AOBPreview originally published online on January 22, 2007
Annals of Botany 2007 99(3):555-560; doi:10.1093/aob/mcl286
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Following the Initiation and Development of Individual Leaf Primordia at the Level of the Shoot Apical Meristem: The Case of Distichous Phyllotaxis in Begonia
1 Institut de recherche en biologie végétale, Jardin botanique de Montréal, 4101 Sherbrooke Est, Montréal, Canada H1X 2B2
2 Department of Biology, University of Prince Edward Island, 550 University Avenue, Charlottetown, PEI, Canada C1A 4P3
3 Laboratoire de Cytologie Expérimentale et Morphogenèse végétale, Université Pierre et Marie Curie, Bât. N2, 4 place Jussieu, 75 252 Paris Cedex 05, France
* For correspondence. E-mail denis.barabe{at}umontreal.ca
Received: 8 September 2006 Returned for revision: 18 October 2006 Accepted: 23 November 2006 Published electronically: 22 January 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Background and Aims: By using the technique of replicas of a developing apex it is possible to obtain a direct measure of phyllotactic parameters (plastochrone and platochronic ratio) involved in the initiation of two successive primordia at the level of the SAM. The goal of this study is to compare, in a real time setting, the value of phyllotactic parameters in distichous sytems using Begonia as a case study, with the value of the same parameters in spiral phyllotactic systems.
Methods: To determine the real-time sequence of events at the level of the SAM, replicas were made of the developing apex at different intervals using previously described techniques. Impression moulds were made at 24-h intervals. The following phyllotactic parameters were measured: plastochrone, angle of divergence, plastochrone ratio and ratio between the diameter of the leaf and the apex.
Results: The time between the appearance of two successive leaves is 15–20 d. The average value of the plastochrone ratio (R) is 1·3, and the ratio of the leaf to the diameter of the apex (
) is 2·5. The angle of divergence varies from 165º to 180º. The speed of advection of the primordium from the apex, varies from 0·28 to 0·37 µm d–1.
Conclusions: The speed of advection of primordia in Begonia is lower than that of Anagalis. This is not in accordance with theoretical simulations that predict the opposite. In Begonia, the plastochrone ratio does not reflect the real time of appearance of two successive primordia. The time separating the appearance of two primordia is not directly related to the distance of these two primordia from the centre of the apex but is related instead to the enlargement of leaves.
Key words: Shoot apex, models, development, phyllotaxis, leaf primordia, Begonia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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In the analysis of phyllotactic organization, one of the main problems is the determination of the time of appearance of primordia in the context of the three general types of phyllotactic patterns: spiral, verticillate and distichous. Phyllotactic systems are generally described by using different parameters (Fig. 1) such as divergence angle (d), plastochrone ratio (R) (the ratio of the distance of two successive primordia from the centre of the apex; Richards 1951), ratio between the radius of a primordium and the radius of the shoot apical meristem (SAM) [parameter b of van Iterson (1907) or
of Douady and Couder (1998)], and number of opposed visible spirals (m, n) called parastichies. For a review of the significance of these parameters in phyllotaxis see Jean (1984). Among all these, parameters R or b constitute an indirect estimation of the time separating the initiation of two successive primordia (Douady and Couder, 1992, 1993).
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Rutishauser (1998) showed empirically that spiral systems are characterized by a lower plastochrone ratio than distichous sytems. These observations correspond to predictions based on theoretical models (Jean, 1984). The experiment of Douady and Couder (1992), which compared leaf primordia with droplets of ferrofluid moving in a magnetic field, simulated this phenomenon adequately and confirmed many of Rutishauser's observations.
In the simulations of Douady and Couder (1992, 1993), the determination of phyllotactic patterns depends on only one dimensionless parameter G = VT/R0, where V represents the speed of advection of droplets, T the periodicity of appearance of droplets and R0 the radius of the zone where particles appear. These authors suggested that, although the dimensions and time scales are not the same between their physical experiment and botanical reality, the value of the parameters can be identical allowing for a comparison between their model and botanical data.
In the model of Douady and Couder (1992, 1993), it was shown that the speed of advection of droplets, which depends on T and R, is higher in distichous systems than in spiral systems. In theoretical and empirical models, it is easy to obtain theoretical values for R. However, the only way to calculate the value of V is to know the real time span separating the initiation of two leaves at the shoot apex.
It is difficult to measure the time of appearance of primordia directly at the shoot apex during the exponential phase of growth. The plastochrone index (Erickson and Michelini, 1957) is the main method used to determine the relative time of appearance of leaves during plant growth. Although this method is very useful in different types of morphological and physiological studies, it does not provide information on what occurs at the shoot apex during the exponential phase of growth. One way to determine the real time sequence of events at the level of the SAM is to make replicas of the developing apex at different intervals using the technique described by Williams et al. (1987), Williams and Green (1988) and Green and Linstead (1990). This method was successfully applied to the analysis of the SAM of Vinca major (Williams, 1991), Graptopetalum paraguayense (Tiwari and Green, 1991), Anagallis arvensis (Green et al., 1991; Kwiatkowska and Dumais, 2003) and Arabidopsis thaliana (Kwiatkowska, 2004) among others. With this method it is possible to obtain a direct measure of the time (plastochrone) interval separating the initiation of two successive primordia at the apex. For example, based on our estimations, the plastochrone corresponds to 5 d in Vinca (Williams, 1991) and to 2 d in Anagallis (Kwiatkowska and Dumais, 2003).
Until now, Green's method (Green and Linstead, 1990) has only been applied to spiral (e.g. Anagallis) or opposite decussate (e.g. Vinca) patterns, not distichous systems like Begonia. It is not surprising to note that there are no studies of distichous or spirodistichous patterns using the sequential replica method because in these phyllotactic patterns (e.g. Zea, Oryza and Begonia) the very small apical meristem is nearly completely enclosed by surrounding leaves (Haccius, 1940; Barabé et al., 1992a, b; Charlton, 1998). Therefore, in contrast to spiral patterns, it is difficult to make many casts of the apex during a long period of time without damaging the apical surface or the surrounding leaves. The development of the leaf of Begonia, which has been analysed in detail in previous studies (Barabé et al., 1991, 1992a, b; McLellan et al., 1993; Jeune and Barabé, 1995; Thibault et al., 1997), represents a good source of data to analyse the sequence of appearance of primordia in distichous systems. As far as is known, this study of Begonia is the first observation of a distichous or spirodistichous phyllotactic pattern in real time.
The general goal of this study is to compare the value of parameters T, R, b and V in distichous sytems using Begonia as a case study to the value of the same parameters in spiral phyllotactic systems to determine if theoretical estimates are valid. The value of T will be determined by direct observations of the developing SAM. Data for spiral patterns will be estimated from a previous study describing the development of the apex of Anagallis in real time (Kwiatkowska and Dumais, 2003). This quantitative comparison will make it possible to determine if the prediction made by theoretical models corresponds to reality, and to discuss the value of theoretical models in the estimation of phyllotactic parameters.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Specimens of Begonia scabrida A. DC. (#2184-57) were obtained from the living collection of the Montreal Botanical Garden (Canada).
Dissection and impression moulds
The shoot tip of living plants was dissected using fine forceps and insect needles (#00, #0, #1) to reveal the shoot apical meristem. Immediately following dissection, a polyvinylsilaxane polymer mixture was applied to the apex with an insect needle and left there to dry for a few minutes to take an impression and prevent any dehydration. The resulting mould was carefully removed by using fine forceps and fixed on a microscope slide with a small quantity of the same polymer. The polymer mixture used consists of one part Kerr's Reflect resin and one part Kerr's Mirror wash catalyst (Kerr UK Ltd., Peterborough, UK); these proportions ensure the desired fluidity to adequately cover all areas of the apical meristem.
Immediately after taking an impression, the shoot apex was sprinkled with water and covered with a 1·5-mL Eppendorf microtube containing some water. The entire plant material was covered with a plastic bag containing paper towels soaked in water to prevent further dehydration. Impression moulds were made at 24- or 48-h intervals. Ten trials were done on different species (B. fagifolia, B. listida, B. roxburghii and B. radicans). However, due to the difficulty associated with making many casts of the same apex without damaging developing primordial, only two specimens yielded data that covered one plastochrone of development.
Specimen reconstruction
Impression moulds were filled with epoxy glue (Devcon Corporation, Wood Dale, USA). Equal amounts of resin and hardener were applied to a microscope slide and gently mixed with a straight dissection needle to avoid the formation of bubbles in the mixture. The epoxy was then applied to the mould using a Pasteur pipette with a rounded tip that was shaped under a flame to a diameter of 0·34 mm.
The moulds were gradually filled by making sure the entire inner surface was covered and no air bubbles formed. Once the epoxy hardened, the impression was removed from the mould.
Microscopy
Epoxy specimens, representing a positive reproduction of the shoot apical meristem at the time of impression, were mounted on metal stubs, sputter coated with a thin layer of gold-palladium, viewed with a Jeol JSM 35 scanning electron microscope operating at an accelerating voltage of 20 or 25 kV, and photographed using 120-mm photographic film. Photographic negatives were scanned at high resolution and the resulting digital images were used to produce the photographic plates with Photoshop version 7.
The centre of leaf primordia was determined by using the median point on the axis of symmetry of the leaf. The diameter of the SAM was determined by using the area enclosed between two leaves. To determine the centre of the SAM the median point of the distance between those two primordial leaves was used.
- d: angle of divergence; the angle between two successive primordia
- r: rate of growth ln(R)/T; rate of displacement of a primordium
- R0: radius of the SAM
- R: plastochron ratio; the ratio of the distance of two successive primordia from the centre of the apex
- T: plastochrone (days); the ‘time’ interval between the initiation of two successive leaves
- V: speed of advection (µm d–1); the speed at which a primordium becomes more distant from the centre of the SAM
- r: rate of growth ln(R)/T; rate of displacement of a primordium
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RESULTS |
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Developmental morphology
Throughout its development, the leaf primordium of Begonia occupies a significant portion of the region of the shoot apex. The stipules at the base of the leaf give the primordium a wide area of insertion (Fig. 2A). The larger of the two stipules is associated with the broader side of the blade (Fig. 2B) and both stipules nearly enclose the next leaf primordium that is initiated on the apex SAM (Fig. 2C, arrow). The blade portion of the leaf assumes an asymmetrical shape during early stages of initiation and maintains this asymmetry throughout development (Figs 2B and 3, days 1–5).
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Figure 3 represents the developmental sequence of a single shoot over a period of 15 d between the initiation of two successive leaves. The area of the SAM is tightly delimited by the base of the leaf primordium which includes the stipules. This area remains relatively unchanged as it becomes progressively enclosed by the developing stipules (days 1–8). By days 9 and 10 in this sequence, the next leaf primordium is initiated as a bump which eventually takes up most of the area of the SAM (days 10–15). The SAM will recover its surface area after the formation of the blade of the leaf (day 1). Based on detailed comparative developmental data, the time between the initiation of two primordia ranges between 15 and 20 d.
Quantitative results
Values of the different parameters under analysis are summarized in Table 1. As noted previously the time between the appearance of two successive leaves is 15–20 d. The average value of the plastochrone ratio (R) is 1·3. By using this value, and the real time separating the formation of two leaves, it is possible to calculate the speed of advection (parameter V) of Douady and Couder (1992, 1993) following their formula G = VT/R0, where T represents the plastochrone and R0 the radius of the SAM. Given that growth near the apex is considered exponential, ln R = G, and V = r x R0, where r is the rate of growth ln(R)/T (Richards, 1951). The speed of advection, which represent the speed of removal of the primordium from the SAM, is therefore equal to 0·28–0·37 µm d–1. The mean value of , which corresponds to the ratio of the diameter of the leaf to the diameter of the SAM, varies from 1·2 to 6·3, depending on the stage of development of the leaf, with an average of 2·5. The angle of divergence varies from 165º to 180º (Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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The value of the plastochrone ratio (R) in Begonia and Anagallis appears, respectively, in the range of distichous systems (1·2–1·67) and spiral systems (1·0–1·67) observed by Rutishauser (1998) for different species (Table 1). However, the plastochrone ratio is strongly inferior to that reported previously by Barabé et al. (1991) for B. scabrida (R = 2·0). This can be explained by the fact that this value of two was obtained using leaves that were in their phase of elongation, instead of taking measurements during the initiation of primordia, as is the case in the present study. During the phase of elongation, the petiole of the oldest leaf is farther away from the centre of the apex than during the phase of initiation (Barabé et al., 1991, fig. 2). According to Douady and Couder (1998), a value of
superior to 1·9 leads to distichy. This is the situation in Begonia where the value of is approximately four times (2·5) that of a spiral system (
0·60) (Table 1). Based on these parameters there appears to be a good concordance beween the theoretical model and empirical data. However, it is not the case for the speed of advection. The speed of advection of primordia (which takes into account the plastochrone in real time) in Begonia is lower than that of Anagalis (Table 1). This is not in accordance with theoretical simulations that predict the opposite relationship. The parameters that were measured and deduced for B. scabrida are in the same range of values in other Begonia species such as B. fagifolia, B. listida and B. roxgurghii (D. Barabé, unpubl. res.).
In the simulation of Douady and Couder (1992) for a divergence angle of 180°, two primordia move quickly away from each other during a period of time sufficient to allow the formation of a new primordium in the opposite position. The time separating the formation of two primordia corresponds to a distance relationship between them and the SAM, and is characterized by the plastochrone ratio. This view of phyllotactic dynamics forms the basis of many theoretical models such as those of van Iterson (1907), Richards (1951), Jean (1994) and Guerreiro and Rothen (1998). In contrast, in Begonia, the latest formed primordium does not initially ‘move away’ from the centre of the SAM (Fig. 4). This is represented by the low value of the speed of advection. To have a speed of advection equivalent to that of the spiral system of Anagalis, the plastochrone ratio would have to be e2·18 ( = 8·85). Obviously, a plant with such a plastochrone ratio has not been reported yet. The long delay between the formation of two successive primordia is due to the formation of an extensive area of insertion of the leaf around the apex, which corresponds to a high value of , a low value of G, and to a leaf that is not displaced from the SAM initially. This is different from theoretical models that predict a strong positive correlation between , R and G. This result indicates that, although the simulation of Douady and Couder (1992, 1993) is analogous to a growing apex in a temporal framework, it does not correspond to the geometry and the growth dynamics involved in a distichous system. Here there is no correlation between the plastochrone (T) and the plastochrone ratio (R) in real time. Among the phyllotactic parameters that were studied, it is that allowed for a better characterization of distichous and spiral systems.
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In the distichous system of Begonia (it is probably the case for many distichous sytems) the establishment of the phyllotactic system is governed by local biological mechanisms linked to the developmental state (diameter) of the last formed leaf and not to its position (distance) with respect to the preceding leaf. This mode of development is difficult to explain using strictly biophysical mechanisms such as the presence of a physical undulating field (buckling) hypothesized by Green et al. (1998). However, the distichous phyllotaxis of Begonia may probably be integrated more easily in models where auxin is required for organ initiation and positioning (Kuhlemeier and Reinhardt, 2001; Smith et al., 2005). Following Reinhardt et al. (2003), one may speculate that only the last formed leaf is effective as a sink of auxin resulting in a distichous phyllotactic pattern. The preceding interpretation remains speculative and more developmental studies are needed to explain the phyllotaxis of Begonia.
It is important to note that in order to make the impression moulds of the apex of Begonia, the stipules of the developing leaf which enclose the apex must be removed. This manipulation probably induces some form of physiological stress at the level of the SAM that leads to a slowing down of growth, and the appearance of a delay in the development of incomimg primordia. However, considering that the apex is a well-integrated entity, it is believed that such a pertubation is not sufficiently strong to affect the general temporal scale that distinguishes between distichous and spiral phyllotactic systems.
Given that R and R0 remain constant during the exponential phase of growth, if the sequential replica method leads to an increase of the plastochrone value, T0 
T1 = 
T0 with > 1. Therefore the unperturbed speed of advection V0 will be reduced proportionally:
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< 8·75 and 1·71 < T0 < 3·38 will be obtained. This indicates that the theoretical unperturbed plastochrone (T0) would need to be <4 d to invalidate the present results. Recent observations of unaltered leaves measuring 1 cm in length as a starting point on specimens of B. cabrida growing in the greenhouses of the Montreal Botanical Garden showed that the plastochrone is not <10 d (D. Barabé, unpubl. res.). Therefore this observation supports the developmental results presented in this paper.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Our results show two points concerning the interpretation of theoretical models in relation to distichous phyllotactic systems: (1) the plastochrone ratio does not always reflect the real time of appearance of two successive primordia; (2) the duration between the appearance of two primordia is not directly related to the distance of these two primordia from the centre of the apex but to the enlargement of leaves. These points should therefore be taken in consideration in the modeling of phyllotactic organization.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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The authors would like to thank Charles Bertrand and Linda Dumont for their technical support for this project. This research was supported by individual discovery grants from the Natural Sciences and Engineering Research Council of Canada to Denis Barabé and Christian Lacroix.
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