JXB Advance Access originally published online on August 1, 2006
Journal of Experimental Botany 2006 57(12):3091-3098; doi:10.1093/jxb/erl072
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RESEARCH PAPER |
The geometry of the forisome–sieve element–sieve plate complex in the phloem of Vicia faba L. leaflets
1Indiana University/Purdue University, Department of Biology, 2101 East Coliseum Boulevard, Fort Wayne, IN 46805-1499, USA
2Institut für Allgemeine Botanik, Justus-Liebig-Universität, Senckenbergstr. 17–21, D-35390 Gießen, Germany
3School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA
*To whom correspondence should be addressed. E-mail: petersw{at}ipfw.edu, knoblauch{at}wsu.edu.
Received 11 April 2006; Accepted 2 June 2006
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Abstract |
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Forisomes are contractile protein bodies that appear to control flux rates in the phloem of faboid legumes by reversibly plugging the sieve tubes. Plugging is triggered by Ca2+ which induces an anisotropic deformation of forisomes, consisting of a longitudinal contraction and a radial expansion. By conventional light microscopy and confocal laser-scanning microscopy, the three-dimensional geometry of the forisome–sieve element–sieve plate complex in intact sieve tubes of leaflets of Vicia faba L. was reconstructed. Forisomes were mostly located close to sieve plates, and occasionally were observed drifting unrestrainedly along the sieve element, suggesting that they might be utilized as internal markers of flow direction. The diameter of forisomes in the resting state correlated with the diameter of their sieve elements, supporting the idea that radial expansion of forisomes is the geometric basis of reversible sieve tube plugging. Comparison of the present results regarding forisome geometry in situ with previously published data on forisome reactivity in vitro makes it questionable, however, whether forisomes are capable of completely sealing sieve tubes in V. faba leaves.
Key words: Ca2+-dependent contractility, contractile protein, forisome, phloem transport, sieve element plugging, sieve tube geometry, Vicia faba L
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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The sieve elements in the phloem of higher plants are highly differentiated cells forming the syncytial sieve tube network that accomplishes the distribution of photo-assimilates throughout the plant body (Evert, 1977; Behnke and Sjolund, 1990; Sjolund, 1997; van Bel and Knoblauch, 2000; van Bel et al., 2002). Phloem-specific protein bodies in the faboid legumes, so-called ‘non-dispersive crystalline P-proteins’ (Behnke, 1991), had previously been regarded as static inclusions of the sieve elements (Sabnis and Sabnis, 1995). However, these P-protein bodies perform rapid Ca2+-regulated conformational changes which lead to the reversible formation of sieve tube plugs (Knoblauch et al., 2001). This reaction occurs in response to stimuli such as shifts in turgor and extracellular osmotic pressure. While its mechanism is not fully understood to date (Pickard et al., 2006), it appears likely to be involved in the well-known transient decreases in phloem transport rate inducible by external stressors in legumes (Pickard and Minchin, 1990, 1992). As the legume P-protein bodies can form plugs in <1 s (Knoblauch et al., 2001, 2003), they provide a rapid sieve tube sealing mechanism that complements the ubiquitous but much slower sieve plate occlusion by callose synthesis (Eschrich, 1975). Their proposed function as sieve tube gates led to the legume P-protein bodies being renamed forisomes (gate-bodies; Knoblauch and Peters, 2004a) which is also apt because forisomes are neither ‘non-dispersive’ nor ‘crystalline’. In the model organism, the broad bean Vicia faba L., forisomes typically are spindle-shaped and
30 µm long. In isolated specimens, Ca2+ caused a shortening of the long axis by some 30% and a >2-fold increase in diameter (Knoblauch et al., 2003). This reaction was reversed upon addition of a chelator, and could be mimicked partly by unphysiologically high or low pH (Knoblauch et al., 2003). As they combine several desirable properties, forisomes appear to be a useful natural prototype for protein-based smart materials with applications in micro/nanotechnology and microfluidics (Knoblauch and Peters, 2004b). There is no shortage of excellent investigations into the ultrastructure of sieve elements containing forisomes in various legume species (Laflèche, 1966; Wergin and Newcomb, 1970; Palevitz and Newcomb, 1971; Fisher, 1975; Lawton, 1978a, b; Arsanto, 1982). However, these studies were conducted before the structural dynamics of forisomes became known, and did not contribute to the discovery of forisome contractility due the static nature of electron micrographs. Moreover, in an extremely sensitive tissue such as the phloem, it is difficult to decide whether structures visible in the electron microscope represent fact or artefact (Weatherley and Johnson, 1968; Evert, 1977; Spanner, 1978; Ehlers et al., 2000). As a consequence, there are no quantitative analyses available to date of the geometry of functional forisomes within transporting sieve elements. Here the three-dimensional structure of forisomes within their cellular context is described, using sieve tubes in the mid-vein of V. faba leaflets as a model. This analysis provides the basis for a future evaluation of the physical consequences of the presence of forisome-like structures in transporting microtubes, which will be helpful in understanding the biophysics of phloem transport and the evolution of reversible sieve tube plugging mechanisms, as well as in developing biomimetic valves in artificial microfluidic systems.
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Materials and methods |
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Vicia faba L. cv. ‘Witkiem major’ (Nunhem Zaden, Haelen, The Netherlands) was grown in a greenhouse as detailed before (Knoblauch and van Bel, 1998). To study forisomes in situ, shallow periclinal cuts were made on the abaxial side of mid-veins of excised leaflets of 6-week-old plants, and the phloem was observed in a Ca2+-free medium (5 mM EGTA, 10 mM KCl, 5 mM NaCl, 50 mM HEPES pH 7.3) using a conventional light microscope (Leica DM LFSA) or a confocal laser-scanning microscope (CLSM; Leica TCS 4D) as described by Knoblauch et al. (2001).
The DM LFSA was equipped with Leica HCX APO water immersion objectives (Leica, Wetzlar, Germany). Images were taken in the bright-field mode using a DC200 digital camera (Leica, Wetzlar, Germany) controlled with Leica's IM50 software. The excitation light for the TCS 4D (Leica, Wetzlar, Germany) was produced with a 75 mW argon/krypton laser (Omnichrome, Chino, CA, USA). No fluorescent dye could be identified that would selectively stain forisomes although numerous commercially available compounds were tested. 5[6]-Carboxy-4',5'-dimethylfluorescein diacetate (CDMFDA) which has been used previously to visualize forisomes by CLSM (Knoblauch et al., 2001) did not allow the distinction to be made between forisomes and membranes as clearly as required in the present study. However, combined application of 10 µg ml–1 each of sulphorhodamine 101 and 3,3-diheptyloxacarbocyanine iodide [DiOC7; Molecular Probes Europe BV, Leiden, The Netherlands; for detection, a fluorescein isothiocyanate (FITC) band pass filter and a 590 nm longpass filter, respectively, were used] allowed pairs of images to be taken in which forisomes and membrane structures, respectively, were highlighted (Fig. 1A, B). Since membranes are only weakly permeable to sulphorhodamine 101, some degree of wounding in the vicinity of the examined cells was unavoidable to allow the dye to enter the sieve tube syncytium. While this dual fluorescence technique enabled forisomes and membraneous structures to be distinguished unequivocally in sieve elements in the transporting state, ambiguous results were obtained where forisomes had formed sieve tube plugs. The fluorescence signals from the plugs were weak and faded without a clear border into the background fluorescence intensity, and could not always be distinguished unambiguously from membranous structures (not shown; for documentation of the typical, poorly defined appearance of V. faba forisome plugs in the CLSM, see Fig. 7 in Knoblauch et al., 2001). Further analysis was therefore restricted to unplugged tubes. To produce three-dimensional reconstructions of forisome–sieve element–sieve plate complexes, forisomes from the sulphorhodamine 101 images, membranes from the DiOC7 images, and structures that unambiguously belonged to the sieve plate in either type of image were combined, as the red, green, and blue channel, respectively, of RGB colour images (Fig. 4).
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Quantitative analysis of micrographs and image processing, including the production of three-dimensional models and animations, was performed using the freeware package ImageJ (v 1.33u; http://rsb.info.nih.gov/ij/). Forisome length, greatest diameter, and distance to sieve plate were determined at least three times on the digital micrographs, in which one pixel corresponded to 0.181 µm. Data used for further analysis were the means of these repeated measurements; measurements of sieve element diameter were repeated at least five times in each specimen. SigmaPlot (version 7.101, SPSS Inc., Chicago IL, USA) was used for statistical analyses. Relationships between two variables, none of which could be considered independent (i.e. free of significant statistical error; in such cases regression analysis does not yield meaningful results), were characterized by the geometric mean functional relationship as described by Draper and Smith (1998).
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Results |
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Observing functional sieve elements is notoriously difficult because of the high sensitivity of living phloem (Weatherley and Johnson, 1968; Lawton, 1978b; Knoblauch and van Bel, 1998; Ehlers et al., 2000). In the phloem of the Faboideae, forisomes respond to various types of disturbance by forming sieve tube plugs (Knoblauch et al., 2001). Gentle preparation methods therefore must be applied to allow observation of forisomes in transporting sieve elements. Water immersion objectives with large working distances were employed to study undamaged sieve tubes embedded in the vascular tissues of complete leaflets of V. faba. Conventional light micrographs obtained from such preparations show poor contrast due to the presence of numerous cell layers between the observed cells and the light source as well as the objective. However, careful examination of serial images with a gradually changed focal plane (Fig. 2A–C) allowed the determination of four characteristic parameters, namely forisome length and greatest diameter, distance from the nearest sieve plate, and diameter of the sieve element at the position of greatest forisome diameter. A total of 82 forisomes in 80 individual sieve elements from nine leaflets collected from five plants were analysed. As not all of the four key parameters could be unambiguously established in all forisomes, 41 complete data sets were obtained; at least one of the four parameters was missing in all other cases.
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Usually, only one forisome per sieve element was found. In two out of 80 sieve elements, two forisomes were detected. As it was difficult to visualize full-length sieve elements (which can be several hundred µm long in this tissue, Fig. 2D), it remains unclear whether every sieve element contained at least one forisome. The majority of forisomes observed (50 out of 82) showed tips that consisted of two or more pointed ends rather than one compact point (Fig. 2A, C). About three-quarters of the forisomes (61 out of 82) were found closer to the proximal end of the sieve element (i.e. close to the distal side of the sieve plate). Interestingly, the minority of forisomes located at the distal end of their sieve elements were not distributed randomly across the data sets. Seventeen of these 21 ‘distal’ forisomes occurred in four clusters comprising six, four, four, and three forisomes, respectively; forisomes of each cluster were observed in close proximity without any ‘proximal’ forisome nearby. In the seven cases in which two forisomes were found in two adjacent sieve elements forming a continuous tube, the forisomes were always located at the same end (six times proximal, once distal) of the sieve elements. Most forisomes were located close to a sieve plate; among the 58 ‘proximal’ forisomes for which the distance could be measured, 49 were found closer than 30 µm (the average length of a forisome in this tissue) to the sieve plate, with 20 of them apparently touching the plate. The situation was similar in ‘distal’ forisomes (Fig. 3).
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On average, forisomes were about 10 times longer than they were wide, and their greatest diameter was roughly a quarter of that of the sieve elements (Table 1). The forisome volume and cross-sectional area were calculated assuming that forisomes were ellipsoids of revolution, and the cross-sectional area of the sieve elements was estimated assuming a circular shape. The results suggested that on average, the greatest cross-sectional area of forisomes was
8% of the sieve element cross-sectional area at the location of the forisome (Table 1). However, the assumption of circular sieve element cross-sections is an oversimplification, and conjectures regarding subcellular geometries based on averaged values of computed geometric parameters may easily be flawed. Therefore, the conclusion was verified by reconstructing the three-dimensional geometry from image stacks obtained by CLSM using dual fluorescence staining (see Materials and methods and Fig. 1 for technical details). Five forisomes were analysed which, as a group, represented the range of typical cases in this tissue (Fig. 4; see also Supplementary files at JXB online). In optical sections, the cross-sectional shape of forisomes frequently resembled rounded polygons rather than circles (Fig. 4). However, the quantitative evaluation of these three-dimensional reconstructions (Table 2), which was based on pixel counting rather than on geometric assumptions, confirmed that forisomes occupied less than one-tenth of the cross-sectional area of their sieve elements.
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Further analysis of the data obtained by conventional light microscopy showed that forisome length correlated neither with forisome diameter nor with sieve element diameter (Fig. 5A, B; a relationship was considered significant if the coefficient of correlation, r, was significant at P <0.05). On the other hand, there was a significant interdependence between the diameters of sieve elements and forisomes (Fig. 5C); thus, larger sieve elements tended to harbour thicker forisomes of variable length. The proportionality between the diameters of forisomes and sieve elements, however, was not preserved: in narrower sieve elements, forisomes tended to be relatively thicker than in larger ones (Fig. 5D).
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Discussion |
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The ultimate goal of the ongoing forisome research programme is to understand the evolution of a reversible plugging mechanism in sieve tubes that is based on the development of ATP-independent contractility in previously non-contractile proteins. To approach this goal, three paths may be followed. Studies of forisome proteins and genes will help to understand the evolutionary sequence of events on the molecular level; ecophysiological investigations will provide insights into selective advantages of possessing forisomes on the whole-plant level; and cell biological and biophysical analyses will elucidate how events on the molecular level translate into whole-plant increases of fitness through improvements of the efficiency and flexibility of the cellular machinery of long-distance transport within the plant. The present study contributes to this third aspect by characterizing the structure of the forisome–sieve element–sieve plate complex in its functional state. While it seemed logical to perform this inquiry using V. faba leaflets, the organ in which forisome dynamics were first observed (Knoblauch et al., 2001), recent comparative studies on forisome morphology (own unpublished results) indicated a significant degree of variation within the Fabaceae, so that conclusions must not be rashly generalized.
Electron microscopic investigations cannot replace light microscopy of the living system when the aim is three-dimensional reconstruction of the forisome in its functional context. This is not only because of the sensitivity of phloem tissue, which gave rise to a continuing debate regarding the extent to which the situation after fixation and preparation for electron microscopy represents the living state (Evert, 1977; Spanner, 1978; Lawton, 1978b; Ehlers et al., 2000). Osmotic pressure in sieve tubes can be as high as 1.5 MPa (Pritchard, 1996), suggesting that turgor is rather significant in these cells, although not necessarily as high as cellular osmotic pressure since the osmolarity of the apoplast solution certainly is not zero. The loss of turgor pressure during tissue preparation is likely to cause slight changes in tissue geometry, which may become sources of significant error in quantitative analyses. Moreover, the Ca2+ response in forisomes of V. faba represents a 3-fold increase in volume within 
0.1 s, and it is fully reversible on the same time scale (Knoblauch et al., 2003; the factor of volume increase can be as high as eight in other species including Glycine max; own unpublished observations). It appears hard to explain such rapid volume changes other than by the uptake and release of water (Pickard et al., 2006), which would seem to imply an involvement of some hydration-related mechanism in forisome action. If so, the shape of forisomes is likely to be influenced by changes in their osmotic environment during preparation for electron microscopy.
The vast majority of forisomes examined were located close to sieve plates (Fig. 3). Occasionally forisomes were observed that drifted along sieve elements, apparently unrestrained by any structures that would fix them at a certain location. In fact, no such structures had been demonstrated by older ultrastructural studies (Laflèche, 1966; Wergin and Newcomb, 1970; Palevitz and Newcomb, 1971; Fisher, 1975; Lawton, 1978a, b; Arsanto, 1982). In a more recent report, minute clamps were described to connect mitochondria, endoplasmic reticulum, phloem-specific plastids, and parietal P-proteins with each other and with the plasma membrane in sieve elements of tomato and V. faba (Ehlers et al., 2000). These authors suggested that the clamp-like structures were necessary to fix sieve element organelles at their parietal position, keeping the sieve tube free for mass flow; intriguingly, no clamps were found on the surface of forisomes. About three-quarters of all forisomes observed in the present study were found on the distal sides of sieve plates (Fig. 3), which is the downstream end of the sieve elements if phloem transport occurs from leaflets towards the stem. Taken together, these facts support the view that forisomes are dragged along passively with the mass flow in the sieve elements. If this inference would hold for the undisturbed plant, an accumulation of forisomes in sink tissues would be mechanically prevented, as it were, by the sieve plates. The location of forisomes relative to sieve plates would then provide a convenient internal marker of the direction of flow within the living phloem. In this context, the positioning at the distal end of the sieve tube (i.e. at the proximal side of the sieve plate) of one-quarter of the forisomes observed indicates flow into the leaflet. The fact that most of these ‘distal’ forisomes occurred in groups within neighbouring sieve elements suggests that their localization was controlled by factors acting on a level above that of the individual sieve element. Whether this phenomenon is of physiological significance or an artefact caused by the preparation of the leaflets for microscopy remains to be established.
Concerning the requirements for efficient micro-tube blockage, the decisive geometric parameter of the tube is its cross-sectional surface area, which, for more or less circular cross-sections, corresponds to the tube's diameter. In V. faba leaflets, sieve element diameter correlated with forisome diameter (Fig. 5C) but not forisome length (Fig. 5B); consequently, forisome diameter and length were independent of each other (Fig. 5A). This result appeared in line with the present expectations, because it was assumed that the Ca2+-induced increase in forisome diameter is the geometric basis of forisome function as a reversible sieve tube plug (Knoblauch and Peters, 2004a); if forisome diameters in the plugging and the resting state differ by a constant factor, and if forisome diameters are adjusted for optimum efficiency, then they should correlate with the diameters of the sieve elements. The correlation was not perfect, however, as smaller sieve elements tended to have relatively thicker forisomes (Fig. 5D).
Unfortunately, forisome plugs in sieve elements of V. faba are practically invisible in the conventional light microscope (Knoblauch et al., 2001), and poorly defined in the CLSM (see Materials and methods). Therefore, they did not lend themselves to three-dimensional analysis, so that it remained unclear whether sieve tube plugs formed by radially expanded forisomes actually are tight. Electron micrographs of forisome plugs in V. faba leaflets suggested that the plugs did not always occlude the tube completely (Knoblauch et al., 2001). The present results seem to support this interpretation. Forisome diameter equalled 27% of the diameter of the sieve element in situ (Table 1), corresponding to an averaged cross-sectional surface area of the forisome of <8% of that of the sieve tube (Fig. 5D). This conclusion drawn from conventional micrographs was corroborated by the three-dimensional reconstructions (Fig. 4; Table 2). It was concluded that the resistance to sieve tube flow offered by forisomes in the resting state is negligible compared with that of the sieve plates. If free Ca2+ triggered an increase in diameter by a factor of 2.2 in situ as it does in vitro (Knoblauch et al., 2003), then the proportion of the forisome's cross-sectional area with respect to the sieve element cross-section would rise to a mere 35%. However, if the radially expanded forisome were freely motile within the sieve element as it appears to be in the resting state, it might become wedged in obliquely on the upstream side of a sieve plate; in this case, its effective cross-sectional area normal to the direction of flow will be increased. Assuming that forisomes are ellipsoids of revolution, and using known average values of forisome length, diameter, and sieve tube diameter (Table 1) as well as the factors for Ca2+-induced decrease in length and increase in diameter (–0.7 and 2.2, respectively; Knoblauch et al., 2003), the conclusion is reached that the maximum angle between the longitudinal axes of sieve element and forisome is 34° under the conditions described. Consequently, the projection of an obliquely oriented, Ca2+-swollen forisome onto the plane perpendicular to the direction of flow along the sieve tube's long axis does not exceed 60% of the sieve element's cross-sectional area. Thus, forisomes certainly are able to reduce the effective sieve tube diameter significantly, but appear incapable of blocking the tubes completely in leaves of V. faba.
However, the latter conclusion may be premature. First, in previous experiments (Knoblauch et al., 2003) which established that forisomes in vitro expanded radially by a factor of 2.2 in response to Ca2+, bathing media were applied that probably had an oxidizing redox potential as they were not redox-buffered and were in equilibrium with atmospheric air. Live phloem tissue, on the other hand, represents a reducing environment with strongly decreased oxygen levels (Darwent et al., 2003; van Dongen et al., 2003). It appears likely that phloem-specific proteins have evolved adaptations to this situation, and that the reactivity of isolated forisomes may be somewhat impaired in oxidizing media. This idea will have to be explored under redox-controlled experimental conditions. Secondly, in the above considerations, it was assumed that the shape of forisomes in the radially expanded state was invariably that of an ellipsoid of revolution. However, if forisomes in this condition were floppy rather than rigid, they might be pressed against the sieve plate on the downstream end of the sieve element and thereby flattened to produce a tight seal. This hypothesis could be tested using atomic force microscopy (Lebedeva et al., 2004) to determine the elasticity modules of forisomes as a basis for modelling their mechanic behaviour within the sieve tube. Generally, it should be kept in mind that forisomes probably co-operate with other factors that might control the effective sieve tube diameter (such as parietal P-proteins) or the viscosity of the sieve tube contents (such as soluble P-proteins), in regulating sieve tube flow.
In any case, with the present results it has become imperative to establish unequivocal experimental proof that forisomes actually do seal sieve elements, in order to substantiate their postulated biological function. Given the technical difficulties in manipulating living phloem tissue, it would appear a promising alternative to study isolated forisomes in artificial microfluidics systems created to mimic sieve tube geometry as defined in the present study.
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Supplementary data |
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Movies providing an animated impression of the three-dimensional structure of a living V. faba sieve element with forisome (complementing Fig. 4E) can be found at JXB online. They show (i), the sieve element along its longitudinal axis; and (ii), the sieve element in the radial direction. The files are animated gifs and can be viewed with any current Internet browser.
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Acknowledgements |
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This study was supported by the Nanobiotechnology program of the BMBF (Federal Ministry of Education and Research, Germany), grant 0312014A,C. We dedicate this work to E. ‘La Donna’ Kesselbacher on the occasion of her 49th birthday.
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Abbreviations |
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CLSM, confocal laser scanning microscope; DiOC7, 3,3-diheptyloxacarbocyanine iodide.
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