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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Isolation of a Crystal Matrix Protein Associated with Calcium Oxalate Precipitation in Vacuoles of Specialized Cells¹

Department of Genetics and Cell Biology (X.L.), School of Biological Sciences (D.Z., V.J.L.-H., V.R.F.), and Institute of Biological Chemistry (T.W.O.), Washington State University, Pullman, Washington 99164–4236

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The formation of calcium (Ca) oxalate crystals is considered to be a high-capacity mechanism for regulating Ca in many plants. Ca oxalate precipitation is not a stochastic process, suggesting the involvement of specific biochemical and cellular mechanisms. Microautoradiography of water lettuce (Pistia stratiotes) tissue exposed to ³H-glutamate showed incorporation into developing crystals, indicating potential acidic proteins associated with the crystals. Dissolution of crystals leaves behind a crystal-shaped matrix "ghost" that is capable of precipitation of Ca oxalate in the original crystal morphology. To assess whether this matrix has a protein component, purified crystals were isolated and analyzed for internal protein. Polyacrylamide gel electrophoresis revealed the presence of one major polypeptide of about 55 kD and two minor species of 60 and 63 kD. Amino acid analysis indicates the matrix protein is relatively high in acidic amino acids, a feature consistent with its solubility in formic acid but not at neutral pH. ⁴⁵Ca-binding assays demonstrated the matrix protein has a strong affinity for Ca. Immunocytochemical localization using antibody raised to the isolated protein showed that the matrix protein is specific to crystal-forming cells. Within the vacuole, the surface and internal structures of two morphologically distinct Ca oxalate crystals, raphide and druse, were labeled by the antimatrix protein serum, as were the surfaces of isolated crystals. These results demonstrate that a specific Ca-binding protein exists as an integral component of Ca oxalate crystals, which holds important implications with respect to regulation of crystal formation.

Large amounts of Ca oxalate crystals can be formed very rapidly (0.5–1 h; Franceschi, 1989) and are associated with specialized subcellular structures, including membranes within the vacuole (for review, see Arnott and Pautard, 1970; Franceschi and Horner, 1980; Horner and Wagner, 1995). These observations and studies of crystal development (Kostman and Franceschi, 2000) indicate that Ca oxalate formation is a highly controlled cellular process rather than a simple precipitation phenomenon and that specialized mechanisms must be present in crystal idioblasts to deal with the large fluxes of Ca. Recent analyses of Medicago truncatula mutants have clearly demonstrated that there is a genetic component to the specificity of Ca oxalate formation involving more than a single gene (Nakata and McConn, 2000, 2003; McConn and Nakata, 2002; Nakata, 2002). The mechanisms that control Ca oxalate formation are not known but are undoubtedly complex and must involve synthesis and transport of oxalic acid (Li and Franceschi, 1990; Horner et al., 2000; Keates et al., 2000; Kostman et al., 2001) and regulation of Ca transport across the plasma membrane, through the cytoplasm and across the tonoplast. We have found that one regulatory step involves the idioblast endoplasmic reticulum (ER) and an ER luminal Ca-binding protein, recently identified as calreticulin, which is localized in specialized ER subdomains and helps buffer cytosolic Ca activity during Ca oxalate formation (Franceschi et al., 1993; Quitadamo et al., 2000; Kostman et al., 2003; Nakata et al., 2003). Another regulatory step must occur within the vacuole where Ca precipitation takes place. Precipitation is not random because crystal growth is coordinated with cell expansion (Kostman and Franceschi, 2000), and the morphology of the crystals produced is specific to a particular species or tissue within a species (Arnott and Pautard, 1970; Franceschi and Horner, 1980).

A fundamental question of Ca oxalate formation concerns how the plant cell is able to regulate Ca precipitation within the vacuole. One level of control may be through regulation of crystal nucleation and the rate and direction of crystal growth by effector molecules, such as those found in animal biomineralization processes (Weiner, 1984, 1986; Addadi and Weiner, 1985; Lowenstam and Weiner, 1989; Mann et al., 1989; Simkiss and Wilbur, 1989; Aizenberg et al., 1995, 1997). For example, various endogenous proteins have been demonstrated to promote or inhibit Ca oxalate crystal growth and aggregation in urinary systems of mammals and in models of this system (Edyvane et al., 1987; Rao et al., 1988; Weiner and Addadi, 1991; Shiraga et al., 1992; Hoyer et al., 2001). Two classes of these proteins, Asp-rich acidic proteins and Ser-rich glycoproteins, are present in mineralized regions of a wide range of organisms and are called matrix proteins (Weiner, 1984; Albeck et al., 1996). Some of these proteins have been found to have strong Ca-binding activity (Addadi and Weiner, 1985; Lanzalaco et al., 1988). Free Asp can also regulate the micromorphology of Ca carbonate crystal formation via a change in the surface properties of the microsteps in the developing crystal (Teng et al., 1998). Although specific matrix proteins have not been characterized in plant biomineralization systems, Webb et al. (1995) have demonstrated that there is a complex organic matrix associated with raphide crystals in grape (Vitis vinifera), which includes a mixture of polysaccharides and proteins of unknown function. More recently, it was demonstrated that protein extracts from tomato (Lycopersicon esculentum) and tobacco (Nicotiana tabacum) crystals can promote nucleation of Ca oxalate and also influence crystal morphology (Bouropoulos et al., 2001), although individual proteins were not characterized.

In view of the strict spatially controlled process, matrix proteins with Ca-binding activity could be critical components in the control of Ca oxalate formation and, thus, bulk Ca regulation in plant tissues. The purpose of this study was to determine if a Ca-binding protein is involved in Ca oxalate formation in water lettuce (Pistia stratiotes), a species for which we have characterized various features of the cell biology of CaOx formation (Franceschi et al., 1993; Keates et al., 2000; Kostman and Franceschi, 2000; Kostman et al., 2001; Volk et al., 2002; Nakata et al., 2003). We developed a procedure for isolation of large amounts of pure Ca oxalate crystals from water lettuce that allowed us to isolate proteins from the crystals that were found to have high Ca-binding activity. Antibodies to these proteins were raised and used to characterize patterns of tissue, cellular, and subcellular location. These results represent the first report, to our knowledge, of specific Ca-binding matrix proteins involved in biomineralization in plants and have significant implications to the biochemical mechanisms involved in this high-capacity Ca regulation system.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Crystal Idioblasts Are Structurally Specialized for Ca Accumulation

Water lettuce forms two types of crystals: needle-shaped raphides and multifaceted druse crystals. We showed previously that the druse crystals occur in the vacuole in association with distinct membranes (Volk et al., 2002), and a single druse conglomerate crystal is formed per cell. Figure 1 illustrates that the raphide crystals occur as bundles of crystals in the vacuole of specialized cells and also in association with membranes. Membranes appear to be a common feature of crystal formation and have been shown to be associated with all crystal types examined in a range of species (for review, see Arnott and Pautard, 1970; Franceschi and Horner, 1980; Horner and Wagner, 1995). The crystals are formed in vacuoles of the specialized cells, or idioblasts, that are scattered in an otherwise homogeneous cell types, such as the mesophyll cells in the leaves. One crystal idioblast contains only one crystal type, raphide or druse. The raphide crystal idioblasts are concentrated at the lower portion of the leaf in tissue partitions between aerenchyma air spaces, whereas the druse crystal idioblasts are usually found at the upper portion of the leaf in compact chlorenchyma above the aerenchyma.

View larger version (167K): [in this window] [in a new window]   Figure 1. Ca oxalate crystal idioblasts of water lettuce have specialized features. A, Phase contrast image of a longitudinal section through a developing raphide crystal idioblast. The Ca oxalate crystals appear as a bundle of needle shaped structures within the vacuole (V) of a greatly elongated idioblast cell. Bar = 10 µm. B, Transmission electron microscopy (TEM) cross section through a developing raphide crystal idioblast. The crystals occur as rows of white rectangular profiles within the vacuole (V). Bar = 2 µm. C, Higher magnification of developing raphide crystals (R) as seen in cross section within the vacuole (V). The crystals occur in files between distinct sheets of parallel membranes (arrowheads). Bar = 0.5 µm.

Acidic Amino Acid Is Incorporated into Ca Oxalate Crystals in Vivo

Matrix proteins in animal biomineralization systems are often enriched in acidic amino acids, so we performed a test to determine if such amino acids are incorporated into developing crystals. After exposing developing plants to tritiated Glu, autoradiographic analysis revealed that Ca oxalate crystals had label associated with them. As seen in Figure 2A, the label was along crystals, indicating it was incorporated into the crystal or along the membranes surrounding the crystals. Label was also found in the cytoplasm, as expected, but not in the vacuolar space surrounding the crystals. Labeling of druses crystals also occurred (not shown). Labeling of crystals was seen in developing crystal idioblasts of young leaves but not already mature idioblasts, indicating that it was not due to a simple absorption effect of the amino acid to the crystal surface. Figure 2B shows an almost mature crystal idioblast, and the crystal bundle has no label associated with it except at the very periphery of the bundle. As shown in Kostman and Franceschi (2000), the crystals at the periphery are the last to form; therefore, this observation is consistent with Glu being incorporated into growing but not mature crystals. It should be noted that samples were prepared in a way that results in loss of all soluble compounds, so the label had to be associated with amino acid incorporated into an insoluble complex, such as a polypeptide or aminoglycan.

View larger version (94K): [in this window] [in a new window]   Figure 2. Acidic amino acids are incorporated into developing Ca oxalate crystals in vivo. A, Microautoradiograph showing exposure of developing plant to 3H-Glu gives rise to labeling (black dots) of vacuolar crystals (arrows) and cytoplasm of idioblasts and cytoplasm but not vacuoles of non-crystal cells. Bar = 10 µm. B, Almost mature crystal idioblast from same plant. The large bundle of crystals (C) in the middle of the vacuole is unlabeled except at the periphery of the bundle where the last crystals are produced (arrows). Note surrounding parenchyma cells are heavily labeled. Bar = 10 µm.

Crystals Contain a Non-Mineral Matrix

When isolated crystals are treated with EDTA, the Ca oxalate is partially or completely dissolved, depending upon the length of treatment, and examination of the samples with TEM shows that a nonmineral matrix remains. As shown in Figure 3, A to C, this material retains the shape of the original crystal; thus, we refer to it as a "crystal matrix ghost." The crystal matrix ghost remains intact during processing of the sample for TEM but is flexible and as seen in Figure 3C can be bent at a 90° angle, unlike the crystal, indicating that it is made of an interconnected macromolecular complex. Often the central region of the raphide crystals do not de-mineralize so that a small block of crystal remains (Fig. 3A). This is the region that we have previously shown is structurally different and the likely point of initial nucleation of the crystals (Kostman and Franceschi, 2000). Partial dissolution with EDTA can also leave behind small "plates" of Ca oxalate along the matrix (Fig. 3B). Figure 3D shows that when a crystal matrix ghost is incubated with Ca and oxalate, a crystal forms with essentially the same shape as the ghost, although often the surfaces are rough or have micro-crystals projecting from them. The druse crystals also have a central core of material, as we have previously shown (Volk et al., 2002), and after dissolution of the mineral with EDTA, this core material can also initiate crystallization, although the crystal morphology is very irregular (data not shown). Microautoradiography of crystals or crystal matrix exposed to radioactive Ca or oxalate further demonstrate the ability of the matrix to bind these ions. Figure 3, E and F, show that the demineralized crystal matrix is capable of binding both Ca and oxalic acid at high levels. The intact mature crystals can also bind substantial Ca but at a lower level than the matrix (Fig. 3G), whereas the intact crystals seem to have little affinity for oxalic acid (Fig. 3H).

View larger version (92K): [in this window] [in a new window]   Figure 3. Ca oxalate crystals have a non-mineral matrix with an affinity for Ca and oxalate. A, EDTA removes most of the Ca oxalate but leaves a flexible matrix "ghost" in the shape of the original crystal. Note that the middle part of the crystal (arrow) has not been dissolved. Bar = 5 µm. B, Higher magnification of the crystal matrix. Some Ca oxalate (arrows) is still present along the matrix ghost. Bar = 0.5 µm. C, Crystal matrix ghost showing a sharp bend (arrow), indicating the integrity and flexibility of the matrix. Bar = 5 µm. D, Incubation of crystal matrix ghost with Ca and oxalate results in precipitation of a new crystal with roughly the same shape as the original. Bar = 2 µm. E, Demineralized crystal matrix incubated with 45Ca. Very strong binding of labeled Ca to the matrix is seen. F, Demineralized crystal matrix incubated with 14C-oxalic acid. The organic matrix binds appreciable amounts of oxalic acid. G, Intact crystal incubated with 45Ca gives rise to labeling but at a lower density than demineralized matrix. H, Intact crystal shows very little binding of 14C-oxalic acid.

Matrix Protein Is Present in Ca Oxalate Crystals

To determine whether proteins are associated with the crystalline matrix of the Ca oxalate crystals, we prepared highly purified samples of crystals from water lettuce, as shown in Figure 4, which were boiled in SDS to remove all contaminating surface proteins. The purified raphide and druse crystals were then dissolved with 0.5 M EDTA to yield the crystal matrix ghosts and a dissolved crystal solution, which did not contain detectable protein (data not shown). The crystal matrix ghosts were essentially insoluble in water but could be dissolved in 78% (v/v) formic acid or 5 M urea. Figure 5A shows the resulting SDS-PAGE of the matrix-associated protein from a raphide crystal sample. Both raphide and druse crystal matrices exhibited the same simple protein profile with a major band at about 55 kD and minor bands at 60 and 63 kD that were just discernible with silver staining.

View larger version (90K): [in this window] [in a new window]   Figure 4. Pure crystal samples isolated from water lettuce leaves. Bars = 50 µm. A, Raphide crystals. B, Druse crystals.

View larger version (76K): [in this window] [in a new window]   Figure 5. Matrix protein is present in Ca oxalate crystals. Purified crystals were dissolved in EDTA, resulting in formation of a precipitate. The precipitate was washed thoroughly, dissolved in 78% (v/v) formic acid, lyophilized, dissolved in 5 M urea, and run on SDS PAGE. A, Crystal matrix protein sample (Matrix) contains a major polypeptide band at about 55 kD and two minor polypeptide bands as determined by silver-stained gel. Total protein was not determined due to the insolubility of the preparation. B, Western immunoblot using antibody raised to the matrix protein. Lane 1, Total protein (27 µg) extracted from young leaves; lanes 2 and 3, 5 and 10 µg of purified matrix protein.

To obtain sufficient amounts of matrix protein for further analysis, preparative amounts of crystals were isolated, yielding about 1 g of mixed raphide and druse crystals from 0.5 kg of leaves. After EDTA dissolution and extensive washing, the resulting insoluble matrix was dissolved with 78% (v/v) formic acid. The formic acid-soluble matrix protein was lyophilized, resulting in a white precipitate that could be dissolved in 5 M urea and then resolved by SDS-PAGE. A protein profile identical to that presented in Figure 5A was observed (data not shown). About 100 µg of protein was recovered from 1 g of crystals, as determined by A₂₁₀ absorbance.

Table I gives the amino acid content of the total matrix protein extracted from Ca oxalate crystals. Amino acid analysis of the matrix protein sample gave values of about 20 mol % for Gln/Glu and Asn/Asp combined (Table I), indicating considerable acidic amino acid residues. Gly, at 13 mol %, was the highest single amino acid residue. The basic amino acids Arg, Lys, and His accounted for about 10.6 mol %. The nonpolar, hydrophobic amino acid residues (Ala, Ile, Leu, Met, Phe, Pro, Trp, and Val) accounted for over 45 mol % of the total amino acids.

Matrix Protein Has Strong Ca-Binding Activity

Matrix protein isolated from some animal biomineralization systems have been found to have strong and specific Ca-binding activity (Addadi and Weiner, 1985). Because an interaction of proteins with Ca in the Ca oxalate crystals was an obvious functional possibility, we assayed the matrix protein from water lettuce crystals for Ca-binding properties. As demonstrated by the Ca-binding dot blot in Figure 6, the matrix protein exhibited strong ⁴⁵Ca²⁺-binding activity, which could be readily competed out by 250-fold excess of unlabeled Ca²⁺. Mg²⁺ was also able to compete against ⁴⁵Ca²⁺, although it was much less effective as compared with Ca²⁺. BSA, used as a control protein on the same blots, did not exhibit detectable Ca²⁺-binding activity (Fig. 6). The matrix protein also stained blue in polyacrylamide gels as viewed by the Stains-all method, a property typically exhibited by Ca-binding proteins (data not shown). These results demonstrate that the matrix protein has strong affinity for Ca and, therefore, may play a significant role in crystal nucleation or growth.

View larger version (69K): [in this window] [in a new window]   Figure 6. Matrix protein from Ca oxalate crystals has strong Ca-binding activity. From 1.0 to 8.0 µg of matrix protein (MP) and bovine serum albumin (BSA) were immobilized onto nitrocellulose paper and then exposed to 45Ca with or without 250-fold excess of unlabeled Ca2+ or Mg2+ as competitors for 45 Ca-binding. Excess Ca completely competes out 45Ca-binding, whereas magnesium gives only partial competition of 45Ca binding.

Matrix Protein Is Specific to Crystal Idioblasts

Crystal matrix proteins are thought to provide a framework upon which crystal nucleation occurs or to control the direction and rate of growth. Thus, the matrix protein should be restricted to cells that are producing crystals. To test this, polyclonal antibodies to the matrix protein from water lettuce were prepared, which, as shown in Figure 5B, recognized the major matrix protein from water lettuce leaf extracts. The antiserum also recognized the isolated matrix protein, as expected, although the isolated matrix protein did not run as a sharp band on the gels due to solubility problems. This antiserum has allowed us to conclusively determine the cellular and subcellular location of the matrix protein in leaves of water lettuce.

Figure 7 documents the localization of the matrix protein in developing crystal idioblasts. Immunolabeling at the light microscope level on resin sections was only found in the vacuoles of raphide and druse crystal idioblasts but not of mesophyll cells of the leaf, and the labeling was primarily associated with the developing crystals. Figure 7B demonstrates the specificity of the reaction because the pre-immune serum did not give rise to labeling of crystals or any other structures in the resin sections. Using electron microscopy immunolocalization, it was found that labeling was restricted to specific structures. The surface of both druse and raphide crystals were labeled as seen in Figures 8, A to C. The central part of the crystals does not infiltrate with the resin and falls out of the sections, so only a thin region at the periphery of the crystals still remains. This appears as a darker material and labeling is clearly associated with it as viewed by both longitudinal (Fig. 8A) and cross (Fig. 8B) sections of raphide crystals and facets of the druse crystals (Fig. 8C). Similar to what was demonstrated with light microscope sections, the preimmune serum does not give rise to any labeling in the resin embedded sections (not shown). Figure 8, A and B, show that the matrix protein antibody also strongly labels the Golgi apparatus, indicating that the Golgi apparatus is involved in transport of the matrix protein to the vacuole.

View larger version (73K): [in this window] [in a new window]   Figure 7. Matrix protein is only expressed in developing Ca oxalate crystal idioblasts. Bars = 20 µm. A, Immune serum. The bundles of crystals (yellow arrowheads) within the vacuoles of two developing crystal idioblasts (arrows) are very heavily labeled (purple product), whereas surrounding cells show no labeling for matrix protein. B, Pre-immune serum. Crystal bundles (yellow arrowheads) in developing crystal idioblasts show no labeling with the control serum.

View larger version (120K): [in this window] [in a new window]   Figure 8. Matrix protein is present along the surface of developing Ca oxalate crystals of water lettuce. Bars = 0.5 µm. A, Longitudinal section through part of a developing raphide crystal idioblast showing crystals (R) within the vacuole (V) as elongate white profiles. Label is distributed along the surface of the crystals. The Golgi bodies (G) in the cytoplasm are also labeled. B, Cross section through a raphide crystal idioblast, with crystals (R) appearing as rectangular profiles within the vacuole (V). Label is specifically associated with material along the crystal surfaces. Inset shows enlargement of a Golgi apparatus and heavy labeling associated with it. C, Section showing part of the edge of a druse crystal (D) within the vacuole (V) of a druse idioblast. The multiple facets of the surface of the druse have label associated with them. D, Whole isolated raphide crystals exposed to matrix protein antiserum have label associated with their surfaces. E, Whole isolated raphide crystals treated with pre-immune serum have no gold particles associated with them.

To ensure that the labeling seen in sectioned material was not artifact, freshly isolated crystals were treated for localization of matrix protein with the same antisera preparations. As seen in Figure 8, D and E, the primary antiserum, but not the preimmune serum, gave specific labeling of the surface of the isolated crystals.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Ca oxalate formation is a basic and important physiological process in many plants, which results in the sequestration of large amounts of Ca as a physiologically and osmotically inactive compound. Here, we show that an organic matrix is involved in crystal formation and that protein is part of this matrix. Crystal growth is carefully regulated to match cell growth rate because the cells and crystals often elongate considerably during tissue maturation. For example, in the case of water lettuce raphide idioblasts, crystals grow bidirectionally in the idioblast vacuole in coordination with the elongation of the cells, which will reach 5 times the length of a typical mesophyll cell (Kostman and Franceschi, 2000). However, little is known about the processes occurring in the vacuole that regulate precipitation and crystal growth rate and direction. The organic matrix shown here is clearly capable of nucleating Ca oxalate precipitation and appears to form a template for such precipitation. Similarly, Webb and Arnott (1983) showed that grape druse crystals have a nonmineralized core material of unknown but presumably organic composition, and Webb et al. (1995) demonstrated that a complex organic matrix was present within the vacuole of grape raphide idioblasts and that this matrix could facilitate crystal formation. Recently, Bouropoulos et al. (2001) found that crystals of tomato and tobacco contain macromolecules that can promote Ca oxalate nucleation. Macromolecular matrix materials can hold important implications with respect to crystal morphology and, as pointed out by Arnott and Webb (2000), crystal stability. However, identification of a specific macromolecular species from the Ca oxalate crystal structure has not been achieved previously. We have now identified protein that appears to be specific to crystal forming cells and associated with the crystals. The protein will be referred to as matrix protein because at least a fraction of it is integrated into the crystal matrix because we isolated the protein only after complete dissolution of detergent washed crystals. Further analysis of this protein will allow us to gain insight into the mechanism by which Ca sequestration in the vacuole may be regulated during Ca oxalate formation.

Most calcified tissues in animal systems undergoing controlled mineralization have been found to have an organic matrix associated with them (Lowenstam and Weiner, 1989), which includes various classes of proteins shown in vitro to be able to control crystal growth and morphology. Such proteins have been found to be integrated into the structure of biominerals of invertebrate organisms such as sponge spicules (Aizenberg et al., 1995, 1996), mollusk shells (Albeck et al., 1996), and sea urchin spines (Aizenberg et al., 1997), and also in Ca oxalate renal stones that form in humans (Homo sapiens; Ryall et al., 2000). Although macromolecules appear to be involved in nucleation and modifying growth patterns (Weiner and Addadi, 1991; Guo et al., 2002), they may also have inhibitory effects in the case of Ca oxalate in the urinary tract (Shiraga et al., 1992; Hoyer et al., 2001). A well-studied model that is particularly relevant to the plant crystal matrix is the larval sea urchin spicules (Wilt, 2002). The calcareous spicules are deposited outside of the cell and have within them a protein matrix but also are enclosed by an extracellular matrix. The matrix materials include proteins and glycoproteins with discrete localizations (Killian and Wilt, 1996; Ameye et al., 1999, 2001) that have been proposed to be involved in controlling the deposition and growth of the spicules by inhibiting or promoting biomineralization depending upon the chemical features of the proteins. For example, antisense suppression of the LSM34 matrix protein, which is in the extracelluar matrix of the spicules, has been shown recently to inhibit biomineralization (Peled-Kamar et al., 2002). The matrix protein we have isolated is found within the crystalline matrix but may also be associated with the surface as indicated by immunolabeling results. Thus, at this time, it is not clear if discrete intracrystalline and extracrystal matrix proteins exist in the plant Ca oxalate crystals.

Amino acid analysis of the total matrix protein indicates it is relatively high in acidic residues (20 mol %), which may be involved in the Ca-binding activity shown here. In addition, there are considerable basic residues (approximately 11 mol %) that might be involved in the oxalate binding seen for demineralized matrix samples. The high amount of nonpolar, hydrophobic residues (45 mol %) may explain the solubility problems experienced with the isolated, Ca-free matrix protein preparations. However, one must take into account not only the total amino acid composition but the relative disposition of the residues within the protein to really understand the physical and chemical properties of the protein. Cloning and analysis of the gene(s) for matrix protein, which is currently under way, will allow for this type of analysis.

It is interesting to note that the acidic proteins from animal matrix have some physical properties similar to our matrix protein, such as poor solubility of some of the animal matrix proteins, a tendency to aggregate, and poor staining on SDS-PAGE (Weiner and Addadi, 1991). We hypothesize that the protein isolated from plant Ca oxalate crystals has a similar function to some of the animal matrix proteins in terms of affecting crystal growth. This is supported by the fact that the crystal matrix protein has Ca-binding properties, which would be important to their integration into the crystalline matrix. The matrix protein with their Ca-binding capacity could act as nucleating agents for Ca oxalate crystal formation. The matrix protein could also potentially affect the direction and rate of crystal growth by interacting in specific orientations or with specific faces of the developing crystal, as suggested by Bouropoulos et al. (2001). Various studies of biocrystals and in vitro studies using animal matrix proteins have shown that this does occur (for review, see Weiner, 1986; Weiner and Addadi, 1991). The acidic proteins in particular are considered to play a major role in controlling crystal initiation and patterns of growth (Weiner and Addadi, 1991). The effect of the Asp-rich proteins on crystal growth is dependent upon the structural conformation of the proteins. Solutions containing such proteins, or related synthetic peptides, are generally inhibitory to growth, whereas immobilized proteins promote ordered crystallization (Termine et al., 1981; Addadi and Weiner, 1985; Campbell et al., 1989; Linde et al., 1989; Shiraga et al., 1992), demonstrating that these proteins can regulate crystal growth rate and morphology.

Our ability to generate antibodies to the matrix proteins has allowed us to confirm that this protein is specifically expressed in Ca oxalate crystal idioblasts and is associated with the developing crystals. It appears that the protein is concentrated at the surface of the crystals and may make up the boundary layer commonly referred to as a membrane or membrane chamber based upon its ultrastructural features. Moreover, in view of its Ca-binding activity, the matrix protein may also be involved in transporting Ca to the surface of the growing crystals. There is a separate membrane system in water lettuce raphide idioblasts that we refer to as the parallel membrane system (Fig. 1C), but such membranes do not contain matrix protein as determined by immunocytochemistry. We are aware of only one other report of a crystal idioblast-specific protein, and in this case, it was found to be associated with an amorphous material at the crystal surface (Trull et al., 1991). It was not determined if this protein had Ca-binding properties; therefore, it is difficult to discuss the nature of the protein with respect to the water lettuce matrix protein.

It has been observed that crystal idioblasts are often enriched in ER, Golgi bodies, and small vesicles (Arnott and Pautard, 1970), which is the case with the developing idioblasts of water lettuce. We have found recently that the crystal idioblasts, and subdomains of their ER, are enriched in calreticulin relative to surrounding cells, supporting a hypothesis that the large amount of ER is needed for internal regulation of Ca fluxes during crystal formation (Kostman et al., 2003; Nakata et al., 2003). The matrix protein is also synthesized on the ER and may be responsible for carrying some Ca with it in transit to the vacuole. Our results indicate that the matrix protein is transported to the vacuole via the Golgi apparatus, which is consistent with mechanisms for transport of proteins to the vacuole (Chrispeels, 1991). We can now assign at least one function for the increased Golgi activity that is commonly seen in developing plant crystal idioblasts, transport of matrix protein to the vacuole, although secretion of wall polymers in support of rapid expansion growth is probably also supported by the large number of Golgi in the idioblasts.

In summary, Ca oxalate crystals were shown to have an organic matrix that has Ca- and oxalate-binding properties. We have isolated a matrix protein that is specific to the cells involved in Ca oxalate formation. The matrix protein has strong Ca-binding properties, and we hypothesize that it is involved in the regulation of Ca oxalate precipitation within the vacuole. Using antibodies against the matrix protein, we have confirmed that it is associated with the developing crystals and transported to the vacuole by the Golgi apparatus. Further studies of the matrix protein will help us to understand the regulation of Ca oxalate formation, an important basic physiological mechanism for sequestering large amounts of Ca in higher plants. To achieve a better understanding of the structure and function of the matrix protein, we have undertaken the cloning of the gene.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material

Water lettuce (Pistia stratiotes) plants were grown in plastic tubs and maintained in a growth chamber programmed for a photoperiod of 16 h at 25°C and a dark period of 8 h at 20°C. Light was provided by a combination of fluorescent and incandescent lamps, giving a photon flux density of about 400 µE m^–2 s^–1 at the plant surface. The nutrient solution (Peter's 20-20-20, W.R. Grace and Co., Foglesville, PA) was replaced weekly.

Transmission Electron Microscopy

Small leaf pieces were fixed for 12 h at 4°C in 1.25% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 50 mM PIPES buffer (pH 7.2), buffer washed, dehydrated with an ethanol series, and embedded in LR White resin. The specimens were sectioned with a diamond knife, and the sections were stained with 1% (w/v) uranyl acetate (aqueous) and 1.5% (w/v) alkaline lead citrate (aqueous) and examined with a Hitachi 600 TEM (Hitachi, Pleasanton, CA).

Microautoradiography

Axenic cultures of water lettuce (Tarlyn et al., 1998) were grown for 2 d on media with 5 mM CaCl₂, for 2 d on media without Ca, then for 1 d on media with 20 mM Ca, 2 mM Glu, and ³H-3,4 Glu to give 20 µCi mL^–1 total activity. Shoot tips were washed with double-distilled water and fixed overnight with 2.0% (v/v) paraformaldehyde and 2.0% (v/v) glutaraldehyde in 50 mM PIPES buffer at 4°C. The specimens were washed three times with 50 mM PIPES buffer, then dehydrated step wise from 30% to 100% (w/v) acetone and infiltrated with Spurr's/100% (v/v) acetone overnight with an increase in Spurr's concentration each day (Spurr's:acetone ratios: 1:3, 1:2, 1:1, and 2:1 [v/v]). The final resin/ethanol mix was replaced with 3 changes of 100% (v/v) Spurr's over 3 d. Individual samples were placed in flat embedding molds, covered with fresh Spurr's, and polymerized at 70°C overnight. Samples were sectioned with glass knives, placed on gelatin-coated slides, and coated with a 1:2 (v/v) dilution of Ilford L4 photographic emulsion and stored at 4°C. Slides were developed at weekly intervals for up to 6 weeks after emulsion coating. They were stained with 1% (w/v) Safranin O for 30 s, coverslipped, and viewed on a Leitz light microscope.

Small-Scale Crystal Isolation

Mature leaves (about 50 g) of water lettuce were homogenized in 4 volumes of water in a blender (Waring, Torrington, CT) for 3 to 5 min. The homogenate was filtered, with washing, through a 205-µm nylon mesh followed by 70-µm nylon mesh. The filtrate was kept on ice for approximately 1 h, at which time the supernatant was collected for raphide isolation and the settled material for druse isolation.

Raphide Isolation
The supernatant was centrifuged at 600g to pellet crystals and debris. The pellets were then incubated in 1% (v/v) Triton X-100 for 1 h, washed, and incubated for 2 to 3 h at 37°C in a digestion medium containing 2% (w/v) cellulase (Worthington Biochemical, Lakewood, NJ), 1% (w/v) Macerase, 1% (w/v) Pectolyase Y-23, 1% (w/v) BSA, 5 mM CaCl₂, 10 mM MES (pH 5.5), and 2% (w/v) -amylase (Sigma, St. Louis). This digestion step effectively removed contaminating cell wall materials and starch grains. After digestion, the samples were cooled, diluted with water, and layered over 2 mL of saturated cesium chloride. Crystals were pelleted by centrifugation for 1 to 2 min at 150g and washed extensively by several cycles of resuspension in distilled water followed by centrifugation.

Druse Isolation
The precipitate from the filtrate was collected by carefully removing the supernate, pelleted, and washed with distilled water. The washed pellet was then resuspended in water, loaded onto 2 mL of saturated cesium chloride, and the crystals were pelleted by centrifugation at 150g for 1 to 2 min. The crystal pellet was suspended in water and washed extensively with water. Some contamination by cell wall materials was present. This was removed by incubation with the digestion medium for 1 h at 37°C, followed by washing and a second cesium chloride centrifugation.

Large-Scale Crystal Isolation

This protocol was designed to isolate preparative amounts of raphide and druse crystal mixture. About 1 kg of leaves was thoroughly washed with tap water and then homogenized, in several batches, in 4 volumes of water with a Waring blender for 3 to 5 min. The homogenate was filtered as described above. The filtrate was then centrifuged at 500g for 3 to 4 min. The pellet was suspended in 4 volumes of 1% (w/v) SDS in water and then centrifuged to pellet the crystals. The resulting crystal pellet was suspended in 1% (w/v) SDS again and boiled for 10 to 20 min, followed by extensive washing with water. This step was repeated several times until a white crystal pellet appeared. The crystal pellet was slightly contaminated by cell wall debris and starch grains, which were digested by incubation with the digestion medium overnight at 37°C. The crystals were washed with distilled water, boiled with 1% (w/v) SDS three times, and finally washed extensively with distilled water.

Visualization of Raphide Crystal Matrix

Crystals isolated as above where incubated in 250 mM EDTA overnight at 4°C to dissolve the Ca oxalate, washed with distilled water, and samples were placed onto Formvar-coated grids. The samples were then examined using a Hitachi 600 transmission electron microscope. Some samples were subsequently incubated in a solution of 5 mM CaCl and 5 mM oxalic acid for 3 d at 4°C on a Labquake rotator, washed with distilled water, then reexamined on the TEM. For light microscope observations ⁴⁵CaCl₂ + oxalic acid and CaCl₂ + ¹⁴ C-oxalic acid were added, respectively. These radioisotope labeling experiments were also performed with intact mature crystal samples at the same time. All samples were incubated at 4°C for 3 d with rotation on a Labquake rotator (Labindustries, Inc., Berkeley, CA).

Isolation of Crystal Matrix Proteins

The purified crystals were dissolved at 4°C in 0.5 M EDTA with a daily change of fresh EDTA. After about a week, the volume of the insoluble materials was reduced by 90% to 95% (w/v). The soluble portion was collected, dialyzed against 10 mM Tris-HCl (pH 8.0), and the volume was reduced using a centrifuge concentrator (Millipore, Billerica, MA). The resulting solution contained no detectable proteins as determined by a variety of protein assays and, therefore, was discarded in subsequent experiments. The insoluble materials were washed thoroughly with distilled water and then extracted with 78% (v/v) formic acid overnight at room temperature. The solution was clarified by centrifugation in a microfuge at full speed. The resulting supernate was lyophilized, giving a white material we refer to as matrix protein, which was subsequently suspended in 5 M urea for further analysis. The protein concentration was determined by the absorbance at A₂₁₀ using BSA as a standard.

Gel Electrophoresis and Western Immunoblotting

SDS-PAGE was performed according to Laemmli (1970) and silver-stained. For immunoblots, the proteins in the gel were transferred to a nitrocellulose membrane and probed with the primary antiserum (1:1,000 [v/v] dilution) as described by Volk et al. (2002). After extensive washing, the bound antibodies were detected with a chemiluminescence system according to the manufacturer (Amersham, Buckinghamshire, UK).

Analysis of Matrix Protein Amino Acid Content

Total matrix protein isolated as described above was used for analysis of amino acid content. Analysis was done by the Washington State University Bioanalytical Center (Pullman) using an Applied Biosystems 475A Protein Sequencer (PE-Applied Biosystems, Foster City, CA). Multiple samples of acid hydrolyzed matrix protein were analyzed, giving very consistent results between runs.

Ca-Binding Assay

Matrix protein and BSA were immobilized on nitrocellulose membranes using a dot-blot apparatus. The membranes were first incubated for 10 min in a binding buffer (60 mM KCl and 10 mM imidazole [pH 7.4]) modified from that of Maruyama et al. (1984). The membranes were then transferred to 10 mL of binding buffer containing 6.25 µM ⁴⁵Ca²⁺ (25 µCi), 6.25 µM ⁴⁵Ca²⁺ + 250x excess of cold Ca²⁺, or 6.25 µM ⁴⁵Ca²⁺ + 250x excess of cold Mg²⁺. After an incubation of 10 min, the membranes were quickly rinsed with binding buffer, washed for 5 min with distilled water, air dried, and then exposed to x-ray film.

Antibody Production and Immunolocalization

Antiserum to the matrix protein was produced using the "Golf" ball technique (Reid et al., 1992). In brief, purified matrix protein (about 200 µg) was immobilized on nitrocellulose membrane that was then homogenized. The homogenate was injected into a plastic whiffle ball that was preimplanted in the rabbit (New Zealand White). Serum was withdrawn from the ball before immunization to serve as control serum ("pre-immune"). After about 4 weeks, a second antigen injection was performed, followed by antiserum harvesting 2 weeks later. The titer of the antiserum was determined as described by Reid et al. (1992).

Immunolocalization on sectioned material was conducted essentially according to Li and Franceschi (1990). After a 1-h blocking incubation in buffer, the 1-µm sections for light microscopy or ultrathin sections for TEM were incubated 2 h with the pre-immune or primary antiserum diluted 1:100 (v/v) in Tris-buffered saline plus Tween 20 (TBST) + BSA (10 mM Tris [pH 7.2], 250 mM NaCl, 0.3% [v/v] Tween 20, and 1% [w/v] BSA). After extensive washing, the sections were incubated with protein A-gold (20 nm; Amersham) for 1.5 h at 1:100 (v/v) dilution, followed by washing. For light microscopy, the gold was silver enhanced using a kit as per the manufacturer's instruction (Amersham), and the sections were imaged using a 1024 laser scanning confocal microscope (Bio-Rad Laboratories, Hercules, CA) operated in a reflected/transmitted mode. The ultrathin sections for TEM were poststained with a freshly prepared 1:3 (v/v) mixture of 1% (w/v) potassium permanganate and 1% (w/v) uranyl acetate and examined with a Hitachi 600 transmission electron microscope.

For immunolocalization of matrix protein on whole crystals, 300 µL of highly purified crystals suspended in distilled water was mixed with 300 µL of TBST + BSA containing 500 mM NaCl, 0.3% (v/v) Tween 20, 10 mM Tris-HCl (pH 7.2), and 1% (w/v) BSA and incubated for 15 min at room temperature, followed by a 2 h of incubation in pre-immune or antimatrix protein antiserum diluted 1:100 (v/v) in the same buffer. After four 10-min washes with TBST + BSA, the crystals were incubated for 1 h with Protein A-gold (20 nm) and diluted 1:100 (v/v) in the same buffer. After extensive rinses with buffer and distilled water, the crystals were resuspended in water, and small drops of the suspension were dried down onto Formvar-coated nickel grids. They were examined and photographed on a Hitachi 600 transmission electron microscope.

	ACKNOWLEDGMENTS

 
Electron microscopy was performed in the Washington State University Electron Microscopy Center (Pullman). We thank Mr. Yujia Wu School of Biological Sciences (Washington State University) for excellent technical help.

Received March 13, 2003; returned for revision May 12, 2003; accepted June 2, 2003.

	FOOTNOTES

 
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.023556.

¹ This work was supported in part by the National Science Foundation (grant nos. MCB 9632027 and MCB 9904562 to V.R.F.) and by a Loyal Davis Fellowship (to X.L.).

² These authors contributed equally to the paper.

^* Corresponding author; e-mail vfrances{at}mail.wsu.edu; fax 509–335–3184.

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