Portal de Periódicos da CAPES

Hydrotropism: Root Bending Does Not Require Auxin Redistribution

2016; Elsevier BV; Volume: 9; Issue: 5 Linguagem: Inglês

10.1016/j.molp.2016.02.001

1674-2052

Doron Shkolnik, Gat Krieger, Roye Nuriel, Hillel Fromm,

Tree Root and Stability Studies

The widely accepted Cholodny-Went theory posits that asymmetrical auxin redistribution is required for tropic growth of plant organs. We demonstrate, using the sensitive auxin-sensor protein DII-VENUS that, in the presence and absence of auxin polar transport inhibitors, root tropism toward moisture (hydrotropism) does not involve auxin redistribution. Moreover, highly specific auxin signaling antagonists accelerate hydrotropism. Thus, hydrotropism is an exception to the Cholodny-Went theory. Plants compensate for their lack of motility by directional growth in response to various environmental stimuli, phenomena termed tropism. Tropic growth is achieved by asymmetrical elongation of cells on opposite sides of an organ. Darwin and Darwin, 1880Darwin C. Darwin F.E. Sensitiveness of Plants to Light: It’s Transmitted Effect. The Power of Movement in Plants. John Murray, London1880: 574-592Google Scholar demonstrated, in coleoptiles, that growth toward light, known as phototropism, requires the transmission of a signal from the tip toward the growing tissue. Subsequently, Cholodny, 1927Cholodny N.G. Wuchshormone und Tropismen bei den Pflanzen.Biol. Zent. 1927; 47: 604-626Google Scholar and Went, 1926Went F.W. On growth-accelerating substances in the coleoptile of Avena sativa.Proc. K Ned. Akad. Wet. 1926; 30: 10-19Google Scholar discovered that a phytohormone is the signal for gravitropism and phototropism, respectively, which led to what is widely accepted as the Cholodny-Went theory (Went, 1926Went F.W. On growth-accelerating substances in the coleoptile of Avena sativa.Proc. K Ned. Akad. Wet. 1926; 30: 10-19Google Scholar, Cholodny, 1927Cholodny N.G. Wuchshormone und Tropismen bei den Pflanzen.Biol. Zent. 1927; 47: 604-626Google Scholar, Went and Thimann, 1937Went F.W. Thimann K.V. Phytohormones. Macmillan, New York1937Google Scholar). The main statement of the Cholodny-Went theory is that redistribution of auxin on opposite sides of the stimulated organ is required for its tropic growth (Gilroy, 2008Gilroy S. Plant tropisms.Curr. Biol. 2008; 18: R275-R277Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Indeed, auxin redistribution was shown in gravitropism (Rashotte et al., 2001Rashotte A.M. DeLong A. Muday G.K. Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth.Plant Cell. 2001; 13: 1683-1697Crossref PubMed Scopus (234) Google Scholar) and phototropism (Friml et al., 2002Friml J. Wisniewska J. Benkova E. Mendgen K. Palme K. Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis.Nature. 2002; 415: 806-809Crossref PubMed Scopus (1066) Google Scholar). Nevertheless, the requirement for redistribution of auxin in tropic responses has been debated over the years (Trewavas, 1992Trewavas A.J. What remains of the Cholodny-Went theory? Introduction.Plant Cell Environ. 1992; 15: 761PubMed Google Scholar). While the mechanisms of sensing gravity and consequent gravitropic growth are well characterized (Toyota and Gilroy, 2013Toyota M. Gilroy S. Gravitropism and mechanical signaling in plants.Am. J. Bot. 2013; 100: 111-125Crossref PubMed Scopus (84) Google Scholar), it is not clear how roots perceive moisture gradients and consequently grow away from low water potential toward higher water potential, a phenomenon termed hydrotropism (Von Sachs, 1887Von Sachs J. Lectures on Physiology of Plants. Clarendon Press, Oxford1887Google Scholar, Jaffe et al., 1985Jaffe M.J. Takahashi H. Biro R.L. A pea mutant for the study of hydrotropism in roots.Science. 1985; 230: 445-447Crossref PubMed Scopus (106) Google Scholar). In gravitropism, bending is a result of asymmetrical increased basipetal auxin flow at the bottom side of the root tip (Rashotte et al., 2001Rashotte A.M. DeLong A. Muday G.K. Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth.Plant Cell. 2001; 13: 1683-1697Crossref PubMed Scopus (234) Google Scholar). Inhibitors of polar auxin transport like N-1-naphthylphthalamic acid (NPA) and 2,3,5-tri-iodobenzoic acid (TIBA) failed to block hydrotropism (Kaneyasu et al., 2007Kaneyasu T. Kobayashi A. Nakayama M. Fujii N. Takahashi H. Miyazawa Y. Auxin response, but not its polar transport, plays a role in hydrotropism of Arabidopsis roots.J. Exp. Bot. 2007; 58: 1143-1150Crossref PubMed Scopus (65) Google Scholar). Therefore either root hydrotropism is mediated by auxin redistribution through a mechanism different from that mediating gravitropic-dependent auxin redistribution, or auxin redistribution is not required in hydrotropism. To date, no evidence for auxin redistribution in hydrostimulated roots has been provided. To address this open question, we investigated hydrostimulated roots expressing the sensitive auxin-sensor protein DII-VENUS and, separately, pDR5rev:erRFP in the presence and absence of auxin polar transport inhibitors, or auxin signaling antagonists, in comparison with gravistimulated roots. Five-day-old Arabidopsis thaliana seedlings were placed in a dry, CaCl2-containing chamber as described in the Supplemental Information and DII-VENUS fluorescence was imaged in primary root tips at several time points following exposure to a moisture gradient (Figure 1A ). Auxin causes the DII-VENUS fluorescent protein to be rapidly degraded (Brunoud et al., 2012Brunoud G. Wells D.M. Oliva M. Larrieu A. Mirabet V. Burrow A.H. Beeckman T. Kepinski S. Traas J. Bennett M.J. A novel sensor to map auxin response and distribution at high spatio-temporal resolution.Nature. 2012; 482: 103-106Crossref PubMed Scopus (530) Google Scholar), thus a low fluorescent signal is indicative of elevated auxin levels. At the start of the experiment (time 0), the DII-VENUS signal was found to be equally distributed on both sides of the root tip (Figure 1A, 1B, and Supplemental Figure 1A). After 2 and 4 h of hydrostimulation, no asymmetrical distribution of auxin was observed (Figure 1A and Supplemental Figure 1A) yet root curvature (the angle was measured at intervals of 20 min) was clearly apparent (Figure 1B, 1C, and Supplemental Figure 1B). In roots that were hydrostimulated for 6, 8 and 10 h, an increased asymmetrical auxin distribution with higher DII-VENUS signal at the concave side of the root tip was observed, indicating the redistribution of auxin on opposite sides of the root tip (Figure 1A, 1B, and Supplemental Figure 1A). Furthermore, quantification of auxin at the lateral root cap of hydrostimulated roots of pDR5rev:erRFP-expressing plants confirmed, yet with less sensitivity than with the DII-VENUS sensor, the lack of auxin differential distribution during the first 2 h of hydrotropism and higher auxin levels at the convex side of the root tip at 6 h of hydrostimulation (Supplemental Figure 2). We suspected that this late auxin redistribution (i.e., after hydrostimulated root bending was initiated) may be the result of gravistimulation of the root following hydrostimulated root curvature. To assess this possibility, we performed a similar hydrotropic assay, however in the presence of the auxin polar transport inhibitor NPA. In the presence of NPA, no differential auxin accumulation on either side of the root tip was visualized at all time points (Figure 1A and Supplemental Figure 1A) yet hydrostimulated root bending was initiated earlier and progressed faster than in control experiments without NPA (Figure 1B, 1C, and Supplemental Figure 1B). Root growth rate was not affected by 1 μM NPA at the time root bending occurred (Supplemental Figure 1C), as previously described (Kaneyasu et al., 2007Kaneyasu T. Kobayashi A. Nakayama M. Fujii N. Takahashi H. Miyazawa Y. Auxin response, but not its polar transport, plays a role in hydrotropism of Arabidopsis roots.J. Exp. Bot. 2007; 58: 1143-1150Crossref PubMed Scopus (65) Google Scholar). The DII-VENUS fluorescent signal decreased in most regions of the root tip with increased duration of exposure to NPA (Figure 1A). In contrast, a fluorescent signal was enhanced in columella cells, which was not observed in the control without NPA (Figure 1A). The DII-VENUS signal at the elongation zone of hydrostimulated roots was found to be relatively weak compared with the signal at the root tip in a pattern that indicates no differential auxin distribution at the root sides (Supplemental Figure 3). A similar pattern was obtained with a different polar auxin transport inhibitor, TIBA (Figure 1C, Supplemental Figure 1B, and 1C). In the presence of these auxin polar transport inhibitors, no gravistimulated curvature could be detected at the root tip of hydrostimulated seedlings (Figure 1B and Supplemental Figure 1B). The predicted auxin flux in hydrostimulated root tips under control conditions or NPA treatment is depicted as green arrows in Figure 1B. In contrast to hydrotropism, when challenging seedlings with gravistimulation, as a control experiment, differential auxin distribution was apparent as early as 2 h of stimulation (Supplemental Figure 4), as expected. Both auxin redistribution and root curvature of gravistimulated roots were abolished in the presence of NPA (Supplemental Figure 4). The lack of auxin redistribution in the hydrotropic response raised questions about the possible roles of auxin in hydrotropism. To address this, we challenged hydrostimulated roots with the highly specific auxin antagonists α-(phenylethyl-2-one)-indole acetic acid (PEO-IAA) and α-(2,4-dimethylethyl-2-oxo)-IAA (auxinole). Interestingly, both PEO-IAA and auxinole remarkably accelerated hydrotropic bending (Figure 1C and Supplemental Figure 5A) without affecting root growth rate (Supplemental Figure 5B) at the tested concentrations of 1 μM. This hydrotropic acceleration was associated with a significant decrease of detectable auxin levels, while no differential auxin distribution was observed (Supplemental Figure 5C–5E). These results imply that if auxin has a role in hydrotropism, it is by negatively regulating it. These results appear to be in contrast to the previously suggested role of auxin in hydrotropism based on the use of a less-specific auxin antagonist P-chlorophenoxyisobutyric acid (PCIB) (Kaneyasu et al., 2007Kaneyasu T. Kobayashi A. Nakayama M. Fujii N. Takahashi H. Miyazawa Y. Auxin response, but not its polar transport, plays a role in hydrotropism of Arabidopsis roots.J. Exp. Bot. 2007; 58: 1143-1150Crossref PubMed Scopus (65) Google Scholar). When root tips confront non-homogeneous low water potentials in their microenvironment, they overcome gravity-directed growth and bend toward regions of higher water potential. Our study shows that this root bending in response to hydrostimulation is independent of auxin redistribution or signaling via TIR proteins. We cannot rule out the possibility of a weak and/or transient auxin signal, undetectable by the DII-VENUS or the pDR5rev:erRFP systems, which may be involved in hydrotropism. However, if such a signal occurred, it would be very different from the auxin signal in gravitropism in its intensity and kinetics, and would not involve NPA- or TIBA-sensitive transport systems. Theoretically, auxin may exert differential growth responses on opposite sides of an organ without its redistribution but rather by differential modulation of auxin-response sensitivities, or by differential accumulation of auxin-response agonists or antagonists (Trewavas, 1992Trewavas A.J. What remains of the Cholodny-Went theory? Introduction.Plant Cell Environ. 1992; 15: 761PubMed Google Scholar and references therein). Furthermore, acceleration of hydrotropic root bending by blocking TIR-dependent auxin signaling suggests an inhibitory function of auxin in hydrotropism. In summary, considering both auxin distribution and signaling, hydrotropism is clearly an exception to the Colodny-Went theory. This research was supported by the I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation (grant no. 757/12).