Phototropic stimulation induces the conversion of glucosinolate to phototropism-regulating substances of radish hypocotyls

The distribution of natural growth inhibitors, the raphanusanins (isomers of 3-(methylthio)methylene-2-pyrrolidinethione) and their precursors (4-methylthio-3-butenyl glucosinolate (MTBG) and 4-methylthio-3-butenyl isothiocyanate (MTBI), between illuminated and shaded halves of radish hypocotyls during phototropic curvature was analyzed using a physicochemical assay. Phototropic stimulation rapidly decreased MTBG content, and abruptly increased contents of MTBI and raphanusanins in the illuminated halves of radish hypocotyls within 30 min after the onset of unilateral illumination. Content in the shaded halves was similar to that in dark controls. When MTBG, MTBI, and raphanusanins at endogenous levels were applied unilaterally to etiolated hypocotyls, MTBI and raphanusanins caused hypocotyls to bend but MTBG showed no activity. Blue illumination promoted myrosinase (thioglucosidase) activity, which releases MTBI from MTBG, in hypocotyls after 10 min, although enzyme activity in dark controls did not change. These results suggest that phototropic stimulation promotes myrosinase activity in the illuminated side of radish hypocotyls, releasing bioactive MTBI from inactive MTBG and simultaneously producing bioactive raphanusanins.     

A gravitropic stimulation-induced growth inhibitor, β-(isoxazolin-5-on-2yl)-alanine,is a possible mediator of negative gravitropic bending of epicotyls in etiolated <Emphasis Type="Italic">Pisum sativum</Emphasis> seedlings

Negative gravitropic bending and its possible mediator in etiolated Alaska pea seedlings were intensively studied in comparison with seedlings of an agravitropic mutant, ageotropum. When 3.5-day-old etiolated Alaska seedlings were horizontally placed, the growth suppression at the upper side of the epicotyls began 10 min after the onset of the gravitropic stimulation, whereas the growth acceleration at the lower side began at 30 min, resulting in negative gravitropic bending. In contrast, no gravitropic bending was observed in the etiolated ageotropum seedlings, for which the epicotyls show an automorphogenesis-like growth. Strenuous efforts to identify a possible mediator that induces the gravitropic bending resulted in successfully identifying β-(isoxazolin-5-on-2yl)-alanine (βIA). The unilateral application of βIA to the etiolated Alaska epicotyls substantially induced epicotyl bending toward the application site, indicating that βIA could act as a growth inhibitor. Analyses of the distribution of βIA in the upper and lower flanks of the etiolated Alaska epicotyls revealed that its content rapidly increased twice in the upper flanks compared with that in the lower ones in response to gravitropic stimulation, whereas its content in the lower flanks was almost equal to that in the vertical control. In etiolated ageotropum epicotyls, an almost equal amount of βIA was distributed in the upper and lower flanks of epicotyls. These results suggest that a gravitropic stimulation increases βIA in the upper flank, resulting in the negative gravitropic bending of epicotyls via the suppression of the growth rate at the upper side of epicotyls in the etiolated Alaska pea seedlings.     

Rapid, bilateral changes in growth rate and curvature during gravitropism of cucumber hypocotyls: implications for mechanism of growth control

Abstract. The growth response of etiolated cucumber ( Cucumis sativus L.) hypocotyls to gravitropic stimulation was examined by means of time-lapse photography and high-resolution analysis of surface expansion and curvature. In comparison with video analysis, the technique described here has five- to 20-fold better resolution; moreover, the mathematical fitting method (cubic splines) allows direct estimation of local and integrated curvature. After switching seedlings from a vertical to horizontal position, both upper and lower surfaces of the stem reacted after a lag of about 11 min with a two- to three-fold increase in surface expansion rate on the lower side and a cessation of expansion, or slight compression, on the upper surface. This growth asymmetry was initiated simultancously along the length of the hypocotyl, on both upper and lower surfaces, and did not migrate basipetally from the apex. Later stages in the gravitropic response involved a complex reversal of the growth asymmetry, with the net result being a basipetal migration of the curved region. This secondary growth reversal may reflect oscillatory and or self-regulatory behaviour of growing cells. With some qualifications, the kinetics and pattern of growth response are consistent with a mechanism involving hormone redistribution, although they do not prove such a mechanism. The growth kinetics require a growth mechanism which can be stimulated by two-to three-fold or completely inhibited within a few minutes.     

Induction of myrosinase gene expression and myrosinase activity in radish hypocotyls by phototropic stimulation

The role of myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) in the phototropic response in radish hypocotyls was investigated. Unilateral illumination with blue light abruptly up-regulated the activity of myrosinase, which releases bioactive 4-methylthio-3-butenyl isothiocyanate (MTBI) from inactive 4-methylthio-3-butenyl glucosinolate (MTBG), in the illuminated halves of radish hypocotyls 10 min after onset of phototropic stimulation, peaking after 30 min and decreasing thereafter. The myrosinase activity in the shaded halves also increased, but was significantly lower than that in the illuminated halves. Furthermore, whether blue light illumination induces myrosinase gene expression was studied. Northern blotting analysis indicated that myrosinase mRNA levels were increased markedly in unilaterally illuminated hypocotyls, reaching maximum signal intensity within 10 min after onset of blue illumination, declining nearly to the control level thereafter. These results suggested that phototropic stimulation promotes myrosinase gene expression and myrosinase activity in the illuminated side, resulting in the conversion of inactive MTBG to active MTBI and simultaneously producing more active raphanusanins, causing a phototropic response.     

Tropic responses of hypocotyls from normal tomato plants and the gravitropic mutant Lazy-1

Abstract Etiolated hypocotyls from normal tomato plants show a negative gravitropic response within 20 min of stimulation. In contrast, etiolated hypocotyls from the gravitropic mutant Lazy-l do not reorientate after gravistimulation. Etiolated hypocotyls from both types of plant are positively phototropic, however, Lazy-l seedlings achieve a greater final angle of bending following phototropic stimulation compared to normal plants. Anatomical studies reveal that etiolated hypocotyls from normal plants contain sedimenting amyloplasts located within the endodermal cells. Such sedimenting amyloplasts are absent in Lazy-l tissue. It is hypothesized that the hypocotyl of Lazy-l is agravitropic since it is unable to perceive a gravistimulus.     

Growth patterns and gravitropic curvature of radish hypocotyls

The rapid growth rate of radish cells makes the hypocotyl particularly valuable in research on growth phenomena and gravitropism. An examination of mean data regarding the growth of aetiolated radish seedlings not subjected to gravitropic stimulation showed that there was an increase in the growth rate from day 3 to day 5. When the hypocotyl was placed horizontally all zones showed an increase in growth of the lower surface of the hypocotyl and generally a decrease in the growth rate in the upper surface. The upper apical part of the gravistimulated hypocotyl grew almost the same as controls. In some cases, in both 4- and 5-day-old seedlings, in the upper median part there was a lower growth rate than in controls. The zones of growth during the development of the hypocotyl were different. Analysis of curvature showed that the growth rate of the different zones was a function of their location in the hypocotyl and that the rate of curvature was different in various parts of the hypocotyl.     

Requirement for the gravity-controlled transport of auxin for a negative gravitropic response of epicotyls in the early growth stage of etiolated pea seedlings

Gravity-controlled transport of auxin was studied for a negative gravitropic response in the early growth stage of etiolated pea (Pisum sativum L. cv. Alaska) seedlings, in which epicotyl bending was observed near the cotyledon nodes of the seedlings grown continuously from seeds germinated in a horizontal or an inclined position. Increased expression of an auxin-inducible gene, PsIAA4/5, was observed in the elongated side of epicotyls grown in a horizontal or an inclined position. Regardless of the conditions of seed germination, polar auxin transport in the proximal side of the first internodes of the seedlings was significantly higher than in the distal side. Polar auxin transport in the proximal side of epicotyls grown in an inclined position was significantly lower than in those grown in a horizontal position. In contrast, lateral auxin distribution from the proximal to distal sides in epicotyls grown in an inclined position was significantly higher than in epicotyls grown in a horizontal position. Accumulation of PsPIN1 mRNA encoding a putative auxin efflux facilitator, which was observed in vascular tissue, cortex and epidermis in the proximal and distal sides of epicotyls, was markedly influenced by gravistimulation. These results strongly suggest that gravistimulation induces changeable polar auxin transport and one-way lateral auxin distribution in epicotyls as well as asymmetric auxin accumulation in the proximal and distal sides of epicotyls, resulting in a negative gravitropic response of epicotyls in the early growth stage of pea seedlings.     

Gravistimulation changes the accumulation pattern of the CsPIN1 auxin efflux facilitator in the endodermis of the transition zone in cucumber seedlings

Cucumber (Cucumis sativus) seedlings grown in a horizontal position develop a specialized protuberance (or peg) on the lower side of the transition zone between the hypocotyl and the root. This occurs by suppressing peg formation on the upper side via a decrease in auxin resulting from a gravitational response. However, the gravity-stimulated mechanism of inducing asymmetric auxin distribution in the transition zone is poorly understood. The gravity-sensing tissue responsible for regulating auxin distribution in the transition zone is thought to be the endodermal cell. To characterize the gravity-stimulated mechanism, the auxin efflux facilitator PIN-FORMED1 (CsPIN1) in the endodermis was identified and the localization of CsPIN1 proteins during the gravimorphogenesis of cucumber seedlings was examined. Immunohistochemical analysis revealed that the accumulation pattern of CsPIN1 protein in the endodermal cells of the transition zone of cucumber seedlings grown horizontally differed from that of plants grown vertically. Gravistimulation for 30 min prompted changes in the accumulation pattern of CsPIN1 protein in the endodermis as well as the asymmetric distribution of auxin in the transition zone. Furthermore, 2,3,5-triiodobenzoic acid inhibited the differential distribution of auxin as well as changes in the accumulation pattern of CsPIN1 in the endodermis of the transition zone during gravistimulation. These results suggest that the altered pattern of CsPIN1 accumulation in the endodermis in response to gravistimulation influences lateral auxin transport through the endodermis, resulting in asymmetric auxin distribution in the transition zone.     

Proof of the subapical differential growth of the flanks in the Chara rhizoid during graviresponse

The displacement of resin spheres attached to the tip of the gravitropically bending Chara rhizoid was measured by means of time-lapse photography. The relative growth of the flanks which at the beginning of stimulation were opposite to each other, was calculated during the earliest response time. The physically upper subapical flank section continues growing after stimulation at a decreasing rate. The growth of the opposite lower flank section is inhibited immediately after stimulation by the position of the statoliths. As soon as the statoliths are displaced in a basal direction, the growth of this section is transiently promoted. The gravitropic downward bending of the Chara rhizoid is by bowing, not by bulging.     

Differential expression of the auxin primary response gene MASSUGU2/IAA19during tropic responses of Arabidopsis hypocotyls

We have examined the expression pattern of an auxin primary response gene, MSG2/IAA19 , during photo- and gravitropic responses of hypocotyls using a transgenic Arabidopsis harboring MSG2/IAA19 promoter::GUS . The upper portion of most etiolated hypocotyls showed uniform β-glucuronidase (GUS) staining with the strongest activity in the pericycle. When hypocotyls were irradiated with unilateral blue light, GUS activity on the concave side of hypocotyls was decreased, resulting in differential GUS staining with a stronger signal on the convex side. The number of differentially stained hypocotyls peaked at 24 h after the onset of the phototropic stimuli, while hypocotyl curvature continued to increase for the entire 36-h experimental period. This result suggests that the MSG2/IAA19 expression precedes the phototropic responses. When seedlings were grown under dim white light, their hypocotyls displayed almost no GUS activity. The light-grown hypocotyls also showed differential GUS staining after phototropic stimuli as result of the increase in GUS activity on the convex side of hypocotyls, especially in the epidermis, the outer cortex and pericycle, although GUS activity was much weaker than that observed in etiolated hypocotyls. Similar but less obvious differential staining was obtained for gravitropic response of hypocotyls. Considering the recent finding that Aux/IAA proteins are immediate targets of the auxin F box receptors, MSG2/IAA19 is likely to act as one of master genes for tropic responses.     

Interaction of gravi- and phototropic stimulation in the response of maize (Zea mays L.) coleoptiles

The influence of gravitropic stimulation upon blue-light-induced first positive phototropism for stimulations in the same (light source and center of gravity opposite to each other) and in opposing directions was investigated in maize cole-optiles by measuring fluence-response patterns. As a result of gravitropic counterstimulation, phototropic bending was transient with maximum curvature occurring 100 min after stimulation. On a horizontal clinostat, however, the seedlings curved for 20 h. Gravistimulation in the opposite direction acted additively upon blue-light curvature. Gravistimulation in the same direction as phototropic stimulation produced a complex behaviour deviating from simple additivity. This pattern can be explained by a gravitropically mediated sensitization of the phototropic reaction, an optimal dependence of differential growth on the sum of photo-and gravistimulation, and blue-light-induced inhibition of gravitropic curvature at high fluences. These findings indicate that several steps of photo-and gravitransduction are separate. Preirradiation with red light desensitized the system independently of applied gravity-treatment, indicating that the site of red-light interaction is common to both transduction chains.Abbreviations BL blue light - G⁺ stimulation by light and gravity in the same direction (i.e. light source and center of gravity opposite to each other) - G^- stimulation by light and gravity in opposing directions     

Effects of hypergravity on the elongation growth in radish and cucumber hypocotyls

The elongation growth of the hypocotyls of radish and cucumber seedlings was examined under hypergravity in a newly developed centrifuge (Kasahara et al. 1995). The effects of hypergravity on elongation growth differed between the two species. The rate of elongation of radish hypocotyls was reduced under basipetal hypergravity (H+2O g) but not under acropetal hypergravity (H-13 g), as compared to growth under the control conditions (C+1 g and C-1 g). In cucumber hypocotyls, elongation growth was inhibited not only by basipetal but also by acropetal hypergravity. Under these conditions, the reduction in the elongation growth of both radish and cucumber hypocotyls was accompanied by an increase in their thickness. Although no distinct differences in relative composition of neutral sugars were found, the amounts of cell-wall components (pectic substances, hemicelluloses and cellulose) per unit length of hypocotyls were increased by exposure to hypergravity.     

Gravitropism of cucumber hypocotyls: biophysical mechanism of altered growth

Abstract. The biophysical basis for the changes in cell elongation rate during gravitropism was examined in aetiolated cucumber ( Cucumis sativus L.) hypocotyls. Bulk osmotic pressures on the two sides of the stem and in the epidermal cells were not altered during the early time course of gravitropism. By the pressureprobe technique, a small increase in turgor (0.3 bar, 30 kPa) was detected on the upper (inhibited) side, whereas there was a negligible decrease in turgor on the lower (stimulated) side. These small changes in turgor and water potential appeared to be indirect, passive consequences of the altered growth and the small resistance for water movement from the xylem, and indicated that the change in growth was principally due to changes in wall properties. The results indicate that the hydraulic conductance of the watertransport pathway was large (.25 h ¹ bar ¹) and the water potential difference supporting cell expansion was no greater than 0.3 bar (30 kPa). From pressureblock experiments, it appeared that upon gravitropic stimulation (1) the yield threshold of the lower half of the stem did not decrease and (2) the wall on the upper side of the stem was not made more rigid by a cross-linking process. Mechanical measurements of the stress/strain properties of the walls showed that the initial development of gravitropism did not involve an alteration of the mechanical behaviour of the isolated walls. Thus, gravitropism in cucumber hypocotyls occurs principally by an alteration of the wall relaxation process, without a necessary change in wall mechanical properties.     

Gravitropic microtubule reorientation can be uncoupled from growth

The causal relationship between gravitropic growth responses and microtubule reorientation has been studied. Growth and microtubule reorientation have been uncoupled during the gravitropic response of maize (Zea mays L.) coleoptiles. Microtubule orientation and growth were measured under three different conditions: (i) a gravitropic stimulation where the growth response was allowed to be expressed (intact seedlings were displaced from the vertical position by 90°), (ii) a gravitropic stimulation where the growth response was suppressed (coleoptiles were attached to microscope slides and kept in a horizontal position), (iii) suppression of growth in the absence of gravitropic stimulation (coleoptiles were attached to microscope slides and kept in a vertical position). It was found that (i) gravitropic stimulation can induce a microtubular reorientation from transverse to longitudinal in the upper (slower growing) flank of the coleoptile, and an inhibition of growth; (ii) the reorientation of microtubules precedes the inhibition of growth; (iii) the gravitropic response of microtubules is weaker, not elevated, when the inhibition of growth is artificially enhanced by attaching the coleoptiles to a slide; and (iv) artificial inhibition of growth in the absence of gravitropic stimulation cannot induce a microtubular response. Thus, the extent of microtubule reorientation is not correlated with the extent of growth inhibition. Moreover, these findings demonstrate that microtubules do not reorient passively after growth changes, but actively in response to gravitropic stimulation. Received: 23 November 1999 / Accepted: 10 May 2000     

Effects of hypergravity on the elongation growth in radish and cucumber hypocotyls

The elongation growth of the hypocotyls of radish and cucumber seedlings was examined under hypergravity in a newly developed centrifuge (Kasaharaet al. 1995). The effects of hypergravity on elongation growth differed between the two species. The rate of elongation of radish hypocotyls was reduced under basipetal hypergravity (H+20g) but not under acropetal hypergravity (H-13g), as compared to growth under the control conditions (C+1g and C-1g). In cucumber hypocotyls, elongation growth was inhibited not only by basipetal but also by acropetal hypergravity. Under these conditions, the reduction in the elongation growth of both radish and cucumber hypocotyls was accompanied by an increase in their thickness. Although no distinct differences in relative composition of neutral sugars were found, the amounts of cell-wall components (pectic substances, hemicelluloses and cellulose) per unit length of hypocotyls were increased by exposure to hypergravity.     

Negative gravitropism in Chara protonemata: A model integrating the opposite gravitropic responses of protonemata and rhizoids

The unicellular protonema of Chara fragilis Desv. was investigated in order to establish a reaction chain for negative gravitropism in tip-growing cells. The time course of gravitropic bending after stimulation at angles of 45 degrees or 90 degrees showed three distinct phases of graviresponse. During the first hour after onset of stimulation a strong upward shift of the tip took place. This initial response was followed by an interval of almost straight growth. Complete reorientation was achieved in a third phase with very low bending rates. Gravitropic reorientation could be completely abolished by basipetal centrifugation of the cells, which lastingly removed conspicuous dark organelles from the protonema tip, thus identifying them as statoliths. Within minutes after onset of gravistimulation most or all statoliths were transported acropetally from their resting position 20-100 micrometers from the cell apex to the lower side of the apical dome. This transport is actin-dependent since it could be inhibited with cytochalasin B. Within minutes after arrival of the statoliths, the apical dome flattened on its lower side and bulged on the upper one. After this massive initial response the statoliths remained firmly sedimented, but the distance between this sedimented complex and the cell vertex increased from 7 micrometers to 22 micrometers during the first hour of stimulation and bending rates sharply declined. From this it is concluded that only statoliths inside the apical dome convey information about the spatial orientation of the cell in the gravitropic reaction chain. After inversion of the protonema the statoliths transiently arranged into a disk-shaped complex about 8 micrometers above the vertex. When this statolith complex tilted towards one side of the apical dome, growth was shifted in the opposite direction and bending started. It is argued that the statoliths intruding into the apical dome may displace a growth-organizing structure from its symmetrical position in the apex and may thus cause bending by bulging. In the positively gravitropic Chara rhizoids only a more stable anchorage of the growth-organizing structure is required. As a consequence, sedimented statoliths cannot dislocate this structure from the vertex. Instead they obstruct a symmetrical distribution of cell-wall-forming vesicles around the structure and thus cause bending by bowing.     

Gravity induces fast electrical field change in soybean hypocotyls

Abstract. Measurement of the electrical field along soybean hypocotyls shows the development of a positive electrical potential in the lower side approximately 1 min after horizontal placement. The time is as fast or faster than the geotropic presentation time of soybean seedlings. The maximum positive electrical field potential is produced in a zone 1–2 cm below the hook, which is the region showing the geotropic curvature.     

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.     

Wall extensibility and gravitropic curvature of sunflower hypocotyls: correlation between timing of curvature and changes in extensibility

Abstract. Gravitropic curvature results from unequal growth rates on the upper and lower sides of horizontal stems. These unequal growth rates cold be due to differences in wall extensibility between the two sides. To test this, the time course of curvature of horizontal sunflower ( Helianthus anmus L.) hypocotyls was determined and compared with the time courses of changes in Instron-measured wall extensibility (PEx) of the upper and lower epidermal layers. As gravicurvature developed, so did the difference in PEx between the upper and lower epidermis. The enhanced growth rate on the lower side during the period of maximum increase in curvature was matched by PEx values greater than those of the vertical control, while the inhibited growth rate on the upper side was accompanied by PEx values below that of the control. The close correlation between changes in growth rates and alterations in PEx demonstrates that changes in wall extensibility play a major role in controlling gravicurvature.     

An analysis of the changes in growth rate occurring during the initial stages of geocurvature in shoots

Abstract A detailed study of the changes in growth rate on the two sides of a shoot prior to, and following geostimulation has been undertaken. Data from etiolated Zea seedlings, and cucumber hypocotyls, and from light grown sunflower hypocotyls are presented. In all cases, the differential growth which brings about geocurvature begins simultaneously along the length of the organ. This is contrary to previous reports in the literature which suggested that curvature begins first at the tip of an organ and progresses basipetally. The feature which is common to all species investigated is that growth of the upper side of the shoot ceases following geostimulation. In some cases there is also a marked acceleration in growth rate on the lower side of the shoot (sunflower) but other species show no such acceleration of growth (cucumber). It has been assumed for many years that the major factor causing upward curvature was an acceleration of growth on the lower side of the organ. The data presented here show that the cessation of growth on the upper side is a major, and in some cases the only factor bringing about geocurvature. The data are discussed in relation to the mechanisms which might control geotropic curvature.