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Spiralizations and tropisms in Arabidopsis roots
Opinion
TRENDS in Plant Science Vol.6 No.12 December 2001
Spiralizations and tropisms in Arabidopsis roots Fernando Migliaccio and Silvia Piconese When Arabidopsis seedlings are grown on a hard-agar plate, their primary roots show characteristic spiralling movements, apparent as waves, coils and torsions, together with a slanting toward the right-hand side. All these movements are believed to be the result of three different processes acting on the roots: circumnutation, positive gravitropism and negative thigmotropism. The basic movement of the roots is described as that of a growing right-handed helix, which, because of the root tip hitting the agar plate, is continuously switched from the right-hand to the left-hand of the growth direction, and vice versa. This movement also produces a slanting root-growth direction toward the right-hand because of the incomplete waves made by the right-handed root to the left-hand. By contrast, the torsions seen in the coils and waves are interpreted as artefacts that form as an adaptation of the three-dimensional root helix to the flat two-dimensional agar surface.
Fernando Migliaccio* Silvia Piconese Institute of Plant Biochemistry and Ecophysiology, Consiglio Nazionale delle Ricerche, Via Salaria Km 29.300, 00016 Monterotondo (Roma), Italy. *e-mail: fernando.migliaccio@ mlib.cnr.it
Spiralizations and symmetry in plants have attracted the attention of botanists since the beginning of modern plant science in the 18th century. The famous poet and naturalist Johann Wolfgang von Goethe even elaborated a spiral theory suggesting that the growth of plants is led both by a straight and spiral tendency: the straight one being the male part and the spiral one the female part1. This anthropomorphizing of nature, in fashion at that time, might contain an element of truth. Leaves frequently initiate growth on a shoot at distances that follow a helix and the rule of the Fibonacci’s series (i.e. each number is the sum of the two smallest numbers immediately preceding it2) – another example of spiraling in plants. However, today we can apply the powerful tools of molecular genetics to find new interpretations3 that are based on facts rather than on speculation. Here we describe the processes seen in the roots of Arabidopsis thaliana, although similarities observed in the growth of different plant roots suggest that the main spiral processes are common to all dicot plants. These spiral structures of plants are strictly speaking helical structures (although mostly the term spiral is used). Understanding the basis of spiralization of roots could bring us a step closer to understanding the molecular basis of symmetry and the spiral structure in plants in general. Waves, coils and torsions in Arabidopsis roots
When Arabidopsis seedlings are grown on a hardagar (1.5%) Petri dish (Fig. 1 a,c) that is set inclined http://plants.trends.com
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at an angle, the primary roots do not grow in a straight line as expected, but grow in waves4. These waves are produced when the ROOT TIP (see Glossary) grows alternately to the right and to the left of the vertical direction. In addition, the tip of the root shows a torsion in the longitudinal cell files, that is left-handed when the root is moving toward the right (when looking at the plant from the shoot apex), and is right-handed when the root is moving to the left (Fig. 2d). This waving was thought to be caused by the root tip hitting the agar surface under the stimulus of positive GRAVITROPISM in a tilted plate. The tip would be forced to move continuously from side to side owing to a negative thigmotropic reaction. This interpretation was supported by the observation that waving is not apparent when the agar plates are set vertically. However, further results seem to complicate the picture, indicating that waving also depends on environmental and nutritional conditions5,6. A short time after these first observations, Carl Simmons and Fernando Migliaccio6 observed another peculiar aspect of Arabidopsis root growth: the roots of the most commonly used ecotypes of Arabidopsis (i.e. Landsberg, Wassilewskija and Columbia) slanted to one side of the agar plate, especially when the plate was tilted at least 60° from the horizontal plane. At first they contemplated phototropism as an initiating factor, but further observation showed that the slanting was a peculiar characteristic of Arabidopsis roots that in some way might be connected with the waving movements. The waving attitude of roots had also been noticed by Charles Darwin; he described it as a form of CIRCUMNUTATION7. Darwin had conducted a similar assay to the test on hard-agar plates. He grew roots of oat, oak and pea in the dark, down an inclined and smoked glass plate (Fig. 1b). On this plate, the roots left a track of waves alternately deep and smooth, which he interpreted to be the tracks produced by roots growing along a three-dimensional spiral path. Taking inspiration from Darwin’s results, waving has been interpreted as the flattening of a right-handed circumnutation spiral on the agar plate6. The right-handedness of the spiral was determined by considering that, when the Arabidopsis plant is seen from the shoot apex, the root appears to grow forward by making frequent clockwise coils. This movement in physics is considered to be right-handed. Although discussion about the precise factors that cause waving and slanting is still ongoing, right-handedness has been accepted by most researchers working on the topic. Some authors4,8 see the process as a sinusoidal pattern, resulting from the alternate hitting of the agar surface by the root tip, and thus as essentially a thigmotropic effect. However, today experts in the field seem to agree on the involvement of circumnutation. Since the publication of Charles Darwin’s The Power of Movement in Plants7
1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02152-5
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Fig. 1. Arabidopsis thaliana (ecotype Wassilewskija) primary root growth patterns on inclined surfaces. (a) Arabidopsis seedlings growing on the surface of a hard-agar (1.5%) Petri dish, which was set at an angle of 60° to the horizontal plane. (b) Charles Darwin’s reproduction of the waving patterns left by oak roots on a smoked and inclined glass plate. (c) Typical waving and right-hand slanting pattern of Arabidopsis roots on an agar plate. (d) Right-handed coiling of Arabidopsis roots growing on a hard-agar surface.
Opinion
TRENDS in Plant Science Vol.6 No.12 December 2001
(a)
Glossary
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(ii)
(iii)
(c)
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Circumnutation: Generally described as the circular or elliptical movement that all plant organs produce around the growth direction. The term was introduced by Charles Darwina in substitution of ‘rotating nutation’ introduced earlier by Julius von Sachsb. However, in Charles Darwin’s view, all the tropic and nastic movements of plants originated phylogenetically from the basic circumnutation of plant organs. Gravitropism: The process by which plant roots grow toward the center of the earth (positive gravitropism) and the shoot grows away from it (negative gravitropism). Thigmotropism: Movement of twining shoots or tendrils toward a support. However, in the case of roots, there is movement away from the obstacle, which Charles Darwin described as ‘an obstacle avoiding movement’a. Because of the lack of a precise term for this movement, we propose to adopt the expression ‘negative thigmotropism’ for roots growing away from obstacles, and positive thigmotropism for the twining shoots and tendrils moving toward a support. G. Massa and S. Gilroy (unpublished) have described this kind of root movement again, and ask the community to name this movement as tropism. Root tip: The root tip (up to a few millimeters long) of the flowering plants is the terminal part of the root, and the only part responsive to tropisms. It comprises the root meristem and the initials of the different tissues that constitute the root. It is covered by the root cap, a protective structure made up of cells rich in amyloplasts or statolyths, which are considered to be the first sensors of gravity in roots. A proximal and a distal elongation zone have been recognized in the root tipc. References a Darwin, C. (1880) The Power of Movement in Plants, J. Murray, London, UK b von Sachs, J. (1887) Lectures on the Physiology of Plants, Clarendon Press, Oxford, UK c Ishikawa, H. and Evans, M.L. (1993) The role of the distal elongation zone in the response of maize roots to auxin and gravity. Plant Physiol. 102, 1203–1210
there is no doubt that roots (like all plant organs) grow by making movements around the growth direction rather than by growing linearly9–12. However, Arabidopsis roots seem peculiar. The roots of the most common laboratory plants (maize, pea and sunflower) circumnutate, but at the end of the process they straighten up and little evidence of circumnutation remains13,14, whereas in the tiny roots (0.2 mm) of Arabidopsis, the waving process is preserved. Arabidopsis roots also make another kind of movement on agar plates, especially when the plates are tilted or horizontally set, which produces coils. http://plants.trends.com
These coils are always right-handed in the wild type and show a strong left-handed torsion like that of a contorted rope15,16 (Figs 1d, 2b). Research with mutants
The root patterns that are produced on hard-agar plates were found in the wild-type Arabidopsis plants of the most commonly studied ecotypes [i.e. Wassilewskija (Ws), Columbia and Landsberg]. However, the root patterns are different in some of the mutants. For example, in some of the wav (waving) mutants, the waving pattern is either barely apparent or is increased4. In the
Opinion
TRENDS in Plant Science Vol.6 No.12 December 2001
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concerned, the spir mutants, which have a strong right-hand torsion in shoots, are probably the most interesting. In spir1, the roots also show slanting and coiling to the left-hand. The inversion of symmetry in the spr mutants is connected with the distribution of microtubule arrays in the shoot and root cells, which at the microscopic level are oriented perpendicular to that of the cortical root cells’ elongation axis. Some drugs that disrupt microtubules can invert the symmetry in the mutants19. Interestingly, the first leaf stalks also show a torsion in mutants named tor1, 2 and 3 (tortifolia). The torsion is right-handed in tor1 (allelic with spir2) and tor2, and left-handed in tor3 (H. Buschmann et al., unpublished). Genes and chirality
(d)
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Fig. 2. (a) Right-handed slanting of wild-type Arabidopsis thaliana (ecotype Wassilewskjia) primary roots, and left-handed slanting of roots from the Arabidopsis thaliana mutant 1-6C. (b) Right-handed coil of a root from A. thaliana Wassilewskija (At Ws) showing a torsion to the left-hand. (c) Left-handed coil of a root from At 1-6C (mutant with left-handed symmetry), showing a torsion to the right-hand. (d) Wave from At Ws showing alternate torsion, left-handed when the root bends to the right, right-handed when the root bends to the left; (e) At Ws hypocotyl showing a torsion to the left-hand. Arabidopsis plants were grown from seeds, set in sterile conditions on agar in a square Petri dish, set vertical or inclined at an angle. They were grown for about two weeks before the pictures were taken. Medium made up of 1.5% agar, 1.0% sucrose and 0.5 Murashige and Skoog medium.
sku (skewing) mutants, the slanting to the right-hand is increased significantly17. In other interesting mutants such as 1-6C (Ref. 18) and spr1,2 3 (spiral), the symmetry is left-handed19 (i.e. slanting, coiling and torsions are in the opposite direction) (Fig. 2a,c). Another mutant, clg1, shows a strong tendency to make coils, even on vertically oriented plates20. However, torsion movements are not limited to the roots. They are also present in the shoots, although they are less visible. Shoot circumnutation movement (unlike the root) can be either to the right-hand or to the left-hand, or mixed (as is the case for most plants7,10,21). Right or left-handed torsions are frequent in wild-type shoots (Fig. 2e), especially if they have been grown in the dark (etiolated) – Julius von Sachs described this phenomenon a century ago22. As far as torsions are http://plants.trends.com
The discovery of mutants of circumnutation and chirality led to the cloning of the genes involved. It is fortunate that we have such a diversity of root and shoot movements in the well characterized model system of Arabidopsis. The full genomic sequence of Arabidopsis is online, and the entire gene pool can now be subjected to accurate analysis. These molecular studies have led to the cloning of genes that characterize some interesting mutants. AUX1, cloned from a mutant that shows irregular waving and no gravitropic response, encodes an auxin influx carrier23. The AGR1 protein (alellic with EIR1, WAV6 and AtPIN1), cloned from a mutant that also shows waving and gravitropic disturbances, is involved in auxin efflux24,25. ASA1, a locus that characterizes a mutant showing abnormal waving, encodes a tryptophan synthase26. RHA1, a gravitropically defective mutant, which is in the process of being cloned in our laboratory, is a heat shock factor and a possible element of the gravitropic signal transduction pathway. By contrast, SPIR1 and 2 and TOR1, 2 and 3 code proteins of unknown function19 (H. Buschmann et al., unpublished). However, these might be involved in symmetry determination, possibly by controlling the synthesis and the distribution of microtubules in cells. More work needs to be done to determine the function of all these genes. However, they seem to support the involvement of auxin in plant movements (gravitropism and circumnutation). There is still much controversy between auxin enthusiasts and auxin sceptics about the involvement of auxin in plant movements27. In the near future, we hope that this discussion will be reduced or placated by new exciting molecular discoveries. Combining the root movements together
In spite of all the different studies, the various Arabidopsis root movements were not reconcilable to one unified theory until recently. This is because the relationships between waving, coiling, slanting
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TRENDS in Plant Science Vol.6 No.12 December 2001
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Right-hand Right-hand
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Fig. 3. Schematic representation of an Arabidopsis primary root growing down an inclined hard-agar (1.5%) surface. (a) Theoretical righthanded helical path followed by roots when free to elongate in air. (b) The alternate movements made to the right- and the left-hand of the root, which produces the slanting to the righthand (arrow). (c) The path resulting from the flattening of the root helix, which appears as a sinusoid on a tilted agar plate. (d) A coil, which results from the flattening of the three-dimensional helix on a two-dimensional agar surface. The torsion in the cell files produced as a consequence of the process described in (d) is shown in Fig. 2b.
and torsions were unclear, even though all these movements were thought to be mainly the consequence of the processes of circumnutation, gravitropism and negative THIGMOTROPISM acting on the plant organs during their elongation. Recently, we elaborated a hypothesis that unifies all the different root movements28,29. If we consider that the three most significant processes involved are circumnutation, gravitropism and negative thigmotropism, we can imagine that the root tip, when free, elongates in the air, following a right-handed helix (Fig. 3a). To date, it has been impossible to prove this for technical reasons. However, when grown on an agar plate, especially if tilted from the vertical, after having described half a circle to the right for instance, the root tip will hit the flat agar surface. At this point, because of the negative thigmotropic reaction, it will appear to revolve to the left and make another half circle by keeping its circumnutational movement until it hits the agar again and revolves to the right. These continuous revolutions (shown by the inversion of the torsion in the tip, reported first by Kiyotaka Okada and Yoshiro Shimura4), together with positive gravitropism, will produce a pattern of half circles (waves) alternately to the right and to the left of the vertical. The general direction of these waves appears to slant to the right-side in the wild type (Fig. 3b). Therefore, the slanting can be explained as a consequence of the intrinsic right-handedness of the Arabidopsis root, which causes the roots to make complete half circles to the right, but incomplete or aborted half circles to the left (the opposite effect occurs in left-handed slanting mutants18,19). The Arabidopsis roots therefore grow helically on an agar plate, but the helix is different to the helix that forms in a homogenous medium, switching direction and symmetry continuously. This hypothesis explains the sinusoidal and slanting pattern of Arabidopsis roots (Fig. 3c) as a consequence of their handedness or chirality. However, we also need an explanation for the torsion in the roots because this shows a helical movement that is the opposite to that of slanting and coiling (a torsion to the same direction would http://plants.trends.com
Fig. 4. Microscopic view of coils made by Arabidopsis primary roots. (a) Root grown on an inclined hard agar surface. (b) Root grown free inside an agar tube avoiding any contact with the tube walls. Scale bars = 300 µm.
not be a problem because it is common in Arabidopsis shoots or climbing plants21). The torsion in the root is left-handed when the root is coiling to the right-hand, or it is right-handed when the coils in some mutants, are made to the left-hand. Instead, in the waves, we have alternate successions of left-handed torsion when the wave is moving to the right-hand and of right-handed torsion when the wave is moving to the left-hand. However, with regard to the coils, and extending the reasoning to the waves, we can explain torsions if we imagine that the coils are right-handed helices that have been flattened on an agar plate (Fig. 3d), and that the flattening of a helix into a two-dimensional spiral needs a torsion to adjust to the flat agar surface (because it results in a reduction of the area inside and an extension of the area outside the helix). Therefore, torsions appear to be an artefact caused by the flattening of the helix. The torsion also has to be in the opposite direction to the coiling to discharge the tension produced by the process. This reasoning does not seem to apply to the spir1 mutant because there is a constitutive right-handed torsion in the whole root and shoot. However, a reduction of the right-handed torsion should be apparent in the waves made by spir1 to the right-hand or in the coils (although to date this has not been confirmed). The artefact hypothesis is supported by the lack of torsion in Arabidopsis roots which, when grown in an agar tube without any contact with the walls, sometimes produce curvatures and coils (Fig. 4). Using Arabidopsis, we are increasing our understanding of the processes of tropisms,
Opinion
Acknowledgements We would like to thank Anders Johnsson (Dept of Physics, Trondheim University, Norway) and Carlo Soave (Dept of Biology, University of Milan, Italy), as well as the referees, for critical reading of the manuscript and the useful suggestions given to improve it.
TRENDS in Plant Science Vol.6 No.12 December 2001
spiralization and symmetry in plants, even though not all the possibilities offered by this plant system have been exploited fully. Forces other than gravitropism, circumnutation and thigmotropism could be involved in the process. We need to discover and study new mutants of waving, slanting and gravitropism, as well as mutants of symmetry and spiral formation, which should offer clues about the processes of movement in plants. Cloning the genes connected with these mutations should provide information about the molecular elements that are involved in the processes and in the connected signal transduction pathways. In addition, another significant avenue of research is now possible using
References 1 Mueller, B. (1989) Goethe’s Botanical Writings, Ox Bow Press, Woodbridge, CT, USA 2 Douady, S. and Couder, Y. (1992) Phyllotaxis as a physical self organized growth process. Phys. Rev. Lett. 68, 2098–2101 3 Hudson, A. (2000) Development of symmetry in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 349–370 4 Okada, K. and Shimura, Y. (1990) Reversible root tip rotation in Arabidopsis seedlings induced by obstacle-touching stimulus. Science 250, 274–276 5 Buer, C. et al. (2000) Growth conditions modulate root-wave phenotypes in Arabidopsis. Plant Cell Physiol. 41, 1164–1170 6 Simmons, C. et al. (1995) Circumnutation and gravitropism cause root waving in Arabidopsis thaliana. J. Exp. Bot. 46, 143–150 7 Darwin, C. (1880) The Power of Movement in Plants, J. Murray, London, UK 8 Okada, K. and Shimura, Y. (1992) Mutational analysis of root gravitropism and phototropism of Arabidopsis thaliana seedlings. Aust. J. Plant Physiol. 19, 439–448 9 Braun, A. (1993) Circumnutations: from Darwin to space flights. Plant Physiol. 101, 345–348 10 Johnsson, A. (1979) Circumnutation. In Encyclopedia of Plant Physiology (New Series: Physiology of Movements) (Vol. 7), pp. 627–646, Springer 11 Johnsson, A. (1997) Circumnutation: results of recent experiments on earth and in space. Planta 203, S147–S158
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the just-assembled International Space Station (ISS) and its plant biology facilities. Microgravity conditions on the ISS are ideal for testing root movements, tropism and spiral processes during exclusion of the force of gravity (see article in this issue by Bratislav Stankovic´ , page 559–593). Circumnutation of Arabidopsis shoots in space showed that circumnutation proceeds in the absence of gravity, although with an interesting alteration in its characteristics11. An overview of these plant movements in roots and shoots, and the dissection of the molecular mechanism(s) that are at the basis of tropisms, symmetry and spiralizations in plants should now be possible.
12 Hart, J.W. (1990) Plant Tropisms, Chapman & Hall 13 Spurny, M. (1966) Spiral feedback oscillations of growing hypocotyl with radicle in Pisum sativum L. Biol. Plant. 15, 167–180 14 Spurny, M. (1974) Interaction of photo and geotropism with periodical oscillation of growing pea roots (P. sativum L.). Biol. Plant. 16, 43–49 15 Maher, E.P. and Martindale, S.J.B. (1980) Mutants of Arabidopsis thaliana with altered responses to auxin and growth. Biochem. Genet. 18, 1041–1053 16 Mirza, J.I. (1987) The effect of light and gravity in the horizontal curvature of roots of gravitropic and agravitropic Arabidopsis thaliana. Plant Physiol. 83, 118–120 17 Rutherford, R. and Masson, P.H. (1996) Arabidopsis thaliana sku mutant seedlings show exaggerated surface-dependent alteration in root growth vector. Plant Physiol. 111, 4090–4096 18 Marinelli, B. et al. (1997) A pleiotropic Arabidopsis thaliana mutant with inverted root chirality. Planta 202, 196–205 19 Furutani, I. et al. (2000) The spiral genes are required for directional control of cell elongation in Arabidopsis thaliana. Development 127, 4443–4453 20 Ferrari, S. et al. (2000) clg1, a new Arabidopsis thaliana root mutant characterised by reduced gravitropism and increased slanting. Plant Sci. 150, 77–85
21 Baillaud, L. (1962) Les mouvements d’ exploration et d’enroulement des plantes volubiles. In Encyclopedia of Plant Physiology; Physiology of Movements (Vol. 17) (Buenning, E., ed.), pp. 637–715, Springer 22 von Sachs, J. (1887) Lectures on the Physiology of Plants, Clarendon Press, London, UK 23 Bennet, M.J. et al. (1997) AUX1 gene: a permeaselike regulator of root gravitropism. Science 273, 948–950 24 Chen, R. et al. (1998) The Arabidopsis thaliana AGRAVITROPIC1 gene encodes a component of the polar auxin-transport efflux carrier. Proc. Natl. Acad. Sci. U. S. A. 95, 15112–15117 25 Palme, K. and Galweiler L. (1999) PIN-pointing the molecular basis of auxin transport. Curr. Opin. Plant Biol. 2, 375–381 26 Rutherford, R. et al. (1998) Mutations in Arabidopsis thaliana genes involved in the tryptophan biosynthesis pathway affect root waving on tilted agar surfaces. Plant J. 16, 145–154 27 Evans, M.L. (1992) What remains of the Cholodny–Went theory. Plant Cell Environ. 17, 767–768 28 Migliaccio, F. et al. (1996) Waving and coiling in Arabidopsis roots. Plant Physiol. Biochem. 19, S02–S04 29 Migliaccio, F. et al. (2001) Investigating the origin and nature of right-handed slanting of primary Arabidopsis roots. J. Gravit. Physiol. 102, 1203–1210
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TRENDS in Plant Science Vol.6 No.12 December 2001
Spiralizations and tropisms in Arabidopsis roots Fernando Migliaccio and Silvia Piconese When Arabidopsis seedlings are grown on a hard-agar plate, their primary roots show characteristic spiralling movements, apparent as waves, coils and torsions, together with a slanting toward the right-hand side. All these movements are believed to be the result of three different processes acting on the roots: circumnutation, positive gravitropism and negative thigmotropism. The basic movement of the roots is described as that of a growing right-handed helix, which, because of the root tip hitting the agar plate, is continuously switched from the right-hand to the left-hand of the growth direction, and vice versa. This movement also produces a slanting root-growth direction toward the right-hand because of the incomplete waves made by the right-handed root to the left-hand. By contrast, the torsions seen in the coils and waves are interpreted as artefacts that form as an adaptation of the three-dimensional root helix to the flat two-dimensional agar surface.
Fernando Migliaccio* Silvia Piconese Institute of Plant Biochemistry and Ecophysiology, Consiglio Nazionale delle Ricerche, Via Salaria Km 29.300, 00016 Monterotondo (Roma), Italy. *e-mail: fernando.migliaccio@ mlib.cnr.it
Spiralizations and symmetry in plants have attracted the attention of botanists since the beginning of modern plant science in the 18th century. The famous poet and naturalist Johann Wolfgang von Goethe even elaborated a spiral theory suggesting that the growth of plants is led both by a straight and spiral tendency: the straight one being the male part and the spiral one the female part1. This anthropomorphizing of nature, in fashion at that time, might contain an element of truth. Leaves frequently initiate growth on a shoot at distances that follow a helix and the rule of the Fibonacci’s series (i.e. each number is the sum of the two smallest numbers immediately preceding it2) – another example of spiraling in plants. However, today we can apply the powerful tools of molecular genetics to find new interpretations3 that are based on facts rather than on speculation. Here we describe the processes seen in the roots of Arabidopsis thaliana, although similarities observed in the growth of different plant roots suggest that the main spiral processes are common to all dicot plants. These spiral structures of plants are strictly speaking helical structures (although mostly the term spiral is used). Understanding the basis of spiralization of roots could bring us a step closer to understanding the molecular basis of symmetry and the spiral structure in plants in general. Waves, coils and torsions in Arabidopsis roots
When Arabidopsis seedlings are grown on a hardagar (1.5%) Petri dish (Fig. 1 a,c) that is set inclined http://plants.trends.com
561
at an angle, the primary roots do not grow in a straight line as expected, but grow in waves4. These waves are produced when the ROOT TIP (see Glossary) grows alternately to the right and to the left of the vertical direction. In addition, the tip of the root shows a torsion in the longitudinal cell files, that is left-handed when the root is moving toward the right (when looking at the plant from the shoot apex), and is right-handed when the root is moving to the left (Fig. 2d). This waving was thought to be caused by the root tip hitting the agar surface under the stimulus of positive GRAVITROPISM in a tilted plate. The tip would be forced to move continuously from side to side owing to a negative thigmotropic reaction. This interpretation was supported by the observation that waving is not apparent when the agar plates are set vertically. However, further results seem to complicate the picture, indicating that waving also depends on environmental and nutritional conditions5,6. A short time after these first observations, Carl Simmons and Fernando Migliaccio6 observed another peculiar aspect of Arabidopsis root growth: the roots of the most commonly used ecotypes of Arabidopsis (i.e. Landsberg, Wassilewskija and Columbia) slanted to one side of the agar plate, especially when the plate was tilted at least 60° from the horizontal plane. At first they contemplated phototropism as an initiating factor, but further observation showed that the slanting was a peculiar characteristic of Arabidopsis roots that in some way might be connected with the waving movements. The waving attitude of roots had also been noticed by Charles Darwin; he described it as a form of CIRCUMNUTATION7. Darwin had conducted a similar assay to the test on hard-agar plates. He grew roots of oat, oak and pea in the dark, down an inclined and smoked glass plate (Fig. 1b). On this plate, the roots left a track of waves alternately deep and smooth, which he interpreted to be the tracks produced by roots growing along a three-dimensional spiral path. Taking inspiration from Darwin’s results, waving has been interpreted as the flattening of a right-handed circumnutation spiral on the agar plate6. The right-handedness of the spiral was determined by considering that, when the Arabidopsis plant is seen from the shoot apex, the root appears to grow forward by making frequent clockwise coils. This movement in physics is considered to be right-handed. Although discussion about the precise factors that cause waving and slanting is still ongoing, right-handedness has been accepted by most researchers working on the topic. Some authors4,8 see the process as a sinusoidal pattern, resulting from the alternate hitting of the agar surface by the root tip, and thus as essentially a thigmotropic effect. However, today experts in the field seem to agree on the involvement of circumnutation. Since the publication of Charles Darwin’s The Power of Movement in Plants7
1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02152-5
562
Fig. 1. Arabidopsis thaliana (ecotype Wassilewskija) primary root growth patterns on inclined surfaces. (a) Arabidopsis seedlings growing on the surface of a hard-agar (1.5%) Petri dish, which was set at an angle of 60° to the horizontal plane. (b) Charles Darwin’s reproduction of the waving patterns left by oak roots on a smoked and inclined glass plate. (c) Typical waving and right-hand slanting pattern of Arabidopsis roots on an agar plate. (d) Right-handed coiling of Arabidopsis roots growing on a hard-agar surface.
Opinion
TRENDS in Plant Science Vol.6 No.12 December 2001
(a)
Glossary
(b) (i)
(ii)
(iii)
(c)
(d)
Circumnutation: Generally described as the circular or elliptical movement that all plant organs produce around the growth direction. The term was introduced by Charles Darwina in substitution of ‘rotating nutation’ introduced earlier by Julius von Sachsb. However, in Charles Darwin’s view, all the tropic and nastic movements of plants originated phylogenetically from the basic circumnutation of plant organs. Gravitropism: The process by which plant roots grow toward the center of the earth (positive gravitropism) and the shoot grows away from it (negative gravitropism). Thigmotropism: Movement of twining shoots or tendrils toward a support. However, in the case of roots, there is movement away from the obstacle, which Charles Darwin described as ‘an obstacle avoiding movement’a. Because of the lack of a precise term for this movement, we propose to adopt the expression ‘negative thigmotropism’ for roots growing away from obstacles, and positive thigmotropism for the twining shoots and tendrils moving toward a support. G. Massa and S. Gilroy (unpublished) have described this kind of root movement again, and ask the community to name this movement as tropism. Root tip: The root tip (up to a few millimeters long) of the flowering plants is the terminal part of the root, and the only part responsive to tropisms. It comprises the root meristem and the initials of the different tissues that constitute the root. It is covered by the root cap, a protective structure made up of cells rich in amyloplasts or statolyths, which are considered to be the first sensors of gravity in roots. A proximal and a distal elongation zone have been recognized in the root tipc. References a Darwin, C. (1880) The Power of Movement in Plants, J. Murray, London, UK b von Sachs, J. (1887) Lectures on the Physiology of Plants, Clarendon Press, Oxford, UK c Ishikawa, H. and Evans, M.L. (1993) The role of the distal elongation zone in the response of maize roots to auxin and gravity. Plant Physiol. 102, 1203–1210
there is no doubt that roots (like all plant organs) grow by making movements around the growth direction rather than by growing linearly9–12. However, Arabidopsis roots seem peculiar. The roots of the most common laboratory plants (maize, pea and sunflower) circumnutate, but at the end of the process they straighten up and little evidence of circumnutation remains13,14, whereas in the tiny roots (0.2 mm) of Arabidopsis, the waving process is preserved. Arabidopsis roots also make another kind of movement on agar plates, especially when the plates are tilted or horizontally set, which produces coils. http://plants.trends.com
These coils are always right-handed in the wild type and show a strong left-handed torsion like that of a contorted rope15,16 (Figs 1d, 2b). Research with mutants
The root patterns that are produced on hard-agar plates were found in the wild-type Arabidopsis plants of the most commonly studied ecotypes [i.e. Wassilewskija (Ws), Columbia and Landsberg]. However, the root patterns are different in some of the mutants. For example, in some of the wav (waving) mutants, the waving pattern is either barely apparent or is increased4. In the
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concerned, the spir mutants, which have a strong right-hand torsion in shoots, are probably the most interesting. In spir1, the roots also show slanting and coiling to the left-hand. The inversion of symmetry in the spr mutants is connected with the distribution of microtubule arrays in the shoot and root cells, which at the microscopic level are oriented perpendicular to that of the cortical root cells’ elongation axis. Some drugs that disrupt microtubules can invert the symmetry in the mutants19. Interestingly, the first leaf stalks also show a torsion in mutants named tor1, 2 and 3 (tortifolia). The torsion is right-handed in tor1 (allelic with spir2) and tor2, and left-handed in tor3 (H. Buschmann et al., unpublished). Genes and chirality
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Fig. 2. (a) Right-handed slanting of wild-type Arabidopsis thaliana (ecotype Wassilewskjia) primary roots, and left-handed slanting of roots from the Arabidopsis thaliana mutant 1-6C. (b) Right-handed coil of a root from A. thaliana Wassilewskija (At Ws) showing a torsion to the left-hand. (c) Left-handed coil of a root from At 1-6C (mutant with left-handed symmetry), showing a torsion to the right-hand. (d) Wave from At Ws showing alternate torsion, left-handed when the root bends to the right, right-handed when the root bends to the left; (e) At Ws hypocotyl showing a torsion to the left-hand. Arabidopsis plants were grown from seeds, set in sterile conditions on agar in a square Petri dish, set vertical or inclined at an angle. They were grown for about two weeks before the pictures were taken. Medium made up of 1.5% agar, 1.0% sucrose and 0.5 Murashige and Skoog medium.
sku (skewing) mutants, the slanting to the right-hand is increased significantly17. In other interesting mutants such as 1-6C (Ref. 18) and spr1,2 3 (spiral), the symmetry is left-handed19 (i.e. slanting, coiling and torsions are in the opposite direction) (Fig. 2a,c). Another mutant, clg1, shows a strong tendency to make coils, even on vertically oriented plates20. However, torsion movements are not limited to the roots. They are also present in the shoots, although they are less visible. Shoot circumnutation movement (unlike the root) can be either to the right-hand or to the left-hand, or mixed (as is the case for most plants7,10,21). Right or left-handed torsions are frequent in wild-type shoots (Fig. 2e), especially if they have been grown in the dark (etiolated) – Julius von Sachs described this phenomenon a century ago22. As far as torsions are http://plants.trends.com
The discovery of mutants of circumnutation and chirality led to the cloning of the genes involved. It is fortunate that we have such a diversity of root and shoot movements in the well characterized model system of Arabidopsis. The full genomic sequence of Arabidopsis is online, and the entire gene pool can now be subjected to accurate analysis. These molecular studies have led to the cloning of genes that characterize some interesting mutants. AUX1, cloned from a mutant that shows irregular waving and no gravitropic response, encodes an auxin influx carrier23. The AGR1 protein (alellic with EIR1, WAV6 and AtPIN1), cloned from a mutant that also shows waving and gravitropic disturbances, is involved in auxin efflux24,25. ASA1, a locus that characterizes a mutant showing abnormal waving, encodes a tryptophan synthase26. RHA1, a gravitropically defective mutant, which is in the process of being cloned in our laboratory, is a heat shock factor and a possible element of the gravitropic signal transduction pathway. By contrast, SPIR1 and 2 and TOR1, 2 and 3 code proteins of unknown function19 (H. Buschmann et al., unpublished). However, these might be involved in symmetry determination, possibly by controlling the synthesis and the distribution of microtubules in cells. More work needs to be done to determine the function of all these genes. However, they seem to support the involvement of auxin in plant movements (gravitropism and circumnutation). There is still much controversy between auxin enthusiasts and auxin sceptics about the involvement of auxin in plant movements27. In the near future, we hope that this discussion will be reduced or placated by new exciting molecular discoveries. Combining the root movements together
In spite of all the different studies, the various Arabidopsis root movements were not reconcilable to one unified theory until recently. This is because the relationships between waving, coiling, slanting
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Fig. 3. Schematic representation of an Arabidopsis primary root growing down an inclined hard-agar (1.5%) surface. (a) Theoretical righthanded helical path followed by roots when free to elongate in air. (b) The alternate movements made to the right- and the left-hand of the root, which produces the slanting to the righthand (arrow). (c) The path resulting from the flattening of the root helix, which appears as a sinusoid on a tilted agar plate. (d) A coil, which results from the flattening of the three-dimensional helix on a two-dimensional agar surface. The torsion in the cell files produced as a consequence of the process described in (d) is shown in Fig. 2b.
and torsions were unclear, even though all these movements were thought to be mainly the consequence of the processes of circumnutation, gravitropism and negative THIGMOTROPISM acting on the plant organs during their elongation. Recently, we elaborated a hypothesis that unifies all the different root movements28,29. If we consider that the three most significant processes involved are circumnutation, gravitropism and negative thigmotropism, we can imagine that the root tip, when free, elongates in the air, following a right-handed helix (Fig. 3a). To date, it has been impossible to prove this for technical reasons. However, when grown on an agar plate, especially if tilted from the vertical, after having described half a circle to the right for instance, the root tip will hit the flat agar surface. At this point, because of the negative thigmotropic reaction, it will appear to revolve to the left and make another half circle by keeping its circumnutational movement until it hits the agar again and revolves to the right. These continuous revolutions (shown by the inversion of the torsion in the tip, reported first by Kiyotaka Okada and Yoshiro Shimura4), together with positive gravitropism, will produce a pattern of half circles (waves) alternately to the right and to the left of the vertical. The general direction of these waves appears to slant to the right-side in the wild type (Fig. 3b). Therefore, the slanting can be explained as a consequence of the intrinsic right-handedness of the Arabidopsis root, which causes the roots to make complete half circles to the right, but incomplete or aborted half circles to the left (the opposite effect occurs in left-handed slanting mutants18,19). The Arabidopsis roots therefore grow helically on an agar plate, but the helix is different to the helix that forms in a homogenous medium, switching direction and symmetry continuously. This hypothesis explains the sinusoidal and slanting pattern of Arabidopsis roots (Fig. 3c) as a consequence of their handedness or chirality. However, we also need an explanation for the torsion in the roots because this shows a helical movement that is the opposite to that of slanting and coiling (a torsion to the same direction would http://plants.trends.com
Fig. 4. Microscopic view of coils made by Arabidopsis primary roots. (a) Root grown on an inclined hard agar surface. (b) Root grown free inside an agar tube avoiding any contact with the tube walls. Scale bars = 300 µm.
not be a problem because it is common in Arabidopsis shoots or climbing plants21). The torsion in the root is left-handed when the root is coiling to the right-hand, or it is right-handed when the coils in some mutants, are made to the left-hand. Instead, in the waves, we have alternate successions of left-handed torsion when the wave is moving to the right-hand and of right-handed torsion when the wave is moving to the left-hand. However, with regard to the coils, and extending the reasoning to the waves, we can explain torsions if we imagine that the coils are right-handed helices that have been flattened on an agar plate (Fig. 3d), and that the flattening of a helix into a two-dimensional spiral needs a torsion to adjust to the flat agar surface (because it results in a reduction of the area inside and an extension of the area outside the helix). Therefore, torsions appear to be an artefact caused by the flattening of the helix. The torsion also has to be in the opposite direction to the coiling to discharge the tension produced by the process. This reasoning does not seem to apply to the spir1 mutant because there is a constitutive right-handed torsion in the whole root and shoot. However, a reduction of the right-handed torsion should be apparent in the waves made by spir1 to the right-hand or in the coils (although to date this has not been confirmed). The artefact hypothesis is supported by the lack of torsion in Arabidopsis roots which, when grown in an agar tube without any contact with the walls, sometimes produce curvatures and coils (Fig. 4). Using Arabidopsis, we are increasing our understanding of the processes of tropisms,
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Acknowledgements We would like to thank Anders Johnsson (Dept of Physics, Trondheim University, Norway) and Carlo Soave (Dept of Biology, University of Milan, Italy), as well as the referees, for critical reading of the manuscript and the useful suggestions given to improve it.
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spiralization and symmetry in plants, even though not all the possibilities offered by this plant system have been exploited fully. Forces other than gravitropism, circumnutation and thigmotropism could be involved in the process. We need to discover and study new mutants of waving, slanting and gravitropism, as well as mutants of symmetry and spiral formation, which should offer clues about the processes of movement in plants. Cloning the genes connected with these mutations should provide information about the molecular elements that are involved in the processes and in the connected signal transduction pathways. In addition, another significant avenue of research is now possible using
References 1 Mueller, B. (1989) Goethe’s Botanical Writings, Ox Bow Press, Woodbridge, CT, USA 2 Douady, S. and Couder, Y. (1992) Phyllotaxis as a physical self organized growth process. Phys. Rev. Lett. 68, 2098–2101 3 Hudson, A. (2000) Development of symmetry in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 349–370 4 Okada, K. and Shimura, Y. (1990) Reversible root tip rotation in Arabidopsis seedlings induced by obstacle-touching stimulus. Science 250, 274–276 5 Buer, C. et al. (2000) Growth conditions modulate root-wave phenotypes in Arabidopsis. Plant Cell Physiol. 41, 1164–1170 6 Simmons, C. et al. (1995) Circumnutation and gravitropism cause root waving in Arabidopsis thaliana. J. Exp. Bot. 46, 143–150 7 Darwin, C. (1880) The Power of Movement in Plants, J. Murray, London, UK 8 Okada, K. and Shimura, Y. (1992) Mutational analysis of root gravitropism and phototropism of Arabidopsis thaliana seedlings. Aust. J. Plant Physiol. 19, 439–448 9 Braun, A. (1993) Circumnutations: from Darwin to space flights. Plant Physiol. 101, 345–348 10 Johnsson, A. (1979) Circumnutation. In Encyclopedia of Plant Physiology (New Series: Physiology of Movements) (Vol. 7), pp. 627–646, Springer 11 Johnsson, A. (1997) Circumnutation: results of recent experiments on earth and in space. Planta 203, S147–S158
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the just-assembled International Space Station (ISS) and its plant biology facilities. Microgravity conditions on the ISS are ideal for testing root movements, tropism and spiral processes during exclusion of the force of gravity (see article in this issue by Bratislav Stankovic´ , page 559–593). Circumnutation of Arabidopsis shoots in space showed that circumnutation proceeds in the absence of gravity, although with an interesting alteration in its characteristics11. An overview of these plant movements in roots and shoots, and the dissection of the molecular mechanism(s) that are at the basis of tropisms, symmetry and spiralizations in plants should now be possible.
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21 Baillaud, L. (1962) Les mouvements d’ exploration et d’enroulement des plantes volubiles. In Encyclopedia of Plant Physiology; Physiology of Movements (Vol. 17) (Buenning, E., ed.), pp. 637–715, Springer 22 von Sachs, J. (1887) Lectures on the Physiology of Plants, Clarendon Press, London, UK 23 Bennet, M.J. et al. (1997) AUX1 gene: a permeaselike regulator of root gravitropism. Science 273, 948–950 24 Chen, R. et al. (1998) The Arabidopsis thaliana AGRAVITROPIC1 gene encodes a component of the polar auxin-transport efflux carrier. Proc. Natl. Acad. Sci. U. S. A. 95, 15112–15117 25 Palme, K. and Galweiler L. (1999) PIN-pointing the molecular basis of auxin transport. Curr. Opin. Plant Biol. 2, 375–381 26 Rutherford, R. et al. (1998) Mutations in Arabidopsis thaliana genes involved in the tryptophan biosynthesis pathway affect root waving on tilted agar surfaces. Plant J. 16, 145–154 27 Evans, M.L. (1992) What remains of the Cholodny–Went theory. Plant Cell Environ. 17, 767–768 28 Migliaccio, F. et al. (1996) Waving and coiling in Arabidopsis roots. Plant Physiol. Biochem. 19, S02–S04 29 Migliaccio, F. et al. (2001) Investigating the origin and nature of right-handed slanting of primary Arabidopsis roots. J. Gravit. Physiol. 102, 1203–1210
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