Calsequestrinlike Calcium-Binding Protein Is Expressed in Calcium-Accumulating Cells of Pistia Stratiotes VINCENT R
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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6986-6990, August 1993 Plant Biology Calsequestrinlike calcium-binding protein is expressed in calcium-accumulating cells of Pistia stratiotes VINCENT R. FRANCESCHI*t, XINGXIANG Lit, DIANZHONG ZHANG*§, AND THOMAS W. OKITA1 *Department of Botany, *Department of Genetics and Cell Biology, and llnstitute of Biological Chemistry, Washington State University, Pullman, WA 99164-4238 Communicated by Clarence A. Ryan, April 19, 1993 (receivedfor review January 20, 1993) ABSTRACT To contend with high calcium (Ca) levels in thereby reducing the apoplastic concentration of this mole- the environment, many plant species contain crystal idioblasts, cule around adjacent cells (12, 13). The importance of these specialized cells which accumulate large amounts of Ca as specialized Ca sink cells is reflected by the fact that greater oxalate crystals. The biochemical processes involved in the than 90% of the total Ca may be present in this physiological accumulation of Ca in crystal idioblasts are unknown, as these and osmotically inert compound (14, 15). Likewise, a value cells constitute only a minor proportion ofthe total plant tissue. of 10% Ca oxalate by dry weight is not uncommon in plants To address how crystal idioblasts buffer cytosolic Ca during (16). crystal formation, we purified these cels from water lettuce There have been numerous developmental and structural and asses their biochemistry. We show here that crystal studies on crystal idioblasts which have described the unique idioblast cells contain three Ca-binding proteins not detectable ultrastructural features presumably involved in Ca transport in mesophyll cells. One of the Ca-binding proteins shares and sequestration into Ca oxalate crystals (reviewed in refs. antigenicity with rabbit calsequestrin, a high-capacity low- 8 and 9). There has been, however, almost no biochemical afnity Ca-binding protein, and is encoded by related nucle- information addressing the mechanisms involved in regulat- otide sequences. Immunocytochemical localization studies fur- ing Ca transport in these specialized cells. One major element ther demonstrate that a calsequestrinlike protein is present that must be operating in crystal idioblasts is a Ca buffering primarily in crystal idioblasts and is preferentially localized in system that will accommodate large fluxes of Ca while enriched in Ca as maintaining biochemical functions within the cytoplasm. In the endoplasmic reticulum, an organelle animal cells which are capable oflarge transitory fluxes ofCa evidenced by vital staining. We thus conclude that crystal (e.g., muscle cells), specialized Ca-binding proteins (CBP) idioblasts possess a buffering system involving calsequestrin- have been found which appear to be involved in buffering Ca like proteins, a process that likely plays an essential role in the activity (17-20). The most extensively studied of these pro- bulk control of Ca in plant cells. teins is calsequestrin, a high-capacity (up to 50 Ca molecules per calsequestrin molecule) low-affinity (Kd = 1 mM) CBP Calcium serves as an important effector molecule in the (21-25). A calsequestrin-like protein has recently been found signal transduction pathways of many physiological and in spinach leaves and Streptanthus tortuosus cell cultures developmental processes in plants (1-4). To serve this role, (26, 27); however, its tissue and subcellular locations have intracellular Ca levels are maintained within strict limits. not been determined. A high-capacity CBP, such as calse- Most plant cells modulate the cytoplasmic levels of Ca by questrin, could play an important role in cells such as the sequestration of this molecule within mitochondria, chloro- crystal idioblasts which are specialized for Ca transport and plasts, and endoplasmic reticulum (ER) or by active pumping compartmentation. of Ca into the apoplast (5, 6). Many plant species, however, To study the biochemical mechanisms involved in the display very little discrimination of Ca in the root zone (7). accumulation and sequestration of Ca in crystal idioblasts, This inability to restrict Ca uptake results in transport of Ca we have developed an efficient procedure for the isolation of throughout the plant via the transpiration stream to levels protoplasts ofthese specialized cells as well as those from the directly proportional to those available in the environment. bulk mesophyll tissue from Pistia stratiotes. Using biochem- As most plants do not excrete appreciable amounts of Ca ical, immunochemical, and molecular techniques, we dem- under normal growth conditions, Ca accumulates in the onstrate that calsequestrin-like proteins are present in crystal apoplast of those organs where evapotranspiration occurs. idioblasts but not in surrounding mesophyll cells of leaves of As up to a 10,000-fold difference in Ca concentration (total) this species. The data indicate that crystal idioblasts possess may occur between the apoplast and cytosol (3), and the a Ca buffering system which is critical to this mechanism of electrical potential difference is inside negative, there is a high-capacity Ca regulation in plants. tremendous driving force for inward diffusion of Ca. Mech- anisms involving sequestration of Ca, as discussed above, have limited capacity and become easily saturated at low METHODS concentrations, especially in those plants subjected to a Isolation of Idioblast and Mesophyll Protoplasts. The adax- continuous and relatively high uptake of Ca. ial surface of young leaves of Pistia stratiotes L. (water To cope with high Ca levels in the environment and, in lettuce) was gently abraded with emery cloth to remove turn, avoid intracellular Ca toxicity, many plants have trichomes. The leaves were cut into 1- to 2-cm2 pieces, placed evolved a mechanism to regulate bulk quantities of Ca by adaxial surface down onto digestion medium [wt/vol: 2% formation ofCa oxalate crystals (8-10). Ca oxalate formation cellulase (Worthington CELF), 1% Macerase (Calbiochem), often occurs in specialized cells called crystal idioblasts (11). 1% pectolyase Y-23 (Seishin Pharmaceutical), and 1% bovine It is thought that the primary function ofthese idioblasts is to serve as a Ca accumulate Ca as oxalate crystals, sink-i.e., Abbreviations: ER, endoplasmic reticulum; CBP, calcium-binding protein. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §Visiting Scientist, Northwestern Agricultural University, Yangling, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Shaanxi, China. 6986 Downloaded by guest on September 28, 2021 Plant Biology: Franceschi et al. Proc. Natl. Acad. Sci. USA 90 (1993) 6987 serum albumin in 5 mM CaCl2/0.4 M mannitol/10 mM tagged with horseradish peroxidase and detected as de- 2-(N-morpholino)ethanesulfonic acid, pH 5.5] and vacuum scribed by the supplier (Sigma). infiltrated for 30 sec. After 1 hr at 34°C in a shaker bath, the RNA Gel Blot Analysis. Poly(A)+ RNA was isolated, frac- digestion was essentially complete. The digest was filtered tionated on an agarose gel, and transferred to a nitrocellulose through 120 ,um pore nylon mesh and the protoplasts were membrane as described by Sambrook et al. (32). The 5' pelleted at 150 x g for 1 min, suspended in cold sucrose buffer coding region (about 800 bp) of the cDNA encoding rabbit [0.4 M sucrose/2 mM CaCl2/5 mM 4-(2-hydroxyethyl)-1- calsequestrin (33) wasinsertedinto Bluescriptplasmid(Strata- piperazine ethanesulfonic acid (Hepes), pH 7.0] and placed gene). The plasmid was used to prepare antisense RNA probe on ice. After 5 min most of the crystal idioblasts settled out, by in vitro transcription using T3 RNA polymerase as de- while mesophyll protoplasts remained suspended. The idio- scribed by the supplier (Promega). Hybridization was per- blast protoplasts were suspended in mannitol buffer (0.4 M formed at 42°C overnight in a solution containing 50% (vol/ mannitol/2 mM CaCl2/5 mM Hepes, pH 7.0) and placed on vol) formamide, 5 x SSPE (32), 3 x Denhardt's solution (32), a step gradient consisting of 2 ml of Percoll (Sigma), 2 ml of 2% SDS, salmon sperm single-stranded DNA at 75 ug/ml, sucrose buffer plus 9.1% dextran T35-50 (United States poly(A)+ RNA at 5 ,ug/ml, and 2 ,ug ofradiolabeled antisense Biochemical), and 2 ml of sucrose buffer. The gradient was or sense strand RNA probe. After hybridization, the mem- centrifuged at 150 x g for 2 min. Raphide idioblast proto- brane was washed sequentially for 15 min each at 42°C, with plasts banded at the top of the Percoll layer, while druse 2x SSPE/0.1% SDS, lx SSPE/0.1% SDS, and 0.5x SSPE/ idioblast protoplasts pelleted at the bottom of the Percoll 0.1% SDS. The membrane was air dried and exposed to x-ray layer. The mesophyll protoplast fraction was placed on a film. similar gradient and after centrifugation was collected at the top of the sucrose buffer plus 9.1% dextran step. Vital Stain for Ca Compartmentation. Protoplasts were RESULTS incubated with vital stains to determine if organelle compart- mentation of Ca could be identified at the light microscope Purification of Idioblast and Mesophyll Protoplasts. Crystal level. The protoplasts were incubated for 15 min with the idioblasts comprise less than 1% of the total leaf cells of following fluorescent probes dissolved in isolation medium: Pistia, and so biochemical dissection of the mechanisms chlortetracycline (10-4 M; Calbiochem) for bound Ca, DiOC6 involved in Ca oxalate formation necessitated the isolation of (5 ,ug/ml; Molecular Probes) for ER, and rhodamine 123 (5 this cell type. As is demonstrated in Fig. 1, our protoplast p,g/ml; Sigma) for mitochondria. After a brief wash with isolation procedure (see Materials and Methods) results in isolation medium, the protoplasts were viewed by using a highly purified preparations of intact idioblast and mesophyll Leitz Aristoplan microscope set up for epifluorescence. protoplasts. 45Ca2+ Overlay Assay.