Journal of Experimental Botany, Vol. 54, No. 393, pp. 2723-2732,
December 1, 2003
© 2003 Oxford University Press
Physiological and biochemical characterization of ethylene-generated gravicompetence in primary shoots of coleoptile-less gravi-incompetent rye seedlings
Received 19 February 2003; Accepted 22 September 2003
1 Botanisches Institut der Universität Bonn, Abteilung Molekularbiologie, Kirschallee 1, D-53115 Bonn, Germany
2 Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, ND 3/48, Universitätsstraße 150, D-44801 Bochum, Germany
3 Institut für Angewandte Physik, Universität Bonn, Wegelerstr. 8, D-53115 Bonn, Germany
4Botanisches Institut der Universität Zu Köln, Gyrhofstr. 15, D-50931, Köln, Germany
* To whom correspondence should be addressed. Fax: +49 221 470 5181. E-mail: h.edelmann{at}uni-koeln.de
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A recent study demonstrated that gravi-incompetent coleoptile-less seedlings of rye exhibit gravi-competence after exogenous application of ethylene. Treatments and conditions which induce and interfere with this phenomenon were analysed in more detail. Aminocyclopropane-1-carboxylic acid (ACC) as a precursor of ethylene has similar gravicompetence-inducing effects and also appropriate conditions of light, which strongly enhances ethylene synthesis. Both effects can be inhibited by the ethylene-perception blocking agent methylcyclopropene (MCP) or inhibitors of ethylene synthesis such as aminovinylglycine (AVG), indicating that light exerts its gravicompetence-generating effect via induced/enhanced ethylene synthesis. Gain in gravicompetence is accompanied by the induced/enhanced occurrence of calreticulin and lipoxygenase as detected by 2D-gels and Q-TOFF-analyses. Previously gravicompetent, light-grown coleoptile-less seedlings are characterized by gravi-incompetent growth during subsequent horizontal gravistimulation when perception of ethylene is inhibited by MCP. The results demonstrate that continuous perception of ethylene is an indispensable step, permanently required for the regulation of gravitropic growth in germinating primary shoots of rye, either within the process of graviperception and/or of the transduction of the gravi-signal.
Key words: Aminocyclopropane-1-carboxylic acid (ACC), aminovinylglycine (AVG), calreticulin, ethylene, light-mediated, gravicompetence, gravitropism, methylcyclopropene, Q-TOFF, rye, Secale cereale, signal transduction.
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Plants orient and (re)direct growth, depending on the gravivector, via changes of the growth rates of opposite organ flanks. This potential is traditionally attributed to three sequential processes, namely the perception of the gravi-signal, the subsequent signal transduction, and the adequate growth response (Chen et al., 1999; and literature cited therein).
According to the starch statolith hypothesis, sedimentation and reorientation of amyloplasts (dense, starch-containing plastids of specialized cells of distinct tissues) represent the gravi-perceiving step yielding positional information (Sack, 1991, 1997). At the end of the induced signal transduction chain, the result is differential organ growth regulated, according to the Cholodny–Went hypothesis, via the lateral redistribution of the growth-promoting hormone, auxin (Chen et al., 1999).
Little is known about what happens between the start and end of this hypothetical chain of distinct events; in fact, the nature of the molecular processes that depend on amyloplast sedimentation eventually inducing differential elongation growth is far from clear (Braun et al., 2002). Whether these plastids interfere with endomembrane systems, as suggested many years ago (Sievers and Volkmann, 1972), or whether their function consists of an informational distortion of cytoskeletal elements is still an enigma (Yoder et al, 2001; Zheng and Staehelin, 2001; Perbal and Driss-Ecole, 2002). Results of recent studies indicate that the actin cytoskeleton plays a crucial role within the mechanism of gravitropic growth regulation (Yamamoto and Kiss, 2002; Hou et al., 2003).
A feature common to previous studies on gravitropic plant growth regulation is the fact that the analysed systems per se were gravicompetent. Exogenously induced changes of graviresponsivity, whether with respect to the velocity or intensity (Legue et al., 1996) of the response or whether in a positive (stimulating) or negative (inhibiting) manner, did not allow definite conclusions to be drawn on the regulatory significance of the processes affected by these treatments. For example, it has been demonstrated that the gravitropic growth of gravi-competent plant organs can be modified either in an inhibiting (Friedman et al., 1998) or a stimulating way (Takahashi et al., 1992) depending on the organ and species of the sample.
Similarly, the inhibition of ethylene synthesis as well as the inhibition of its perception has been demonstrated to affect the response of per se gravicompetent organs negatively, such as the stems of graviresponding cocklebur (Wheeler and Salisbury, 1980). Such inhibiting effects range from 4% to 30% after many hours of inhibitor application (e.g. Fig. 1 in Wheeler and Salisbury, 1980). They therefore seem to interfere somehow with the studied phenomenon rather than to affect causal steps in the regulation mechanism of gravitropic growth.
Unlike in previous studies, it has recently been demonstrated (Edelmann, 2002) that ethylene generates gravicompetence in growing, yet gravi-incompetent primary shoots of coleoptile-less rye seedlings. Thus, ethylene perception apparently acts as a cardinal switch cabinet triggering the processes relevant for the regulation of gravitropic differential growth of primary shoots of rye.
In the present study, the parameters and treatments which also induce or eliminate a gravicompetence-generating effect on a physiological and biochemical level have been analysed in more detail.
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Plants
Seeds of rye (Secale cereale cv. Marder II) were germinated for 2 d in the dark at 25±2 °C in moist vermiculite in perspex trays of 20x20x9 cm (lengthxwidthxheight). In some cases, dry seeds were pushed with forceps at an angle of roughly 45° (embryo at the bottom half on the top side of the seed) into the watered vermiculite in order to obtain seedlings with straight growing seedling axes. Seedlings, which by then had, on average, 1.5–2 cm long coleoptiles and enclosed 1–1.5 cm long primary leaves, were harvested and the coleoptiles removed, as described earlier (Edelmann, 1996). In brief, the basal part of the coleoptiles was slightly traced tangentially with a sharp blade. Cautious circular bending resulted in a circumferential crack in the coleoptile but not in the enclosed more elastic primary leaves. The coleoptile could then be removed by carefully pulling it away from the kernel between the thumb and the forefinger. Residual basal parts of the coleoptiles attached to the seed were removed with forceps. Such coleoptile-less ‘primary shoot seedlings’ were then placed horizontally in rows on water-imbibed filter paper and treated as indicated in the figures.
Ethylene treatment was conducted by applying solutions of ethephon or of its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), also as indicated in the figures. Methylcyclopropene (MCP) was applied by dissolving 1, 3 or 5 mg of these compounds in a total volume of 5 ml distilled water in Petri dishes immediately before closing the incubation boxes. Aminovinylglycine (AVG) was added to the incubation solution to give a final concentration of 0.2 mM.
White light (Osram L 36/21-840-1 Lumilux Plus Cool White) with different intensities, as indicated in the figures, was applied by placing the seedlings enclosed in the perspex boxes in defined places in a growth chamber, the light intensities of which were determined with a luxmeter (LI-COR, Modell LI 185B, USA, Lincoln).
Gravicompetence measurements
For statistical analyses of gravitropic competence, three to six independent parallel batches of 10–12 2-d-old freshly prepared primary shoot seedlings were placed horizontally on moist filter paper with an area of 20x20 cm in closed perspex boxes of 20x20x9 cm (lengthxwidthxheight). They were then placed either in the dark or under laboratory light conditions or in growth chambers with defined light- and temperature regimes, as indicated in the figures. For quantitative characterization of the gravitropic growth response two parameters were taken into account (a) the number of seedlings showing a deviation of their organ axes from the horizontal (expressed as a percentage of the total number of seedlings) and (b) the average value of the deviation angles of the responding seedlings from the horizontal. In all gravitropically responding seedlings the region of differential growth was recorded. Length increases were analysed by subdividing organs of similar lengths at the beginning of the experiments into similar sections labelled a, b, c, d, and e, from the basal part to the tip.
For documentation purposes pictures were taken at specific time points with a RICOH digital camera.
Ethylene measurement
Ethylene production was measured in real-time with a photoacoustic laser spectrometer consisting of a line-tunable CO2 laser and two resonant photoacoustic cells (Bessler et al., 1998). Caryopses were fixed on filter-paper and placed on Petri dishes in a 2.0 l glass cuvette which received a constant flow of 33 ml min–1 air. Ambient air was drawn over a platinum catalyst at 450 °C prior to entering the cuvette to remove hydrocarbons. After passing through the cuvette, the air was pushed through a liquid-N2 cooling trap to remove CO2 and H2O before entering the photoacoustic cell in which ethylene concentrations were measured every 3 min (where applicable: emissions from treatment and control were measured simultaneously in the two cells). To identify and quantify ethylene unambiguously, measurements were taken at three different laser lines. The detection limit for this experiment was about 500 ng l–1; calibration was performed with certified gas samples of ethylene in N2 (Praxair, Biebesheim am Rhein, Germany).
Protein separation analyses
For protein analyses, regions characterized by differential growth of the graviresponding seedlings and the appropriate organ parts of gravi-incompetent non-differentially growing seedlings, respectively, were cut out with a blade and forceps and stored in liquid nitrogen. After thawing, samples were homogenized in mini-potters with 1 µl Roti-Load Sample buffer (Roth, Germany) including 0.035 µl protease inhibitor (for plant cell extracts; from Sigma) mg–1 fresh weight, vortexed in Eppendorf tubes, and centrifuged for 5 min at 10 000 rpm on a bench centrifuge. The pellet was discarded and the supernatant was precipitated with a chloroform/methanol mixture according to Wessel and Flügge (1984), and centrifuged at 4 °C at 14 000 rpm. The upper phase was discarded, the interphase and the resulting pellet were vortexed and again centrifuged. The resulting pellet was resuspended in rehydration solution, kept at 30 °C in a heating block and, frozen at –80 °C for later use. After the samples had been thawed at 30 °C in a thermomixer, 200 µl aliquots of the resuspended solutions were applied to IPG-strips (Ready Strip, 7 cm pH range 3–10, from BioRad) and the IEF was carried out overnight employing a BioRad Protean IEF Cell. The strips with the focused sample were then placed onto 12% SDS-PAGE-gels and separated employing Miniprotean 3 electrophoresis cell (BioRad) at a constant current of 130 V. Gels were stained with Coomassie brilliant blue R 250 or Serva Blue G fixation solution and subsequently de-stained in 20% methanol/10% acetic acid.
In-gel digestion
Coomassie-stained protein spots were digested in the gel, as described by Jensen et al. (1998), using sequencing grade modified trypsin (Promega, Mannheim, Germany). After extraction from the gel, peptides were desalted using ZipTips C18 (Millipore, Eschborn, Germany).
De novo sequencing by mass spectrometry
Mass spectrometric measurements were carried out on a quadrupole/time-of-flight hybrid mass spectrometer (Q-TOFF, Micromass, Manchester, UK). The sample was introduced as nanospray in positive ion mode. Doubly or triply charged molecules were selected for fragmentation in MS/MS mode. Interpretation of MS/MS spectra was aided by the MaxEnt3 algorithm and the BioLynx software-package of MaxLynx 3.4 (Micromass, Manchester, UK).
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As recently demonstrated, removal of the coleoptiles of dark-germinating rye seedlings results in the loss of gravicompetence, despite pronounced elongation growth within the basal primary shoot region (Edelmann, 1996). Exogenous supply of ethylene generates gravicompetence within these systems (Edelmann 2002). For a detailed and statistical analysis of this effect, ‘primary shoot seedlings’ (i.e. seedlings with coleoptiles removed) were horizontally gravistimulated in batches of 10–12 on wet filter paper in the dark and the deviation of single seedling axes from the horizontal, as well as the percentage of responding seedlings, were analysed. As Fig. 1a demonstrates, after 24 h under the conditions employed some of the water-incubated seedlings apparently exhibited negative gravitropic responses which, in most cases, again decreased after 48 h. After 96 h 36% of the seedlings showed an average angle of 28° relative to the horizontal (Fig. 2a). The movements of the primary shoot seedlings were very heterogeneous, especially during the early phases of the incubation period, conceivably due to some traumatic effects which appeared more pronounced the less smoothly the coleoptiles were removed. [In addition, various other factors, such as preparation accuracy with razor blades and forceps or the influence of fungi occasionally developing during the incubation period apparently interfered with the subsequent growth responses (in order to avoid any possible impact of fungicides its use was avoided). Pronounced bending or deliberate injuring of the seedlings’ primary leaves by scratching with razor blades, which may happen during preparation, resulted in a higher percentage of apparently graviresponding seedlings as compared to batches of seedlings not injured; data not shown.]
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Germinating the seedlings on filter paper in solutions of ACC as a precursor of ethylene synthesis (Bleeker and Kende, 2000) resulted, on average, in a doubling of the number of responding plants as well as a doubling of the average angle of the gravitropic growth response which, apart from day one, was generally enhanced (Fig. 1b). Compared with water-incubated seedlings, ethylene, applied via solutions of ethephon, induced pronounced differential gravitropic growth responses both with respect to the number of seedlings responding, which was four to five times as high, and to the angle of gravitropic growth (Figs 1c, 2b). This pronounced ethylene-induced graviresponse was totally inhibited by MCP (methylcyclopropene), which has been newly introduced as an inhibitor of ethylene perception (Serek et al., 1994; Sisler and Serek, 1997). As shown in Fig. 1d, such seedlings were characterized by temporary non-directional movements lasting one day at most. (A video camera revealed that these movements were random in character; according to the documentation protocol they apparently mimicked gravitropic growth and were therefore categorized as ‘graviresponsive’; data not shown.) The primary leaves thus ‘crawled’ over the surface of the filter paper in a gravi-incompetent manner.
To test whether light has any effect on the capacity for gravitropic growth, primary shoot seedlings were incubated under laboratory light conditions. As demonstrated in Fig. 1e, in water, such seedlings showed only slightly enhanced graviresponses compared with dark-germinating water-controls (Fig. 1a) during a period of 96 h, both with respect to the degree of gravitropic bending as well as the number of responding seedlings (see also, for 96 h, Fig. 2c). ACC-treated seedlings were characterized by an increased percentage of responding seedlings with, on average, a similar degree of graviresponse compared with dark controls (Fig. 1f). In the presence of laboratory light, ethylene-treated seedlings were characterized by enhanced gravitropic competence. The effect was most pronounced after 96 h, especially with respect to the average extent of the graviresponse (Figs 1g, 2d). Compared with dark-grown seedlings (Figs 1c, 2b), samples growing under laboratory light conditions had a tendency to show a more homogeneous, steady increase in the number of graviresponding seedlings within single parallels. Similar to its effect on seedlings gravistimulated in darkness, MCP efficiently inhibited gravitropic growth of light-grown seedlings during the time period analysed.
Compared with the growth responses observed in parallels of dark-growing seedlings, the graviresponsive growth of seedlings growing under laboratory light conditions in some cases varied significantly between different parallels. Since the temperature of the laboratory was fairly constant, one conceivable parameter on which the observed heterogeneity in the gain of gravicompetence also depended were the heterogeneous weather/sunlight conditions interfering with the light regime within the laboratory. Seedlings were therefore subjected to defined growing conditions in growth chambers, both with respect to light and temperature. As demonstrated in Fig. 3a and b, in water-incubated seedlings illuminated with 15 µE and 30 µE during a 18/6 h light/dark regime, respectively, the number of graviresponding seedlings was similar over 4 d, resembling the results presented in Fig. 1e, apart from slight differences in the extent of the graviresponse. By contrast, seedlings grown under light conditions with 65 µE during a similar daily light regime exhibited a pronounced, continuously increasing gravitropic growth response, both with respect to the number of the seedlings as well as to the extent of this response (Figs 2e, 3c). Additional application of ethephon at a concentration of 300 mg l–1 to seedlings exposed to 65 µE only slightly enhanced the number and the extent of graviresponding seedlings. A further increase in the concentration of ethephon, however, had no additional effect (data not shown). This may imply that light has an additional ethylene-independent enhancing influence on gravicompetence. However, treatment of light-exposed, either water-incubated or ethylene-treated seedlings treated with MCP at a concentration of 3 g l–1 resulted in total inhibition of any graviresponse during the first 3 d (Fig. 3e). Only after 96 h, during which time the compound may either be destroyed by light, or diluted, or otherwise made ineffective, 20% of the seedlings showed some response at an average angle of 30° (Fig. 2f).
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The findings imply that the light-induced effect on gravicompetence is mediated via ethylene synthesis triggered thereby. Therefore, the influence of light on ethylene synthesis was tested by measuring ethylene emission in primary shoot seedlings incubated in sealed measuring cuvettes with a 15/9 h light/dark regime. Similar to previously published results, in which ethylene emission was measured in intact seedlings subject to light/dark cycles (Edelmann et al., 2002), primary shoot seedlings released increasing amounts of ethylene at the onset of the light phases, showing a decreasing trend just prior to the next dark phases, during which the emission rate decreased in general (Fig. 4). Water-incubated primary shoot seedlings exposed to such a light/dark cycle developed gravicompetence, comparable to the results shown in Fig. 2c (data not shown). To test whether effects on gravicompetence similar to the inhibition of ethylene perception by MCP can be achieved by inhibiting endogenous synthesis of ethylene, primary shoot seedlings were incubated together with AVG (aminoethoxyvinylglycine), an inhibitor of ACC-synthase which converts ACC to ethylene (Bleeker and Kende, 2000). Following an initial peak which was observed in most of the measurements, independent of the light conditions (apparently representing an inevitable traumatic response due to the preparation procedure), treatment of the seedlings with AVG resulted in a complete inhibition of endogenous ethylene release (Fig. 4). Along with this effect, AVG-treated seedlings were characterized by a total inhibition of gravitropic growth, similar to the gravitropic growth observed in MCP-treated seedlings (Fig. 2f).
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Analysis of the gravitropic growth stimulated either by light, ACC or ethylene treatment showed that the negative graviresponse was not restricted to a special organ or a special organ part of the primary shoot seedlings (Fig. 5). In about half of the samples differential growth occurred within the developing mesocotyl, apparently independent of the treatment (i.e. in water-incubated, ACC-treated or ethephon incubated seedlings), whereas in the other half of the seedlings bending occurred mainly within the lower basal part of the leaves and/or the mesocotyl and the node. The location of differential growth also appeared independent of whether the seedlings were gravistimulated in darkness or under light conditions.
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Figures 2 and 5 show that light had a strong inhibiting effect on elongation growth of the mesocotyl. Compared with dark-grown seedlings, mesocotyls of light-grown seedlings were, on average, 4–5 times shorter, confirming previously published results on the light-inhibiting effect on mesocotyl growth (Schopfer et al., 2001).
In addition, the overall elongation growth of the entire primary shoot of the seedlings was generally affected in a way that was dependent on the various treatments. In water- and MCP-treated seedlings length increases were similar, whereas in ethephon- and ACC-treated specimens growth was inhibited by an average of 20–35%, respectively. Light had a general growth-inhibiting effect of 30–35% on average, irrespective of the treatment of the samples (Fig. 6).
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The stringent necessity for continuous ethylene perception in order to accomplish a differential gravi-dependent growth of primary shoots is also illustrated in Fig. 7. Primary shoot seedlings were horizontally gravistimulated under laboratory light conditions for a period of 8 d, during which time all of them gained a gravi-dependent vertical orientation. Subsequently, the seedlings were again gravistimulated horizontally by shifting their orientation by an angle of 90° without (Fig. 7a) and with MCP (Fig. 7b). As illustrated, control seedlings showed a pronounced graviresponse during the following 72 h. By contrast, inhibition of ethylene perception in seedlings treated with MCP prevented gravitropic differential growth despite pronounced elongation growth which was similar to the growth of graviresponding controls. Previously gravi-competent primary shoot seedlings of rye are, therefore, no longer able to accomplish gravitropic growth once ethylene perception is inhibited. This indicates that ethylene perception itself is an indispensable step closely connected with the regulation mechanism of gravitropic growth.
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Employing 2-D gel electrophoresis, differences were searched for in the protein patterns inherent in the gravicompetence-generating effect in seedlings treated for 96 h with ACC as the precursor of ethylene, compared with MCP-treated seedlings which have been demonstrated to be totally gravi-incompetent (Fig. 1f, h). The ACC-treated seedlings were chosen because the ethylene synthesis thus mediated appeared to be more compatible with the physiological conditions. As visualized in representative Coomassie-stained gels (Fig. 8), a range of various gravicompetence-associated proteins could be detected by this method. Concentrating on the reproducible, most obvious and prominent ACC-inducible proteins of graviresponding seedlings, Q-TOFF analyses resulted in the identification of calreticulin and lipoxygenase, both of which appeared induced by or at least strongly enhanced within the appropriate tissue of graviresponding seedlings (Table 1).
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Discussion |
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The present study demonstrates the perception of ethylene as an indispensable step for the regulation of gravitropic growth within germinating, per se gravi-incompetent primary shoots of rye seedlings. Data from numerous previous studies (also quoted in an excellent paper by Smalle and Van Der Straeten, 1997) on the relevance of ethylene for the mechanism of gravitropic growth regulation are contradictory (Jackson, 1979; Kaufman et al., 1985; Harrison and Pickard, 1986; Wheeler and Salisbury, 1980; Friedman et al., 1998) and recent studies analysing mutants of tomato attribute ‘multiple non-primary roles’ for the regulation of gravitropic growth (Madlung et al., 1999) to ethylene perception. In previously employed systems immanent gravicompetence was modified, i.e. gravitropic growth was inhibited by a certain percentage after the application of ethylene perception or ethylene action inhibitors, inhibiting values ranging after 24 h, for example, from 5–30% in stems of cocklebur (Wheeler and Salisbury, 1980). In addition, the exogenous application of ethylene has been reported to reduce the absolute, but not the relative, magnitude of the graviresponse of four strains of Arabidopsis (Kiss et al., 1999). By contrast, primary shoots of germinating rye are per se gravi-incompetent, at least below certain light intensities for many days (Edelmann, 1996), as also demonstrated in the present study and to be discussed later. The novel feature of the present system consists in the fact that the competence for gravitropic growth can be generated as well as eliminated, depending on whether or not ethylene perception takes place (Fig. 1). Compared with these data, therefore, earlier results on the effects of ethylene synthesis or ethylene action inhibitors, in principle, only allow the deduction of a certain tendency to interfere in the process(es) thus affected, i.e. the inhibiting effect may be specific or not. It can now be shown that in the case of rye ‘primary shoot seedlings’ ethylene perception is a cardinal, indispensable process for the regulation of gravitropic growth.
As demonstrated in Figs 1 and 2, the gain in the gravicompetence of this system is greatly affected by the light conditions which induce gravitropic growth and which, as shown in Fig. 4, have an enhancing effect on the seedlings’ ethylene emission. During this developmental phase, inhibition of ethylene synthesis and of its perception eliminates the effect of light. So it is conceivable that, within this system, light-induced gravicompetence is gained via induced ethylene synthesis. It has been demonstrated in a number of studies (Subramaniam et al., 1996) that light enhances ACC-synthase which converts ACC into ethylene and, in a number of earlier studies, the enhancing effects of light on gravitropic growth were demonstrated (Feldman, 1983; Mandoli et al., 1984). It is therefore possible that the reported promoting effects of light, for example on starch-deficient tobacco mutants (Vitha et al., 1998), may (also) be due to induced ethylene synthesis, contributing to the enhanced graviresponse. Similarly, restored gravitropic sensitivity in starch-deficient mutants of Arabidopsis due to hypergravity may be due to induced ethylene synthesis, which has been demonstrated to increase as a result of enhanced stress (Morgan and Drew, 1997; Müller et al., 2000). It would therefore be interesting to test whether these effects could also be mimicked by ethylene application and/or could also be inhibited by inhibiting ethylene perception.
Further data supporting the physiological relevance of ethylene perception for the successful negative gravitropic growth of rye seedlings include (a) the gravicompetence-generating effect of exogenously supplied ACC (Fig. 1b), implying either constitutively existing or treatment-induced ACC-oxidase activity, (b) the inhibition of this effect by inhibitors of ethylene synthesis such as AVG (Fig. 4), as well as (c) its inhibition by ethylene perception inhibitors such as MCP (Figs 1d, h, 2f, 3e).
With respect to ethylene release, an interesting phenomenon is the synchronized oscillation of 30 (or in some cases 50) individual specimens incubated in the measuring chambers (Fig. 4). The graph illustrates that enhanced ethylene synthesis strictly coincides with the dark/light shift, whereas a decrease in ethylene release can be observed before the light/dark shift. Ethylene synthesis and the emission thereby mediated out of the seedlings seems to follow some additional circadian rhythm which has also been demonstrated for Sorghum by Finlayson et al. (1998). In accordance with this study’s findings, a recently conducted transcription profiling the early gravitropic response in Arabidopsis revealed that the level of the gene transcripts of several ethylene-responsive element-binding factors (Moseyko et al., 2002) was increased.
In support of the physiological relevance of ethylene perception for the regulation of gravitropic growth, altered ethylene emissions have been measured in excised longitudinal halves of previously graviresponding snap-dragon spikes (Friedman et al., 1998). These authors, seemingly under some compulsion, relate the differential occurrence of ethylene to auxin gradients, which are generally regarded as the means for the regulation of gravitropic growth. From these results, however, the significance of such a gradient for the regulation of differential growth is not obvious. By contrast, if such an ethylene gradient were necessary for gravi-stimulated differential growth, its homogeneous application via the gas phase or within the incubation solution as carried out in the present study would inevitably eliminate any gravitropic differential growth. This is not the case, as demonstrated in Figs 1 and 2. Based on these results, the relevance of ethylene perception for the accomplishment of gravitropic differential growth rather seems to consist in the realization and/or processing of an existing differential profile that depends on the gravistimulus. Yet, although this aspect has, to our knowledge, never been taken into account in previous studies, it could, in principle, also be directly or indirectly involved in the mechanism of graviperception.
Based on our findings, the reported inhibition of a gradient of ethylene production by LaCl3, which also inhibits gravitropic bending may be a consequence of the LaCl3-treatment rather than the cause for the inhibiting effect on gravitropic growth (Friedman et al., 1998). LaCl3 also blocks calcium channels (Tester, 1990) and thus interferes with calcium homeostasis. In numerous previous studies on gravitropic growth regulation, the functional significance of calcium (which in principle plays two roles, one as second messenger and one within the wall) has been demonstrated by Takahashi et al. (1992), and critically discussed by Sinclair and Trewavas (1997). In addition, results of recent studies employing LaCl3 argue that a role is played by Ca2+ within the mechanism of gravitropic growth regulation (Friedman et al., 1998).
In accordance with these results, our data demonstrate that ethylene-generated gravicompetence is characterized by a strongly increased occurrence of calreticulin protein. As suggested by a number of studies (Crofts and Denecke, 1998; Michalak et al., 1999), calreticulin is an ER- and Golgi-localized protein involved in the regulation of calcium homeostasis that can act as a chaperone and also a store for Ca2+-ions (Napier et al., 1995). Overexpression of maize calreticulin has been shown to perturb Ca2+-homeostasis in transgenic tobacco cells (Persson et al., 2001). Interestingly, Wyatt et al. (2002) demonstrated that, in Arabidopsis, overexpression of calreticulin, when grown on low calcium medium, has an enhanced response to gravity compared with controls (http://www.cals.ncsu. edu/nscort/pdf/2000.pdf). In addition, the transcripts coding for calreticulin have been demonstrated to go up 5-fold within 15 min of gravistimulation of maize pulvini (Heilmann et al., 2001). The induction or strong increase of calreticulin protein together with the gain in gravicompetence of primary shoot seedlings of rye therefore supports the relevance of this protein within the regulation mechanism of gravitropic differential growth, although molecular details on the causal relationships have yet to be elucidated. It is conceivable that coating amyloplasts with calreticulin may be of graviperception-related relevance as indicated by the results of Volkmann et al. (1998), who also demonstrated that calreticulin occurs at the plasmodesmata (Baluska et al., 1999).
Similar to calreticulin, the ethylene-perception dependent, enhanced occurrence of lipoxygenase was measured (Table 1). In a number of studies, lipoxygenase has been shown to be increased by jasmonate (Ehret et al., 1994; Hause et al., 1999). Therefore, in preliminary experiments, it was tested whether the ethylene effect could be imitated by applying jasmonate to gravi-incompetent primary shoot seedlings. Despite a pronounced inhibiting effect on growth, the first results indicate that exogenously supplied jasmonic acid also induces gravitropic growth within these systems. The enhanced occurrence of this hormone may therefore represent not an unspecific side effect of ethylene application but a step within or somehow related to the signal transduction of the gravistimulus necessary for the realization of differential growth.
With respect to the organ specificity of the graviresponse, our results indicate that there is no specific organ part accomplishing differential gravi-dependent growth, as Fig. 5 shows. Irrespective of whether the seedlings were grown under light conditions with different growth rates or were dependent on different treatments, gradients of gravi-related elongation growth were observed in the developing mesocotyl and/or the nodes, as well as within the still enrolled leaves, implying a complicated growth pattern within the differently oriented areas of the leaf plane relative to the gravi-vector. Quite obviously, the anatomically very different organs of the seedlings (Fig. 5), show similar graviresponses. This is remarkable, since it also implies that different mechanistic relations have to be co-ordinated differently in order to get similar curvatures. As yet the reasons for the variability are not known; it may also indicate that some cross-talk exists between the responding and non-responding organs. Furthermore, it should be pointed out that the gravitropic bending capacity of these seedlings does not depend on growth velocity as, for example, affected by light conditions (Figs 2, 5).
Based on its pronounced elongation growth this system would, in principle, have the capacity to respond gravitropically, i.e. it would be able to translate transduced signals into different elongation rates. A gain in gravicompetence due to ethylene perception of endogenously synthesized or exogenously supplied ethylene in a homogeneous manner may, therefore, in principle, trigger its effect in the two previous processes generally ascribed to the mechanism of gravitropic growth; it therefore overcomes incompetence in relation to graviperception or insufficient transduction of the perceived gravi-signal (Volkmann and Sievers, 1979; Wilkins, 1979; Sack, 1991), or both. Experiments, aimed at discriminating between the two possibilities, are currently being carried out.
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Conclusions |
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In fast-growing primary shoots of rye, ethylene perception represents an indispensable step in realizing gravitropic differential growth which can occur within the developing mesocotyl, the node between the mesocotyl and the primary leaf, as well as within the enrolled primary leaf itself. Light stimulates the gain in gravicompetence, the effect of which can be inhibited by inhibitors of ethylene synthesis as well as of ethylene perception inhibitors. The effect of light may therefore be mediated via induced or enhanced ethylene synthesis, at least during this developmental stage. A gain of gravicompetence dependent on ethylene perception is accompanied by enhanced levels of calreticulin and of lipoxygenase.
Whether the gravi-relevant processes affected by ethylene perception are intrinsically part of the mechanism of graviperception or of the transduction of the gravi-signal cannot yet be decided.
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Acknowledgements |
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This work was supported by the Fonds der Chemischen Industrie, which is gratefully acknowledged. We also thank M Serek (University of Hannover, Germany) for the supply of MCP.
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References |
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