Corn Coleoptile Segments Research Articles

IAA-glucopyranoside stimulation of corn coleoptiles elongation

It has been previously suggested that 1-O-IAGIuc growth stimulation occurs as the effect of its hydrolysis into a free IAA. In present experiments castanospermine, a known β-glucosidase inhibitor, was included. 1-O-IAGluc in the presence of castanospermine stimulated growth of corn coleoptiles segments even stronger then free IAA. So, it seems that 1-O-IAGluc itself, is responsible for the observed stimulation of corn coleoptile segments elongation.

Molecular and Biochemical Evidence for the Involvement of Calcium/Calmodulin in Auxin Action

The use of (35)S-labeled calmodulin (CaM) to screen a corn root cDNA expression library has led to the isolation of a CaM-binding protein, encoded by a cDNA with sequence similarity to small auxin up RNAs (SAURs), a class of early auxin-responsive genes. The cDNA designated as ZmSAUR1 (Zea mays SAURs) was expressed in Escherichia coli, and the recombinant protein was purified by CaM affinity chromatography. The CaM binding assay revealed that the recombinant protein binds to CaM in a calcium-dependent manner. Deletion analysis revealed that the CaM binding site was located at the NH(2)-terminal domain. A synthetic peptide of amino acids 20-45, corresponding to the potential CaM binding region, was used for calcium-dependent mobility shift assays. The synthetic peptide formed a stable complex with CaM only in the presence of calcium. The CaM affinity assay indicated that ZmSAUR1 binds to CaM with high affinity (K(d) approximately 15 nM) in a calcium-dependent manner. Comparison of the NH(2)-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic alpha-amphiphilic helix similar to the CaM binding region of ZmSAUR1. CaM binds to the two synthetic peptides from the NH(2)-terminal regions of Arabidopsis SAUR-AC1 and soybean 10A5, suggesting that this is a general phenomenon for all SAURs. Northern analysis was carried out using the total RNA isolated from auxin-treated corn coleoptile segments. ZmSAUR1 gene expression began within 10 min, increased rapidly between 10 and 60 min, and peaked around 60 min after 10 microM alpha-naphthaleneacetic acid treatment. These results indicate that ZmSAUR1 is an early auxin-responsive gene. The CaM antagonist N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride inhibited the auxin-induced cell elongation but not the auxin-induced expression of ZmSAUR1. This suggests that calcium/CaM do not regulate ZmSAUR1 at the transcriptional level. CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the post-translational level. Our data provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction.

(Aminooxy)acetic acid inhibits petunia growth and gibberellin- and cytokinin-stimulated growth in bioassays

(Aminooxy)acetic acid (AOA) was applied to greenhouse-grown petunias and was used in bioassays for three plant growth hormones so that its growth regulator properties could be studied. In greenhouse studies foliar sprays of 4.8–12 mm AOA inhibited vegetative growth of petunia seedlings (Petunia xhybrida Vilm. ‘White Flash’). When gibberellin A 3 (GA3) was applied to shoot tips previously treated with AOA, plant growth was stimulated, but there was no AOA x GA3 interaction. Some changes in petunia leaf morphology induced by AOA were reversed by GA3. AOA inhibited elongation of corn coleoptile segments (Zea mays L. B73 x Mol7) whether or not 10 μm indole-3-acetic acid (IAA) was present, but there was no AOA x IAA interaction. AOA reduced lettuce hypocotyl (Lactuca sativa L. ‘Grand Rapids’) elongation induced by GA3 and radish cotyledon (Raphanus sativus L. ‘Champion’) expansion induced by benzyladenine (BA). We propose that AOA interferes with postsynthetic metabolism of plant hormones during cell elongation induced by GA3 and cell expansion induced by BA.

Autoregulation of Auxin Transport in Corn Coleoptile Segments

Polar transport of auxin decreased and its net uptake increased in maize (corn) coleoptile segments preincubated in buffer following excision from the intact seedling. Reduced rate of cellular efflux of auxin accounted for these changes in preincubated segments where the endogenous auxin level was depleted. The difference in net uptake of auxin between freshly excised and preincubated tissues was completely abolished by the phytotropin N-1-naphthylphthalamic acid. Studies on in vitro binding of NPA to membrane vesicles isolated from the tissue indicated that the number of binding sites on the plasma membrane was likewise reduced following excision of the tissue. Presence of exogenous auxin in the preincubation medium prevented the decay of both auxin transport and the putative auxin efflux carriers. These results suggest that polar auxin transport in corn coleoptiles is autoregulated by endogenous auxin level through its influence on the auxin efflux carrier.

Sensitivity of ATPases to silver ions suggests that silver acts outside the plasma membrane to block ethylene action

The possibility of inhibition of ATPases, when silver salts are used to inhibit ethylene action in plant tissues, was considered. The vanadate-sensitive ATPase in a plasma membrane preparation was sensitive to 1 μM silver nitrate. Inhibition was reversed by a 20-fold excess of sodium thiosulphate, or dithiothreitol, but not by EDTA. This ATPase was also inhibited by silver nitrate in the presence of a four- or eight-fold excess of sodium thiosulphate. Mitochondrial ATPases appeared to be less sensitive than the plasma membrane enzymes to silver nitrate, but more sensitive to silver thiosulphate mixtures. Dilution of silver thiosulphate increased its inhibitory effect. Silver above 10 μM promoted ion and sugar efflux from corn coleoptile segments, but tissue respiration was only inhibited at 500 μM silver. It is suggested that ethylene action is inhibited by silver acting outside the plasma membrane.

Control of Apoplast pH in Corn Coleoptile Segments. I: The Endogenous Regulation of Cell Wall pH

Summary The endogenous control of cell wall pH of Zea mays L. coleoptiles has been investigated using abraded as well as non-abraded segments. Regardless of the initial medium pH, coleoptile segments characteristically change the external pH: after about 2 h a pH maximum ( ) around 6 is reached, followed by a slow pH decrease that levels off in a steady state at 4.8 after 7 h ( ). This basic behavior is independent of medium composition, but critically requires O 2 ; it is not altered when the number of coleoptiles per volume is increased to over 10 col./mL. Thus, experimental conditions can be chosen that eliminate the time needed to establish an equilibrium medium pH as a major source of error. When IAA is added at only slight effects on medium pH are observed, but when added at , IAA exerts rapid and reversible acidification. Since pH development through the segments is rather insensitive to different external conditions and the pH is fully recovered after IAA induced acidification, we conclude that the apoplast pH is endogenously regulated. Furthermore, since the response to IAA is only weak if added at , we suggest that such experiments should be started during .

Control of Apoplast pH in Corn Coleoptile Segments. II: The Effects of Various Auxins and Auxin Analogues

Using abraded Zea mays L. coleoptile segments, proton extrusion has been measured in the presence of growth promoting auxins and their inactive structural analogues. Prior to application, the segments spontaneously acidified the external medium to pH 4.8 within 7h. Indole-3-acetic acid (IAA) concentration dependently decreases external pH to 4.2, fusicoccin (FC) to below 3.5. Whereas the IAA-effect spontaneously recovers and can be repeated, the FC-effect is final and irreversible. The growth promoting auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), indole-3-butyric acid (IBA) and 1-naphthylacetic acid (1-NAA) all stimulate proton extrusion similar to IAA. The growth inactive structural analogues 2,3-dichlorophenoxyacetic acid (2,3-D) and 2-naphthylacetic acid (2-NAA) also acidify the external medium, but quantitatively to a lesser extent. p-chlorophenoxyisobutyric acid (PCIB) does not inhibit the IAA response. No effects were obtained with deactivated H+-ATPase (anoxia) and in the presence of benzoic acid.

Wall‐protein antibodies as inhibitors of growth and of autolytic reactions of isolated cell wall

Antiserum raised against protein prepared from corn (Zea mays L. hybrid B73 X Mo17) seedling cell wall exhibits antigrowth activity and inhibits autolytic reactions of isolated cell wall. The growth of corn coleoptile segments pretreated in wall‐protein antiserum was inhibited 35% compared to tissue which had been exposed to normal serum. Since the cuticle constituted a barrier to the entry of serum protein the coleoptile surface was abraded prior to exposure to the serum. Growth inhibition observed in nonabraded sections (8 to 10%) was apparently the result of the entry of serum protein via the cut ends of the tissue. Subsections recut from the central region of antiserum‐treated, nonabraded coleoptiles responded normally in growth experiments. Antiserum brought in contact with isolated cell wall prior to its use with intact coleoptiles was less effective as a growth inhibitor, which suggested that the antibodies which impede growth responses bind to cell wall.Wall‐protein antiserum also strongly inhibited autolytic reactions of isolated cell wall. Wall treated in normal serum or buffer liberated sugars at a rate of 8.6 μg‐ (mg wall dry‐weight)−1·h−1 while antiserum‐treated wall autolyzed at a rate of 2.6 μg·mg−1·h−1 representing a 70% reduction in autolytic potential. After 24 h yields from antiserum‐treated wall were 50% lower than yields obtained from control wall. The antiserum had no effect on the molecular‐weight distribution of the autolytically‐liberated products, and direct assays of wall‐bound and solubilized endo‐ and exo‐β‐d‐glucanases involved in autolytic reactions demonstrated no significant impairment of their catalytic activity. However, the addition to antiserum‐treated wall of a wall‐protein preparation containing autolytic enzymes resulted in a significant enhancement (132%) in autolytic potential. The evidence suggests that enzyme mobility may be critical in the autolytic hydrolytsis of plant cell wall.

An Improved Method for Detecting Auxin-induced Hydrogen Ion Efflux from Corn Coleoptile Segments

Conditions necessary to detect maximal auxin-induced H(+) secretion using a macroelectrode have been investigated using corn coleoptile segments. Auxin-induced H(+) secretion is strongly dependent upon oxygenation or aeration when the tissue to volume ratio is high. Cuticle disruption or removal is also necessary to detect substantial auxin-induced H(+) secretion. The auxin-induced decrease in pH of the external medium is stronger when the hormone is applied to tissue in which the cuticle has been disrupted with an abrasive than when the hormone is applied to tissue from which the cuticle and epidermis have been removed by peeling. The lower detectable acidification of the external medium when using peeled segments appears to be due in part to the leakage of buffers into the medium and in part to the removal of the auxin-sensitive epidermal cells.The sensitivity of corn coleoptile segments to auxin, as measured by H(+) secretion, increases about 2-fold during the first 2 hours after excision. This change in apparent sensitivity to auxin as reflected by H(+) secretion is paralleled by a time-dependent change in the growth response to auxin. Under optimal conditions for detecting H(+) efflux (oxygenation, abrasion, hormone application 2 hours after excision), the latent period in auxin-induced H(+) efflux (about 7 or 8 minutes) is only half as great as the latent period in auxin-induced growth (about 18 to 20 minutes). These observations are consistent with the acid growth hypothesis of auxin action.

Phytochrome Modifies Blue-light-induced Electrical Changes in Corn Coleoptiles

Unilateral blue light administered to corn coleoptile segments produces no alteration of transmembrane potential on the light side, and only a small and slow hyperpolarization on the dark side. Red light causes a 5-15 millivolt depolarization in cells on the light side causes and somewhat smaller effects on the dark side. Blue given after red causes a rapid hyperpolarization on both sides of the coleoptile. The effect of the potentiating red preirradiation is probably due to phytochrome, being largely abolished by far-red given after red, but before the blue light. The effect of prior red irradiation decays in the dark, showing a half-time of about 45 minutes at room temperature. This rapid cooperativity between phytochrome and the phototropic pigment may indicate a common locale, possibly in a membrane.

Nonhormonal induction of H + efflux from plant tissues and its correlation with growth

When the esterase substrate alpha-naphthyl acetate is added to segments from coleoptiles of oat (Avena sativa L., var. Victory) or corn (Zea mays L., Bear hybrid WF 9 x 38) or to roots of lentil (Lens culinaris Med.), it is rapidly taken up and hydrolyzed to alpha-naphthol and acetic acid. This technique was used to generate intracellular acid and to study the effect of its efflux on growth. With corn coleoptile segments, treatment with alpha-naphthyl acetate induced more rapid and more prolonged growth than did exposure to external acidic buffers. Dose-response studies of alpha-naphthyl acetate induction of H(+) secretion and growth indicated the existence of a distinct threshold rate of H(+) efflux below which growth is not enhanced. The rate of H(+) efflux induced by optimal levels of auxin appears to be at or below this threshold level in corn coleoptile tissue. These data indicate that the relationship between external acidification and elongation is not a simple one and that the effectiveness of H(+) entering the cell wall may depend on factors not directly correlated with the appearance of H(+) in the external medium.

The Nature of Spontaneous Changes in Growth Rate in Isolated Coleoptile Segments

About 4 hours after they are cut from the seedling, corn (Zea mays L.) coleoptile segments mounted vertically show a strong increase in growth rate. This increase occurs in water or various buffers near pH 7 and is not accompanied by the accumulation of a growth promoter in the medium. The increase in growth rate is prevented by 1 mmp-fluorophenylalanine and is strongly inhibited by 0.1 mmp-chlorophenoxyisobutyric acid.The increased growth rate is accompanied by a 95% increase in the ability of tissue extracts to catalyze the conversion of (14)C-tryptophan to (14)C-indole-3-acetic acid and by a nearly 3-fold increase in indole-3-acetic acid oxidase activity. The increase in growth rate is also observed in segments from coleoptiles grown aseptically.The spontaneous increase in growth rate is completely but reversibly inhibited by 1 mum indole-3-acetic acid. Cytokinins have little effect on the spontaneous growth response, whereas gibberellic acid is observed to extend the latent period and reduce the magnitude of the response. It is tentatively concluded that the increase in endogenous growth rate may result from increased auxin production upon derepression of the auxin biosynthesis pathway after isolating the tissue from the normal supply of auxin from the tip.

Linear Velocity of Cyclic Adenosine 3′,5′-Monophosphate Transport in Corn Coleoptiles

The transport of cyclic adenosine 3', 5'-monophosphate in corn coleoptile segments is very rapid. The linear velocity of basipetal transport is 183 millimeters per hour, while the velocity of acropetal transport is 79 millimeters per hour. Transport velocity as well as intensity thus appear to be polar in the corn coleoptile. Application of metabolic inhibitors such as cyanide, ouabain, and 2,4-dinitrophenol increase rather than decrease the velocity and intensity of transport. The mechanism of transport in light of these data is discussed.

Rapid-growth responses of corn root segments: Effect of auxin on elongation

The effect of indole-3-acetic acid (IAA) on the elongation rates of 2 mm corn (Zea mays L.) root segments induced by citrate-phosphate buffer (or unbuffered) solutions of pH 4.0 and 7.0 was studied. At pH 7.0, auxin initially reduced the elongation rate in both buffered and unbuffered solutions. Only in buffer at pH 7.0 was auxin at a concentration of 0.1 μM found to promote the elongation rate though briefly. THis promoted rate represented only ca. 20% of the rate achieved with only buffer at pH 4.0. Auxin in pH 4.0 buffered and unbuffered solutions only served to reduce the elongation rates of root segments. Some comparative experiments were done using 2 mm corn coleoptile segments. Auxin (pH 6.8) promoted the elongation rate of coleoptile segments to a level equal or greater than the maximal H ion-induced rate. The two responses of root segments to auxin are compared to auxin action in coleoptile growth.

Relationship Between Metabolism and the Lateral Transport of IAA in Corn Coleoptiles

The lateral movement of IAA in coleoptiles of Zea mays has been investigated under aerobic and anaerobic conditions. The IAA-1-(14)C was supplied asymmetrically to the apical end of the segment. The results were as follows: A) In air more (14)C was found in the lower half of horizontal segments supplied with an upper donor than in the half opposite the donor in vertical segments. The enhanced lateral movement of (14)C in geotropically stimulated segments of corn coleoptiles under aerobic conditions has thus been confirmed. B) This increased lateral movement of (14)C in geotropically stimulated segments is greatly reduced, but is not completely abolished, under anaerobic conditions. C) The lateral movement of (14)C in vertical segments is significantly less under anaerobic conditions than in air. D) Under anaerobic conditions, the lateral movement of (14)C in horizontal segments can be reduced to the level found in vertical segments by pre-soaking the tissue in a 1 mm solution of the metabolic inhibitor sodium fluoride for 2 hours. The inhibitor has no effect on lateral movement of (14)C in vertical anaerobic segments. E) In air, sodium fluoride has no effect on the lateral movement of (14)C in either vertical or horizontal segments.On the assumption that the movement of (14)C reflects the movement of IAA, these results show that the enhanced polarized lateral movement of IAA observed in horizontal corn coleoptile segments is totally abolished only when both aerobic and anaerobic metabolism are arrested. In addition, there appears to be a small metabolically dependent component in the lateral movement of IAA in vertical segments supplied with an asymmetric donor in air.

The Effect of 2,4-Dichlorophenoxyacetic Acid on the Respiration of Etiolated Pea Seedlings.

Since the report of Brown (6) that 2,4-dichlorophenoxyacetic acid (2,4-D) increased the rate of carbon dioxide production from intact bean plants, many reports on the influence of 2,4-D on respiration have appeared in the literature. Smith (14) and Avery (1) have reviewed the literature which appeared before 1950. Some of the more recent papers are listed in the Literature Cited section of this paper (7, 8, 12, 17, 18). French and Beevers (7) suggest that the increase in respiration induced by 2,4-D, indoleacetic acid (IAA), and other plant growth substances is a result of the increase in growth induced by these substances. An increase in growth with an attendant increase in anabolic reactions would increase the supply of high energy phosphate acceptors, and thus, presumably, increase the rate of respiration. This suggestion was based, in part, on the findings that these plant growth substances, in the concentration range that increased the elongation of corn coleoptile segments, increased the respiration of the segments, while 2,4-dinitrophenol, which inhibited growth, also caused a marked increase in the rate of respiration. The work of Kelly and Avery (12) lends support to this interpretation. These workers found that, although the respiration of young pea stem tissue was markedly stimulated by 2,4-D, the respiration of tissue slices from older pea stem segments which had ceased to elongate was not increased by 2,4-D. Bonner and Bandurski (4), in their discussion of the action of IAA on respiration and growth, are inclined to a view similar to that of French and Beevers (7). This paper reports the results of an investigation on the phenomenon of respiratory stimulation by 2,4-D with respect to the qualitative and quantitative changes in the respiratory substrate. It is postulated that 2,4-D increases respiration by causing more glucose to be catabolized via the pentose phosphate path-