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     

Uridine and the Control of Phototropism in Oat (Avena sativa L.) Coleoptiles

The short-term growth response of oat (Avena sativa L.) coleoptiles to exogenously applied uridine was studied both in excised apical segments and in the intact seedlings. In both cases growth of coleoptile tissue was inhibited by uridine. The inhibition of coleoptile growth consistently occurred 20–30 min after uridine treatment, which is within the lag period of their phototropic response. Asymmetric application of uridine to coleoptiles in the intact seedlings resulted in their bending toward the direction to which uridine was applied in the absence of light stimulus. These findings suggest that uridine or its metabolites, plays an important role in the phototropism of oat coleoptiles and provide support to the Bruinsma–Hasegawa theory as an alternative to the Cholodny–Went theory for explaining phototropism.     

Phototropism and Protein Phosphorylation in Higher Plants: Unilateral Blue Light Irradiation Generates a Directional Gradient of Protein Phosphorylation Across the Oat Coleoptile

Blue light induces the phosphorylation of a 116 kDa oat protein found in plasma membrane preparations from coleoptile tips. We developed a very sensitive in vitro method that allowed us to determine the tissue distribution of protein phosphorylation after applying unilateral and bilateral blue light pulses in vivo. We found that following unilateral in vivo irradiation the degree in phosphorylation of the 116 kDa protein is significantly higher at the irradiated than at the shaded side of the coleoptile tip. This asymmetry can be considered as previously missing criterion that protein phosphorylation represents an early event within the transduction chain for phototropism.     

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.     

Hormonal Relations in the Phototropic Response: III. The Movement of C-labeled and Endogenous Indoleacetic Acid in Phototropically Stimulated Zea Coleoptiles

The effect of phototropic stimulation of Zea coleoptile tips on the distribution of both endogenous indoleacetic acid (IAA) and applied C¹⁴-labeled IAA was determined. The tips rested on bisected agar blocks. More IAA was found in the blocks under the shaded side of the coleoptile tips than those under the irradiated side. However, no significant difference was observed between the total amounts of IAA, endogenous or labeled, in the irradiated and shaded sides of the experimental system. In addition, less endogenous auxin was found in the shaded tissues than in their irradiated counterparts. It is suggested that phototropism following unilateral irradiation with first positive radiant densities might be a consequence of lateral inequalities in the ability of the irradiated and shaded tissues to transport auxin basipetally.     

Distribution of Endogenous Indole-3-acetic Acid and Growth Inhibitor(s) in Phototropically Responding Oat Coleoptiles

The relationship between the flank growth of oat (Avena sativaL. cv. Victory) coleoptiles and the distribution of endogenousindole-3-acetic acid (IAA) and growth inhibitor(s) in the coleoptileswas studied for the second positive phototropic curvature inducedby a continuous unilateral illumination with white light (0.1W.m^–2). The phototropic curvature was caused by growthinhibition at the lighted side and growth promotion at the shadedside. Using electron capture detection gas chromatography, weanalyzed the distribution of endogenous IAA in phototropicallyresponding oat coleoptiles and found that the IAA was evenlydistributed over the lighted and shaded sides during the phototropicresponse; there was also no detectable difference in the amountsof IAA between phototropically stimulated and non-irradiatedcoleoptiles. By contrast, oat coleoptile straight-growth testresults showed that the amount of unknown acidic growth inhibitor(s),different from abscisic acid, increased in the lighted halfof the coleoptiles and decreased in the shaded half, as comparedto the amount in the non-irradiated half. These data suggestthat the phototropic curvature of oat coleoptile is inducedby a difference in lateral flank growth through a lateral gradientof endogenous growth inhibitor(s) rather than of IAA. (Received February 10, 1988; Accepted July 29, 1988)     

Red light causes a reduction in IAA levels at the apical tip by inhibiting de novo biosynthesis from tryptophan in maize coleoptiles

When maize coleoptiles were unilaterally exposed to red light (7.9 μmol m⁻²s⁻¹ for 5 min), 3 h after treatment IAA levels in coleoptiles decreased in all regions, from top to basal, with levels about 60% of dark controls. Localized irradiation in the 5 mm top zone was sufficient to cause the same extent of IAA reduction in the tips to that in the tips of whole irradiated shoots. When coleoptiles were treated with N-1-naphthylphthalamic acid (NPA), an accumulation of IAA in the tip and a decrease of diffusible IAA from tips were simultaneously detected. IAA accumulation in red-light treated coleoptiles by NPA was much lower than that of dark controls. NPA treatment did not affect the content of conjugated IAA in either dark or light treated coleoptile tips. When ¹³C₁₁ ¹⁵N₂-tryptophan (Trp) was applied to the top of coleoptiles, substantial amounts of stable isotope were incorporated into free IAA in dark and red-light treated coleoptile tips. The ratio of incorporation was slightly lower in red-light treated coleoptile tips than that in dark controls. The label could not be detected in conjugated IAA. The rate of basipetal transport of IAA was about 10 mm h⁻¹ and the velocity was not affected by red light. These results strongly suggest that red light does not affect the rates of conversion of free IAA to the conjugate form or of the basipetal transport, but just reduces the IAA level in the tips, probably inhibited by IAA biosynthesis from Trp in this region.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

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.     

High-resolution measurement of growth during first positive phototropism in maize†

Abstract Growth redistribution which occurs as a result of phototropic stimulation was studied in red light-grown, maize (Zea mays L.) seedlings. The pattern of elongation of small areas (0.1mm²) of coleoptile epidermis on intact plants was analysed from time-lapse, photomicrographic records. Growth following unilateral, pulse irradiation with blue light was depressed on the illuminated side and was stimulated on the shaded side. The time at which the change in growth rate occurred, on both illuminated and shaded sides, was significantly earlier in apical patches than it was in basal patches. Both kinds of change in the growth rate (stimulation and depression) occurred rapidly such that a new, constant growth rate was often established within five minutes. Micrographic, time-lapse records were also obtained of growth changes induced by sub-apical, unilateral application of a spot of an indole-3-acetic acid (IAA) and lanolin mixture. Growth on the side of the coleoptile to which IAA had been applied was similar to the growth on shaded sides of phototropically stimulated plants. The distance between apical and basal patches and the elapsed time between their changes in growth rate gave a velocity at which the growth response moved basipetally. Calculation of this velocity for blue light and auxin treatment gave values that were not significantly different. Thus, basipetal movement of a transverse auxin gradient could mediate growth changes that cause curvature of the coleoptile towards first positive fluences of blue light.     

Phytochrome-controlled extension growth of Avena sativa L. seedlings

The effects of continuous red and far-red light and of brief light pulses on the growth kinetics of the mesocotyl, coleoptile, and primary leaf of intact oat (Avena sativa L.) seedlings were investigated. Mesocotyl lengthening is strongly inhibited, even by very small amounts of P_fr, the far-red light absorbing form of phytochrome (e.g., by [P_fr]0.1% of total phytochrome, established by a 756-nm light pulse). Coleoptile growth is at first promoted by P_fr, but apparently inhibited later. This inhibition is correlated in time with the rupturing of the coleoptile tip by the primary leaf, the growth of which is also promoted by phytochrome. The growth responses of all three seedling organs are fully reversible by far-red light. The apparent lack of photoreversibility observed by some previous investigators of the mesocotyl inhibition can be explained by an extremely high sensitivity to P_fr. Experiments with different seedling parts failed to demonstrate any further obvious interorgan relationship in the light-mediated growth responses of the mesocotyl and coleoptile. The organspecific growth kinetics, don't appear to be influenced by P_fr destruction. Following an irradiation, the growth responses are quantitatively determined by the level of P_fr established at the onset of darkness rather than by the actual P_fr level present during the growth period.Abbreviation P_fr far-red light absorbing form of phytochrome     

High-irradiance responses induced by far-red light in grass seedlings of the wild type or overexpressing phytochrome A

The occurrence of phytochrome-mediated highirradiance responses (HIR), previously characterised largely in dicotyledonous plants, was investigated in Triticum aestivum L., Zea mays L., Lolium multiflorum Lam. and in both wild-type Oryza sativa L. and in transgenic plants overexpressing oat phytochrome A under the control of a 35S promoter. Coleoptile growth was promoted (maize, ryegrass) or inhibited (wild-type rice) by continuous far-red light (FRc). However, at equal fluences, hourly pulses of far-red light (FRp) were equally effective, indicating that the growth responses to FRc were not true HIR. In contrast, in maize and rice, FRc increased anthocyanin content in the coleoptile in a fluence-rate dependent manner. This response was a true HIR as FRp had reduced effects. In maize, anthocyanin levels were significantly higher under FRc than under continuous red light. In rice, overexpression of phytochrome A increased the inhibition of coleoptile growth and the levels of anthocyanin under FRc but not under FRp or under continuous red light. The effect of FRc was fluence-rate dependent. In light-grown rice, overexpression of phytochrome A reduced leaf-sheath length, impaired the response to supplementary far-red light, but did not affect the response to canopy shade-light. In grasses, typical HIR, i.e. fluence-rate dependent responses showing reciprocity failure, can be induced by FRc. Under FRc, overexpressed phytochrome A operates through this action mode in transgenic rice.Abbreviations FR far-red light - FRc continuous far-red light - FRp pulses of far-red light - HIR high-irradiance responses - LFR low-fluence responses - OPHYA transgenic rice overexpressing oat phytochrome A - Pfr far-red light-absorbing form of phytochrome - phyA phytochrome A - R red light - Rc continuous red light - VLFR very low-fluence responses - WT wildtype We thank Marcelo J. Yanovsky for his help with the photographs and Professor Rodolfo A. Sanchez for providing a reprint of the paper by P.J.A.L. de Lint. This work was supported by grants from UBA (AG041) and Fundacion Antorchas (A-13218/1-15) to J.J.C.     

Effects of the steroidal alkaloid tomatine in auxin bioassays and its interaction with indole-3-acetic acid

Summary The steroidal alkaloid tomatine did not enhance elongation of oat coleoptile and first internode sections, or of wheat coleoptile sections. Higher concentrations of the alkaloid inhibited elongation and interacted antagonistically with IAA. Although 10^-4 M tomatine alone did not influence elongation of oat coleoptile sections, it did reduce growth response to exogenous IAA. Tomatine concentrations less than 10^-4 M did not influence response to IAA. The auxin activity of tomatine, reported by Vendrig, was therefore not confirmed.     

Inhibition of the synthesis of cell wall polysaccharides in oat coleoptile segments by jasmonic acid: Relevance to its growth inhibition

The inhibitory mode of action of jasmonic acid (JA) on the growth of etiolated oat (Avena sativa L. cv. Victory) coleoptile segments was studied in relation to the synthesis of cell wall polysaccharides using [¹⁴C]glucose. Exogenously applied JA significantly inhibited indoleacetic acid (IAA)-induced elongation of oat coleoptile segments and prevented the increase of the total amounts of cell wall polysaccharides in both the noncellulosic and cellulosic fractions during coleoptile growth. JA had no effect on neutral sugar compositions of hemicellulosic polysaccharides but substantially inhibited the IAA-stimulated incorporation of [¹⁴C]glucose into noncellulosic and cellulosic polysaccharides. JA-induced inhibition of growth was completely prevented by pretreating segments with 30 mm sucrose for 4 h before the addition of IAA. The endogenous levels of UDP-sugars, which are key intermediates for the synthesis of cell wall polysaccharides, were not reduced significantly by JA. Although these observations suggest that the inhibitory mode of action of JA associated with the growth of oat coleoptile segments is relevant to sugar metabolism during cell wall polysaccharide synthesis, the precise site of inhibition remains to be investigated.Abbreviations JA jasmonic acid - ABA abscisic acid - IAA indoleacetic acid - T ₀ minimum stress relaxation time - TFA trifluoroacetic acid - TCA trichloroacetic acid - HPLC high-performance liquid chromatography - EtOAc ethyl acetate - TLC thin-layer chromatography - JA-Me methyl jasmonate - GLC-SIM gas-liquid chromatography-selected ion monitoring     

The relationship between pathogenic fungal metabolities (fusaric and picolinic acid), endogenous ethylene evolution and the development of ethylene-like symptoms

Summary The effects of fusaric acid (5-n-butylpicolinic acid), picolinic acid (2-pyridine carboxylic acid), and picloram (4-amino-3, 5, 6-trichloropicolinic acid) on endogenous ethylene production by tomato cuttings and elongation growth of oat coleoptile sections were measured. Ethylene production by tomato cuttings was substantially stimulated by treatment with 1×10⁻³ and 1×10⁻⁵ M picoloram and to a lesser extent by 1×10⁻³ M fusaric acid; picolinic acid had little effect. The ethylene levels produced in response to fusaric acid are not high enough to account for the ethylene injury observed in Fusarium wilt. Fusaric acid inhibited oat coleoptile extension, picolinic acid had little effect, and picloram promoted growth.     

PHYSIOLOGICAL AND MORPHOLOGICAL MODIFICATIONS OF PLANTAGO MAJOR (PLANTAGINACEAE) IN RESPONSE TO LIGHT CONDITIONS

Physiological and morphological differences between Plantago major L. (Plantaginaceae) growing in full sunlight and shaded conditions were examined. Photosynthesis of isolated leaves was saturated by irradiance around 300 μE m^−-2 sec^−-1 and 170 μE m^−-2 sec^−-1, respectively. In contrast to previous studies of sun/shade leaf responses, initial slopes of curves from shaded plants are significantly less than those taken from full-sun plants. Within the 400–500 nm and 600–700 nm ranges, leaves 5.0 cm or longer are essentially opaque, transmitting less than 1.25% of incident light. Chlorophyll content per unit leaf area is nearly equivalent for leaves from plants growing under the two extremes in light levels. Morphometric comparisons indicate shaded plants bear fewer leaves, have less leaf overlap, lower total leaf area, and longer petioles than full-sun plants. Leaf elongation rates are lower and the duration between the emergence of successive leaves is longer in shaded plants. Computer analyses of both types of rosette morphology reveal shaded plants have an equal or greater capacity to intercept light than full-sun plants, principally because of the minimization of leaf overlap and the large variation in the deflection angles of leaves in shaded rosette morphologies. Simulations, calculated on the basis of light interception, and taking into account the transition between photosynthate-importing and -exporting leaves, predict relative growth rates for full-sun and shaded rosette morphologies that are in reasonable agreement with empirically determined leaf growth rates. However, the data indicate that significant physiological and morphological differences exist among leaves from a single rosette, and among developmentally comparable leaves from rosettes growing under different ambient light environments. Differences among leaves on a single plant must be accommodated in computerized techniques attempting to simulate light interception and its consequences on potential growth rates.     

Growth distribution during first positive phototropic curvature of maize coleoptiles*

Abastract Measurements of growth increments on the shaded and the irradiated sides of phototropically stimulated maize (Zea mays L.) coleoptiles, obtained over the entire fluence range of the first positive curvature, indicate that the curvature is induced by growth stimulation on the shaded side and compensating inhibition on the irradiated side (length increments on the coleoptile flanks were determined 100 min after 30 s phototropic induction with blue light). At high fluences of blue light, overall stimulation of growth takes place, but this tendency is largely eliminated when only the tip of the coleoptile is irradiated. Time courses for growth increments obtained for the maximum first positive response show that the growth stimulation on the shaded side and the growth inhibition on the irradiated side commence almost simultaneously 20-30 min after the phototropic induction. The growth on the irradiated side almost ceases, but the growth rate on the shaded side is doubled, relative to the control rate. The onset of differential growth migrates basipetally from the tip at a velocity similar to that for polar auxin transport. The first positive phototropic response of the coleoptile is concluded to be the consequence of lateral redistribution of growth, which is not necessarily accompanied by changes in the net growth. The results are consonant with the Cholodny-Went theory of tropisms, in which lateral redistribution of auxin is considered to be the cause of tropic responses.     

Optimization of red light-induced elongation in Avena coleoptile sections and properties of the phytochrome-mediated growth response*

Abstract Optimal conditions for studying the elongation response to a 1 mmol m^?2, 2-min pulse of red light in subapical coleoptile sections from dark-grown oat (Avena sativa L. ev. Lodi) seedlings have been determined. A technique for obtaining standard-length coleoptile sections without exposing either seedlings or sections to any light has been developed, and is described. The optimal conditions found were: sampling time, 12 h after irradiation; buffer conditions, 5 mol m^?3 potassium phosphate with 5% (w/v) sucrose (pH 5.9). The optima were determined by obtaining the time course for light-induced growth under various conditions. The red light-induced growth response is linear until 12 h after irradiation, when it undergoes an interruption. Optimal incubation conditions were determined by varying the buffer contents systematically and measuring the responses at the optimal lime determined. The results indicate a distinct difference between auxin-induced and light-induced growth responses. Even with variations of basal growth rate and several incubation conditions, the red light-induced elongation appears to be of a constant magnitude, to persist for a constant time period. and to exhibit a constant lag period between irradiation and the onset of response. The use of sections that were produced and handled in complete darkness yielded an unusual response to fusicoccin. A linear, high growth rate in response to I mmol m^?3 FC was observed for more than 12 h, both in the irradiated sections and in the dark controls.     

Photoreversible calcium fluxes induced by phytochrome in oat coleoptile cells

The chromometallic dye murexide was used to measure photoreversible Ca fluxes in apical tips of etiolated oat coleoptiles and in suspension cultures of protoplasts derived from the coleoptile segments. Phytochrome presence in the protoplasts was indicated by a repeatably photoreversible ΔA(725 - 800 nm) of >0.001 A centimeters⁻¹, recorded on a dual wavelength spectrophotometer. Concentrations of Ca in the solution bathing the cells were observed to change photoreversibly, red irradiation inducing an increase in the medium Ca concentration and subsequent farred irradiation inducing a decrease down to near dark control levels. These changes could be measured in media with or without exogenously added Ca. Protoplasts from green primary leaves of oat, which had no spectro-photometrically detectable phytochrome, showed no photoreversible Ca fluxes when measured by the same method. These data imply that red light induces an efflux of Ca from phytochrome-containing cells and that far red light can reverse this change by promoting a Ca reentry into these cells.     

Action of red light on indole-3-acetic-acid status and growth in coleoptiles of etiolated maize seedlings

Brief irradiation of intact etiolated seedlings of maize (Zea mays L.) with red light (R; 30 W cm^-2, 10 min) reduces the amounts of diffusible and free (solvent-extractable) indole-3-acetic acid (IAA) obtainable from excised coleoptile tips. The effect is transient, the lowest level (30% of the dark control) occurring at about 3 h after irradiation. The free-IAA content of the whole coleoptile and the diffusible-IAA yield from the base of the same organ are similarly reduced, whereas the conjugated-IAA content of the coleoptile is not affected. These results support the view that R inhibits the production of IAA at the coleoptile tip. It is further shown that R inhibits biosynthesis of [³H]IAA from [³H]tryptophan supplied to the coleoptile tip. The shapes of the fluence-response curves obtained for the reduction of the diffusible-IAA yield by R and far-red light (FR) indicate the participation of two photoreactive systems. One has thresholds at 10^-3 W s cm² of R, five orders of magnitude less than the minimum required for the appearance of spectrophotometrically measurable far-red-absorbing form of phytochrome (Pfr) in vivo, and 10^-1 W s cm^-2 of FR; its response is linear to the logarithm of fluence exceeding five orders of magnitude. The other system is seen above 10² W s cm^-2 as an increase in the slope of the fluenceresponse curve; its response is FR reversible and related to the Pfr level of total photoreversible phytochrome. Both systems inhibit biosynthesis of IAA from tryptophan. Elongation of the coleoptile is stimulated by R; the stimulation is most apparent in the apical region, and is saturated with a fluence at which bo detectable pfr is formed. Farred light can also saturate this response. Since the endogenous IAA concentration in the coleoptile appears not to be in the inhibitory range, it is concluded that the stimulation of coleoptile elongation is not the result of changes in free-IAA levels.Abbreviations FR far-red light - IAA indole-3-acetic acid - Pfr phytochrome in the far-red-absorbing form - Pr phytochrome in the red-absorbing form - R red light     

Auxin-stimulated RNA Synthesis in Oat Coleoptile Cells

Using oat coleoptile segments the following results were obtained. Ten mg/l auxin (indole-3-acetic acid) increased the incorporation of uracil-2-¹⁴C and orthophosphate-³²P into RNA fraction during a relatively short incubation period. Stimulation of ³²P incorporation due to auxin was found only in the region heavier than ribosomal RNA, probably in the messenger RNA region. The stimulation of uracil-2-¹⁴C incorporation into RNA caused by auxin was not influenced by the presence of 0.3 M mannitol which prevents osmotically the water absorption of cells. It is concluded that auxin primarily stimulates the biosynthesis of RNA, possibly messenger, in oat coleoptile cells.