Control by pH of Cell Wall Peroxidase Activity Involved in Lignification

pH affected both conyferyl alcohol (CA) oxidation and NADH-dependentH₂O₂ formation catalysed by cell wall-bound peroxidase fromlupin. The kinetics of CA oxidation shown at pH 5.0 was of theMichaelian-type, independently of the physical state (boundor soluble) of the enzyme in the cell wall, whereas at pH 8.0,it was cooperative. The affinity of the enzyme to H₂O₂and therate of H₂O₂ formation were higher at pH 8.0 than at pH 5.0.The kinetics of H₂O₂ formation at pH 8.0 was strongly cooperative.These results are discussed with respect to the important roleof pH in the regulation of cell wall peroxidase activities involvedin lignification. (Received December 12, 1988; Accepted December 7, 1988)     

Cell Growth and the Structure and Mechanical Properties of the Wall in Internodal Cells of Nitella opaca: I. WALL STRUCTURE AND GROWTH

This is the first of two papers dealing with the relationshipbetween growth and the mechanical properties of the wall ininternodal cells of Nitella opaca L. The submicroscopic structure of the cell wall of this alga,as determined by chemical analysis, X-ray crystallography, polarizingmicroscopy, electron microscopy, swelling measurements, andinfra-red spectrography, is described in detail and the changesduring growth are recorded. It has been found that the wallcontains cellulose in the form of cellulose I (type B). Theconstituent microfibrils are preferentially oriented, usuallyin slow helices with considerable angular dispersion about thecommon direction. They are arranged in discrete layers withpectic substances providing an amorphous matrix between microfibrillar-reinforcedlaminations. It is shown that, as the cell elongates, both thestreaming direction in the cell and the mean microfibrillarorientation in the wall change in such a way as to allow thepossibility of a causal connexion between streaming and microfibrillarorientation in a new wall lamella. The orientation in such alamella is undoubtedly modified by subsequent passive extensionmuch as implied in the multi-net growth hypothesis of Roelofsen.     

Proton-Metal Cation Exchange in the Cell Wall of Nitella flexilis

When protons are exchanging for bivalent cations (Cu²⁺, Zn²⁺, or Ca²⁺) on the carboxylic groups of Nitella flexilis cell wall, the values of the respective global equilibrium constants do not change up to a protonation degree of 80%. These values drastically increase at higher proton concentrations and tend to 3.4, which is the intrinsic pK value of the constitutive α-d-galacturonic acid monomer. These data suggest that the electric field in the matricial polymer and the cation bridges between pairs of negative sites have disappeared.     

Cell Growth and the Structure and Mechanical Properties of the Wall in Internodal Cells of Nitella opaca: III. SPIRAL GROWTH AND CELL WALL STRUCTURE

The internodal cells of the alga Nitella opaca L, which arein the form of long thin cylinders, exhibit the phenomenon ofspiral growth, i.e. as the cells elongate they also twist abouttheir longitudinal axis. It has been shown in an earlier paper(Probine and Preston, 1962) that the cell wall is mechanicallyanisotropic. In this paper the moduli necessary to describethe elastic behaviour of a material possessing this sort ofsymmetry are considered. It is pointed out that if the Nitellacell is regarded as a thin-walled cylinder built of a materialpossessing orthorhombic elastic symmetry, then there can bea coupling between shear and extension which will produce atorsional twist as the cylinder is pressurized. It is suggested that this is the basic mechanism of spiral growth.Experimental evidence is presented which supports this view.     

Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

Acidification of the extracellular and/or intracellular environment is involved in many aspects of cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca²⁺/calmodulin-dependent kinase that regulates translation elongation by phosphorylating and inhibiting eEF2. Here we show that extracellular acidosis elicits activation of eEF2K in vivo, leading to enhanced phosphorylation of eEF2. We identify five histidine residues in eEF2K that are crucial for the activation of eEF2K during acidosis. Three of them (H80, H87, and H94) are in its calmodulin-binding site, and their protonation appears to enhance the ability of calmodulin to activate eEF2K. The other two histidines (H227 and H230) lie in the catalytic domain of eEF2K. We also identify His108 in calmodulin as essential for activation of eEF2K. Acidification of cancer cell microenvironments is a hallmark of malignant solid tumors. Knocking down eEF2K in cancer cells attenuated the decrease in global protein synthesis when cells were cultured at acidic pH. Importantly, activation of eEF2K is linked to cancer cell survival under acidic conditions. Inhibition of eEF2K promotes cancer cell death under acidosis.     

Ionic Diffusion Through the Nitella Cell Wall in the Presence of Calcium

Diffusion potentials (concentration and bi- or multi-ionic potentials)in KC1, NaCl, or LiCl solutions have been measured across anisolated cell wall of Nitella, with or without the same concentrationof CaCl² on either side of the cell wall. The absolute valueof the potentials decreases as the external Ca²⁺concentrationincreases and it may happen that an inversion of the sign ofthe concentration potentials results when the external Ca²⁺solution reaches 1 mM. Dosages of K⁺ and Ca²⁺ in the cell wallhave shown that Ca²⁺ easily displaces the monovalent ion fromthe exchange sites and tends to neutralize the cationic exchanger.However, in most cases, the measured potentials are still morenegative than the theoretical potentials which would be setup by a neutral-site membrane in the same conditions. Theseresults suggest that Ca²⁺ largely reduces the discriminationproperties of the cell wall between cations and anions.     

Al Binding in the Epidermis Cell Wall Inhibits Cell Elongation of Okra Hypocotyl

Al inhibits root elongation at micromolar concentrations, butthe mechanisms leading to this process are unknown. In thesestudies, Al-induced inhibition of cell elongation was examinedusing hypocotyl of okra (Abelmoschus esculentus Moench cv. ClemsonSpineless) as an experimental model. One-h exposure to Al (0.5mM A1Cl₃) in the presence of 10 µM auxin in 0.5 mM CaCl₂,pH 4.0 significantly inhibited auxin-induced cell elongationof okra hypocotyl segments. Elongation was further suppressedwith increasing Al concentrations up to 1 mM. Treatment of thehypocotyl with 1 mM citrate for 10 minutes after 2-h exposureto Al resulted in significant recovery of elongation. The amountof Al in the cell wall relative to the total in the tissue was96.0, 96.2, and 85.4%, respectively, following 1-, 2-, and 3-hexposure to the Al solution. The total and cell wall Al contentwas decreased by half after the citrate desorption treatment.Further-more, 95% of Al was found in the epidermis, and 95%of the Al in the epidermis was associated with the cell wall.Experiments using split hypocotyl segments showed that Al exposureincreased the outward bending of hypocotyl segments, suggestingthat the epidermis elongation was specifically inhibited byAl. Al inhibited the autolysis of epidermis by about 20%, buthad little effect on the autolysis of core tissue. Taken together,these results suggest that Al binding in the epidermal cellwall inhibits critical components in cell wall loosening mechanism,resulting in inhibition of cell elongation.     

The Capacity for Acid-Induced Wall Loosening as a Factor in the Control of Avena Coleoptile Cell Elongation

The rate of acid-induced wall extension depends not only onthe pH of the wall solution, but on the capacity of the wallsto undergo acid-induced wall loosening (CAWL). CAWL has beenevaluated by incubating sections in vivo under various conditions,freezing them, and then measuring the rate of acid-induced extensionwhen the thawed sections are subjected to a constant stressand a constant acidic pH. CAWL varies in response to auxin andfusicoccin (FC). While the control CAWL declines steadily, auxincauses the CAWL to increase slowly so that after 10 h auxin-treatedsections undergo acid-induced wall loosening at twice the rateof the controls, given the same pH and stress. This increasein CAWL is unlikely to be a response to the elongation becauseno such increase occurs in the presence of FC. With FC, boththe CAWL and the growth rate decrease sharply after about 2h, suggesting that in this case the changes in CAWL might bethe primary factor controlling the growth rate. The CAWL maybe a function of the proteins present in the wall rather thanthe polysaccharides, since the protein synthesis inhibitor cycloheximidecauses the CAWL to decrease while sucrose has no effect on theCAWL. Key words: Avena sativa, Acid-growth, Auxin, Coleoptile, Wall loosening     

Conformational Transitions and Modifications in Ionic Exchange Properties Induced by Alkaline Ions in the Nitella flexilis Cell Wall

The decrease in the cationic exchange capacity (CEC) concomitantto the replacement in the Nitella wall of adsorbed Mn²⁺ ionswas measured in different mixtures of alkaline ions. At lowexternal concentrations, the loss of CEC is enhanced in presenceof Li⁺ ions but is weaker when Na⁺ ions are present in the exchangemixtures. The relative affinity of the wall exchange sites foralkaline ions was Na⁺>K⁺>Rb⁺Cs⁺>Li⁺. As the CEC isprogressively reduced, the wall discrimination between the differentalkaline ions tends to cancel out except for the Na⁺-K⁺ pair.The wall preference for K⁺ is then increased. A diminution ofthe effective pK of the polygalacturonic acids constitutiveof the wall is also observed, while increasing the CEC loss.The simple disruption of divalent cation crosslinks cannot fullyexplain the CEC leakage at low monovalent concentrations. Itis suggested that the alkaline ions also cleave H bonds or solvatation-likebonds between the cell wall polyuronides and then cause a concomitantunfolding of the short pectic chains which involves their solubilization. (Received May 6, 1993; Accepted November 8, 1993)     

Photoinhibition of Stem Elongation by Blue and Red Light : Effects on Hydraulic and Cell Wall Properties

The underlying mechanism of photoinhibition of stem elongation by blue (BL) and red light (RL) was studied in etiolated seedlings of pea (Pisum sativum L. cv Alaska). Brief BL irradiations resulted in fast transient inhibition of elongation, while a delayed (lag approximately 60 minutes) but prolonged inhibition was observed after brief RL. Possible changes in the hydraulic and wall properties of the growing cells during photoinhibition were examined. Cell sap osmotic pressure was unaffected by BL and RL, but both irradiations increased turgor pressure by approximately 0.05 megapascal (pressure-probe technique). Cell wall yielding was analyzed by in vivo stress relaxation (pressure-block technique). BL and RL reduced the initial rate of relaxation by 38 and 54%, while the final amount of relaxation was decreased by 48 and 10%, respectively. These results indicate that RL inhibits elongation mainly by lowering the wall yield coefficient, while most of the inhibitory effect of BL was due to an increase of the yield threshold. Mechanical extensibility of cell walls (Instron technique) was decreased by BL and RL, mainly due to a reduction in the plastic component of extensibility. Thus, photoinhibitions of elongation by both BL and RL are achieved through changes in cell wall properties, and are not due to effects on the hydraulic properties of the cell.     

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.     

Correlation Between the Weight Loss Induced by Alkaline Ions and the Cationic Exchange Capacity of the Nitella Cell Wall

There is a linear relationship between loss of weight and lossof cationic exchange capacity of Nitella cell walls, inducedby treatment with alkaline ions. As the rate of CEC loss dependsto a much greater extent on the ionic species than does themaximal loss quantity, it is suggested that the alkaline ionscompete with the bivalent ions initially adsorbed for a particulartype of site. These are different from the carboxylate exchangesites since the equivalent fractions of the alkaline ions adsorbedon to these sites are about the same for external concentrationswhere the effect of the different ions on weight and CEC lossis, nevertheless, quite different. Such sites give to the matrixpectin its structural coherence by preventing the action ofelectrostatic repulsive forces. In fact, it also appears thatCEC loss is a linear function of the inverse of the square rootof the internal ionic strength, a parameter proportional tothe Debye length. Key words: Cationic exchange capacity, cell wall, Nitella flexilis     

Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of Lactuca sativa L.

Four 13-hydroxygibberellins, gibberellin A₁ (GA₁), 3-epi-GA₁,GA₁₉ and GA₂₀ were identified by full-scan GC/MS in extractsof lettuce seedlings (Lactuca sativa L. cv. Grand Rapids). Theresults suggest that the early-13-hydroxylation biosyntheticpathway to GA₁ functions in the lettuce seedlings. It was alsofound that GA₁ is active per se in the control of hypocotylelongation in lettuce seedlings. To investigate the relationshipbetween control by light of hypocotyl elongation and levelsof endogenous GAs in lettuce, endogenous levels of GAs werequantified by radioimmunoassay in seedlings that had been grownfor 5 days in the dark (5D) and in those that had been grownfor 4 days in the dark and then under white light for 1 day(4D1L). The endogenous level of GA₁ in the upper and lower partsof hypocotyls in 5D seedlings was about four times higher thanthat in 4D1L seedlings. The response of explants (hypocotylsegments with cotyledons) from dark-grown seedlings to GA₁ isknown to be similar in the dark and under white light when theexplants are treated with inhibitors of the biosynthesis ofGA. Therefore, the above information suggests that the highlevel of GA₁ in hypocotyls of dark-grown seedlings is responsiblefor the rapid elongation of hypocotyl, while irradiation bywhite light decreases the endogenous level of GA₁ in the hypocotylswith a resultant decrease in the rate of hypocotyl elongation. (Received March 13, 1992; Accepted May 21, 1992)     

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)     

Inhibition of Cell Elongation in Avena Coleoptile by Hydroxyproline

A study has been made of the hydroxyproline-induced inhibition of elongation of Avena coleoptile tissues. The isomers of 4-hydroxyproline differ in their effectiveness; only the L isomers are growth inhibitors with the cis form (allohydroxyproline) being more effective than the trans form (hydroxyproline).Hydroxyproline differs from other amino acid antagonists and protein synthesis inhibitors in respect to 2 characteristics of the growth inhibition. First, a certain increment of auxin-induced elongation must take place following addition of hydroxyproline before the growth is inhibited. In contrast, pretreatment with other amino acid antagonists or protein synthesis inhibitors completely eliminates the ability of Avena coleoptile sections to respond to auxin. Secondly, sucrose markedly increases the magnitude of the hydroxyproline inhibition; i.e., sucrose acts to inhibit rather than promote growth when in the presence of hydroxyproline.It appears that hydroxyproline is a specific inhibitor for the synthesis of some factor which is utilized in elongation. Following addition of hydroxyproline, auxin-induced elongation continues until the pool of this factor is exhausted; then elongation is inhibited.     

Control of Wheat Root Elongation Growth: I. EFFECTS OF IONS ON GROWTH RATE, WALL RHEOLOGY AND CELL WATER RELATIONS

Pritchard, J., Tomos, A. D. and Wyn Jones, R. G. 1987. Controlof wheat root elongation growth. I. Effects of ions on growthrate, wall rheology and cell water relations.—J. exp.Bot. 38: 948–959. The nature of the ions in the bathing medium of hydroponicallygrown wheat seedlings strongly influenced root growth rate.In 0·5 mol m^–3 CaSO₄ the growth rate was 32 mm24 h^–1 (used as 100% control rate). K⁺ and SO^– ions(10 mol m^–3) each inhibited extension growth (to about40% and 70% of the control value respectively). In the absenceof K⁺, Cl^– greatly reduced the inhibition due to SO₄^2–.Measurement of tissue plasticity and elasticity in the expandingzone with an Instron-type tensiometer indicated that both werea function of growth rate although relationship of plasticityto growth rate was the steeper and the more pronounced. Turgor pressure at the proximal end of the expanding zone wasnot correlated to growth, being approximately 0·65 MPain all treatments. In mature tissue turgor pressure varied withtreatment, but was also not related to growth rate. Cell membranehydraulic conductivity (5 x 10^–7 ± 1·3 (10)m s^–1 MPa^–2) was not influenced by the presenceof K⁺. We propose that K⁺ and SO₄^{2 –} influence root growthrates by modulating the rheological properties of the wallsof the expanding cell. The physiological significance of these properties is discussed. Key words: Growth, wall extensibility, turgor pressure, wheat roots