Anatomical Changes and Immunolocalization of Cellulase ... - NCBI

Plant Physiol. (1990) 93, 160-165 0032-0889/90/93/01 60/06/$01 .00/0

Received for publication September 8, 1989 and in revised form December 11, 1989

Anatomical Changes and Immunolocalization of Cellulase during Abscission as Observed on Nitrocellulose Tissue Prints Philip D. Reid*, Elena del Campillo, and Lowell N. Lewis Department of Biological Sciences, Smith College Northampton, Massachusetts 01063 (P.D.R.) and Department of Plant Biology, University of California, Berkeley, California 94720 (E.dC., L.N.L) ABSTRACT

for this form of the enzyme and lack cross reactivity with other cellulases from bean abscission zones (5, 7). It has recently been shown that anatomical tissue prints can be made on NC' membranes (3, 6, 10, 15). This work suggested the possibility of using the technique to study the anatomical and biochemical changes that occur during abscission. Treatment of such tissue prints with antibodies specific for abscission regulating enzymes would provide an anatomical and biochemical picture of abscission on the same slide.

A fundamental event in abscission is the breakdown of cell wall material in a discrete zone of cells known as the separation layer. Three dimensional images produced by viewing tissue prints of abscission zones on nitrocellulose (NC) membranes with incident illumination showed changes in the tissue integrity taking place in the separation layer as the process of abscission proceeded. The cell softening which occurs due to the dissolution of the cell wall appeared in the tissue prints as a diffuse line at the anatomical transition between the pulvinus and petiole and was easily observed on NC tissue prints of either longitudinal or serial cross-sections through abscission zones. In bean leaf abscission the dissolution of cell walls has been correlated with the appearance of a form of cellulase with an isoelectric point of pH 9.5. Antibodies specific for this enzyme were used to study the localization of 9.5 cellulase in the distal abscission zone of Phaseolus vulgaris L., cv Red Kidney after tissue printing on NC. It was found that 9.5 cellulase was localized in the separation layer but also occurred in the vascular tissue of the adjacent pulvinus. No antibody binding was observed in nonabscising tissue or preimmune controls. These results confirm previous biochemical studies and demonstrate that immunostaining of nitrocellulose tissue prints is a fast and reliable method to localize proteins or enzymes in plant tissue.

MATERIALS AND METHODS Plant Material

Phaseolus vulgaris L., cv Red Kidney, seeds were obtained from W. Atlee Burpee Co., Warminster, PA, planted in vermiculite, and grown in a plant growth chamber for 12 to 14 d. Light was supplied by both incandescent and fluorescent lamps at an intensity of 300 ,E/m2/s for 12 h photoperiods. Explants were prepared by removing the primary leaf blades and the cotyledons, and the stems were cut just above the planting medium, leaving the main axis plus the petioles including the distal abscission zones of the primary leaves as a unit. Sections for tissue printing were made from distal abscission zones which included the pulvinus plus 5 mm of petiole. Explants were incubated in air by placing the explant stem in a beaker of distilled water for the duration of the

experiment.

Various cultivars of bean, Phaseolus vulgaris L., have provided the system of choice to study abscission for many years. Anatomical changes which accompany the abscission process have been thoroughly described using fixed tissues with both light and electron microscopy (2, 17, 18) and the study of abscission in beans as well as other plants has been recently reviewed (14). Interestingly, these studies revealed that the cells that will eventually divide and form a separation layer do not differentiate from adjacent cells until after abscission has been induced. These anatomical descriptions have been complimented by more recent biochemical studies which have shown that an increase in cellulase activity occurs as abscission proceeds (8, 19), and that these changes can be affected by auxin and ethylene (1, 16). In bean, two or more forms of cellulase can be extracted from abscission zones (4, 8, 1 1), but only one form, a 51 kD protein which has an isoelectric point of pH 9.5, increases as abscission proceeds. Antibodies raised against pure 9.5 cellulase have been shown to be specific

Tissue Prints A 2 x 5 cm piece of dry NC membrane (0.45 ,um pore size) obtained from Schleicher & Schuell, Inc., Keene, NH was placed on two layers of paper toweling. Tissue sections 200 to 500 ,um thick were placed on the membrane, covered by a piece of protective glascine paper, and pressed into the NC by finger pressure. The tissue was removed from the membrane with forceps and the membrane taped to a glass microscope slide for viewing and processing with antibodies.

Photography Tissue prints were photographed using transmitted light with an Olympus BH-2 microscope, usually with a 4X objec'Abbreviations: NC, nitrocellulose; DMF, N,N-dimethylformamide 160

' imp'a~ S

CELLULASE AND ABSCISSION ON NITROCELLULOSE TISSUE PRINTS

161

Figure 1. Anatomical tissue prints showing arrangement of tissue in sagittal-longitudinal section (b), cross-section of pulvinus (a) and crosssection of petiole (c). Bar, 500 Am.

well1

Figure 2. 25x magnification of vascular tissue in the pulvinus using incident (a) or transmitted (b) illumination. Altering the light makes visible different tissues: arrow indicates phloem in b. These sections indicate that the resolution is less than 5 M~mwhich is the average thickness of a cell wall in a xylem element in this tissue Bar 250 m

tive lens and a 2.5X photo-occular. Incident illumination was provided by a Nicholas type illuminator placed next to the microscope at the level of the stage. Antibody Purification

Rabbit serum (6 mL) containing 9.5 cellulose antibodies obtained as described by Koehler et al. (7) was purified on a DEAE Affi-gel blue agarose beads column, (1.5 x 12.5 cm). The enriched IgG fraction was eluted with a buffer containing 20 mm Tris-HCl (pH 8.0), 28 mM NaCi, and 0.02% Na Azide. Immunolocalization of Antigens NC blots containing the tissue imprint were rinsed in 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, (TBS), for 10 min. The NC sheets were then immersed in a blocking solution containing 3% gelatin in TBS and incubated at room temperature

for 1 h on a rocking platform. The sheets were then transferred to a fresh TBS solution containing 0.05% Tween 20 (w/v) (TTBS) and the 9.5 cellulose antibody in a 1:500 dilution. The blots were incubated at room temperature for 2 h. Detection of the primary antibody was achieved using alkaline phosphatase conjugated goat antibodies raised against the heavy chain of rabbit IgG. The blots were washed twice with TTBS before the second antibody was added. The second antibody was diluted 1:20,000 in TTBS and incubation continued for 2 h at room temperature. Before alkaline phosphatase detection, the NC membranes were washed 2 times in TTBS for 15 min at room temperature and 2 times in 100 mM Tris-HCI (pH 9.5), 100 mM NaCl, 5 mM MgCl2 (APB) for 15 min at 370C. Detection of alkaline phosphatase was achieved by addition of 75 mL of APB containing 25 mg of nitro blue tetrazolium (dissolved in 90% DMF) and 12.5 mg of 5 bromo-4 chloro-3 indolyl-phosphate (solubilized in DMF) as substrates.

162

REID ET AL.

._ ._ = . -_r. vs

Plant Physiol. Vol. 93,1990

Figure 3. Time course showing development of the abscission layer at 0 h (a), 24 h (b), 48 h (c), 53 h (d) after excission. Bar, 1 mm. RESULTS

Anatomy of the Distal Abscission Zone of Bean Hand cut sections of plant tissue made with a sharp razor blade and pressed into NC membranes provide a low resolution view of the anatomical detail in the segment. Incident illumination of the tissue prints results in a three-dimensional image which is useful in studying anatomical changes which occur in the tissue over time. Figure 1 shows longitudinal and cross-sections through regions of the distal abscission zone of Phaseolus vulgaris. Figure 2 shows a 25x magnification of the vascular stele tissue print from the pulvinar region of the distal abscission zone with both incident (a) and transmitted (b) illumination. Resolution of anatomical detail using this technique is less than 5 ,um. Transmitted illumination provides a clearer image of the phloem and endodermal region of the stele, but cellular detail is lacking, presumably due to the consistency of the cell walls in this region. Figure 3 shows tissue prints which illustrate anatomical changes which occur in the abscission zones as the process of abscission proceeds from 0 to 53 h. The abscission layer is clearly visible after 24 h and by 48 h the cell softening which occurs, presumably due to the action of cellulose and other wall hydrolyzing enzymes, appears in the tissue print as a diffuse line near the anatomical transition between pulvinus and petiole. By 53 h, most abscission zones break, even with gentle handling. Serial cross-sections through the abscission layer illustrate

the transition between pulvinar and petiolar anatomy (Fig. 4). Because of wall softening in the separation layer, cellular detail is lost in the cortex ofseparation layer segments. Section 4c is cut obliquely through the separation layer indicated by an arrow in the longitudinal frontal plane (4d). It has been reported that abscission in some plants begins on the abaxial side of the petiole and proceeds adaxially through the cortex, while in other tissues the process appears to begin on the adaxial side and then proceeds abaxially around the cortex (7). In either case, a tissue print of a frontal section through the abscission zone should show the abscission layer on both sides of the stele. It is clear from Figure 4d that wall dissolution is uniform on both sides of the vascular stele. 9.5 Cellulase Localization NC membranes are routinely used for the transfer of proteins which have been separated in analytical gel systems followed by detection of specific ligands using antibodies. When tissue sections are placed on NC membranes, protein from the tissue is transferred to the membrane mainly by diffusion and is then immobilized in the NC matrix. An attempt was made to localize 9.5 cellulose on the anatomical tissue prints of distal abscission zones. Localization of the enzyme was based on the binding of rabbit antibodies raised against pure 9.5 cellulose. The primary antibody was detected by a secondary antibody raised against rabbit IgG and linked to alkaline phosphatase as a marker. Previous biochemical studies have shown that 9.5 cellulose is synthesized de novo


163

Figure 4. Serial cross-sections in the region of the abscission layer and proximal to that. Printed sections were estimated to be about 300 Am thick. Arrows in d indicate the region of the cross sections shown in a, b, and c. Bar, 500 Am.

following induction of abscission (8, 16). Thus, tissue prints of abscission zones made prior to induction should show no binding of the 9.5 cellulose antibody and hence no alkaline phosphatase reaction. Similarly, preimmune serum treated NC tissue prints should show no alkaline phosphatase reaction. Figure 5 shows NC tissue prints of abscission zones prepared before (zero time) and after (48 h) abscission develops which were immunolabeled with 9.5 cellulose antibody. There was no label in the zero time prints (a, b) while prints made 48 h after excission show specific labeling in the separation layer and some label in the vascular tissue of the pulvinus (c, d) Figure 5 (e, f) shows pure 9.5 cellulose spotted on NC membrane and immunostained as described above. These spots show that the appearance of the stain differs when viewed with transmitted or incident light. This difference results in a color change as observed in the microscope from dark red (transmitted light) to blue (incident light). Tissue prints immunolabeled with preimmune sera showed no label and the 9.5 cellulose could be washed out of the tissue by vacuum infiltration as described by Reid et al. (1 1) (data not shown). DISCUSSION Tissue printing on NC membranes has provided a useful technique to study the anatomical and biochemical changes which accompany the abscission process and reveal a remarkable amount of anatomical detail. In the pulvinus the vascular

system is arranged in a central core surrounded by the parenchymatous motor cells of the cortex. A distinct anatomical transition occurs between the pulvinus and petiole where the vascular tissue is arranged in bundles with a clearly defined central pith region. Xylem cells are easily recognized on the anatomical tissue prints; however, the phloem cells are not clearly distinguishable. The cell softening which occurs due to the action of 9.5 cellulose and other wall hydrolysing enzymes appears in the tissue print as a diffuse line near the anatomical transition between the pulvinus and petiole. The ease and speed with which sections can be prepared is a distinct advantage over fixed sections, and while some resolution is lost by the technique, artifacts due to fixation are eliminated. The use of incident illumination allows for visualization of changes in cell wall rigidity during abscission while the use of transmitted light through the same section tissue print provides better anatomical detail. Because chemical as well as anatomical information is transferred to the NC membrane during the printing process, it was possible to use antibodies raised against purified 9.5 cellulose to localize the enzyme in the separation layer. Antibodies to 9.5 cellulose have previously been used to study the localization of 9.5 cellulose in sections of proximal abscission zones (12, 13) but these preparations have shown limited resolution due to background staining in the stele with preimmune serum. Antibody stained tissue prints on NC membranes allows

164

Plant Physiol. Vol. 93,1990

REID ET AL.

"or,

f,

"W ..,Ov0770,

,.,!.

"Op

0-15

1%

.1

55W Figure 5. Induction of 9.5 cellulose after excission of the leaf blade. Tissue prints were made with sagittal sections at time zero and 48 h after excission and immunolabeled with 9.5 cellulose antibody. 0 h (a, incident illumination), 0 h (b, transmitted illumination), 48 h (c, incident illumination), 48 h (d, transmitted illumination). Pure 9.5 cellulose was spotted and immunolabeled as described, (e, incident illumination), (f, transmitted illumination). Bar, 500 flm.

for detailed study of enzyme occurrence, distribution, and localization. Refinement of the procedures by making chemical prints on buffer treated membranes may enhance the resolution (3). The data presented here confirm previous work describing the appearance of 9.5 cellulose during abscission (5) and show that 9.5 cellulose also appears in the pulvinar stele of the distal abscission zone as was similarly shown for the proximal zone (13). In a subsequent paper we analyze the localization of cellulose in ethylene-induced abscission and the occurrence of cellulose in cells other than those of the separation layer (4a). ACKNOWLEDGMENTS We are grateful to Drs. Joseph Varner, Otto Stein, and Roy Sexton for their helpful suggestions.

LITERATURE CITED 1. Abeles FB, Leather GR (1971) Abscission: Control of cellulose

secretion by ethylene. Planta 97: 87-91 2. Brown HS, Addicott FT (1950) The anatomy of experimental leaflet abscission in Phaseolus vulgarism Am J Bot 37: 650-656 3. Cassab G, Varner JE (1987) Immunocytolocalization of extensin in developing soybean seed coats by immunosilver staining and by tissue printing on nitrocellulose paper. Cell Biol 105: 2581-2588 4. del Campillo E, Durbin M, Lewis LN (1988) Changes in two forms of membrane-associated cellulose during ethylene-incuced abscission. Plant Physiol 88: 904-909 4a.del Campillo E, Reid PD, Sexton R, Lewis LN (1990) Occurrence and localization of 9.5 cellulose in abscising and nonabscising tissues. Plant Cell 2: 245-254 5. Durbin ML, Sexton R, Lewis LN (1981) The use of immunolog-


6. 7.

8.

9.

10.

11.

ical methods to study the activity of cellulase isozymes in bean leaf abscission zones. Plant Cell Environ 4: 67-73 Jacobson JV, Knox RB (1973) Cytochemical localization and antigenicity of a-amylase in barley aleurone tissue. Planta 112: 213-224 Koehler D, Lewis LN, Shannon LM, Durbin ML (1981) purification of a cellulase from kidney bean abscission zones. Phytochemistry 20: 409-412 Lewis LN, Varner JE (1970) Synthesis of cellulase during abscission of Phaseolus vulgaris leaf explants. Plant Physiol 46: 194199 Lewis LN, Linkins AE, O'Sullivan S, Reid PD (1975) Two forms of cellulase in bean plants. In S Kikuchi, ed, Proceedings of the 8th International Conference of Plant Growth Substances, Hirokawa Pub Co, Tokoyo, pp 708-718 Reid PD, del Campillo E (1989) Anatomy and cytochemistry of abscission zones by microscopical examination of nitrocellulose tissue prints In GW Bailey, ed, Proceedings of the 47th Annual Meeting of the Electron Microscope Society of America, San Francisco Press, San Francisco, pp 750-751 Reid PD, Strong HG, Lew F, Lewis LN (1974) Cellulase and abscission in the red kidney bean (Phaseolus vulgaris). Plant Physiol 53: 732-737

165

12. Sexton R, Durbin ML, Lewis LN, Thompson WW (1980) Use of cellulase antibodies to study leaf abscission. Nature 283: 873-874 13. Sexton R, Durbin ML, Lewis LN, Thompson WW (1981) The immunocytochemical localization of 9.5 cellulase in abscission zones of bean (Phaseolus vulgaris cv. Red Kidney). Protoplasma 109: 335-347 14. Sexton R, Roberts JA (1982) Cell biology of abscission. Annu Rev Plant Physiol 33: 133-162 15. Spruce J, Mayer AM, Osborne DJ (1987) A simple histochemical method for locating enzymes in plant tissue using nitrocellulose blotting. Phytochemistry 26: 2901-2903 16. Tucker ML, Sexton R, del Campillo E, Lewis LN (1988) Bean abscission cellulase. Characterization of a cDNA and regulation of gene expression by ethylene and auxin. Plant Physiol 88:1257-1262 17. Webster BD (1968) Anatomical aspects of abscission. Plant Physiol 43: 1512-1544 18. Webster BD (1973) Ultrastructural studies of abscission in Phaseolus: ethylene effects on cell walls. Am J Bot 60: 436-447 19. Wright M, Osborne DJ (1974) Abscission in Phaseolus vulgaris. Positional differentiation and ethylene induced growth of specialized cells. Planta 120: 163-170