Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles

Distribution of endogenous diffusible auxin into agar blocks from phototropically stimulated maize coleoptile tips was studied using a bioassay and a physicochemical assay, to clarify whether phototropism in maize coleoptiles involves a lateral gradient in the amount of auxin. At 50 min after the onset of phototropic stimulation, when the phototropic response was still developing, direct assay of the blocks with the Avena curvature test showed that the auxin activity in the blocks from the shaded half-tips was twice that of the lighted side, at both the first and second positive phototropic curvatures. However, physicochemical determination following purification showed that the amount of indole-3-acetic acid (IAA) was evenly distributed in the blocks from lighted and shaded coleoptile half-tips at both the first and second positive phototropic curvatures. The even distribution of the IAA was also confirmed with the Avena curvature test following purification by HPLC. These results indicate that phototropism in maize coleoptiles is not caused by a lateral gradient of IAA itself and thus cannot be described by the Cholodny-Went theory. Furthermore, the lower auxin activity in the blocks from the lighted half-tips suggests the presence of inhibitor(s) interfering with the action of auxin and their significant diffusion from unilaterally illuminated coleoptile tips.     

Control of growth by auxin and its specific neutral inhibitor

Evidence for the existence of a neutral inhibitor specific for auxin has been in the literature for 45 years. The inhibitor is demonstrable by its effect in causing positive curvature of theAvena coleoptile. The growth of the mesocotyl of oat and corn seedlings in darkness and its inhibition by light are determined by the neutral inhibitor as is the phototropic response of the sunflower stem. Production of the inhibitor is promoted by red and fluorescent light. Irradiance at 730 nm promotes auxin production while irradiance at 660 nm promotes production of the inhibitor. The positive curvature induced by 2,3,5-triiodobenzoic acid can be used to quantify the neutral inhibitor. Since the benzoic acid is more effective than iodoacetate in reacting with the sulfhydryl of cysteine, a sulfhydryl group is indicated to bo one reaction site for auxin and to be the basis of polar transport.     

Conversion of isatin to isatate as related to growth promotion in Avena coleoptile and pisum stem sections

Isatin, (indole 2,3-dione), which promotes elongation of Pisum stem sections at concentrations exceeding 0.1 mm, promotes elongation of Avena coleoptile sections only at higher concentrations, exceeding 1 mm. Aged isatin solutions are more active than fresh solutions, due to the slow, spontaneous conversion to isatate (o-aminophenylglyoxylate). A concentration of 0.1 mm aged isatin is as active in Avena coleoptile sections as in peas. Isatate has been independently synthesized and its auxin activity in both Avena coleoptile and Pisum stem sections confirmed. The synthetic isatate is more effective than isatin in both systems. This suggests that the auxin activity of isatin is due to its conversion to isatate.     

A DESEEDED AVENA TEST METHOD FOR SMALL AMOUNTS OF AUXIN AND AUXIN PRECURSORS

The main results presented in this article may be summarized as follows: 1. A test method with deseeded Avena seedlings for small concentrations of auxin and precursors of auxin has been described. 2. This method makes possible quantitative determinations of about ten times as low concentrations of hormone as can be obtained with the standard method, (a) Through an increase in the time of the test, so that nearly all the hormone applied can be utilized. (b) Through an increase in sensitivity of deseeded plants to unilaterally applied small concentrations of hormone. 3. The effect of deseeding in relation to curvature growth is primarily the prevention of auxin regeneration through the removal of the material for auxin synthesis, and in addition the prevention of physiological aging. 4. The mechanism of auxin synthesis in the tip of the coleoptile and the mechanism of auxin regeneration in the new physiological tip have been shown to be identical. 5. The application of the deseeded method is illustrated by determinations of auxin in primary leaves and coleoptile sections of Avena seedlings. 6. The deseeded method has been used as a test method for precursors of auxin obtainable from the coleoptile and from other sources. The method further makes possible a distinction between auxins and these substances which may become activated by the plant. 7. Evidence for the existence of a precursor of auxin in the plant is given (a) indirectly by determinations of the decrease in auxin synthesis in deseeded plants. (b) Directly by its isolation from the plant. 8. Precursors of hetero-auxin are demonstrated; their chemical nature and activation are briefly considered.     

Correlations between net auxin and secondary xylem development in young Populus deltoides

Stems of cottonwood ( Populs deltoides Bartr. ex Marsh.) plants grown under different conditions were examined to determine the relation between net endogenous auxin yields and the acropetal advance of the primary-secondary vascular transition zone (TZ). In all treatments, the internode yielding maximum net auxin activity, as determined by the Avena curvature bioassay, closely corresponded with the internode in which the TZ occurred. Under short-day (SD) dormancy-inducing conditions, auxin yield declined steadily while the maximum auxin peak and the TZ shifted toward younger internodes. Auxin yields from these plants were extremely low after 5 weeks of SD compared with those from long-day (LD) plants. The only consistent auxin yield was obtained from internodes subtending young leaves beneath the apical bud. Plants placed in SD for 3 weeks and then returned to LD conditions showed an immediate increase in auxin yield in the stem, and the progressive acropetal advance of the TZ under SD was reversed. Therefore, within 7 LD the positions of the TZ and peak auxin yield corresponded to those observed before the imposition of SD Fully dormant plants placed in LD showed a dramatic rise in auxin yields during the first 2 weeks of renewed growth. Although low levels of auxin were found in the newly developing shoots after 6 LD, yields increased rapidly after 9 and 14 LD. The position of the TZ corresponded with the peak of net auxin activity after 9 and 14 LD.     

Rapid Inhibition of Auxin-induced Elongation of Avena Coleoptile Segments by Cordycepin

The effects of cordycepin (3'-deoxyadenosine), an RNA synthesis inhibitor, on auxin-induced elongation in Avena coleoptile segments were studied with a position-sensing transducer. Cordycepin rapidly inhibited auxin-stimulated growth in the coleoptile segments whether added before, at the same time as, or after, the 2 mum auxin treatment. Midcourse additions of 100, 50, and 25 mug/ml cordycepin inhibited auxin-promoted elongation in an average of 18, 22, and 35 minutes, respectively. Additions of cordycepin before or at the same time as the auxin treatment partially inhibited the magnitude of the subsequent auxin-promoted growth but did not appreciably alter the latent period of the auxin response. It was concluded that if cordycepin is inhibiting the synthesis of RNA required for growth, the decay time for this RNA may be considerably shorter than that suggested in the literature from actinomycin D experiments. Preliminary kinetic evidence indicated that cordycepin does not inhibit auxin-induced elongation by acting as a respiratory inhibitor. Studies in mung bean shoot mitochondria demonstrated that cordycepin has no effect on respiration, respiratory control, or ADP/oxygen ratios.     

Oxindole-3-acetic Acid: Physical Properties and Lack of Influence on Growth

The auxin activity of OAA was studied. The assays used were the Avena coleoptile curvature and section tests, and the first internode test. OAA was completely inactive to these assays. Physical tests indicated no color reaction with modified Salkowski reagent (Gordon-Weber) and no activity of any of the biochromatograms run with OAA. These data indicate that OAA is inactive as an auxin due to its characteristics of structural difference from IAA and further lends some speculation as to the site of activity of IAA as an auxin.     

Auxin Balance in the Avena Coleoptile

Coleoptiles of Avena possessed the capacity to degrade infiltrated indole-3-acetic acid (IAA). This activity decreased along the length of the coleoptile from apex to base on the bases of fresh weight, dry weight and protein; the apical 1 cm segment degraded more IAA than segments from other parts of the coleoptile. The naturally occurring inhibitor of the IAA oxidase activity increased in concentration up to 20 mm from the coleoptile apex; beyond, it decreased gradually towards the base. The spatial distribution of this inhibitor does not explain the gradient in IAA oxidase activity. Growth in length of the coleoptile and the IAA inactivating capacity of the apical 1 cm segment, increased 5- and 4,4-fold, respectively, between the ages of 70 and 130 h; but auxin secretion into agar platelets by the apical 2 mm of the coleoptile registered only a 2.7-fold increase. Deseeding and derooting the seedlings reduced the subsequent growth, diffusible auxin content and the IAA oxidase activity of the coleoptiles; derooting proved to be more deleterious than deseeding. A parallel reduction was evident in auxin content and IAA degrading activity following these treatments. Application of the cytokinin 6-benzylaminopurine (BAP) to coleoptiles of derooted seedlings failed to influence their capacity to degrade IAA. Nor was the activity of the aldehyde oxidase, which converts indole-3-acetaldehyde (IAAld) to IAA, affected by such treatment.     

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.     

Effect of red light on coleoptile growth

The effects of red light in reducing the growth of the oat (Avena sativa L.) coleoptile and the synthesis of auxin in the coleoptile tip are detectable 2 hours after treatment and become more pronounced with time. When the coleoptile tip is supplied with additional tryptophan the synthesis of auxin is doubled both in darkness and when exposed to red light. Treatment of the tip with gibberellic acid or pyridoxal phosphate overcomes the reduction of auxin synthesis caused by red light. The uptake of exogenous indoleacetic acid, at pH 6.5, by coleoptile tissue is doubled by exposure to red light. The effect of red light on coleoptile growth appears to be mediated by phytochrome in the cell membrane which delocalizes the tryptophan utilized for auxin synthesis.     

Revision of the theory of phototropism in plants: a new interpretation of a classical experiment

Went's classical experiment on the diffusion of auxin activity from unilaterally illuminated oat coleoptile tips (Went 1928), was repeated as precisely as possible. In agreement with Went's data with theAvena curvature assay, the agar blocks from the illuminated side of oat (Avena sativa L. cv. Victory) coleoptile tips had, on an average, 38% of the auxin activity of those from the shaded side. However, determination of the absolute amounts of indole-3-acetic acid (IAA) in the agar blocks, using a physicochemical assay following purification, showed that the IAA was evenly distributed in the blocks from the illuminated and shaded sides. In the blocks from the shaded and dark-control halves the amounts of IAA were 2.5 times higher than the auxin activity measured by theAvena curvature test, and in those from the illuminated half even 7 times higher. Chromatography of the diffusates prior to theAvena curvature test demonstrated that the amounts of two growth inhibitors, especially of the more polar one, were significantly higher in the agar blocks from the illuminated side than in those from the shaded side and the dark control. These results show that the basic experiment from which the Cholodny-Went theory was derived, does not justify this theory. The data rather indicate that phototropism is caused by the light-induced, local accumulation of growth inhibitors against a background of even auxin distribution, the diffusion of auxin being unaffected.Abbreviation IAA indole-3-acetic acid     

Evidence for the presence of a native,non-auxinic rooting promoter in avocado (Persea americana Mill.)

The presence of a rooting promoter in paratially purified extracts of avocado (Persea amricana Mill.) organs has been demonstrated using the mung bean rooting bioassay. Extraction with 80% methanol was followed by partition into diethyl ether, paper chromatography (PC) and 3 steps of thin layer chromatography (TLC). The number of roots induced by the rooting promoter in the absence of exogenous auxin was 5 to 7 times higher than that of the water control and 50% higher than by 4-(indol-3-yl) butyric acid (IBA) at its reported optimum concentration. Rooting of tomato, Coleus and young avocado cuttings was also enhanced by the rooting promoter. The rooting promoter was inhibitory in the wheat coleoptile section elongation bioassay for auxins and had slight inhibitory activity in the split pea stem curvature test.The biological properties of the avocado rooting promoter may be comparable to those of -(p-chlorophenoxy) isobutyric acid (PCIB) which acts as an anti-auxin in certain bioassays and, nevertheless, promotes the rooting of mung bean cuttings.     

The occurrence of auxins in the old leaves of apple trees

A relatively high auxin level was found in old leaves from the lower part of long shoots of young apple trees using the straight growth bioassay on wheat coleoptile segments. The auxin level remains almost constant during summer and autumn.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Light interacts with auxin during leaf elongation and leaf angle development in young corn seedlings

Modern corn ( Zea mays L.) varieties have been selected for their ability to maintain productivity in dense plantings. We have tested the possibility that the physiological consequence of the selection of the modern hybrid, 3394, for increased crop yield includes changes in responsiveness to auxin and light. Etiolated seedlings in the modern line are shorter than in an older hybrid, 307, since they produce shorter coleoptile, mesocotyl, and leaves (blade as well as sheath). Etiolated 3394 seedlings, as well as isolated mesocotyl and sheath segments, were less responsive to auxin and an inhibitor of polar auxin transport, N-1-naphthylphthalamic acid (NPA). Reduced response of 3394 to auxin was associated with less reduction of elongation growth by light (white, red, far-red, blue) than in 307, whereas the activity of polar auxin transport (PAT) and its reduction by red or far-red light was similar in both genotypes. NPA reduced PAT in etiolated 3394 seedlings much less than in 307. A characteristic feature of 3394 plants is more erect leaves. In both hybrids, light (white, red, blue) increases leaf declination from the vertical, whereas NPA reduces leaf declination in 307, but not in 3394. Our results support findings that auxin and PAT are involved in elongation growth of corn seedlings, and we show that light interacts with auxin or PAT in regulation of leaf declination. We hypothesize that, relative to 307, more erect leaves in the modern hybrid may be primarily a consequence of a reduced amount of auxin receptor(s) and reduced responsiveness to light in etiolated 3394 plants. The more erect leaves in 3394 may contribute to the tolerance of the modern corn hybrid to dense planting.     

Physical strain-mediated microtubule reorientation in the epidermis of gravitropically or phototropically stimulated maize coleoptiles

During gravitropic and phototropic curvature of the maize coleoptile, the cortical microtubules (MTs) adjacent to the outer epidermal cell wall assume opposite orientations at the two sides of the organ. Starting from a uniformly random pattern during straight growth in darkness, the MTs reorientate perpendicularly to the organ axis at the outer (faster growing) side and parallel to the organ axis at the inner (slower growing) side. As similar reorientations can be induced during straight growth by increasing or decreasing the effective auxin concentration, it has been proposed that these reorientations may be used as a diagnostic test for assessing the auxin status of the epidermal cells during tropic curvature. This idea was tested by determining the MT orientations in the coleoptile of intact maize seedlings in which the gravitropic or phototropic curvature was prevented or inversed by an appropriate mechanical counterforce. Forces that just prevented the coleoptile from curving in a gravity or light field prevented reorientations of the MTs. Forces strong enough to overcompensate the tropic stimuli by enforcing curvature in the opposite direction induced reorientations of the MTs opposite to those produced by tropic stimulation. These results show that the MTs at the outer surface of the coleoptile respond to changes in mechanical tissue strain rather than to gravitropic or phototropic stimuli and associated changes at the level of auxin or any other element in the signal transduction chain between perception of tropic stimuli and asymmetric growth response. It is proposed that cortical MTs can act as strain gauges in a positive feed-back regulatory circle utilized for amplification and stabilization of environmentally induced changes in the direction of elongation growth.     

The Effect of Gibberellin on Tryptophan Conversion and Elongation of the Avena Coleoptile

Gibberellic acid (GA₃) enhanced directly the release of ¹⁴CO₂ from tryptophan-1-^l4C by cell free preparations of Avena coleoptile tips. The rate of tryptophan metabolism in the presence of GA₃ was increased by approximately 100 per cent. The addition of auxin synthesis inhibitors to incubation flasks nullified the enhancement effect of GA₃ on elongation of the coleoptile tips. These studies implicate tryptamine as an intermediate in the formation of auxin from tryptophan. The possibility of GA₃-IAA interaction in the elongation processes was also investigated. Combination treatments of these growth-promoting substances did not induce a synergistic growth response by the coleoptile tissue.     

Selective effects of victorin on growth and the auxin response in Avena

Victorin, the pathotoxin from the host-specific pathogen, Helminthosporium victoriae, promotes the growth of coleoptile segments when given at concentrations that are high but which still show selective effects on susceptible and resistant tissue. The latent period in the growth response of both susceptible and resistant tissue is about 3.6 minutes compared to 11.0 minutes in the response of these tissues to auxin. The victorinpromoted rate of elongation of 8-millimeter segments is about 0.2 millimeter per hour in susceptible tissue and about 0.1 millimeter per hour in resistant tissue compared to about 0.4 millimeter per hour in response to auxin. At low concentrations, the toxin has no growth-promoting effect in either susceptible or resistant coleoptile segments. Over a wide range of concentrations, victorin inhibits the growth response of susceptible tissue to auxin completely while having no effect on the response of resistant tissue to auxin.     

Exposure of oat seedlings to blue light results in amplified phosphorylation of the putative photoreceptor for phototropism and in higher sensitivity of the plants to phototropic stimulation

Dark recovery of blue light-induced in vitro phosphorylation in oat (Avena sativa L.) seedlings after in vivo preirradiation with blue light revealed different recovery kinetics for the coleoptile base and tip. Although, in both cases, maximum in vitro phosphorylation was observed 90 min after in vivo blue light treatment, the phosphorylation levels for the entire base were about 3-fold higher than those found in nonpreirradiated plants. The tip response only slightly exceeded that of the dark controls. The fluence applied during preirradiation determined the extent of the increase in phosphorylation. Consequently, unilateral irradiation and subsequent dark incubation resulted in a more pronounced increase in phosphorylation in the irradiated than in the shaded side of the coleoptile base. Furthermore, blue light-irradiation conditions, known to induce neither first- nor second-positive curvature in nonpreirradiated plants, stimulated both asymmetric distribution of protein phosphorylation and second-positive phototropic curvature in the coleoptile base when administered to blue light-pretreated plants. Based on these data, we conclude that photosensitivity of the coleoptile base increases upon exposure to blue light in a time-and fluence-dependent manner, providing an excellent explanation of the invalidity of the Bunsen-Roscoe reciprocity law for second-positive phototropism.