Relationship of Pectic Enzymes to Abscission

A comprehensive review of work pertinent to changes in the separation zone prior to abscission is presented. Since these changes are largely pectic in nature the involvement of pectic enzymes is suggested. The results obtained for the role of pectin methylesterase ( PME) in abscission arc as follows: ( 1 ) A localization of PME activity in the abscission zone of tobacco pedicels was established; ( 2) Pollination of tobacco flowers is followed by an increase in PME activity in the abscission zone while PME activity remains static after prevention of pollination (the flower abscises within a week if not pollinated); (3) Application of indole acetic acid to unfertilized tobacco ovaries stimulated an increase in PME activity of the abscission zone of the pedicel. Early work with abscission was primarily anatomical although there was some speculation concerning the regulation of abscission. Von Mohl (1860) noted a definite separation layer in which abscission always occurred. Within this layer cells separated from one another with their walls still intact. Wiesner ( 1871) confirmed some of von Mohl's observations and theorized that the dissolution of the intercellular substances of the separation layer was caused by organic acids developed in the leaves. Molisch ( 1886) suggested that a gum ferment might be the cause of the dissolution process. Kubart (1906) concurred with Weisner (1905) that an organic acid was responsible for the dissolution of the middle lamella, but he admitted the possibility of enzymatic im·olvement in the abscission process. At the turn of the century a conflict developed concerning the manner in which abscission occurs. Tison ( 1900) reported dissolution of the primary cell wa1ls of the separation layer in addition to the middle lamella. This was confirmed by later v.;orkers (Lloyd 1914, Dutt 1928). Howe\·er, Lee (1911) concurred with von Mohl (1860) and reported that abscission of leaves involved a dissolution of the middle lamella between the primary walls of adjacent layers. This type of abscission was also noted in several types of flowers by Hannig (1913). In addition, Hannig (1913) and Myers ( 1940) observed disintegration of cells during abscission. More recently this disagreement was still evident as Yager (1957) noted dissolution of the middle lamella while Lineweber and Hall (1959) reported hydrolysis of the primary wall as well as the middle lamella. Addicott and Lynch ( 1951) concluded that the critical 1 Paper presented as a portion of the Symposium on Development in Plants. 2 Department of Biology, University of Northern Iowa. The assistance of Dr. Robert Muir, Department of Botany, University of Iowa is gratefully acknowledged. 45 1 Smith: Relationship of Pectic Enzymes to Abscission Published by UNI ScholarWorks, 1969 46 IOWA ACADEMY OF SCIENCE [Vol. 76 phase of leaf abscission was the dissolution of the entire cell. However, Addicott ( 1965) modified this somewhat by stating that in some cases little more than the midd1e lamella was digested. Rubinstein and Leopold (1964) expressed the opinion that anatomical changes during abscission vary somewhat from plant to plant. Species variation in the manner in which abscission occurs had been noted in 1928 by Pfeiffer. A recent extensive examination of the anatomical aspects of abscission by Webster ( 1968) coupled with the electron microscopy work of Morre (1968) permit a more definitive view of the subject. Appaveintly local breakdown of intercellular materials between adjacent cells of the separation layer is initiated in the middle lamella. However, subsequent dissolution of cell walls and digestion of cellular content may occur. Morre (1968) is of the opinion that dissolution of the intercellular cement is necessary, and perhaps sufficient, for formation of the separation layer during abscission. Knowledge of changes in the middle lamella that occur during abscission would be quite useful in an elucidation of the abscission process. The pectinaceous nature of the middle lamella has been established for some time (Mangin 1889). In fact, Mangin's work served as a basis for some workers referring to the abscission process as a dissolution of the protopectin and calcium pectate of the middle lamella. Lloyd (1916) spoke of the dissolution of the middle lamella as a process of hydrolysis and apparently assumed that enzymes were involved. As early as 1918 Kendall reported the appearance of pectin as abscission occurred in tobacco flowers and assumed that enzymes were involved in the dissolution process. Sampson (1918) determined that the final stage of changes in the middle lamella prior to cell separation was the breakdown of calcium pectate. Facey ( 1950) using histochemical procedures in the study of the abscission zone of Fraxinus cuttings demonstrated a shift from calcium pectate to pectic acid and then to water soluble pectin. She suggested that esterification of pectic acid to the form of pectin does occur during abscission, since pectin is the only form that is water soluble. Rasmussen ( 1965) and Morre ( 1968) reported similar results. Morre considered that the observed increase in solubility of pectins of the middle lamella might arise from removal of polyvalent cations, methylation of carboxyl groups and/or depolymerization. The appearance of pectin lends support to the idea of methylation of carboxyl groups. However, in vitro evidence exists (McClendon 1964, Yager 1960, Zaitlin and Coltrin 1964) to support the thought that depolymerization of pectic substances or removal of ions will stimulate natural separation. That esterification may be involved in abscission has been emphasized by the work of Yager and Muir (1958 a,b). They observed that methionine, a methyl donor, caused a very rapid acceleration of abscission of un2 Proceedings of the Iowa Academy of Science, Vol. 76 [1969], No. 1, Art. 8 http://scholarworks.uni.edu/pias/vol76/iss1/8 1969] PECTIC ENZYMES TO ABSCISSION 47 fertilized ovaries of tobacco. Methionine and certain other amino ~cids have been found to contribute methyl groups for esterificat1on of pectin and other substances of the abscission zone by Nelson (1960) and Valdovinos and Muir (1965). Recently work concerning enzymes involved in abscission has focused on pectic enzymes. However, various other enzymes have been reported to be associated with the abscission process. Sampson (1918) found oxidases to be present in all tissue of the abscission zone except the xylem; however in the stem and petiole, oxidases were present only in epidermal and phloem tissue. In 1919 Heinicke noted that catalase was more active in the abscission zone than in contiguous areas. Kertesz (1943) suggested that perioX:idase may act to change pectic compounds in plant tissue. Cams (1951) applied inhibitors of specific respiratory enzymes to explants and found that abscission was retarded. Cellulase has been specifically associated with wall changes in the abscission zone (Horton and Osborne 1967). Since abscission is initiated in the middle lamella with the dissolution of pectinaceous material, the action of pectic enzymes should be relevant. However, there is a general lack of information concerning pectic enzymes other than pectin methylesterase ( PME) . Much of the work with pectic enzymes has been done in relation to fruit ripening (Bateman and Millar 1966, Demain and Phaff 1957, Kertesz 1951, Lineweaver and Jansen 1951, Sterling and Kalb 1959), a process which may be analagous to the abscission process. Bonner ( 1936), speculating from the work of Sampson ( 1918) , suggested that protopectinase was responsible: for leaf abscission although its existence has been debated. Pectin-polygalacturonase is probably the best known of the pectic enzymes other than PME. Demain and Phaff (1954, 1957) reported a complex of enzymes instead of a single pectin-polygalacturonase. The complex consisted of several exo-polygalacturonases. specific for pectins of different degrees of esterification and size of polymer and a single endogalacturonase. Schubert (1952), Dingle et al. (1953) and Ayres et al. (1952) indicated that the endo-polygalacturonase is a complex of enzymes. Pectinase is frequently used to describe pectinpolygalacturonase and is also used to designate pectic enzyme mixtures. The complexity in the identification of specific pectinpolygalacturonases may explain some of the conflicting reports as as well as the problems arising from nomenclature. McClendon and Somers ( 1956) reported the occurrence of two pectin-glycosidases with different conditions required for activity. Kertesz ( 1951) postulated the probable occurrence of additional pectic enzymes in higher plants. Sato, Byerrum, and Ball ( 1957) reported the bio3 Smith: Relationship of Pectic Enzymes to Abscission Published by UNI ScholarWorks, 1969 48 IOWA ACADEMY OF SCIENCE [Vol. 76 synthesis of methyl esters of pectinic acid through transmethylation from methionine with a preparation of radish tissue. Albershcim, Neudom, and Deuel ( 1960) isolated a hydrolytic enzyme that acts as a transcliminase by splitting methylated pectin. Albersheim and Killias ( 1962) established the presence of the enzyme in higher plants (pea) and Albersheim ( 1963) found that it could be inhibited by 2, 4-D and indoie acetic acid (IAA). Yager (1960) reported the in vitro effects of pectinase and a pectin glycosidase on tobacco tissue. These two enzymes caused dissolution of the middle lamella and separation of cells of the abscission zone as well as other cells of the tissue slice through the pediccl. Methionine and low concentrations of IA.A increased the dissolution activity of the two enzymes whi1e high concentrations of IAA retarded their activity. Morre ( 1968) found that pectinase activity which was low or absent at the time of excission, rose to a maximum at 72 hours and then declined. Appearance of activity coincided closely with separation layer formation and declined when separation was complete. The pectinase of the bean explant was assumed to be an endopolygalacturonase. Pectin methylcsterase (PME) has been studied more extensively than the other pectic enzymes. It is widely distributed in higher plants and appears to be associated with cell walls. Its action is generally considered to be that of catalyzing the hydrolysis of the methyl ester bonds of pectinic acid and pectin (Kertesz 1951). Lineweaver and Jansen (1951) suggest that PME may act only on ester bonds that are adjacent to free carboxyl groups or may act on these mo11e rapidly than on other ester bonds. Much of the work has been in relation to plant growth (Bryan and Newcomb 1954, Glasziou 1959 and Jansen, Jang and Bonner 1960). Osborne (1958) found that PME activity decreases with the age of the bean leaf. The greatest drop in activity was found in pulvinus tissue as compared with other parts of the blade or petiole. Application of ethylene accelerated abscission while application of such compounds as 2, 4-D sustained activity of the enzyme. Yager ( 1960) found PME activity to be high in the abscission region of tobacco pedicels. In addition, high concentrations of IAA which delayed abscission, increased PME activity and methionine, an accelerator of abscission, decreased PME activity. LaMotte, etc. ( 1960) compared PME activity in various portions of attached leaves and abscissing petioles and confirmed some of the earlier findings of Osborne ( 1958) and Yager ( 1960) . The team found that PME activity in both Coleus and bean plants was lower m the distal portion of abscission zones of abscissing petioles than m attached leaves. Also, auxin treatment of debladed petioles of Coleus prevented abscission and J1esulted in small increases m PME activity in abscission zones and most of the other regions sampled. 4 Proceedings of the Iowa Academy of Science, Vol. 76 [1969], No. 1, Art. 8 http://scholarworks.uni.edu/pias/vol76/iss1/8 1969] PECTIC ENZYMES TO ABSCISSION 49 The above relationship of the activity of pectic enzymes to auxins coupled with the effect of auxins on abscission provides fuel for some interesting speculation concerning the physioJogical control of abscission. Many workers have found evidence that an endogenous regulator is responsible for controlling abscission. Among the first was Laibach (1933) who reported orchid pollen (a rich source of auxin) could effectively delay abscission of debladed Coleus petioles. This observation was confirmed by LaRue ( 1936) who found that the retarding effect of pure indoleacetic acid compared with the intact leaf. Myers (1940) related the amount of auxin in a leaf blade to its inhibition of abscission. Wetmore and Jacobs (1953) found a smiliar correlation between the normal longevity of intact leaves and their content of diffusible auxin. Since auxins affect abscission and pectic enzymes arie associated with abscission the question arises as to how they are related. Osborne (1958) found that 2, 4-D sustained activity of PME. Yager ( 1960) observed the following in vitro effect of varying IAA concentration: low concentrations of IAA, which accelerated abscission, caused a decrease in PME activity of tissue macerates and an increase in action of pectinanse and pectin-glycosidase whereas higher concentrations of IAA, which retarded abscission, increased PME activity but inhibited pectinase and pectin-glycosidase. This correlates well the work of Gaur and Leopold ( 1955) and Biggs and Leopold (1958) who found that dilute concentrations of auxin accelerated abscission, whereas higher concentrations. were inhibitory. Investigators using other types of plant tissue have observed auxin induced increases in PME activity (Neely, etc. 1950, Bryan and Newcomb 1954). The dual abilities of auxin either to inhibit or promote abscission somewhat complicate the issue. Rubinstein and Leopold ( 1964) suggest the auxin effects may operate through mechanisms involving changes in membrane permeability, changes in pectic enzyme activities, or changes in the production of ethylene by the petiole tissue. The following is a report of the results of a series of investigations in an attempt to evaluate the role of PME in abscission of tobacco flowers, Nicotiana tabacum L. cultivar Lizard's Tail. The relative activity of the enzyme was determined by an increase in free carboxyl groups as suggested originally by Kertesz ( 193 7) . The results are reported in three parts: ( 1) distribution of PME in the tobacco pedicel; (2) comparison of PME in the abscission zones of pollinated and unpollinated flowers; ( 3) cffoct of IAA treatment of tobacco ovaries on the PME activity of the abscission zones of tobacco pedicels. The localization of PME activity in the pcdicels of tobacco flowers was examined by comparing tissue of the abscission zone to 5 Smith: Relationship of Pectic Enzymes to Abscission Published by UNI ScholarWorks, 1969 50 IOWA ACADEMY OF SCIENCE [Vol. 76 tisue of similar weight from the region of the pedicels just below the receptacle. It is evident from the data of Table 1 that the PME activity is higher in the abscission zone of the pedicel than it is in the distal region. This confirms the eariler work of Yager ( 1960) . As can be seen there is considerable variation in the PME activity of the abscission zones from pedicels of flowers at an thesis.