Mechanism of specific inhibition of phototropism by phenylacetic Acid in corn seedling

Using geotropism as a control for phototropism, compounds similar to phenylacetic acid that photoreact with flavins and/or have auxin-like activity were examined for their ability to specifically inhibit phototropism in corn seedlings using geotropism as a control. Results using indole-3-acetic acid, napthalene-1-acetic acid, naphthalene-2-acetic acid, phenylacetic acid, and β-phenylpyruvic acid suggest that such compounds will specifically inhibit phototropism primarily because of their photoreactivity with flavins and not their auxin activity. For example, strong auxins, indole-3-acetic acid and naphthalene-1-acetic acid, affected both tropic responses at all concentrations tested whereas weak auxins, phenylacetic acid and naphthalene-2-acetic acid, exhibited specific inhibition. In addition, the in vivo concentration of phenylacetic acid required to induce specificity was well below that required to stimulate coleoptile growth. Estimates of the percentage of photoreceptor pigment inactivated by phenylacetic acid (>10%) suggest that phenylacetic acid could be used to photoaffinity label the flavoprotein involved in corn seedling phototropism.     

Role of carotenoids in the phototropic response of corn seedlings

The herbicide, 4 chloro-5-(methylamino)-2-(α,α,α,-trifluoro-m-tolyl)-3 (2H)-pyridazinone (SAN 9789), which blocks the synthesis in higher plants of colored carotenoids but not of flavins, was used to examine the involvement of carotenoids in corn seedling phototropism. It was concluded that “bulk” carotenoids are not the photoreceptor pigment based on the results that increasing concentrations of SAN 9789 (up to 100 micromolar) did not alter the phototropic sensitivity to 380 nanometers light (using geotropism as a control) and did not increase the threshold intensities of fluence response curves for both 380 and 450 nanometers light even though carotenoid content was reduced to 1 to 2% of normal. SAN 9789 treatment, however, did reduce seedling sensitivity toward 450 nanometers light indicating that carotenoids are involved in phototropism. Carotenoids, which are located mainly in the primary leaves, may act in phototropism as an internal screen, enhancing the light intensity gradient across the seedling and thus contributing to the ability of the seedling to perceive light direction. These results indicate that the action spectra for phototropic responses can be significantly affected by the absorbance of screening pigments in vivo thus altering its shape from the in vitro absorption spectrum of the photoreceptor pigment.     

Wirkung von Vorbestrahlung mit Rot- oder Blaulicht auf die geotropische Empfindlichkeit von Maiskoleoptilen

The early geotropic downward bending of corn (Zea mays L.) coleoptiles was found to be influenced by red and blue light. The coleoptiles were illuminated from above and kept in the dark for defined intervals; afterwards they were positioned horizontally and their curvature was monitored for 40 min. After illumination with red light and a 120 min interval and early upward bending instead of an early downward bending was found. This effect was nullified by a far-red illumination administered immediately after exposure to red light. These results indicate that the phytochrome system influences the geotropic reaction. After illumination with blue light and a 30 min interval little downward bending was found. This result corresponds well with the findings of earlier authors who measured the late geotropic reaction, on the basis of the hypothesis that the strength of the early downward bending is a measure of the geotropic sensitivity. The dose-effect curve of the blue light influence on geotropic sensitivity, measured by early downward bending, is very similar to the dose-effect curve of the phototropic curvature of corn coleoptiles. This fact together with the earlier finding of similar adaptation times of about 30 min suggests the existence of some common transducers in the reaction chain of phototropism and geotropism.

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Abkürzungen HR Hellrot - DR Dunkelrot - D Dunkel - WT Wartezeit - DK Dunkelkontrolle     

The Rice COLEOPTILE PHOTOTROPISM1 gene encoding an ortholog of Arabidopsis NPH3 is required for phototropism of coleoptiles and lateral translocation of auxin

We isolated a mutant, named coleoptile phototropism1 (cpt1), from gamma-ray-mutagenized japonica-type rice (Oryza sativa). This mutant showed no coleoptile phototropism and severely reduced root phototropism after continuous stimulation. A map-based cloning strategy and transgenic complementation test were applied to demonstrate that a NPH3-like gene deleted in the mutant corresponds to CPT1. Phylogenetic analysis of putative CPT1 homologs of rice and related proteins indicated that CPT1 has an orthologous relationship with Arabidopsis thaliana NPH3. These results, along with those for Arabidopsis, demonstrate that NPH3/CPT1 is a key signal transduction component of higher plant phototropism. In an extended study with the cpt1 mutant, it was found that phototropic differential growth is accompanied by a CPT1-independent inhibition of net growth. Kinetic investigation further indicated that a small phototropism occurs in cpt1 coleoptiles. This response, induced only transiently, was thought to be caused by the CPT1-independent growth inhibition. The 3H-indole-3-acetic acid applied to the coleoptile tip was asymmetrically distributed between the two sides of phototropically responding coleoptiles. However, no asymmetry was induced in cpt1 coleoptiles, indicating that lateral translocation of auxin occurs downstream of CPT1. It is concluded that the CPT1-dependent major phototropism of coleoptiles is achieved by lateral auxin translocation and subsequent growth redistribution.     

Reception of far-ultraviolet light in Phycomyces: antagonistic interaction with blue and red light

Phototropism experiments were done with sporangiophores of the fungus Phycomyces blakesleeanus to characterize the interaction between far-UV, blue and red light. Far-UV light elicits negative phototropism (bending away from the light source) while blue light elicits positive phototropism (bending toward the light source). In contrast, red light above 600 nm is phototropically inert. Phototropism was analyzed with light regimens of bilateral or unilateral irradiation with far-UV and blue light. Under bilateral irradiation, in which the two light sources were facing each other, blue light partially inhibited the far-UV-elicited phototropism. A fluence-response curve for this inhibition showed that blue light was maximally effective at fluence rates which exceeded 3 to 57 times that of the far-UV. Tonic red light, which was given from above, abolished to a large extent the antagonistic action of blue light. With a regimen of unilateral irradiation, i.e. when far-UV and blue light were given from the same side, a phototropic balance could be achieved with approximately equal fluence rates of blue and UV light. Above or below this critical balance point the bending was either negative or positive. In this setup the effect of tonic red light was complex. First, it caused an enhancement of the positive or negative bending, and second, it caused at some fluence rates a sign reversal from positive to negative phototropism. The balance point itself was only marginally affected. The data cannot be explained on the basis of a single photoreceptor and support the previous notion of separate far-UV and blue-light receptors. The antagonism between these two receptors probably occurs on the level of a red-light-absorbing receptor intermediate. Received: 16 November 1997 / Accepted: 18 December 1997     

Characterization of a Membrane Fraction Containing a b-type Cytochrome

The various components obtained from etiolated corn (Zea mays L.) coleoptiles were fractionated by differential or sucrose gradient centrifugation. The endoplasmic reticulum, proplastids, Golgi, and mitochondria were localized by enzymic or other markers in the various fractions. A fifth fraction was also characterized. It contains glucan synthetase II activity, binding sites for N-naphthylphthalamic acid, NADH dehydrogenase activity which is both antimycin A- and cyanide-insensitive, and a b-type cytochrome. It is possible that this fraction is plasma membrane and that it may contain the blue-ultraviolet photoreceptor for phototropism in corn.     

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.     

Analysis of microsomal flavoproteins from Phycomyces sporangiophores: Candidates for the blue-light photoreceptor

To help identify components of the blue-light photoreceptor system for phototropism in Phycomyces blakesleeanus Bgff., proteins from a microsomal fraction obtained from synchronous sporangiophores were studied. By two-dimensional gel electrophoresis, two proteins (FP₁, FP₂) with covalently bound flavins were found. FP₁ has a molecular weight of 71 000 and an isoelectric point of 6.6; FP₂ has a molecular weight of 59 000 and an isoelectric point of 6.5. These flavoproteins were purified by column chromatography and gel filtration while assaying for flavins by fluorescence. The relative concentrations of FP₁ and FP₂ were affected by light applied during growth. These flavoproteins are likely components of the blue-light photoreceptor complex mediating phototropism in Phycomyces.Abbreviations 10 k pellet 10 000-g pellet - 100 k pellet 100 000-g pellet - FP₁, FP₂ proteins with covalently bound flavins having molecular weights of 71 000 and 59 000 and isoelectric points of 6.6 and 6.5, respectively     

Blue-light regulation of ZmPHOT1 and ZmPHOT2 gene expression and the possible involvement of Zmphot1 in phototropism in maize coleoptiles

Main conclusion

ZmPHOT1 and ZmPHOT2 are expressed differentially in maize coleoptiles and leaves, with Zmphot1 possibly involved in first-positive phototropic curvature of red-light-adapted maize coleoptiles exposed to pulsed low-fluence blue light. Unilateral blue-light perception by phototropin(s) is the first event of phototropism, with the subsequent signal causing lateral transport of auxin at the coleoptile tip region of monocots. In this study, we analyzed the behavior of two maize phototropin genes: ZmPHOT1 and ZmPHOT2, the latter identified from the maize genome database and newly characterized. Quantitative real-time PCR analysis demonstrated that ZmPHOT1 was abundantly expressed in etiolated coleoptiles, while lower expressions of both ZmPHOT1 and ZmPHOT2 were observed in young leaves. Interestingly, these genes were not specifically expressed in the coleoptile tip region, a key position for photoperception in phototropism. Exposure to pulsed low-fluence blue light (LBL) (0.33 µmol m^?2 s^?1 × 8 s) and continuous high-fluence blue light (HBL) (10 µmol m^?2 s^?1) rapidly decreased ZmPHOT1 gene expression in coleoptiles, with levels of ZmPHOT2 not significantly altered in that tissue. In young leaves, no drastic expression changes were induced in either ZmPHOT1 or ZmPHOT2 by LBL or HBL irradiation. The Zmphot1 protein was investigated by Western blot analysis with anti-Osphot1 antibodies. Zmphot1 was detected in microsomal fractions, with higher levels in coleoptiles than in leaves. HBL caused rapid phosphorylation of the protein, whereas no phot1 phosphorylation was induced by LBL. The involvement of Zmphot1 in LBL-induced phototropic curvature of maize coleoptiles is discussed.     

Phytochrome-controlled phototropism of protonemata of the moss Ceratodon purpureus: physiology of the wild type and class 2 ptr–mutants

Phototropism and polarotropism in protonemata of the moss Ceratodon purpureus are controlled by the photoreceptor phytochrome. One class of phototropism mutants is characterised by growing randomly when kept for a prolonged time (5 d or longer) in unilateral red light. It was found that a subclass of these mutants grows faster than the wild type, the rate of cell division and the length of the cells being increased. This difference is found for light-grown and dark-grown filaments. It is therefore suggested that the mutant phenotype neither results from a defect in phytochrome photoconversion nor from a defect in phytochrome-gradient formation. Instead, it is possible that a factor which is involved in both signal transduction of phototropism and regulation of cell size and cell division is deregulated. If dark-grown mutant filaments are phototropically stimulated for 24 h, they show a weak phototropic response. Phototropism and polarotropism fluence-rate effect curves for mutants were flattened and shifted to higher fluence rates compared with those for the wild type. With wild-type filaments, a previously unreported response was observed. At a low fluence rate, half of the filaments grew positively phototropically, while the other half grew negatively phototropically. It seems that under these conditions, a phytochrome gradient with two maxima for the far-red-absorbing form of phytochrome (Pfr) within the cross-section of the cell is displayed by the response of the filaments. At higher fluence rates, all filaments of the wild type grew towards the light. These data and results from microbeam irradiation experiments and from phototropism studies with filaments growing within agar, indicate that light refraction plays an important role in the formation of the Pfr gradient in phototropism of Ceratodon. Received: 10 September 1998 / Accepted: 30 December 1998     

Mechanism of detecting light direction in first positive phototropism in Zea mays L.

Abstract. The carotenoid content of corn seedlings was reduced by 80–90% with the herbicide SAN 9789 or by using carotenoidless mutants. This caused a decrease in 'first positive' phototropism by about 50% without affecting geotropism. This reduction in phototropism is attributed to the decreased light gradient across the albino shoot. Decreased screening should increase the response if a focusing mechanism is used to measure the light gradient, but should decrease the response if a screening mechanism is used. Thus, these data support the hypothesis that screening establishes the light gradient used to measure light direction in 'first positive' phototropism.     

Inhibition of auxin transport by isoquinolinedione herbicides

2-(p-carbethoxyphenyl)-1,3(2H,4H)-isoquinolinedione (CEPIQ), an experimental herbicide, caused effects on geotropism, which are often indicative of an effect on auxin transport, in a whole plant herbicidal screen. However, it showed little or no activity in an in vitro binding assay in corn coleoptiles for the auxin-transport inhibitor,N-1-naphthylphthalamic acid (NPA). Other active isoquinolinedione analogues of this compound did, however, exhibit significant in vitro activity. Direct measurements of auxin transport in corn coleoptiles were undertaken in an attempt to resolve the apparent discrepancy between herbicidal and binding activities. In all cases examined, compounds that were highly active on whole plants were good inhibitors of auxin transport, and compounds that were weak as herbicides showed little or no effect on auxin transport. Therefore, it is concluded that the mode of action of these isoquinolinedione herbicides is the inhibition of auxin transport. Ring-opened analogues of several isoquinolinediones were synthesized and assayed in both the transport and binding assays, in order to test whether compounds in this class express their herbicidal activity by undergoing ring-opening in vivo, yielding products that are more straightforward analogues of NPA with free carboxyl groups. The homophthalamic acids had little or no activity in both assays. On the other hand, thep-ethyl- andp-ethoxy-phenyl phthalamic acids showed auxin transport inhibition comparable to the parent isoquinolinediones, but with markedly increased binding activity. These results support the possible role of ring-opening in the generation of biological activity. However, thep-carbethoxyphenyl phthalamic acid, analogous to CEPIQ, was very weak in both assays. Thus, ring-opening in vivo cannot alone account for the biological activity of this class of compounds.     

Phototropism: mechanisms and ecological implications

Abstract. Phototropism in seed plants, either etiolated or de-etiolated, is mediated by unidentified photoreceptor(s) sensitive to blue and near-UV regions of the light spectrum. Green plants may have an additional phototropic system sensitive to red light. Fluence-response studies of the blue light-sensitive phototropism, initially made on oat coleoptiles, have indicated the occurrence of multiple response types. Of those, two are found to be general: the first pulse-induced positive phototropism (fPIPP), or the so-called first positive curvature, and the time-dependent phototropism (TDP) or the second positive curvature. The fPIPP, elicited by a pulse stimulus shorter than a few minutes, is characterized by a bell-shaped fluence-response curve and the validity of reciprocity. The TDP, elicited by prolonged irradiation, is characterized by its dependence on the exposure time and the invalidity of reciprocity. Studies made on these two response types have revealed the following: (1) plants acquire directional light information for phototropism by sensing internal light gradients created by light scattering and absorption; (2) phototropism results from redistribution of growth, i.e. inhibition on the irradiated side and compensating stimulation on the shaded side; (3) lateral movement of growth regulators, the principle of the Cholodny-Went theory, can account for the growth redistribution, and auxin is clearly the mediating regulator in maize coleoptiles. This review further describes some mechanistic implications of fPIPP. Experimental results indicate that (1) fPIPP is mediated by a single step of photoreaction, (2) the responsiveness, reflected in the height of the fluenceresponse curve, is reduced by pre-irradiation with blue light and recovers gradually afterward, and (3) the light sensitivity, reflected in the position of the fluence-response curve along the log fluence axis, is also reduced by the pre-irradiation and recovers gradually. Analyses of these results, based on kinetic models, suggest that the bell-shaped fluence-response curve is caused by the difference in the amounts of a photoproduct between irradiated and shaded sides, and that fPIPP represents a mechanism of TDP. It is also indicated that phytochrome in the red-absorbing form exerts two separate effects on phototropism: reduction of the light sensitivity and enhancement of the responsiveness. Along with the discussion of the mechanisms of phototropism, their ecological implications are considered.     

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     

Photogravitropic equilibrium in <Emphasis Type="Italic">Avena</Emphasis> coleoptiles: fluence rate–response relationships and dependence on dark adaptation and clinostating

Phototropism of Avena coleoptiles was measured in response to blue-light irradiation lasting between 2 and 24 h. During this time the coleoptiles established a bending angle of photogravitropic equilibrium that was dependent on the time of irradiation and also on the pretreatment in light or darkness prior to stimulation. The absolute threshold for the photogravitropic equilibrium in response to blue light was 10⁻⁸ μmol m⁻² s⁻¹. Photon fluence rate–response curves, which were generated after several hours of dark adaptation, had a characteristic shape with a prominent optimum in the middle of the dynamic range. Curves which were generated without prior dark adaptation displayed no such optimum. Clinostating dark-adapted coleoptiles caused an increase of sensitivity and responsiveness during a 2-h period of unilateral irradiation. The advantages and the drawbacks of long-term irradiation experiments for the investigation of phototropism and the generation of action spectra are discussed. Received: May 14, 2001 / Accepted: December 7, 2001     

The role of mutants in the search for the photoreceptor for phototropism in higher plants

Early attempts to identify the chromophore of the photoreceptor for phototropism are reviewed. Carotenoids and flavins were the principal candidates, but studies with grass coleoptiles devoid of carotenoids suggest that at least in these organs carotenoids are most unlikely to play that role. The status of characterization of a gene for a putative photoreceptor protein is also reviewed. As the action spectrum for phototropism resembles the absorption spectrum of a flavoprotein, flavoproteins are attractive candidates at present, especially since the CRY1 photoreceptor in Arabidopsis thaliana that mediates blue light-dependent hypocotyl growth suppression has flavin adenine dinucleotide as one of its two chromophores. As the second chromophore appears to be pterin, pterins should not be ruled out as candidate chromophores for the photoreceptor for phototropism.     

Auxin Redistribution during First Positive Phototropism in Corn Coleoptiles : Microtubule Reorientation and the Cholodny-Went Theory

In red-light grown corn (Zea mays L. cv Brio42.HT) coleoptiles, cortical microtubules adjacent to the outer cell wall of the outer epidermis reorient from transverse to longitudinal in response to auxin depletion and after phototropic stimulation in the lighted side of the coleoptile. This was used as an in situ assay of cellular auxin concentration. The fluence-response relation for the blue light-induced reorientation is compared with that for first positive phototropism and the dose-response relationship for the auxin-dependent reorientation. The result supports the theory by Cholodny and Went, claiming that phototropic stimulation results in auxin displacement across the coleoptile. In terms of microtubule orientation, this displacement becomes even more pronounced after preirradiation with a weak blue light pulse from above.     

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.     

Inhibition of hypocotyl elongation by ultraviolet-B radiation in de-etiolating tomato seedlings. I. The photoreceptor

Broad-band UV-B radiation inhibited hypocotyl elongation in etiolated tomato ( Lycopersicon esculentum Mill. cv. Alisa Craig) seedlings. This inhibition could be elicited by < 3 μmol m⁻² s⁻¹ of UV-B radiation provided against a background of white light (> 620 μmol m⁻² s⁻¹ between 320 and 800 nm), and was similar in wild-type and phytochrome-1-deficient aurea mutant seedlings. These observations suggest that the effect of UV-B radiation is not mediated by phytochrome. An activity spectrum obtained by delivering 1 μmol m⁻² s⁻¹ of monochromatic UV radiation against a while light background (63 μmol m⁻² s⁻¹ showed maximum effectiveness around 300 nm, which suggests that DNA or aromatic residues in proteins are not the chromophores mediating UV-B induced inhibition of elongation. Chemicals that affect the normal (photo)chemistry of flavins and possibly pterins (KI, NaN, and phenylacetic acid) largely abolished the inhibitor) effect of broad-hand UV-B radiation when applied to the root zone before irradiation. KI was effective at concentrations < 10⁻⁴ M , which have been shown in vitro to be effective in quenching the triplet excited stales of flavins but not fluorescence from pterine or singlet states of flavins. Elimination of blue light or reduction of UV-A, two sources of flavin excitation, promoted hypocotyl elongation, but did not affect the inhibition of elongation evened by UV-B. Kl applied after UV-B irradiation had no effect on the inhibition response. Taken together these findings suggest that the chromophore of the photoreceptor system invoked in UV-B perception by tomato seedlings during de-etiolation may be a flavin.     

Far-red light irradiation of intact corn seedlings affects mitochondrial and calmodulin-dependent microsomal Ca2+ transport

Microsomal fractions isolated from coleoptiles of dark or far-red light grown corn show ATP-dependent Ca²⁺ accumulation. The microsomal transport from dark but not from far-red light grown tissue could be stimulated by calmodulin. Ca²⁺ accumulation into mitochondria from coleoptiles of far-red light grown corn in also inhibited as compared to the dark controls. Light irradiation of isolated microsomes and mitochondria had no effect on either Ca²⁺ uptake nor efflux.