The SPIRAL genes are required for directional control of cell elongation in Arabidopsis thaliana

There are numerous examples of left-right asymmetries in biological systems. Although the development of left-right asymmetries in vertebrates recently began to yield to molecular analysis (Capdevila et al., 2000), cellular and molecular mechanisms of handedness in plants have not been explored at all. Many species of twining plants show apparent handedness, either consistently forming right- or left-handed helices as they climb. Here, the direction of a helix is defined as right-handed when it matches the appearance of a right-handed corkscrew. For example, runner bean and bindweed make right-handed helices, whereas hop and honeysuckle produce left-handed helices (Coen, 1999). Helical growth is not restricted to tendrils of climbing plants. Darwin surveyed dozens of plant species and discovered that oscillating growth of plant organs is widespread and introduced the term ‘circumnutation’ (Darwin, 1875; Darwin and Darwin, 1880). Roots, hypocotyls, shoots, branches and flower stalks may oscillate either in a clockwise or anticlockwise direction, strictly in a growth-dependent manner. More recently circumnutations have been found not only in dicots and monocots but also in gymnosperms, fungi and algae (Brown, 1993). A ‘wavy’ growth pattern of Arabidopsis thaliana roots on inclined agar plates (Okada and Shimura, 1990) is also interpreted to result from circumnutation and gravitropism (Simmons et al., 1995). Although much descriptive work has been carried out for several decades to characterize the kinematics of circumnutations, models describing the underlying mechanisms remain speculative.

There are further examples of handedness in plants. The petals of several species are arranged like fan blades that all seem to twist in the same direction, forming either a clockwise or an anticlockwise appearance. Clockwise-rotating petals are found, for example, in oleander, whereas petals of the greater periwinkle are arranged in an anticlockwise fashion (Coen, 1999). Plant species of the families Guttiferae, Malvaceae and Oxalidaceae, however, generally develop clockwise- and anticlockwise-rotating flowers with equal frequency. Therefore, either a chance event or a genetically determined developmental program appears to produce flowers with distinct handedness.

The patterned arrangement of organs such as leaves around the shoot axis (phyllotaxy) represents another example of handedness in plants. A spiral/helical phyllotactic pattern where leaves are formed 137.5° apart predominates in nature (Williams, 1975). In Arabidopsis thaliana, the handedness of the generative spiral is maintained throughout vegetative development and can be either clockwise or anticlockwise in equal frequency (Callos and Medford, 1994). This stochastic phyllotactic handedness can be traced back to the asymmetry in the developmental distances between the two cotyledons during Arabidopsis embryo development (Woodrick et al., 2000).

Genetically controlled handedness should be amenable to molecular genetic studies. In this study, we have screened Arabidopsis thaliana seedlings for mutants with consistent right-handed skewing of root epidermal cell files. Furthermore, we have also found that treatment of seedlings with drugs that compromise microtubule (MT) functions produces a lefthanded helical growth. We propose a model in which anisotropic growth is controlled by antagonizing effects between a MT-organizing process and SPR genes.

Arabidopsis thaliana seeds were sterilized in 5% sodium hypochlorite and were allowed to germinate on plates containing 0.5× Arabidopsis nutrient solution (Haughn and Somerville, 1986), 2% sucrose and 1.5% agar, unless otherwise noted. After 2 days at 4°C, plates were incubated in a near vertical position at 22°C with a 16 hour light/8 hour dark cycle, unless otherwise noted. Day 0 of growth is defined as the time when plates were transferred to 22°C. To prepare stock solutions, oryzalin (AccuStandard, New Haven, CT), propyzamide (Wako, Osaka, Japan), and taxol (Nacalai tesque, Kyoto, Japan) were dissolved in dimethyl sulfoxide. Final concentrations of dimethyl sulfoxide in the media were lower than 0.3%, at which concentration the growth of seedlings was not affected.

The spr1-1 and spr2-1 mutant alleles were isolated from fastneutron mutagenized M2 seeds of the Landsberg erecta (Ler) ecotype (Lehle Seeds, Round Rock, TX), whereas spr1-4 was recovered from gamma-ray mutagenized M2 seeds of the Wassilewskija ecotype (Lehle Seeds). spr1-2 and spr1-3 in the Columbia background were isolated, respectively, by J. Schiefelbein from enhancer trap lines produced by T. Jack (ABRC stock center), and by K. Nakamura from fast-neutron mutagenized M2 seeds (Lehle Seeds). tortifolia1 (Enkheim ecotype) was obtained from T. Schäffner, and convoluta (S95 ecotype) was obtained from ABRC stock center. spr1-1 and spr2-1 alleles were out-crossed at least three times and twice, respectively, to the Ler wild type before being used in this study.

For mapping, homozygous spr1-1 and spr2-1 plants were crossed with Col wild-type plants, and selfed to give F₂-mapping populations. Seedlings homozygous for the spr mutation were selected and used to extract DNA for mapping with CAPS and SSLP markers (http://www.arabidopsis.org/aboutcaps.html).

The spr1-1spr2-1 double mutant was selected in F₂ populations, and the homozygous double mutants in the F₃ generation were used for phenotypic analysis. Spiral phenotypes in root, petiole and hypocotyl were used to distinguish the spr1-1 and spr2-1 mutations. A 0.6-kb deletion in the SPR1 gene was also used to confirm the spr1-1 mutation by genomic PCR (I. F., H. Tachimoto and T. H., unpublished).

Spiral phenotypes of whole seedlings were analyzed using an Olympus stereoscope SZX12 equipped with an Olympus digital camera DP10. For measurement of length and width of root cells, seven-day-old seedlings grown at 22°C were cleared with chloral hydrate, and viewed under Nomarski optics. Optical sections of root cells at the differentiation zone were made at a series of focal planes, and the maximal cell width of epidermis, cortex and endodermis was determined. Distance from the quiescent center, cell length and skewed angle of the long cell axis from the central axis of a primary root were likewise measured on the root epidermal cells of ten-day-old seedlings grown at 14°C.

For histological analysis, hypocotyls of five-day-old etiolated seedlings were fixed and embedded in Technovit 7100 (Kulzer, Hereaus), essentially as described (Scheres et al., 1994). A series of 6 μm thick logitudinal sections and 8 μm thick transverse sections were made with a rotary microtome HM325 (Microm). Transverse sections were serially aligned from the shoot tip to the rapidly elongating region (approximately 1 mm away from the tip), so that the distance from the tip could be assigned for given sections. Sections were stained with toluidine blue O and photographed on an Olympus BX50 microscope equipped with a PM-20 camera.

Replica images of seedlings were made using polyvinylsiloxane impression material (Extrude; Keer Co., Romulus, MI) and epoxy glue (Araldite; Ciba Geigy), coated with Pt, and examined with scanning electron microscopy N3200 (Hitachi).

The protocol for fixing and immunostaining of seedling roots was as described (Wasteneys et al., 1998; Sugimoto, 2000 – PhD Thesis, The Australian National University.) except that the cold methanol treatment was not used and we used anti-α-tubulin (N356; Amersham) diluted 1:1000 in wash buffer as a primary antibody.

Hypocotyls of five-day-old etiolated seedlings were fixed, rinsed and embedded in 5% agar. Fixed hypocotyls in the agar blocks were cut longitudinally into 100 μm thick sections with a DTK-1500 microslicer (Dohan EM, Kyoto, Japan). Buffers, fixation solutions and immunolabeling procedures were the same as those used for roots.

Stained cells were optically sectioned at several different focal planes with a confocal laser-scanning microscope LSM510 (Zeiss). The orientation of cortical MTs adjacent to the outer tangential wall of each epidermal cell was measured at upper, middle and lower regions of the longest cell axis with an image processing software MacSCOPE (Mitani, Fukui, Japan), and the three measurements were averaged to represent the MT angle of the examined cell. In a majority of epidermal cells, the orientation of cortical MTs was uniform within the cell.

When wild-type Arabidopsis thaliana seedlings (Ler ecotype) were grown vertically on a hard-agar surface, the direction of root growth deviated slightly to the left of vertical (when the seedlings are viewed from above the agar surface; Fig. 1A). The spiral (spr) mutants were isolated primarily on the basis of their tendency to bend to the right (Fig. 1A). After test crosses, these mutants were classified into two non-complementation groups. We identified four alleles of spr1 (spr1-1 to spr1-4), and one allele of spr2 (spr2-1). The phenotypic similarity of spr2 to previously reported mutants prompted us to conduct complementation tests between them, which revealed that spr2 is allelic to tortifolia1 (Bürger, 1971) and convoluta (Relichova, 1976). A pleiotropic arabidopsis mutant that partially resembles spr mutants and was interpreted to result from two independent mutations was also reported (Marinelli et al., 1997), but was not tested here for the allelism. Upon outcrossing the spr alleles to wild type, all F1 seedlings were phenotypically wild-type, and the F1 plants produced 25% mutant seedlings upon selfing, indicating that spr1 and spr2 mutations are recessive.

Mapping against molecular markers in a mapping population of F2 spr seedlings revealed that SPR1 is tightly linked to m246 on the top of chromosome 2 (no recombination among 282 chromosomes), and that SPR2 is positioned 0.27 cM south of PG11 and 1.5 cM north of mi123 on chromosome 4, in accordance with linkage of TORTIFOLIA1 to cer2 and d104 in that region (Fabri and Schäffner, 1994).

Since all four spr1 alleles showed very similar phenotypes, the spr1-1 allele was characterized in detail although the results obtained were essentially reproduced in the spr1-2 allele. When grown on a vertically positioned hard-agar surface, spr1-1 seedling roots grew sharply skewed to the right, while spr2 roots grew almost vertically or slightly skewed to the right (Fig. 1A). Under these conditions, both spr roots showed typical wavy growth paths as observed in wild-type roots. The spr1 root meristem appeared normal but, at the distal elongation zone, epidermal cell files formed right-handed helices (Fig. 1C). The right-handed helix extended from the differentiation zone to the base of the primary root, and was observed in the lateral roots as well. Physical contact with the agar surface did not affect the helical phenotype since the spr1 roots projecting into the air or growing within the agar also showed the constitutive helical epidermis (not shown). When spr1 roots penetrated the agar, the submerged roots grew directly downwards (Fig. 1F), indicating that the skewing to the right on the hard agar surface was caused by right-handed torsion at the root tip generated by the constitutive right-handed helix of spr1 epidermal cell. The helical epidermal cell files were not apparent in wild-type and spr2 roots (Fig. 1B,D).

When grown in white light, hypocotyls of wild-type and spr1 seedlings were indistinguishable, whereas the epidermal cell files of spr2 hypocotyl skewed slightly to the right (Fig. 2A-C). The cotyledon petioles showed right-handed skewing in spr2, resulting in anticlockwise rotation of cotyledons when plants were viewed from above the plate (see Fig. 6D). It is known that dark-grown hypocotyls after 3 days growth elongate at the basal-mid regions, and that the elongation zone moves up the hypocotyl with time; at day 5, the apical third of the hypocotyl is rapidly elongating (Gendreau et al., 1997). We therefore examined etiolated hypocotyls at day 3 and day 5. The epidermal cell files of three-day-old dark-grown spr1 and spr2 hypocotyls skewed to the right, especially at the basalmid regions (Fig. 2F,G). When the seedlings were grown for a further 2 days in the dark, epidermal skewing strongly intensified at the apical one-third of the spr1 hypocotyl (Fig. 2J,N), whereas no such enhancement was observed in spr2 (Fig. 2K,O).

Vegetative and reproductive development of spr1 and spr2 plants were mostly normal, and these plants were fully fertile. In spr2, rosette leaves and petals showed anticlockwise twisting (Fig. 3C,G), and cauline leaves also tended to curl in an anticlockwise direction (not shown). While inflorescence stems of light-grown spr mutants looked the same as wild type, those of dark-grown plants had right-handed helices in epidermal cell files; the epidermal defect was especially strong in spr1 plants (not shown). In general, the spr1 twisting phenotype is stronger in dark-grown central axis, including stem, hypocotyl and root, whereas spr2 phenotype is most apparent in lateral appendages, such as petioles, cauline leaves and petals.

The spr1-1spr2-1 double mutant showed strong synergistic defects in all aspects of the spr1 and spr2 phenotypes. The spr1spr2 seedling root was shorter and wider, grew more strongly skewed to the right when grown on the agar and had epidermal cell files that skewed more strongly to the right than expected from simple addition of the spr1 and spr2 phenotypes (Fig. 1A,E). The hypocotyl epidermis of light-grown spr1spr2 seedlings also showed right-handed helix more strongly skewed than the light-grown spr2 hypocotyl epidermis, and included many deformed cells (Fig. 2D). Hypocotyls of etiolated double mutant seedlings were much shorter than those of either parental mutants (Fig. 2H,L), and consisted of round epidermal cells with highly reduced anisotropic growth in the upper hypocotyl region (Fig. 2P). Characteristic protrusions or bumps were often observed at the central region of the expanded epidermal cells in both light- and dark-grown hypocotyls (arrowheads in Fig. 2D,P). Rosette leaves of spr1spr2 plants were smaller than the leaves of either parental plants (Fig. 3D).

To address cellular defects in spr mutants, longitudinal and transverse sections of plastic-embedded seedlings were made at the upper region of etiolated hypocotyls (Fig. 4). The longitudinal sections that cut hypocotyls just at their mid planes (Fig. 4A-D) were used to measure the distance of each cell in the inner cortex cell file from the shoot apex (Fig. 5). Inner cortex cells of wild-type hypocotyls gradually elongated as the distance from the shoot apex increased, and became much longer, starting from the fifth cortex cell approximately 450 μm distal from the apex. Inner cortex cells of spr1, spr2 hypocotyls elongated at wild-type rates up to the fifth cortex cells, but thereafter did not keep up with the progressively faster elongation; the elongation defect was more pronounced in spr1 than in spr2. Inner cortex cells of spr1spr2 hypocotyls expanded even more slowly than spr1 or spr2 rates in the longitudinal direction.

Inner and outer cortex cells of etiolated wild-type hypocotyls were polyhedral in transverse section (Fig. 4A,F). The shape of individual cells was more or less uniform within each cell layer of ground tissue (endodermis and two cortex layers), and this stereotypical cell arrangement continued from the shoot apical region to the basal region. The transverse sections of spr1 hypocotyls up to approximately 400 μm distal from the apex looked the same as wild type, although a few cells in each ground tissue layer were occasionally somewhat enlarged (not shown). Cell expansion was clearly abnormal in spr1 hypocotyls at the region starting approximately 500 μm distal from the apex (Fig. 4B), which corresponds to the fifth cell from the apex (Fig. 5). A series of transverse sections from 600 μm to 680 μm (Fig. 4H-I), combined with longitudinal sectioning analysis (Fig. 4B), showed that the cells in ground tissue were only weakly elongated. The same sections also showed that the spr1 cells in epidermis and stele kept substantially normal appearance, compared with the ground tissue cells. In spr2 hypocotyls, there was no clear difference in cell shape and arrangement up to approximately 400 μm, but the shape of cortex cells became somewhat irregular starting approximately 600 μm from the apex (Fig. 4J). In the spr1spr2 double mutant, the defect in anisotropic expansion was stronger in ground tissue than it was in the parental mutants while epidermal and stele tissues remained relatively normal in appearance (Fig. 4D; transverse sections not shown).

Interestingly, we found that the addition of a MT-depolymerizing drug propyzamide at 3 μM in the agar medium induced a left-handed helical growth to the epidermal cell files of light-grown wild-type seedling roots and petioles, resulting in root growth skewed to the left and a clockwise twisting of cotyledons (Fig. 6A,E). Etiolated wild-type hypocotyls were more sensitive to propyzamide; 3 μM propyzamide inhibited cell elongation and produced distorted epidermal cell expansion (not shown). In light-grown spr1 and spr2 seedlings, propyzamide at 3 μM reversed the direction of helical epidermal cell files from right-handed to left-handed, resulting in root growth skewed to the left on agar plates and a clockwise twisting of cotyledons, as in similarly treated wild-type seedlings (Fig. 6A,F,G). Thus, propyzamide-induced left-handed helical growth is dominant over right-handed helical growth of spr1 and spr2.

We quantified the concentration-dependent effects of propyzamide on the angle of root bending, root length and the direction of cotyledon twisting in light-grown seedlings (Fig. 7A). Propyzamide up to 0.3 μM did not affect wild-type seedlings on the three characteristics examined; 1 μM propyzamide exaggerated slightly the tropic movement of roots to the left; and 3 μM propyzamide induced a clockwise twisting of cotyledons and a strong left-handed helical growth of roots with a concomitant decrease in root length. spr1 seedlings responded to as low a concentration of propyzamide as 0.1 μM. In a concentration-dependent manner, the rightward bending of spr1 roots was decreased and at 3 μM a strong left-handed helical growth was observed. In etiolated hypocotyls of spr1, 1 μM propyzamide completely suppressed helical growth of epidermal cell files and the highly isotropic expansion of endodermis and cortex cells (compare panels B and E; and panels G-I and K, in Fig. 4). For spr2, roots gradually reversed their bending direction from right to left in response to increasing concentrations of propyamide, but even at 3 μM, the leftward bending was not as pronounced as in wild-type or spr1 roots, indicating that spr2 roots had reduced responsiveness to propyzamide in this assay. Cotyledon twisting in light-grown spr2 seedlings and the helical epidermis in etiolated spr2 were suppressed by 1 μM propyzamide. Oryzalin, another class of MT-depolymerizing drug, also decreased the degree of right-handed helical growth of spr1 and spr2 seedling roots when tested up to 0.1 μM (not shown). However, oryzalin at concentrations above 0.1 μM significantly inhibited seedling growth, and complete reversal of helical handedness in spr roots and petioles was not attained with this drug.

The MT-stabilizing drug taxol (paclitaxel) was also tested (Fig. 7B). In wild-type seedlings, 0.3 μM taxol had no effect on root helical growth but induced a clockwise twisting of cotyledons (Fig. 6H), and 1 μM taxol induced a strong left-handed bending and reduced root growth. Reduced bending of spr1 roots was already apparent with 0.3 μM taxol. spr2 roots responded similarly to increasing concentrations of taxol, although their response to taxol appeared to be more sensitive than the wild-type response. Petiole elongation was inhibited more severely by 1 μM taxol in spr2 seedlings (Fig. 6J) than in wild-type (Fig. 6H) or spr1 (Fig. 6I) seedlings. In etiolated seedlings, the strong right-handed helix of spr1 hypocotyls was suppressed at 0.3 μM taxol, and the weak right-handed helix of spr2 hypocotyls reversed to a weak left-handed helix at 0.2 μM taxol.

The effects of propyzamide on anisotropic cell expansion were quantified in different root cell layers of light-grown seedlings (Fig. 7C). In wild type, anisotropic cell growth was not affected by 1 μM propyzamide, whereas radial cell expansion in ground tissue, but not in the epidermis, was promoted by 3 μM propyzamide. Anisotropic cell growth, normally impaired in ground tissue of spr1, completely recovered to wild-type levels at 1 μM. A higher concentration (3 μM), which induced left-handed helical growth, caused radial cell expansion of ground tissue, but not of the epidermis, in spr1 roots. In spr2 roots, defective anisotropic cell expansion of the ground tissue was marginal in the absence of the drug or at 1 μM. Propyzamide at 3 μM induced only weak radial cell expansion of ground tissue.

The cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile and the actin filament disrupting drug cytochalasin D were tested at low concentrations up to levels that significantly inhibited seedling growth, but neither drug induced helical growth of wild-type seedlings nor did they affect the skewing phenotype of spr1 and spr2 seedlings.

Although spr1 leaves usually do not twist when grown at 22°C, we noticed that they often twisted in an anticlockwise direction when grown in a greenhouse during winter. We, therefore, examined the effect of temperature on the skewing phenotype. Epidermal cell files in the upper hypocotyl of etiolated spr1 seedlings, although skewed at 22°C, were nearly straight at 29°C (Fig. 8B), whereas at 15°C the hypocotyl was short with an exaggerated helical growth (Fig. 8E). Prolonged incubation in the dark at 4°C produced extensive outgrowth of spr1 hypocotyl epidermal cells, forming abundant hair-like protrusions in the upper hypocotyl region (Fig. 8G). The helical growth of spr2 hypocotyls, however, was not affected by shifting the growth temperature between 29°C and 15°C (Fig. 8C,F), although at 4°C, etiolated spr2 hypocotyls occasionally produced similar hair-like protrusions (not shown). spr1 roots grew slightly to the right at 29°C, bent even more to the right when the temperature was shifted down to 22°C, and then returned to the weak rightward growth when the temperature was shifted back to 29°C (Fig. 8H). Similar temperature-dependency was not observed in spr2 roots.

Reduced growth anisotropy and the effects of MT-interacting drugs in spr mutants prompted us to examine cortical MT arrays. First, whole-mount MTs of seedling roots grown at 22°C were stained using immunocytochemistry. The method used stained MTs of root epidermal cells from the root apex to the distal end of elongation zone; differentiated root cells rarely stained. Cortical MTs underneath the outer tangential wall were examined in root epidermal cells at the end of elongation zone where, in spr1 and drug-treated wild-type roots, epidermal cell files were already skewed (Fig. 9A,B,D,E). Wild-type cells had cortical MT arrays that were aligned almost transverse to the long axis of the cell (Fig. 9A), while the arrays in spr1 epidermis were oblique (Fig. 9B). The MT arrays underneath the inner wall of the same cell as in Fig. 9B showed an oblique alignment of opposite slant (Fig. 9C), indicating that the cortical MTs in spr1 epidermis form left-handed helical arrays. In contrast, wild-type roots treated with 1 μM taxol had right-handed helical arrays (Fig. 9D). Wild-type root cells treated with 3 μM propyzamide had mostly transversely oriented cortical MTs, but occasionally included arrays with right-handed helices (Fig. 9E). In spr2 roots, the cortical MT arrays appeared to be transverse as in wild-type roots (not shown).

Next, the seedling roots were grown at 14°C, and both the length and angle relative to the long axis of primary root of epidermal cells were plotted with respect to the distance from the quiescent center (Fig. 10A). At 14°C, spr1 showed an exaggerated phenotype and we could observe clear differences in MT organization between wild type and spr1, even in the early phase of cell elongation. Wild-type epidermal cell lengths increased up to approximately 1.2 mm from the quiescent center. spr1 epidermal cell lengths continued to increase similarly, at least by 0.6 mm, although considerable skewing of spr1 epidermis prevented accurate measurements beyond this distance (Fig. 10B). Wild-type epidermal cells were aligned essentially parallel to the long axis of primary root, whereas spr1 cells began to skew to the right starting 550 μm distal to the quiescent center. Thereafter, the skewing angle remained at approximately 20° to the right (Fig. 10C). The orientation of cortical MTs underneath the outer tangential wall was examined in the root epidermal cells positioned between 200 μm and 600 μm from the quiescent center. This region defines the early elongation zone and marginally precedes the helical growth in spr1. Cortical MT arrays in wild type were positioned transverse to the long axis of the cell (Fig. 10D). In spr1, deviation of the helical pitch was wider and shifted 13° on average from the transverse axis of cells to form left-handed helices (Fig. 10E).

Finally, inner cortex cells were examined at the upper region of etiolated hypocotyls. Wild-type cortex cells were expanding anisotropically and had cortical MTs aligned at right angles to the long axis of the cell (Fig. 9F). In contrast, the orientation of cortical MT arrays was irregular in spr1 cortex cells with highly reduced anisotropic growth (Fig. 9G,H). The arrays were often parallel in localized regions of a cell, but most cells contained a mixed population of longitudinal, transverse and oblique arrays.

Since helical growth phenotypes in spr mutants and drug-treated seedlings are remarkably similar (though of opposite handedness), we discuss the phenomena collectively. Root and shoot meristems appear to be normal in skewing seedlings. Helical epidermal cell files and the cell elongation defect in ground tissue develop concomitantly at the mid/basal elongation zone where the elongation rate of root (Beemster and Baskin, 1998) and etiolated hypocotyl (Gendreau et al., 1997) becomes progressively accelerated compared with a more apical region. The growth-rate dependency of the helical phenotype is most apparent in the hypocotyls of five-day-old dark-grown spr1 seedlings, which showed most severe mutant phenotype in the upper quarter of the hypocotyl – the region of most rapid growth (Gendreau et al., 1997). Moreover, helical epidermal cell files in the elongation zone accompany the reduced length of the skewing organs.

Beneath the helical epidermal cell files of the spr mutants, cells in ground tissue are more or less isodiametric. This was most obvious in dark-grown spr1 hypocotyls. In the epidermis, cell files were skewed but radial expansion was scarcely observed. Thus, anisotropic growth of ground tissue is more severely affected than that of epidermal cells in skewing tissues. Taken together with the fact that the skewing angle of cell files progressively decreases from the epidermis through the inner cell layers, reduced anisotropic growth in ground tissue may be the primary cause of helical cell files in the epidermis (Fig. 11A). According to this model, radially expanding ground tissue cell files are shorter along the longitudinal axis than normally elongating epidermal cell files. Since extra cell production is apparently not induced in ground tissue, the outer cell layers must be skewed to reduce the length along the longitudinal axis to a level comparable with that of the inner cells.

Considerable evidence indicates that in most plant cells elongating with polar diffuse growth, the cortical MTs are involved in the orientation of cellulose microfibrils during the synthesis of new cell walls (Giddings and Staehelin, 1991), and thus are essential for establishing the polarity of cell elongation. Several compounds are known to affect polymerization of plant MTs. Propyzamide (also known as pronamide) and oryzalin, two structurally different classes of herbicides, are thought to bind tubulin and promote depolymerization of MTs (Morejohn, 1991), whereas taxol binds the β;-tubulin subunit and stabilizes MTs (Bokros et al., 1993). When applied to intact plants at concentrations above certain thresholds, these drugs severely inhibit shoot and root growth, and cause extensive radial swelling in some types of treated cells; however, helical epidermal cell files have not been documented (Morejohn, 1991; Baskin et al., 1994; Hasenstein et al., 1999; and references cited therein). At the drug concentrations used in these and previous studies, cortical MTs are often disorganized, fragmented or depleted by treatment with depolymerizing drugs, or are bundled by taxol treatment. We also observed similar defects in MT organization and radial swelling of root epidermal cells at the propyzamide concentrations much higher than those reported in this study (I. F. and T. H., unpublished). Instead, propyzamide at 3 μM and taxol at 1 μM induced rather weak right-handed helices on the MT arrays in root epidermal cells but the arrays otherwise appeared to be normal. At these relatively low and possibly crucial concentrations, ground tissue might be more sensitive to the drugs than epidermis with regard to the integrity of MT organization and anisotropic growth. In etiolated spr1 hypocotyls, cortical MT arrays are more irregularly oriented in cortex cells than in epidermal cells, reflecting differential radial swelling between the two cell types. Distinct arrangement or responses of cortical MTs in different cell types have been sporadically noted, such as in cortex and epidermis of oryzalin-treated maize roots (Hasenstein et al., 1999), and in several cell types of cold-treated and untreated maize roots (Baluska et al., 1992, 1993).

Propyzamide and taxol caused helical growth of identical handedness in arabidopsis seedlings, despite their opposite effects on MT polymerization. At the concentrations effective for inducing helical growth, the gross MT organization in root epidermal cells appeared to be normal, indicating that these drugs might be influencing a MT function other than simple polymerization/depolymerization status. Taxol treatment of plant cells can also induce radial swelling without changing overall alignment of MT arrays (Weerderburg and Seagull, 1988; Baskin et al., 1994). Taxol and MT-depolymerizing drugs vinblastine and vincristine at their lowest effective concentrations appear to block mitosis in cultured mammalian cells by kinetically stabilizing spindle MTs and not by changing the mass of polymerized MTs (Jordan et al., 1993; Dhamdharan et al., 1995). Similarly, at concentrations that reportedly cause little change in the polymer level of MTs, taxol, vinblastine and another MT-depolymerizing drug, nocodazole, all dramatically decrease the rate of locomotion of fibroblasts, probably by suppressing MT dynamics (Liao et al., 1995). Therefore, it is possible that MT dynamics is also crucial in the function of the cortical array in plant cells and in proper anisotropic growth.

Propyzamide and taxol affected the helical growth in spr seedlings. The most dramatic effect is exemplified in highly efficient suppression of almost isotropic cell expansion in the spr1 hypocotyls and roots by low doses of these drugs. Moreover, spr1 roots clearly responded to lower concentrations of MT-interacting drugs that did not visibly affect wild-type roots, by reducing their right-handed root skewing. Another feature of spr1 phenotype is its temperature sensitivity: growth at 29°C tends to suppress the mutant phenotype, whereas growth at low temperatures exaggerates the phenotype. Cold destabilizes MTs and will lead to their depolymerization unless they are stabilized by associated proteins (Bokros et al., 1996). A large fraction of MT-related yeast mutants shows cold sensitivity (e.g. Richards et al., 2000). Collectively, these results suggest that spr1 MTs are destabilized to some extent, or are defective in proper functioning in the tissues in which anisotropy is impaired.

spr2 seedlings show reduced response to the radial cell expansion effect of propyzamide, and increased response to the effect of taxol as compared to wild type. Although the spr2 helical growth phenotype is not significantly altered by growth temperature, prolonged incubation of spr2 seedlings at 4°C in the dark induces hair-like protrusions at the upper hypocotyl region (as in spr1 epidermis under the same conditions), indicating that anisotropic growth of spr2 epidermal cells is rendered somewhat cold-sensitive under certain conditions. Interestingly, transgenic Arabidopsis plants that overexpress a fusion protein between green-fluorescent protein and α-tubulin show anticlockwise arrangement of petals, cotyledons, and young rosette leaves in otherwise normal morphology and development, thus mostly phenocopying spr2 (Ueda et al., 1999). Taken together, these data suggest that spr2 disrupts an MT-dependent process as well. It should be noted, however, that spr1 and spr2 phenotypes are distinct in affected tissues, drug responses and cold sensitivity. The synergistic enhancement of anisotropic growth defects in the spr1spr2 double mutant is in accordance with a model that SPR1 and SPR2 act on a similar process but in separate pathways in the control of MT-dependent anisotropic cell elongation.

Drug-induced helical growth and the constitutive helical growth of spr mutants differ in their chirality; drug-induced helices are always left-handed, whereas helices in spr1 and spr2 mutants are invariably right-handed. Any model for helical growth needs to account for the fixed handedness. According to Fig. 11A, epidermal cells are predicted to exert a strong influence on the handedness. Although cellular defects in spr1 are most obvious in ground tissue, epidermal cells under certain conditions show moderate radial swelling or local outgrowth of bulges, suggesting that the mutant epidermis has a compromised physical property and becomes partially defective in anisotropic growth under conditions that enhance the mutant phenotype. Likewise, low concentrations of propyzamide or taxol induce similar local bulges on the hypocotyl epidermis of light-grown wild-type seedlings (T. H., unpublished). Here, we refer to the compromised state of epidermal cells that potentially give rise to a right-handed helical cell file as ‘R’, the opposite state as ‘L’, and the state that results in straight elongation parallel to the organ’s long axis as ‘N’ (Fig. 11B). The R and L states may correspond to the cellular states before rapid elongation in which anisotropic cell expansion is partially impaired and may potentially give rise to a substantial loss of anisotropic expansion, as in the spr1 ground tissue. The helical handedness induced by propyzamide or taxol suggests that intact dynamics of MTs pushes the state from L to N. Partial disruption of MT function by these drugs (possibly through suppression of dynamic instability) overrides the spr mutations, indicating that SPR1 and SPR2 function in the processes that prevent cells to proceed further from N to R in a MT-dependent pathway. SPR1 and SPR2 may represent a R-to-L reverse pathway, may act to suppress the L-to-R pathway or may stabilize the N state to prevent overshooting to R. Considering that the driving force for helical growth should come from radial swelling of inner cells and that elongating epidermis may function passively just to determine the direction of skewing, the difference between N and L, and between N and R are not necessarily large but should be consistent in a majority of elongating epidermal cells.

The orientation of cortical MT arrays in epidermal cells of L, N and R states showed right-handed helix (Z-helix), transverse and left-handed helix (S-helix), respectively. As studied in detail in the R state of spr1 root epidermis, the helical pitch was not steep but of consistent handedness in a population of elongating cells. Assuming that the load-bearing innermost cellulose microfibrils are deposited along the helical MT arrays, the resulting microfibril arrangement on the outer tangential wall would then generate a polar cell growth in the direction perpendicular to the helical pitch of the arrays. Thus, a right-handed MT helical array in L is expected to give rise to left-handed helical growth, and a left-handed array in R would produce right-handed helical growth (Fig. 11B). If epidermal cells and neighboring cortex cells possess the same handedness of MT helix, a criss-cross cellulose microfibril pattern would be formed on the wall between them. The criss-cross pattern would seem to reduce the skewing force generated on that wall. In total, the helical pattern of the newly synthesized microfibrils on the outer wall of epidermis presumably has the strongest influence on the handedness of helical growth.

The model in Fig. 11B may be interpreted as a concept that the configurations of the cortical MT array can change from an S-helix to a Z-helix via transverse orientation, and that SPR1 and SPR2 act to partially suppress this transition. Oblique MTs are commonly observed in plant cells. In cases in which the handedness of helical arrays was studied, the orientation changes progressively from a flat S-helix to a steep Z-helix and then to a flat S-helix in differentiating tracheids (Abe et al., 1995), and the array in a given region of maize and arabidopsis root cells takes the same handedness, either an S- or Z-helix (Liang et al., 1996; Baskin et al., 1999). When MT orientation is synchronized in nonelongating epidermal cells of azuki bean epicotyls, the MT array can be induced to cycle between longitudinal and transverse via oblique orientation by treatments with plant hormones (Mayumi and Shibaoka, 1996; Takesue and Shibaoka, 1999). Reorientation can be suppressed when MTs are stabilized by taxol in differentiating tracheary elements (Falconer and Seagull, 1985). Thus, differentiation and a variety of internal and external stimuli appear to change the helical pitch and orientation of cortical MT arrays, and the SPR genes might be involved in some of these processes.

The model proposes that normal epidermal cells elongate parallel to the longitudinal axis of organs because SPR1 and SPR2 suppress the excess right-handed helical activity. Notably, wild-type arabidopsis roots of the ecotypes WS and Ler normally tend to bend somewhat to the left on a vertical agar surface, while seedling roots of Col ecotype grow almost straight to the gravity vector (Fig. 1A; Rutherford and Masson, 1996). A slight defect in MT function or somewhat stronger SPR activity in WS and Ler, compared with Col, might explain the observation. We also predict that tubulin mutations that partially compromise MT functions will cause left-handed helical growth and exaggerated left-slanting root growth on a vertical agar surface. Arabidopsis mutants with such a phenotype have been reported (Rutherford and Masson, 1996).

Circumnutation is an oscillating growth pattern of plant axial organs that has attracted the attention of plant biologists since the era of Charles Darwin. A mechanistic model for circumnutation must explain the salient features of seedlings’ circumnutational behavior (Brown, 1993). (1) Gravity is not required but can influence circumnutation. (2) Mechanical perturbations often have an immediate effect on the oscillations. (3) Circumnutations are absolutely growth dependent. Because circumnutation is clearly advantageous to the plant only in a small minority of cases (such as in twining plants) but is universally found in the plant kingdom, it is believed that some fundamental growth process underlies the behavior (Brown, 1993). Our MT-dependent helical growth model (Fig. 11) may explain circumnutation. The model is independent of gravity, but gravitropic responses of plant organs involve differential growth patterns on the convex and concave sides of the gravi-stimulated tissues (Hart, 1990), and thus are potentially capable of influencing the anisotropic expansion process. Mechanical forces imposed on a plant cell have been frequently implicated in having a profound influence on the alignment of cortical MTs (Cyr, 1994). Finally, the model is intrinsically growth dependent. The period of circumnutation considerably decreases at higher temperature (Johnsson, 1979). The proposed stabilization of MTs at higher temperature possibly stabilizes the N state, thereby decreasing the slanting angle of the elongating epidermis. Because a variety of plant hormones influences the organization of MTs (Shibaoka, 1994), physiological changes in cellular hormonal status may cause inconsistent oscillations of plant organs during extended periods of growth. In fact, the ethylene pathway strongly affects the helical phenotype in spr1 mutant and the drug-treated seedlings (T. H., unpublished). A variation in the periodicity and handedness of circumnutation in a given plant has been well documented (Johnsson, 1979). It would be interesting to see whether circumnutation behavior is altered in spr mutants and in drug-treated seedlings.

We thank J. Schiefelbein and K. Nakamura for spr1-2 and spr1-3 alleles, respectively, and T. Schäffner for tortifolia1. We would like to acknowledge Arabidopsis Biological Resource Center for providing convoluta seeds; K. Ueda, H. Tachimoto, K. Tuchihara, J. Uchida and K. Hayashi for technical assistance; and K. Sugimoto for assistance in immunostaining techniques. Helpful comments on the manuscript were kindly contributed by G. Wasteneys. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (‘Molecular Mechanisms Controlling Multicellular Organization of Plant’) for the Ministry of Education, Science, Sports and Culture, to T. H.