Novel software for analysis of root gravitropism: comparative response patterns of Arabidopsis wild-type and axr1 seedlings

In an earlier study (Evans, Ishikawa & Estelle 1994, Planta 194, 215-222) we used a video digitizer system to compare the kinetics of auxin action on root elongation in wild-type seedlings and seedlings of auxin response mutants of Arabidopsis thaliana (L.) Heynh. We have since modified the system software to allow determination of elongation on opposite sides of vertical or gravistimulated roots and to allow continuous measurement of the angle of orientation of sequential subsections of the root during the response. We used this technology to compare the patterns of differential growth that generate curvature in roots of the Columbia ecotype and in the mutants axr1-3, axr1-12 and axr2, which show reduced gravitropic responsiveness and reduced sensitivity to inhibition by auxin. The pattern of differential growth during gravitropism differed in roots of wild-type and axr1 seedlings. In wild-type roots, initial curvature resulted from differential inhibition of elongation in the distal elongation zone (DEZ). This was followed by an acceleration of elongation along the top side of the DEZ. In roots of axr1-3, curvature resulted from differential stimulation of elongation whereas in roots of axr1-12 the response was variable. Roots of axr2 did not exhibit gravitropic curvature. The observation that the pattern of differential growth causing curvature is dramatically altered by a change in sensitivity to auxin is consistent with the classical Cholodny-Went theory of gravitropism which maintains that differential growth patterns induced by gravistimulation are mediated primarily by gravi-induced shifts in auxin distribution. The new technology introduced with this report allows automated determination of stimulus response patterns in the small but experimentally popular roots of Arabidopsis.     

Ethylene modulates flavonoid accumulation and gravitropic responses in roots of Arabidopsis

Plant organs change their growth direction in response to reorientation relative to the gravity vector. We explored the role of ethylene in Arabidopsis (Arabidopsis thaliana) root gravitropism. Treatment of wild-type Columbia seedlings with the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC) reduced root elongation and gravitropic curvature. The ethylene-insensitive mutants ein2-5 and etr1-3 had wild-type root gravity responses, but lacked the growth and gravity inhibition by ACC found in the wild type. We examined the effect of ACC on tt4(2YY6) seedlings, which have a null mutation in the gene encoding chalcone synthase, the first enzyme in flavonoid synthesis. The tt4(2YY6) mutant makes no flavonoids, has elevated indole-3-acetic acid transport, and exhibits a delayed gravity response. Roots of tt4(2YY6), the backcrossed line tt4-2, and two other tt4 alleles had wild-type sensitivity to growth inhibition by ACC, whereas the root gravitropic curvature of these tt4 alleles was much less inhibited by ACC than wild-type roots, suggesting that ACC may reduce gravitropic curvature by altering flavonoid synthesis. ACC treatment induced flavonoid accumulation in root tips, as judged by a dye that becomes fluorescent upon binding flavonoids in wild type, but not in ein2-5 and etr1-3. ACC also prevented a transient peak in flavonoid synthesis in response to gravity. Together, these experiments suggest that elevated ethylene levels negatively regulate root gravitropism, using EIN2- and ETR1-dependent pathways, and that ACC inhibition of gravity response occurs through altering flavonoid synthesis.     

The roles of phytochromes in elongation and gravitropism of roots

Gravitropic orientation and the elongation of etiolated hypocotyls are both regulated by red light through the phytochrome family of photoreceptors. The importance of phytochromes A and B (phyA and phyB) in these red light responses has been established through studies using phy mutants. To identify the roles that phytochromes play in gravitropism and elongation of roots, we studied the effects of red light on root elongation and then compared the gravitropic curvature from roots of phytochrome mutants of Arabidopsis (phyA, phyB, phyD and phyAB) with wild type. We found that red light inhibits root elongation approximately 35% in etiolated seedlings and that this response is controlled by phytochromes. Roots from dark- and light-grown double mutants (phyAB) and light-grown phyB seedlings have reduced elongation rates compared with wild type. In addition, roots from these seedlings (dark/light-grown phyAB and light-grown phyB) have reduced rates of gravitropic curvature compared with wild type. These results demonstrate roles for phytochromes in regulating both the elongation and gravitropic curvature of roots.     

Light-enhanced perception of gravity in stems of intact pea seedlings

Dark-grown, 6-d-old pea seedlings (Pisum sativum L. cv. Alaska) do not respond gravitropically to brief (approx. 3 min) horizontal presentations, but seedlings given a pulse of red light (R) 16–24 h earlier respond to such stimuli by vigorous curvature of the epicotyl. With continuous horizontal stimulation (approx. 100 min), the kinetics and extent of the gravitropic response are almost identical in irradiated and dark-control plants. Prior R thus increases graviperception without altering the rate-limiting steps underlying the generation of curvature. This effect of R on graviperception develops slowly; seedlings studied only a few hours after R show differences in the kinetics of the gravitropic response, but not in presentation time. Neither the kinetics nor the extent of gravitropic curvature should be used as criteria for establishing changes in primary processes in gravitropism.     

The massugu1 mutation of Arabidopsis identified with failure of auxin-induced growth curvature of hypocotyl confers auxin insensitivity to hypocotyl and leaf.

Unilateral application of indole-3-acetic acid (IAA) in a lanolin base to hypocotyls of partially etiolated seedlings of wild-type Arabidopsis thaliana induced growth curvature in a dose-dependent manner. The effects of IAA in concentrations from 1 to 1000 microM were studied, with maximum IAA-induced curvature at 100 microM. Three IAA-insensitive mutants were isolated and are all in the same locus, massugu1 (msg1). They did not undergo hypocotyl growth curvature at any of the IAA concentrations tested. msg1 is recessive and is located on chromosome 5. msg 1 hypocotyl growth is resistant to 2,4-dichlorophenoxyacetic acid (2,4-D), but the roots are as sensitive to 2,4-D as the wild type. Growth of the hypocotyl was inhibited to essentially the same extent as the wild type by 6-benzylaminopurine, abscisic acid, and 1-aminocyclopropane-1-carboxylate, an ethylene precursor. The msg1 leaves were also resistant to 2,4-D-induced chlorosis. The gravitropic response of the msg1 hypocotyl takes much more time to initiate and achieve the wild-type degree of curvature, whereas the msg1 roots responded normally to gravity. The mature plants and the etiolated seedlings of msg1 were generally wild type in appearance, except that their rosette leaves were either epinastic or hyponastic. msg1 is the first auxin-insensitive mutant in which it effects are mostly restricted to the hypocotyl and leaf, and msg1 also appears to be auxin specific.     

Effects of the myosin ATPase inhibitor 2,3-butanedione monoxime on amyloplast kinetics and gravitropism of Arabidopsis hypocotyls

The actin cytoskeleton is a crucial component in plant gravitropism, and studies confirm that alterations to actin filaments (F-actin) can have dramatic effects on gravitropic curvature in roots and shoots. Many models for gravisensing in higher plants suggest that the key to gravity perception and signal transduction lies in intimate interactions between F-actin and amyloplasts. In this study, we investigated gravitropism in hypocotyls by analyzing the effect of myosin inhibition on gravitropic curvature in order to clarify the role of the actomyosin system in shoot gravitropism. To study amyloplast movement in endodermal cells (i.e., gravity-perceiving statocytes) of living seedlings, we repositioned a confocal laser scanning microscope (CLSM) so that its rotatable stage was oriented vertically. Seedlings containing green fluorescent protein-labeled endodermal amyloplasts were incubated with the ATPase inhibitor 2,3-butanedione monoxime (BDM) and then mounted on the stage so that the hypocotyls were vertical. Using CLSM, we imaged the endodermal amyloplasts, while the hypocotyls were oriented vertically and also after they were reoriented by 90°. Our results show that BDM reduces gravitropic curvature in a concentration-dependent manner. In addition, BDM increases amyloplast movement in hypocotyls of vertical seedlings, but reduces amyloplast movement in hypocotyls of reoriented seedlings, suggesting that myosin may participate in the intracellular transport of amyloplasts in statocytes. These results can be explained in the context of amyloplasts as both noise indicators and gravity susceptors, with BDM producing less coherent amyloplast movement that results in an increased signal-to-noise ratio, which may account for at least part of the observed reduction in gravitopic curvature.     

Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.     

Gravitropic response of primary maize rootlets as influenced by light and temperature

Abstract. The gravitropic curvature of primary maize rootlets was measured as a function of temperature, both in the presence and absence of light. In two different cultivars, light strongly increased the downward curvature of roots developing from horizontally-oriented embryos. At 15–20°C, the bending angle was in the range of 70–80° in the light, and 25–50° in the dark, depending on the cultivar. When the temperature was increased above the 15–20°C range, marked differences were found between the two cultivars in their response to light. In one variety tested, JX180, the effect of light was relatively small at 30–35°C. Gravitropic curvature in another variety, Halamish, depended strongly on light throughout the temperature range tested. In both cultivars, gravitropic curvature was only slightly temperature dependent when germination and growth were in total darkness. In the dark, the extent of gravitropic curvature also depended on whether the kernels were oriented with their embryos facing upwards or downwards. Under continuous light, the gravitropic bending of roots of cultivar Halamish did not show a marked temperature dependence. When the seedlings were subjected to only a 15 min illumination, their gravitropic response was partial, and the dependence on temperature somewhat increased. In cultivar JX180, a combination of temperature and light modulates gravitropism. The gravitropic response of different maize cultivars thus differs considerably in its combined dependence on light and temperature.     

Gravitropic response of inflorescence stems in Arabidopsis thaliana.

We have characterized the gravitropic response of inflorescence stems in Arabidopsis thaliana. When the inflorescence stems were placed horizontally, they curved upward about 90 degrees within 90 min in darkness at 23 degrees C, exhibiting strong negative gravitropism. Decapitated stem segments (without all flowers, flower buds, and apical apices) also showed gravitropic responses when they included the elongation zone. This result indicates that the minimum elements needed for the gravitropic response exist in the decapitated inflorescence stem segments. At least the 3-min gravistimulation time was sufficient to induce the initial curvature at 23 degrees C after a lag time of about 30 min. In the gravitropic response of inflorescence stems, (a) the gravity perception site exists through the elongating zone, (b) auxin is involved in this response, (c) the gravitropic curvature was inhibited at 4 degrees C but at least the gravity perception step could occur, and (d) two curvatures could be induced in sequence at 23 degrees C by two opposite directional horizontal gravistimulations at 4 degrees C.     

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     

PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism

Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.     

Isolation of genes involved in gravitropism.

When upright growing wild type barley seedling are placed to a horizontal position they will respond to this gravistimulation by a curvature against the gravitational force. The mutant serpentina, however, shows gravitropic response only in an early stage of development. Older seedlings, gravistimulated 92 h after germination are agravitropic. They will grow horizontally. We try to identify genes involved in gravitropism by comparing gene expression of i) non-gravistimulated and gravistimulated wild type barley seedlings and of ii) gravistimulated wild type and serpentina.     

The role of calmodulin in the gravitropic response of the Arabidopsis thaliana agr-3 mutant

Calmodulin, a primary plant calcium receptor, is known to be intimately involved with gravitropic sensing and transduction. Using the calmodulin-binding inhibitors trifluoperazine, W7 and calmidazolium, gravitropic curvature of Arabidopsis thaliana (L.) Heynh, ecotype Landsberg, roots was separable into two phases. Phase I was detected at very low concentrations (0.01 μM) of trifluoperazine and calmidazolium, did not involve growth changes, accounted for about half the total curvature of the root and may represent the specific contribution of the cap to gravity sensing. Phase II commenced around 1.0 μM and involved inhibition of both growth and curvature. The agr-3 mutant exhibited a reduced gravitropic response and was found to lack phase I curvature, suggesting that the mutation alters either use or expression of calmodulin. The sequences of wild-type and agr-3 calmodulin (CaM-1) cDNAs, which are root specific were completely determined and found to be identical. Upon gravitropic stimulation, wild-type Arabidopsis seedlings increased calmodulin mRNA levels by threefold in 0.5 h. On the other hand, gravitropic stimulation of agr-3 decreased calmodulin mRNA accumulation. The possible basis of the two phases of curvature is discussed and it is concluded that agr-3 has a lesion located in a general gravity transmission sequence, present in many root cells, which involves calmodulin mRNA accumulation.     

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.     

The Role of Starch in the Gravitropic Response of the Lentil Root

It is well accepted that the amyloplasts of the cap are responsible for gravisensing in primary roots. However, roots with starch-depleted plastids are able to respond to gravistimulus, but their curvature is slower than that of roots containing amyloplasts. The goal of our experiment was to analyse the effects of natural variations of statolith starch in the gravitropic response of lentil roots to a stimulation in the horizontal position. In lentil seedlings grown in the vertical position for 26 h, the volume of the amyloplasts in the statocytes differed between individual roots. The amount of starch in the cap was determined parallel to the rate of gravitropic curvature. There was no statistical correlation between the intensity of the gravitropic response and the starch content in the statocytes. Lentil roots were treated with gibberellic acid (GA₃) at 32°C in order to reduce the volume of starch in the statoliths. There was 53% less starch in the cap of GA₃treated roots as compared to the cap of control roots. But there was no relationship between starch content in the cap and the responsiveness of the root to a gravistimulus, except when the amount of starch was small.     

The Arabidopsis WAVY GROWTH 2 protein modulates root bending in response to environmental stimuli

To understand how the direction of root growth changes in response to obstacles, light, and gravity, we characterized an Arabidopsis thaliana mutant, wavy growth 2 (wav2), whose roots show a short-pitch pattern of wavy growth on inclined agar medium. The roots of the wav2 mutant bent with larger curvature than those of the wild-type seedlings in wavy growth and in gravitropic and phototropic responses. The cell file rotations of the root epidermis of wav2-1 in the wavy growth pattern were enhanced in both right-handed and left-handed rotations. WAV2 encodes a protein belonging to the BUD EMERGENCE 46 family with a transmembrane domain at the N terminus and an alpha/beta-hydrolase domain at the C terminus. Expression analyses showed that mRNA of WAV2 was expressed strongly in adult plant roots and seedlings, especially in the root tip, the cell elongation zone, and the stele. Our results suggest that WAV2 is not involved in sensing environmental stimuli but that it negatively regulates stimulus-induced root bending through inhibition of root tip rotation.     

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.     

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.     

Interaction of light and gravitropism with nutation of hypocotyls of Arabidopsis thaliana seedlings

Etiolated seedlings of Arabidopsis thaliana nutated under conditions of physiological darkness while about ten percent of monitored individuals exhibited regular elliptical nutation, circumnutation. Pre-irradiation with red light prevented occurrence of circumnutation without having an effect on the average rate of the nutational movement. Phototropic response of seedlings to unilateral blue light appeared to be superimposed over nutation. Throughout gravitropism, some seedlings continued to exhibit nutation suggesting that these two processes are independently controlled. Based on these results, we suggest that nutation in Arabidopsis probably is not controlled by the mechanism predicted by the theory of gravitropic overshoots.