Phytochrome is required for the occurrence of time-dependent phototropism in maize coleoptiles

Time-dependent phototropism (TDP), sometimes called second positive curvature, occurs when the duration of phototropic stimulation with blue light (B) exceeds a few minutes. TDP was characterized in maize (Zea mays L.) coleoptiles raised under continuous red light (R). Subsequently, coleoptiles adapted to darkness were used to investigate the effect of R on TDP. It was found that TDP, which is induced in R-grown coleoptiles, does not occur in dark-adapted coleoptiles and that dark-adapted coleoptiles begin to show TDP after treatment with R. The TDP responsiveness became maximal 1-2 h after treatment with a R pulse and decreased during the next few hours. At least 10 min was required after a short pulse of R before the coleoptile began to respond to B for the induction of TDP. The effect of R in establishing the TDP responsiveness was totally suppressed by a pulse of far-red light given immediately after an inductive pulse of R. It is concluded that the mechanism of TDP requires for its establishment a R signal perceived by phytochrome. The TDP of R-grown and R-pretreated coleoptiles showed relationships to stimulation times and fluence rates that are similar to those reported for oat coleoptiles, except that TDP of maize showed a sharp increase in its magnitude within a narrow range of stimulation times as short as 5-10 min.     

Opposing roles of phytochrome A and phytochrome B in early cryptochrome-mediated growth inhibition

The cryptochrome 1 (cry1) photoreceptor is responsible for the majority of the inhibitory effect of blue light on hypocotyl elongation, but phytochrome photoreceptors also contribute to the response through a phenomenon known as coaction. In Arabidopsis thaliana the participation of phytochromes A and B (phyA and phyB) in the early phase of cry1 action was investigated by determining the effects of phyA, phyB and hy1 mutations on a cry1-dependent membrane depolarization, which is caused by the activation of plasma-membrane anion channels within seconds of blue light treatment. High-resolution growth measurements were also performed to determine the timing of the requirement for phytochrome in cry1-mediated growth inhibition, which is causally linked to the preceding anion-channel activation. A null mutation in PHYA impaired the membrane depolarization and prevented the early cry1-dependent phase of growth inhibition as effectively and with the same time course as mutations in CRY1. Thus, phyA is necessary for cry1/cry2 to activate anion channels within the first few seconds of blue light and to suppress hypocotyl elongation for at least 120 min. This finding furthers the notion of an intimate mechanistic association between the cry and phy receptors in mediating light responses. The absence of phyB did not affect the depolarization or growth inhibition during this time frame. Instead, double mutant analyses showed that the phyB mutation suppressed the early growth phenotypes of both phyA and cry1 seedlings. This result is consistent with the emerging view that the prevailing growth rate of a stem is a compromise between light-dependent inhibitory and promotive influences. It appears that phyB opposes the cry1/phyA-mediated inhibition by promoting growth during at least the first 120 min of blue light treatment.     

Both phytochrome A and phytochrome B are required for the normal expression of phototropism in Arabidopsis thaliana seedlings

The role of phytochrome A (phyA) and phytochrome B (phyB) in phototropism was investigated by using the phytochrome-deficient mutants phyA-101 , phyB-1 and a phyA/phyB double mutant. The red-light-induced enhancement of phototropism, which is normally observed in wild-type seedlings, could not be detected in the phyA/phyB mutant at fluences of red light between 0.1 and 19 000 μmol m⁻². The loss of phyB has been shown to have no apparent effect on enhancement, while the loss of phyA resulted in a loss of enhancement only in the low fluence range (Janoudi et al. 1997). The conclusions of the aforementioned study can now be modified based on the current results which indicate that phototropic enhancement in the high fluence range is mediated by either phyA or phyB, and that other phytochromes have no role in enhancement. First positive phototropism was unaffected in phyA-101 and phyB-1 However, the magnitude of first positive phototropism in the phyA/phyB mutant was significantly lower than that of the wild-type Landsberg parent. Thus, the presence of either phyA or phyB is required for normal expression of first positive phototropism. The time threshold for second positive phototropism is unaltered in the phyA-101 and phyB mutants. However, the time threshold in the phyA/phyB mutant is about 2 h, approximately six times that of the wild type. Finally, the magnitude of second positive phototropism in both phyA-101 and phyB-1 is diminished in comparison with the wild-type response. Thus, phyA and phyB, acting independently or in combination, regulate the magnitude of phototropic curvature and the time threshold for second positive phototropism. We conclude that the presence of phyA and phyB is required, but not sufficient, for the expression of normal phototropism.     

Phytochrome regulation of pea phototropin

Type 1 phototropin, one of the blue light receptors responsible for phototropism, is encoded in peas by at least two genes, PsPHOT1A and PsPHOT1B (formerly PsPK4 and PsPK5), both of which are more similar to Arabidopsis PHOT1 than to Arabidopsis PHOT2. We show here that PsPHOT1B encodes a full-length phototropin, whose expression pattern suggests that Psphot1b is the predominant phot1-type phototropin in etiolated seedlings. The gene encoding the other type 1 phototropin, PsPHOT1A, is expressed at low levels, with its highest levels in the leaves and stems of more mature, light-grown plants. Studies with phyA, phyB and the phyAphyB double mutants show that phyA and phyB have partially redundant roles in the reduction of PsPHOT1B expression under red light.     

Effects of red light on the fluence–response relationship for pulse-induced phototropism of maize coleoptiles

Dark-adapted coleoptiles of maize (Zea mays L.) were treated with red light (3min at 10.5 μmol m^?2S^?1) and were Stimulated, after a dark interval, with a pulse of unilateral blue light to induce phototropism. Phototropic fluence-response curves were obtained in this way for different dark intervals. It was confirmed that the bell-shaped fluence-response curve for the first pulse-induced positive phototropism (FPIPP) shifts to higher fluences following the red-light treatment, the maximal shift being achieved at a dark interval of 2h. We found, however, that the two arms of the Fluence-response curve do not shift synchronously. The shift of the descending arm to higher fluences began at 15 min. The ascending arm showed a slight shift to lower fluences before a greater shift to higher flucnces. the change of the shift direction occurring at 30–40min. Accordingly, the fluence-response curve obtained for a 30 min dark interval was comparatively wide. Although dark-adapted coleoptiles showed only fPIPP, another bell-shaped fluence-response curve, representing the second pulse-induced positive phototropism (sPIPP), appeared gradually after the red-light treatment. These changes of the phototropic fluence– respnse curve following exposure to red light are likely to have adaptive values because they favour phototropism under brighter light.     

PKS1 plays a role in red-light-based positive phototropism in roots

Aerial parts of plants curve towards the light (i.e. positive phototropism), and roots typically grow away from the light (i.e. negative phototropism). In addition, Arabidopsis roots exhibit positive phototropism relative to red light (RL), and this response is mediated by phytochromes A and B (phyA and phyB). Upon light stimulation, phyA and phyB interact with the phytochrome kinase substrate (PKS1) in the cytoplasm. In this study, we investigated the role of PKS1, along with phyA and phyB, in the positive phototropic responses to RL in roots. Using a high-resolution feedback system, we studied the phenotypic responses of roots of phyA, phyB, pks1, phyA pks1 and phyB pks1 null mutants as well as the PKS1-overexpressing line in response to RL. PKS1 emerged as an intermediary in the signalling pathways and appears to promote a negative curvature to RL in roots. In addition, phyA and phyB were both essential for a positive response to RL and act in a complementary fashion. However, either photoreceptor acting without the other results in negative curvature in response to red illumination so that the mode of action differs depending on whether phyA and phyB act independently or together. Our results suggest that PKS1 is part of a signalling pathway independent of phyA and phyB and that PKS1 modulates RL-based root phototropism.     

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.     

Lag period for phototropism inPilobolus crystallinus sporangiophores

The lag period for the second positive curvature was examined inPilobolus crystallinus sporangiophores. The lag period for curvature development was 20–30 min at lower fluence rates than 6.32 nmol/m²s but greatly extended at higher fluence rates. When a 20-min symmetrical irradiation with blue light was applied before a 20-min unilateral blue light irradiation, sporangiophores bent as much as those unilaterally and continuously irradiated for 40 min. However, when a 20-min unilateral irradiation was followed by a 20-min symmetrical irradiation, sporangiophores did not show any curvature. That is, the reaction during the first 20 min of the lag period is independent of light direction. This light-direction-independent lag period is considered to be the duration required for adaptation. The lag period for phototropism was also extended when fluence rate was reduced after the start of irradiation. These results suggested that an adaptation process is involved in phototropism ofPilobolus.     

Phytochrome A is an irradiance-dependent red light sensor

Plants perceive red (R) and far-red (FR) light signals using the phytochrome family of photoreceptors. In Arabidopsis thaliana, five phytochromes (phyA-phyE) have been identified and characterized. Unlike other family members, phyA is subject to rapid light-induced proteolytic degradation and so accumulates to relatively high levels in dark-grown seedlings. The insensitivity of phyA mutant seedlings to prolonged FR and wild-type appearance in R has led to suggestions that phyA functions predominantly as an FR sensor during the early stages of seedling establishment. The majority of published photomorphogenesis experiments have, however, used <50 micromol m(-2) sec(-1) of R when characterizing phytochrome functions. Here we reveal considerable phyA activity in R at higher (>160 micromol m(-2) sec(-1)) photon irradiances. Under these conditions, plant architecture was observed to be largely regulated by the redundant actions of phytochromes A, B and D. Moreover, quadruple phyBphyCphyDphyE mutants containing only functional phyA displayed R-mediated de-etiolation and survived to flowering. The enhanced activity of phyA in continuous R (Rc) of high photon irradiance correlates with retarded degradation of the endogenous protein in wild-type plants and prolonged epifluorescence of nuclear-localized phyA:YFP in transgenic lines. Such observations suggest irradiance-dependent 'photoprotection' of nuclear phyA in R, providing a possible explanation for the increased activity observed. The discovery that phyA can function as an effective irradiance sensor, even in light environments that establish a high Pfr concentration, raises the possibility that phyA may contribute significantly to the regulation of growth and development in daylight-grown plants.     

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.     

Phytochrome-mediated phototropism in maize seedling shoots

Unilateral irradiation with red light (R) or blue light (BL) elicits positive curvature of the mesocotyl of maize (Zea mays L.) seedlings raised under R for 2 d from sowing and kept in the dark for 1 d prior to curvature induction. The fluenceresponse curve for R-induced mesocotyl curvature, obtained by measuring curvature 100 min after phototropic induction, shows peaks in two fluence ranges, designated first positive range (from the threshold to the trough), and second positive range (above the trough). The fluence-response curve for BL is similar to that for R but shifted two orders of magnitude to higher fluences. Blue light elicits the classical first positive curvature of the coleoptile, whereas this response is not found with R. Positive mesocotyl curvature induced by either R or BL is eliminated by R given from above just before the unilateral irradiation, whereas BL-induced coleoptile curvature is not eliminated. The above results collectively offer evidence that phototropic curvature of the mesocotyl is induced by R-sensitive photosystem(s). Mesocotyl curvature in the second positive range is reduced by vertical far-red light (FR) applied after phototropic induction with R, but is not affected by FR applied before R. Unilateral irradiation with FR following vertical irradiation with a high R fluence leads to negative curvature of the mesocotyl. It is concluded that mesocotyl curvature in the second positive range results from a gradient in the amount of the FR-absorbing form of phytochrome (Pfr) established across the plant axis. Mesocotyl curvature in the first positive range is inhibited by vertical FR given either before or after phototropic induction with R. Since the FR used here is likely to produce more Pfr than the very low fluences of R eliciting the mesocotyl curvature in the first positive range, it is assumed that FR reduces the response in this case by adding Pfr at both sides of the plant axis. By rotating seedlings on a clinostat with its axis horizontal, the kinetics of mesocotyl curvature can be studied in the absence of a counteracting gravitropic response. On the clinostat, the R-induced mesocotyl curvature develops after a lag, through two successive phases having different curvature rates, the late phase is slower than the early phase. Negative curvature of the coleoptile can be induced by either R or BL; the BL-induced negative curvature is found at fluences higher than those giving positive curvature. The clinostat experiments show that the negative coleoptile curvature induced by either R or BL is a gravitropic compensation for positive mesocotyl curvature.Abbreviations BL blue light - FR far-red light - Pfr phytochrome in the far-red-absorbing form - Pr phytochrome in the red-absorbing form - R red light C.I.W.-D.P.B. Publication No. 824     

Unexpected roles for cryptochrome 2 and phototropin revealed by high-resolution analysis of blue light-mediated hypocotyl growth inhibition

Blue light (BL) rapidly and strongly inhibits hypocotyl elongation during the photomorphogenic response known as de-etiolation, the transformation of a dark-grown seedling into a pigmented, photoautotrophic organism. In Arabidopsis thaliana, high-resolution studies of hypocotyl growth accomplished by computer-assisted electronic image capture and analysis revealed that inhibition occurs in two genetically independent phases, the first beginning within 30 sec of illumination. The present work demonstrates that phototropin (nph1), the photoreceptor responsible for phototropism, is largely responsible for the initial, rapid inhibition. Signaling from phototropin during the curvature response is dependent upon interaction with NPH3, but the results presented here demonstrate that NPH3 is not necessary for phototropin-dependent growth inhibition. Activation of anion channels, which transiently depolarizes the plasma membrane within seconds of BL, is an early event in the cryptochrome signaling pathway leading to a phase of growth inhibition that replaces the transient phototropin-dependent phase after approximately 30 min of BL. Surprisingly, cry1 and cry2 were found to contribute equally and non-redundantly to anion-channel activation and to growth inhibition between 30 and 120 min of BL. Inspection of the inhibition kinetics displayed by nph1 and nph1cry1 mutants revealed that the cryptochrome phase of inhibition is delayed in seedlings lacking phototropin. This result indicates that BL-activation of phototropin influences cryptochrome signaling leading to growth inhibition. Mutations in the NPQ1 gene, which inhibit BL-induced stomatal opening, do not affect any aspect of the growth inhibition within the first 120 min examined here, and NPQ1 does not affect the activation of anion channels.     

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.     

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.     

Control of hypocotyl phototropism by phytochrome in a dicotyledonous seedling (Sesamum indicum L.)

Abstract The phototropic response in stems of higher plants is brought about by blue/UV light. The problem studied here is to what extent long-wavelength light, which is absorbed by phytochrome, affects the phototropic response. A refined measurement of phototropism — a curvature index — was applied to the hypocotyl of the sesame seedling (Sesamum indicum L.). The time course of the phototropic response was followed in continuous unilateral weak blue light (B, 460 nm, 8 mW m^?2). Long term red light (R) pretreatments, operating through phytochrome, strongly increase the rate and extent of the phototropic response once it is elicited by unilateral B, while the pretreatments decrease the sensitivity towards B. If a R pulse is given immediately prior to the onset of unilateral B, the rate of the response is strongly reduced compared to the time course of curvature observed when the pretreatment was terminated with a long wavelength far-red light (FR) pulse. R and FR were then applied simultaneously with unilateral B to manipulate the status of the phytochrome system during actual curvature. It was found that a low Pfr/P ratio (established by FR) stimulates the phototropic response far above the control (B alone), while a high Pfr/P ratio (established by R) reduces the response below the control. During bending a positive effect of phytochrome on the rate and extent of the phototropic response, which is saturated at a low level of Pfr, appears to be counteracted by an inhibitory effect which dominates at higher levels of Pfr, such as established by omnilateral R. However, if R is applied unilaterally from the same direction as B, R increases the rate of curvature. Apparently the sesame seedling is capable of detecting the direction of R relative to the direction of B. While a mechanistic explanation of these effects cannot be advanced at present, it is clear that the seedling is capable of super-imposing information about the actual light conditions during bending on a ‘memory’ of the light conditions prior to the onset of bending. Thus, the previous as well as the actual light conditions determine its phototropic responsiveness.     

Mutants of Arabidopsis thaliana with altered phototropism

Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and second positive phototropism. However, while the amplitude for first positive phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in first positive phototropism are shifted to higher fluence by a factor of 20–30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 mol·m^-2 to 2700 mol·m^-2, but shows normal gravitropism. Strain JK345 shows no first positive phototropism, and reduced gravitropism and second positive phototropism. Strain JK229 shows no measurable first positive phototropism, but normal gravitropism and second positive phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. first positive and second positive phototropism contain at least one common element; and 3. first positive phototropism can be substantially altered without any apparent alteration of second positive phototropism.Abbreviation WT wild-type     

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.     

Kinetic modelling of phototropism in maize coleoptiles

Blue-light-induced phototropism of maize (Zea mays L.) coleoptiles was studied with a view to kinetic models. Red-light-grown plants were used to eliminate complication arising from the activation by blue light of phytochrome-mediated phototropism. In the first part, mathematical models were developed to explain the phototropic fluence-response data, which were obtained for the responses induced by a single unilateral pulse (30 s) and those induced by a unilateral pulse (30 s) given immediately after a bilateral pulse (30 s, fixed fluences). These data showed bell-shaped fluence-response curves, characteristic of first positive curvature. Modelling began with the assumptions that the light gradient plays a fundamental role in phototropism and that the magnitude of the response is determined by the gradient, or the concentration difference, in a photoproduct between the irradiated and the shaded sides of the tissue. Minimal mathematical models were then derived, by defining chemical kinetics of the photoreaction and introducing the minimum of parameters needed to correlate the incident fluencerate to the functional fluence-rates within the tissue, the functional fluence-rate to the rate constant of the photoreaction, and the photoproduct concentration difference to the curvature response. The models were tested using a curve-fitting computer program. The model obtained by assigning first-order kinetics to the photoreaction failed to explain the fluence-response data, whereas application of second-order kinetics led to a successful fit of the model to the data. In the second part, temporal aspects of the photosystem were examined. Experimental results showed that a high-fluence bilateral pulse eliminated the bell-shaped fluence-response curve for an immediate unilateral pulse, and that the curve gradually reappeared as the time for unilateral stimulation elapsed after the bilateral pulse. The model based on a second-order photoreaction could be extended to explain the results, with assumed changes in two components: the concentration of the reactant for the photoproduct, and the light-sensitivity of the reaction. The reactant concentration, computed with the curvefitting program, showed a gradual increase from zero to a saturation level. This increase was then modelled in terms of regeneration of the reactant from the photoproduct, with an estimated first-order rate constant of about 0.001·s^-1. The computed value for the constant reflecting the light-sensitivity showed a sharp decline after the high-fluence pulse, followed by a gradual return to the initial level. From these analytical results, the appearance of second positive curvature was predicted.Abbreviations FPC first positive curvature - SPC second positive curvature CIW-DPB publication No. 884     

Phytochrome effects on leaf growth and chlorophyll content in Petunia axilaris

Abstract Changes in the phytochrome status at the end of the daily photosynthetic period result in several plant responses. To understand the causal relations among these responses it is useful to investigate species or experimental conditions where the most common correlations among responses are broken. A step in this direction is presented here with Petunia axilaris, where FR-treated plants showed low chlorophyll content and erect leaves, but- contrary to other species-higher leaf area and plant dry weight. Differences in area expansion were related to the late phase of leaf growth and were due, at least in part, to larger cells in FR-treated plants. Effects on length/width ratio, specific leaf area, net assimilation rate, shoot/root ratio and leaf number were small or non-existent. It is suggested that the lower chlorophyll content in FR-treated plants was not a consequence of scarcity of assimilates.