Isolation of Protoplasts and Vacuoles from Sugarcane Suspension and Stem Parenchyma Cells

THE VACUOLE is a compartment within sugarcane cells in which most of the sucrose is stored. Despite its importance, the sugarcane vacuole is not well understood, because it has been very difficult to examine from sucroseaccumulating stem cells. It is only separated from other cell components by a single membrane which is generally broken during attempts to isolate the vacuole for study. One new approach to sugarcane improvement is to direct novel enzymes into the vacuole to convert sucrose into higher value biomaterials. Unfortunately, the vacuole is very acidic and it also contains proteolytic enzymes that seem to degrade most introduced enzymes before they can perform useful bioconversions. This new potential led us to revisit the challenge of vacuole isolation from mature sugarcane stems. Our immediate interest was to use the isolated vacuoles to understand how to stabilise introduced enzymes. They would also be very useful in studies to understand how sugarcane achieves its exceptional stored sugar concentrations. A first step to vacuole isolation is the removal of the cellulose walls surrounding sugarcane cells, yielding protoplasts. So we developed a method to isolate protoplasts from sugarcane immature and mature stem parenchyma cells by: (i) using an enzyme solution containing cellulase onozuka RS, macerozyme, pectinase and driselase; (ii) adjusting the solution to match the internode osmotic strength; (iii) pretreating internodes at 4C for 1–2 days before the enzyme treatment; (iv) vacuum infiltration of tissues in enzyme solution and changing the solution after 1–2 hours of incubation; (v) purification of protoplasts in Ficoll gradients. However, osmotic lysis of these protoplasts yielded less than 2% of vacuoles in most trials and polybase-induced lysis did not yield any vacuoles. Introduction Sugarcane accumulates sucrose at concentrations up to 62% dry weight (16–25% fresh weight) in the storage parenchyma cells of the mature culm. A large vacuole found in these storage cells contains most of the stored sucrose (Moore et al., 1997). Hence, the ability to direct foreign proteins to the sugarcane vacuole and retain biological activity of such proteins in the vacuole is of substantial practical relevance to the sugarcane industry to synthesise valuable novel products and compounds other than sucrose. There is no published method for isolation of vacuoles from sugarcane mature stem parenchyma cells, and their physiology is not well understood. For example, the nature of any proteolytic enzymes (enzymes that may inactivate introduced proteins) in the sucrose storage Gnanasambandam, A. & Birch, R.G. Proc. Aust. Soc. Sugar Cane Technol., Vol. 28: 2006 __________________________________________________________________________ 318 vacuoles has not been reported. Hence, we would like to isolate vacuoles from sugarcane stem parenchyma cells for characterisation of proteolytic enzymes and for compartmentation analysis to identify and characterise foreign proteins that are targeted to the vacuole. The first step towards vacuole isolation is generally to isolate intact cells from which the cellulose walls have been removed. These wall-free cells, called protoplasts, are generally obtained by treatment of plant tissues with enzymes to dissolve the cell walls. Methods to isolate protoplasts from sugarcane cells grown in tissue culture are well established (Srinivasan and Vasil, 1986; Taylor et al., 1992; Matsuoka et al., 1996; Aftab and Iqbal, 1999). However, only a few reports are available for isolation of protoplasts from sugarcane plants; and these are limited to young tissues such as the spindle region (Krishnamurthy, 1973) and immature stalk tissue (Thom et al., 1983; Maretzki and Thom, 1987). Unfortunately, mature tissues generally develop hardened cell walls which are more difficult to remove without damaging the cells. Here we report a method for efficient isolation of protoplasts from sugarcane mature stem parenchyma cells, as a step towards the isolation of the vacuoles from these cells. Materials and methods Embryogenic callus and cell suspension culture Embryogenic callus and cell suspension cultures of commercial sugarcane cultivar Q63 were initiated and maintained as described by Franks and Birch (1991) and Taylor et al. (1992), respectively. Solutions used 1. Enzyme solution A: 2% cellulase onozuka RS, 0.2% pectinase, 0.1% driselase and 0.2% macerozyme in wash solution A [pH 5.9, 3 mM MES and 0.7 mM NaH2PO4 H2O with 0.2 M each of mannitol and sorbitol]. 2. Enzyme solution B: 2% cellulase onozuka RS, 0.2% pectinase, 0.1% driselase and 0.2% macerozyme in wash solution B (wash solution A with 0.25 M each of mannitol and sorbitol). 3. Enzyme solution C: 2.5% cellulase onozuka RS, 0.2% pectinase, 0.1% driselase and 1% macerozyme in wash solution C (wash solution A with 0.3 M each of mannitol and sorbitol). 4. Lysis buffer A or B or C: 0.1 or 0.2 or 0.13 M KH2PO4/K2HPO4, respectively, 2 mM EDTA-Tris (pH 8) and 2.5 mM DTT. 5. Lysis buffer D: 0.2 M sorbitol, 10% Ficoll 400 and 10 mM Hepes-KOH (pH 7.5). Protoplast enzyme solution The enzyme solution used for isolation of protoplasts was prepared as described by Maas et al. (1995) and Dombrowski et al. (1994). The enzymes [cellulase onozuka RS (from mutant strain of Trichoderma viride) and macerozyme (from Rhizopus sp.) (Yakult Manufacturing Co., Japan); pectinase (from Aspergillus niger) and driselase (from Basidiomycetes sp.) (Sigma)] were slowly dissolved in millipore-filtered sterile water and stirred for 2 to 3 hours at 24C. Care was taken not to create foaming, which may result in the inactivation of the enzymes in solution. Then, CaCl2, NaH2PO4, MES, mannitol and sorbitol were added in that order as required from stock solutions, and the pH was adjusted to 5.9. When dissolved, the enzyme solution was slowly filter sterilised using a 0.22 !m filter, and stored in 20 mL aliquots at –20C. Gnanasambandam, A. & Birch, R.G. Proc. Aust. Soc. Sugar Cane Technol., Vol. 28: 2006 __________________________________________________________________________ 319 Protoplast isolation from suspension cells Protoplasts were isolated from suspension culture cells as described by Srinivasan and Vasil (1986). Five mL of the suspension was mixed with 25 mL of filter sterilised enzyme solution A in a Petri dish and incubated on a shaker for 4 to 5 hours at 60 r/min in darkness at 27C. Protoplasts were filtered through two 200 !m nylon mesh [Swiss Screens (Australia) Pty. Ltd., Brisbane, Australia], pelleted by centrifugation at 100 g for 5 min and washed three times in wash solution A. Protoplast isolation from stem parenchyma cells The green peripheral part of the sugarcane stem (or culm), where vascular bundles are closely packed, was peeled off. Internode cores were then cut into 1–2 mm thick slices in Petri dishes containing the wash solution B or C. Tissue pieces were then transferred to the enzyme solution B or C and incubated for 1 to 4 hours on an orbital shaker at 60 r/min in darkness at 27C. Internode numbering was done as described in Moore (1987) such that numbers increase down the stem, with the internode below the node attached to the top visible dewlap leaf being designated as 1. For internode one, enzyme solution B and wash solution B were used. For internodes 2 to 18, enzyme solution C and wash solution C were used. For storage at 4C, stem internodes were kept for 24 hours or more in a polythene bag. After tissue digestion, released protoplasts were filtered through two 200 !m nylon mesh over two layers of cheese cloth, washed in wash solution B or C by sedimenting at 100 g for 5 min. The protoplasts were further purified twice on Ficoll 400 (Pharmacia) gradients using the procedure of Boller and Kende (1979) as given in Wagner (1987). Protoplasts were gently mixed with one volume of 15–20% (w/v) Ficoll prepared in wash solution B or C. This solution is overlayered with 8, 6 and 0% (w/v) Ficoll zones (10 mL), each containing mannitol and sorbitol and buffers as in wash solution B or C. After centrifugation at 1000 g for 10–20 min at 4C, purified protoplasts were gently collected from the 0–6% interface and diluted into wash solution B or C and collected from this suspension by sedimentation at 100 g for 5 min. Isolation of vacuoles Osmotic lysis of protoplasts involved rapid (5 sec) dilution of 1 or 2 mL suspension of protoplasts in wash solution A or B or C in a round-bottomed glass tube with 10 or 20 mL of lysis buffer A/B/C/D. The suspension was gently stirred (about 2 min) or pipetted up and down using a glass pipette to effect emergence of vacuoles from lysing protoplasts (Wagner, 1987). In polybase-induced lysis of protoplasts, washed protoplasts were layered onto a three-step gradient containing the following: first step, 5 mL medium of 25 mM Tris-MES buffer, pH 6.5, 0.6 M mannitol containing Ficoll 2%, DEAE dextran 4 mg/ml; second step, 2 mL medium of 25 mM Tris-MES buffer, pH 8.0, 0.6 M mannitol (medium D) containing Ficoll 5%, dextran sulfate 4 mg/ml; third step, 2 mL medium D containing Ficoll 20%. The gradient was centrifuged for 30 min at 2000 g at 17C and the unlysed protoplasts were collected at the interface between the second and third steps (Wagner, 1987). Neutral red staining Fifteen !L of 10 mg/mL neutral red was added per 12 mL of wash solution containing the protoplasts to visualise the vacuoles. Sugarcane suspension cells and friable Gnanasambandam, A. & Birch, R.G. Proc. Aust. Soc. Sugar Cane Technol., Vol. 28: 2006 __________________________________________________________________________ 320 calli cells (obtained from suspension cells) were suspended either in MSC3 medium or 50 mM sodium phosphate buffer (pH 7) for neutral red staining. Results Examination of sugarcane suspension cells, including staining to identify vacuoles We attempted to visually distinguish cytosolic and vacuolar regions in suspension cells. When sugarcane Q63 cells were examined without any staining, the distinction between the vacuole and cytosol was visible only in a few cells (Figure 1A). In a few cells, the nucleus was visible (arrow in Figure 1C). Hence, to confirm the vacuolar region, neutral red staining was used. When the Q63 suspension cells in MSC3 medium were stained with the neutral red, the dye did not stain the vacuoles, probably due to low pH of the medium. In some of the cells, only the nucleus (arrow in Figure 1D) and the nucleolus (dotted arrow in Figure 1D) were preferentially stained. However, when the cells were stained after transferring them to 50 mM sodium phosphate buffer (pH 7.0), the vacuoles accumulated the neutral red readily and the cytosolic and the vacuolar regions were clearly identified in many cells (Figure 1B). In these cells, the vacuoles occupied more than 80% of the cell region. Isolation of protoplasts from suspension cells Protoplasts were successfully isolated from sugarcane suspension cells using enzyme solution A (Figure 1E). In these protoplasts, vacuoles were visible without any staining under phase contrast microscope (Figure 1E). With neutral red staining, most of the vacuoles accumulated the dye and appeared pink to red (Figure 1F). However, some protoplasts showed a reversed staining pattern, with pink to red cytosol and unstained or faintly stained vacuole (dotted arrows in Figure 1F). Isolation of vacuoles from suspension cell protoplasts Isolation of the vacuoles from suspension cell protoplasts using osmotic and polybase-induced lysis proved to be difficult. Several attempts using polybase-induced lysis could not yield any vacuoles. In contrast, osmotic lysis of protoplasts yielded a few vacuoles in most of the trials (arrows in Figure 1F and I). However, in this method, addition of 10 mL of lysis buffer A to the protoplasts could not lyse most of the protoplasts (Figure 1F), while addition of 20 mL of lysis buffer A caused bursting of the majority of the protoplasts (Figure 1I). Reproducible and efficient lysis of most of the protoplasts to yield vacuoles was not accomplished. Isolation of protoplasts from stem cells When slices from immature internode cores of sugarcane cultivars Q63, Q117, Q155 and Pindar were incubated in enzyme solution A (used for isolating protoplasts from cell suspension culture), a lot of cell debris was found after 4 hours. The pH of the solution (pH 5.9) in the three replicate plates had dropped to 2.77, 3.86 and 3.69 indicating lysis of cells. Increasing the mannitol and sorbitol concentration from 0.2 M to 0.25 M in the enzyme solution (enzyme solution B) yielded protoplasts from internode 1 for all four cultivars. Within 2 hours of incubation in enzyme solution B, all the tissues were completely digested and yielded the protoplasts (Figure 1H, K and L). In these protoplasts, neutral red staining indicated that the vacuole has occupied more than 90% of the cell volume (Figure 1K). In some protoplasts, the nucleus can be identified (arrow in Figure 1L). Gnanasambandam, A. & Birch, R.G. Proc. Aust. Soc. Sugar Cane Technol., Vol. 28: 2006 __________________________________________________________________________ 321 Fig. 1—Protoplasts and vacuoles from sugarcane suspension and stem parenchyma cells. Sugarcane suspension cells without any staining (A and C) and with neutral red staining (B and D). Nuclei are shown by black arrows in C, D and L and nucleolus is shown by dotted arrow in D. Protoplasts from suspension cells without any staining (E – phase contrast micrograph) and with neutral red staining (F and I); Isolated vacuoles are shown by black arrows in F and I. Protoplasts showing a reverse staining pattern, with stained cytosol and unstained or faintly stained vacuole are shown by dotted arrows in F. Protoplasts (H, K-L) and vacuole (M) from sugarcane stem cells (G – unstained transverse section). Protoplasts after polybase induced lysis (J). Bar = 25 mm (except in B and K-M where it equals 5 mm). Gnanasambandam, A. & Birch, R.G. Proc. Aust. Soc. Sugar Cane Technol., Vol. 28: 2006 __________________________________________________________________________ 322 Efficient isolation and purification of protoplasts from internodes 2 and 3 of the four cultivars was obtained by: (i) Increasing the mannitol and sorbitol concentration to 0.3 M each and increasing the cellulase and macerozyme concentrations to 2.5% and 1%, respectively (enzyme solution C). (ii) Slicing the tissues under wash solution C and vacuum infiltration of the tissues with enzyme solution C at the start of the incubation (Upon slicing, the spaces between the cells are filled with air that hinders the digestive enzymes coming into contact with the cell walls. This difficulty is avoided by infiltrating the tissues under vacuum or by slicing the tissue under wash solution, draining the strips and transferring them to the incubation medium). (iii) Replacing old enzymes with new enzyme solution after 1 or 2 hours of incubation. (iv) Purification of protoplasts in Ficoll 400 gradients. Incubation of stem tissues (Figure 1G) from internode 4 in enzyme solution C yielded no protoplasts. However, storage of stem internodes in a polythene bag at 4C for 24 hours or more prior to enzyme digestion, yielded protoplasts with enzyme solution C. The stem segments stored in this way were able to yield protoplasts for up to 3 weeks in enzyme solution B (for internodes 1 to 3) or C (for internodes 4 to 18). Attempts to isolate vacuoles from stem protoplasts Some of the trials attempted for vacuole isolation from stem cells of cultivars Q63, Q117, Q155 and Pindar are given here. A flotation technique was used for polybase-induced lysis of protoplasts from stem cells as the protoplasts required flotation during purification in Ficoll gradients. In contrast, a sedimentation technique was used for protoplasts from suspension cells. Trial 1: The protoplasts were chilled on ice for 30 minutes and were subjected to osmotic lysis using lysis buffer C. Addition of 10 mL of lysis buffer C at 27C or 38C yielded less than 1% of vacuoles/protoplasts. Addition of 10 mL of lysis buffer C at 55C did not yield any vacuoles. Trial 2: Addition of 10 mL or 20 mL of lysis buffer A resulted in less than 1% of vacuoles/protoplasts. The yield of vacuoles increased to 2% of vacuoles/protoplasts when 20 mL of prewarmed lysis buffer A at 42C was added. No vacuoles were obtained when 20 mL of lysis buffer B was added. Trial 3: Polybase-induced lysis of protoplasts was tried several times with no success of isolating vacuoles (Figure 1J). Trial 4: The method of Dombrowski et al. (1994), involving addition of lysis buffer D, yielded less than 1% of vacuoles/protoplasts (Figure 1M).