Radioautographic study of cell wall deposition in growing plant cells.

Segments cut from growing oat coleoptiles and pea stems were fed glucose-(3)H in presence and absence of the growth hormone indoleacetic acid (IAA). By means of electron microscope radioautography it was demonstrated that new cell wall material is deposited both at the wall surface (apposition) and within the preexisting wall structure (internally). Quantitative profiles for the distribution of incorporation with position through the thickness of the wall were obtained for the thick outer wall of epidermal cells. With both oat coleoptile and pea stem epidermal outer walls, it was found that a larger proportion of the newly synthesized wall material appeared to become incorporated within the wall in the presence of IAA. Extraction experiments on coleoptile tissue showed that activity that had been incorporated into the cell wall interior represented noncellulosic constituents, mainly hemicelluloses, whereas cellulose was deposited largely or entirely by apposition. It seems possible that internal incorporation of hemicelluloses plays a role in the cell wall expansion process that is involved in cell growth.

Ethylene and carbon dioxide: mediation of hypocotyl hook-opening response.

Ethylene at low concentrations inhibits the light-induced opening of the bean hypocotyl hook; auxin inhibits the opening by inducing production of ethylene. Light causes a decrease in ethylene production and an increase in the production of carbon dioxide. Hook opening appears to be a response in which ethylene serves as a natural growth regulator and in which carbon dioxide may be involved also as a growth regulator through its antagonism of the action of ethylene.

Cooperative action of beta-glucan synthetase and UDP-xylose xylosyl transferase of Golgi membranes in the synthesis of xyloglucan-like polysaccharide.

Golgi membranes of pea seedling tissue contain a UDP xylose:polysaccharide xylosyl transferase, the action of which is stimulated by UDP glucose. In the presence of both nucleotide-sugars a heteropolysaccharide containing both xylose and glucose (xyloglucan) is produced. Transfer of xylose and glucose units is presumed to be due to separate enzymes, because their properties differ in a number of respects. Xylosyl units appear to be transferred to a glucan core polysaccharide that is produced from UDP glucose by beta-1,4-glucan synthetase. This, rather than cellulose biosynthesis, is inferred to be the in vivo role of Golgi membrane beta-1,4-glucan synthetase.

Timing of the auxin response in coleoptiles and its implications regarding auxin action.

The timing of the auxin response was followed in oat and corn coleoptile tissue by a sensitive optical method in which the elongation of about a dozen coleoptile segments was recorded automatically. The response possesses a latent period of about 10 min at 23 degrees C, which is extended by low concentrations of KCN or by reducing the temperature, but is not extended by pretreatments with actinomycin D, puromycin, or cycloheximide at concentrations that partially inhibit the elongation response. Analysis of the data indicates that auxin probably does not act on the elongation of these tissues by promoting the synthesis of informational RNA or of enzymatic protein. Not excluded is the possibility that auxin acts at the translational level to induce synthesis of a structural protein, such as cell wall protein or membrane protein. While the data do not provide direct support for this hypothesis, the speed with which cycloheximide inhibits elongation suggests that continual protein synthesis may be important in the mechanism of cell wall expansion.

A 55 kDa plasma membrane-associated polypeptide is involved in beta-1,3-glucan synthase activity in pea tissue.

By glycerol gradient centrifugation of a detergent-solubilized plasma membrane fraction from pea tissue, we find a polypeptide of 55 kDa that copurifies with beta-1,3-glucan synthase activity. An antiserum against this polypeptide adsorbs glucan synthase activity and the 55 kDa polypeptide from digitonin-solubilized plasma membrane. These results indicate that the 55 kDa polypeptide is involved in pea beta-1,3-glucan synthase activity.

Genus-specific detection of salmonellae using the polymerase chain reaction (PCR).

Oligonucleotide primers for the polymerase chain reaction (PCR) that enable genus-specific detection of members of the genus Salmonella were developed. The primers amplify a 496-bp genetic sequence of members of the genus Salmonella. Amplification of DNA extracted from all other genera of the family Enterobacteriaceae and various other gram-positive aerobic and anaerobic bacteria yielded negative results. Applications of the PCR using these genus-specific primers are discussed.

Salmonella enteritidis immune leukocyte-stimulated soluble factors: effects on increased resistance to Salmonella organ invasion in day-old Leghorn chicks.

Cytokines, derived from either concanavalin A-stimulated Salmonella enteritidis-immune chicken T lymphocytes [SE-immune Lymphocyte Stimulated Soluble Factor (LSSF)] or lipopolysaccharide-stimulated SE-immune chicken macrophages [SE-immune Macrophage Stimulated Soluble Factor (MSSF)], were evaluated for their ability to increase resistance to SE organ invasion in day-old Leghorn chicks. In Trial 1, day of hatch chicks were injected i.p. with either SE-immune LSSF or SE-nonimmune LSSF (control). In Trial 2, chicks were similarly injected with either SE-immune MSSF, SE-nonimmune MSSF, or SE-immune LSSF (positive control). Thirty minutes postinjection, all chicks were gavaged with an invasive dose of SE. Twenty-four hours later, livers and spleens from all chicks were cultured for SE. In Trial 1, SE-immune LSSF caused a rapid and marked protection (P < .01) against SE infection as determined by the number of chicks that were culture positive regardless of challenge dose. In Trial 2, SE-immune MSSF was not associated with protection against SE organ infection. These experiments demonstrate that SE-immune LSSF, but not MSSF, are able to confer protection against SE organ invasion in day-old Leghorn chicks. Thus, it appears that the stimulated immune T cell, and not the macrophage, is responsible for producing the soluble products that protected the chicks.

Involvement of Macromolecule Biosynthesis in Auxin and Fusicoccin Enhancement of beta-Glucan Synthase Activity in Pea.

In pea stem segments whose cuticle has been made permeable by abrading it, actinomycin D (ActD) and 80S ribosomal protein synthesis inhibitors such as cycloheximide (CHI) inhibit enhancement by indole 3-acetic acid (IAA) of the activity of the cell wall biosynthetic enzyme, glucan synthase I (GS). This supersedes earlier, negative results with inhibitors, obtained with segments having an intact cuticle, which prevents adequate inhibitor uptake. Since these inhibitors also block IAA-stimulated H(+) extrusion, which according to earlier results is involved in the GS response, the significance of these inhibitions would be ambiguous without additional evidence. ActD does not inhibit fusicoccin (FC) enhancement of GS activity, which indicates existence of a post-transcriptional control mechanism for GS, but does not preclude involvement of transcription in the GS response to IAA. Although protein synthesis inhibitors such as CHI do not block FC-stimulated H(+) extrusion, they do inhibit FC enhancement of GS activity, indicating an involvement of protein synthesis in the GS response to FC, and presumably also to IAA. However, protein synthesis inhibitors (but not ActD) by themselves paradoxically elevate GS activity, less strongly than IAA does but resembling the IAA enhancement in several characteristics. These results suggest that IAA may enhance GS activity at least in part by inhibiting the synthesis or action of a labile repressor of the transcription of, or a labile destabilizer of, mRNA for GS or some polypeptide that enhances GS activity. However, resemblances between the IAA and FC effects on GS suggest that IAA also has a posttranscriptional GS-enhancing action like that of FC. Lipid biosynthesis may be involved in this aspect of the response since both IAA and FC enhancements of GS activity are inhibited by cerulenin.

Auxin and Fusicoccin Enhancement of beta-Glucan Synthase in Peas : An Intracellular Enzyme Activity Apparently Modulated by Proton Extrusion.

Fusicoccin (FC), like indoleacetic acid (IAA), causes Golgi-localized beta-1,4-glucan synthase (GS) activity to increase when applied to pea third internode segments whose GS activity has declined after isolation from the plant. This suggests that GS activity is modulated by H(+) extrusion; in agreement, vanadate and nigericin inhibit the GS response. The GS response is not due to acidification of the cell wall. Treatment of tissue with heavy water, which in effect raises intracellular pH, mimics the IAA/FC GS response. However, various treatments that tend to raise cytoplasmic pH directly, other than IAA- or FC-induced H(+) extrusion, failed to increase GS activity, suggesting that cytoplasmic pH is not the link between H(+) extrusion and increased GS activity. Although FC stimulates H(+) extrusion more strongly than IAA does, FC enhances GS activity at most only as much as, and often somewhat less than, IAA does. This and other observations indicate that GS enhancement is probably not due to membrane hyperpolarization, stimulated sugar uptake, or changes in ATP level, but leave open the possibility that GS is controlled by H(+) transport-driven changes in intracellular concentrations of ions other than H(+).

Auxin-binding Sites of Maize Coleoptiles Are Localized on Membranes of the Endoplasmic Reticulum.

Sites in maize (Zea mays L.) coleoptile homogenates that reversibly bind naphthalene-1-acetic acid with high affinity and may represent receptor sites for auxins are located primarily on cellular membranes that show the enzymic and buoyant density characteristics of membranes of the rough endoplasmic reticulum. The sites remain attached to the endoplasmic reticulum (ER) membranes after the ribosomes have been stripped off them. Binding sites for naphthylphthalamic acid, an inhibitor of auxin transport, are located on membranes different from those that carry the naphthalene-1-acetic-acid (NAA)-binding sites, and which are probably plasma membrane. The two kinds of binding sites can be largely separated by appropriate density gradient centrifugation. The results raise the possibility that primary auxin action occurs at ER membranes and could represent facilitation of the transfer of hydrogen ions and nascent secretory protein into the ER lumen followed by secretory transport of these products to the cell exterior via the Golgi system.

Specificity of Auxin-binding Sites on Maize Coleoptile Membranes as Possible Receptor Sites for Auxin Action.

Dissociation coefficients of auxin-binding sites on maize (Zea mays L.) coleoptile membranes were measured, for 48 auxins and related ring compounds, by competitive displacement of (14)C-naphthaleneacetic acid from the binding sites. The sites bind with high affinity several ring compounds with acidic side chains 2 to 4 carbons long, and much more weakly bind neutral ring compounds and phenols related to these active acids, most phenoxyalkylcarboxylic acids, and arylcarboxylic acids except benzoic acid, which scarcely binds, and triiodobenzoic acids, which bind strongly. Specificity of the binding is narrowed in the presence of a low molecular weight "supernatant factor" that occurs in maize and other tissues. Activity of many of the analogs as auxin agonists or antagonists in the cell elongation response was determined with maize coleoptiles. These activities on the whole roughly parallel the affinities of the binding sites for the same compounds, especially affinities measured in the presence of supernatant factor, but there are some quantitative discrepancies, especially among phenoxyalkylcarboxylic acids. In view of several factors that can cause receptor affinity and biological activity values to diverge quantitatively among analogs, the findings appear to support the presumption that the auxin-binding sites may be receptors for auxin action.

Regulation of beta-Glucan Synthetase Activity by Auxin in Pea Stem Tissue: I. Kinetic Aspects.

Treatment of pea stem segments with indoleacetic acid (IAA) causes within 1 hour a 2- to 4-fold increase in activity of particulate uridine diphosphoglucose-dependent beta-glucan synthetase obtainable from the tissue. The IAA effect is observable in tissue from all parts of the elongation zone of the pea stem, and also in older tissue that is not capable of a cell enlargement response to IAA. A large increase in activity is caused by IAA only if synthetase activity in the isolated tissue has first been allowed to fall substantially below the intact plant level, and only if sucrose is supplied along with IAA. Treatment of tissue with sucrose alone after a period of sugar starvation causes a transient rise of synthetase activity. The decline in synthetase activity in absence of IAA, the rise caused by IAA, and the transient rise caused by sucrose are all strongly temperature-dependent. IAA and sucrose do not affect the activity of isolated synthetase particles. Synthetase activity in vivo is sensitive to as low as 0.1 mum IAA and is increased by IAA analogues that are active as auxins on elongation but not by nonauxin analogues. Activity begins to rise 10 to 15 minutes after exposure to IAA, which places this among the most rapid enzyme effects of a plant growth regulator heretofore demonstrated, and among the most rapid known metabolic effects of auxins. The effect is seen also with polysaccharide synthetase activity using uridine diphosphate-galactose or uridine diphosphate-xylose as substrates, and to a lesser extent with guanosine diphosphoglucose-dependent glucan synthetase activity. Glucan synthetase from IAA-treated tissue appears to have a higher affinity for uridine diphosphate-glucose than the control.

Regulation of beta-Glucan Synthetase Activity by Auxin in Pea Stem Tissue: II. Metabolic Requirements.

The 2- to 4-fold rise in particle-bound beta-glucan synthetase (uridine diphosphate-glucose: beta-1, 4-glucan glucosyltransferase) activity that can be induced by indoleacetic acid in pea stem tissue is not prevented by concentrations of actinomycin D or cycloheximide that inhibit growth and macromolecule synthesis. The rise is concluded to be a hormonally induced activation of previously existing, reversibly deactivated enzyme. The activation is not a direct allosteric effect of indoleacetic acid or sugars. It is blocked by inhibitors of energy metabolism, by 2-deoxyglucose, and by high osmolarity, but not by Ca(2+) at concentrations that inhibit auxin-induced elongation and prevent promotion of sugar uptake by indoleacetic acid, and not by alpha, alpha'-dipyridyl at concentrations that inhibit formation of hydroxyproline. Regulation of the system could be due either to an ATP-dependent activating reaction affecting this enzyme, or to changes in levels of a primer or a lipid cofactor.

Molecular size and separability features of pea cell wall polysaccharides : implications for models of primary wall structure.

Relative molecular size distributions of pectic and hemicellulosic polysaccharides of pea (Pisum sativum cv Alaska) third internode primary walls were determined by gel filtration chromatography. Pectic polyuronides have a peak molecular mass of about 1100 kilodaltons, relative to dextran standards. This peak may be partly an aggregate of smaller molecular units, because demonstrable aggregation occurred when samples were concentrated by evaporation. About 86% of the neutral sugars (mostly arabinose and galactose) in the pectin cofractionate with polyuronide in gel filtration chromatography and diethylaminoethyl-cellulose chromatography and appear to be attached covalently to polyuronide chains, probably as constituents of rhamnogalacturonans. However, at least 60% of the wall's arabinan/galactan is not linked covalently to the bulk of its rhamnogalacturonan, either glycosidically or by ester links, but occurs in the hemicellulose fraction, accompanied by negligible uronic acid, and has a peak molecular mass of about 1000 kilodaltons. Xyloglucan, the other principal hemicellulosic polymer, has a peak molecular mass of about 30 kilodaltons (with a secondary, usually minor, peak of approximately 300 kilodaltons) and is mostly not linked glycosidically either to pectic polyuronides or to arabinogalactan. The relatively narrow molecular mass distributions of these polymers suggest mechanisms of co- or postsynthetic control of hemicellulose chain length by the cell. Although the macromolecular features of the mentioned polymers individually agree generally with those shown in the widely disseminated sycamore cell primary wall model, the matrix polymers seem to be associated mostly noncovalently rather than in the covalently interlinked meshwork postulated by that model. Xyloglucan and arabinan/galactan may form tightly and more loosely bound layers, respectively, around the cellulose microfibrils, the outer layer interacting with pectic rhamnogalacturonans that occupy interstices between the hemicellulose-coated microfibrils.

Changes in molecular size of previously deposited and newly synthesized pea cell wall matrix polysaccharides : effects of auxin and turgor.

Effects of indoleacetic acid (IAA) and of turgor changes on the apparent molecular mass (M(r)) distributions of cell wall matrix polysaccharides from etiolated pea (Pisum sativum L.) epicotyl segments were determined by gel filtration chromatography. IAA causes a two- to threefold decline in the peak M(r) of xyloglucan, relative to minus-auxin controls, to occur within 0.5 hour. IAA causes an even larger decrease in the peak M(r) concurrently biosynthesized xyloglucan, as determined by [(3)H]fucose labeling, but this effect begins only after 1 hour. In contrast, IAA does not appreciably affect the M(r) distributions of pectic polyuronides or hemicellulosic arabinose/galactose polysaccharides within 1.5 hours. However, after epicotyl segments are cut, their peak polyuronide M(r) increases and later decreases, possibly as part of a wound response. Xyloglucan also undergoes IAA-independent changes in its M(r) distribution after cutting segments. In addition, the peak M(r) of newly deposited xyloglucan increases from about 9 kilodaltons shortly after deposition to about 30 kilodaltons within 0.5 hour. This may represent a process of integration into the cell wall. A step increase in turgor causes the peak M(r) of previously deposited xyloglucan (but not of the other major polymers) to increase about 10-fold within 0.5 hour, returning to its initial value by 1.5 hours. This upshift may comprise a feedback mechanism that decreases wall extensibility when the rate of wall extension suddenly increases. IAA-induced reduction of xyloglucan M(r) might cause wall loosening that leads to cell enlargement, as has been suggested previously, but the lack of a simple relation between xyloglucan M(r) and elongation rate indicates that loosening must also involve other wall factors, one of which might be the deposition of new xyloglucan of much smaller size. Although the M(r) shifts in polyuronides may represent changes in noncovalent association, and for xyloglucan this cannot be completely excluded, xyloglucan seems to participate in a dynamic process that can both decrease and increase its chain length, possible mechanisms for which are suggested.

Phospholipid-synthesizing Enzymes Associated with Golgi Dictyosomes from Pea Tissue.

Golgi dictyosomal membranes isolated from pea (Pisum sativum) stem tissue, using a combination of rate zonal and isopycnic sucrose density centrifugation, were shown to bear cytidine diphosphate-choline:diglyceride phosphorylcholinetransferase, CDP-ethanolamine:diglyceride phosphorylethanolaminetransferase, and CTP:phosphorylcholine cytidyltransferase activities. Although the majority of the activity of the phospholipid-synthesizing enzymes was associated with the endoplasmic reticulum, the activity found in the Golgi system was about 25% of the total activity. These results suggest that Golgi dictyosomes probably synthesize at least part of the membrane phospholipids that they may need for their secretory function and for dictyosomal proliferation during cell growth, rather than importing this material entirely from the endoplasmic reticulum.

Characterization of naphthaleneacetic Acid binding to receptor sites on cellular membranes of maize coleoptile tissue.

Characteristics of and optimum conditions for saturable ("specific") binding of [(14)C]naphthaleneacetic acid to sites located on membranous particles from maize (Zea mays L.) coleoptiles are described. Most, if not all, of the specific binding appears to be due to a single kinetic class of binding sites having a K(D) of 5 to 7 x 10(-7)m for naphthalene-1-acetic acid (NAA). Binding of NAA is insensitive to high monovalent salt concentrations, indicating that binding is not primarily ionic. However, specific binding is inhibited by Mg(2+) or Ca(2+) above 5 mm. Specific binding is improved by organic acids, especially citrate. Binding is heat-labile and is sensitive to agents that act either on proteins or on lipids. Specific binding is reversibly inactivated by reducing agents such as dithioerythritol; a reducible group, possibly a disulfide group, may be located at the binding site and required for its function. The affinity of the specific binding sites for auxins is modified by an unidentified dialyzable, heat-stable, apparently amphoteric, organic factor ("supernatant factor") found in maize tissue.

Rapid Auxin-induced Decrease in Free Space pH and Its Relationship to Auxin-induced Growth in Maize and Pea.

A pH microelectrode has been used to investigate the auxin effect on free space pH and its correlation with auxin-stimulated elongation in segments of pea (Pisum sativum) stem and maize (Zea mays var. Bear Hybrid) coleoptile tissue. Auxin induces a decrease in free space pH in both tissues. In maize coleoptiles, free space pH begins to fall within about 12 minutes of exposure to auxin and decreases by about 1 pH unit by approximately 30 minutes. In pea, pH begins to decrease within an average of 15 to 18 minutes of exposure to auxin and falls by about 0.9 pH unit by approximately 40 minutes. Auxin-stimulated elongation, measured in the same two tissues similarly prepared, appears in maize at the earliest 18 minutes after auxin application, while in pea it appears at the earliest 21 to 24 minutes after auxin application. The auxin analogs p-chlorophenoxyisobutyric acid and phenylacetic acid do not stimulate elongation above control levels in maize or pea tissue segments and do not cause a decrease in free space pH in either tissue. These findings are consistent with the acid secretion theory of auxin action.

Nature of cell-to-cell transfer of auxin in polar transport.

By application of agar blocks ("side blocks") against the inner and outer epidermis of maize (Zea mays L.) coleoptiles whose cuticle has been abraded it is found that radioactive auxin in the polar transport stream exchanges rapidly with the tissue's free space and therefore does not move confined within the symplast. Polar transport of IAA is demonstrable in Avena coleoptile segments plasmolyzed in 0.5 and 0.7 M mannitol, in which most of the plasmodesmatal connections between successive cells in the polar transport pathway appear to have been broken. We conclude that during polar transport IAA probably moves from cell to cell by crossing the plasmalemmas and the free space between successive cells, rather than via plasmodesmata. Auxin in the polar transport stream exchanges rapidly with side blocks by a cyanide-and azide-insensitive, presumably passive, process. A similarly passive uptake takes place into the cells from an external donor. NPA almost completely inhibits efflux from the polar transport stream even though it does not inhibit uptake; its inhibition of efflux is completely reversed by azide or cyanide. These findings are compatible either with the traditional model of polar transport as passive uptake combined with an active basal efflux pump for IAA, or with the model of purely passive polar transport driven by pH and/or potential differences across the plasma membrane, provided certain ad hoc assumptions are made about the characteristics of the IAA anion carrier that would be operating in either model.

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.