Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles

Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl₁Glc₁), xyloglucan heptasaccharides (Xyl₃Glc₄), and octasaccharides (Gal₁Xyl₃Glc₄). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles.     

An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls

Plant cell walls expand considerably during cell enlargement, but the biochemical reactions leading to wall expansion are unknown. McQueen-Mason et al. (1992, Plant Cell 4, 1425) recently identified two proteins from cucumber (Cucumis sativus L.) that induced extension in walls isolated from dicotyledons, but were relatively ineffective on grass coleoptile walls. Here we report the identification and partial characterization of an oat (Avena sativa L.) coleoptile wall protein with similar properties. The oat protein has an apparent molecular mass of 29 kDa as revealed by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Activity was optimal between pH 4.5 and 5.0, which makes it a suitable candidate for acid growth responses of plant cell walls. The oat protein induced extension in walls from oat coleoptiles, cucumber hypocotyls and pea (Pisum sativum L.) epicotyls and was specifically recognized by an antibody raised against the 29-kDa wall-extension-inducing protein from cucumber hypocotyls. Contrary to the situation in cucumber walls, the acid-extension response in heat-inactivated oat walls was only partially restored by oat or cucumber wall-extension proteins. Our results show that an antigenically conserved protein in the walls of cucumber and oat seedlings is able to mediate a form of acid-induced wall extension. This implies that dicotyledons and grasses share a common biochemical mechanism for at least part of acid-induced wall extensions, despite the significant differences in wall composition between these two classes of plants.Abbreviations ConA concanavalin A - CM carboxymethyl - DEAE diethylaminoethyl - DTT dithiothreitol - Ex29 29-kDa expansin     

Role of Expansin in Cell Enlargement of Oat Coleoptiles (Analysis of Developmental Gradients and Photocontrol)

Expansins are wall proteins that mediate a type of acid-induced extension in isolated plant cell walls (S. McQueen-Mason, D.M. Durachko, D.J. Cosgrove [1992] Plant Cell 4: 1425-1433). To assess the role of these proteins in the process of cell enlargement in living tissues, we compared the spatial and temporal growth patterns of oat (Avena sativa L.) coleoptiles with four wall properties related to expansin action. These properties were (a) the ability of isolated walls and living segments to extend in acidic buffer, (b) the ability of heat-inactivated walls to extend upon application of expansins, (c) the amount of immunologically detectable expansin in wall protein extracts, and (d) the extractable expansin activity of walls. Growth rate was maximal in the apical half of dark-grown coleoptiles and negligible in the basal region. This growth pattern correlated with properties a and b; in contrast, the amount and activity of extractable expansin (properties c and d) were reduced only in the most basal region. Upon exposure to white light, coleoptiles abruptly ceased elongation at 8 to 10 h after start of irradiation, and this cessation correlated with reductions in properties a to c. The growth cessation at 8 to 10 h also coincided with the loss of growth response to exogenous auxin and fusicoccin in excised coleoptile segments. These results lend correlative support to the hypothesis that expansin action is important for growth responses of living oat coleoptiles (e.g. responses to acidic buffers, auxin, fusicoccin, aging, and light). Our results suggest that changes in the susceptibility of the wall to expansin action, rather than changes in expansin activity, may be a key determinant of the growth patterns in oat coleoptiles.     

pH-Dependence of Extension Growth in Avena Coleoptiles and Its Implications for the Mechanism of Auxin Action

The pH-dependence of acid-induced growth in excised segments of Avena sativa coleoptiles has been reinvestigated in the pH range 3 to 7. In contrast to previous reports (e.g. DL Rayle [1973] Planta 114: 63-73), only acidic buffers with a pH below 5.0 induce an extension response. A pH of 3.5 to 4.0 is required to mimic auxin-mediated growth. Very similar pH-response curves are obtained with both intact (abraded) and peeled coleoptiles. These results agree with the recent finding of a similarly low sensitivity to protons in maize coleoptiles. It is shown that the apparently much higher sensitivity to protons previously reported for peeled Avena coleoptiles is due to incubating the tissue in buffer of pH 6.8 between peeling and measuring the effect of acidic buffers. Neutral pH reversibly inhibits the spontaneous extension burst originating on release from tissue tension after removing the epidermis. Reversal of this inhibition can be achieved by buffers of pH 5.0 to 6.0 (or distilled water), thereby simulating an acid-induced growth response in this pH range. It is concluded that true acid-induced wall-loosening generally does not take place above pH 5.0 and that a pH considerably below 4.0 is required in order to stimulate growth to an extent comparable to that obtained in response to auxin. The “acid-growth theory,” which requires an acid-mediated loosening of the cell wall in the pH range 5 to 6, this pH being established by auxin-induced proton excretion, can therefore also not be substantiated in Avena.     

Distribution of pectins in cell walls of maize coleoptiles and evidence against their involvement in auxin-induced extension growth

Summary Aiming to elucidate the possible involvement of pectins in auxin-mediated elongation growth the distribution of pectins in cell walls of maize coleoptiles was investigated. Antibodies against defined epitopes of pectin were used: JIM 5 recognizing pectin with a low degree of esterification, JIM 7 recognizing highly esterified pectin and 2F4 recognizing a pectin epitope induced by Ca²⁺. JIM 5 weakly labeled the outer third of the outer epidermal wall and the center of filled cell corners in the parenchyma. A similar labeling pattern was obtained with 2F4. In contrast, JIM 7 densely labeled the whole outer epidermal wall except the innermost layer, the middle lamellae, and the inner edges of open cell corners in the parenchyma. Enzymatic de-esterification with pectin methylesterase increased the labeling by JIM 5 and 2F4 substantially. A further increase of the labeling density by JIM 5 and 2F4 and an extension of the labeling over the whole outer epidermal wall could be observed after chemical de-esterification with alkali. This indicates that both methyl- and other esters exist in maize outer epidermal walls. Thus, in the growth-controlling outer epidermal wall a clear zonation of pectin fractions was observed: the outermost layer (about one third to one half of wall thickness) contains unesterified pectin epitopes, presumably cross-linked by Ca²⁺ extract. Tracer experiments with³H-myo-inositol showed rapid accumulation of tracer in all extractable pectin fractions and in a fraction tightly bound to the cell wall. A stimulatory effect of IAA on tracer incorporation could not be detected in any fraction. Summarizing the data a model of the pectin distribution in the cell walls of maize coleoptiles was developed and its implications for the mechanism of auxin-induced wall loosening are discussed.Abbreviations CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid - CWP cell-wall pellet - IAA indole-3-acetic acid - LSE low-salt extract - TCA trichloroacetic acid; Tris tris-(hydroxy-methyl)aminoethane     

Role of Polysaccharide Synthesis in Elongation Growth and Cell Wall Loosening in Intact Rice Coleoptiles

2,6-Dichlorobenzonitrile (DCB) inhibited only increases in levelsof the cellulosic polysac-charides while monensin and galactoseinhibited increases in levels of both the cellulosic and thematrix polysaccharides in intact rice coleoptiles that weresubmerged in water. Elongation growth of rice coleoptiles wassuppressed by DCB at 10^–6 M, by monensin at 10^–7M, and by galactose at 3 ? 10^–3 M and above. Thus, thesynthesis of both the cellulosic and the matrix polysaccharidesis essential for the elongation of intact rice coleoptiles.These inhibitors increased the minimum stress-relaxation timeand the relaxation rate and they decreased the mechanical extensibilityof the cell wall, indicating that they inhibited cell wall loosening.The concentrations of the inhibitors required for inhibitionof cell wall loosening were higher than those for suppressionof elongation. The data suggest that polysaccharides synthesisplays two roles in elongation. It keeps the cell wall in a "loosened"condition by producing new extensible cell walls, while itsother role is probably related to the fixation or extensionof polymers already present in the cell wall. (Received November 15, 1990; Accepted May 23, 1991)     

In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical (^·OH)-mediated cell wall loosening during plant seed germination and seedling growth. We used electron paramagnetic resonance spectroscopy to show that ^·OH is generated in the cell wall during radicle elongation and weakening of the endosperm of cress (Lepidium sativum; Brassicaceae) seeds. Endosperm weakening precedes radicle emergence, as demonstrated by direct biomechanical measurements. By ³H fingerprinting, we showed that wall polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic ^·OH in radicles and endosperm caps: the production and action of ^·OH increased during endosperm weakening and radicle elongation and were inhibited by the germination-inhibiting hormone abscisic acid. Both effects were reversed by gibberellin. Distinct and tissue-specific target sites of ^·OH attack on polysaccharides were evident. In vivo ^·OH attack on cell wall polysaccharides were evident not only in germinating seeds but also in elongating maize (Zea mays; Poaceae) seedling coleoptiles. We conclude that plant cell wall loosening by ^·OH is a controlled action of this type of reactive oxygen species.     

Effects of multivalent cations on cell wall-associated Acid phosphatase activity

Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu²⁺, Mg²⁺, Zn²⁺, and Mn²⁺; unaffected by Ba²⁺, Cd²⁺, and Pb²⁺; and inhibited by Al³⁺. The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg²⁺. On the other hand, in the case of corn root cell walls (CCW), only inhibition of the bound acid phosphatase by Al³⁺ and Hg²⁺ was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg²⁺. In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca²⁺ significantly reduced the effects of Hg²⁺ or Al³⁺, but not Mg²⁺, to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg²⁺ or Al³⁺ which caused a Ca²⁺-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure.     

Absence of auxin-induced stored growth in Avena coleoptiles and its implication concerning the mechanism of wall extension

Summary We have reinvestigated the ability of Avena coleoptiles to undergo auxin-induced stored growth (stored growth is defined as the ability of a cell to store up a potential for extension during periods of reduced turgor which can be converted into extra extension upon restoration of normal turgor). We could detect little or no stored growth, with either moderate (1–2 bar) or more severe (3–5 bar) reductions in turgor, and with varying periods (10–100 min) of reduced turgor. Earlier reports of a stored growth potential (e.g., Cleland and Bonner, 1956) are shown to be in error, in that the apparent growth potential is probably an artifact of the use of argon or nitrogen as an inhibitor of auxin action. The absence of stored growth reported here is not due to a direct inhibitory effect of the osmoticum itself on auxin action, since coleoptiles can extend in response to auxin even in the presence of mannitol if an external force is applied to the section to replace the normal turgor. These results show that the two components of cell-wall extension, wall loosening and wall extension, usually are inseparable. Two possible explanations are considered; the walls may be extending by the process of chemical creep, or the wall loosening may only occur when the load-bearing bonds are under tension.     

Loss of capacity for acid-induced wall loosening as the principal cause of the cessation of cell enlargement in light-grown bean leaves

Cell enlargement in primary leaves of bean (Phaseolus vulgaris L.) can be induced, free of cell divisions, by exposure of 10-d-old, red-light-grown seedlings to white light. The absolute rate of leaf expansion increases until day 12, then decreases until the leaves reached mature size on day 18. The cause of the reduction in growth rate following day 12 has been investigated. Turgor calculated from measurements of leaf water and osmotic potential fell from 6.5 to 3.5 bar before day 12, but remained constant thereafter. The decline of growth after day 12 is not caused by a decrease in turgor. On the other hand, Instron-measured cell-wall extensibility decreased in parallel with growth rate after day 12. Two parameters influencing extensibility were examined. Light-induced acidification of cell walls, which has been shown to initiate wall extension, remained constant over the growth period (days 10–18). Furthermore, cells of any age could be stimulated to excrete H⁺ by fusicoccin. However, older tissue was not able to grow in response to fusicoccin or light. Measurements of acid-induced extension on preparations of isolated cell walls showed that as cells matured, the cell walls became less able to extend when acidified. These data indicate that it is a decline in the capacity for acid-induced wall loosening that reduces wall extensibility and thus cell enlargement in maturing leaves.Abbreviations and symbols FC fusicoccin - P turgor pressure - RL red light - WEx wall extensibility - WL white light - P _w leaf water potential - P _s osmotic potential     

Hydroxyl radical-induced cell-wall loosening in vitro and in vivo: implications for the control of elongation growth

Hydroxyl radicals (OH) are capable of unspecifically cleaving cell-wall polysaccharides in a site-specific reaction. I investigated the hypothesis that cell-wall loosening underlying the elongation growth of plant organs is controlled by apoplastically produced OH attacking load-bearing cell-wall matrix polymers. Isolated cell walls (operationally, frozen/thawed, abraded segments from coleoptiles or hypocotyls, respectively) from maize, cucumber, soybean, sunflower or Scots pine seedlings were pre-loaded with catalytic Cu or Fe ions and then incubated in a mixture of ascorbate + H2O2 for generating OH in the walls. This treatment induced irreversible wall extension (creep) in walls stretched in an extensiometer. The reaction could be promoted by acid pH and inhibited by several OH scavengers. Generation of OH by the same reaction in living coleoptile or hypocotyl segments caused elongation growth. Auxin-induced elongation growth of maize coleoptiles could be inhibited by OH scavengers. Auxin promoted the production of superoxide radicals (O2(-)), an OH precursor, in the growth-controlling outer epidermis of maize coleoptiles. It is concluded that OH fulfils basic criteria for a wall-loosening factor acting in auxin-mediated elongation growth of plant species with widely differing cell-wall polysaccharide compositions.     

Use of a pH-response curve for growth to predict apparent wall pH in elongating segments of maize coleoptiles and sunflower hypocotyls

To determine the relationship between apparent pH of the wall solution and shoot segment elongation, curves for the initial growth rates as a function of pH of the external solution were determined for maize (Zea mays L.) coleoptiles and sunflower (Helianthus annuus L.) hypocotyls and used to predict apparent wall pH in segments responding to indole-3-acetic acid (IAA) and fusicoccin (FC). When a solution having a pH predicted for walls of coleoptile segments responding to IAA was applied to the segments in the presence of IAA, this pH was not maintained. However, when the same was done for coleoptile segments responding to FC, the predicted pH was maintained in the external solution. Sunflower hypocotyl tissue did not maintain the external pH at the predicted value in the presence of either IAA or FC. The results indicate that wall loosening in coleoptiles caused by IAA may not be solely controlled by pH in the wall, yet growth (wall loosening) caused by FC apparently is directly related to wall pH. In sunflower the growth response to neither IAA nor FC appears to be directly correlated with wall pH.     

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as ''acid growth'', by which we mean they are more extensible at low pH (< 5) ¹. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism ^{2, 3}. Auxin activates a H⁺ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins ⁴, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This ''acid growth'' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins ⁵. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 °C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.Open in a separate windowClick here to view.^{(58M, flv)}     

beta-d-Glucan Antibodies Inhibit Auxin-Induced Cell Elongation and Changes in the Cell Wall of Zea Coleoptile Segments

Antiserum was raised against the Avena sativa L. caryopsis β-d-glucan fraction with an average molecular weight of 1.5 × 10⁴. Polyclonal antibodies recovered from the serum after Protein A-Sepharose column chromatography precipitated when cross-reacted with high molecular weight (1→3), (1→4)-β-d-glucans. These antibodies were effective in suppression of cell wall autohydrolytic reactions and auxin-induced decreases in noncellulosic glucose content of the cell wall of maize (Zea mays L.) coleoptiles. The results indicate antibody-mediated interference with in situ β-d-glucan degradation. The antibodies at a concentration of 200 micrograms per milliliter also suppress auxin-induced elongation by about 40% and cell wall loosening (measured by the minimum stress-relaxation time of the segments) of Zea coleoptiles. The suppression of elongation by antibodies was imposed without a lag period. Auxin-induced elongation, cell wall loosening, and chemical changes in the cell walls were near the levels of control tissues when segments were subjected to antibody preparation precipitated by a pretreatment with Avena caryopsis β-d-glucans. These results support the idea that the degradation of (1→3), (1→4)-β-d-glucans by cell wall enzymes is associated with the cell wall loosening responsible for auxin-induced elongation.     

Effects of gibberellic Acid, calcium, kinetic, and ethylene on growth and cell wall composition of pea epicotyls

The influence of gibberellic acid (GA), calcium, kinetin, and ethylene on growth and cell-wall composition of decapitated pea epicotyls (Pisum sativum L. var. Alaska) was investigated. Calcium, kinetin, and ethylene each caused an inhibition of GA-induced elongation of pea stems. Gibberellic acid did not reverse the induction of swelling by Ca²⁺, kinetin, or ethylene. Both Ca²⁺ and ethylene significantly inhibited the stimulatory effects of GA on the formation of residual wall material. Although GA promoted the development of walls relatively low in pectic substances and pectic uronic acid, Ca²⁺, kinetin, and ethylene favored the formation of walls rich in these constituents. Calcium, kinetin, and GA, alone or in combination, had no effect on the production of ethylene by pea epicotyls.     

Uptake and Metabolic Fate of Glucose, Arabinose, and Xylose by Zea mays Coleoptiles in Relation to Cell Wall Synthesis

According to the acid-growth hypothesis, auxin-induced secretion of hydrogen ions activate “wall loosening” enzymes that change the rheological properties of the cell wall. The wall loosening process may yield monosaccharides by the enzymic cleavage of load-bearing polysaccharides. Our study was initiated to determine the metabolic fate of such sugars when released from the major hemicellulosic polysaccharides of the cell walls of Zea mays coleoptiles.     

Cellular and subcellular localization of calcium in gravistimulated oat coleoptiles and its possible significance in the establishment of tropic curvature

Light—and electron-microscopic studies of the distribution of calcium in gravitropically responding oat (Avena sativa L. cv. “Garry”) coleoptiles are described. A modification of the antimonate precipitation procedure was used to localize tissue calcium in situ. An accumulation of Ca in the upper halves of horizontal, gravistimulated coleoptiles is seen within 10 min of stimulus onset. A pronounced redistribution of Ca to the upper side occurs within 30 min; although the localization of this cation is not uniform along the organ axis and in the apical region, Ca appears to accumulate along the lower side. The observed asymmetric distribution of Ca in these tissues precedes large-scale visible bending by 20–30 min, but is temporally well-correlated with differential growth responses in the coleoptile, as measured by more sensitive quantitative techniques. Gravitropic curvature is well developed by 3 h and is accompanied by further redistribution of Ca to tissues along the upper coleoptile half, centered around the bend. Ultrastructural localization studies indicate that Ca asymmetry results primarily from changes in the distribution of Ca within the apoplastic compartment. Large amounts of Ca accumulate at the cuticle in epidermal cell walls and in the walls of the underlying parenchyma cells at the upper side of the organ in the region of maximal bending. The differential growth response resulting in the establishment of gravitropic curvature may largely be the consequence of antagonistic effects of Ca on auxin-mediated cell wall loosening and elongation growth processes at the upper side of the organ.     

Does indoleacetic acid promote growth via cell wall acidification?

Growth of Triticum aestivum L. cv. Cappelle Desprez coleoptiles is promoted by 5.7×10^–5 M indole acetic acid (IAA) as effectively in pH 3.4 buffer as in water, but IAA is not effective in the presence of buffer at pH 3.0 or 3.2 A combination of 5.7×10^–5 M IAA and pH 3.4 buffer promotes growth to a greater extent than pH 3.2 buffer alone, which is optimal for acid-induced growth. IAA employed at 10^–7 M is still effective at promoting growth in the presence of pH 3.4 buffer, moreover, IAA at 10^–7 M interacts synergistically with the acidic buffer to promote growth. It is concluded that IAA and acid promote growth via separate mechanisms, and that IAA does not promote cell wall loosening by rendering the cell wall more acid.Abbreviation IAA Indoleacetic acid     

Cyanide Inhibition of Acid-induced Growth in Avena Coleoptile Segments

The comparative effects of metabolic inhibitors on acid- and auxininduced growth in oat (Avena sativa L. var. Victory) coleoptile segments have been examined. Acid (pH 4)-induced growth in both peeled and unpeeled segments is inhibited by 1 millimolar KCN when added at the time of acidification. KCN inhibits total acid-induced growth by 59 and 76%, respectively, in peeled and nonpeeled segments during the first 60 minutes. The growth rate of cyanide-treated tissue drops to zero or near zero in both peeled and nonpeeled segments during this period. Cyanide inhibition of total acid-induced growth in peeled segments at pH 5 is even more severe, amounting to about 80% during the first 60 minutes. The possibility that inhibition by cyanide may be caused by some nonspecific effect of the inhibitor on a process other than respiration, e.g. turgor reduction due to membrane damage, has not been ruled out. Acid-induced growth is also inhibited by 3 millimolar sodium fluoride and by anoxia. In unpeeled segments total pH 4-induced growth is inhibited 73% by sodium fluoride and 38% by anoxia during the 1st hour. Possible corrections to the above inhibition percentages which may be necessary due to the sensitivity of basal growth to inhibitors are discussed. Cyanide was found to inhibit auxin-induced growth much more rapidly than acid-induced growth. These data suggest that acid growth may be dependent on respiratory metabolism but to a lesser degree than is auxin-induced growth. If the acid growth theory of auxin action is correct, it appears that there may be two steps in the growth process which are dependent on respiratory metabolism: (a) auxin-induced proton pumping which is highly sensitive to respiratory inhibitors; and (b) acid-mediated wall loosening which is moderately and perhaps indirectly sensitive to respiratory inhibitors.     

Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth

Hydroxyl radicals (*OH), produced in the cell wall, are capable of cleaving wall polymers and can thus mediate cell wall loosening and extension growth. It has recently been proposed that the biochemical mechanism responsible for *OH generation in the cell walls of growing plant organs represents an enzymatic reaction catalyzed by apoplastic peroxidase (POD). This hypothesis was investigated by supplying cell walls of maize ( Zea mays L.) coleoptiles and sunflower ( Helianthus annuus L.) hypocotyls with external NADH, an artificial substrate known to cause *OH generation by POD in vitro. The effects of NADH on wall loosening, growth, and *OH production in vivo were determined. NADH mediates cell wall extension in vitro and in vivo in an H2O2-dependent reaction that shows the characteristic features of POD. NADH-mediated production of *OH in vivo was demonstrated in maize coleoptiles using electron paramagnetic resonance spectroscopy in combination with a specific spin-trapping reaction. Kinetic properties and inhibitor/activator sensitivities of the *OH-producing reaction in the cell walls of coleoptiles resembled the properties of horseradish POD. Apoplastic consumption of external NADH by living coleoptiles can be traced back to the superimposed action of two enzymatic reactions, a KCN-sensitive reaction mediated by POD operating in the *OH-forming mode, and a KCN-insensitive reaction with the kinetic properties of a superoxide-producing plasma-membrane NADH oxidase the activity of which can be promoted by auxin. Under natural conditions, i.e. in the absence of external NADH, this enzyme may provide superoxide (O2*-) (and H2O2 utilized by POD for) *OH production in the cell wall.