Polarity induction versus phototropism in maize: Auxin cannot replace blue light

In a previous study (Nick and Schäfer 1991, Planta 185, 415–424), unilateral blue light had been shown, in maize coleoptiles, to induce phototropism and a stable transverse polarity, which became detectable as stable curvature if counteracting gravitropic stimulation was removed by rotation on a horizontal clinostat. This response was accompanied by a reorientation of cortical microtubules in the outer epidermis (Nick et al. 1990, Planta 181, 162–168). In the present study, this stable transverse polarity is shown to be correlated with stability of microtubule orientation against blue light and changes of auxin content. The role of auxin in this stabilisation was assessed. Although auxin can induce reorientation of microtubules it fails to induce the stabilisation of microtubule orientation induced by blue light. This was even true for gradients of auxin able to induce a bending response similar to that ellicited by phototropic stimulation. Experiments involving partial irradiation demonstrated different perception sites for phototropism and polarity induction. Phototropism starts from the very coleoptile tip and involves transmission of a signal (auxin) towards the subapical elongation zone. In contrast, polarity induction requires local action of blue light in the elongation zone itself. This blue-light response is independent of auxin.This work was supported by the Deutsche Forschungsgemeinschaft and two grants of the Studienstiftung des Deutschen Volkes and the Human Frontier Science Program Organization to P.N.     

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.     

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.

     

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.     

Physical strain-mediated microtubule reorientation in the epidermis of gravitropically or phototropically stimulated maize coleoptiles

During gravitropic and phototropic curvature of the maize coleoptile, the cortical microtubules (MTs) adjacent to the outer epidermal cell wall assume opposite orientations at the two sides of the organ. Starting from a uniformly random pattern during straight growth in darkness, the MTs reorientate perpendicularly to the organ axis at the outer (faster growing) side and parallel to the organ axis at the inner (slower growing) side. As similar reorientations can be induced during straight growth by increasing or decreasing the effective auxin concentration, it has been proposed that these reorientations may be used as a diagnostic test for assessing the auxin status of the epidermal cells during tropic curvature. This idea was tested by determining the MT orientations in the coleoptile of intact maize seedlings in which the gravitropic or phototropic curvature was prevented or inversed by an appropriate mechanical counterforce. Forces that just prevented the coleoptile from curving in a gravity or light field prevented reorientations of the MTs. Forces strong enough to overcompensate the tropic stimuli by enforcing curvature in the opposite direction induced reorientations of the MTs opposite to those produced by tropic stimulation. These results show that the MTs at the outer surface of the coleoptile respond to changes in mechanical tissue strain rather than to gravitropic or phototropic stimuli and associated changes at the level of auxin or any other element in the signal transduction chain between perception of tropic stimuli and asymmetric growth response. It is proposed that cortical MTs can act as strain gauges in a positive feed-back regulatory circle utilized for amplification and stabilization of environmentally induced changes in the direction of elongation growth.     

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

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

Unilateral reorientation of microtubules at the outer epidermal wall during photo- and gravitropic curvature of maize coleoptiles and sunflower hypocotyls

Auxin (indole-3-acetic acid) controls the orientation of cortical microtubes (MT) at the outer wall of the outer epidermis of growing maize coleoptiles (Bergfeld, R., Speth, V., Schopfer, P., 1988, Bot. Acta 101, 57-67). A detailed time course of MT reorientation, determined by labeling MT with fluorescent antibodies, revealed that the auxin-mediated movement of MT from the longitudinal to the transverse direction starts after less than 15 min and is completed after 60 min. This response was used for a critical test of the functional involvement of auxin in tropic curvature. It was found that phototropic (first phototropic curvature) as well as gravitropic bending are correlated with a change of MT orientation from transverse to longitudinal at the slower-growing organ flank whereas the transverse MT orientation is maintained (or even augmented) at the faster-growing organ flank. These directional changes are confined to the MT subjacent to the outer epidermal wall. The same basic results were obtained with sunflower hypocotyls subjected to phototropic or gravitropic stimulation. It is concluded that auxin is, in fact, involved in asymmetric growth leading to tropic curvature. However, our results do not allow us to discriminate between an uneven distribution of endogenous auxin or an even distribution of auxin, the activity of which is modulated by an unevenly distributed inhibitor of auxin action.     

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.     

Lateral movement of auxin in phototropism

Lateral movement of indoleacetic acid-1-¹⁴C in corn coleoptiles was measured as radioactivity moving laterally following unilateral application of the auxin. The data suggest that there is an endogenous lateral movement of auxin, and that phototropic stimulation of the coleoptile depresses lateral movement towards the light and enhances lateral movement away from the light. The lateral movement was found to be principally as indoleacetic acid. In experiments using sunflower hypocotyl sections, evidence is also presented to support the suggestion that lateral redistribution of auxin may be effected by a deflection of auxin around a barrier to basipetal transport.     

Reduced phototropism in pks mutants may be due to altered auxin‐regulated gene expression or reduced lateral auxin transport

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1‐ and phot2‐mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi‐reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.     

IAA transport during the phototropic responses of intact Zea and Avena coleoptiles

Summary Transport of indolyl-3-acetic acid (IAA) was studied during the phototropic responses of intact shoots and detached coleoptiles of Zea mays L. and Avena sativa L. The use of a high specific activity [5-³H]IAA and glass micropipettes enabled asymmetric application of the IAA to be made to individual coleoptiles with minimal tissue damage.A unilateral stimulus of 2.59×10^-11 einstein cm^-2 of blue light, probably in the dose range of the first positive phototropic response, caused significant net lateral movement of radioactivity from [5-³H]IAA away from the illuminated side of intact shoots and detached coleoptile apices of both Avena and Zea. The magnitude of the net lateral movement was 15.3% in Zea seedlings and 12.3% in Avena seedlings. Chromatographic analyses indicated that the movement of radioactivity reflected that of IAA. A phototropic stimulus of 1.24×10^-7 einstein cm^-2, which was probably in the second positive dose range, caused significant lateral movement of radioactivity in intact shoots and detached coleoptiles of Zea but not of Avena.In intact Zea seedlings, neither phototropic dosage affected the longitudinal transport of IAA. In intact Avena seedlings, first positive stimulation inhibited longitudinal transport only when the IAA was applied to the illuminated side of the coleoptile, but second positive stimulation inhibited basipetal movement of IAA regardless of the side of application.Exposing the intact seedlings to red light before phototropic stimulation abolished lateral transport after a first positive stimulus in Zea and in Avena.Phototropic stimulation can thus induce a lateral transport of IAA towards the shaded side of the coleoptiles of both Zea and Avena seedlings, and can affect longitudinal movement of IAA in the coleoptile of Avena. However, since phototropic curvature was observed under certain conditions in the absence of either of these effects, the extent to which they are involved in the induction of asymmetric growth in a stimulated coleoptile has yet to be resolved.     

Plasmodesmata, Tropisms, and Auxin Transport

Attempts were made to disrupt the plasmodesmata between oatcoleoptile cells (Avena saliva L. cv. Victory) by severe plasmolysis.Coleoptiles, allowed to regain turgor after plasmolysis, wereable to execute geotropic and phototropic curvatures and segmentswould grow in response to applied auxin. In coleoptiles similarlytreated, studies with [¹⁴C]IAA have shown that longitudinal,basipetal transport of auxin still takes place and, as in controls,IAA is preferentially redistributed laterally within coleoptilesorientated horizontally. Physical continuity of the symplast of oat coleoptile cellsmay not always be disrupted by severe plasmolysis. Nevertheless,functional continuity appears to be interrupted. Despite this,all the processes involved in the execution of tropistic curvaturesremain intact, including transport of hormones. Plasmodesmatalcontinuity between oat coleoptile cells appears not to be anecessary requirement for auxin transport.     

Phototropic Stimulation can Shift the Gradient of Crown Root Emergence in Maize

The crown roots in the coleoptilar node of maize emerge asymmetrically: emergence at the dorsal flank of the node (opposite to the caryopsis) precedes emergence at the ventral flank (facing the caryopsis). This asymmetry can be altered by phototropic stimulation: emergence of crown roots is delayed in the lighted flank and promoted in the shaded flank causing an inversion of the endogenous asymmetry. The curvature induced by the phototropic stimulation is transient, the effect on crown root emergence, in contrast, persists. This stable effect is not a consequence of curvature per se and becomes irreversibly fixed between one and two hours after stimulation. The emergence of crown roots depends on directional signalling from the coleoptile to the node. The data are discussed in terms of a stable blue light induced transverse polarity of the coleoptile that can imprint a stable asymmetry upon the coleoptilar node guiding the emergence of crown roots.     

D6PK AGCVIII Kinases Are Required for Auxin Transport and Phototropic Hypocotyl Bending in Arabidopsis

Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.     

玉米胚芽鞘向光性运动的一些特性

利用云母片分隔、ＨＰＬＣ分析等方法研究了玉米胚芽鞘向光性运动的特性。云母片阻隔生长素的移动后并不能阻止胚芽鞘的向光性变弯曲。     

Unequal Distribution of 6-methoxy-2-benzoxazolinone (MBOA) is the Main Reason for Phototropism in Maize Coleoptiles

The distribution of 6-methoxy-2-benzoxazolinone (MBOA) which is induced by blue light stimulation in maize ( Zea mays L. ) coleoplile was investigated by HPLC analysis. The results showed that: 1. The MBOA content on the irradiated side of the coleoptile was 1.5 fold more than that on the shaded side. 2. There was little change of IAA on both sides of the coleoptile which was treated with phototropic stimulation. 3. The growing coleoptile bent towards the side which was treated with MBOA, 5,6-dimethoxy-2-benzoxazolinone (DMBOA) or 2-chloro-5,6-dimethoxy-2-benzoxazolinone ( C1-DMBOA). The above results indicated that the phototropic bending of the coleoptile was attributed to unequal distribution of MBOA.     

NPH3- and PGP-like genes are exclusively expressed in the apical tip region essential for blue-light perception and lateral auxin transport in maize coleoptiles

Phototropic curvature results from differential growth on two sides of the elongating shoot, which is explained by asymmetrical indole-3-acetic acid (IAA) distribution. Using 2 cm maize coleoptile segments, 1st positive phototropic curvature was confirmed here after 8 s irradiation with unilateral blue light (0.33 μmol m(-2) s(-1)). IAA was redistributed asymmetrically by approximately 20 min after photo-stimulation. This asymmetric distribution was initiated in the top 0-3 mm region and was then transmitted to lower regions. Application of the IAA transport inhibitor, 1-N-naphthylphthalamic acid (NPA), to the top 2 mm region completely inhibited phototropic curvature, even when auxin was simultaneously applied below the NPA-treated zone. Thus, lateral IAA movement occurred only within the top 0-3 mm region after photo-stimulation. Localized irradiation experiments indicated that the photo-stimulus was perceived in the apical 2 mm region. The results suggest that this region harbours key components responsible for photo-sensing and lateral IAA transport. In the present study, it was found that the NPH3- and PGP-like genes were exclusively expressed in the 0-2 mm region of the tip, whereas PHOT1 and ZmPIN1a, b, and c were expressed relatively evenly along the coleoptile, and ZmAUX1, ZMK1, and ZmSAURE2 were strongly expressed in the elongation zone. These results suggest that the NPH3-like and PGP-like gene products have a key role in photo-signal transduction and regulation of the direction of auxin transport after blue light perception by phot1 at the very tip region of maize coleoptiles.     

Interaction of auxin, light, and mechanical stress in orienting microtubules in relation to tropic curvature in the epidermis of maize coleoptiles

Summary Changes in the orientation of cortical microtubules (longitudinal vs. transverse with respect to the long cell axis) at the outer epidermal wall of maize coleoptile segments were induced by auxin, red or blue light, and mechanical stresses (cell extension or compression produced by bending). Immunofluorescent techniques were used for the quantitative determination of frequency distributions of microtubule orientation. Detailed kinetic studies showed that microtubule reorientations are temporally correlated with the simultaneously measured changes in growth rate elicited by auxin, red light, or blue light. Growth inhibition induced by depletion of endogenous auxin produces a longitudinal microtubule pattern that can be changed into a transverse pattern in a dose-dependent manner by applying exogenous auxin. A mid-point pattern with equal frequencies of longitudinal and transverse microtubules was adjusted at 2 mol/1 auxin. Bending stress applied under these conditions adjusts permanent, maximally longitudinal and transverse microtubule orientations at the compressed and extended segment sides, respectively, quantitatively mimicking the responses to differential flank growth during phototropic and gravitropic curvature. During tropic curvature the changes in microtubule pattern reflect the distribution of growth rather than the distribution of auxin. The microtubule pattern responds to auxin-dependent growth changes and mechanical stress in a synergistic manner, confirming the functional equivalence of these factors in affecting microtubule orientation. Similar results were obtained when segment growth was altered by blue or red light instead of auxin in the presence or absence of mechanical stress. It is concluded from these results that growth changes, elicited by auxin, light, etc., and mechanical stress affect microtubule orientation through a common signal perception and transduction chain.Abbreviations IAA indole-3-acetic acid (auxin) - MT cortical microtubule     

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.     

Blue‐light‐induced PIN3 polarization for root negative phototropic response in Arabidopsis

Root negative phototropism is an important response in plants. Although blue light is known to mediate this response, the cellular and molecular mechanisms underlying root negative phototropism remain unclear. Here, we report that the auxin efflux carrier PIN‐FORMED (PIN) 3 is involved in asymmetric auxin distribution and root negative phototropism. Unilateral blue‐light illumination polarized PIN3 to the outer lateral membrane of columella cells at the illuminated root side, and increased auxin activity at the illuminated side of roots, where auxin promotes growth and causes roots bending away from the light source. Furthermore, root negative phototropic response and blue‐light‐induced PIN3 polarization were modulated by a brefeldin A‐sensitive, GNOM‐dependent, trafficking pathway and by phot1‐regulated PINOID (PID)/PROTEIN PHOSPHATASE 2A (PP2A) activity. Our results indicate that blue‐light‐induced PIN3 polarization is needed for asymmetric auxin distribution during root negative phototropic response.