Journal of Experimental Botany, Vol. 51, No. 344, pp. 587-594,
March 2000
© 2000 Oxford University Press
Low temperature sensing in tulip (Tulipa gesneriana L.) is mediated through an increased response to auxin
1 Agrotechnological Research Institute, Bornsesteeg 59, PO Box 17, 6700 AA Wageningen, The Netherlands
2 Bulb Research Center, Vennestraat 22, PO Box 85, 2160 AB Lisse, The Netherlands
3 Agricultural University of Wageningen, Department of Plant Physiology, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
4 University of Utrecht, Department of Molecular Cell Biology, PO Box 80.056, 3508 TB Utrecht, The Netherlands
Received 5 May 1999; Accepted 30 September 1999
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Abstract |
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Tulip (Tulipa gesneriana L.) is a bulbous plant species that requires a period of low temperature for proper growth and flowering. The mechanism of sensing the low temperature period is unknown. The study presented in this paper shows that the essential developmental change in tulip bulbs during cold treatment is an increase in sensitivity to the phytohormone auxin. This is demonstrated using a model system consisting of isolated internodes grown on tissue culture medium containing different combinations of the phytohormones auxin and gibberellin. Using mathematical modelling, equations taken from the field of enzyme kinetics were fitted through the data. By doing so it became apparent that longer periods of low temperature resulted in an increased maximum response at a lower auxin concentration. Besides the cold treatment, gibberellin also enhances the response to auxin in the internodes in this in vitro system. A working model describing the relationship between the cold requirement, gibberellin action and auxin sensitivity is put forward. Possible analogies with other cold-requiring processes such as vernalization and stratification, and the interaction of auxin and gibberellin in the stalk elongation process in other plant species are discussed.
Key words: Tulip, cold treatment, auxin, gibberelin, mathematical model.
Key words: DMSO, dimethyl sulphoxide, STS, silver thiosulphate.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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A period of low temperature is an essential part of the life cycle of many bulbous plant species. In tulip bulbs (Tulipa gesneriana L.) flower development is induced at higher temperatures (20–25 °C), but subsequent elongation of the flower stalk and proper flowering is dependent on an extended period of low temperature (<10 °C)(De Hertogh and Le Nard, 1993
). This dependence of flowering on low temperatures is a well-known phenomenon and the involvement of gibberellins in this cold requirement has been implicated for many cold-requiring plant species such as winter canola (Brassica napus cv. Crystal; Zanewich and Rood, 1995), chicory (Cichorium intybus L.; Joseph et al., 1983), winter wheat (Triticum aestivum L.; Reda et al., 1978), Thlaspi arvense L. (Hazebroek et al., 1993), radish (Raphanus sativus L.; Michniewicz et al., 1981; Suge, 1970), and also tulip (De Hertogh and Le Nard, 1993). In tulip bulbs, GA application can partly substitute for the required cold treatment (Van Bracht and Zijlstra, 1971; Hanks, 1982; Saniewski and De Munk, 1981). Inhibition of GA biosynthesis greatly reduces stem elongation, both in whole flowering tulip bulbs (Shoub and De Hertogh, 1974; Saniewski, 1989) as well as in isolated sprouts grown in vitro (Rebers et al., 1994). This inhibition is reversed by the application of various gibberellins, but no significant increase in endogenous gibberellins is found during cold treatment compared to untreated bulbs (Rebers et al., 1995).
Auxins are well-known factors involved in stem elongation in several plant species; a quantitative relationship exists between endogenous indole-3-acetic acid (IAA) levels and growth rate in different genetic lines of garden pea (Pisum sativum, Law and Davies, 1990; McKay et al., 1994), in bean stem (Phaseolus vulgaris L.; Bialek et al., 1983) and in lupin hypocotyls (Lupinus albus L.; Ortuño et al., 1990). Other studies show that exogenously applied IAA promotes stem elongation in light-grown garden pea seedlings (Yang et al., 1993), in hypocotyls of sunflower and marrow (Helianthus annuus and Cucurbita pepo; Tamini and Firn, 1985) and watermelon (Citrullus vulgaris; Carington and Esnard, 1988). However, in the latter two studies, the auxin-induced elongation is short-lived and applied auxin even becomes inhibitory. In tulips, auxin is also necessary for stalk elongation. Removal of the flower bud and leaves, both major auxin sources, before the rapid elongation of the floral stalk reduces floral stalk elongation considerably, whereas application of IAA reverses this effect (Saniewski and de Munk, 1981; Okubo and Uemoto, 1985; Saniewski, 1989). The auxin-induced floral stalk elongation is inhibited by application of paclobutrazol, a GA biosynthesis inhibitor (Saniewski, 1989), suggesting that GA biosynthesis is also necessary for auxin-induced floral stalk elongation. Therefore, interaction of gibberellins and auxins is a prerequisite for stalk elongation in tulips (a view strengthened by the work of Okubo and Uemoto, 1985; Okubo et al., 1986; Saniewski, 1989). Also in other species this interaction seems to be important for stem elongation, for example, cucumber (Cucumis sativus L.) hypocotyls (Katsumi and Kazama, 1978), cowpea (Vigna unguiculata L.; Okamoto et al., 1995) and garden pea (Yang et al., 1996).
In the work reported here the mechanism of low temperature sensing is addressed and an attempt is made to clarify the role and possible interaction of both auxin and gibberellin in the regulation of cold-induced stalk elongation in tulip. For this a simple in vitro system of tulip bottom internodes placed on tissue culture medium supplemented with various concentrations of the two hormones was used (a system adapted from Gabryszewska and Saniewski, 1983). The internodes were isolated from tulip bulbs that have been cooled for different periods of time to study the effect of a low temperature treatment on the ability to respond to the different hormones. The obtained dose–response curves are described in terms of a mathematical equation, adopted from the field of enzyme kinetics (as suggested by Weyers et al., 1987). The equation is fitted to the data using regression to obtain a working model for the regulation of stalk elongation by gibberellin, auxin and low temperature treatment. Possible analogies with stalk elongation in other plant species are discussed.
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Materials and methods |
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Plant material
In vitro experiments were performed with stalks from field-grown bulbs (Tulipa gesneriana L. cv. Apeldoorn) harvested in July 1995 (experiment A) and July 1994 (experiment B). Bulbs (circumference 11–12 cm for experiment A and 12–13 cm for experiment B) were stored in dark ventilated rooms at 20 °C.
Internodes were isolated from tulip bulbs by isolating whole sprouts. These were surface sterilized (ethanol rinse for 30 s followed by rinsing in 1% NaClO for 30 min and three consecutive washes with sterilized demineralized water). In a sterile flow cabinet bottom internodes were then isolated from the sprouts and placed inverted (topside in the medium) in glass tubes with 10 ml of solid medium. All internodes were incubated in the dark at 24 °C.
For experiment A, the bulbs were transferred on September 22 1995 to dark ventilated rooms at 17 °C. After 12 weeks the cold treatment began by transferring the bulbs to dark ventilated rooms at 5 °C. Internodes were isolated from the bulbs and placed on medium as described above, after 0 (22 December 1995, start of the treatment), 4 (19 January 1996), 8 (16 February 1996) or 12 weeks (15 March 1996) of cold treatment.
For experiment B, the bulbs were transferred on September 23 1994 to dark ventilated rooms at 5 °C for the cold treatment, or 17 °C for the non-cooled controls. After 12 weeks internodes were isolated from the bulbs and placed on medium as described above.
Growth medium and auxin and gibberellin application
Internodes were incubated on standard Murashige and Skoog (Murashige and Skoog, 1962) medium (Duchefa, Haarlem, the Netherlands), with 30 g l-1 sucrose added and 0.8% agar (Duchefa). The medium was autoclaved for 20 min. To prevent possible ethylene effects, filter-sterilized (0.22 µm disposable filter, Amicon) silver thiosulphate (STS) solution was added to the medium to a final concentration of 50 µM. Indole-3-acetic acid (IAA, Duchefa) was added from a filter-sterilized stock-solution (10 mM). GA3 (Duchefa,) stock solution was prepared by dissolving it in dimethylsulphoxide (DMSO) to a final concentration of 100 mM. For the lower GA3 concentrations a 10 times diluted stock was used. DMSO concentration in the media was, if necessary, corrected by adding DMSO to a final concentration of 0.1% (v/v). STS, IAA and GA3 were added after autoclaving.
In experiment A internodes from 0, 4, 8, and 12 weeks cooled bulbs were grown on media containing a range of eight concentrations of IAA (0, 0.1, 0.3, 1, 3, 10, 20, and 30 µM) combined with two concentrations of GA3 (0 and 100 µM). For every hormone combination 10 internodes were used. In experiment B, internodes from both cooled and non-cooled bulbs were grown on media containing IAA as above combined with a range of six concentrations GA3 (0, 0.3, 1, 3, 30, and 100 µM). Per combination 10 internodes were used, both for cooled and uncooled bulbs.
Data analysis
The elongation of the internodes was monitored for 4 weeks. Sometimes internodes did not show a growth response, and later started to show a callus-like growth, especially at higher IAA concentrations. These internodes and any infected ones were discarded. On average 2.7 internodes per hormone combination were discarded, resulting in average 7.3 usable samples per hormone combination. The lengths of internodes for every IAA–GA3 combination were averaged for every time point and a logistic growth-curve was fitted through them from which the maximum elongation rate (mm d-1) during these 4 weeks was determined. If a logistic growth curve could not be fitted (at low IAA concentrations and in zero or short cold treatments) the maximum elongation rate was determined as the maximum rate of change between two data points. The maximum rate is normally reached within the first week of growth. The maximum elongation rate, i.e. the slope at the steepest point in the growth curve, throughout this study is referred to as the response of the internodes to a combination of hormones (as suggested by Weyers et al., 1987).
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Results |
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Adapting an in vitro system for studying hormonal regulation of low temperature-dependent stalk elongation
The tulip flower stalk is only able to elongate rapidly after a minimally required cold treatment (De Hertog and Le Nard, 1993
). In field-grown bulbs, the largest relative change in internode elongation and elongation rate after different periods of low temperature treatment occurs in the bottom internode, which is the first to begin elongating after planting (data not shown).
To investigate this further, bottom internodes were placed inverted on solid tissue culture medium supplemented with auxin (1 µM), gibberellic acid (GA3, 10 µM) or both. At these hormone concentrations the elongation rate of the bottom internode is mainly determined by auxin, with an enhancing effect by low temperature treatment of the bulbs and the presence of gibberellic acid (Fig. 1). Both latter factors are themselves not sufficient to induce a significant increase in elongation compared to the control. The endogenous hormone level in the bottom internodes is not sufficient for induction of a rapid elongation rate.
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The in vitro system was used for a detailed evaluation of the hormonal regulation of low temperature-dependent internode elongation. Experiment B shows that both the cold treatment and the addition of GA3 to the medium enhance the elongation response of the internodes to auxin and shift the maximum response to lower auxin concentrations (Fig. 2
C and D versus A and B, and A and C versus B and D, respectively). The combination of cold storage of the bulbs and addition of GA3 to the medium has the largest effect on the elongation.
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The auxin response is analysed as the maximum observed elongation rate over the time-course of the elongation curve (as suggested by Weyers et al., 1987
).
Auxin dose–response curves fit equations for uncompetitive inhibition of enzyme kinetics
Interaction between a hormone and its receptor can be considered as substrate enzyme interactions (Weyers et al., 1987), as long as the same assumptions and simplifications are applied as in the Michaelis–Menten model for enzyme kinetics. At higher IAA concentrations, elongation was inhibited and this inhibition was independent of ethylene (data not shown). Therefore, equations describing different kinds of inhibition were fitted to the dose–response curves. The equations are derived from the Michaelis–Menten model for saturated enzyme kinetics. The equation that fits best is an adapted equation describing uncompetitive inhibition (Segel, 1975). The reaction scheme is as follows:
 | (a) |
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Replacing the terms substrate and enzyme with hormone and receptor, respectively, and replacing the term inhibitor by hormone, results in the following scheme:
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 | (2) |
Cold treatment gradually enhances auxin response
In experiment A the elongation curves of the bottom internodes at different auxin concentrations were recorded and the response to auxin was determined as described above after 0, 4, 8 or 12 weeks of cold storage. Auxin dose–response curves were fitted using the equation described previously. Increasing the length of the cold storage has a significant influence on the response of the internodes to auxin (Fig. 3A). The maximum response level of the internodes increases with increasing length of the cold treatment. This is further referred to as the responsiveness of the internodes to IAA. The corresponding IAA concentration at which the maximum response is reached (IAAmax) is further referred to as the sensitivity. Thus, a lower IAAmax means a higher sensitivity. The maximum response and corresponding IAAmax can be calculated using the estimated parameters of the fitted equations (Table 1). The cold treatment enhances the responsiveness dependent on its duration, leading to a 3-fold increase after 12 weeks of treatment. The cold treatment also increases the sensitivity of the internodes, although this effect is more pronounced during the first 8 weeks of the treatment.
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---), 8 weeks at 5 °C (–·–
–·–), 12 weeks at 5 °C (---
---). Per data point an average of 7.3 internodes was used, so per curve an average of 58.4 internodes was used.
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GA3 in the medium enhances the response to auxin independent of low temperature treatment
The addition of GA3 enhances both the responsiveness and sensitivity of the internodes to auxin (Fig. 3B; Table1). However, after 12 weeks of cold treatment the effect of GA3 in the medium on both parameters is minimal, although a general broadening of the dose–response curve can be observed (Fig. 3B versus 3A).
To determine the effect of different GA3 concentrations in the medium on the auxin response of bottom internodes, the elongation curves at different auxin and gibberellin concentrations were recorded and analysed as described above. Increasing amounts of GA3 result in a gradual shift of the dose–response curves. Both the responsiveness and the sensitivity are increased, for both cooled or non-cooled tulip bulbs (Table 2). The addition of >30 µM of GA3 to the medium does not lead to an additional effect, either on the maximum response or on the IAAmax. These results confirm those of a similar experiment performed in the season 1993/1994 (data not shown).
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Discussion |
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Rapid floral stalk elongation in tulips only occurs with bulbs that have received a low temperature treatment. This study clearly shows that the effect of the cold treatment is on the response to auxin, which is the main hormonal factor involved in the induction of the elongation in vitro. Upon cooling, the internodes show a higher responsiveness and sensitivity to auxin (Table 1; Figs 2, 3). This change in the response of the bottom internodes to auxin gradually increases with the length of the cold treatment. However, gibberellin without auxin is not able to cause a significant elongation, and thus auxin is the main factor determining the elongation rate (Figs 2, 3). At higher auxin concentrations in the medium (IAA >10 µM) the elongation rates dropped (Fig. 3). Ethylene was eliminated as an interacting factor because no significant ethylene production could be detected during elongation and the addition to the medium of silver thiosulphate (STS), an inhibitor of ethylene action, had no effect on the reduction of elongation rates at higher auxin concentrations (data not shown).
The role of gibberellic acid
Adding gibberellin to the growth medium has an effect on the auxin response that is independent of, but very similar to the effect of a cold treatment. A higher gibberellin concentration results in a higher responsiveness and higher sensitivity to auxin (Table 2). At GA3 concentrations higher than 30 µM a plateau in the effect of GA3 on the dose–response curves is observed. That gibberellin is a necessary component in the elongation process, is illustrated in vitro by the effect of paclobutrazol application on the elongation of complete sprouts (Rebers et al., 1994). It reduces stem elongation severely, and its effect is reversible by gibberellin application. This need for gibberellin during the elongation phase is also present in vivo as the addition of gibberellin biosynthesis inhibitors after planting inhibits elongation, with its main effect in the bottom internode (Shoub and De Hertogh, 1974; Saniewski, 1989; Suh et al., 1992). The site for GA biosynthesis appears to be the growing root system as removal of the roots inhibits stalk elongation, which could be reversed by the addition of GA3 (Kawa-Miszczak et al., 1992).
The maximum response levels and optimum auxin concentration in Tables 1 and 2 cannot be compared in an absolute way because the experiments were performed in two different seasons. The main difference between the data in the two tables is the magnitude of the gibberellin effect. The effect of GA3 on the response levels and optimum auxin concentration in Table 2 is higher (than in Table 1). The cold storage for the experiment resulting in the data of Table 1 ended later in the season than for the experiment resulting in the data of Table 2. Also, in vivo, stalk elongation is reported to be more rapid later in the season, suggesting a reduced need for gibberellins at that time (Saniewski, 1989).
It is postulated that a cooling period is needed to increase the sensitivity of the tulip sprout for gibberellins (Hanks, 1982). However, the results in this paper show that gibberellins are not the main factor involved in cold-induced stem elongation, although it is clear that de novo synthesis of gibberellic acid is a prerequisite for stalk elongation in tulip bulbs. The hypothesis for this study is that in vivo the auxin response in the internodes of non-cooled bulbs is not sufficient for proper elongation. Only after proper cold treatment and the resulting shift in the auxin response are the internodes able to elongate at a proper rate, but only in the presence of certain amounts of gibberellins. After cold treatment, GA3 is able to induce an even greater enhancement of the auxin response, but late in the season this effect is less clear. GA3 alone is not able to induce the change in the auxin response that can be induced by an optimal cold treatment. Thence gibberellins and cold treatment may not operate via the same modi. They affect the auxin response in separate ways, but with a similar result.
The involvement of gibberellins in vernalization and stratification is well known in many plant species (Metzger, 1995; Rock and Quatrano, 1995), but until now little has been reported about a decisive role for auxin sensitivity in low temperature-induced processes. Interaction of auxin and gibberellins as being important for stem elongation is suggested also for other species, as described in the Introduction. In one study in particular on garden pea, the stimulating effect of GA is mainly attributed to an increase in cell number indicating that GA stimulates cell division pea (Yang et al., 1996). It was shown that, in tulip, the elongation of the floral stalk after planting is due to cell elongation only (Gilford and Rees, 1973).
Mathematical modelling
The curve-fitting procedure used in this study is based on suggestions made by Weyers et al. (Weyers et al., 1987). They suggest that hormone receptor interaction can be considered as substrate enzyme interaction. This technique has been applied by other authors to describe the effect of ethylene on flower senescence (Woltering et al., 1993), of nitrate on light-induced germination (Hilhorst, 1990) and of abscisic acid on stomatal closure (Weyers and Paterson, 1992). Although several different equations taken from the field of enzyme kinetics were used for curve fitting, the best results were obtained with the one adapted from the model for uncompetitive inhibition (data not shown). In all dose–response curves the inhibitory effect of IAA at high concentrations is evident. However, it is only possible to speculate on the significance of this for the molecular mechanism of auxin action, although the results exclude the involvement of ethylene. It is suggested that reaction scheme b is best interpreted in such a way that the equation term RH is not considered as the real hormone–receptor complex, but as a general term describing all the steps from the hormone binding to its receptor to the final changes in the cells that cause the elongation. The inhibition by IAA can take place on any of these events in the chain leading from receptor binding, but also on events preceding this binding, such as activities of conjugating enzymes or transport of exogenous hormone.
The most important reason to describe the dose–response curves by mathematical equations is to be able to compare the curves in a quantitative way and thus compare the effects of low temperature and gibberellin on auxin perception. In this study it is not meant to elucidate what the molecular mechanism is of auxin perception itself.
Low temperature sensing by a change in the response to auxin
Figure 4 depicts a hypothetical model for low temperature induction of stalk elongation in tulip. It is shown that auxin is the hormone responsible for the elongation of the bottom internode in tulip, with both cold treatment and gibberellins enhancing the response to auxin. In the model the auxin perception complex must be interpreted as all steps between binding of auxin to its receptor to the actual elongation, and thus includes signal transduction events, primary and later gene induction events resulting in elongation. These data and data from the literature suggest that this change in auxin response is the main factor responsible for the different growth behaviour in vivo of cold-treated versus untreated tulip bulbs and thus that low temperature sensing in tulip is mediated by this change in auxin response. It is a clear example of modulating plant development by changing the sensitivity for a plant growth regulator (as presented by Trewavas, 1981, 1982). However, the results show that auxin acts as a real plant hormone, contrary to the view described in these papers. The effective range of the dose–response curves spans only two orders of magnitude and auxin can thus effectively regulate elongation. Internode elongation in tulip is thus a case of combined control (a mechanism proposed by Weyers et al., 1995). The low temperature changes the response to auxin, but the actual elongation only occurs when auxin is supplied (for example by the leaves or flower bud).
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In tulip, stalk elongation is dependent on low temperature, but flower formation is not. In some other bulbous species like iris, both the initiation of flower development and stalk elongation are dependent on reduced temperatures (De Munk and Schipper, 1993
). This is also true for many vernalizable plant species, and it would be interesting to investigate if increased auxin sensitivity occurs also in these species. Presently, the tulip is being used as a model system for the isolation of genes involved in low temperature regulation and auxin sensitivity. The advantage of this system is the absence of interfering processes such as flower development as is the case in many vernalizable species, especially in crucifers like Thlaspi arvense (Hazebroek et al., 1993). In conclusion, cold treatment and gibberellin positively enhance the auxin response in the bottom internode in tulip, and thus its potential to elongate rapidly. It is hypothesized that in vivo rapid elongation only occurs when the sensitivity to auxin is increased sufficiently by a low temperature treatment and that for optimal elongation, interaction of auxin and gibberellins is important.
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Acknowledgments |
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We thank Dr E Gabryszewska and R Luyten for their assistance during the initial part of this work. We also thank Drs EJ Woltering and GJA Rouwendal for critically reading of the manuscript. This work was supported in part by the Life Sciences Foundation (SLW), which is subsidized by the Netherlands Organisation for Scientific Research (NWO).
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Notes |
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5 Present address and to whom corespondence should be sent. Genetwister Technologies BV, PO Box 193, 6700 AD Wageningen, The Netherlands. Fax: +31 317 475347. E-mail:a.d.deboer{at}genetwister.nl
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