Effect of Temperature on Growth and Phototropism of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Seedlings

Seedlings of Arabidopsis thaliana grown at 25°C responded to a change in growth temperature by changing their elongation rate within the next 150 min. Regardless of whether the new temperature was higher or lower than 25°C, the seedlings grew slower after the transfer at all tested temperatures. When the seedlings were grown for 2 days at 11.5°C, 17.9°C, and 23.5°C and then transferred to the range of temperatures between 4°C and 38°C they exhibited maximum elongation in the temperature range between 18°C and 23°C. The kinetics of first positive phototropism in seedlings transferred from 25°C to 15°C differed from the kinetics exhibited by seedlings transferred from 25°C to 28°C. At 15°C, measurable curvature began 40–50 min after the blue light (BL) pulse and no straightening was evident within 150 min after the BL pulse. Seedlings transferred to 28°C exhibited kinetics of phototropism similar to the phototropic response of plants maintained at 25°C except that straightening began slightly faster in the seedlings at 28°C. Based on these results, it is concluded that changes in temperature conditions affect both the elongation rate of seedlings and a first positive phototropism and that phototropic curvature and subsequent straightening are independently controlled. In memory of Radomir Konjević (1 August 1946–22 July 2006), plant physiologist, teacher, mentor, and friend.     

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.     

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.     

Kinetics for Phototropic Curvature by Etiolated Seedlings of Arabidopsis thaliana

An infrared-imaging system has been used to study the influence of gravity on the kinetics of first positive phototropism. The development of phototropic curvature of etiolated seedlings of Arabidopsis thaliana was measured in the absence of visible radiation. Following a pulse of blue light, stationary seedlings curved to a maximum of approximately 16° about 80 minutes after stimulation. The seedlings then curved upward again or straightened by about 6° during the subsequent 100 minutes. Seedlings rotated on a clinostat reached a similar maximum curvature following photostimulation. These seedlings maintained that curvature for 30 to 40 minutes before subsequently straightening to the same extent as the stationary seedlings. It is concluded that straightening is not a consequence of gravitropism, although gravity has some effect on the phototropism kinetics.     

8-Epixanthatin, a light-induced growth inhibitor, mediates the phototropic curvature in sunflower (Helianthus annuus) hypocotyls

The distribution of the endogenous auxin-inhibiting substance, 8-epixanthatin, was determined in the lighted and shaded sides of phototropically-stimulated, de-etiolated sunflower ( Helianthus annuus L. cv. Taiyo) hypocotyls. From 40 min after the onset of phototropic stimulation, the growth rate at the lighted side was inhibited, whereas that at the shaded side showed no change. In the lighted side, 8-epixanthatin increased by 20 min after the onset of unilateral illumination and, after 40 min, reached a 3-fold larger concentration than that in the shaded side. Unilateral application of 8-epixanthatin suppressed the growth of etiolated hypocotyls at the applied side only, causing the hypocotyls to bend at the site of application. It is concluded that phototropic curvature in sunflower hypocotyls is caused by a lateral gradient of the auxin-inhibiting substance 8-epixanthatin.     

Role of leaves in phototropism

Experiments with green seedlings of sunflower (Helianthus annuns L.) indicate the existence of a phototropic mechanism which involves the leaves or cotyledons, and which can produce an asymmetry of auxin content without the involvement of lateral auxin transport, the classic explanation of phototropism in etiolated seedlings. The basic lines of evidence for the leaf-mediated tropism are: 1) darkening of one cotyledon will cause curvature of the stem toward the lighted cotyledon: 2) the darkened cotyledon sustains an enhanced growth rate in the stem below it: 3) conversely, light suppresses the growth-stimulating effects of a single cotyledon: and 4) more diffusible auxin is obtained from the stem below darkened cotyledons than below lighted ones.     

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.     

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.     

Phototropism in Higher Plants — Controversies and Caveats1

Recent literature on light-induced changes in the distribution of growth and of endogenous growth regulators in the development of phototropic curvature is reviewed. It is concluded that in a great many cases the Cholodny-Went hypothesis is sufficient to account for the growth changes bringing about curvature, though in certain dicotyledonous seedlings differential effects of light on the synthesis of growth inhibitors across the tissue from the lighted to the shaded side are implicated. The problems in the interpretation of results from experiments in which more than one photoresponse may be simultaneously induced are discussed and methods of circumventing them considered. Action spectroscopy suggests that dicotyledonous seedlings may have the same phototropic photoreceptor as monocotyledonous seedlings.     

Differential roles of auxin efflux carrier PIN proteins in hypocotyl phototropism of etiolated Arabidopsis seedlings depend on the direction of light stimulus

In a recent study, we demonstrated that although the auxin efflux carrier PIN-FORMED (PIN) proteins, such as PIN3 and PIN7, are required for the pulse-induced first positive phototropism in etiolated Arabidopsis hypocotyls, they are not necessary for the continuous-light-induced second positive phototropism when the seedlings are grown on the surface of agar medium, which causes the hypocotyls to separate from the agar surface. Previous reports have shown that hypocotyl phototropism is slightly impaired in pin3 single mutants when they are grown along the surface of agar medium, where the hypocotyls always contact the agar, producing some friction. To clarify the possible involvement of PIN3 and PIN7 in continuous-light-induced phototropism, we investigated hypocotyl phototropism in the pin3 pin7 double mutant grown along the surface of agar medium. Intriguingly, the phototropic curvature was slightly impaired in the double mutant when the phototropic stimulus was presented on the adaxial side of the hook, but was not impaired when the phototropic stimulus was presented on the abaxial side of the hook. These results indicate that PIN proteins are required for continuous-light-induced second positive phototropism, depending on the direction of the light stimulus, when the seedlings are in contact with agar medium.     

Analysis of growth during phototropic curvature of cress hypocotyls

Abstract. Growth rates throughout an organ curving phototropically under continuous, unilateral while light were monitored by lime-lapse photography of cress hypocotyls marked into 1 mm sections by two rows of ion-exchange beads. Curvature resulted from an integrated sequence of changes in growth rate on each side of the organ, but the actual patterns of change and, therefore rales of curvature, differed within even this one species, depending upon the immediate pretreatment of the seedlings. Transference of seedlings from darkness to unilateral irradiation gave immediate growth inhibition on both sides of the organ. Curvature resulted from differential recovery of growth rate, being seen first on the shaded side, most prominently in the apical regions; only 2h after initial exposure to light did growth recover on the lit (lower) side. On the other hand, transfer of seedlings from omnilateral to unilateral irradiation of the same intensity resulted in simultaneous growth inhibition on the irradiated side and stimulated growth on the shaded side: this growth stimulation of the shaded side was greater than occurred in totally darkened control plants.     

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.     

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.

     

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)     

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.     

Isolation and identification of a light-induced growth inhibitor in diffusates from blue light-illuminated oat (Avena sativa L.) coleoptile tips

The amounts of two growth inhibitors in diffusates from illuminatedhalves of phototropically stimulated oat (Avena sativa L.)coleoptile tips were larger than those from shaded halves. The less polarinhibitor was isolated from diffusates from oat coleoptile tips illuminatedwithblue light, and identified as uridine from ¹H NMR spectrum. Thedistribution of endogenous uridine in diffusates from the illuminated andshadedsides of coleoptile tips unilaterally exposed to blue light for 3, causing a first positive phototropic curvature, and fromdark-control tips, was determined using a physicochemical assay. The uridineconcentration was significantly higher in the diffusates from the illuminatedside than in those from the shaded side and the dark-control. Uridine inhibitedthe growth of etiolated oat coleoptile tips at concentrations of 30 and above. These results suggest that uridine plays a role inthe phototropism of oat coleoptiles.     

Role of the Cotyledons in the Phototropic Response of Lavatera cretica Seedlings

Young seedlings of Lavatera cretica L. exhibit positive phototropism. The hypocotyl perceives unilateral illumination with blue light and curves towards the light source by unequal growth. In addition, the cotyledonary laminas perceive the vectorial component of unilateral illumination with blue light and reorient normal to the beam by creating a turgor differential in their pulvini. Excision of one cotyledon resulted in negative organotropic curvature of the hypocotyl, away from the remaining cotyledon. Illumination of the cotyledonary lamina did not participate in the phototropic curvature of the hypocotyl, so long as the lamina was free to reorient to face the beam. When the lamina was continuously exposed to vectorial photoexcitation, elongation of the hypocotyl on the side carrying the cotyledon could be enhanced, or inhibited, depending on the direction of the beam striking its lamina.     

A Common Fluence Threshold for First Positive and Second Positive Phototropism in Arabidopsis thaliana

The relationship between the amount of light and the amount of response for any photobiological process can be based on the number of incident quanta per unit time (fluence rate-response) or on the number of incident quanta during a given period of irradiation (fluence-response). Fluence-response and fluence rate-response relationships have been measured for second positive phototropism by seedlings of Arabidopsis thaliana. The fluence-response relationships exhibit a single limiting threshold at about 0.01 micromole per square meter when measured at fluence rates from 2.4 × 10⁻⁵ to 6.5 × 10⁻³ micromoles per square meter per second. The threshold values in the fluence rateresponse curves decrease with increasing time of irradiation, but show a common fluence threshold at about 0.01 micromole per square meter. These thresholds are the same as the threshold of about 0.01 micromole per square meter measured for first positive phototropism. Based on these data, it is suggested that second positive curvature has a threshold in time of about 10 minutes. Moreover, if the times of irradiation exceed the time threshold, there is a single limiting fluence threshold at about 0.01 micromole per square meter. Thus, the limiting fluence threshold for second positive phototropism is the same as the fluence threshold for first positive phototropism. Based on these data, we suggest that this common fluence threshold for first positive and second positive phototropism is set by a single photoreceptor pigment system.     

Genetic separation of phototropism and blue light inhibition of stem elongation

Blue light-induced regulation of cell elongation is a component of the signal response pathway for both phototropic curvature and inhibition of stem elongation in higher plants. To determine if blue light regulates cell elongation in these responses through shared or discrete pathways, phototropism and hypocotyl elongation were investigated in several blue light response mutants in Arabidopsis thaliana. Specifically, the blu mutants that lack blue light-dependent inhibition of hypocotyl elongation were found to exhibit a normal phototropic response. In contrast, a phototropic null mutant (JK218) and a mutant that has a 20- to 30-fold shift in the fluence dependence for first positive phototropism (JK224) showed normal inhibition of hypocotyl elongation in blue light. F1 progeny of crosses between the blu mutants and JK218 showed normal phototropism and inhibition of hypocotyl elongation, and approximately 1 in 16 F2 progeny were double mutants lacking both responses. Thus, blue light-dependent inhibition of hypocotyl elongation and phototropism operate through at least some genetically distinct components.     

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.