Mathematical modelling of morphogenesis in fungi: a key role for curvature compensation ('autotropism') in the local curvature distribution model

The assumption that the mushroom stem has the ability to undergo autonomic straightening enables a mathematical model to be written that accurately mimics the gravitropic reaction of the stems of Coprinus cinereus . The straightening mechanism is called curvature compensation here, but is equivalent to the 'autotropism' that often accompanies the gravitropic reactions of axial organs in plants. In the consequently revised local curvature distribution model, local bending rate is determined by the difference between the 'bending signal' (generated by gravitropic signal perception systems) and the 'straightening signal' (proportional to the local curvature at the given point). The model describes gravitropic stem bending in the standard assay with great accuracy but has the virtue of operating well outside the experimental data set used in its derivation. It is shown, for example, that the mathematical model can be fitted to the gravitropic reactions of stems treated with metabolic inhibitors by a change of parameters that parallel the independently derived physiological interpretation of inhibitor action. The revised local curvature distribution model promises to be a predictive tool in the further analysis of gravitropism in mushrooms.     

Elongation growth and gravitropic curvature in the Flammulina velutipes (Agaricales) fruiting body

Differential elongation of stipe hyphae drives the gravitropic reorientation of Flammulina velutipes (Agaricales) fruiting bodies. The gravitropic curvature is strictly dependent on the presence of the transition zone between pileus and stipe. Elongation growth, providing the driving force for curvature, is also promoted by the pileus. Gravitropic curvature is successfully suppressed by clinostatic rotation, but the elongation rate is not affected. Explantation of fruiting body stipes lowers curvature and elongation rates corresponding to explant size reduction. In Flammulina, 25 mm length of transition zone explants is an efficient size for reproducible curvature and elongation during 48- to 72-h curvature tests. Submersion of specimens in aqueous medium causes cessation of the gravitropic curvature, but does not affect elongation. Thus the involvement of a diffusible factor in transmission of the curvature signal is probable. Splitting the fruiting body stipe in segments of 1/8 diameter does not suppress the gravitropic response, and the segments are individually reoriented to the vertical. It is concluded that the graviresponse of the Flammulina fruiting body is based on cellular perception of the gravistimulus and that a differential growth signal is transmitted in the stipe by a soluble factor that regulates hyphal elongation.     

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.     

Gravitropism of the basidiomycete Flammulina velutipes: morphological and physiological aspects of the graviresponse

The fruiting body of the basidiomycete Flammulina velutipes shows a distinct negative gravitropic response. Maturing fruiting bodies in the rapid elongation phase become graviresponsive with basidiospore differentiation. Lateral gravistimulation by horizontal arrangement of the fruiting body results in unilateral growth regulation. Elongation in the upper Stipe side decreases to 40% during gravitropic reorientation of the fruiting body. Overshooting of the gravitropic response during reorientation is precisely regulated. The graviresponsiveness is concentrated to the apical area of the stipe, the transition zone between pileus and stipe, which features a prominent elongation capability. The small size and low vacuolization of the transition zone hyphae compared with differentiated basal stipe hyphae correspond with this physiological function on the light and electron microscopical levels. Curvature experiments using intact and explanted fruiting bodies demonstrated the graviperceptive role of the transition zone. The excision of various amounts of pilear tissue, even the disruption of the whole pileus, had no severe effect on gravitropic curvature, until the transition zone was damaged. Removal of the transition zone resulted in a dramatic loss of graviresponse, whereas the decrease of elongation was less drastic.     

Proton Efflux from the Outer Layer of the Peduncle of Tulip in Gravitropism and Circumnutation*

Epidermal plus hypodermal peels from tulip peduncles produced bands of acidity on agar containing bromocresol purple. Peels from horizontally oriented peduncles gave rise to an acidity band which corresponded to the lower side of the peduncle. The band began 3–6 cm beneath the flower and extended basipetally within the region of gravitropic bending. No corresponding band appeared in an agar layer laid on the cortical surface exposed by peeling. Peduncles growing in the normal vertical position showed circumnutations with a period in the range of 4 h. The peels from these stalks produced one or two bands more acid than the remaining part of the peel. Since the acidity band in horizontally positioned stalks corresponds to the zone of faster growth causing gravitropic bending, we infer that the band(s) produced by vertical stalks also correspond to zones of differential growth involved in circumnutation. On the basis of a previous finding that tulip leaves give rise to an oscillating acidity pattern, we infer that vertical stalks also show such a pattern. This inference fits the model proposing the involvement of an internal oscillator in circumnutation. However, the ratio of the circumnutation period to the gravitropic lag phase in tulip peduncles is such as predicted by the gravitropic-feedback model of circumnutation.     

Kinetics of stem gravitropism in Coprinus cinereus: determination of presentation time and "dosage-response" relationships using clinostats

The sensitivity to gravitational stimulation of excised stems of the mushroom fruit body of Coprinus cinereus was determined using clinostat rotation to remove partially-stimulated stems from the normal unidirectional gravitational field. For the strain and conditions tested, the presentation time (the minimum time of stimulation required to elicit a gravitropic reaction) was determined to be 9.6 min. This is the first time the presentation time has been determined for a fungal gravitropic response. Constructional details are given of the clinostats employed in the research and their further use is discussed.     

A major factor in gravitropism in radish hypocotyls is the suppression of growth on the upper side of hypocotyls

The changes in length on the two opposite sides of etiolated radish (Raphanus sativus) hypocotyls prior to, and following gravitropic stimulation, were measured using an infrared-imaging system. It was observed that the growth suppression on the upper side began first at least 10 min after the onset of gravitropic stimulation, and after 30 min the acceleration in growth on the lower side started. The gravitropic curvature was steadily induced from 10 min. When radish hypocotyls were switched from a vertical to horizontal position for different durations and then replaced to the vertical position, the growth suppression on the gravity-stimulated (upper) side was observed in all cases, but the acceleration in growth on the opposite (lower) side appeared only in continuously gravity-stimulated seedlings, although it occurred later than the growth suppression on the upper side. These results suggest that the suppression in growth on the upper side of the hypocotyls is a direct effect of gravitropic stimulation, but not the acceleration on the lower side. When 4-methylthio-3-butenyl isothiocyanate (4-MTBI), which has an inhibitory activity against radish hypocotyl growth, was applied on the one side of radish hypocotyls and then the 4-MTBI-applied side or opposite side was placed in a horizontal position, the former showed greater bending than the control, suggesting that the growth suppression on the upper side is enhanced and maintained with MTBI application there. In the latter case, the seedlings showed less bending than the control, suggesting a decrease in growth on the lower side with MTBI application. All the results suggest that gravitropism of radish hypocotyls may be caused by an increase in growth-inhibiting substance(s) induced with gravitropic stimulation in the upper side, inducing growth inhibition there.     

Characterization of gravitropic inflorescence bending in brassinosteroid biosynthesis and signaling Arabidopsis mutants

The interaction between the plant hormones, brassinosteroids and auxins has been documented in various processes using a variety of plants and plant parts. In this study, detached inflorescences from brassinosteroid biosynthesis and signaling Arabidopsis mutants were evaluated for their gravitropic bending in response to epibrassinolide (EBR) and indole-3-acetic acid (IAA). EBR supplied to the base of detached inflorescences stimulated gravitropic bending in all BR biosynthetic mutants but there was no effect on the BR signaling mutant or wild type plants. When IAA was supplied to the base of BR mutant inflorescences both natural and EBR-induced gravitropic bending was inhibited. Treatment with the auxin inhibitors also decreased both natural and EBR-induced gravitropic bending. No gravitropic bending was observed when the apical tips of BR mutant inflorescences were removed. IAA treatment to the tips of decapitated BR mutant inflorescences restored gravitropic bending to values observed in the inflorescences with an apical tip, however, EBR applied to the tip had no effect. When decapitated inflorescences from BR mutants were treated with IAA to the base and either gel, EBR or IAA was applied to the tip; there was no gravitropic bending. These results show that brassinosteroids have a role in the gravitropic bending response in Arabidopsis and mutants serve to uncover this hidden contributor.     

Effects of MAPKK inhibitor PD98059 on the gravitropism of primary roots of maize

Gravity plays a fundamental role in plant growth and development, yet the molecular details of gravitropism is not fully understood. Here, we report the effects of PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase kinase, on the gravitropism of primary roots of maize. Unilateral application of PD98059 to horizontal roots led to different gravitropic growth. Placing PD98059-containing agar on the upper side of the root tips accelerated gravitropic curvature, whereas placing the agar on the lower side inhibited gravitropic curvature. However, no effect was detected when asymmetric application of PD98059 to vertical roots. Global application of maize primary root with PD98059 suppressed root gravitropism. Furthermore, the effects of H₂O₂ on horizontal root gravitropism and vertical root bending were compromised by pretreatment with PD98059. These results suggest an involvement of MAP kinase pathway(s) in gravitropism of maize roots.     

Asymmetric cytokinin signaling opposes gravitropism in roots

Plants depend on gravity to provide the constant landmark for downward root growth and upward shoot growth. The phytohormone auxin and its cell‐to‐cell transport machinery are central determinants ensuring gravitropic growth. Statolith sedimentation toward gravity is sensed in specialized cells. This positional cue is translated into the polar distribution of PIN auxin efflux carriers at the plasma membrane, leading to asymmetric auxin distribution and consequently, differential growth and organ bending. While we have started to understand the general principles of how primary organs execute gravitropism, we currently lack basic understanding of how lateral plant organs can defy gravitropic responses. Here we briefly review the establishment of the oblique gravitropic set point angle in lateral roots and particularly discuss the emerging role of asymmetric cytokinin signaling as a central anti‐gravitropic signal. Differential cytokinin signaling is co‐opted in gravitropic lateral and hydrotropic primary roots to counterbalance gravitropic root growth.     

The role of actin filaments in the gravitropic response of snapdragon flowering shoots

The involvement of the actin and the microtubule cytoskeleton networks in the gravitropic response of snapdragon ( Antirrhinum majus L.) flowering shoots was studied using various specific cytoskeleton modulators. The microtubule-depolymerizing drugs tested had no effect on gravitropic bending. In contrast, the actin-modulating drugs, cytochalasin D (CD), cytochalasin B (CB) and latrunculin B (Lat B) significantly inhibited the gravitropic response. CB completely inhibited shoot bending via inhibiting general growth, whereas CD completely inhibited bending via specific inhibition of the differential flank growth in the shoot bending zone. Surprisingly, Lat B had only a partial inhibitory effect on shoot bending as compared to CD. This probably resulted from the different effects of these two drugs on the actin cytoskeleton, as was seen in cortical cells. CD caused fragmentation of the actin cytoskeleton and delayed amyloplast displacement following gravistimulation. In contrast, Lat B caused a complete depolymerization of the actin filaments in the shoot bending zone, but only slightly reduced the amyloplast sedimentation rate following gravistimulation. Taken together, our results suggest that the actin cytoskeleton is involved in the gravitropic response of snapdragon shoots. The actin cytoskeleton within the shoot cells is necessary for normal amyloplast displacement upon gravistimulation, which leads to the gravitropic bending.     

Phototropic bending of non-elongating and radially growing woody stems results from asymmetrical xylem formation

Active phototropic bending of non-elongating and radially growing portion of stems (woody stems) has not been previously documented, whereas negative gravitropic bending is well known. We found phototropic bending in woody stems and searched for the underlying mechanism. We inclined 1-year-old Quercus crispula Blume seedlings and unilaterally illuminated them from a horizontal direction perpendicular to ('normal' illumination) or parallel to ('parallel' illumination) the inclination azimuth. With normal illumination, active phototropic bending and xylem formation could be evaluated separately from the negative gravitropic response and vertical deflection resulting from the weight of the seedlings. One-year-old stems with normal illumination bent significantly, with asymmetrical xylem formation towards the illuminated upper surface and side of the stem, whereas those with parallel illumination showed non-significant lateral bending, with asymmetrical xylem formation only on the upper side. A mechanical model was built on the assumption that a bending moment resulted from the asymmetrical xylem formation during phototropic bending of the woody stems. The model fitted the relationship between the observed spatial distributions of the xylem and the observed lateral bending, and thus supported the hypothesis that phototropic bending of woody stems results from asymmetrical xylem formation, as such occurs during gravitropism.     

Investigations into the possible regulation of negative gravitropic curvature in intact Avena sativa plants and in isolated stem segments by ethylene and gibberellins

Using Avena sativa L. cv. Victory oat seedlings and excised p-1 stem segments (including the p-1 and p-2 internodes) the effect of exogenously supplied ethylene and the removal of ethylene on internodal extension and gravitropic bending was assessed. Similarly, the ability of the excised system to respond to gravistimulation was assessed in the presence of inhibitors of ethylene action (AgNO₃) and ethylene synthesis (3,5-diiodo-4-hydroxybenzoic acid and benzyl isothiocyanate; BITC). The production of ethylene from both intact and excised systems was also measured from 0 to 48 h after gravistimulation, relative to vertical controls. Although gravitropic curvature is initiated, and indeed enters the most rapid phase of upward bending during the first 6 h, there is no difference in ethylene production between vertical and geostimulated plants during this period. The ethylene production of gravistimulated plants rises sharply to a maximum at 24 h, then decreases steeply to almost the control level by 48 h, at which time the rate of upward curvature is diminishing. Neither the addition nor removal of ethylene, nor the addition of inhibitors affecting ethylene-action (AgNO₃) or synthesis (DIHB) influence gravitropic bending or internodal extension in excised segments. Although the ethylene synthesis inhibitor BITC showed down the rate of upward bending, this effect could not be reversed by addition of ethylene. We conclude that the burst in ethylene production that develops in leaf-sheath bases (pulvini) after they have started to curve upwards is not primary to the induction of curvature. We further suggest that ethylene has no major effect or role in the induction of upward bending after gravistimulation. The metabolism of high specific activity gibberellin A₁ ([³H]-GA₁) in the excised system was assessed during 1, 2 and 4 h of gravistimulation. Changes in endogenous GAs and GA metabolism have been shown previously to be correlated (at the later stages) with gravistimulated bending in intact Avena shoots. The excised segments ‘leaked’ free [³H]-GAs and [³H]-GA glucosyl conjugate-like substances into the bathing medium, and this was a confounding factor. Nevertheless, gravistimulated stem segments, and especially the bottom half of the segment, were significantly less leaky then vertical segments. Thus, just 1 h after gravistimulation, bottom segment halves retained 22% more precursor [³H]-GA₁, 36% more free [³H]-GA-like metabolites, and 48% more [³H]-GA glucosyl conjugate-like metabolites than vertical segments. In contrast, the 1 h gravistimulated top halves retained slightly less (1–4%) precursor [³H]-GA and free [³H]-GA metabolites, but 21% more [³H]-GA glucosyl conjugate-like radioactivity than vertical segments.     

The response to auxin of rapeseed (Brassica napus L.) roots displaying reduced gravitropism due to transformation by Agrobacterium rhizogenes

It has recently been documented that, compared to untransformed controls, the roots of oilseed rape (Brassica napus L. CV CrGC5) seedlings transformed by Agrobacterium rhizogenes A4 show a reduced gravitropic reaction (Legué et al. 1994, Physiol Plant 91: 559–566). After stimulation at 90°C or 135°, the transformed root tips curve, but never reach a vertical orientation. In the present study, we investigated the causes of reduced gravitropic bending observed in stimulated transformed root tips. First, we localized the gravitropic curvature in normal and in transformed roots after 1.5 h of stimulation. The cells involved in root curvature (target cells) corresponded at the cellular level to the apical part of the zone of increasing cell length. In transformed roots grown in the vertical position, these cells showed a reduction in cell length compared to controls. Because auxin is considered to be the gravitropic mediator, the response of normal and transformed roots to exogenous auxin was studied. Indole-3-acetic acid (IAA) was applied along the first 3 mm using resin beads loaded with the hormone. In comparison to normal roots, transformed roots showed reduced bending toward the bead at all points of bead application. Moreover, the cells which responded to IAA corresponded to the target cells involved in the gravitropic reaction. The level of endogenous IAA was lower in transformed roots. Thus, it was concluded that the modified behavior of transformed roots during gravitropic stimulation could be due to differences either in IAA levels or in reactivity of the target cells to the message from the cap.Abbreviations DEZ distal elongation zone - ELISA enzymelinked immunosorbent assay - T-DNA DNA transferred from Agrobacterium rhizogenes to the plant genome This work was supported by the Centre National d'Etudes Spatiales.     

Nitric oxide mediates gravitropic bending in soybean roots

Plant roots are gravitropic, detecting and responding to changes in orientation via differential growth that results in bending and reestablishment of downward growth. Recent data support the basics of the Cholodny-Went hypothesis, indicating that differential growth is due to redistribution of auxin to the lower sides of gravistimulated roots, but little is known regarding the molecular details of such effects. Here, we investigate auxin and gravity signal transduction by demonstrating that the endogenous signaling molecules nitric oxide (NO) and cGMP mediate responses to gravistimulation in primary roots of soybean (Glycine max). Horizontal orientation of soybean roots caused the accumulation of both NO and cGMP in the primary root tip. Fluorescence confocal microcopy revealed that the accumulation of NO was asymmetric, with NO concentrating in the lower side of the root. Removal of NO with an NO scavenger or inhibition of NO synthesis via NO synthase inhibitors or an inhibitor of nitrate reductase reduced both NO accumulation and gravitropic bending, indicating that NO synthesis was required for the gravitropic responses and that both NO synthase and nitrate reductase may contribute to the synthesis of the NO required. Auxin induced NO accumulation in root protoplasts and asymmetric NO accumulation in root tips. Gravistimulation, NO, and auxin also induced the accumulation of cGMP, a response inhibited by removal of NO or by inhibitors of guanylyl cyclase, compounds that also reduced gravitropic bending. Asymmetric NO accumulation and gravitropic bending were both inhibited by an auxin transport inhibitor, and the inhibition of bending was overcome by treatment with NO or 8-bromo-cGMP, a cell-permeable analog of cGMP. These data indicate that auxin-induced NO and cGMP mediate gravitropic curvature in soybean roots.     

Actin cytoskeleton rearrangements during the gravitropic response of Arabidopsis roots

Gravitropic response is a plant growth response against changing its position relative to the gravity vector. In the present work we studied actin cytoskeleton rearrangements during Arabidopsis root gravitropic response. Two alternative approaches were used to visualize actin microfilaments: histochemical staining of fixed roots with rhodamine-phalloidin and live imaging of microfilaments in GFP-fABD2 transgenic plants. The curvature of actin microfilaments was shown to be increased within 30–60 min of gravistimulation, the fraction of axially oriented microfilaments decreased with a concomitant increase in the fraction of oblique and transversally oriented microfilaments. Methodological issues of actin cytoskeleton visualization in the study of Arabidopsis root gravitropic response, as well as the role of microfilaments at the stages of gravity perception, signal transduction and gravitropic bending formation are discussed. It is concluded that the actin cytoskeleton rearrangements observed are associated with the regulation of basic mechanisms of cell extension growth by which the gravitropic bending is formed.     

Role of xyloglucan in gravitropic bending of azuki bean epicotyl

The mechanism of the gravitropic bending was studied in azuki bean epicotyls. The cell wall extensibility of the lower side became higher than that of the upper side in the epicotyl bending upward. The contents of matrix polysaccharides of the cell wall (pectin and xyloglucan in hemicellulose-II) in the lower side became smaller than those in the upper side. The molecular mass of xyloglucans in the lower side decreased. After an epicotyl was fixed to a metal rod to prevent the bending, gravistimulation was applied. Fundamentally the same results were obtained with respect to rheological and chemical characteristics of the cell wall as those of epicotyls showing gravitropic bending. The present results suggested that the initial gravitropic bending was caused by the increase in extensibility of the lower side and the decrease in extensibility of the upper side via the change of the cell wall matrix, especially xyloglucans.     

Inhibition of the gravitropic bending response of flowering shoots by salicylic acid.

The upward gravitropic bending of cut snapdragon, lupinus and anemone flowering shoots was inhibited by salicylic acid (SA) applied at 0.5 mM and above. This effect was probably not due to acidification of the cytoplasm, since other weak acids did not inhibit bending of snapdragon shoots. In order to study its mode of inhibitory action, we have examined in cut snapdragon shoots the effect of SA on three processes of the gravity-signaling pathway, including: amyloplast sedimentation, formation of ethylene gradient across the stem, and differential growth response. The results show that 1 mM SA inhibited differential ethylene production rates across the horizontal stem and the gravity-induced growth, without significantly inhibiting vertical growth or amyloplast sedimentation following horizontal placement. However, 5 mM SA inhibited all three gravity-induced processes, as well as the growth of vertical shoots, while increasing flower wilting. It may, therefore, be concluded that SA inhibits bending of various cut flowering shoots in a concentration-dependent manner. Thus, at a low concentration SA exerts its effect in snapdragon shoots by inhibiting processes operating downstream to stimulus sensing exerted by amyloplast sedimentation. At a higher concentration SA inhibits bending probably by exerting general negative effects on various cellular processes.