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Growth Conditions Modulate Root-Wave Phenotypes in Arabidopsis

Growth Conditions Modulate Root-Wave Phenotypes in Arabidopsis Abstract The cause for the wave-like growth of Arabidopsis thaliana roots on semi-solid medium remains unclear. Researchers have hypothesized a gravity-induced touch-response, circumnutation, or combinations thereof act as the major stimuli. Our data demonstrate that the gaseous environment within the Petri dish can override gravitational effects. Furthermore, we show that medium ion concentrations and gelling polymers modify the wave response. Although the mechanisms driving our wide-ranging wild-type phenotypes are currently unknown, these results are of immediate significance for interpreting genetic and physiological modifications of environmentally and genetically induced characteristics. (Received April 14, 2000; Accepted July 28, 2000). Due to their experimental amenability (Smyth 1990, Koncz et al. 1992, Meyerowitz and Somerville 1994), arabidopsis seedlings have been used for studies of root morphogenesis and gravity responses. In many of these studies, seedlings are grown on the surface of sterile semi-solid media (such as agar or gellan gum) in Petri dishes allowing precise and non-invasive observations. The roots are in contact with the medium on one side (allowing nutrient uptake) and exposed to air on the other. Using this experimental set-up, Okada and Shimura (1990) first described the root-wave phenomenon, wherein roots generated a repeating right-to-left undulation across the vertical growth axis as they elongated, producing a regular sinusoidal pattern. They hypothesized the stimulus was a gravity-induced touch-response to the medium surface and the root tip changed direction in an obstacle-avoidance manoeuvre. Subsequently, other authors have proposed a combination of gravitropism and circumnutation (natural oscillating growth) induced the response (Simmons et al. 1995, Mullen et al. 1998). The stimulus remains uncertain. Experiments to induce root waving were implemented in our laboratory. Our normal growth conditions did not induce root waving [using Phytagel as the gelling agent, full-strength Murashige and Skoog (MS) basal nutrient salts, and Nescofilm (NF) as the plate wrapping material]. In this article we identify several environmental factors that profoundly influence root waving. If not carefully controlled, these factors may confound interpretation of mutant phenotypes, and therefore they require more careful consideration during experiments. Arabidopsis thaliana (L.) Heynh. (ecotype Landsberg erecta) seeds were sterilized by washing in 70% ethanol for 2 min, rinsed, then immersed in 1.25% hypochlorite plus 0.02% Triton X-100 and agitated for 5 min; and then rinsed three times with sterile water. Seeds were sown 16 per Petri dish (ϕ = 9 cm, polystyrene) spaced 8 mm apart in two rows 2.5 cm apart onto the medium surface. The gelling agents were 1.5% (w/v) Bacto-agar (Difco Laboratories, Detroit, MI, U.S.A.) or 1.0% (w/v) Phytagel (Sigma Chemical Co., St. Louis, MO, U.S.A.). Basal salts were MS [Sigma M-0564 macronutrients and M-0529 micronutrients (1× = MS or 0.5× = MS/2)], 3.0% sucrose (1.5% at MS/2, 0% for zero salts), vitamins (Sigma M-6896) at the recommended level (MS), halved (MS/2), or zero with zero nutrients. OS salts were as defined by Okada and Shimura (1990). MES (2-[N-morpholino]ethanesulfonic acid, 0.05%, w/v) was added to all media formulations as a buffer. All media pH were set at 5.7 before adding the gelling agent and autoclaving. The plates were sealed with NF (Azwell Inc., Osaka, Japan) or wrapped with porous Micropore surgical tape (ST; 3M Health Care, St. Paul, MN, U.S.A.) and immediately placed in a Conviron (Controlled Environments, Pembina, ND, U.S.A.) growth chamber oriented vertically (0°), 20°, or 45° from vertical, as required. Some plates were shifted between the orientations to determine gravity effects. Each condition contained three replicates in each round of experiments and was repeated independently. Additional growth conditions included continuous light (100 µmol m–2 s–1, cool white fluorescent and incandescent bulbs) and 80% relative humidity at 21°C. The position of each root tip was marked on the plate’s base daily to quantify growth rates (the first mark was 60 h following sowing). Images of the Petri dishes were generated by scanning the individual plates, through the bottom of the plate and the medium, on a flatbed scanner into Adobe PhotoShop at 600 pixels in–1. Photomontages of roots were assembled from captured video images using NIH’s Scion Frame Grabber. Measurements of root lengths over time were accomplished using the NIH Scion Image software program (Windows version Beta 3b), available at the URL http://www.scioncorp.com. Wave quantification (illustrated in Fig. 1d) included wavelength (λ), amplitude, wave tangent angle (WTA, as defined by Rutherford et al. 1998), and wave frequency (waves mm–1). Non-waving roots were counted in the waving percentages in Table 1. Means between categories were compared by the independent Student’s t-Test at the 95% confidence level using Microcal’s Origin 6.0 software package. We use the term loop to describe the two-dimensional arc the roots formed under some conditions, comprising part of or a complete circle. This differs from previous literature (circles: Okada and Shimura 1990; coils: Simmons et al. 1995, Mullen et al. 1998). A coil is generally three-dimensional, forming a structure such as a coil spring. A circle implies the loop is closed, which was not always the case. Wide-ranging phenotypes resulted between NF- and ST-wrapped plates under different nutrient treatments, altered angle (α) between the gravity vector and the root elongation axis, and with different gelling agents. Root elongation rates increased when the plates were wrapped with ST versus NF under all conditions (Table 1). Roots in the few plates that developed a small crack in the NF reverted to the respective ST phenotype (not shown). These small cracks would not allow similar water loss within the plates compared to ST wrapping, but would allow gas exchange. Plants in NF-wrapped plates showed signs of ethylene exposure. The roots in NF-wrapped plates were thicker and hairier (as also observed by Baskin and Williamson 1992) in side-by-side comparisons to ST-wrapped plates (Fig. 1, compare c versus d). The cotyledons exhibited epinasty (compare Fig. 2a versus c; Van Der Straeten et al. 1999). The epinasty appears less pronounced in plates at 45° as the hypocotyl turned upward due to gravity. Understandably, tiny cotyledons resulted when using zero nutrient ions, but cotyledons were larger in ST-wrapped plates (Fig. 2–4) than in NF-wrapped plates. With nutrient ions in the media, the cotyledons grew more, but they were larger still with ST wrapping than with NF. Ethylene was shown to inhibit leaf expansion in A. thaliana (Kieber et al. 1993, and references therein for other species). Plate aeration could also affect other gas concentrations within the petri dish. Limiting the medium nutrient ion concentration for seedlings grown on agar modulated root waving differentially, depending on plate aeration (Fig. 2). As the nutrient ion concentration was decreased in NF-sealed plates, the wavelengths progressively shortened, the wave amplitudes generally decreased, and the waving frequency increased (Fig. 2a, e, i and b, f, j). Roots exhibited the most extreme waving on zero nutrient ion media with NF (Fig. 1c and 2i–j), had the shortest wavelengths, the greatest wave tangent angles, and occurred with an increased frequency (Table 1). In contrast, roots in plates wrapped with ST looped on MS basal salts (Fig. 2c, d) and as nutrient salt concentrations were decreased, the looping was replaced by waving (Fig. 2g, k and h, l). Wrapping plates with ST increased the wave characteristics unless the seedlings were grown on zero ion concentrations, wherein the wave-response decreased (Fig. 1c versus d; Fig. 2i versus k, j versus l). Whether this is a general phenomenon associated with overall nutrient depletion or whether it is associated with specific nutrients is unknown, though several could be expected to play important roles in the response, with protein cofactors obvious candidates. The angle (α) between the gravity vector and the root elongation axis influenced waving but, again, this effect was dependent on the plate wrapping and media ion concentrations (Fig. 3). At the start, the plates were oriented at 20°, 60 h post-sowing the orientation was changed to 45° (first mark), after 48 h the plates were shifted to 0° (black triangles), and 48 h later the orientation was returned to 45° (white triangles). Under Okada and Shimura (1990) defined conditions (hereafter denoted OS), roots responded quickly and predictably to changes in plate orientations when using NF (Fig. 3a). The roots waved until the plates were shifted to vertical, then they grew nearly straight, and waved again after the shift back to 45°. Under other treatments, however, the roots lost the ability to adjust to a change in α. After the shift to the vertical orientation (Fig. 3e), roots on zero nutrient salt media in plates wrapped with NF continued to wave at nearly the same frequency. Roots on zero nutrient salts in plates wrapped with ST had a more random, reduced waving phenotype at the second turn to 45° (Fig. 3f). Plates with OS nutrient salts and MS/2 salts and wrapped with ST produced roots that generally did not wave at the second turn to 45° (Fig. 3b, d; lateral root formation obscures initial waving). Roots in plates oriented vertically were generally indistinguishable from those tilted at 20° (Table 1) except that they often lost contact with the medium and became dehydrated. Root waving was not completely suppressed at vertical plate orientations in our experiments, but it occurred irregularly. A wave response at vertical orientations was also noted by Simmons et al. (1995). Vertical growth reduces the gravity-influenced touch-response against the medium and any frictional forces against root growth, implying other factors are involved in the waving stimulus. The role of other factors is also implied by the phenotype differences observed between roots on Phytagel and agar medium formulations at a constant α. The gelling agent itself influenced root waving. On Phytagel plates wrapped with NF, root waving was virtually nonexistent (Fig. 4a–b), but random waving was generally observed when plates were wrapped with ST (Fig. 4c–d). Roots of seedlings grown in NF-wrapped plates were shorter and exhibited an increased root hair density, resulting in a thicker appearance (Fig. 4a–b). Growth rates were further inhibited when plates were shifted from 20° to 45° on plates sealed with NF (Fig. 4a–b), but increased when ST was used (Fig 4c–d). Increasing α to 45° shortened the wavelength and increased the frequency of waves only slightly on Phytagel (Table 1; Fig. 4). The seedlings on agar produced the most orderly and consistent root-wave phenotypes (compare Fig. 1 versus 4). Phytagel-solidified media produced other differences as well. Roots on Phytagel exhibited increased hair densities compared to agar plates (Fig. 1, compare a versus b). The increased hairiness was also evident on ST-wrapped Phytagel plates (Fig. 4c–d) suggesting that hairiness is attributable to some property of the gelling agent per se. Among the literature involving the root-wave phenomenon, only Rutherford and Masson (1996) mentioned the agar brand used, only Mullen et al. (1998) and Luschnig et al. (1998) specified the Petri dish wrapping material, and only Simmons et al. (1995) and Mullen et al. (1998) cited the initial media pH. Mirza (1987) did not state what basal nutrient salts were used. Plate wrapping materials especially, and other growth conditions occasionally, are taken for granted, and it is possible phenotypes separated for mutational analyses are profoundly enhanced or suppressed by growth conditions that may confound interpretation of the function of the gene affected by a particular genetic lesion. If mutants and wild-type plants are grown in the same plate, allelopathic effects can occur (not shown). Responses on tightly sealed plates could differ from responses on loosely wrapped plates. As the basis for phenotype assessment is against wild-type plants, our wide-ranging phenotypes indicate that growth conditions require careful attention. Almost certainly, different genes are switched on or off depending on variations in growth media and plate ventilation. These environmental effects induced unexpected changes in the root-wave phenotypes, but could also influence the expression of other seedling phenotypes that have been reported in arabidopsis. Agar plates wrapped with ST produced the slanting root growth mentioned previously (Simmons et al. 1995, Rutherford and Masson 1996, Mullen et al. 1998). This response was almost nonexistent on Phytagel plates and much reduced on plates wrapped with NF. We demonstrate that root waving is sensitive to characteristics of the microenvironment related to the extent of the gas exchange between the inside and the outside of the Petri dish and to the chemical and structural properties of the growth medium. Our results suggest the mechanisms controlling root waving are complex and imply that gene expression stimulated by mechanical parameters is influenced by several environmental factors. The nature of these characteristics and the underlying reasons are unknown, but our data implicate mechanisms partly independent of gravitropism. Acknowledgments Graham Farquhar is thanked for critical comments regarding the manuscript. 3 Corresponding author: E-mail, [email protected]; Fax, +61-2-6249-4919. (Fax, +61-2-6251-4919 from 1 January 2001) View largeDownload slide Fig. 1 Micrographs of typical root phenotypes on 1.0% Phytagel or 1.5% Bacto-agar, in Petri dishes sealed with NF or ST, grown on MS or zero basal nutrient salts, and tilted 20° or 45° from vertical. An illustration indicating wave quantification parameters is shown (d). View largeDownload slide Fig. 1 Micrographs of typical root phenotypes on 1.0% Phytagel or 1.5% Bacto-agar, in Petri dishes sealed with NF or ST, grown on MS or zero basal nutrient salts, and tilted 20° or 45° from vertical. An illustration indicating wave quantification parameters is shown (d). View largeDownload slide Fig. 2 Phenotype changes of A. thaliana, Landsberg erecta, seedlings on 1.5% Bacto-agar at varying nutrient conditions (marked in the column margin): MS basal nutrient salts (a–d), MS/2 basal nutrient salts (e–h), or zero nutrient salts (i–l). The wrapping method and plate orientation are noted in the row margin: NF, 20° (a, e and i); NF, 45°, (b, f and j); ST, 20° (c, g and k); and ST, 45° (d, h and l). All plates were scanned the seventh day after sowing except the zero salt condition (10 d). The horizontal scores along the root axis are the growth measurement marks at 24 h intervals. View largeDownload slide Fig. 2 Phenotype changes of A. thaliana, Landsberg erecta, seedlings on 1.5% Bacto-agar at varying nutrient conditions (marked in the column margin): MS basal nutrient salts (a–d), MS/2 basal nutrient salts (e–h), or zero nutrient salts (i–l). The wrapping method and plate orientation are noted in the row margin: NF, 20° (a, e and i); NF, 45°, (b, f and j); ST, 20° (c, g and k); and ST, 45° (d, h and l). All plates were scanned the seventh day after sowing except the zero salt condition (10 d). The horizontal scores along the root axis are the growth measurement marks at 24 h intervals. View largeDownload slide Fig. 3 Effects on the root-wave phenomenon of A. thaliana, Landsberg erecta, seedlings by changing the plate angle from 45° to 0° (black arrow) and back to 45° (white arrow) on different basal salt media concentrations after 10 d. The conditions are shown in the row margin: OS nutrient salts (a, b), MS/2 basal nutrient salts (c, d), and zero salts (e, f), all without sucrose. Plates in column one are wrapped with NF and column two with ST. View largeDownload slide Fig. 3 Effects on the root-wave phenomenon of A. thaliana, Landsberg erecta, seedlings by changing the plate angle from 45° to 0° (black arrow) and back to 45° (white arrow) on different basal salt media concentrations after 10 d. The conditions are shown in the row margin: OS nutrient salts (a, b), MS/2 basal nutrient salts (c, d), and zero salts (e, f), all without sucrose. Plates in column one are wrapped with NF and column two with ST. View largeDownload slide Fig. 4 Differences in root morphology in A. thaliana, Landsberg erecta, on 1.0% Phytagel, MS basal nutrient salts, NF (a, b) or with ST (c, d) wrapping, and tilted 20° or 45° from vertical. Images were recorded the seventh day after sowing. View largeDownload slide Fig. 4 Differences in root morphology in A. thaliana, Landsberg erecta, on 1.0% Phytagel, MS basal nutrient salts, NF (a, b) or with ST (c, d) wrapping, and tilted 20° or 45° from vertical. Images were recorded the seventh day after sowing. Table 1  Comparisons of Arabidopsis thaliana, ecotype Landsberg erecta, root growth and wave characteristics on varying gelling agents, nutrient salt concentrations, plate orientations, and plate wrapping material Media formulation and plate orientation  Wavelength (mm)  Wave amplitude (mm)  Wave frequency (waves mm–1)  Wave tangent angle (°)  Growth rates (mm h–1)  Waving roots (%); + = 100  Looping roots (%)  Loop diameter (mm)  1.0% Phytagel  MS salts (two independent experiments)  NF  0°  3.9 ± 0.7a  0.19 ± 0.05j  0.071 ± 0.024a  18.5 ± 3.5a  0.138 ± 0.019a  22.8  ―      20°  3.4 ± 1.0ad  0.23 ± 0.07ab  0.083 ± 0.035a  21.2 ± 4.6b  0.124 ± 0.022bc  16.7  ―      45°  2.9 ± 0.6bdijm  0.23 ± 0.08b  0.079 ± 0.037a  23.5 ± 7.1d  0.103 ± 0.026f  24.7  ―    ST  20°  3.4 ± 1.0ac  0.29 ± 0.10c  0.138 ± 0.049b  26.0 ± 6.3c  0.272 ± 0.033d  +  ―      45°  3.3 ± 0.7cf  0.36 ± 0.12fh  0.160 ± 0.037ce  35.7 ± 9.4ij  0.294 ± 0.031g  +  2.3  2.05 ± 0.21a  1.5% Bacto-agar  MS salts (three independent experiments)  NF  0°  3.2 ± 0.9abc  0.26 ± 0.08a  0.133 ± 0.063b  20.5 ± 6.0ab  0.121 ± 0.017b  +  ―      20°  3.1 ± 0.9bef  0.29 ± 0.11c  0.101 ± 0.050ad  26.3 ± 9.5cd  0.137 ± 0.027ae  +  ―      45°  2.9 ± 0.8ehi  0.44 ± 0.14gh  0.237 ± 0.056f  42.6 ± 12.1m  0.139 ± 0.025ac  +  2.7  1.24 ± 0.23abcd  ST  20°  2.8 ± 0.8gi  0.41 ± 0.15e  0.199 ± 0.056g  40.7 ± 11.2f  0.209 ± 0.036h  +  30.9  1.53 ± 0.33b    45°  2.5 ± 0.9kn  0.42 ± 0.16eg  0.229 ± 0.062f  47.9 ± 15.3l  0.195 ± 0.033i  +  96.8  1.41 ± 0.27c  MS/2 salts (three independent experiments)  NF  20°  3.0 ± 0.8bgh  0.30 ± 0.09cd  0.144 ± 0.055bc  30.3 ± 7.6n  0.150 ± 0.022j  +  ―      45°  2.2 ± 0.4lno  0.36 ± 0.08k  0.319 ± 0.047h  39.0 ± 7.1fh  0.102 ± 0.011k  +  ―    ST  20°  2.5 ± 0.9jk  0.33 ± 0.13fg  0.171 ± 0.043de  37.6 ± 7.8ghi  0.314 ± 0.031l  +  ―      45°  2.5 ± 1.1kmo  0.45 ± 0.18l  0.246 ± 0.053f  51.5 ± 11.9kl  0.261 ± 0.036d  +  56.5  1.72 ± 0.68abd  Zero salts (five independent experiments)  NF  20°  1.1 ± 0.3q  0.28 ± 0.06ci  0.521 ± 0.079i  55.9 ± 11.4e  0.079 ± 0.020m  +  ―      45°  1.0 ± 0.3r  0.30 ± 0.07c  0.616 ± 0.065j  63.1 ± 11.9°  0.090 ± 0.017f  +  ―    ST  20°  2.1 ± 0.6l  0.28 ± 0.09c  0.218 ± 0.11df  37.4 ± 8.0gj  0.122 ± 0.033b  +  5.3  1.47 ± 0.07 abc    45°  1.5 ± 0.6p  0.30 ± 0.08cd  0.247 ± 0.11f  55.5 ± 11.4e  0.129 ± 0.026bce  +  4.7  1.00 ± 0.03d  OS salts (two independent experiments)  NF  45°  1.7 ± 0.3p  0.32 ± 0.08dfi  0.422 ± 0.10k  51.4 ± 11.8k  0.113 ± 0.037b  +  ―    Media formulation and plate orientation  Wavelength (mm)  Wave amplitude (mm)  Wave frequency (waves mm–1)  Wave tangent angle (°)  Growth rates (mm h–1)  Waving roots (%); + = 100  Looping roots (%)  Loop diameter (mm)  1.0% Phytagel  MS salts (two independent experiments)  NF  0°  3.9 ± 0.7a  0.19 ± 0.05j  0.071 ± 0.024a  18.5 ± 3.5a  0.138 ± 0.019a  22.8  ―      20°  3.4 ± 1.0ad  0.23 ± 0.07ab  0.083 ± 0.035a  21.2 ± 4.6b  0.124 ± 0.022bc  16.7  ―      45°  2.9 ± 0.6bdijm  0.23 ± 0.08b  0.079 ± 0.037a  23.5 ± 7.1d  0.103 ± 0.026f  24.7  ―    ST  20°  3.4 ± 1.0ac  0.29 ± 0.10c  0.138 ± 0.049b  26.0 ± 6.3c  0.272 ± 0.033d  +  ―      45°  3.3 ± 0.7cf  0.36 ± 0.12fh  0.160 ± 0.037ce  35.7 ± 9.4ij  0.294 ± 0.031g  +  2.3  2.05 ± 0.21a  1.5% Bacto-agar  MS salts (three independent experiments)  NF  0°  3.2 ± 0.9abc  0.26 ± 0.08a  0.133 ± 0.063b  20.5 ± 6.0ab  0.121 ± 0.017b  +  ―      20°  3.1 ± 0.9bef  0.29 ± 0.11c  0.101 ± 0.050ad  26.3 ± 9.5cd  0.137 ± 0.027ae  +  ―      45°  2.9 ± 0.8ehi  0.44 ± 0.14gh  0.237 ± 0.056f  42.6 ± 12.1m  0.139 ± 0.025ac  +  2.7  1.24 ± 0.23abcd  ST  20°  2.8 ± 0.8gi  0.41 ± 0.15e  0.199 ± 0.056g  40.7 ± 11.2f  0.209 ± 0.036h  +  30.9  1.53 ± 0.33b    45°  2.5 ± 0.9kn  0.42 ± 0.16eg  0.229 ± 0.062f  47.9 ± 15.3l  0.195 ± 0.033i  +  96.8  1.41 ± 0.27c  MS/2 salts (three independent experiments)  NF  20°  3.0 ± 0.8bgh  0.30 ± 0.09cd  0.144 ± 0.055bc  30.3 ± 7.6n  0.150 ± 0.022j  +  ―      45°  2.2 ± 0.4lno  0.36 ± 0.08k  0.319 ± 0.047h  39.0 ± 7.1fh  0.102 ± 0.011k  +  ―    ST  20°  2.5 ± 0.9jk  0.33 ± 0.13fg  0.171 ± 0.043de  37.6 ± 7.8ghi  0.314 ± 0.031l  +  ―      45°  2.5 ± 1.1kmo  0.45 ± 0.18l  0.246 ± 0.053f  51.5 ± 11.9kl  0.261 ± 0.036d  +  56.5  1.72 ± 0.68abd  Zero salts (five independent experiments)  NF  20°  1.1 ± 0.3q  0.28 ± 0.06ci  0.521 ± 0.079i  55.9 ± 11.4e  0.079 ± 0.020m  +  ―      45°  1.0 ± 0.3r  0.30 ± 0.07c  0.616 ± 0.065j  63.1 ± 11.9°  0.090 ± 0.017f  +  ―    ST  20°  2.1 ± 0.6l  0.28 ± 0.09c  0.218 ± 0.11df  37.4 ± 8.0gj  0.122 ± 0.033b  +  5.3  1.47 ± 0.07 abc    45°  1.5 ± 0.6p  0.30 ± 0.08cd  0.247 ± 0.11f  55.5 ± 11.4e  0.129 ± 0.026bce  +  4.7  1.00 ± 0.03d  OS salts (two independent experiments)  NF  45°  1.7 ± 0.3p  0.32 ± 0.08dfi  0.422 ± 0.10k  51.4 ± 11.8k  0.113 ± 0.037b  +  ―    Numbers are the means ± SD of three replicates within the noted number of independent experiments. Similar superscript letters within a column indicates the means are not significantly different. Statistical analyses were performed by the Student’s t-test at the 95% confidence level. s Looping hampered calculations. View Large Abbreviations MS Murashige and Skoog NF Nescofilm OS Okada and Shimura ST surgical tape. References Baskin, T.I. and Williamson, R.E. ( 1992) Ethylene, microtubules and root morphology in wild-type and mutant Arabidopsis seedlings. Curr. Top. Plant Biochem. Physiol.  11: 118–130. Google Scholar Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A. and Ecker, J.R. ( 1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases. Cell  72: 427–441. Google Scholar Koncz, C., Chua, N.-H. and Schell, J. (eds) ( 1992) Methods in Arabidopsis Research. World Scientific, Singapore. Google Scholar Luschnig, C., Gaxiola, R.A., Grisafi, P. and Fink, G.R. ( 1998) EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes Dev.  12: 2175–2187. Google Scholar Meyerowitz, E.M. and Somerville, C.R. (eds) ( 1994) Arabidopsis. (Monograph No. 27) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Google Scholar Mirza, J.I. ( 1987) The effects of light and gravity on the horizontal curvature of roots of gravitropic and agravitropic Arabidopsis thaliana L. Plant Physiol.  83: 118–120. Google Scholar Mullen, J.L., Turk, E., Johnson, K., Wolverton, C., Ishikawa, H., Simmons, C., Söll, D. and Evans, M.L. ( 1998) Root-growth behavior of the Arabidopsis mutant rgr1.Roles of gravitropism and circumnutation in the waving/coiling phenomenon. Plant Physiol.  118: 1139–1145. Google Scholar Okada, K. and Shimura, Y. ( 1990) Reversible root tip rotation in Arabidopsis seedlings induced by obstacle-touching stimulus. Science  250: 274–276. Google Scholar Rutherford, R. and Masson, P.H. ( 1996) Arabidopsis thaliana sku mutant seedlings show exaggerated surface-dependent alteration in root growth vector. Plant Physiol.  111: 987–998. Google Scholar Rutherford, R., Gallois, P. and Masson, P.H. ( 1998) Mutations in Arabidopsis thaliana genes involved in the tryptophan biosynthesis pathway affect root waving on tilted agar surfaces. Plant J.  16: 145–154. Google Scholar Simmons, C., Söll, D. and Migliaccio, F. ( 1995) Circumnutation and gravitropism cause root waving in Arabidopsis thaliana. J. Exp. Bot.  46: 143–150. Google Scholar Smyth, D.R. ( 1990) Arabidopsis thaliana:a model plant for studying the molecular basis of morphogenesis. Aust. J. Plant Physiol.  17: 323–331. Google Scholar Van Der Straeten, D., Smalle, J., Bertrand, S., De Paepe, A., De Pauw, I., Vandenbussche, F., Haegman, M., Van Caeneghe, W. and Van Montagu, M. ( 1999) Ethylene signaling: more players in the game. In Biology and Biotechnology of the Plant Hormone Ethylene II. 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Growth Conditions Modulate Root-Wave Phenotypes in Arabidopsis

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Oxford University Press
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0032-0781
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1471-9053
DOI
10.1093/pcp/pcd042
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Abstract

Abstract The cause for the wave-like growth of Arabidopsis thaliana roots on semi-solid medium remains unclear. Researchers have hypothesized a gravity-induced touch-response, circumnutation, or combinations thereof act as the major stimuli. Our data demonstrate that the gaseous environment within the Petri dish can override gravitational effects. Furthermore, we show that medium ion concentrations and gelling polymers modify the wave response. Although the mechanisms driving our wide-ranging wild-type phenotypes are currently unknown, these results are of immediate significance for interpreting genetic and physiological modifications of environmentally and genetically induced characteristics. (Received April 14, 2000; Accepted July 28, 2000). Due to their experimental amenability (Smyth 1990, Koncz et al. 1992, Meyerowitz and Somerville 1994), arabidopsis seedlings have been used for studies of root morphogenesis and gravity responses. In many of these studies, seedlings are grown on the surface of sterile semi-solid media (such as agar or gellan gum) in Petri dishes allowing precise and non-invasive observations. The roots are in contact with the medium on one side (allowing nutrient uptake) and exposed to air on the other. Using this experimental set-up, Okada and Shimura (1990) first described the root-wave phenomenon, wherein roots generated a repeating right-to-left undulation across the vertical growth axis as they elongated, producing a regular sinusoidal pattern. They hypothesized the stimulus was a gravity-induced touch-response to the medium surface and the root tip changed direction in an obstacle-avoidance manoeuvre. Subsequently, other authors have proposed a combination of gravitropism and circumnutation (natural oscillating growth) induced the response (Simmons et al. 1995, Mullen et al. 1998). The stimulus remains uncertain. Experiments to induce root waving were implemented in our laboratory. Our normal growth conditions did not induce root waving [using Phytagel as the gelling agent, full-strength Murashige and Skoog (MS) basal nutrient salts, and Nescofilm (NF) as the plate wrapping material]. In this article we identify several environmental factors that profoundly influence root waving. If not carefully controlled, these factors may confound interpretation of mutant phenotypes, and therefore they require more careful consideration during experiments. Arabidopsis thaliana (L.) Heynh. (ecotype Landsberg erecta) seeds were sterilized by washing in 70% ethanol for 2 min, rinsed, then immersed in 1.25% hypochlorite plus 0.02% Triton X-100 and agitated for 5 min; and then rinsed three times with sterile water. Seeds were sown 16 per Petri dish (ϕ = 9 cm, polystyrene) spaced 8 mm apart in two rows 2.5 cm apart onto the medium surface. The gelling agents were 1.5% (w/v) Bacto-agar (Difco Laboratories, Detroit, MI, U.S.A.) or 1.0% (w/v) Phytagel (Sigma Chemical Co., St. Louis, MO, U.S.A.). Basal salts were MS [Sigma M-0564 macronutrients and M-0529 micronutrients (1× = MS or 0.5× = MS/2)], 3.0% sucrose (1.5% at MS/2, 0% for zero salts), vitamins (Sigma M-6896) at the recommended level (MS), halved (MS/2), or zero with zero nutrients. OS salts were as defined by Okada and Shimura (1990). MES (2-[N-morpholino]ethanesulfonic acid, 0.05%, w/v) was added to all media formulations as a buffer. All media pH were set at 5.7 before adding the gelling agent and autoclaving. The plates were sealed with NF (Azwell Inc., Osaka, Japan) or wrapped with porous Micropore surgical tape (ST; 3M Health Care, St. Paul, MN, U.S.A.) and immediately placed in a Conviron (Controlled Environments, Pembina, ND, U.S.A.) growth chamber oriented vertically (0°), 20°, or 45° from vertical, as required. Some plates were shifted between the orientations to determine gravity effects. Each condition contained three replicates in each round of experiments and was repeated independently. Additional growth conditions included continuous light (100 µmol m–2 s–1, cool white fluorescent and incandescent bulbs) and 80% relative humidity at 21°C. The position of each root tip was marked on the plate’s base daily to quantify growth rates (the first mark was 60 h following sowing). Images of the Petri dishes were generated by scanning the individual plates, through the bottom of the plate and the medium, on a flatbed scanner into Adobe PhotoShop at 600 pixels in–1. Photomontages of roots were assembled from captured video images using NIH’s Scion Frame Grabber. Measurements of root lengths over time were accomplished using the NIH Scion Image software program (Windows version Beta 3b), available at the URL http://www.scioncorp.com. Wave quantification (illustrated in Fig. 1d) included wavelength (λ), amplitude, wave tangent angle (WTA, as defined by Rutherford et al. 1998), and wave frequency (waves mm–1). Non-waving roots were counted in the waving percentages in Table 1. Means between categories were compared by the independent Student’s t-Test at the 95% confidence level using Microcal’s Origin 6.0 software package. We use the term loop to describe the two-dimensional arc the roots formed under some conditions, comprising part of or a complete circle. This differs from previous literature (circles: Okada and Shimura 1990; coils: Simmons et al. 1995, Mullen et al. 1998). A coil is generally three-dimensional, forming a structure such as a coil spring. A circle implies the loop is closed, which was not always the case. Wide-ranging phenotypes resulted between NF- and ST-wrapped plates under different nutrient treatments, altered angle (α) between the gravity vector and the root elongation axis, and with different gelling agents. Root elongation rates increased when the plates were wrapped with ST versus NF under all conditions (Table 1). Roots in the few plates that developed a small crack in the NF reverted to the respective ST phenotype (not shown). These small cracks would not allow similar water loss within the plates compared to ST wrapping, but would allow gas exchange. Plants in NF-wrapped plates showed signs of ethylene exposure. The roots in NF-wrapped plates were thicker and hairier (as also observed by Baskin and Williamson 1992) in side-by-side comparisons to ST-wrapped plates (Fig. 1, compare c versus d). The cotyledons exhibited epinasty (compare Fig. 2a versus c; Van Der Straeten et al. 1999). The epinasty appears less pronounced in plates at 45° as the hypocotyl turned upward due to gravity. Understandably, tiny cotyledons resulted when using zero nutrient ions, but cotyledons were larger in ST-wrapped plates (Fig. 2–4) than in NF-wrapped plates. With nutrient ions in the media, the cotyledons grew more, but they were larger still with ST wrapping than with NF. Ethylene was shown to inhibit leaf expansion in A. thaliana (Kieber et al. 1993, and references therein for other species). Plate aeration could also affect other gas concentrations within the petri dish. Limiting the medium nutrient ion concentration for seedlings grown on agar modulated root waving differentially, depending on plate aeration (Fig. 2). As the nutrient ion concentration was decreased in NF-sealed plates, the wavelengths progressively shortened, the wave amplitudes generally decreased, and the waving frequency increased (Fig. 2a, e, i and b, f, j). Roots exhibited the most extreme waving on zero nutrient ion media with NF (Fig. 1c and 2i–j), had the shortest wavelengths, the greatest wave tangent angles, and occurred with an increased frequency (Table 1). In contrast, roots in plates wrapped with ST looped on MS basal salts (Fig. 2c, d) and as nutrient salt concentrations were decreased, the looping was replaced by waving (Fig. 2g, k and h, l). Wrapping plates with ST increased the wave characteristics unless the seedlings were grown on zero ion concentrations, wherein the wave-response decreased (Fig. 1c versus d; Fig. 2i versus k, j versus l). Whether this is a general phenomenon associated with overall nutrient depletion or whether it is associated with specific nutrients is unknown, though several could be expected to play important roles in the response, with protein cofactors obvious candidates. The angle (α) between the gravity vector and the root elongation axis influenced waving but, again, this effect was dependent on the plate wrapping and media ion concentrations (Fig. 3). At the start, the plates were oriented at 20°, 60 h post-sowing the orientation was changed to 45° (first mark), after 48 h the plates were shifted to 0° (black triangles), and 48 h later the orientation was returned to 45° (white triangles). Under Okada and Shimura (1990) defined conditions (hereafter denoted OS), roots responded quickly and predictably to changes in plate orientations when using NF (Fig. 3a). The roots waved until the plates were shifted to vertical, then they grew nearly straight, and waved again after the shift back to 45°. Under other treatments, however, the roots lost the ability to adjust to a change in α. After the shift to the vertical orientation (Fig. 3e), roots on zero nutrient salt media in plates wrapped with NF continued to wave at nearly the same frequency. Roots on zero nutrient salts in plates wrapped with ST had a more random, reduced waving phenotype at the second turn to 45° (Fig. 3f). Plates with OS nutrient salts and MS/2 salts and wrapped with ST produced roots that generally did not wave at the second turn to 45° (Fig. 3b, d; lateral root formation obscures initial waving). Roots in plates oriented vertically were generally indistinguishable from those tilted at 20° (Table 1) except that they often lost contact with the medium and became dehydrated. Root waving was not completely suppressed at vertical plate orientations in our experiments, but it occurred irregularly. A wave response at vertical orientations was also noted by Simmons et al. (1995). Vertical growth reduces the gravity-influenced touch-response against the medium and any frictional forces against root growth, implying other factors are involved in the waving stimulus. The role of other factors is also implied by the phenotype differences observed between roots on Phytagel and agar medium formulations at a constant α. The gelling agent itself influenced root waving. On Phytagel plates wrapped with NF, root waving was virtually nonexistent (Fig. 4a–b), but random waving was generally observed when plates were wrapped with ST (Fig. 4c–d). Roots of seedlings grown in NF-wrapped plates were shorter and exhibited an increased root hair density, resulting in a thicker appearance (Fig. 4a–b). Growth rates were further inhibited when plates were shifted from 20° to 45° on plates sealed with NF (Fig. 4a–b), but increased when ST was used (Fig 4c–d). Increasing α to 45° shortened the wavelength and increased the frequency of waves only slightly on Phytagel (Table 1; Fig. 4). The seedlings on agar produced the most orderly and consistent root-wave phenotypes (compare Fig. 1 versus 4). Phytagel-solidified media produced other differences as well. Roots on Phytagel exhibited increased hair densities compared to agar plates (Fig. 1, compare a versus b). The increased hairiness was also evident on ST-wrapped Phytagel plates (Fig. 4c–d) suggesting that hairiness is attributable to some property of the gelling agent per se. Among the literature involving the root-wave phenomenon, only Rutherford and Masson (1996) mentioned the agar brand used, only Mullen et al. (1998) and Luschnig et al. (1998) specified the Petri dish wrapping material, and only Simmons et al. (1995) and Mullen et al. (1998) cited the initial media pH. Mirza (1987) did not state what basal nutrient salts were used. Plate wrapping materials especially, and other growth conditions occasionally, are taken for granted, and it is possible phenotypes separated for mutational analyses are profoundly enhanced or suppressed by growth conditions that may confound interpretation of the function of the gene affected by a particular genetic lesion. If mutants and wild-type plants are grown in the same plate, allelopathic effects can occur (not shown). Responses on tightly sealed plates could differ from responses on loosely wrapped plates. As the basis for phenotype assessment is against wild-type plants, our wide-ranging phenotypes indicate that growth conditions require careful attention. Almost certainly, different genes are switched on or off depending on variations in growth media and plate ventilation. These environmental effects induced unexpected changes in the root-wave phenotypes, but could also influence the expression of other seedling phenotypes that have been reported in arabidopsis. Agar plates wrapped with ST produced the slanting root growth mentioned previously (Simmons et al. 1995, Rutherford and Masson 1996, Mullen et al. 1998). This response was almost nonexistent on Phytagel plates and much reduced on plates wrapped with NF. We demonstrate that root waving is sensitive to characteristics of the microenvironment related to the extent of the gas exchange between the inside and the outside of the Petri dish and to the chemical and structural properties of the growth medium. Our results suggest the mechanisms controlling root waving are complex and imply that gene expression stimulated by mechanical parameters is influenced by several environmental factors. The nature of these characteristics and the underlying reasons are unknown, but our data implicate mechanisms partly independent of gravitropism. Acknowledgments Graham Farquhar is thanked for critical comments regarding the manuscript. 3 Corresponding author: E-mail, [email protected]; Fax, +61-2-6249-4919. (Fax, +61-2-6251-4919 from 1 January 2001) View largeDownload slide Fig. 1 Micrographs of typical root phenotypes on 1.0% Phytagel or 1.5% Bacto-agar, in Petri dishes sealed with NF or ST, grown on MS or zero basal nutrient salts, and tilted 20° or 45° from vertical. An illustration indicating wave quantification parameters is shown (d). View largeDownload slide Fig. 1 Micrographs of typical root phenotypes on 1.0% Phytagel or 1.5% Bacto-agar, in Petri dishes sealed with NF or ST, grown on MS or zero basal nutrient salts, and tilted 20° or 45° from vertical. An illustration indicating wave quantification parameters is shown (d). View largeDownload slide Fig. 2 Phenotype changes of A. thaliana, Landsberg erecta, seedlings on 1.5% Bacto-agar at varying nutrient conditions (marked in the column margin): MS basal nutrient salts (a–d), MS/2 basal nutrient salts (e–h), or zero nutrient salts (i–l). The wrapping method and plate orientation are noted in the row margin: NF, 20° (a, e and i); NF, 45°, (b, f and j); ST, 20° (c, g and k); and ST, 45° (d, h and l). All plates were scanned the seventh day after sowing except the zero salt condition (10 d). The horizontal scores along the root axis are the growth measurement marks at 24 h intervals. View largeDownload slide Fig. 2 Phenotype changes of A. thaliana, Landsberg erecta, seedlings on 1.5% Bacto-agar at varying nutrient conditions (marked in the column margin): MS basal nutrient salts (a–d), MS/2 basal nutrient salts (e–h), or zero nutrient salts (i–l). The wrapping method and plate orientation are noted in the row margin: NF, 20° (a, e and i); NF, 45°, (b, f and j); ST, 20° (c, g and k); and ST, 45° (d, h and l). All plates were scanned the seventh day after sowing except the zero salt condition (10 d). The horizontal scores along the root axis are the growth measurement marks at 24 h intervals. View largeDownload slide Fig. 3 Effects on the root-wave phenomenon of A. thaliana, Landsberg erecta, seedlings by changing the plate angle from 45° to 0° (black arrow) and back to 45° (white arrow) on different basal salt media concentrations after 10 d. The conditions are shown in the row margin: OS nutrient salts (a, b), MS/2 basal nutrient salts (c, d), and zero salts (e, f), all without sucrose. Plates in column one are wrapped with NF and column two with ST. View largeDownload slide Fig. 3 Effects on the root-wave phenomenon of A. thaliana, Landsberg erecta, seedlings by changing the plate angle from 45° to 0° (black arrow) and back to 45° (white arrow) on different basal salt media concentrations after 10 d. The conditions are shown in the row margin: OS nutrient salts (a, b), MS/2 basal nutrient salts (c, d), and zero salts (e, f), all without sucrose. Plates in column one are wrapped with NF and column two with ST. View largeDownload slide Fig. 4 Differences in root morphology in A. thaliana, Landsberg erecta, on 1.0% Phytagel, MS basal nutrient salts, NF (a, b) or with ST (c, d) wrapping, and tilted 20° or 45° from vertical. Images were recorded the seventh day after sowing. View largeDownload slide Fig. 4 Differences in root morphology in A. thaliana, Landsberg erecta, on 1.0% Phytagel, MS basal nutrient salts, NF (a, b) or with ST (c, d) wrapping, and tilted 20° or 45° from vertical. Images were recorded the seventh day after sowing. Table 1  Comparisons of Arabidopsis thaliana, ecotype Landsberg erecta, root growth and wave characteristics on varying gelling agents, nutrient salt concentrations, plate orientations, and plate wrapping material Media formulation and plate orientation  Wavelength (mm)  Wave amplitude (mm)  Wave frequency (waves mm–1)  Wave tangent angle (°)  Growth rates (mm h–1)  Waving roots (%); + = 100  Looping roots (%)  Loop diameter (mm)  1.0% Phytagel  MS salts (two independent experiments)  NF  0°  3.9 ± 0.7a  0.19 ± 0.05j  0.071 ± 0.024a  18.5 ± 3.5a  0.138 ± 0.019a  22.8  ―      20°  3.4 ± 1.0ad  0.23 ± 0.07ab  0.083 ± 0.035a  21.2 ± 4.6b  0.124 ± 0.022bc  16.7  ―      45°  2.9 ± 0.6bdijm  0.23 ± 0.08b  0.079 ± 0.037a  23.5 ± 7.1d  0.103 ± 0.026f  24.7  ―    ST  20°  3.4 ± 1.0ac  0.29 ± 0.10c  0.138 ± 0.049b  26.0 ± 6.3c  0.272 ± 0.033d  +  ―      45°  3.3 ± 0.7cf  0.36 ± 0.12fh  0.160 ± 0.037ce  35.7 ± 9.4ij  0.294 ± 0.031g  +  2.3  2.05 ± 0.21a  1.5% Bacto-agar  MS salts (three independent experiments)  NF  0°  3.2 ± 0.9abc  0.26 ± 0.08a  0.133 ± 0.063b  20.5 ± 6.0ab  0.121 ± 0.017b  +  ―      20°  3.1 ± 0.9bef  0.29 ± 0.11c  0.101 ± 0.050ad  26.3 ± 9.5cd  0.137 ± 0.027ae  +  ―      45°  2.9 ± 0.8ehi  0.44 ± 0.14gh  0.237 ± 0.056f  42.6 ± 12.1m  0.139 ± 0.025ac  +  2.7  1.24 ± 0.23abcd  ST  20°  2.8 ± 0.8gi  0.41 ± 0.15e  0.199 ± 0.056g  40.7 ± 11.2f  0.209 ± 0.036h  +  30.9  1.53 ± 0.33b    45°  2.5 ± 0.9kn  0.42 ± 0.16eg  0.229 ± 0.062f  47.9 ± 15.3l  0.195 ± 0.033i  +  96.8  1.41 ± 0.27c  MS/2 salts (three independent experiments)  NF  20°  3.0 ± 0.8bgh  0.30 ± 0.09cd  0.144 ± 0.055bc  30.3 ± 7.6n  0.150 ± 0.022j  +  ―      45°  2.2 ± 0.4lno  0.36 ± 0.08k  0.319 ± 0.047h  39.0 ± 7.1fh  0.102 ± 0.011k  +  ―    ST  20°  2.5 ± 0.9jk  0.33 ± 0.13fg  0.171 ± 0.043de  37.6 ± 7.8ghi  0.314 ± 0.031l  +  ―      45°  2.5 ± 1.1kmo  0.45 ± 0.18l  0.246 ± 0.053f  51.5 ± 11.9kl  0.261 ± 0.036d  +  56.5  1.72 ± 0.68abd  Zero salts (five independent experiments)  NF  20°  1.1 ± 0.3q  0.28 ± 0.06ci  0.521 ± 0.079i  55.9 ± 11.4e  0.079 ± 0.020m  +  ―      45°  1.0 ± 0.3r  0.30 ± 0.07c  0.616 ± 0.065j  63.1 ± 11.9°  0.090 ± 0.017f  +  ―    ST  20°  2.1 ± 0.6l  0.28 ± 0.09c  0.218 ± 0.11df  37.4 ± 8.0gj  0.122 ± 0.033b  +  5.3  1.47 ± 0.07 abc    45°  1.5 ± 0.6p  0.30 ± 0.08cd  0.247 ± 0.11f  55.5 ± 11.4e  0.129 ± 0.026bce  +  4.7  1.00 ± 0.03d  OS salts (two independent experiments)  NF  45°  1.7 ± 0.3p  0.32 ± 0.08dfi  0.422 ± 0.10k  51.4 ± 11.8k  0.113 ± 0.037b  +  ―    Media formulation and plate orientation  Wavelength (mm)  Wave amplitude (mm)  Wave frequency (waves mm–1)  Wave tangent angle (°)  Growth rates (mm h–1)  Waving roots (%); + = 100  Looping roots (%)  Loop diameter (mm)  1.0% Phytagel  MS salts (two independent experiments)  NF  0°  3.9 ± 0.7a  0.19 ± 0.05j  0.071 ± 0.024a  18.5 ± 3.5a  0.138 ± 0.019a  22.8  ―      20°  3.4 ± 1.0ad  0.23 ± 0.07ab  0.083 ± 0.035a  21.2 ± 4.6b  0.124 ± 0.022bc  16.7  ―      45°  2.9 ± 0.6bdijm  0.23 ± 0.08b  0.079 ± 0.037a  23.5 ± 7.1d  0.103 ± 0.026f  24.7  ―    ST  20°  3.4 ± 1.0ac  0.29 ± 0.10c  0.138 ± 0.049b  26.0 ± 6.3c  0.272 ± 0.033d  +  ―      45°  3.3 ± 0.7cf  0.36 ± 0.12fh  0.160 ± 0.037ce  35.7 ± 9.4ij  0.294 ± 0.031g  +  2.3  2.05 ± 0.21a  1.5% Bacto-agar  MS salts (three independent experiments)  NF  0°  3.2 ± 0.9abc  0.26 ± 0.08a  0.133 ± 0.063b  20.5 ± 6.0ab  0.121 ± 0.017b  +  ―      20°  3.1 ± 0.9bef  0.29 ± 0.11c  0.101 ± 0.050ad  26.3 ± 9.5cd  0.137 ± 0.027ae  +  ―      45°  2.9 ± 0.8ehi  0.44 ± 0.14gh  0.237 ± 0.056f  42.6 ± 12.1m  0.139 ± 0.025ac  +  2.7  1.24 ± 0.23abcd  ST  20°  2.8 ± 0.8gi  0.41 ± 0.15e  0.199 ± 0.056g  40.7 ± 11.2f  0.209 ± 0.036h  +  30.9  1.53 ± 0.33b    45°  2.5 ± 0.9kn  0.42 ± 0.16eg  0.229 ± 0.062f  47.9 ± 15.3l  0.195 ± 0.033i  +  96.8  1.41 ± 0.27c  MS/2 salts (three independent experiments)  NF  20°  3.0 ± 0.8bgh  0.30 ± 0.09cd  0.144 ± 0.055bc  30.3 ± 7.6n  0.150 ± 0.022j  +  ―      45°  2.2 ± 0.4lno  0.36 ± 0.08k  0.319 ± 0.047h  39.0 ± 7.1fh  0.102 ± 0.011k  +  ―    ST  20°  2.5 ± 0.9jk  0.33 ± 0.13fg  0.171 ± 0.043de  37.6 ± 7.8ghi  0.314 ± 0.031l  +  ―      45°  2.5 ± 1.1kmo  0.45 ± 0.18l  0.246 ± 0.053f  51.5 ± 11.9kl  0.261 ± 0.036d  +  56.5  1.72 ± 0.68abd  Zero salts (five independent experiments)  NF  20°  1.1 ± 0.3q  0.28 ± 0.06ci  0.521 ± 0.079i  55.9 ± 11.4e  0.079 ± 0.020m  +  ―      45°  1.0 ± 0.3r  0.30 ± 0.07c  0.616 ± 0.065j  63.1 ± 11.9°  0.090 ± 0.017f  +  ―    ST  20°  2.1 ± 0.6l  0.28 ± 0.09c  0.218 ± 0.11df  37.4 ± 8.0gj  0.122 ± 0.033b  +  5.3  1.47 ± 0.07 abc    45°  1.5 ± 0.6p  0.30 ± 0.08cd  0.247 ± 0.11f  55.5 ± 11.4e  0.129 ± 0.026bce  +  4.7  1.00 ± 0.03d  OS salts (two independent experiments)  NF  45°  1.7 ± 0.3p  0.32 ± 0.08dfi  0.422 ± 0.10k  51.4 ± 11.8k  0.113 ± 0.037b  +  ―    Numbers are the means ± SD of three replicates within the noted number of independent experiments. 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Plant and Cell Physiology – Oxford University Press

Published: Oct 15, 2000

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