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The occurrence of enzymes associated with bean leaf abscission was investigated in bean (Phaseolus vulgaris) flower reproductive organs in which catabolic cell wall events are essential during anther and pistil development. Cellulase activity was detected in high levels in both pistil and anthers of bean flowers before anthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified a protein in anthers and pistil with the same size (51 kilodaltons) and serologically closely related to the abscission cellulase. The accumulation of 9.5 cellulase protein in the anther is developmentally regulated and increases from undetectable levels at very young stages of anther development to high levels as the anther matures. In the pistil, the 9.5 cellulase was localized in the upper part of the pistil where the stigma and the stylar neck reside and was detected in the youngest developmental stage analyzed. Antibodies against basic chitinase, which accumulates to high levels in abscission zones after exposure to ethylene, identified a protein with the same size (33 kilodaltons) and serologically closely related, in both anthers and upper portion of the pistil. In contrast, a 45-kilodalton protein and the basic beta-1,3-glucanase associated with abscission were undetected in bean reproductive organs. Interestingly, beta-1,3-glucanase activity was detected in young bean anthers and decreased at anthesis, but the anther beta-1,3-glucanase is serologically unrelated to the basic beta-1,3-glucanase. Thus, it appears that the basic cellulase and chitinase occur in combination in many plant processes that require major cell wall disruption, whereas hemicellulases such as beta-1,3-glucanase are specific to each process.

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.

A cellulase (β(1:4)4-glucan hydrolase) has been purified 700-fold by cellulose affinity chromatography from anthers of Lathyrus odoratus L. Two forms are present. The major species has a molecular weight of 49 kDa and a p I value of 8.0; the minor a molecular weight of 51 kDa and a p I value of 7.8. Both proteins are recognized by antibodies raised against the ethylene/IAA regulated bean ( Phaseolus vulgaris L.) abscission zone cellulase which has a more basic p I value and some unique antigenic determinants. Ethylene induced Lathyrus flower bud abscission zones contain two forms of cellulase apparently identical to those in the anthers. The possible role of cellulase in the development of anthers is discussed.

Nitrocellulose tissue prints immunoblotted with 9.5 cellulase antibody were used to demonstrate areas of cellulase localization within Phaseolus vulgaris explants on exposure to ethylene. The 9.5 cellulase was induced in the distal and proximal abscission zone and in the stem. In both abscission zones, the 9.5 cellulase was found in the cortical cells of the separation layer, which develops as a narrow band of cells at the place where fracture occurs. The enzyme was also found associated with the vascular traces of the tissues adjacent to the separation layer extending through the first few millimeters at each side of the separation layer. The two abscission zones differed in the way that cellulase distributed through the separation layer as abscission proceeded. In the distal zone, cellulase appeared first in the cells of the separation layer adjacent to vascular traces and extended toward the periphery. In the proximal zone, 9.5 cellulase accumulated first in the cortical cells that lie in the adaxial side and then extended to the abaxial side. In response to ethylene, 9.5 cellulase was also induced in the vascular traces of the stem and the pulvinus without developing a separation layer. The role of 9.5 cellulase in the vascular traces is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified the same 51-kilodalton protein in both abscising and nonabscising tissues. Therefore, the determinant characteristic of the abscission process is the induction of 9.5 cellulase by cortical cells in the separation layer, and this implies that these cells have a unique mechanism for initiating 9.5 cellulase synthesis.

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10(-5) or 10(-2)m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10(-2)m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10(-5)m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.

The purification of a cellulase isoenzyme with a pI of 9.5 from kidney bean abscission zones is described. An important step in the purification involved the adsorption of the cellulase isoenzyme onto an affinity column of CF-11 cellulose and the subsequent elution with cellobiose. Native and SDS polyacrylamide gel electrophoresis established that there was only one component in the purified cellulase samples. Antibodies raised against the purified pI 9.5 cellulase precipitated this isoenzyme from crude or purified solutions but did not cross react with pI 4.5 cellulase from 2,4-D-treated abscission zones. The antibody was shown to be monospecific by immunoelectrophoresis and by the fact that it precipitated only a single 14C-labeled protein from an abscission zone extract heavily labeled with 14C amino acids.

Antibodies raised against 9.5 cellulase (β 1,4 glucan-4-glucanohydrolase) were used to investigate the distribution of the enzyme in abscising bean petioles (Phaseolus vulgaris L. cv. Red Kidney).

Membranes isolated from abscission zones of Phaseolus vulgaris L., cv. Red Kidney, contained cellulase activity. This particulate activity was enhanced 10- to 20-fold by treatment with Triton X-100. Sucrose density gradient analyses of cell fractions showed that the membranes with which cellulase was associated had a peak equilibrium density of 1.16 to 1.17 g/cm(3) which coincided with that of ion-activated ATPase, a marker for plasma membranes. The membrane fraction having the highest cellulase activity also contained a high proportion of plasma membranes as shown by electron microscopy of sucrose density gradient fractions after staining by periodic acid-chromic acid-phosphotungstic acid. It was concluded that the particulate cellulase was associated with the plasma membrane.

Optimum conditions for the extraction and purification of avocado cellulase have been established. The best quantiative recovery of the cellulase from avocado tissue was obtained with a 0.04M acetate buffer, pH 5.0, containing 0.4M NaCl and 0.25% (v/v) Triton X-100. The isoelectric point of the enzyme was 4.7 and the molecular weight, based on SDS gel electrophoresis, was 49,000. The enzyme was purified on a cellulose column to a single protein band on SDS gel electrophoresis. Rabbit antibody produced to the cellulase gave further proof of the enzyme's purity.

The changes in the levels of two different isozymes of cellulase (EC 3.2.1.4) have been followed during the abscission of the primary leaves of bean (Phaseolus vulgaris c.v. Red Kidney), using antibodies raised against the 9.5 form of the enzyme. Data from both radioimmune and direct assay show that the 9.5 form of cellulase is undetectable prior to the induction of abscission. After a 12 h lag this isozyme increases in activity, the increase preceding a decrease in integrity of the abscission zone cell walls. The results are consistent not only with the view that this specific isozyme is involved in wall hydrolysis but also with previous data which showed that cellulase is synthesized ‘de novo’. The 4.5 isozyme of cellulase is more widely spread throughout the plant, being most active in young tissues. During abscission the activity of this isozyme in the abscission layer falls and consequently it is not thought to be involved directly in the abscission process.

Cotyledons of germinating kidney beans contain two forms of a carboxy methyl cellulase which can be separated by ammonium sulfate fractionation and isoelectric focusing. The two cellulases are similar in their molecular weight but differ in isoelectric points, pH and temperature optimum, pH and temperature stability and sensitivity to thiol inhibitors and metal ions. One cellulase (isoelectric point 4.8) has been purified 100-fold to give a major protein band on acrylamide gel electrophoresis.

A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.

Publisher Summary Research on cellulase in the bean plant, Phaseolus vulgaris L. cv. Red Kidney, has led to the discovery of a family of cellulases with differing forms and functions. The development of methods for differentiating cellulase activities made it possible to begin sorting out the functions of the various cellulases. With the purification of the 9.5 cellulase and subsequent production of antibodies to it, the amount of 9.5 cellulase activity vs acidic pl cellulases could be quantitated. It was found that 9.5 cellulase is synthesized de novo in the abscission zone of bean leaves in response to ethylene. Synthesis of 9.5 cellulase is confined to a very narrow band of cells in the abscission zone. The acidic forms of cellulase occur throughout the plant, particularly in young rapidly expanding tissue. These forms appear to be auxin regulated and involved in growth and differentiation. The affinity of 9.5 cellulase has been exploited for its substrate and purified the enzyme on a cellulose column. Hydrolysis of the cellulose does occur to some extent, as reducing sugars are found in the peak fractions containing the purified enzyme. As the acidic cellulases have only been partially purified, their substrate specificity has not been firmly established.

Plasma membranes from abscission zones of kidney bean seedlings were isolated on sucrose density gradients using cellulase and KCl-stimulated ATPase as marker enzymes. Following treatment of the seedling explants with 50 microliters per liter ethylene, an increase in the buoyant density of the plasma membrane fraction from 1.165 to 1.175 grams per cubic centimeter was observed. Such a change is consistent with the loss of lipids from the senescent cells of the abscission zone.

Cellulase (beta-1, 4-glucan-glucanohydrolase EC 3.2.1.4) activity in the abscission zone of red kidney bean (Phaseolus vulgaris) was previously shown to exist in at least two different molecular forms. The form of the enzyme which has an isoelectric point of 4.5 is present in both abscising and nonabscising tissue and requires grinding for extraction. Another form of the enzyme which has an isoelectric point of 9.5 is present only in tissue in which the abscission process has been induced. Further, much of this form of cellulase can be removed from the tissue by vacuum infiltration with buffer. Time course studies indicate that while the increase in measurable cellulase activity in tissue which is actively undergoing abscission was due primarily to the appearance of cellulase 9.5, this form of the enzyme cannot be removed by vacuum infiltration until after the breakstrength of the abscission zone has decreased nearly to zero. The intracellular localization of these two forms of cellulase is discussed.

When abscission in leaf explants from Phaseolus vulgaris, cultivar Red Kidney, was allowed to proceed while the explants were in (2)H(2)O, a 1.25% increase in the buoyant density of cellulase in a cesium chloride gradient was observed. These data indicate that the increase in cellulase activity during abscission is a result of the synthesis of new protein. Two differentially soluble forms of cellulase are present in the abscission zone. The form which is soluble only in a high salt buffer seems more closely related to the abscission process than the form which is soluble in dilute buffer. The correlation between changes in pull force and increase in cellulase activity and the effects of several hormones on cellulase activity are discussed.

The primary leaves of the bean (Phaseolus vulgaris c.v. red kidney) are shed as the result of cell wall breakdown in the abscission zones found at the base of the lamina and at the stem–petiole junction. Horton and Osborne1 suggested that cellulase (B l : 4 glucan 4-glucanohydrolase) was involved in this wall hydrolysis because its activity increased considerably during abscission. Although this result has been repeated on a wide range of plant material2, critical scrutiny of the data has led several authors3,4 to question the direct involvement of the enzyme in wall weakening. The following objections have been raised: (1) cellulase may be present at relatively high levels before abscission is induced5, (2) although wall breakdown is confined to two or three layers of cells, cellulase is usually found over a wider area of the petiole, (3) the increase in cellulase which accompanies abscission may not take place until cell separation is essentially complete3, (4) cellulase may increase without concomitant breakdown of the abscission layer4. One possible explanation for these anomalies is that these assays failed to distinguish between the various isozymes of cellulase, of which only one is postulated to be involved in abscission6. Recently, the specific isozyme thought to participate in bean leaf abscission (9.5 cellulase) has been purified and monospecific rabbit antibodies raised against it7. These antibodies do not cross-react with the other form (s) of cellulase and thus can be used both to quantify changes in 9.5 cellulase during abscission and to determine cytochemically the distribution of the enzyme. Using these antibodies, we have now made observations consistent with the participation of cellulase in the wall hydrolysis which leads to fracture.