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.     

Phototropism in Hypocotyls of Radish: IV. Flank Growth and Lateral Distribution of cis- and trans-Raphanusanins in the First Positive Phototropic Curvature

The first positive phototropic curvature induced by a pulse of unilateral white irradiation (0.1 watt per square meter, 30 seconds) of etiolated and de-etiolated Sakurajima radish (Raphanus sativus var hortensis f. gigantissimus Makino) hypocotyls was analyzed in terms of differential growth and growth inhibitor contents of the hypocotyls. In both etiolated and de-etiolated hypocotyls, the growth rates at the lighted sides were suppressed whereas those at the shaded ones showed no change. De-etiolation treatment induced a larger difference between the growth rates at the lighted and shaded sides of the hypocotyls, resulting in a larger curvature of de-etiolated seedlings than of etiolated ones. The contents of growth inhibitors, cis- and trans-raphanusanins, increased in the lighted but not in the shaded halves of the hypocotyls of etiolated seedlings. In de-etiolated seedlings, the two inhibitors increased due to the de-etiolation treatment. When de-etiolated seedlings were exposed to a pulse of unilateral irradiation the level of the two inhibitors remained high along the lighted side for 1 h following the light pulse, whereas at the shaded side the contents of the inhibitors abruptly decreased upon transfer to the dark, the difference between their amounts in the lighted and shaded sides being larger than in etiolated seedlings. Another growth inhibitor, raphanusamide, did not respond to the phototropic stimulus, although its amounts increased by the de-etiolation treatment. These data suggest that cis- and trans-raphanusanins are involved in the first positive phototropic response of radish hypocotyls, and that de-etiolation magnifies the phototropic response through induction of a larger lateral gradient of the raphanusanins in the hypocotyls by the phototropic stimulus.     

Growth Distribution during Phototropism of Arabidopsis thaliana Seedlings

The elongation rates of two opposite sides of hypocotyls of Arabidopsis thaliana seedlings were measured during phototropism by using an infrared imaging system. In first positive phototropism, second positive phototropism, and red light-enhanced first positive phototropism, curvature toward the light source was the result of an increase in the rate of elongation of the shaded side and a decrease in the rate of elongation of the lighted side of the seedlings. The phase of straightening that followed maximum curvature resulted from a decrease in the elongation rate of the shaded side and an increase in the elongation rate of the lighted side. These data for the three types of blue light-induced phototropism tested in this study and for the phase of straightening are all clearly consistent with the growth rate changes predicted by the Cholodny-Went theory.     

Phototropism in Hypocotyls of Radish : III. Influence of Unilateral or Bilateral Illumination of Various Light Intensities on Phototropism and Distribution of cis- and trans-Raphanusanins and Raphanusamide

When etiolated radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls were subjected to a continuous unilateral illumination with white fluorescent light at 0.05, 0.1, or 1 watt per square meter, the suppression of the growth rate on the lighted side depended on the light intensity. The growth rate at the shaded side was only a little affected by the illumination at 0.05 and 0.1 watt per square meter but considerably suppressed by that at 1 watt per square meter. Upon a continuous unequal bilateral illumination, the growth rate was more strongly suppressed on the side of the higher intensity than on the side of the lower one, resulting in phototropic curvature toward the light source of the higher intensity. It was calculated from correlation analysis of light intensity and growth rate that, on an average, 6.9% of the irradiation applied to one side reached the opposite side. The amounts of cis- and trans-raphanusanins and raphanusamide in hypocotyls subjected to unilateral or unequal bilateral illumination increased much more at the side of the lighted or the higher intensity than at the opposite side. The present study demonstrates that phototropism in radish hypocotyl is correlated with and we conclude caused by a gradient of growth inhibition in the hypocotyl, depending on irradiation-induced amounts of cis- and trans-raphanusanins and raphanusamide.     

Redistribution of growth during phototropism and nutation in the pea epicotyl

First positive phototropism of the third internode of intact, 5-d-old pea (Pisum sativum L.) seedlings, grown under continuous, dim red light, showed maximal response following a photon fluence of 3 mol·m^-2 blue light. Greater or lesser fluences (with irradiation time 100 s or less) caused less bending, no response being detectable above 300 or below 0.03 mol·m^-2. Bilateral irradiation with blue light caused no detectable inhibition of growth rate over that range of fluences. The linear nutation of the pea third internode was shown to be driven by a balanced oscillation of growth rate such that the overall growth rate was little changed during the oscillation. Phototropic stimulation changed neither the amplitude nor the period of nutation. Nutation and phototropism probably regulate growth independently. Phototropism in response to the optimal blue light fluence was caused by concomitant depressed growth on the irradiated side and stimulated growth on the shaded side of the bending internode. These results are consistent with the Cholodny-Went hypothesis which states that unilateral blue light induces a lateral redistribution of a growth regulator.Abbreviations R red light - BL blue light Carnegie Institution, Department of Plant Biology paper No. 921     

Regional growth patterns in the hypocotyls of etiolated and green cress seedlings in light and darkness

Abstract. A system is described whereby seedling development can be analysed in terms of growth rates of specific 1 mm regions of the hypocotyl. The technique involves time-lapse photography of marked hypocotyls in a specially designed chamber which accommodates seedlings in various orientations with respect to gravity, and under irradiation regimes differing in light quality, quantity and direction. The results of a preliminary study of the upward growth of etiolated or green cress seedlings in darkness or overhead while light are reported. Highest growth rates in etiolated seedlings were observed in zones in the upper one-third of ihe hypocotyl. In green seedlings, growth was more prominent within the subapical zones. Light further restricted growth of the median and basal zones in both types of seedling. However, in their immediate responses to the onset of irradiation, green and etiolated seedlings differed markedly. In etiolated seedlings, recovery of growth at the apex was accompanied by the development of inhibition in the median-basal regions; green seedlings showed a transient inhibition of growth in the apical zone together with a strong immediate inhibition in the median-basal regions.     

Evidence Inconsistent with the Blaauw Model of Phototropism

The Blaauw model of phototropism equates the inhibition of growthat the illuminated side of a unilaterally illuminated organwith the blue light inhibition of overall organ extension evidentwhen some shoots are exposed to uniform blue light However,a study of the growth responses of Avena coleoptiles exposedto omnilateral, equal bilateral, unequal bilateral and unilateralblue light has revealed some light induced growth rate changeswhich cannot be explained by the Blaauw model. The growth responsesof cells at the illuminated and shaded sides of phototropicallystimulated coleoptiles seem to depend on the existence of alight gradient across the whole organ rather than the absolutelevels of light at either side. Key words: Phototropism, Avena coleoptile, Blaauw hypothesis, Blue light, Growth inhibition     

Effects of microbeam light on growth and phototropism ofPilobolus crystallinus sporangiophores

A small blue-light beam (50 μm in diam) was used to examine light-growth response and phototropism inPilobolus crystallinus sporangiophores. Continuous irradiation by microbeam of a region 100–150 μm from the apex promoted the growth of a dark-adapted sporangiophore for about 15 min after a lag period of 1–2 min. After the promotion, the growth rate fell below that before the irradiation. Irradiation of the apex of sporangiophore slightly promoted the growth but strongly inhibited the growth after the promotion. A smaller light beam (10 μm in diam) applied continuously at grazing incidence along one side of the sporangiophore caused bending toward the shaded side, implying that the irradiated side grew more rapidly than the shaded side and that the lens effect is involved in the phototropism of young sporangiophores ofP. crystallinus. The involvement of the lens effect was confirmed by the fact that a carotenoid-less mutant was 1.5–2 times more sensitive to unilateral blue light than the wild type, probably because of a smaller intracellular light attenuation during passage through the mutant cell.     

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.     

Osmotic relations during elongation growth in hypocotyls of Helianthus annum L.

The relationship between growth, change in cell osmotic pressure and accumulation of osmotic solutes was investigated in hypocotyls of sunflower (Helianthus annum L.) seedlings. During growth in darkness the osmotic pressure decreased by 50% between days 2 and 6 after sowing. After irradiation of dark-grown seedlings with continuous white light (WL) an inhibition of hypocotyl growth was measured, but the osmotic pressure of the growing cells was not lower than in the dark-grown control. Growth in darkness and after WL irradiation was accompanied by an increase in the amount of osmotic substances (soluble sugars) which was proportional to the increase in length of the organ. During growth in continuous WL the cell osmotic pressure decreased by 45 % between days 2 and 6 after sowing. The transfer of WL-grown seedlings to darkness (“re-etiolation”) resulted in a rapid acceleration of hypocotyl growth, but the cell osmotic pressure was the same as that of the WL grown control. Growth in continuous WL was accompanied by a corresponding accumulation of osmotic substances (soluble sugars). The transition from WL to darkness resulted in an enhanced accumulation of osmotica and an increase in cell-wall extensibility. The results indicate that the relative maintenance of cell osmotic pressure during rapid hypocotyl growth in darkness is caused by an enhanced accumulation of soluble sugars into the growing cells of the organ.     

Diffusible gibberellins and phototropism in Helianthus annuus

Summary Endogenous gibberellins were obtained in agar from the lower cut surface of upright sunflower shoot-tips. Exposure to unilateral light of the tips standing on agar, with the lower cut ends bisected by a vertical glass barrier at right angles to incident light, resulted in approximately 8 times the quantity of gibberellins moving into the agar below the shaded side than into the agar below the illuminated side. These results are similar to those reported earlier for gibberellins and geotropism in sunflower shoots, and suggest than the development of both light-and gravity-induced growth curvatures involve an asymmetry in gibberellin distribution across elongating internodes.     

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.     

Gravitational compensation and the phototropic response of oat coleoptiles

Avena seedlings were germinated and grown while continuously rotated on the horizontal axis of a clinostat. The coleoptiles of these gravity-compensated plants were phototropically more responsive than those of plants rotated on a vertical axis. When the plants were compensated after unilateral irradiation, phototropic curvature of the shoot progressed for the next 6 hours, with the rate of curving decreasing about 3 hours after irradiation. The decrease in rate was less in the plants gravity-compensated before irradiation than in those vertically rotated. In the period 70 to 76 hours after planting, the growth rate of the compensated coleoptiles was significantly less than that of the vertically rotated seedlings. The greater phototropic curvature, the decreased growth rate, and the slower rate of straightening of the curved, compensated shoot can be correlated with several consequences of compensation: an increase in sensitivity to auxin, a lowering of auxin content in the coleoptile tip, and possibly, from an interaction between compensation and phototropic stimulation, an enhanced difference in auxin transport between the illuminated and shaded halves of the unilaterally irradiated shoot.

The phototropic response of the vertically rotated seedling was significantly different from that of the vertical stationary, indicating the importance of vertically rotated controls in clinostat experiments.

     

Phototropism in Hypocotyls of Radish : II. Role of cis- and trans-Raphanusanins, and Raphanusamide in Phototropism of Radish Hypocotyls

When etiolated radish (Raphanus sativus var. hortensis f. gigantissimus Makino) hypocotyls were subjected to a continuous unilateral illumination with white fluorescent light (0.1 watt per square meter), the growth rate at the lighted side was strongly inhibited for the first 2 hours, while that at the shaded side showed no change. After 2.5 hours growth on the lighted side recovered gradually, while that on the shaded side was slightly inhibited. The neutral growth inhibitors, cis- and trans-raphanusanins and raphanusamide, were determined in the lighted and shaded sides from 1 hour before until 2 hours after the start of unilateral illumination. In the lighted side, cis- and trans-raphanusanins increased by 0.5 hour after the start of illumination, reached 3 to 3.5-fold greater concentrations than in the shaded side after 1 hour, and then decreased gradually. Raphanusamide increased in the lighted side to a 3-fold greater concentration than that in the shaded one 2 hours after the start of the illumination. Unilateral applications of cis- and trans-raphanusanins and raphanusamide suppressed the growth of the hypocotyl on the applied side more than that on the opposite one, causing the hypocotyls to bend towards the site of application. The data suggest that phototropic curvature in radish is caused by the light-induced synthesis of growth-inhibiting cis- and trans-raphanusanins, and raphanusamide at the site of illumination.     

Positive and negative tropic curvature induced by microbeam irradiation of protonemal tip cells of the moss Ceratodon purpureus

The photoreceptor phytochrome mediates tropic responses in protonemata of the moss Ceratodon purpureus. Under standard conditions the tip cells grow towards unilateral red light, or perpendicular to the electrical vector of polarized light. In this study the response of tip cells to partial irradiation of the apical region was analysed using a microbeam apparatus. The fluence response curve gave an unexpected pattern: whereas a 15-min microbeam with light intensities around 3 micro mol m (-2) s (-1) induced a growth curvature towards the irradiated side, higher light intensities around 100 micro mol m (-2) s (-1) caused a negative response, the cells grew away from the irradiated side. This avoidance response is explained by two effects: the light intensity is high enough to induce photoconversion into the active Pfr form of phytochrome, not only on the irradiated but also on the non-irradiated side by stray light. At the same time, the strong light on the irradiated side acts antagonistically to Pfr. As a result of this inhibition, the growth direction is moved to the light-avoiding side. Such a Pfr-independent mechanism might be important for the phototropic response to distinguish between the light-directed and light-avoiding side under unilateral light.     

Ethylene is not involved in the blue light-induced growth inhibition of red light-grown peas

Although the growth of intact plants is inhibited by irradiation with blue light, the growth rate of isolated stem segments is largely unaffected by blue light. We hypothesized that this loss of responsiveness was a result of ethylene production as part of the wounding response. However, we found no interaction between ethylene- and blue light-induced growth inhibition in dark- or red light-grown seedlings of pea (Pisum sativum L.). Inhibition of growth begins in dark-grown seedlings exposed to blue light within 3 min of the onset of blue light, as was known for red light-grown seedlings. By contrast, ethylene-induced inhibition of growth occurs only after a lag of 20 to 30 min or more (dark-grown seedlings) or 60 min (red light-grown seedlings). Also, the inhibition response of red light-grown seedlings is the same whether ethylene is present from the onset of continuous blue-light treatment or not. Finally the spatial distribution of inhibition following blue light was different from that following ethylene treatment.     

Photomodulation of mesocotyl elongation in maize seedlings: Is there a correlative relationship between phytochrome,auxin and cell wall extensibility?

Three h white light irradiation of etiolated maize seedlings ( Zea mays L. cv. Jubilee) inhibited mesocotyl elongation and caused a sharp decrease in cell wall plastic extensibility as measured by the Instron technique. The plastic extensibility following white light irradiation (3 h) was photomodulated by phytochrome. Although the photomodulation of the plastic extensibility was correlated with growth during 20 h, no such correlation was observed at shorter times. The addition of indole-3-acetic acid to light-inhibited intact seedlings, or seedlings from which the coleoptile and inner leaves were excised, resulted in a stimulation of growth. However, none of the IAA concentrations could reverse light inhibition. The possibility of a correlative relationship between phytochrome, auxin and cell wall extensibility is discussed.     

Photoregulation of hypocotyl growth: geotropic evidence for the operation of two photosystems

Abstract. The rate of curvature of etiolated cress ( Lepi-dium sativum L. ) hypocotyls in response to gravity (negative geotropism) was retarded by red or blue light; far-red irradiation was without effect. The timing of the irradiation period in relation to the presentation for geostimulus markedly affected the response. When seedlings were irradiated during the 1–2 h period of geostimulus, blue light was more effective than red at retarding curvature; when seedlings were irradiated prior to geostimulus, only red light affected geocurvature. These results are interpreted as a further example of the kinetically distinct effects of red and blue light on hypocotyl development. Blue light elicited a rapid, immediate response effective only during the period of irradiation; red light induced a response characterized by a lag period and persistence in subsequent darkness. Etiolated mustard seedlings showed similar responses to light and gravity. The results are discussed in relation to the possibility that two photosystems operate in hypocotyl growth.     

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.