Activity of DNA topoisomerase I and quiescence of embryo cells in seeds of Pisum sativum L.

DNA topoisomerase activity is present at a very early stageof germination in nuclear extract of pea root meristems. Theactivity of this enzyme changes before the onset of replicativeDNA synthesis, thus suggesting the existence of a correlationbetween DNA topoisomerase I and events taking place during therelease of cells from a quiescent state. An antibody preparedagainst a human topoisomerase I is able to immuno-precipitatepart of the DNA topoisomerase activity present in pea nuclearextracts, and recognizes a protein with a molecular weight of45 kDa. We suggest that the 45 kDa protein is a DNA topoisomeraseI; its presence during embryogenesis and its storage in dryseeds would explain the presence of DNA topoisomerase I activityduring early stages of germination. Key words: Pisum sativum L, DNA topoisomerase, nuclei, quiescence, proliferation     

Nuclear Proteins During the Onset of Cell Proliferation in Pea Root Meristems

The DNA: proteins ratio in nuclei of root meristems of pea (Pisumsativum L. cv. Lincoln) changed considerably during germinationas cells moved from a quiescent to an actively proliferatingstate with a higher protein content in nuclei of the latter.Electrophoretic patterns of nuclear proteins extracted at differenttimes of germination were used to examine qualitative changes.The various patterns presented a substantial similarity butthere were some proteins whose content increased or decreasedand others which disappeared or appeared during germination.The bulk of these variations occurred between 24 and 48 h ofgermination, suggesting that they might be correlated to thetransition from quiescence to the proliferating state. The patternof nuclear proteins obtained from adult differentiated roottissue was also examined. We tried to purify five differentnuclear protein components from intact nuclei by a multi-stepextraction procedure using a series of different buffers toascertain the nature of proteins presenting major interestingvariations. Most of these proteins purified with the nuclearsap or ribosomal components. Key words: Cell proliferation, electrophoresis, Pisum sativum L, root meristems     

Embryology of Early Abortion Due to Limited Maternal Resources in Pisum sativum L.

In most flowering plants, many embryos are aborted early intheir development due to limited maternal resources. The kin-conflictinterpretation of plant embryology predicts these abortionsshould be under maternal control. In a study of the abortionprocess in Pisum sativum, we found the first visible indicationof abortion was formation of a weak hypostase. Callose was locallydeposited around the chalazal endosperm haustorium, and ligninalong the outer cell walls of the remnant nucellar tissue. Thenucellus was compressed by proliferating adjacent inner integumentalcells. The endosperm haustorium's cytoplasm was forced backinto the embryo sac cavity. With suppression of haustorial activitythe endosperm nuclei gradually enlarged followed by enlargementof the embryo and suspensor nuclei. Finally, nuclei and cytoplasm throughout the endosperm and embryolost stainability and broke down. Four successive stages wererecognized in seed abortion. In seeds developing to maturity,no hypostase was developed and the haustorium continued to digestboth the remnant nucellus and the proliferated inner integumentalcells. These observations are consistent with the kin-conflicthypothesis. Pisum sativum, garden pea, ovule abortion, histology, hypostase, kin-conflict hypothesis     

Time Course and Polarity of Primary Phloem Fibre Differentiation in Pisum sativum

Primary phloem fibres of Pisum sativum deposit lignified cellwalls, 2 and 5 days after germination in root and stem, respectively.Fibre bundles reach their final size within 4–6 days.The differentiation of the bundles as a whole is faster in thestem compared with the root. In the special diverging bundlesof the lower internodes of the stem the peripheral portionsmature earlier than the inner portions. A wound in the rootas well as in the stem, interrrupts the differentiation of fibresdirectly below it. At least one bundle below a wound is disturbedand often a whole bundle is missing. The meaning of these findingsconcerning the control of cell size is discussed. Pisum sativum, differentiation, induction, phloem fibres, polarity, time course     

Ultrastructural localization ofl -fucose residues in nuclei of root primordia of the green peaPisum sativum

Summary Sugars have been demonstrated in animal cell nuclei, but only a few studies have mentioned their presence in plant cell nuclei. In this studyl-fucose residues were localized at the ultrastructural level, usingUlex europeaus agglutinin I lectin, during the early stages of germination ofPisum sativum and in mature root tip cells. This sugar was present after 1 h of germination, and its concentration was found to vary during 3 to 6 h imbition; after 72 h of imbition its concentration had more than doubled. Furthermore, labelling was particularly abundant in the nucleolus, nucleolus-associated bodies and dense nuclear bodies. The possibility that some of thel-fucose residues are associated with proteins is discussed.     

Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content

• Background and Aims Flow cytometry (FCM) is extensivelyused to estimate DNA ploidy and genome size in plants. In orderto determine nuclear DNA content, nuclei in suspension are stainedby a DNA-specific fluorochrome and fluorescence emission isquantified. Recent studies have shown that cytosolic compoundsmay interfere with binding of fluorochromes to DNA, leadingto flawed data. Tannic acid, a common phenolic compound, maybe responsible for some of the stoichiometric errors, especiallyin woody plants. In this study, the effect of tannic acid onestimation of nuclear DNA content was evaluated in Pisum sativumand Zea mays, which were chosen as model species. • Methods Nuclear suspensions were prepared from P. sativumleaf tissue using four different lysis buffers (Galbraith's,LB01, Otto's and Tris.MgCl₂). The suspensions were treated withtannic acid (TA) at 13 different initial concentrations rangingfrom 0·25 to 3·50 mg mL^–1. After propidiumiodide (PI) staining, samples were analysed using FCM. In additionto the measurement of nuclei fluorescence, light scatter propertieswere assessed. Subsequently, a single TA concentration was chosenfor each buffer and the effect of incubation time was assessed.Similar analyses were performed on liquid suspensions of P.sativum and Z. mays nuclei that were isolated, treated and analysedsimultaneously. FCM analyses were accompanied by microscopicobservations of nuclei suspensions. • Key Results TA affected PI fluorescence and light scatterproperties of plant nuclei, regardless of the isolation bufferused. The least pronounced effects of TA were observed in Tris.MgCl₂buffer. Samples obtained using Galbraith's and LB01 bufferswere the most affected by this compound. A newly described ‘tannicacid effect’ occurred immediately after the addition ofthe compound. With the exception of Otto's buffer, nuclei ofP. sativum and Z. mays were affected differently, with pea nucleiexhibiting a greater decrease in fluorescence intensity. • Conclusions A negative effect of a secondary metabolite,TA, on estimation of nuclear DNA content is described and recommendationsfor minimizing the effect of cytosolic compounds are presented.Alteration in light scattering properties of isolated nucleican be used as an indicator of the presence of TA, which maycause stoichiometric errors in nuclei staining using a DNA intercalator,PI.     

Stomata on the Primary Root of Pisum sativum L.

The first record of stomata on a non-specialized root was obtainedby scanning electron microscopy of 4-d-old Pisum sativum L.In some cases subsidiary cells were trichoblasts. Stomata andthe root triarch vascular structure were simultaneously presentin transverse sections through the root. Pisum sativum, pea, root stomata, guard cells, trichoblasts     

Chromosome Aberrations and Ageing Root Meristems

Changes in mitotic activity and frequency of spontaneous chromosomeaberrations have been determined for primary roots of Zea muys,Pisum sativum, Vicia faba var. green Windsor and var. minor,and Allium cepa. Mitotic activity was found to decline withage in all root apices. In the primary root apices immediately after germination, therewas a high frequency of spontaneous chromosome aberrations whichdeclined with time after germination. However, In the case ofV. faba var. green Windsor, Z mays and P. sativum there wasa return to the high levels of spontaneous chromosome aberrationon ageing of the root apex. root meristems, mitotic activity, chromosome aberrations, ageing     

The Effect of Paclobutrazol on Physiological and Biochemical Changes in the Primary Roots of Pea

Primary roots of pea (Pisum sativum L. cv. Taichung No. 11)were treated with 0, 10 and 50 mg dm^–3 paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)-4, 4-dimethyl-2-(l,2,4-triazol-l-yl)pentan-3-ol]for 1 h at 48 h after germination. Paclobutrazol treatment inhibitedroot extension, promoted swelling (cell expansion was radialrather than longitudinal), and increased cell volume and theactivity of catalase and peroxidase enzymes. Paclobutrazol alsodecreased root respiration and ethylene production. However,under non-stressed conditions, paclobutrazol treatment did notaffect soluble carbohydrate content, water potential, osmoticpotential or water loss. Under osmotic stress with polyethyleneglycol (PEG), paclobutrazol diminished the increase of waterpotential and decreased the rate of water loss caused by theimposed stress, but had no effect on osmotic potential. Catalaseand peroxidase activity were increased in osmotically-stressedroots of treated plants. Key words: Root growth, paclobutrazol, pea, Pisum sativum, water shortage     

Abscisic Acid and Assimilate Partitioning to Developing Seeds: III. DOES ABSCISIC ACID INFLUENCE SUGAR RELEASE FROM ATTACHED EMPTY SEED COATS IN AN ABA-DEFICIENT PISUM SATIVUM MUTANT?

Influence of abscisic acid on the release of sucrose from surgicallymodified Pisum sativum ovules was studied with the use of awilty pea mutant and variations in the amount of available assimilates.Phloem import was unaffected by applied ABA, independent ofthe endogenous ABA level and source-limited conditions. Key words: Pisum sativum, abscisic acid, ABA-deficient (wil) mutant, assimilate partitioning, empty-seed-coat technique     

Amino Acid Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing ovules of Viciafaba L. and Pisum sativum L., seed-coat exudates were collectedand the amino acid fraction of the exudate was analyzed. InV. faba, alanine was the most important compound of the aminoacid fraction. In P. sativum, alanine and glutamine were thetwo most important components, whereas only small amounts ofasparagine were present. Comparison with published data suggeststhat seed-coat exudates may differ from phloem sap in the relativeimportance of these amino acids. Pisum sativum, pea, Vicia faba, broad bean, amino acid transport, amino acid unloading, seed-coat exudate, seed development     

Cryopreservation of Somatic Embryos of Four Species With and Without Cryoprotectant Pre-treatment

The development of cryostorage procedures for somatic embryosproduced from the tissues of plants that normally propagateby means of desiccation- and (often) chilling-sensitive seeds,and that are unstorable by conventional means, offers a viablealternative to the conservation of this otherwise recalcitrantgermplasm. A cryopreservation procedure utilizing cryoprotectantsand partial dehydration was previously developed for hydratedand germinating Pisum sativum embryonic axes. The present contributionapplies that technology to the somatic embryos of a range ofspecies, viz., Coffea arabica (coffee), Manihot esculenta (cassava),Phoenix dactylifera (date palm) and Pisum sativum (pea) andcompares it with results for material that was partially dehydrated,then very rapidly frozen. Cassava, coffee and date palm showedsimilar recovery from cryopreservation irrespective of the procedure.Pea somatic embryos, on the other hand, recovered best fromcryopreservation when pre-treated with the cryoprotectants,glycerol and sucrose, and then subjected to partial dehydration.Copyright1995, 1999 Academic Press Coffea arabica, Manihot esculenta, Phoenix dactylifera, Pisum sativum, cryopreservation, somatic embryos     

Topoisomerase II is a structural component of mitotic chromosome scaffolds

We have obtained a polyclonal antibody that recognizes a major polypeptide component of chicken mitotic chromosome scaffolds. This polypeptide migrates in SDS PAGE with Mr 170,000. Indirect immunofluorescence and subcellular fractionation experiments confirm that it is present in both mitotic chromosomes and interphase nuclei. Two lines of evidence suggest that this protein is DNA topoisomerase II, an abundant nuclear enzyme that controls DNA topological states: anti-scaffold antibody inhibits the strand-passing activity of DNA topoisomerase II; and both anti-scaffold antibody and an independent antibody raised against purified bovine topoisomerase II recognize identical partial proteolysis fragments of the 170,000-mol-wt scaffold protein in immunoblots. Our results suggest that topoisomerase II may be an enzyme that is also a structural protein of interphase nuclei and mitotic chromosomes.     

Comparative Studies on the Properties of Two Ferredoxins from Pisum sativum L.

Two ferredoxins in approximately equal amounts were isolatedfrom 3 week old Pisum sativum L. seedlings. Both ferredoxinshad identical absorption spectra with maxima at 276, 327, 424,and 468 nm in the oxidized state, and each possessed a single2Fe-2S active centre. The isoelectric points of the two ferredoxinswere both at pH 3·3, and mixtures could not be separatedby isoelectric focusing on polyacrylamide gels. The midpointredox potentials of the ferredoxins were close to –415mV, but they differed slightly in their biological activity.Ferredoxin I was slightly the more active of the two in catalysingNADP⁺ photoreduction by Pisum or Hordeum chloroplasts whereasferredoxin II was more active in catalysing the oxidative cleavageof pyruvate by extracts of Clostridium pasteurianum. Thoughthe molecular weights of the ferredoxins determined by ultracentrifugationwere the same within experimental error, the amino acid compositionsshowed marked differences. The N-terminal 40 amino acid residuesof ferredoxins I and II were determined by means of an automaticsequencer. There were 15 differences, suggesting that gene duplicationhad occurred early in evolutionary time. Ferredoxin I appearsto be more closely related to the other angiosperm ferredoxinssince it differed in only 6 positions compared with the correspondingsequence for Medicago sativa (alfalfa) ferredoxin. The ratioof the two ferredoxins in Pisum sativum was shown to be dependenton the age of the seedlings and environmental growth conditions.     

Water Deficit in Pea Root Tips: Effects on the Cell Cycle and on the Production of Dehydrin-like Proteins

Dehydrin-like proteins have been detected in nuclei and cytoplasmof meristematic root tip cells from pea seedlings subjectedto slow dehydration at 90% relative humidity for 48 h or more.Evidence was gained from Western blotting and immunocytochemicalexperiments using an antibody raised against the conserved domainof dehydrin proteins. Flow cytometer analysis has shown thatcycling cells of root tip meristems from dehydrated seedlingsare mostly arrested in G2 phase. Other stress treatments thoughtto involve water depletion (osmotic stress, cold treatment)or to modulate cell response to water deficit (abscisic acid)gave less clear-cut results with all treatments lowering theproportion of cells entering the S phase, but without a definiteand persistent arrest in any preferential phase of the cycle.Possible interrelationships between G2 arrest and dehydrin productionare discussed. Cell cycle; dehydrins; flow cytometry; nuclei; pea; Pisum sativum L.; water stress     

Salinity and in vitro Ageing Effects on Primary Photosynthetic Processes of Thylakoids Isolated from Pisum sativum and Spinacia oleracea

The effects of in vitro ageing and salinity of the reactionmedium on the primary photochemistry of photosystem II and thepattern of energy distribution within the photochemical apparatusof thylakoids isolated from Pisum sativum and Spinacia oleraceaare described. Analyses of the low temperature induction curvesof fluorescence emission at 695 and 735 nm and of low temperatureabsorption and fluorescence emission spectra were used to examinethese processes. In vitro ageing over short periods reducedthe photochemical activity and changed the energy distributionwithin thylakoids of P. sativum, but had little effect on thylakoidsof S. oleracea. A synergistic effect of in vitro ageing andsalinity of the reaction medium was observed for P. sativumthylakoids. Ageing effects could be minimized by addition of100 mM NaCl to the resuspension medium. Changes in NaCl concentrationin the reaction medium produced large and similar changes inthe primary photochemical functioning of thylakoids from P.sativum and S. oleracea, which could be attributed mainly tothe cation species, Na^$; however, experiments using mannitolto produce osmotic stress indicated some small osmotically inducedchanges in photofunction of the thylakoids. Optimal primaryphotochemical activity of photosystem II, for both species,was observed with 200 mM NaCl. Cation-induced regulation ofexciton distribution appears to be facilitated by controllingthe degree of energy coupling between the light-harvesting chlorophylla/b complex and the two photosystems, I and II, and not by regulationof coupling between photosystems I and II.     

The Rate of Morphogenesis of Embryos and Seeds in Four Species of Grain Legumes

Comparative embryo development has been studied histologicallyin Lupinus albus, Lupinus mutabilis, Vicia faba, Pisum sativumand Latkyrus latifolius. The detailed histology of the stagesof embryo formation up to the early differentiation of tissuesof the seed is reported. The rate of embryogenesis has beentimed through 15 stages of development from anthesis and comparativerates of tissue formation established between the species. Themain observation was the slow rate of morphogenesis of embryosand seeds in Lupinus albus in comparison with the very rapidrate observed in Pisum sativum. A long period at the globularembryo stage, when embryo morphogenesis was inactive contributedto the extended development time of embryos and seeds in Lupinusalbus. Slow differentiation of reproductive tissues in L. albusdetermines late maturity in seeds and pods. Lupinus albus, white lupin, L. mutabilis, tarwi, Vicia faba, faba bean, Pisum sativum, pea, Lathyrus latifolius, everlasting pea, embryo development     

Effects of Extreme Acceleration on the Germination, Growth and Cell Wall Composition of Pea Epicotyls

Pea (Pisum sativum) seedlings were germinated and grown in darknessfor 5 d whilst being subjected to continuous accelerations ofbetween 1 ? g and 1054 ? g. Increased acceleration retardedthe elongation of both epicotyls and roots, and inhibited lateralroot growth. Changes in the cell wall carbohydrate and lignincomponents were also recognized. Nevertheless, increasing accelerationfrom 1 ? g to 1054 ? g had no effect on germination and peaswere able to germinate and grow successfully at up to approximately10 000 ? g. Key words: Acceleration, cell walls, Pisum