Isozymes of Cellulase in the Abscission Zone of Phaseolus vulgaris

Lewis, Lowell N.; Lew, Francis; Reid, Philip D.; Barnes, J. E.

doi:10.1007/978-3-642-65406-0_32

Cited by 11 publications

(19 citation statements)

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“…Based on studies of plants treated with auxin or ethylene, it appears that plant cellulase occurs in different forms (12), de-grades cellulose differentially (17), and exists in different parts of the cell (1,4). Lewis et al.…”

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Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L.

Linkins¹,

Lewis²,

Palmer³

1973

Plant Physiol.

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Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10' or 102 M indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (102 M) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10-5 M) Lewis and Varner (13). Seedlings 10-to 11-days-old were debladed, the laminar pulvinus and cotyledons were removed, 1 This work was supported by National Science Foundation Grant GB 17850. and the remaining plant was cut off 4 to 5 cm below the cotyledonary node. The intact primary axis with the first leaf node petioles were then placed in cups of water and treated as follows: explants had either 10'2 M or 10' M indoleacetic acid in lanolin paste or pure lanolin paste applied to the acropetal cut petiolar surfaces (Fig. 1). In selected experiments the explants were exposed to 15 u1/I ethylene (Matheson Co.) for the final 20 to 24 hr of IAA treatment. At the termination of treatment periods, the lanolin paste was removed from the control explants or explants treated with auxin or ethylene or both, and the 5 mm area below the treated surface was taken for anatomical study or cellulase extraction (Fig. 1).In experiments where ethylene was used as the sole treatment, the main stem was severed immediately below the basipetal end of the nodal pulvinus of the first leaf axis (Fig. 1). Lanolin paste was then applied to the cut surface of the stem before exposure to 5 ,ul/l ethylene in air-tight chambers. This modification was necessary to circumvent the premature cessation of experiments using petiole tissue due to the abscission of the petiole at the nodal axis after 36 to 48 hr treatment.Cellulase Extraction and Purification. Cellulase was extracted by grinding the excised 5-mm segments for 1 min in a VirTis homogenizer with 0.2 M phosphate buffer, pH 6.1, 1.0 M NaCl, and 3% insoluble polyvinylpyrrolidone. The homogenate was passed through a 50 , mesh nylon cloth. The pellet was resuspended in the above buffer less the polyvinylpyrrolidone, ground for 1 min, and filtered again. The filtered extracts were pooled and centrifuged at 10,000g for 15 min; the resultant supematant solution was centrifuged at 100,OOOg for 1 hr. The final supernatant solution was then dialyzed against two changes of 1 % glycine, pH 6.1, for 2 hr each.The solution was then subjected to isoelectric focusing according to the procedure of Hagland (6) to separate acidic pl (isoelectric point) and basic pl cellulase. Samples were focused for 18 to 24 hr at 300 v.Determination of Ethylene Generated by Explants. Petioles prepared as described were removed from the explants, treated at their acropetal cut surface with either pure lanolin, or lanolin with 10' to 10'2 M IAA. The treated petioles were supported by agar in the bottom of a 50-ml flask which was then sealed with a serum cap. Ethylene measurements were determined on 1-...

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confidence: 99%

Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L.

Linkins¹,

Lewis²,

Palmer³

1973

Plant Physiol.

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“…In the abscission zone of kidney beans, two forms of cellulase are also found based on their salt solubility (12,13). We have termed the buffer-soluble and salt-soluble cellulases as cellulase A and cellulase B, respectively (the properties of which are summarized in Table V).…”

Section: Discussionmentioning

confidence: 99%

“…Cellulolytic enzymes increase in activity in some fruits during ripening (7,9,10,17), in abscission zones of petioles prior to leaf abscission (1,3,12,13), in rapidly expanding epicotyls after auxin treatment (8,16,19), and appear to be involved in pollen tube elongation (22 15), an increase in cellulase activity has been detected only after auxin treatment (16,19,20). The present work provides evidence that in the expanding pea epicotyl, auxin and ethylene each appear to control the development of a different form of cellulase and each enzyme possesses different requirements for activity and apparently differing subcellular locations and isoenzyme complements.…”

Section: Introductionmentioning

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“…Evidence suggests that more than one molecular form of cellulase (EC 3.2.1.4) may be involved in these diverse physiological plant processes. For example, based on differential salt extraction (12,16) and isoelectric focusing (13), there are at least two molecular forms of cellulase in abscising bean petioles and pea epicotyls. Superimposed on these controls over molecular form appears to be a hormonal regulation of the development of cellulase isoenzymes (14,18) and of cellulase compartmentation (3) in abscission zones of bean petioles.…”

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Extraction and Partial Characterization of Cellulases from Expanding Pea Epicotyls

Ferrari

Arnison

1974

Plant Physiol.

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Etiolated pea (Pisum sativum) epicotyls synthesize a buffersoluble cellulase (cellulase A) and a salt-soluble cellulase (cellulase B) (EC 3.2.1.4) after treatment with high (0.5%) auxin levels. Only cellulase A increased in activity after treatment with low (0.005 % ) auxin. Cellulase A was released into the supernatant after homogenization of tissue in dilute buffer (buffer-soluble), had a pH optimum at 5.5, was relatively thermostable, and its activity was inhibited by NaCl. Cellulase B was released by 1 M NaCl (salt-soluble) from excised tissue segments or from the insoluble residue remaining after removal of the buffer-soluble form. It had a pH optimum at 7.0, was thermolabile, and required salt for maximum activity. When subjected to polyacrylamide gel electrophoresis, the cellulase fraction released by NaCl from excised segments showed two bands of celiulase activity compared to several for the buffer-soluble fraction. Electrophoretic analysis of the buffer and salt-soluble fractions for marker enzymes indicated the presence of malate dehydrogenase activity in all fractions and glutamate dehydrogenase activity in the buffer-soluble fraction only.Exposure of intact pea epicotyls to 70 ,ml/I of ethylene gas for 3 days did not affect cellulase A activity, but caused a 5-fold increase in cellulase B activity (enzyme released by salt from the buffer-insoluble residue). We concluded that ethylene and auxin generate different forms of cellulase.Cellulolytic enzymes increase in activity in some fruits during ripening (7,9,10,17), in abscission zones of petioles prior to leaf abscission (1,3,12,13), in rapidly expanding epicotyls after auxin treatment (8,16,19), and appear to be involved in pollen tube elongation (22 15), an increase in cellulase activity has been detected only after auxin treatment (16,19,20). The present work provides evidence that in the expanding pea epicotyl, auxin and ethylene each appear to control the development of a different form of cellulase and each enzyme possesses different requirements for activity and apparently differing subcellular locations and isoenzyme complements. MATERIALS AND METHODSAfter 7 to 8 days growth, etiolated pea (Pisum sativum) seedlings were decapitated and the epicotyls were treated with auxin (indoleacetic acid 0.5%, except where indicated) as described previously (8). Two to 4 days later, salt-soluble and buffersoluble cellulases were extracted from apical, swollen tissue segments which were approximately 10 mm in length. Tissue samples (1 g) were homogenized with mortar and pestle in 20 mm sodium phosphate buffer (pH 6.5, 1 g/ 2 ml of medium) containing 0.02% NaN. (as a precaution against microbial contamination). The homogenate was centrifuged (37,000g, 10 min), and the supernatant containing the buffer-soluble cellulase fraction was saved. The pellet (buffer-insoluble residue) was washed twice in homogenization medium (1 ml/g fresh weight of tissue), and after centrifugation, the supernatants were combined. (In the experiment presented in Table I, t...

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β-Glucanases in Higher Plants: Localization, Potential Functions, and Regulation

Verma

Kumar

Maclachlan

1982

Cellulose and Other Natural Polymer Systems

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Isozymes of Cellulase in the Abscission Zone of Phaseolus vulgaris

Cited by 11 publications

References 7 publications

Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L.

Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L.

Extraction and Partial Characterization of Cellulases from Expanding Pea Epicotyls

β-Glucanases in Higher Plants: Localization, Potential Functions, and Regulation

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