Plasma Membrane Redox System in Guard Cell Protoplasts of Pea (Pisum sativum L.)

Guard cell protoplasts (GCP) from leaves of pea (Pisum sativum)were capable of reducing/oxidizing the membrane impermeableelectron carriers, ferricyanide/NADH. The redox activity ofGCP required the presence of both ferricyanide and NADH, althoughsome ferricyanide reduction occurred even in the absence ofNADH. The GCP preferred NADH to NADPH during ferricyanide reductionand the reduction was slow with DCPIP or cytochrome c. A stoichiometryof about 2 existed between moles of ferricyanide reduced andNADH oxidized by GCP. The redox activities of GCP were severaltimes greater than those of mesophyll protoplasts from pea leaves.The ferricyanide reduction or NADH oxidation by GCP was unaffectedby abscisic acid or sodium orthovanadate and fusicoccin indicatingthe non-involvement of plasma membrane ATPase in these redoxreactions.The redox activities were markedly inhibited by chloroquineor 8-hydroxyquinoline. The findings are discussed in relationto the possible regulatory role of a guard cell plasma membraneredox system in stomatal function. Key words: Plasma membrane redox system, mesophyll protoplasts, pea, guard cell protoplasts, stomatal function     

Effect of Vanadate on Microsomal ATPase Activity, Acidification of the Medium and Auxin-stimulated Growth in Pea and Cucumber

The relationship between ATPase activity, medium acidificationand auxin-stimulated growth in segments of pea stem (Pisum sativumL., cv. Alaska) and cucumber hypocotyl (Cucumis sativus L.,cv. Long Green Ridge) was investigated using sodium orthovanadate,widely used as a selective inhibitor of plasma membrane-associatedATPase activity. ATPase activity of cucumber microsomal preparationswas about seven times lower than similar preparations from pea(on a mg microsomal protein basis) and was much more effectivelyinhibited by vanadate. Similarly, acidification of the mediumby abraded cucumber segments occurred to a lesser extent thanwith pea and showed a greater inhibition by vanadate. Both growthin controls and auxin-stimulated growth of cucumber segmentswere strongly inhibited by vanadate, whereas in pea auxin-stimulatedgrowth was reduced by only half and controls showed little inhibition.Acidification of the medium by segments of both species wasfound to occur readily even in controls and showed little promotionin the presence of IAA, although growth in both species wasrapidly and significantly promoted by IAA. These results indicatethat acidification is brought about by a plasma membrane-associatedATPase, and suggest that while acidification is an essentialfactor for auxin-stimulated growth it may not be the mechanismby which the growth rate is controlled. ATPase, Cucumis sativus, indole-3-acetic acid, Pisum sativum, vanadate     

Functional Significance of Acid Phosphatase Distribution During Embryo Development in Pisum sativum L

The distribution of P₁, ester P and acid-insoluble nucleic acidP has been studied in relation to acid phosphatase activity(EC 3. 1. 3. 2) in the component parts of developing pea seeds(Pisum sativum L.). Despite the favourable pH of the liquidcontents of the embryo sac (pH 5.5), only very low acid phosphataseactivity was detected in this fluid (c. 0.01 units per seed).Potential substrates for phosphatase action were in fact absentfrom the secretion, the only form of P present being P_i, inconcentrations up to 8 mM. The data support the hypothesis thatthe high acid phosphatase activities which develop in the seed-coatsare involved in regulating the supply of P as P₁ to the developingembryo. Pisum sativum L., pea, embryo development, acid phosphatase, phosphorus, seed-coats, seed development     

Gibberellic Acid Controls the Secretion of Acid Phosphatase in Aleurone Layers and Isolated Aleurone Protoplasts of Avena fatua

The role of gibberellic acid (GA₃) in controlling the secretion(across the plasma membrane) and release (through the cell wall)of acid phosphatase (E.C. 3.1.3.2 [EC] .) from Avena aleurone layershas been investigated. Evidence from this comparative studywith intact aleurone layers and isolated aleurone protoplastsreveals that the secretion of acid phosphatase is under GA₃control. The mechanism underlying secretion and release of theenzyme from aleurone cells is discussed. Key words: Avena fatua, Acid phosphatase, Aleurone protoplasts, Gibberellic acid, Secretion     

Identification of Shoot Apical Meristem Cells Committed to Form Vascular Elements in Pisum sativum L. and Vicia faba L.

A cytochemical study of naphthol AS-D esterases in vegetativeshoot apices of Pisum sativum and Vicia faba L. has shown thepresence of carboxyl esterases (E.C. 3.1.1.1 [EC] .) in those meristemcells already committed to form vascular elements. These cellsform a sequence linking the morphologically identifiable procambiumto the cells of the tunica layers at a site either already identifiableas the next primordium or which will form the next primordium.The implications of this result are briefly discussed in relationto the control of primordia formation and procambial cell development. Pisum sativum, Vicia faba, determination, vascular tissue, shoot apex, cytochemistry     

Lack of Influence of the Epidermis on Underlying Cell Development in Leaflets ofPisum sativumvar.argenteum(Fabaceae)

We explored whether epidermal pressure regulates cell and organgrowth in leaflets ofPisum sativumvar.argenteum,a mutant cultivarof the garden pea characterized by reduced adhesion betweenthe epidermis and subjacent mesophyll. Developing leaflets ofleaves arising at three positions on the seedling axis werepeeledin situand grown to maturity in humidity chambers. Themature anatomy and morphology could be accurately assessed becausewound responses normally associated with peeling were preventedby theArgmutation that permitted peeling without damage to themesophyll and by the humidity chambers that protected peeledareas from desiccation. The mesophyll cell size, state of differentiation,and layering pattern as well as the overall morphology of mature,peeled leaflets were indistinguishable from those of mature,intact leaflets grown under the same conditions. The epidermisexerted no detectable regulatory effect on the expansion ofthe leaflets as a whole or on the tissue layers and cells withinthe leaflets.Copyright 1999 Annals of Botany Company. Biomechanics, compression, epidermis, leaf development, mesophyll, pressure, wound response,Pisum sativumvar.argenteum.     

Signal Integration by ABA in the Blue Light-Induced Acidification of Leaf Pavement Cells in Pea (Pisum sativum L. var. Argenteum)

Leaf pavement cell expansion in light depends on apoplastic acidification by a plasma membrane proton-pumping ATPase, modifying cell wall extensibility and providing the driving force for uptake of osmotically active solutes generating turgor. This paper shows that the plant hormone ABA inhibits light-induced leaf disk growth as well as the blue light-induced pavement cell growth in pea (Pisum sativum L.). In the phytochrome chromophore-deficient mutant pcd2, the effect of ABA on the blue light-induced apoplastic acidification response, which exhibits a high fluence phase via phytochrome and a low fluence phase via an unknown blue light receptor, is still present, indicating an interaction of ABA with the blue light receptor pathway. Furthermore, it is shown that ABA inhibits the blue light-induced apoplastic acidification reversibly. These results indicate that the effect of ABA on apoplastic acidification can provide a mechanism for short term, reversible adjustment of leaf growth rate to environmental change.Key Words: ABA, apoplastic acidification, blue light, epidermal pavement cell growth, leaf growth, pea (Pisum sativum L.), signal integration     

The Effects of Conditions of Growth, Extraction and Reaction on a Nucleotide Phosphatase Activity Obtained from the Shoot-Root Axes of Pisum sativum var. Alaska Seedlings

Nucleotide phosphatase activity in extracts from etiolated peaseedlings (Pisum sativum var, Alaska) was studied. A markedincrease followed rupture of the seed coat by the elongatingradicle, indicating a dependence on aerobic respiration fordevelopment of the activity. Activity from the shoot-root axiswas directly correlated with axis size, inhibition of growthresulting in a reduced activity. Growth in continuous illuminationalso reduced activity, as did imbibition of phenylalanine, tyrosine,glutathione, and sodium phosphate. The activity was greaterin the shaft regions of the axis than in the tip regions. About30 percent of the activity was associated with the nuclear—mitochondral—cellwall pellet, while the reminder was more or less evenly distributedbetween a membranee fraction of high specific activity and asoluble fraction. The effects of substractes, ions, and inhibitorson the activities of membrane and soluble fractions was quitedifferent.     

Plasma Membrane Permeability of Root-Tip Cells Following Temporary Exposure to Al Ions Is a Rapid Measure of Al Tolerance among Plant Species

No correlations were recognized between Al tolerance among fourplant species, rice (Oryza sativa L.), maize (Zea mays L.),pea (Pisum sativum L.), and barley (Hordeum vulgare L.), inrank order of Al tolerance, and cation exchange capacities ofroot-tip (0-1 cm) cells or of their cell walls. The plasma membraneof root-tip of Al sensitive plant species (pea and barley) wasconsiderably permeabilized with elongation of root in Al-freesolution following 0.5 h pretreatment with Al. K⁺ release fromand Al permeation into the protoplasts isolated from the root-tipof Al-sensitive plant species were more significant than thosefor Al-tolerant plant species (rice and maize) on 10 or 30 mintreatment with Al. The permeability of the plasma membrane forprotoplasts isolated from Al sensitive plant species was considerablyincreased by treatment with hy-potonic Al-free control solutionfollowing 10 min pretreatment with Al. To our knowlege, theseare the most rapid responses to Al ions reported to date, i.e.,within 0.5 h in whole plant and within 10 min in protoplast.These results suggest that a temporary contact with Al ionsirreversibly alters the plasma membrane of root-tip cells ofAl-sensitive plant species: the cells become more leaky andrigid due to binding of Al ions to the plasma membrane. (Received January 5, 1998; Accepted February 26, 1998)     

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     

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     

Correlative Inhibition of Lateral Bud Growth in Pisum sativum L. and Phaseolus vulgaris L.: Studies of the Role of Abscisic Acid

The possibility has been investigated that abscisic acid (ABA)might act as a correlative inhibitor of lateral bud growth inPisum sativum and Phaseolus vulgaris. Application of ABA insmall quantities (2µg) to axillary buds on decapitatedplants of P. sativum caused appreciable inhibition of theirgrowth, and induced a compensatory growth of the bud on an adjacentnode. Application of this same quantity of ABA to axillary budson decapitated plants of Phaseolus vulgaris was without effect,but a high concentration in lanolin (1 mg g^–1) did substantiallyreduce bud outgrowth. Endogenous ABA-like substances in Phaseolusvulgaris, detected by bioassay and electron capture g.l.c.,were present in similar concentrations in shoot tips, lateralbuds on intact plants and lateral buds on plants decapitated24 h earlier. The effects of applied ABA suggested that it might be involvedin the mechanism of correlative inhibition in Pisum sativum,but it was not possible to test this hypothesis by determiningendogenous ABA levels in axillary buds because of their smallsize. The evidence presented here suggests that ABA is not acorrelative inhibitor in Phaseolus vulgaris even though at highconcentration it can inhibit the growth of axillary buds.     

A Biolchemical and Cytochemical Study of Peroxidase Activity in Roots of Pisum sativum: I. A COMPARISON OF DAB-PEROXIDASE AND GUAIACOL-PEROXIDASE WITH PARTICULAR EMPHASIS ON THE PROPERTIES OF CELL WALL ACTIVITY

Peroxidase activity in roots of Pisum sativum has been examinedusing both guaiacol and 3,3'-diaminobenzidine (DAB) as hydrogendonors. Biochemically, differences were observed between thetwo donors with respect to the pH optimum (6–9 and 4–0,respectively), and in response to added NaCl (guaiacol-peroxidasewas unaffected while the DAB-peroxidase was markedly inhibited).Both reactions showed highest specific activity in a high speedsupernatant fraction, and, of nine anionic bands demonstratedby gel electrophoresis with DAB, only six were visible withguaiacol. Histochemically, similar staining patterns were observedwith both donors. Cell wall fractions prepared by bead filtration contained 2%and 3.5% of the total peroxidase and acid phosphatase activitiesrespectively. 50% and 27% of these activities were ionicallybound, as indicated by salt treatment In addition, washing withsalt solutions produced a marked stimulation of peroxidase activityat high salt concentrations: this affect was not observed withthe supernatant peroxidase or with cell wall acid phosphatase.Possible functions of cell wall peroxidase are discussed     

Labeling of the Plasma Membrane of Pea Cells by a Surface-localized Glucan Synthetase

When radioactive UDP-glucose is supplied to 1-millimeter-thick slices of pea (Pisum sativum) stem tissue, radioactive glucose becomes incorporated into membrane-bound polysaccharides. Evidence is given that this incorporation does not result from breakdown of UDP-glucose and utilization of the resultant free glucose, and that the incorporation most likely takes place at the cell surface, leading to a specific labeling of the plasma membrane. The properties of the plasma membrane that are indicated by this method of recognition, including the association of K⁺-stimulated ATPase activity with the plasma membrane, resemble properties inferred using other approaches. The membrane-associated polysaccharide product formed from UDP-glucose is largely 1,3-linked glucan, presumably callose, and does not behave as a precursor of cell wall polymers. No substantial amount of cellulose is formed from UDP-glucose in this procedure, even though these cells incorporate free glucose rapidly into cellulose. This synthetase system that uses external UDP-glucose may serve for formation of wound callose.     

Effect of Brefeldin A on the synthesis and transport of cell wall polysaccharides and proteins in pea root seedlings

The in vivo effect of Brefeldin A (BFA) on the synthesis andtransport of cell wall polysaccharides and proteins in the rootsof pea seedlings (Pisum sativum L. cv. Alaska) was investigated.BFA (10µgml^—1 inhibited the synthesis of cell wallmatrix polysaccharides by approximately 43%. Under the sameconditions, cellulose synthesis was inhibited by approximately77%. The percentage of incorporation of L—[U—¹⁴C]leucineand L-[U-14C]proline into cytosolic, membrane and cell wallproteins was only slightly changed in the presence of BFA. Inaddition, the drug did not change the pattern of newly synthesizedproteins in the three fractions as judged by SDS—PAGEfluorography. Double labelling of proteins and cell wall polysaccharidesconfirmed the above reported data. All these results showedthat the synthesis and transport of proteins to the cell wallwas only slightly affected by BFA under similar conditions tothose which brought about a strong inhibition of the synthesisof matrix and cellulosic polysaccharides. BFA had no effecton the activity of membrane-bound and digitonin-solubilizedmannan and glucomannan synthase isolated from the third internodeof pea seedlings. This would exclude an effect of BFA at thelevel of the catalytic site of the synthases. The inhibitionof polysaccharide synthesis by the drug was rapidly eliminatedafter its removal. It is concluded that the effect of BFA onthe biosynthesis of cell wall polysaccharides could be causedby an interaction of the drug with the topological organizationof the synthase complexes in the membranes. This effect wouldprecede the action of the drug at the level of vesicle transportto the walls. Key words: Brefeldin A, cell wall polysaccharides (synthesis and transport), Pisum sativum L, polysaccharide synthases, proteins (synthesis and transport)     

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     

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     

An analysis of seed development in Pisum sativum XIX. Effect of mutant alleles at the r and rb loci on starch grain size and on the content and composition of starch in developing pea seeds

The effects of mutant alleles at the r and rb loci on starchgrain size and the levels of starch and amylose in developingpea (Pisum sativum L.) seeds have been examined. Four lines,near-isogenic except for genes at these loci, have been usedto show that both mutations reduce levels of starch throughoutembryo development and reduce levels still further when combinedin the ‘double mutant’. The reduction in starchcontent was due, at least in part, to a reduction in starchgranule size. Although the proportion of starch in mature embryoswas similar in the rrRbRb and RRrbrb lines, the starch contentdiffered between these two lines during development, as a percentageof embryo dry weight. This difference was due to a reductionin the absolute growth of the embryo caused by the rb mutation.Lines homozygous for the mutant r allele with either wild-type(RbRb) or mutant (rbrb) alleles at the rb locus contained increasedproportions of amylose in their starch throughout development,due to a reduced production of amylopectin. The presence ofthe rb mutation, however, also reduced the amount of amylosein relation to the reduction in total starch levels. Mutantalleles at both loci also reduced starch levels in the testaduring development, the reduction due to rb being more extreme.Reciprocal crosses showed a maternal effect of the rb mutationon final seed size and on the absolute amount of starch in theembryo. Key words: Pisum sativum L., seed, starch, development, mutant     

An antibody against a sequence of human topoisomerase II gives different immunofluorescence patterns in quiescent and proliferating nuclei of Pisum sativum L.

Immunofluorescence with an antibody against a C-terminal sequenceof human topoisomerase II has been performed on nuclei releasedfrom different tissues of Pisum sativum L. All the nuclei labelledand preincubation of the antibody with the corresponding immunogenpeptide strongly decreased the fluorescence. The labelling pattern(particularly nucleolar) was different in quiescent and proliferatingnuclei and changed during germination. In prophase nuclei, thelabelling was at the periphery of the condensing chromosomes,and in metaphase chromosomes, a characteristic labelling atpericentromeric regions was found. A computer search indicatedthat, apart from mammalian topoisomerase II, the immunogen peptidedid not match any other sequenced protein which could reasonablybe present in plant nuclei. The possible relation between theantigen recognized and topoisomerase II is discussed. Key words: Topoisomerase II, seed germination, Pisum sativum, immunofluorescence, nuclear proteins     

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