results illustrate the trade‐offs associated with a reduced wax trait in peas but also show that certain combinations of reduced wax and gross morphology lead to reduced pea aphid populations and yields similar to those of normal wax peas.     

Transformation and Regeneration of Two Cultivars of Pea (Pisum sativum L.)

A reproducible transformation system was developed for pea (Pisum sativum L.) using as explants sections from the embryonic axis of immature seeds. A construct containing two chimeric genes, nopaline synthase-phosphinothricin acetyl transferase (bar) and cauliflower mosaic virus 35S-neomycin phosphotransferase (nptII), was introduced into two pea cultivars using Agrobacterium tumefaciens-mediated transformation procedures. Regeneration was via organogenesis, and transformed plants were selected on medium containing 15 mg/L of phosphinothricin. Transgenic peas were raised in the glasshouse to produce flowers and viable seeds. The bar and nptII genes were expressed in both the primary transgenic pea plants and in the next generation progeny, in which they showed a typical 3:1 Mendelian inheritance pattern. Transformation of regenerated plants was confirmed by assays for neomycin phosphotransferase and phosphinothricin acetyl transferase activity and by northern blot analyses. Transformed plants were resistant to the herbicide Basta when sprayed at rates used in field practice.     

Studies upon the copper fungicides. IX. Investigations with the exudate from fungus spores

Determinations have been made of the copper dissolved from pure copper compounds by water, by a standardized solution of the exudate of Neurospora sitophila spores, by standard solutions of malic and succinic acids and their sodium salts, and by glycine and mannitol.
The results compared with the known fungicidal performances of the copper compounds show that the amounts of copper yielded to water and to the standardized spore extract or glycine solution provide an indication of fungicidal properties.
Although soluble copper in excess of that dissolved by water can only appear from alkaline Bordeaux deposit by complex ion formation, results show that this is not necessarily the only means by which copper can dissolve from other copper compounds.
In a chemical investigation of the exudate of N. sitophila spores, mannitol, succinate and fumarate have been isolated. None of these materials can dissolve an appreciable amount of copper from alkaline Bordeaux deposit. Malate, found by other workers to be a constituent of the exudate of N. sitophila spores cultured on potato dextrose agar, has not been detected in such a solution from spores grown on malt agar. The quantity of mannitol in spore exudate has been shown to depend on the nutrient medium on which the fungus is grown.     

Full, reversible copper removal from ascorbate oxidase

Anaerobic treatment with cyanide of reduced ascorbate oxidase causes total depletion of copper. No significant amount of the metal is reincorporated when the apo-enzyme is incubated with cupric ions, but it is upon incubation with a stoichiometric amount (eight mol per mol of native enzyme) of a Cu(I) complex stable in air [Cu(I)(thiourea)3]Cl. The yield in reconstituted protein is higher under anaerobic conditions (85-90%) than in air (70-75%). By treatment with less than stoichiometric amounts of [Cu(I)(thiourea)3]Cl the apo-protein binds copper preferentially at the blue copper site. As a consequence the recovery of enzymatic activity is percentually lower than copper reincorporation.     

The analogous mechanisms of enzymatic inactivation induced by ascorbate and superoxide in the presence of copper

The mechanism of enzymatic inactivation of purified and membrane-bound acetylcholine esterase by ascorbate and copper was investigated. While the exposure of the enzyme to either ascorbate or copper did not cause enzymatic inactivation, the incubation of the enzyme with a combination of both ascorbate and copper resulted in a loss in acetylcholine esterase activity, which was time dependent. The enzymatic inactivation required either molecular oxygen or hydrogen peroxide under anaerobic conditions. Scavengers of hydroxyl radicals at concentrations of up to 100 mM did not provide protection to acetylcholine esterase. Only mannitol at very high concentrations (above 1 M) efficiently prevented the inactivation of the enzyme. The kinetics of the aerobic oxidation of reduced ascorbate in the presence of acetylcholine esterase and copper closely followed the rate of enzyme inactivation. Addition of the chelating agents EDTA and diethylenetriaminepentaacetic acid prevented both the oxidation of ascorbate and the inactivation of the enzyme. In the presence of low concentrations of histidine (0.5-2.0 mM), which forms high affinity complexes with copper, the rate of ascorbate oxidation was similar to that recorded in its absence. On the other hand, no enzyme inactivation was indicated in the presence of histidine. Low temperature EPR measurements have demonstrated the binding of copper to the enzyme, and have shown the reduction of the cupric enzyme to the corresponding cuprous complex. In view of these results, a general "site-specific" mechanism for biological damage can be offered, in which copper(II) ions are bound to enzymes or other biological macromolecules. Ascorbate plays a dual role: it reduces the cupric complex to the corresponding cuprous state and serves as a source for H2O2, which, in turn, reacts with the reduced copper complex, in a Fenton reaction. In this reaction, secondary hydroxyl radicals are site specifically formed, and react preferentially with the protein, at the site of their formation, causing its inactivation. This mechanism is analogous to that previously proposed (Samuni, A., Chevion, M., and Czapski, G. (1981) J. Biol. Chem. 256, 12632-12635) for the enhancement of the biological damage caused by superoxide in the presence of copper.     

Light-Sensitivity of Hazel Seeds with Respect to the Breaking of Dormancy

Light breaks the dormancy of hazel (Corylus avellana L.) seedswhen the testa is removed. Light, acting via phytochrome, alsopromotes the germination of hazel seeds when the embryonic axisis surgically uncovered. In such seeds with the axis uncoveredremoval of the testa is antagonistic to the light promotionof germination. (Received January 17, 1982; Accepted April 25, 1983)     

Early-season aerial adult colonization and ground activity of pea leaf weevil (Coleoptera: Curculionidae) in pea as influenced by tillage system

The pea leaf weevil, Sitona lineatus (L.) (Coleoptera: Curculionidae), is an important pest of pea, Pisum sativum L., in northern Idaho. Previous research revealed greater relative pea leaf weevil abundance and feeding damage in peas grown using conventional-tillage compared with no-tillage practices. However, the effects of tillage practices on early season colonization and activity by the pea leaf weevil on pea are not fully understood. Aerial traps and pitfall traps were used to assess adult colonization and relative density of adult pea leaf weevil into conventional-tillage and no-tillage pea in northern Idaho during 2005 and 2006. Feeding damage to the crop also was evaluated. During both years, aerial traps captured more pea leaf weevil in May, when crop establishment and early growth occurred, than in later months. Significantly more adult pea leaf weevils were captured in aerial traps in conventional-tillage than in no-tillage plots in May of both years. Significantly more pea leaf weevil were captured in pitfall traps in conventional-tillage plots than in no-tillage plots during the period immediately after peak aerial adult colonization in late May and early June. Crop feeding damage was significantly greater in conventional-tillage than in no-tillage plots in late May and early June. The patterns suggest that more adult pea leaf weevil colonize conventional-tillage pea than no-tillage pea. Pea plants in conventional-tillage emerged earlier and were larger than those in no-tillage during the pea leaf weevil colonization period, possibly accounting for the differences in colonization rates. This leads to greater early season pea leaf weevil infestation of conventional-tillage plots at a critical period for pea development that might ultimately influence crop yield.     

Response to water deficit and high temperature of transgenic peas (Pisum sativum L.) containing a seed-specific alpha-amylase inhibitor and the subsequent effects on pea weevil (Bruchus pisorum L.) survival

The effects of water deficit and high temperature on the production of alpha-amylase inhibitor 1 (alpha-AI-1) were studied in transgenic peas (Pisum sativum L.) that were developed to control the seed-feeding pea weevil (Bruchus pisorum L., Coleoptera: Bruchidae). Transgenic and non-transgenic plants were subjected to water-deficit and high-temperature treatments under controlled conditions in the glasshouse and growth cabinet, beginning 1 week after the first pods were formed. In the water-deficit treatments, the peas were either adequately watered (control) or water was withheld after first pod formation. The high-temperature experiments were performed in two growth cabinets, one maintained at 27/22 degrees C (control) and one at 32/27 degrees C day/night temperatures, with the vapour pressure deficit maintained at 1.3 kPa. The plants exposure to high temperatures and water deficit produced 27% and 79% fewer seeds, respectively, than the controls. In the transgenic peas the level of alpha-AI-1 as a percentage of total protein was not influenced by water stress, but was reduced on average by 36.3% (the range in two experiments was 11-50%) in the high-temperature treatment. Transgenic and non-transgenic pods of plants grown at 27/22 degrees C and 32/27 degrees C were inoculated with pea weevil eggs to evaluate whether the reduction in level of alpha-AI-1 in the transgenic pea seeds affected pea weevil development and survival. At the higher temperatures, 39% of adult pea weevil emerged, compared to 1.2% in the transgenic peas grown at the lower temperatures, indicating that high temperature reduced the protective capacity of the transgenic peas.     

An examination of the importance of ethanol in causing injury to flooded plants

Abstract. We have examined the widely held theory that ethanol toxicity is a prime cause of the injury and death of plants in soil flooded with water. The tests were made on peas ( Pisum sativum L.) at the early flowering or fruiting stages, when they are known to be severely injured by flooding.
Supplying ethanol in aerobic or anaerobic nutrient solution at similar concentrations to those we found in flooded soil (up to 3.9 mol m⁻³) or in the xylem sap of flooded pea plants (up to 2.1 mol m⁻³) caused no injury. One hundred times these concentrations gave little extra effect and failed to simulate flooding injury. Isolated leaf protoplasts and detached leaves were also resistant to damage by ethanol at these concentrations.
Other published measurements of ethanol concentrations in flooded plants are similar to or less than those we report for pea plants. Exceptions include root nodules and germinating pea seeds. Reports by others of responses to applied ethanol in a wide variety of circumstances confirm that in flooded plants the amounts are probably too small to explain the observed injury. Alternative mechanisms are discussed.     

The Deleterious Effect of Water and Low Temperature on Germination of Pea Seed

Some pea seeds were killed or damaged by soaking in water, andthe damage was aggravated by low temperatures. Low-vigour seedlots were more sensitive to injury than high-vigour lots. Reducedwater uptake in osmotic solutions resulted in less damage andmost injury occurred during the initial phase of imbibition. More electrolytes exuded from dead and low-vigour seeds thanfrom high-vigour seeds and increased exudation at low temperaturewas associated with a higher incidence of dead seeds. Death is thought to be caused by a sudden inrush of water whichdisrupts the sub-cellular organization and membranes of a proportionof seeds predisposed to injury.