TY - JOUR
AU - Suzuki, Suechika
AB - Abstract The columella cells of soybean roots grown under gravity and simulated microgravity induced by a clinostat were examined using potassium pyroantimonate (PA) and quantitative X-ray microanalysis of cryosections to determine the role of Ca in the regulation of the gravitropic response. Amyloplasts in the columella cells were localized exclusively at the bottom under gravity, but diffusely distributed in the cytoplasmic matrix under simulated microgravity, thus supporting the statolith theory. In the columella cells, PA precipitates containing Ca were diffusely distributed in the cytoplasmic matrix under gravity. Under simulated microgravity, however, they decreased in number and size in the cytoplasmic matrix, whereas increased only in number in the vacuole, indicating that Ca moved from the cytoplasmic matrix into the vacuole. The vacuole of columella cells contained mostly electron-dense granular structures localized along the inner surface of tonoplasts, which closely resembled the tannin vacuole reported in Mimosa pulvinar motor cells. Under simulated microgravity, their configuration changed dramatically from a granular shape to a flat plate. The quantitative X-ray microanalysis of cryosections showed that the vacuolar electron-dense structures contained a large amount of Ca. Under simulated microgravity, the concentration of Ca increased conspicuously in these vacuolar electron-dense structures, concomitantly with a marked decrease of K in the vacuoles and an increase of K in the cell walls. These results suggest that the release of Ca2+ from, and uptake by, the vacuolar electron-dense structures is closely related to the signal transmission in the gravitropic response and that Ca movement occurs opposite to that of K. soybean root, columella cell, gravitropism, calcium, X-ray microanalysis, cryosection Introduction The gravitropic response in plant root is considered to occur in three phases: graviperception of the gravitational stimulus, its signal transmission and resulting root growth in the direction of earth's gravitational force [1]. The starch–statolith hypothesis of Nemec–Haberlandt is the most persuasive explanation for the first phase of graviresponse, in which amyloplasts function as statoliths [2]. In the second phase, many factors such as Ca2+ and other messengers have been suggested to link graviperception with the growth movement (for review, see [2–5]). However, the details of this linking process remain unknown. In the third phase, hormones such as auxin have been suggested to affect the elongation growth of root cortex cells surrounding the columella cells [6,7]. However, Ca2+ and other ions also possibly act as signal messengers from the columella cells to the surrounding cortex cells. Sievers and Volkmann [8] reported that, in the columella cells, a complex of endoplasmic reticulum (ER) found at the cell bottom did not move even when the root was tilted, and the graviperception is caused by a differential pressure on the distal ER of the statocytes. The graviresponse of plant roots is completely suppressed by various kinds of Ca chelators [9,10]. Furthermore, Sievers et al. [3] revealed that the adenosine triphosphate (ATP)-dependent Ca2+ accumulation occurs in the vesicular membrane fraction corresponding to the ER vesicles, isolated from the Lepidium root. Analogous to the intracellular Ca localization and its translocation in skeletal muscles [11,12], the localization of Ca in, and release from, the ER might help regulate the second phase [3,9]. The pyroantimonate (PA) method has been used to demonstrate the intracellular localization of Ca and its translocation during muscle contraction [12,13]. Using the PA method, Dauwalder et al. [14] reported that, in corn roots after gravistimulation, the PA precipitates were localized exclusively along the upper surface in horizontally positioned roots. Furthermore, they reported that, in cortical cells, the PA precipitates were mainly localized along the membrane of mitochondria, plastids and Golgi apparatus, but gravistimulation did not change the localization of PA precipitates in those organelles. Moore and Evans [2] revealed that the predominant localization of PA precipitates in the columella tissue of corn roots changed from the plasma membrane to the cell wall of columella cells when the root orientation was changed from vertical to horizontal. Belyavskaya [15] also observed that the PA precipitates were localized in various organelles of pea columella cells, and the number of these precipitates decreased after the treatment of the root with LiCl, which is an inhibitor of Ca2+ influx. Although these studies have revealed the Ca localization in columella cells and/or the Ca distribution change in root tips during the graviresponse, the details of intracellular Ca translocation remain unknown. In this study, we examined the intracellular Ca translocation of columella cells of soybean roots using the PA method. Quantitative X-ray microanalysis of cryosections has been well established to determine the intracellular Ca localization and its dynamic translocation during the contraction-relaxation cycle in skeletal muscles [16–18]. It has revealed the opposite movements of K and Ca during the gravicurvature in horizontally oriented roots [19,20] and also that, in the corn root tips, the Ca concentration is slightly increased in the cytoplasmic matrix under simulated microgravity induced by a clinostat [21]. In the present study, using quantitative X-ray microanalysis of cryosections, we measured the intracellular Ca concentration in various regions or organelles of columella cells in soybean roots grown under gravity and simulated microgravity and examined whether Ca distribution is changed by gravistimulation in order to determine the role of Ca in linking the graviperception with the growth movement in the second phase of the graviresponse. Material and methods Seeding and gravistimulation Soybean seeds, Glycine max Merr., were soaked overnight in distilled water at room temperature. Then, they were mechanically fixed with fine pins on a thin layer of wet absorbent cotton on silicone rubber at the bottom of small Petri dishes. As a control, Petri dishes with fixed seeds were put vertically in an incubator. On the other hand, for experimental roots stimulated by simulated microgravity, some Petri dishes with fixed seeds were placed on the rotating cylinder of a clinostat set in an incubator. Previously, we constructed a clinostat by modifying a commercial shaking machine (YAYOI YGG-1 type, YAYOI, Inc., Japan), which could stir and mix powder and/or fluid by a double rotation system (self-rotation and arm-rotation) and evaluated its efficiency for producing simulated microgravity for electron microscope observations of amyloplast movements in columella cells of Arabidopsis (unpublished) and corn roots [21]. All seeds were incubated for 3 days in the dark at 18°C. Conventional electron microscopy and Ca cytochemistry Soybean roots were cut off at 5 mm from the end of the root caps and immersed in distilled water in small test tubes. Air in the root tissues was sucked up with a water vacuum suction system. For conventional electron microscopy, the specimens were fixed with a 3% glutaraldehyde solution (pH 7.2 by 0.1 M phosphate buffer) and postfixed with a 2% osmium tetroxide (OsO4) solution, at 4°C overnight. The fixed specimens were dehydrated with a graded series of acetone and embedded in resin Quetol 812. For the Ca cytochemistry, soybean roots were fixed with a 1% OsO4 solution (pH 6.8 by 0.01 N acetic acid) containing 2% potassium (K[Sb(OH)6]) for 24 h at 4°C, dehydrated with a graded series of ethanol and embedded in Quetol 812. Ultrathin sections with a silver color (∼70 nm in thickness) and a blue color (∼150 nm in thickness) and thick sections (0.9 μm in thickness) were cut on an ultramicrotome (Ultracut-N; Reichert, Vienna, Austria). To observe the fine structure, ultrathin sections with a silver color were stained with uranyl acetate and lead citrate. For Ca cytochemistry, ultrathin sections with a blue color were observed without any electron staining. All ultrathin sections were examined with a JEM 1230 transmission electron microscope (JEOL, Akishima, Tokyo, Japan) equipped with an energy-dispersive X-ray microanalyzer (MiniCup/EX-14033JTP; JEOL, Akishima, Tokyo, Japan) and operated at an accelerating voltage of 100 kV. For cytochemical identification of the precipitate of PA salts, ultrathin sections with a blue color were analyzed using the X-ray microanalyzer. The thick sections (0.9 μm) were stained with 1% toluidine blue solution and examined with a light microscope. Rapid freezing and cryosection preparation Root tips were cut off at 2 mm from the end of the root cap and further cut longitudinally along the plane including the central axis. They were inserted into the small hole of a specimen carrier supplied for a high-pressure freezing machine (EM-PACT; Leica, Austria), as the planes cut longitudinally were faced upward. Then, roots were rapidly frozen in liquid nitrogen (liquid N2, −196°C) using the high-pressure freezing machine and stored in liquid N2. Longitudinal cryosections of ∼200 nm thickness were cut from the frozen root at −140°C using a cryoultramicrotome (Ultracut UCT/EM FCS; Leica, Austria). Then, the cryosections were carefully sandwiched between two cooled Ni-grids coated with thin carbon films in the cryochamber kept at −145°C. Subsequently, the Ni-grid sandwich was transferred into a small hole of a cooled copper container for freeze-drying. The mass of the freeze-drying container was sufficient to maintain temperatures below −95°C for 24 h in a vacuum chamber. Then, the freeze-drying container was cooled further to −196°C with liquid N2 and put into the vacuum chamber of a freeze-drying machine (VDF300S; Vacuum Device, Inc., Mito, Japan), in which the cryosections were freeze-dried at 10−6 Torr (1.3 × 10−4 Pa) and −95°C for 24 h. After the freeze-drying procedure, the Ni-grid sandwich was split apart, and the cryosections were lightly evaporated in vacuo with carbon for quantitative X-ray microanalysis. Electron probe X-ray microanalysis Electron probe X-ray microanalysis was carried out using an analytical electron microscope (JEM 1230; JEOL, Akishima, Tokyo, Japan) equipped with an energy-dispersive X-ray microanalyzer (MiniCup/EX-14033JTP; JEOL, Akishima, Tokyo, Japan). Initially, the cold trap attached to the electron microscope column was cooled with liquid N2 to avoid specimen contamination. Freeze-dried cryosections on the Ni-grid were mounted on a Be-stage of the cryotransfer holder (G626DH; Gatan, Tokyo, Japan) and put into the electron microscope. These cryosections were then cooled to −130°C with liquid N2 for the cryotransfer holder. The transmission system of the analytical electron microscope was operated at an accelerating voltage of 80 kV. For X-ray microanalysis (spot analysis), the electron beam with a current density of 75.0 pA/cm2 in a small viewing screen was focused on a fixed area (diameter, 0.1 μm) under a magnification of 15 000×. X-ray emissions from the cryosection were collected for a period of 200 s. To examine whether or not mass-loss and/or contamination in the cryosection occurred during the collection of X-ray emissions, we monitored the detection sensitivity of the microanalyzer at dead time ∼20%, count per second (cps) ∼2000 and confirmed the stability of detection. The concentration of elements was calculated from X-ray spectra in mmol−1 kg dry wt, on the basis of Hall's quantitative equation [16,18,22]. X-ray emissions of up to 10 keV were collected. The characteristic X-ray count of each spectral peak was calculated as an intensity of the peak (peak intensity P), and the integral X-ray count in a region of 4.5–5.5 keV was used as a representative intensity of X-ray continuum (background intensity B). On the basis of the P/B intensity ratio, the concentration of each element was calculated using software NORAN System SIX (Thermo Electron Co., Middleton, WI, USA). Results Physiological effect of gravity disturbance on soybean seedlings Figure 1 shows soybean seedlings grown under gravity (Fig. 1a) and simulated microgravity (Fig. 1b), 3 days after germination. Under gravity, roots were 20–30 mm in length and extended straight toward the ground. On the other hand, under simulated microgravity, every root wound with a marked curvature, although the root length was similar to that of roots grown under gravity. The root curvature indicated that the graviresponse of seedlings was completely disturbed by simulated microgravity induced by the clinostat. Fig. 1. View largeDownload slide Elongation of soybean roots under gravity (a) and simulated microgravity (b), after 3 days from germination. Note the root curvature in (b). Fig. 1. View largeDownload slide Elongation of soybean roots under gravity (a) and simulated microgravity (b), after 3 days from germination. Note the root curvature in (b). Ultrastructure of columella cells and the distribution change of organelles under simulated microgravity Columella cells in soybean roots grown under gravity and simulated microgravity were observed by conventional electron microscopy (Fig. 2). They were generally cylindrical and ∼20–30 μm in height. The nucleus was always located in the central region of the cytoplasm and many vacuoles were small, indicating that those cells were still young. Many amyloplasts, ∼3 μm in diameter, were also found in the columella cells. There was no difference in structural features of those organelles, including mitochondria, Golgi apparatus and others, between the columella cells of roots grown under gravity and simulated microgravity. However, there was a clear difference in the intracellular distribution of amyloplasts and ER, and the distribution of vacuolar inclusions (vacuolar electron-dense structures) between them. Fig. 2. View largeDownload slide Electron micrographs of the columella cells in roots grown under gravity (a–c) and simulated microgravity (d–f). Amyloplasts in the columella cells are observed at the bottom of cells in (a) and contact the ER membrane in (b), while they are diffusely distributed in (d). The contact of amyloplasts with the ER membrane is not clear in (e). Vacuolar electron-dense structures localized along the inner surface of tonoplasts are granular in (c), but plate-like in (f). Bars: (a, d) 5 μm, (b, e) 1 μm and (c, f) 1 μm. Fig. 2. View largeDownload slide Electron micrographs of the columella cells in roots grown under gravity (a–c) and simulated microgravity (d–f). Amyloplasts in the columella cells are observed at the bottom of cells in (a) and contact the ER membrane in (b), while they are diffusely distributed in (d). The contact of amyloplasts with the ER membrane is not clear in (e). Vacuolar electron-dense structures localized along the inner surface of tonoplasts are granular in (c), but plate-like in (f). Bars: (a, d) 5 μm, (b, e) 1 μm and (c, f) 1 μm. In the columella cells of roots grown under gravity, amyloplasts were exclusively localized at the bottom of these cells, indicating sedimentation as a result of the response to gravity (Fig. 2a). Furthermore, many ER with a fragmented shape were also found around the bottom, and the outer membrane of amyloplast envelopes attached frequently to the membrane of ER (Fig. 2b). Many small vacuoles were found mostly around the nucleus (Fig. 2a), and they contained many granular electron-dense structures localized along the inner surface of the tonoplasts, like the beads of a rosary (Fig. 2c). In the columella cells of roots grown under simulated microgravity, amyloplasts were diffusely distributed in these cells and did not show sedimentation phenomena (Fig. 2d), which is in contrast with the columella cells stimulated by gravity (Fig. 2a). Furthermore, the ER was not always concentrated at the bottom of the cell (Fig. 2e). In vacuoles, the electron-dense structures localized along the inner surface of tonoplasts were conspicuously changed in configuration, from a granular shape to a flat plate, and covered the inner surface of tonoplasts (Fig. 2f). Cytochemical observation of the intracellular Ca localization and its translocation under simulated microgravity Soybean roots grown under gravity and simulated microgravity were fixed cytochemically using a 1% OsO4 solution containing 2% potassium PA. In the columella cells of roots grown under gravity, many electron-dense PA precipitates were diffusely distributed in the cytoplasmic matrix (Fig. 3a). In the vacuole, the localization of precipitates was not conspicuous. On the other hand, in the columella cells of roots grown under simulated microgravity, the PA precipitates decreased in number and size in the cytoplasmic matrix, whereas increased only in number in those vacuoles (Fig. 3b), indicating the Ca movement from the cytoplasmic matrix to the lumen of vacuole. PA precipitates were also found in mitochondria, ER and cell wall regions in roots grown under both gravity and simulated gravity, but were fewer than in the cytoplasmic matrix and the vacuolar lumen. Fig. 3. View largeDownload slide Electron micrographs of the columella cells in roots grown under gravity (a) and simulated microgravity (b), showing the intracellular localization of PA precipitates. V, vacuoles. Bars: (a, b) 5 μm. Fig. 3. View largeDownload slide Electron micrographs of the columella cells in roots grown under gravity (a) and simulated microgravity (b), showing the intracellular localization of PA precipitates. V, vacuoles. Bars: (a, b) 5 μm. The PA method is an efficient cytochemical approach to demonstrate Ca localization and its translocation during various cell movements [13]. However, PA binds not only with Ca2+, but also with other cations such as Na+, K+ and Mg2+ [13,23,24]. Therefore, electron probe X-ray microanalysis is suitable for demonstration of the existence of Ca in the PA precipitate. Figure 4 shows an example of X-ray spectra obtained from the spot analysis of PA precipitates observed in the cytoplasmic matrix of the columella cells of roots grown under gravity. All X-ray spectra of PA precipitates observed in the present study had a spectral peak at 3620 eV. Since the spectral peak at 3620 eV has been interpreted as a form caused by a combination of Sb-Lα (3600 eV) and Ca-Kα (3690 eV) emissions [13], the PA precipitates observed in the present study contained Ca. Fig. 4. View largeDownload slide A typical X-ray spectrum of PA precipitates found in the cytoplasmic matrix of columella cells in roots grown under gravity, showing a distinct peak (Sb–Ca) at 3620 eV resulting from the combination of the Sb-Lα (3600 eV) and the Ca-Kα (3690 eV) emissions. The ordinate shows the number of X-ray events (counts), and the abscissa shows the X-ray energy in keV. Fig. 4. View largeDownload slide A typical X-ray spectrum of PA precipitates found in the cytoplasmic matrix of columella cells in roots grown under gravity, showing a distinct peak (Sb–Ca) at 3620 eV resulting from the combination of the Sb-Lα (3600 eV) and the Ca-Kα (3690 eV) emissions. The ordinate shows the number of X-ray events (counts), and the abscissa shows the X-ray energy in keV. X-ray microanalysis of cryosections Typical electron micrographs of cryosections cut longitudinally at the columella region in roots grown under gravity or simulated microgravity are shown in Fig. 5. Although all cryosections were prepared without any chemical fixation, typical cell organelles such as nuclei, mitochondria and plastids were observed well in the columella cells. The nucleus was always located at the central region of the columella cells. The change in intracellular amyloplast distribution from sedimentation to dispersion, caused by the gravity disturbance, was confirmed again in these cryosections. Without any electron staining, it was very difficult to recognize the change in ER distribution. The vacuolar electron-dense structures in columella cells observed in the chemically fixed materials were also found along the inner surface of tonoplasts. However, the electron density of these structures became very low, and there was little configuration change in the columella cells of roots grown under simulated microgravity. Fig. 5. View largeDownload slide Cryosection images of the columella cells in soybean roots grown under gravity (a) and simulated microgravity (b). The nucleus (N) is always located at the central region of columella cells. In columella cells, amyloplasts with electron-dense starch grains are localized at the bottom in (a), but diffusely distributed in (b). V, vacuoles. Vacuolar electron-dense structure (arrowheads). Starch grain in amyloplast (*). Bars: (a, b) 2 μm. Fig. 5. View largeDownload slide Cryosection images of the columella cells in soybean roots grown under gravity (a) and simulated microgravity (b). The nucleus (N) is always located at the central region of columella cells. In columella cells, amyloplasts with electron-dense starch grains are localized at the bottom in (a), but diffusely distributed in (b). V, vacuoles. Vacuolar electron-dense structure (arrowheads). Starch grain in amyloplast (*). Bars: (a, b) 2 μm. Spot analysis was carried out at the cell wall, the cytoplasmic matrix, the vacuolar lumen and the vacuolar electron-dense structure localized along the inner surface of tonoplasts. Figure 6 shows typical X-ray spectra obtained from these regions of the columella cells. The X-ray spectra exhibited distinct peaks of K-Kα (3312 eV) and K-Kβ (3589 eV) emissions. On the other hand, distinct peaks of Ca-Kα (3690 eV) and Ca-Kβ (4012 eV) emissions were exhibited only in the X-ray spectra obtained from the spot analysis of vacuolar electron-dense structures found in the columella cells of roots grown under simulated microgravity (Fig. 6h). These spectral peaks were conspicuously high, indicating the existence of a large amount of Ca. In addition, possible peaks combining K-Kβ and Ca-Kα emissions were recognized in the X-ray spectra obtained from the spot analysis of cell walls in the columella cells of roots grown under both gravity and simulated microgravity (Fig. 5a and e), although the contribution of Ca-Kα emissions to the combined peaks seemed to be very small. Fig. 6. View largeDownload slide X-ray spectra of four regions analyzed in the columella cells of roots grown under gravity (a–d) and simulated microgravity (e–f). Four regions are the cell wall (a, e), the cytoplasmic matrix (b, f), the vacuolar lumen (c, g) and the vacuolar electron-dense structure (d, h). Labels indicate the general element-emission line of respective elements. The ordinate shows the number of X-ray events (counts), and the abscissa shows the X-ray energy in keV. Fig. 6. View largeDownload slide X-ray spectra of four regions analyzed in the columella cells of roots grown under gravity (a–d) and simulated microgravity (e–f). Four regions are the cell wall (a, e), the cytoplasmic matrix (b, f), the vacuolar lumen (c, g) and the vacuolar electron-dense structure (d, h). Labels indicate the general element-emission line of respective elements. The ordinate shows the number of X-ray events (counts), and the abscissa shows the X-ray energy in keV. Distinct peaks of P-Kα (2013 eV) emissions were observed in the X-ray spectra from the spot analysis of the cytoplasmic matrix and the vacuolar electron-dense structure in the columella cells of roots grown under gravity (Fig. 6b and d) and simulated microgravity (Fig. 6f and h). The spectral peak of P-Kα emissions was also distinct in the X-ray spectra obtained from the spot analysis of the cell wall in the columella cells of roots grown under simulated microgravity (Fig. 6e), but it was scarcely observed in the X-ray spectra obtained from the spot analysis of the cell wall in the columella cells of roots grown under gravity (Fig. 6a). Distinct peaks of Mg-Kα (1253 eV) emissions were exhibited in the X-ray spectra obtained from the spot analysis of the vacuolar electron-dense structures in the columella cells of roots grown under both gravity and simulated microgravity (Fig. 6d and h). In all X-ray spectra, the background derived from continuum X-ray emissions was nearly constant in intensity and its spectral configuration, that is, the Bremsstrahlung curve, indicating that analyses were well performed without any serious fluctuation. Table 1 shows the concentrations of various elements including Ca in the cell wall, the cytoplasmic matrix, the vacuolar lumen and the vacuolar electron-dense structure, of which typical X-ray spectra are shown in Fig. 5. In the roots grown under gravity, the K concentration ([K]) of cell walls was 374.23 ± 75.97 mmol kg−1 dry wt (mean ± SD, N = 10) and was approximately three times higher than that of cytoplasmic matrices and vacuolar lumens (138.92 ± 45.46 and 130.99 ± 35.21 mmol kg−1 dry wt, N = 10, respectively). In addition, a large quantity of [K] was detected from the vacuolar electron-dense structures (900.38 ± 434.20 mmol kg−1 dry wt, N = 10). On the other hand, in the roots grown under simulated microgravity, there was a tendency for extracellular [K] to increase and intracellular [K] to decrease. The [K] of cell walls was 645.37 ± 131.40 mmol kg−1 dry wt (N = 10) and was approximately twice that detected from the roots grown under gravity. In contrast, the [K] in vacuolar electron-dense structures and vacuolar lumens decreased to half (652.47 ± 238.08 and 75.25 ± 32.25 mmol kg−1 dry wt, N = 10, respectively). The [K] in cytoplasmic matrices (136.68 ± 20.17 mmol kg−1 dry wt, N = 10) was similar to that detected from the roots grown under gravity. Table 1. Concentrations of various elements including Ca in the cell wall, the cytoplasmic matrix, the vacuolar lumen and the vacuolar electron-dense structure     Mg  P  K  Ca  Cell wall  Gravity  33.91 ± 22.10  39.61 ± 16.93  374.23 ± 75.93  16.54 ± 15.39  Simulated microgravity  45.37 ± 24.13*  42.00 ± 30.85*  645.37 ± 131.40****  18.39 ± 16.19*  Cytoplasmic matrix  Gravity  24.08 ± 12.34  117.59 ± 37.75  138.92 ± 45.46  7.86 ± 6.37  Simulated microgravity  19.21 ± 16.39*  82.68 ± 42.40**  136.68 ± 20.17*  7.48 ± 6.50*  Vacuolar lumen  Gravity  21.46 ± 11.99  23.28 ± 8.56  130.99 ± 35.21  8.66 ± 8.09  Simulated microgravity  26.44 ± 16.50*  16.35 ± 10.65*  75.25 ± 32.25****  5.34 ± 5.35*  Vacuolar electron-dense structure  Gravity  206.07 ± 108.17  804.53 ± 392.34  900.38 ± 434.20  45.43 ± 45.05  Simulated microgravity  245.00 ± 185.58*  1462.84 ± 516.38***  652.47 ± 238.08***  2012.34 ± 651.54****      Mg  P  K  Ca  Cell wall  Gravity  33.91 ± 22.10  39.61 ± 16.93  374.23 ± 75.93  16.54 ± 15.39  Simulated microgravity  45.37 ± 24.13*  42.00 ± 30.85*  645.37 ± 131.40****  18.39 ± 16.19*  Cytoplasmic matrix  Gravity  24.08 ± 12.34  117.59 ± 37.75  138.92 ± 45.46  7.86 ± 6.37  Simulated microgravity  19.21 ± 16.39*  82.68 ± 42.40**  136.68 ± 20.17*  7.48 ± 6.50*  Vacuolar lumen  Gravity  21.46 ± 11.99  23.28 ± 8.56  130.99 ± 35.21  8.66 ± 8.09  Simulated microgravity  26.44 ± 16.50*  16.35 ± 10.65*  75.25 ± 32.25****  5.34 ± 5.35*  Vacuolar electron-dense structure  Gravity  206.07 ± 108.17  804.53 ± 392.34  900.38 ± 434.20  45.43 ± 45.05  Simulated microgravity  245.00 ± 185.58*  1462.84 ± 516.38***  652.47 ± 238.08***  2012.34 ± 651.54****  Values are mmol kg−1 dry wt (means ± SD). Significances of difference from the value under gravity and simulated microgravity are also shown. *P > 0.1. **P < 0.1. ***P < 0.05. ****P < 0.01. View Large The concentration of Ca ([Ca]) was low in the cell wall of the columella cells in roots grown under gravity (16.54 ± 15.39 mmol kg−1 dry wt, N = 10) and simulated microgravity (18.39 ± 16.19 mmol kg−1 dry wt, N = 10). Furthermore, the [Ca] was much less in cytoplasmic matrices (7.86 ± 6.37 and 7.48 ± 6.50 mmol kg−1 dry wt, N = 10, respectively) and vacuolar lumens (8.66 ± 8.09 and 5.34 ± 5.35 mmol kg−1 dry wt, N = 10, respectively) of the columella cells in roots grown under gravity and simulated microgravity. These values indicated that no significant change in [Ca] occurred as a result of the gravity disturbance. On the other hand, a conspicuously high concentration of Ca was detected from vacuolar electron-dense structures in the columella cells of roots grown under gravity (45.43 ± 45.05 mmol kg−1 dry wt, N = 10). Furthermore, the [Ca] in the vacuolar electron-dense structure was markedly increased ∼40 times (2012.34 ± 651.54 mmol kg−1 dry wt, N = 10) by the gravity disturbance and had a correlation with the significant decrease in [K]. In the columella cells of roots grown under gravity, the concentration of P ([P]) was fairly low in the cell wall (39.61 ± 16.93 mmol kg−1 dry wt, N = 10), although it was higher than the [Ca] detected. However, the [P] was significantly high in the cytoplasmic matrix (117.59 ± 37.75 mmol kg−1 dry wt, N = 10) and conversely low in the vacuolar lumen (23.23 ± 8.56 mmol kg−1 dry wt, N = 10). In soybeans grown under simulated microgravity, no change in [P] occurred in these regions. In contrast, the [P] in the vacuolar electron-dense structure was remarkably high (804.53 ± 392.34 mmol kg−1 dry wt, N = 10) and was increased ∼1.5 times by the gravity disturbance. The concentration of Mg ([Mg]) was low in the cell wall, in the cytoplasmic matrix and in the vacuolar lumen (33.91 ± 22.10, 24.08 ± 12.34 and 21.46 ± 11.99 mmol kg−1 dry wt, N = 10, respectively), whereas it was very high in the vacuolar electron-dense structures (206.07 ± 108.17 mmol kg−1 dry wt, N = 10) in the columella cells of roots grown under gravity. The [Mg] differed with the region analyzed, but simulated microgravity did not cause any significant change in it. The concentrations of the four elements shown in Table 1 correspond to those reported in corn roots by Moore et al. [20], except for the unusually high Ca concentration of the vacuolar electron-dense structure, which is a necessary result caused under simulated gravity. The [K] in the cell walls may be a suitable marker, as a similar examination was performed on extracellular [Na] in animals [18]. Discussion Amyloplasts function as statoliths The intracellular localization of amyloplasts was changed by the disturbance of gravity. Amyloplasts were localized exclusively at the bottom of columella cells of roots grown under gravity, whereas they were diffusely distributed in the cytoplasmic matrix of those cells in the plants grown under simulated microgravity. These results support the starch–statolith hypothesis of Nemec–Haberlandt (for review, see [2,4]). The sedimentation of amyloplasts under gravity coincided well with the reports on the columella cells of roots in various plants [1,2,8,15]. Although it cannot be ruled out that other cell organelles function as statoliths [25,26], or as other factors for gravitropism [27], at least in the soybean roots, the amyloplasts in the columella cells may play a significant role as statoliths to perceive the direction of gravity. Possible role of ER in the release of signaling Ca for graviresponse The results of this study showed that numerous ER existed exclusively at the bottom of the columella cells in roots grown under gravity, and the outer membrane of amyloplast envelope attached frequently to the membrane of ER (Fig. 2a and b). This is in agreement with the ultrastructural characteristics of ER reported in the columella cells of Lepidium roots [8] and supports the view that Ca2+ released from the ER might be caused by the contact with the sedimented amyloplast to mediate graviperception [3,28]. In the present study, we could not obtain a direct evidence of the Ca2+ release from ER after the contact of amyloplasts. However, as discussed in the following section, the intracellular distribution change of PA precipitates under simulated microgravity suggested the possibility of Ca translocation in the columella cells, probably from the ER lumen to the vacuole, passing through the cytoplasmic matrix. Intracellular Ca translocation under simulated microgravity In the columella cells, many electron-dense PA precipitates were diffusely distributed in cytoplasmic matrix under gravity (Fig. 3a). On the other hand, PA precipitates decreased in number and size in the cytoplasmic matrix, whereas increased only in number in those vacuoles under simulated microgravity (Fig. 3b). These results suggested that the intracellular Ca translocation from the cytoplasmic matrix to the vacuolar lumen might be caused by gravity disturbance. This view was further supported by the quantitative X-ray microanalysis of cryosections. In the soybean roots grown under gravity, the [Ca] was commonly low in the cell wall, the cytoplasmic matrix and the vacuolar lumen, but relatively high in the vacuolar electron-dense structure. Under simulated microgravity, the [Ca] was conspicuously increased only at the vacuolar electron-dense structure. These results suggest that the increased Ca at the vacuolar electron-dense structure is transported in succession from other regions, such as the vacuolar lumen, the cytoplasmic matrix and the cell wall. The transport of Ca2+ from the cell wall to the cytoplasmic matrix may be carried out through Ca-permeable channels localized in the plasma membrane [29]. In turn, entered Ca2+ may be taken up into the vacuolar lumen by the Ca2+-ATPase and/or H+/Ca2+ exchanger, which are generally believed to exist in the tonoplast [30]. Eventually, it may be that, in the vacuole, excess Ca2+ binds rapidly with the vacuolar electron-dense structure. In soybean roots fixed with glutaraldehyde and OsO4, the vacuolar electron-dense structure was clearly observed in the vacuole of columella cells (Fig. 2). Furthermore, its configuration changed from a granular shape to a flat plate under simulated microgravity. Similar electron-dense structures have been observed in Mimosa pulvinar motor cells, and their configurations changed with the turgor pressure change of motor cells [31]. Analogous to the “tannin vacuole” in Mimosa pulvinar motor cells [32], it is likely that the vacuolar electron-dense structure observed in the present study is mainly composed of tannin, although it is difficult to identify it as the tannin vacuole because its limiting membrane has not been confirmed in the present study. Generally, it is believed that tannin in the vacuole effectively adsorbs not only Ca2+, but also various other cations [31]. In the Mimosa pulvinar motor cells, it has also been well established that a large amount of Ca binds to, or is released from, the electron-dense tannin vacuole, during the seismonastic response of leaf [32]. Thus, in the columella cells of soybean roots, the vacuolar electron-dense structure may also function as a significant Ca storage site. The [Ca] detected in the vacuolar electron-dense structure was ∼47 mmol kg−1 dry wt under gravity. This value was slightly lower than the [Ca] measured in the lower half of horizontally oriented corn roots [19] and corresponds to the [Ca] in the SR lumen of scorpionfish swimbladder muscle [18]. Therefore, we considered that the concentration of Ca binding to the vacuolar electron-dense structures under gravity indicates the physiological Ca levels found generally in plants and animals. On the other hand, under simulated microgravity, the [Ca] in the vacuolar electron-dense structure increased to ∼2000 mmol kg−1 dry wt, which was ∼40 times higher than the value obtained under normal gravity. It is generally accepted that apoplastic Ca2+ transport occurs freely in various kinds of plants [10,26,33]. Assuming that Ca2+ is always supplied by apoplastic transport, under simulated microgravity, plants may continuously cause Ca2+ influx and subsequent Ca2+ uptake into vacuoles, and ultimately Ca binding to the vacuolar electron-dense structures, to avoid cytotoxic damage induced by the total increase of free Ca2+ concentration in the columella zone [34,35]. The results of this study support the view that the vacuole plays a significant role in the regulation of [Ca] in cytoplasmic matrixes [36,37] and that Ca2+ functions as the intracellular secondary messenger for the graviresponse [29,30]. Ca translocation coupled with the movement of other cations As discussed previously, under simulated microgravity, Ca2+ moves toward the central zone of columella cells, eventually to the vacuolar electron-dense structure, from the outer surrounding regions, whereas K+ moves in the opposite direction (Fig. 6, Table 1). Under simulated microgravity, the [K] decreased conspicuously in the vacuolar electron-dense structure and the vacuolar lumen and increased markedly in the cell wall, although there was no change in the cytoplasmic matrix. A similar increase of [K] in the cell walls around the columella cells after gravistimulation, in which the root orientation was changed to the horizontal, has been reported in Zea mays L., although the region showing an increase in [K] was restricted to the lower part of the columella zone, and the [Ca] in the cell walls was slightly increased by the apoplastic Ca2+ movement [19]. Under simulated microgravity, the [K] in the vacuole of the corn columella cells was decreased to ∼50% of that detected under gravity [21]. Thus, we assume that the increase of extracellular [K] and the efflux of K+ from the vacuole via the cytoplasmic matrix are common physiological responses to various gravistimulations. The [K] in the vacuole and the cell wall changes dramatically in the pulvinar motor cells of Mimosa pudica L. during the seismonastic petiole movement [31,32]. It has been considered that, in the pulvinus, K+ acts as a trigger for the influx of water to cause swelling of motor cells [38], and the movement of K+ and water is related to the change of [Ca] in the vacuolar lumen [31,32]. These factors and the correlated changes of [Ca] and [K] in the present study suggest that, in the columellar cells of soybean roots, an antiport system for Ca2+/K+ translocation plays a significant role in linking graviperception with the growth movement of roots. The [P] was increased markedly only in the vacuolar electron-dense structure under simulated microgravity (Table 1). The increase in [P] was caused by excess P included in the vacuole adsorbed by the vacuolar electron-dense structure, like Ca binding. In contrast with other cations, simulated microgravity did not cause any significant change in [Mg], indicating that the contribution of Mg2+ to the graviresponse was negligible. Concluding remarks Ultrastructural observations revealed that, in the columella cells of soybean roots grown under simulated microgravity, amyloplasts changed their intracellular localization to a diffuse distribution from the sedimentation at the cell bottom found generally in plants grown under gravity. It has also been confirmed that, under gravity, numerous ER exist exclusively at the bottom of the columella cells and are frequently attached to by the amyloplasts. In the vacuole of columella cells, the electron-dense structure closely resembled the tannin vacuoles found in Mimosa pulvinar motor cells that change their configuration from a granular shape to a flat plate. Ca cytochemistry by the PA method showed that, in the columella cells, the number of PA precipitates decreased in cytoplasmic matrix and increased in the vacuole under simulated microgravity. The quantitative X-ray microanalysis of cryosections revealed that the Ca concentration was very high in the vacuolar electron-dense structure under gravity, and increased conspicuously under simulated microgravity, concomitantly with a significant decrease of K in the vacuoles and a significant increase of K in the cell walls. The results of ultrastructural observations support a view that amyloplasts function as statoliths for the graviresponse, and Ca2+ is released from the ER by the contact of amyloplasts. 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TI - Cytochemical and electron probe X-ray microanalysis studies on the distribution change of intracellular calcium in columella cells of soybean roots under simulated microgravity
JF - Journal of Electron Microscopy
DO - 10.1093/jmicro/dfr095
DA - 2011-12-08
UR - https://www.deepdyve.com/lp/oxford-university-press/cytochemical-and-electron-probe-x-ray-microanalysis-studies-on-the-pZF3c508HG
SP - 57
EP - 69
VL - 61
IS - 1
DP - DeepDyve
ER -