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

Abstract

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-...

Association of Latent Cellulase Activity with Plasma Membranes from Kidney Bean Abscission Zones

“…An intracellular form with a p13 of 4.5 was present at all times, and an extracellular form with a pl of 9.5 increased during abscission or after treatment with ethylene (10). The 4.5-pI form was buffer-soluble whereas the 9.5-pI form was extracted from cell walls by high salt.…”

Comparative Study of Cellulases Associated with Adventitious Root Initiation, Apical Buds, and Leaf, Flower, and Pod Abscission Zones in Soybean

“…However, other tissues known to have cellulase activity were not screened for expression of these particular genes. Linkins et al (1973) reported that a cellulase associated with auxin-induced adventitious rooting in bean had a basic pI similar to that of the pI 9.5 cellulase associated with bean leaf abscission. In addition, they concluded that ethylene synthesis induced by the auxin treatment was essential to the increase in the basic pI cellulase associated with root initiation.…”

“…Cellulases (endo-1,4-P-~-glucanases) constitute one class of such cell wall hydrolases. Cellulase activity has been identified in various tissues including leaf abscission zones (Lewis and Koehler, 1979; Tucker et al, 1988), fruit abscission zones (Greenberg et al, 1975), ripening fruit (reviewed by Fischer and Bennett, 1991), senescing cotyledons (Lew and Lewis, 1974), and styles and anthers de1 Campillo and Lewis, 1992), in stems during adventitious root initiation (Linkins et al, 1973), and in tissues undergoing cell division and expansion (Fan and Maclachlan, 1967). Not a11 of these cellulases have the same pI, suggesting that there are different cellulases with distinct cellular functions.…”

Extraction and Partial Characterization of Cellulases from Expanding Pea Epicotyls

“…With pea epicotyls, as with the abscission zone of bean (14,18), ethylene alone caused a rise in cellulase B but not cellulase A activity (Fig. 2).…”

“…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.…”