The effect of 2,4,5-TP on the activity of cellulase, pectin methylesterase and polygalacturonase in bean explants

The effect of 2,4,5-TP on the activity of cellulase, pectin methylesterase and polygalacturonase in bean explants J.E. Watts and O.T. de Villiers Department of Botany, University of Stellenbosch, Stellenbosch and Department of Agronomy and Pastures, University of Stellenbosch, Stellenbosch

Bean explants were used as a model system to study the effect of 2-(2,4,5-trichlorophenoxy) propionic acid {2,4,5-TP) on certain enzymes which are involved in abscission. An increase in cellulase activity in pulvini, petioles and abscission zones during the course of abscission was significantly inhibited by 2,4,5-TP. During abscission the gradient of pectin methylesterase (PME) activity in control explants decreased significantly, but 2,4,5-TP maintained this gradient by increasing PME-activity in abscission zones and maintaining it in pulvini and petioles. The activity of exo-polygalacturonase (exo-PG) was not affected by 2,4,5-TP. However, the activity of endo-PG increased from zero to a maximum in control explants, with 2,4,5-TP maintaining the activity at zero. It is concluded that 2,4,5-TP prevents abscission in bean explants by maintaining a form(s) of pectin constituting a more rigid complex in middle lamellae. S. Afr. J. Bot. 1986, 52: 241-245

Die invloed van 2-(2,4,5-trichlorofenoksi)-propioonsuur (2,4,5-TP) op sekere ensieme wat by afsnyding betrokke is, is met behulp van boontjie-eksplante as modelsisteem ondersoek. 'n Toename in sellulase-aktiwiteit in pulvini, blaarstele en afsnydingsones gedurende die verloop van afsnyding is betekenisvol deur 2,4,5-TP ge"inhibeer. Die gradient van pektienmetielesterase (PME)-aktiwiteit in kontrole-eksplante het gedurende afsnyding betekenisvol afgeneem, maar 2,4,5-TP het die gradient in stand gehou deur PME-aktiwiteit in afsnydingsones te verhoog en dit in pulvini en blaarstele op 'n konstante vlak te hou. Die aktiwiteit van ekso-poligalakturonase (ekso-PG) is nie deur 2,4,5-TP be"invloed nie. Die aktiwiteit van endo-PG het egter toegeneem vanaf zero tot 'n maksimum in kontroleeksplante, terwyl geen aktiwiteit in die teenwoordigheid van 2,4,5-TP waargeneem is nie. Die gevolgtrekking word gemaak dat 2,4,5-TP afsnyding in boontjie-eksplante voorkom deur die behoud van 'n vorm(s) van pektien wat 'n stewiger kompleks in die middellamella verteenwoordig. S.-Afr. Tydskr. Plantk. 1986, 52: 241-245

Keywords: Abscission, bean explants, enzymes, 2,4,5-TP

J.E. Watts* Department of Botany, University of Stellenbosch , Stellenbosch, 7600 Republic of South Africa O.T. de Villiers Department of Agronomy and Pastures, University of Stellenbosch, Stellenbosch, 7600 Republic of South Africa *To whom correspondence should be addressed Accepted 29 October 1985

Introduction

Poor fruit-set in Packham's Triumph pears (Pyrus communis L.) is a serious problem in the Western Cape (Strydom eta/. 1973). However, optimum set can be achieved by spraying with 2-(2,4,5-trichlorophenoxy) propionic acid (2,4,5-TP) during the latter half of April (Strydom et a/. 1966). Little is known about the mechanism whereby 2,4,5-TP exerts its effect on fruit-set. Previous studies (Watts & De Villiers 1979; Watts 1984) indicated that source- sink relationships between the different plant parts of Packham's Triumph pear trees are not modified by 2,4,5-TP. It was concluded that 2,4,5-TP probably exerts an effect by acting in an auxin-like manner via changes in the abscission layers. In a study to determine the effect of 2,4,5-TP on abscission in bean ex plants the results indicated that its effect corresponded to known effects of IAA on abscission (Watts 1984). It was concluded that 2,4,5-TP prevented abscission in bean explants by maintaining a favourable auxin gradient across abscission zones. The same mechanism may also prevent fruit drop in Packham's Triumph pears since direct applications of 2,4,5-TP to deflowered pedicels and defruited fruit stalks prevented their abscission (Watts 1984). Unfavourable auxin gradients across abscission zones may induce the formation of abscission layers via certain biochemical changes that lead to a stimulated synthesis of the enzymes actively involved in abscission (Morn~ 1968; Addicott 1982). The activities of several enzymes increase during the period immediately prior to abscission. Horton & Osborne (1967) found a correlation between the activity of cellulase and abscission in bean explants. Such correlations were also found for cotton and Coleus (Abeles 1969) as well as for Citrus (Greenberg et a!. 1975). According to Horton & Osborne (1967) and Abeles (1969) the increased cellulase activity is involved in cytolysis and cell disintegration during abscission. However, electron micrographs clearly indicated that cell separation is caused entirely by the dissolution of middle lamellae and that the hydrolysis of cellulose is not involved (Valdovinos & Jensen 1968; Sexton 1976). Since middle lamellae consist virtually entirely of pectic substances (Albersheim & Killias 1963), pectic enzymes may play a primary role in abscission. A decrease in the activity of pectin methylesterase (PME) during abscission was observed for explants of Phaseolus (Osborne 1958), Nicotiana (Yager 1960) and Coleus (Moline eta/. 1972). These results imply that abscission is promoted by an increase in esterification of pectic substances. However, several authors could not confirm this relationship between abscission and PME activity (Ratner et a/., 1969; Hanisch ten

242 Cate eta/. 1975). An increase in the activity of the endo-form of polygalacturonase (PG) in abscission zones of bean plants shortly before abscission was observed by Morn~ (1968). The exo-form of PG, however, showed no increase in activity during abscission in bean explants (Berger & Reid 1979). The prevention of fruit drop in Packham's Triumph pears may be mediated through effects on the above-mentioned enzymes. This study was therefore undertaken to determine whether 2,4,5-TP prevents abscission by affecting the activities of cellulase, PME and PG (exo- and endo-forms). For several reasons bean explants were used as a model system. According to Addicott (1982), explants, especially bean explants, are ideal experimental material to study the abscission process as such, because all external influences are eliminated. A great advantage is that large numbers of uniform explants may be prepared, treated and kept under controlled conditions. Such explants respond in a quick and uniform manner to treatments (Morre et a!. 1969). Materials and Methods Bean explants (Phaseolus vulgaris L. cv. Top Crop) were prepared, mounted, treated and incubated as described previously (Watts 1984). Groups of explants were treated distally with 2 mm 3 of a solution containing 50 mg dm - 3 Na-2,4,5-TP while control explants were treated with 2 mm 3 water. At set times after treatments, groups of explants were used for enzyme extraction and the percentage abscission of each group determined. For the determination of PME- and cellulase activities the explants were divided into pulvini, petioles and abscission zones. This was done to determine the effect(s) of 2,4,5-TP on (a) the gradient of PME-activity across abscission zones and (b) the part of cellulase activity probably related to the formation of protective layers. The division was performed by excising the petiole and pulvinus so that I mm of each remained on either side of the abscission line. Cellulase was extracted at 4 oc and determined according to the method of Horton & Osborne (1967). Groups of 60 petiole sections, 60 pulvini and 100 abscission zones were ground separately in a mortar with 5 cm 3 sodium phosphate buffer (0, 1 mol dm - 3 ; pH 6,0) containing 0,07.0Jo L-cysteine hydrochloride. A small amount of acid-washed sand was used to facilitate grinding. The mixture was centrifuged at 5000 x g for 15 min and the clear supernatant served as enzyme source. The ability of the e·nzyme solution to decrease the viscosity of a buffered solution of carboxymethylcellulose at 25°C (sodium salt-BDH) was taken as a measure of enzyme activity. Enzyme activity was calculated as the percent change in viscosity per 2 h according to the formula of Norkrans & Hammarstrom (1963):

p = to tx 100 to - tw · 1 where P to tx

Percent change in viscosity Initial flow-time of reaction mixture Flow-time following the relevant incubation period (2 h) tw Flow-time of water All flow-times were noted in seconds. Viscosity determinations were carried out in duplicate on each of four samples per treatment. The extraction and determination of PME were performed by a modified procedure of Osborne (1958). Groups of 80 sections of pulvini, petiole and abscission zones were ground separately with 7 cm 3 glass-distilled water in a mortar containing a small amount of acid-washed sand. The extracts were

S.-Afr. T ydskr. Plantk. , 1986, 52(3)

then centrifuged at 5000 x g for 10 min. The activity of PME was determined by adding 2,5 cm 3 of the supernatant to 2 cm 3 of I OJo (m/ v) citrus pectin (Grade I, Sigma) in 0,1 mol dm - 3 NaCl containing 0,00175% methyl red. The pectinindicator solution was previously adjusted to pH 6,0 with 0,1 mol dm - 3 NaOH. Blank reaction mixtures were prepared with boiled enzyme extracts. The reaction mixtures were incubated for 1 hat 25 °C in a shaking water bath, whereafter increases in their absorbance at 420 nm were determined. Enzyme activity was calculated as the change in ~2o per g (fresh mass) of tissue per 100 min in the standard pectin solution under the above-mentioned conditions. All determinations were done in duplicate on each of four samples per treatment. The activity of the exo-form of PG was determined according to the procedure of Berger & Reid (1979). Eighty abscission zones were ground in a mortar containing 10 cm 3 sodium phosphate buffer (50 mmol dm - 3; pH 7,5) containing 6% (NH4)2S04 (m/ v) and a small amount of acid-washed sand. The following steps (centrifugation, precipitation with (NH4)zS04 and dialysis) were performed as described by Berger & Reid (1979). Reaction mixtures for the colorimetric determination of reducing end groups consisted of 0,5 cm 3 of the enzyme extract and 0,5 cm 3 0,25% (m/ v) sodium polypectate (Grade II, Sigma) in 50 mmol dm - 3 sodium acetate buffer (pH 5,0) containing 1,0 mmol dm - 3 sodium hydrosulphite. A unit of enzyme activity was taken as the amount that catalyzed the release of 1,0 f.! mol D-galacturonic acid in 1 h. Purified D-galacturonic acid (Sigma) was used as a standard. Results were expressed per 80 abscission zones. All determinations were done in duplicate on each of six samples per treatment. Endo-PG was extracted by the method of Mussell & Morre (pers. comm.). Groups of 300 explants were surface-sterilized by immersion in a mixture of I OJo Na-hypochlorite and 1OJo ethanol (1: 1; v/ v) for 1 min, rinsed in sterilized glass-distilled water and blotted dry. They were then placed vertically in 1OJo agar in petri dishes up to a depth of 4 mm so that the pulvini still protruded above the agar. Distal application of 2,4,5-TP (or water for control groups) was performed as described (Watts 1984). The petri dishes were closed and kept in the dark at 27°C. At set times percentage abscission was determined on groups of explants. Each group was then ground in a mortar with 15 cm 3 sodium phosphate-citrate buffer (0,05 mol dm - 3 ; pH 5,0) and a small amount of acidwashed sand. The resulting extracts were filtered through a layer of 'Miracloth' (Calbiochem) to obtain the enzyme source. Enzyme activity was determined according to the method of Mussel & Morre (1969). The method is based on the ability of the extract to weaken middle lamellae in cucumber pericarp sections with a resulting loss of cells and a consequent decrease in mass which is then measured accurately. Determinations were performed in triplicate on each of four samples per treatment. Enzyme activity was expressed as f..lg pectinase em - 3 , using a standard curve obtained with commercial pectinase (Sigma). Results and Discussion The activity of cellulase in pulvini, petioles and abscission zones did not change appreciably during the first 24 h after the respective treatments (Figure 1). During the next 12 h cellulase activity increased significantly in control groups, with the largest increase shown in abscission zones. This increase coincided approximately with the commencement of abscission and continued to a maximum as abscission proceded. The

243

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percentage abscission increased in direct proportion to the increase in cellulase activity in abscission zones. Treatment with 2,4,5-TP led to a significant inhibition of cellulase activity in pulvini, abscission zones and petioles, whereas the activity in controls remained at the initial level. Horton & Osborne (1967) also found increases in cellulase activity [inhibited by 2,4,5-T(2,4,5-trichlorophenoxyacetic acid)] during the course of abscission in bean explants, but these increases preceded abscission. Abeles (1969) observed similar increases (inhibited by IAA) but did not determine whether they preceded abscission or not. These authors suggested that cellulase is involved in the hydrolysis of cell wall components and, in so doing, contributes actively to abscission. However, this view is questionable on account of the finding that increases in cellulase activity in bean explants did not precede decreases in the pull-force of abscission zones,

but proceded parallel to it (Lewis & Varner 1970). Hanisch ten Cate eta!. (1975) even found increases in cellulase activity (in pedicels of Begonia) after abscission was already complete. These results, as well as those of the present study (namely parallel increases in cellulase activity and abscission) oppose the view of a causal relationship between cellulase and abscission. The fact that IAA and synthetic auxins such as 2,4,5-TP inhibit cellulase activity as well as abscission, indicates a possible role for cellulase in abscission . According to Addicott (1982) the increase in cellulase activity may be involved in the formation of protective layers immediately after cell separation has taken place. The formation of such layers is accompanied by considerable cell enlargement that may be facilitated by cellulases (Albersheim et a!. 1977). This finding offers a possible explanation for the increase in cellulase activity in pulvini and petioles in the present study. The implication is then that 2,4,5-TP (and other auxins) inhibits abscission without a direct effect on cellulase. 2,4,5-TP will therefore inhibit abscission via other enzymes and cellulase activity should remain more or less constant, as was found in the present study. PME-activity was initially the highest in pulvini, somewhat lower in abscission zones and the lowest in petioles (Figure 2). In pulvini of control groups the activity of PME showed a significant sharp decrease, even before any visible signs of abscission could be observed. Similar but smaller decreases in activity occured in abscission zones, but in petioles no significant changes in PME activity occurred during the course of abscission. These decreases in PME activity preceded the commencement of abscission so that (in contrast to cellulase) a causal relationship between PME activity and abscission seems plausible. These results verify the view of Osborne (1958) that abscission in bean explants is caused by a decrease in the gradient of PME activity across abscission zones . Treatment with 2,4,5-TP resulted in the activity of PME in pulvini and petioles remaining relatively constant at the initial level, but led to a significant increase in activity in abscission zones. 2,4,5-TP therefore maintained the gradient of PME-activity across abscission zones. It can be postulated that 2,4,5-TP, by maintaining (or increasing) PME activity, is able to sustain a form(s) of pectin with more free carboxyl groups. According to Addicott (1970) such forms of pectin will contain more calcium bridges and constitute a more rigid complex in middle lamellae. The action of 2,4,5-TP may therefore lead to stronger binding between cells and an inhibition of abscission. The activity of the exo-form of PG did not change significantly during the course of abscission (Figure 3). This is in accordance with the results of Rasmussen (1973) and Berger & Reid (1979) with bean explants. Treatment with 2,4,5-TP had no significant effect on PG-activity (exo-form). The activity of the endo-form of PG in control groups increased steadily from zero to a maximum and then decreased slightly with the increase preceding the first visible signs of abscission (Figure 4). Similar results were obtained by Morn~ (1968) with bean explants. Treatment with 2,4,5-TP maintained the activity at zero during the whole experimental period. These results indicate that exo-forms of PG are not actively involved in the abscission of bean explants. McClendon (1964) and Bateman & Millar (1966) concluded that, unlike endoforms, exo-forms of PG do not macerate tissues. A crude enzyme extract (as used for the determination of endo-PG) is actually a macerase that may contain a variety of pectic enzymes, including exo- and endo-forms of PO. The fact that

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macerase act1v1ty was initially absent but subsequently increased, suggests a possible de novo synthesis of a specific endo-form(s) of PG. Since no enzyme activity was found after treatment, 2,4,5-TP may act via an inhibition of the particular endo-form(s) of PG.

Conclusion It is concluded that 2,4,5-TP prevents abscission in bean explants by sustaining a form(s) of pectin constituting a more rigid complex in middle lamellae. This may be accomplished by the maintenance of the gradient of PME activity across abscission zones and the complete inhibition of endo-PGactivity. The prevention of abscission of Packham's T~iumph pears may therefore also be mediated in the same way since direct applications of 2,4,5-TP to pedicels and fruit stalks prevented their abscission (Watts 1984).

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S. Afr. J. Bot., 1986, 52(3) McCLENDON, J.H. 1964. Evidence for the pectic nature of the middle lamella of potato tuber cell walls based on chromatography of macerating enzymes. Am. J. Bal. 51: 628-633. MOLINE, H .E ., LAMOTTE, C.E., GOCHNAVER, C. & McNAMER, A. 1972. Further comparative studies of pectin esterase in relation to leaf and flower abscission . Pl. Physiol. 50: 655-659. MORRE, D.J. 1968. Cell wall dissolution and enzyme secretion during leaf abscission. Pl. Physiol. 43: 1545 - 1559. MORRE, D.J., RAU, B., VIEIRA, R., STANCEU, T. & DION, T. 1969. Environmental regulation of experimental leaflet abscission. Proc. Indiana A cad. Sci. 78: 146 - 160. MUSSELL, H.W. & MORRE, D.J. 1969. A quantitative bioassay specific for polygalacturonases. Ann. Biochem. 28: 353 - 360. NORKRANS, B. & HAMMARSTROM, A. 1963. Studies on growth of Rhizina undulata Fr. and its production of cellulase and pectin-decomposing enzymes. Physiol. Plant. 16: I - 10. OSBORNE, D.J. 1958. Changes in the distribution of pectin methylesterase across leaf abscission zones of Phaseolus vulgaris. J. exp. Bot. 9: 446-457. RASMUSSEN, G.K. 1973. Changes in cellulase and pectinase activities in fruit tissues and separation zones of Citrus treated with cycloheximide. Pl. Physiol. 51: 626- 628.

245 RATNER, A., GOREN, R. & MONSELISE, S.P. 1969. Activity of pectin esterase and cellulase in the abscission zone of Citrus leaf ex plants. Pl. Physiol. 44: 1717 - 1723. SEXTON, R. 1976. Some ultrastructural observations on the nature of foliar abscission in lmpaliens sui/ani. Plan/a 128: 49 - 58. STRYDOM, O.K., HONEYBORNE, G .E. & ALLISON, E. 1973. Packham's Triumph-vrugsetstudies. Die Saglevrugteboer 23: 134 - 137. STRYDOM, O.K., VAN ZYL, H.J . & GINSBURG, L. 1966. Die gebruik van 2,4,5-TP-bespuitings om die vrugset van Packham's Triumph-peerbome te verhoog. Die Sagtevrugteboer 16: 102 - 103. VALDOVINOS, J.G. & JENSEN, T.E. 1968 . Fine structure of abscission zones. II. Cell wall changes in abscising pedicels of tobacco and tomato flowers. Plan/a 83: 295 - 302. YAGER, R.E . 1960. Possible role of pectic enzymes in abscission. Pl. Physiol. 35: !57- 162. WATTS, J.E. 1984. Die opname, metabolisme en meganisme van werking van 2,4,5-TP tydens vrugset in Pyrus communis L. cv. Packham's Triumph. Ph.D.-thesis, Univ. Stellenbosch. WATTS, J .E. & DE VILLIERS, O.T. 1979. The effect of 2,4,5-TP on the concentration of sorbitol, sugars and starch in the leaves, stems and fruits of Packham's Triumph pear trees during early fruit development. Agroplantae II: 75 - 77.