Characterisation of glutathione transferases and glutathione peroxidases in pea (Pisum sativum)

Glutathione peroxidases (GPOXs) and glutathione transferases, also termed glutathione S-transferases (GST, EC 2.5.1.18), with activities toward a range of xenobiotic substrates including herbicides, have been characterized in etiolated pea (Pisum sativum L. cv. Feltham's First) seedlings. Crude extracts showed high activity toward a range of GST substrates including 1-chloro-2,4-dinitrobenzene (GSTC activity) and the herbicide fluorodifen (GSTF) but low activities toward chloroacetanilides and atrazine. Treatment of the pea seedlings with the herbicide safener dichlormid selectively increased the activity of GSTC and the GST which detoxified atrazine. This induction was restricted to the roots and was not observed with any of the other GST or GPOX activities. In contrast, treatment with CuCl₂ increased GPOX activity in the root but had no effect on any GST activity, while treatment of epicotyls with elicitors of the phytoalexin response increased GST activity toward ethacrynic acid, but had no effect on other GST or GPOX activities. The major enzymes with GSTC, GSTF and GPOX activities were purified from pea epicotyls 3609-fold, 1431-fold and 1554-fold, respectively. During purification by hydrophobic interaction chromatography and affinity chromatography using S-hexyl-glutathione as ligand all three activities co-eluted but could be partially resolved by anion exchange chromatography and gel filtration chromatography. Both GSTC and GPOX had a molecular mass of 48 kDa and their activities were associated with a similar 27.5-kDa subunit but distinct 29-kDa subunits. GSTF could be resolved into two isoenzymes with molecular masses of 49.5 and 54 kDa. GSTF activity was associated with a unique 30-kDa subunit in addition to 27.5- and 29-kDa peptides, suggesting that the two isoenzymes were composed of differing subunits. These results demonstrate that peas contain multiple GST isoenzymes some of which have GPOX activity and that the various activities are differentially responsive to biotic and abiotic stress.     

Regeneration of shoots from pea (Pisum sativum) hypocotyl explants

A sequential indole-3-acetic acid (IAA)-zeatin treatment was applied to Pisum sativum hypocotyl explants, resulting in shoot formation from 50% of the explants. Shoots were easily rooted and transplantable plants could be obtained in 3 months. The method has been applicable to the 5 cultivars tested. Histological examination of explants suggests the shoots to be of de novo origin, which would make the system suitable for transformation experiments.     

Differential reponses to paraquat‐induced oxidative injury in a pea (Pisum sativum) protoplast system

Antioxidant responses to varying degrees of paraquat stress in freshly isolated photosynthesizing pea (Pisum sativum L.) protoplasts from cultivars Progress and Nugget were studied. Leaves of comparable maturity were used for protoplast isolation. Nugget protoplasts were more resistant to paraquat in the micromolar range under our conditions. In Nugget, a non-bleaching paraquat concentration (10 µM) inhibited CO₂-dependent O₂ evolution ca 50% during the first 40 min, remaining at that rate (“coping behavior”) for up to 100 min. In contrast, Progress protoplasts treated with the same concentration of paraquat did not exhibit coping behavior. Antioxidant enzyme activities were unaltered throughout the time course of the experiment in treated protoplasts from Nugget and in chloroplasts isolated from them. Thus, the coping behavior of Nugget protoplasts cannot be attributed to changes in activities of the three antioxidant enzymes tested. Paraquat treatment did not affect antioxidant enzyme activities in Progress protoplasts nor in chloroplasts isolated from them. When higher doses of paraquat were used (12 h, 0.1 mM paraquat), protoplasts from both cultivars were rapidly bleached and total protein decreased to ca 30% of pre-stress levels. Glutathione reductase (GR, EC 1.6.4.2) activity dropped in protoplasts from both cultivars under the severe stress conditions in concert with declines in protein levels. However, superoxide dismutase (SOD, EC 1.15.1.1) activity remained constant over the first 9 h of the time course, increasing to ca 150& of original levels by the final, 12-h time point. The activity of the plastid Cu,Zn isoform, expressed as a percentage of total SOD activity, declined over the time course of the experiment while that of mitochondrial MnSOD appeared to increase. This change in activity of MnSOD correlated with cell decline, therefore, and was not correlated with protection. These data are in agreement with some earlier reports and are compatible with the hypothesis that SOD activity levels increase in response to reactive oxygen species levels, even under conditions leading to cell death.     

A chemically induced new pea (Pisum sativum) mutant SGECdt with increased tolerance to, and accumulation of, cadmium

BACKGROUND AND AIMS: To date, there are no crop mutants described in the literature that display both Cd accumulation and tolerance. In the present study a unique pea (Pisum sativum) mutant SGECd(t) with increased Cd tolerance and accumulation was isolated and characterized. METHODS: Ethylmethane sulfonate mutagenesis of the pea line SGE was used to obtain the mutant. Screening for Cd-tolerant seedlings in the M2 generation was performed using hydroponics in the presence of 6 microm CdCl2. Hybridological analysis was used to identify the inheritance of the mutant phenotype. Several physiological and biochemical characteristics of SGECd(t) were studied in hydroponic experiments in the presence of 3 microm CdCl2, and elemental analysis was conducted. KEY RESULTS: The mutant SGECd(t) was characterized as having a monogenic inheritance and a recessive phenotype. It showed increased Cd concentrations in roots and shoots but no obvious morphological defects, demonstrating its capability to cope well with increased Cd levels in its tissues. The enhanced Cd accumulation in the mutant was accompanied by maintenance of homeostasis of shoot Ca, Mg, Zn and Mn contents, and root Ca and Mg contents. Through the application of La(+3) and the exclusion of Ca from the nutrient solution, maintenance of nutrient homeostasis in Cd-stressed SGECd(t) was shown to contribute to the increased Cd tolerance. Control plants of the mutant (i.e. no Cd treatment) had elevated concentrations of glutathione (GSH) in the roots. Through measurements of chitinase and guaiacol-dependent peroxidase activities, as well as proline and non-protein thiol (NPT) levels, it was shown that there were lower levels of Cd stress both in roots and shoots of SGECd(t). Accumulation of phytochelatins [(PCcalculated) = (NPT)-(GSH)] could be excluded as a cause of the increased Cd tolerance in the mutant. CONCLUSIONS: The SGECd(t) mutant represents a novel and unique model to study adaptation of plants to toxic heavy metal concentrations.     

Molecular analysis of a null mutant for pea (Pisum sativum L.) seed lipoxygenase-2

A mutant line of Pisum fulvum was identified that lacked seed lipoxygenase-2 (LOX-2). The mutant phenotype was introgressed into a standard Pisum sativum cv. Birte to provide near-isogenic lines with or without seed LOX-2. Genetic analyses showed the mutation to behave as a single, recessive Mendelian gene. Northern and dot-blot analyses showed a large reduction in LOX-2 mRNA from developing seeds of the LOX-2-null mutant. A restriction fragment length polymorphism associated with the 5 end of the LOX-2 gene(s) co-segregated with the null phenotype, indicating that the reduction of LOX-2 mRNA was neither a consequence of deletion of the LOX genes nor a consequence of the action of a genetically distant regulatory gene. Analysis of the 5-flanking sequences of LOX-2 genes from Birte and the near-isogenic LOX-2-null mutant revealed a number of insertions, deletions and substitutions within the promoter from the LOX-2-null mutant that could be responsible for the null phenotype. Incubation of crude seed LOX preparations from Birte and the LOX-2-null mutant showed that the latter generated relatively less 13-hydroperoxides and also produced relatively more hydroxy- and ketoacid compounds that have implications for the fresh-frozen pea industry.     

Foliar uptake of Cd by pea (Pisum sativum) and sugar beet (Beta vulgaris)

Pea ( Pisum sativum L. cv. Fenomen) and sugar beet ( Beta vulgaris L. cv. Monohill) were cultivated in nutrient media without or with 10 μM CdCl₂. Leaves of the same size and stage of development, detached or still attached to the intact plants, were submerged into redistilled water containing 1 to 250 μM CdCl₂. The uptake experiments were run for 1 to 8 h at pH 3.6 and 5.1. Cuticular transpiration rate, density of leaf and density of stomata were also measured. Percentage of open stomata was studied at different pH.
Foliar uptake of Cd into the leaf is evident since Cd is transported from the exposed part of the pea leaves, through the petioles and into the stipules, and since the Cd concentration of the leaves increases with time and external Cd concentration. The foliar uptake depends on the permeability of the cuticular membrane, which is increased by a high intrinsic Cd level, which in turn enhances the foliar uptake of Cd in sugar beet. Higher cuticular permeability in pea than in sugar beet is shown by a 2.5 times higher cuticular transpiration rate and a 4 times lower density of leaf for pea, which causes a 7 times higher foliar uptake in pea than in sugar beet. Low pH decreases the net uptake of Cd, probably by an exchange reaction in the cutin and pectin of the cuticular membrane. Stomata are not directly involved in the Cd uptake, and the differences in the sum total of stomatal aperture area per unit leaf area is not related to differences in foliar uptake of Cd. Percentage of open stomata, calculated as average of both sides of the leaves, was not affected by changes in pH: but especially at high pH. proportionally more stomata were open on the adaxial than on the abaxial side.     

Isolation and properties of a lectin from the roots of Pisum sativum (Garden pea)

Abstract The roots of pea (Pisum sativum L. ev. Feltham First) seedlings contained haemagglutinating activity and a protein which reacted with antibodies directed against pea seed lectin. This protein was shown to be present on the surface of root hairs and in the root cortical cells by immunofluorescence. Lectin (haemagglutinin) was purified from pea seedling roots by both immunoaffinity chromatography and affinity chromatography on Sephadex G-100. The pea root lectin was similar to the seed lectin when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was antigenically identical: however, the isoelectric focussing band patterns of the proteins differed. The sugar specificity of the root lectin differed from that of the seed lectin, and the haemagglutinating activity of the root lectin was less than the seed lectin. These results are discussed with reference to the hypothesis that lectins mediate in the symbiotic association of legume and Rhizobium through their carbohydrate-binding properties.     

Protochlorophyllide transformations and chlorophyll accumulation in epicotyls of pea (Pisum sativum)

Low-temperature fluorescence emission spectra of epicotyls of 6.5-day-old dark-grown seedlings of pea ( Pisum sativum L.) showed the dominance of short-wavelength protoch lorophyllide forms with emission maxima at 629 and 636 nm, respectively. The presence of long-wavelength protochlorophyllide with emission maxima around 650 nm was just detectable. Accordingly, irradiation with millisecond flashes gave a minute formation of chlorophyllide. The chlorophyll(ide) formation varied along the epicotyl. Irradiation with continuous light for 1.5 h resulted in an evident accumulation of chlorophyll(ide) in the upper part of the epicotyl. Only small amounts accumulated in the middle section. The conversion of protochlorophyllide to chlorophyllide was temperature dependent and almost arrested at 0°C. The chlorophyll(ide) formed had one dominating fluorescence peak at 681 nm. Irradiation for 24 h gave almost 100 times more chlorophyll in the upper part of the epicotyl than in the lower part. Electron micrographs from the upper part of the epicotyl irradiated for 6 h showed plastids with several developing thylakoids, while the plastids in the lower part of the epicotyl had only a few thylakoids. The dominance of short-wavelength protochlorophyllide forms indicated the presence of protochlorophyllide not bound to the active site of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33). The inability of the short-wavelength form to transform into chlorophyllide with flash light denotes a dislocation from the active site. The time and temperature dependence of the chlorophyll(ide) formation in continuous light indicates that a relocation is required of the short-wavelength protochlorophyllide before chlorophyllide formation can occur.     

Effects of enhanced UV-B radiation on pea (Pisum sativum L.) grown under field conditions in the UK

A new modulated lamp system is described. This system has successfully provided an ultraviolet-B (UV-B) supplement in proportion to ambient UV-B. The modulated system was used to simulate the UV-B environment resulting from an annual mean reduction of 15% in the stratospheric ozone under UK field conditions, but taking account of seasonal variation in depletion. The effects of this enhanced level of UV-B on the growth, physiology and yield of four cultivars of pea were assessed. Enhanced UV-B resulted in small reductions in the number of stems and total stem length per plant (respectively 4.7 and 8.7%). There were also significant decreases in the dry weight of peas (10.1%), pods (10.3%) and stems (7.8%) per plant. UV-B treatment had no effect on the number of peas per pod or average pea weight, but did significantly reduce (12.1%) the number of pods per plant. This decrease in pod number was partly due to enhanced abscission of pods during the final month of plant growth. UV-B treatment had no significant effect on chlorophyll fluorescence characteristics or CO₂assimilation rate per unit leaf area. These results are consistent with previous controlled environment experiments, and suggest that reduction in yield may be due to direct effects of UV-B on plant growth rather than a decrease in photosynthetic capacity per unit leaf area.     

Paraquat toxicity in Pisum sativum: Effects on soluble and membrane-bound proteins

The effects of paraquat (PQ) on Pisum sativum L. proteins were investigated in vivo in a new experimental system utilizing 10-day-old plant cuts.A marked decrease in the specific activity of membrane-bound Ca²⁺-dependent ATPase was recorded, while that of Mg²⁺-dependent ATPase remained unchanged. Concurrently with a drop in the total plant protein, the specific activities of the three cytoplasmic enzymes, malate dehydrogenase, hydroxypyruvate reductase and triose-phosphate isomerase, were also found to decrease. The effect on various enzymes involved in cellular defense mechanisms was also studied: glutathione reductase and superoxide dismutase activities increased, while ascorbate peroxidase was not affected.
These findings shed light on the selectivity of PQ-induced injurious processes, focusing on protein homeostasis mechanisms in the membrane and cytoplasmic compartments at the cellular level, as well as on the prominent role played by enzymatic defense systems against PQ poisoning.     

Effects of apoplastic sucrose on carbohydrate pools and sucrose efflux of mesophyll protoplasts of pea (Pisum sativum)

The effects of the apoplastic, i.e. external, concentration of sucrose (0–30 m M ) on O₂ evolution, O₂ consumption, starch, sucrose, glucose and fructose content, and uptake and efflux of sucrose in mesophyll protoplasts of Pisum sativum L. cv. Fenomen were studied. Neither photosynthesis, dark respiration, sucrose nor starch content change with increased apoplastic sucrose concentration. The contents of glucose and fructose in the protoplasts increase with increased apoplastic sucrose concentration. The sucrose efflux increases with increased external sucrose concentrations between 1 and 5 m M , but above this range the efflux decreases with increased external sucrose concentrations. These findings indicate that although external sucrose does not enter the protoplasts, there is a relationship between the external sucrose pool and the internal pools of sugars in the mesophyll protoplasts. The results suggest an active sucrose efflux from the protoplasts at physiological concentrations of apoplastic sucrose (max 5 m M ) and a simple diffusion mechanism at higher concentrations.     

Influence of selenium on uptake and toxicity of copper and cadmium in pea (Pisum sativum) and wheat (Triticum aestivum)

The detoxifying effect of selenium on animals toxicated with heavy metals is well known. In this study we examine if there is a similar effect in plants. Wheat ( Triticum aestivum L. cv. Sunny) and pea ( Pisum sativum L. cv. Fenomen) were grown for 21 days on a nutrient solution based on the nutrient proportions in healthy plants. Nutrients along with cadmium, copper, selenite, selenate or selenite and selenate in combinations with copper or cadmium were supplied in small amounts with a daily incremental increase of 0.12 (wheat) and 0.20 (pea). The metal and selenium uptake and distribution in the plants as well as the effects on growth were investigated.
The results show that selenium does not reduce the toxicity of heavy metals to plants. Instead, selenium enhances metal uptake and toxicity, especially in peas grown in the presence of metal and selenate. Selenite increased cadmium concentrations of pea roots up to 300% and selenate that of wheat shoots up to 50%.     

Chloroplast ultrastructure changes in Pisum sativum associated with supplementary ultraviolet (UV-B) radiation

Pea plants (Pisum sativum L. cv. Greenfeast) were grown and exposed to supplementary UV-B radiation from day 17 after planting under growth cabinet conditions. The effects of this exposure on the ultrastructure of chloroplasts and the total soluble sugar and starch concentrations were estimated. Supplementary UV-B radiation was shown to damage the structure of chloroplasts, as manifested by dilation of thylakoid membranes, a progressive disruption of the thylakoid structure and disintegration of the double membrane envelope surrounding the chloroplast, accompanied by the accumulation of large starch grains. Diurnal changes observed in starch concentration suggest that the higher concentration of starch in supplementary UV-B-treated leaves is due to its immobilization, rather than to any increase in starch synthesis: soluble sugars accumulated and remained at a higher level and then later declined.     

孟德尔豌豆基因克隆的研究进展及其在遗传学教学中的应用

150多年前, 孟德尔进行了豌豆7对相对性状的杂交试验, 发现了遗传学的两个基本规律。1900年, 孟德尔定律被重新发现以后, 人们从生理生化、细胞和分子水平等不同层次上对豌豆的这7个性状进行了深入研究。近年, 随着分子生物学技术的发展, 已有种子形状(R)、茎的长度(Le)、子叶颜色(I)和花的颜色(A)等4个性状的基因被克隆; 未成熟豆荚的颜色(Gp)、花的着生位置(Fa)和豆荚形状(V)的基因已被定位在各自的连锁群上。4个孟德尔基因的鉴定和克隆加深了人们对基因概念的理解：如基因功能的多样性、在分子水平上基因变异原因的多样性、显性和隐性的分子实质等。在遗传学教学中, 把孟德尔基因克隆和研究的最新进展介绍给学生, 在分子水平上诠释经典遗传规律, 有助于提高学生的学习兴趣, 帮助学生全面把握从形式遗传学到分子遗传学的内容和遗传学的发展方向。