Differential effect of Calcium and Magnesium on Mechanical Properties of Pea Stem Cell Walls

Replacement of calcium ion with magnesium ion in the cell wallof the pea epicotyl makes the wall more extensible. A possiblerole of this differential effect of Ca²⁺ and Mg^2– in regulatingcell elongation in pea epicotyl is discussed. The ratio of the content of calcium ion to that of magnesiumion (Ca²⁺/Mg²⁺) in the walls decreased markedly in the orderof the first > the second > the third internodes of thepea epicotyl. The capacity of the walls for cation exchangeincreased in the same order, whereas the calcium-magnesium ionexchange selectivity of the walls was virtually constant. Ourresults indicate that the changes in the Ca²⁺/Mg²⁺ ratio amongthe internodes is not attributable to the ion exchange propertiesof the walk per se, but is due to other physiological conditionswhich regulate the activities of free calcium and magnesiumions in the environment with which the walls are in equilibrium. ¹ Present address: Wakayama Research Laboratories, Kao SoapCo., Ltd., Wakayama 640-91, Japan ² Present address: Foold Development Laboratories, Meiji SeikaKaisha Ltd., Kawasaki 210, Japan (Received May 1, 1981; Accepted August 26, 1981)     

Equilibrium Analysis of Calcium Binding to Cell Walls Isolated from Plant Tissue

Primary cell walls were isolated and purified from potato tubersand carrots via a Parr N₂ bomb technique. Calcium binding topurified cell walls was measured with both calcium selectiveelectrode and use of the metallochromic indicator, ArsenazoIII. The cell walls used in this study were biologically activeand presumably approached the physiological cell wall. Aliquotsof the untreated cell walls (control) were then salt-extractedor EDTA-treated and binding properties were compared to thecontrols. In addition, the binding properties of freshly preparedcell walls were compared to cell walls which were stored for1 week at 2°C. Both simple Scatchard plot analysis and anelectrostatic interaction model were used to evaluate calciumbinding parameters. The controls from the two tissue types hadinherently different calcium binding properties and these propertieswere affected by treating the cell walls with salt or EDTA.Cold storage treatment drastically changed the binding propertiesof carrot cell walls but had negligible effect on potato tubercell walls. (Received January 28, 1992; Accepted April 3, 1992)     

Properties and Possible Physiological Significance of Cell Wall Calcium Binding in Etiolated Pea Epicotyls

Baydoun, E. A-H. and Brett, C. T. 1988. Properties and possiblephysiological significance of cell wall calcium binding in etiolatedpea epicotyls.—J. exp. Bot. 39: 199–208. The binding of ⁴⁵Ca²⁺ ions to cell walls prepared from pea epicotylswas examined in young and old parts of the epicotyl, and wasfound to be considerably greater, on a carbohydrate basis, inthe older, non-growing cells. A similar comparison between light-and dark-grown stems showed greater binding in the dark-grownstems. The polygalacturonase-insensitive component of the bindingcontained at least three types of binding with different affinities,and had an apparent pK of 4.3. The specificity of the bindingfor calcium ions was examined and a considerable degree of specificitywas observed. The specificity of inhibition by calcium of epicotylelongation was similar to the specificity of calcium binding.A specific calcium chelator, EGTA, when present at a concentrationof above 10 mol m^–3, promoted the extension of matureregions of the epicotyl, while inhibiting extension of youngertissue. Key words: Cell wall, calcium, pea epicotyl     

Autolysis Promotes the Extension Capacity of Zea mays Coleoptile Cell Walls in Response to Acid pH Solutions

The relationship between autolytic degradation of ß(1–3),(1–4)-D-glucanand acid pH-induced extension of isolated Zea mays cell wallshas been investigated using a constant-load extension technique.Acidic buffer (4.5) was able to induce an additional extension(E_a) on cell walls already extended at pH 6.8 buffer under a20 g-mass load, indicating that the additional extension (E_a)was the parameter that better represented the effect of thedifferent treatments on the mechanical properties of maize coleoptilecell walls. The additional extension in response to acidic pHwas higher when cell walls had been previously autolysed for24 h at pH 5.5. Furthermore, the acid-pH effect was dependenton the presence during the constant load extension of some thermo-labilefactors, suggesting the participation of expansins. Acid pHincreased E_a of native cell walls through an increase in theplastic extension (E_p) in agreement with a one step mechanismleading directly to irreversible (plastic) wall extension assuggested by Cosgrove (1977). The autolytic degradation of ß(1–3),(1–4)-D-glucan was also able to modify the mechanicalproperties of maize coleoptile cell walls increasing its elasticextension (E_e) in response to pH 4.5 buffer but that modificationonly leads to an increase in wall extension when expansins areactive, suggesting a cooperation between ß-glucanturnover and expansin action. (Received August 5, 1998; Accepted March 16, 1999)     

Evidence Against the Involvement of Ionically Bound Cell Wall Proteins in Pea Epicotyl Growth

Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.     

Effect of GA3 on the Molecular Mass of Polyuronides in the Cell Walls of Alaska Pea Roots

The role of cell wall matrix polysaccharides in gibberellin-regulatedroot growth is unknown. We examined pectic polysaccharides frompea roots treated with or without gibberellin A₃ (GA₃) in thepresence of ancymidol, an inhibitor of gibberellin biosynthesis.Pectic polymers solubilized by CDTA (trans-l,2-cyclohexanediamine-N,N,N',N'-tetraaceticacid) at 23°C and subjected to gel permeation analysis exhibitedhigh polydispersity with a molecular mass in excess of 500 kDa.Subsequent extraction of cell walls with CDTA at 100°C solubilizedpolymers with an average mol mass of 10 to 40 kDa. Subjectingthe high molecular mass pectic polymers extracted at 23°Cto 70–100°C for 2h generated 10 to 40 kDa fragments,similar in size distribution to those solubilized directly fromcell walls by CDTA solutions at 100°C. Pectic polymers from(GA₃+Anc)-treated roots were of higher average mol mass thanthose from Anc-treated roots in both the elongation zone andin the basal maturation zone. Since (GA₃+Anc)-treated rootselongate more quickly than Anc-treated roots [Tanimoto (1994)Plant Cell Physiol. 35:1019], the slender, GA₃-treated rootsmay produce and deposit highly integrated pectins more rapidlythan the thicker, Anc-treated roots in the elongating or elongatedcell walls. ²Present address: Horticultural Sciences Department, POB 110690IFAS, University of Florida, Gainesville, FL 32611-0690 U.S.A.     

The Epidermis of the Pea Epicotyl Is Not a Unique Target Tissue for Auxin-Induced Growth

Previous research has suggested that the epidermis of dicotyledonous stems is the primary site of auxin action in elongation growth. We show for pea (Pisum sativum L.) epicotyl sections that this hypothesis is incorrect. In buffer (pH 6.5), sections from which the outer cell layers were removed (peeled) elongated slowly and to the same extent as intact sections. Addition of 10 micromolar indoleacetic acid to this incubation medium caused peeled sections to grow to the same extent and with the same kinetics as auxin-treated nonpeeled sections. This indicates that both epidermis and cortical tissues have the ability to respond rapidly to auxin and that the epidermis is not the sole site of auxin action in dicotyledonous stems. Previous reports that peeled pea sections respond poorly to auxin may have resulted from an acid extension of these sections due to the use of distilled water as the incubation medium.     

Effect of Calcium on the Structure and Template Activity of Pea Chromatin

The absorption spectrum of native pea chromatin solubilizedunder minimal shearing conditions changed with increasing Ca²⁺concentration; the ratio of maximum to minimum absorption decreasedand the maximum absorption peak shifted to a longer wavelength.The concentration of Ca²⁺ to cause half complete sedimentationof chromatin was much lower for the solubilized native chromatin(more condensed and larger in size) than for the sonicated chromatin(less condensed and smaller in size). Solubilized native chromatinshowed a two-step melting profile in the absence of Ca²⁺. In the presence of Ca²⁺ the two T_ms disappeared and a new higherT_m appeared. Template activity of solubilized native chromatinincreased 3-fold upon dispersion and fragmentation by sonication.Addition of a small amount of ethylene glycol-bis (ß-aminoethylether)-N, N'-tetraacetic acid (EGTA) promoted the template activityof solubilized native chromatin, but not that of sonicated ordenatured DNA. The effect of EGTA was reversed by Ca²⁺. Thechromatin reconstituted in the presence of EGTA showed a lowerT_m than the chromatin reconstituted in the presence of Ca²⁺.The relationship between chromatin structure and its templateactivity is discussed in relation to Ca²⁺. (Received August 12, 1985; Accepted December 7, 1985)     

The Incorporation of Leucine-C14 into Microsomal Particles and other Subcellular Components of the Pea Epicotyl

Incorporation of leucine-C¹⁴ into subcellular fractions of the apical section of pea seedlings has been studied as a function of the length of incubation. The specific activity of the microsomes was higher than that of the supernatant for short but not for long incubations, in agreement with observations on other systems. In this developing tissue the nuclei and especially the mitochondria appear to incorporate amino acid very rapidly. An insoluble fraction of the microsome pellet, which is presumably a liponucleoprotein complex, was found to possess, after 1 hour of incubation, a specific activity much greater than that of the purified microsomal particles or the supernatant fraction. Ninety-eight per cent of the leucine-C¹⁴ in the purified microsomal particles has been shown to possess bound amino groups, presumably in peptide linkages, by the DNP-end group method. These particles liberate but little peptide or protein of very high specific activity when they are destroyed by removal of Mg or by hydrolysis of RNA. Microsomal particles were fractionated into an RNA fraction and five protein fractions by means of density gradient centrifugation. By this method 95 per cent of the RNA can be separated from 90 per cent of the protein of the particle. Furthermore, the RNA fraction has been shown to contain very little protein of high specific activity. A particular protein fraction which contains the remaining 5 per cent of the RNA, possessed after 1 hour of incubation a specific activity 2 to 9 times higher than the protein of the other fractions.     

Expansive Growth vs. pH Reflects a Poisson Point Process of Binding/Unbinding Events in Plant Cell Walls

Journal of Plant Growth Regulation - The paramount role of $$\mathrm{pH}$$ and temperature $$\left(T\right)$$ in the expansive growth of a plant coleoptile/hypocotyl non-meristematic zone or plant...     

Binding of Deoxyribonucleic Acid by Cell Walls of Transformable and Nontransformable Streptococci

Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.     

Effect of Gibberellic Acid on Rate of Extension and Maturation of Pea Internodes

Gibberellic acid (GA) does not delay maturation of pea internodes;on the con-trary, maturation (i.e. cessation of extension) takesplace slightly earlier. Thus the increased length of internodesresulting from GA treatment is due entirely to increased rateof extension. In this experiment, GA treatment of plants accelerated the visibleproduction of the first flower bud by about 4 days: the nodebearing the first flower was not altered. The total number offlower buds produced by the end of the experiment was increasedas a result of GA treatment, but many of those first formedon plants receiving high doses (I-IOµg.) withered beforeopening.     

Lectin Binding Studies on the Cell Walls of Soybean Rust (Phakopsora pachyrhizi Syd.)

Walls of uredospores, infection structures, intercellular hyphae and haustoria of the soybean rust fungus (Phakopsora pachyrhizi) were studied by electron microscopy using gold-labeled wheat germ lectin (WGL) and Concanavalin A (ConA) as cytochemical probes. Receptors for WGL (probably chitin) were detected in all fungal walls included in this study. WGL-binding occurred throughout the entire walls (uredospores, appressorial cone, penetration hyphae, haustorial mother cells) or only to the inner wall layers (germ tubes, appressoria, intercellular hyphae).     

Serum Binding of Calcium and the Red Cell Membrane in Cystic Fibrosis

THERE is evidence for a factor in the serum of cystic fibrosis patients which may play a role in the pathological manifestation of the genetic syndrome: serum of patients with cystic fibrosis alters the cilia movement of rabbit trachea in vitro¹ and of oyster cilia². This activity is apparently associated with the immunoglobulin fraction^3,4. Balfe et al.⁵ reported a modification of sodium flux in red cells obtained from patients with cystic fibrosis. In the course of searching for evidence of interaction of serum with red cells in cystic fibrosis, we found that there is a substantial increase in serum calcium binding in patients with cystic fibrosis. This seems to be associated with a modified membrane protein electrophoretic pattern of cystic fibrosis red cells in specified experimental conditions.     

The Effect of Calcium Antagonists on Auxin-induced Elongation and on the Expression of Two Auxin-Regulated Genes in Pea Epicotyls

Calcium has been implicated in various regulatory roles in plantcells including auxin-induced cell elongation. Treatment ofpea epicotyl segments with the calcium chelators, EGTA and chlorotetracycline(CTC), the calcium ionophore, A23187 [GenBank] , and channel blocker, D-600,inhibits auxin-induced cell elongation. Depletion of tissuecalcium either by EGTA or EGTA and a calcium ionophore doesnot interfere with the induction of the early auxin induciblemRNAs pIAA4/5 and pIAA6. Similarly, an increase in cytosoliccalcium with calcium and calcium ionophore neither induces thehormonally regulated mRNAs nor interferes with their inductionby auxin. The calcium channel blocker, D-600, is without effecton the auxin-regulated mRNA induction. The results indicatethat calcium is not involved in the rapid induction of IAA4/5and IAA6 genes in pea tissue. However, a possible role for calciumin the translation of these mRNAs, or in the expression of otherauxin-regulated genes, is not excluded. ³Present Address: Department of Biology, Tokyo MetropolitanUniversity, Tokyo, Japan. (Received April 8, 1988; Accepted July 30, 1988)