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Peroxidases in the separation zone during ethylene-induced bean leaf abscission
Pergamon
Phytochem stry, Vol. 35, No. 3, pp. 567-572, 1994 Copynght Q 1994 Elsevier Science Ltd Printed in Great Britain . All rights reserved
0031-9422/94 $6 .00+0.00
PEROXIDASES IN THE SEPARATION ZONE DURING ETHYLENE-INDUCED BEAN LEAF ABSCISSION MICHAEL T . MCMANUS •
Department of Biochemistry, Royal Holloway and Bedford New College, University of London, Surrey TW20 OEX, U .K. (Received in revised form 22 June 1993)
Key Word Index-Phaseolus vulgaris; Leguminosae; leaf abscission; peroxidase isoenzymes .
Abstract-To determine if peroxidase activity is induced specifically in the abscission zone during ethylene-induced cell separation, the spectrum of isoenzymes and activity of the enzyme in extracts from the distal pulvinus, the pulvinus : petiole abscission zone and the petiole of Phaseolus vulgaris leaves were compared . For the cationic isoenzymes, the onset of ethylene-induced cell separation correlated with an increase in the expression of two isoenzymes that are present in both the pulvinus and the abscission zone . In terms of the anionic isoenzymes, two isoenzymes were induced at the time of ethylene-induced cell separation . Both of these are expressed preferentially in the zone, while another was induced predominantly in the petiole . No peroxidase isoenzymes could be detected that were induced specifically in the abscission zone at the time of pulvinus separation . When total enzyme activity was compared, ethylene treatment increased the measurable activity in all tissues, although the highest was at the zone at the time of cell separation . When specific activity was compared, these differences were not as marked. Together, these results show that while peroxidase activity is induced at the abscission zone during ethylene-induced cell separation, changes in specific isoenzymes are observed both in the zone and in neighbouring (non-abscission) tissue . These changes in the spectrum of isoenzymes observed in neighbouring tissue, therefore, should not be ignored when postulating a role for peroxidase in mediating cell-cell separation at the abscission zone .
INTRODUCTION
Peroxidases (donor: hydrogen peroxide oxidoreductase, EC 1 .11 .1 .7) have been implicated in the regulation of a number of biochemical and physiological processes in higher plants [1, 2]. Of these, the role of specific isoenzymes in regulating the terminal steps of lignification, or as indole-3-acetic acid (IAA) oxidases has received considerable attention . Anionic isoenzymes of peroxidase have been implicated in the terminal steps of lignification by mediating the polymerization of substituted cinnamyl alcohols [3-6] . These isoenzymes have also been implicated in the crosslinking of extension monomers and feruloylated polysaccharides in cell walls [7], and in wound healing through suberization [8, 9] . Cationic isoenzymes are implicated as IAA oxidases. While the molecular mechanism of IAA oxidation by horse-radish peroxidase, in vitro, has been established [10-13], the significance of IAA catabolism by peroxidase, in vivo, is still unclear, particularly since the enzyme can also oxidize IAA-amino acid conjugates [14]. A number of studies have now been published concerning the correlation of peroxidase activity with leaf abscis*Present address : Plant Molecular Genetics Laboratory, Grasslands Research Centre, AgResearch, Private Bag 11008, Palmerston North, New Zealand .
sion, although as yet, there is little consensus on the specific role of this enzyme in the mechanism of cell-cell separation [15] . Using histochemical staining procedures, intense activity of the enzyme has been localized in the wall and middle lamella region of cells at the separation layer in bean leaves [16-18], the lower (pedicel :fruit) zone in sweet cherry [19] and in tobacco flower pedicels [20-22] . As well as studies on the specific cellular localization of peroxidase activity at cell separation, changes in the isoenzyme profile of the enzyme at the onset of abscission in the zone have also been documented . In most tissues examined so far, only quantitative changes in the isoenzyme profile are observed [16, 23, 24], although qualitative changes have been recorded during leaf and cotyledon abscission in cotton [25]. In general though, these isoenzyme studies have only concentrated on changes specifically in the abscission zone in an attempt to link the enzyme with the cell separation process . However, it is known that ethylene can induce peroxidase activity in a number of other tissues, including the leaves and storage roots of sweet potato [26, 27], and in senescent cucumber cotyledons [28]. The identification of abscission zone-specific peroxidase isoenzymes is of interest because other ethylene-induced proteins (e.g. B-1,4-glucanase and chitinase) have been identified in bean leaf abscission zones, and their function in mediating cell--cell separation considered [29]. In order to implicate peroxidase in this process, a comparison of 567
568
M . T . MCMANUS
ethylene-induced isoenzymes in both the abscission zone and in neighbouring tissue is necessary . In this study, the profile of peroxidase isoenzymes has been monitored during ethylene-induced cell separation at the distal leaf pulvinus :petiole abscission zone in Phaseolus vulgaris L., to determine whether there are any that are abscission cell-specific . Further, the induction of enzyme activity in these tissues has also been investigated, in order to determine if any increase in total peroxidase activity during abscission is confined precisely to the zone. RESULTS
Changes in the spectrum of peroxidase isoenzymes during ethylene-induced cell separation
Cell extracts from the pulvinus, abscission zone and petiole were prepared at the time of explant excision (0 hr) and after 36 hr in ethylene (the time of pulvinus separation). Using diaminobenzidine as substrate, the peroxidase isoenzyme profile was determined for each extract . On separation of the cationic isoenzymes, no qualitative differences could be discerned between the extracts at the time of excision (Fig. IA) and at pulvinus separation (Fig. 1B). However, the concentration of two of the prevalent isoenzymes (C1, C2) increased with the onset of cell separation in both the pulvinus and the abscission zone . With separation of anionic isoenzymes, several changes in the staining intensity of the existing isoenzymes could be identified with the onset of cell separation (Fig . 2). Of these, two isoenzymes in particular, A3 and A7 (Fig . 2B, lane f), have increased in concentration, most notably in
the abscission zone, although A3 has increased to a lesser degree in the pulvinus (Fig . 2B, lane d). Isoenzyme A7 can also be detected on the pulvinus surface immediately adjacent to the abscission zone, suggesting that it is secreted (Fig . 2B, lane e). One isoenzyme, A5, has increased in the petiole (Fig . 2B, lane g) during the onset of cell separation, while isoenzyme A l has decreased in intensity in the pulvinus after 36 hr in ethylene (cf. Fig. 2, lane a with d) . In common with changes observed in the cationic isoenzyme profile during cell separation, no qualitative changes in anionic iscenzymes were observed (A7 is present in 0 hr zone, albeit at very low concentrations) . Overall, these results indicate that during the onset of ethylene-induced cell separation at the distal pulvinus :petiole abscission zone in P. vulgaris, the use of diaminobenzidine as substrate does reveal that quantitative changes in the peroxidase isoenzyme profile occur, but these are not restricted solely to the zone. Changes in peroxidase activity during ethylene-induced cell separation
Using guaiacol (o-methoxyphenol) as substrate, total and specific peroxidase activity in the abscission zone and surrounding tissue was measured at 0 hr and after ethylene treatment to induce pulvinus separation (36 hr) . For these experiments, it was convenient to divide each tissue extract into two fractions, a water-soluble fraction and an ionically bound (high salt-soluble) cell wall fraction . In terms of total activity (expressed as enzyme units), an increase was observed with the onset of cell separation in the soluble and ionically bound fractions from all three tissues. However, activity was highest in both abscission
C1
C2
Fig. 1. Separation of cationic isoenzymes. Extracts were subjected to polyacrylamide gel electrophoresis using the method of Reisfeld, Lewis and Williams [33] and the separated peroxidase isoenzymes visualized using 3',3'-diaminobenzidine . A. Extracts from tissues at 0 hr, a = leaf pulvinus, b = abscission zone, c = petiole . B . Extracts from tissue after 36 hr in ethylene ; d = leaf pulvinus, e = abscission zone, 1= petiole .
Peroxidase in bean leaf abscission
569
a b c
-A1 -A2 -A3 - A4 -A -A - A7 - A8 Fig. 2 . Separation of anionic isoenzymes . Extracts were subjected to polyacrylamide gel electrophoresis using the method of Davis [32] . Visualization of separated peroxidase isoenzymes was achieved using 3',3'-diaminobenzidine . A. Extracts from tissues at 0 hr, a =leaf pulvinus, b = abscission zone, c = petiole . B. Extracts from tissue after 36 hr in ethylene ; d = leaf pulvinus, a=leaf pulvinus surface adjacent to the abscission zone, f= abscission zone, g = petiole .
zone fractions when compared with neighbouring tissue (Table 1) . Of the two abscission fractions, activity is greater in the ionically bound preparation . In terms of specific activity, the highest activity in any tissue at the time of cell separation (36 hr) was again detected in the abscission zone. This occurs both in the soluble and ionically bound fractions, although the difference between the abscission zone and neighbouring tissue is now not as marked as with comparisons made for total activity . This is particularly so for the soluble fraction, and presumably reflects an increase in the relative protein content of the abscission zone after ethylene treatment, particularly with the induction of cell wall hydrolases . These results confirm that during ethylene-induced cell separation, peroxidase activity is highest in the abscission zone, but the enzyme is also induced in neighbouring tissues. Kinetic parameters of peroxidase induction
The increase in enzyme activity in the abscission zone with the onset of cell separation was investigated in terms of two kinetic parameters, V., and K . for the substrate guaiacol (Table 2) . The induction of enzyme activity is accompanied by the significant increase in VV for the PHYTO 35 :3-C
enzyme at 36 hr in both fractions, when compared with 0 hr. However, the K . for guaiacol has not decreased significantly over this time-course . These results suggest that for this substrate at least, the observed increase in peroxidase activity at the abscission zone is mediated essentially through an overall increase in enzyme synthesis and not through any significant change in the specificity for the substrate . DISCUSSION
This study has sought to identify isoenzymes of peroxidase which are induced specifically in the distal pulvinus :petiole abscission zone of bean leaves at the onset of ethylene-induced cell separation. Cytochemical staining [16, 17] has identified peroxidase as preferentially expressed in the abscission zone . Poovaiah and Rasmussen [16] also identified specific anionic isoenzymes that were induced (or decreased) in the abscission zone and compared these with changes 2 cm below the zone. The results presented here show that, in common with observations made by other workers, the concentration of several isoenzymes in the abscission zone does increase with ethylene-induced cell separation, but many of these changes also occur in neighbouring
570
M. T. MCMANUS
Table 1 . Peroxidase activity in various tissue extracts from bean leaf abscission explants at the time of excision (0 hr, A) and after incubation for 36 hr in ethylene (B)
Time of assay (hr) A
0 hr
B
36 hr
Specific activity (nkat mg - ' protein)
Extract
Enzyme unit (nkat)
Soluble Pulvinus Abscission zone Petiole
0.19±0.005* 0.15±0 .005 0.09±0 .002
0.31±0.070 0.43±0 .100 0.03±0.003
lonically bound Pulvinus Abscission zone Petiole
0.41+0 .005 0.22+0 .003 0.04±0 .001
3.96+0.040 2.47+_0.036 0.91 ±0.017
Soluble Pulvinus Abscission zone Petiole
0.17+0 .003 0.63+0 .020 0.23±0 .004
1 .24±0 .009 1 .52±0.042 0.78±0.013
lonically bound Pulvinus Abscission zone Petiole
0.19±0 .011 1 .42±0 .100 0.15±0 .010
5.67±0.298 15.12±0 .622 3.65+0.244
Soluble Pulvinus Abscission zone Petiole
0.18±0 .005 0 .20±0 .007 0.12+0 .005
0.30±0.050 0.60±0.040 0.10±0.005
lonically bound Pulvinus Abscission zone Petiole
0.35±0 .055 0.40±0 .070 0.08 ±0.005
3.70±0.050 3 .50±0.120 1 .42 ±0.060
*Values are+S .E.M. Peroxidase activity was determined using constant substrate concentrations of 25 mM guaiacol and I mM H202 in 50 mM phosphate buffer, pH 6 at 25°, with the accumulation of tetraguaiacol monitored at 470 nm . Table 2 . K,. and V., for the substrate guaiacol of peroxidase extracted from the abscission zone at the time of explant excision (0 hr) and at cell separation (36 hr)
These observations, therefore, suggest which isoenzymes, of those induced during separation of the pulvinus, may have either a more direct involvement in the cell separation process, or in post-abscission events .
Time of assay (hr) Ohr 36 hr
The two anionic isoenzymes identified here could be Extract
K,,, (mM)
V., (nkat)
Soluble lonically bound Soluble lonically bound
8.75+ _ 0.65* 8.60+1 .20 7.20± 1 .10 7.30±2 .10
0.23+_0.024 0.24±0 .025 1 .05±0 .075 1 .85±0 .115
involved simply in post-abscission lignification and suberization reactions, or may have a more direct role (e .g . the detoxification of lipid peroxides) which may result from modifications to cell membranes during cell separation . The observed changes in the concentration of specific
*Values are + S.E.M. and V., determinations for the substrate guaiacol were performed at 25' in the presence of a constant concentration of 1 mM H 2 0 2 in 50 mM phosphate buffer, pH 6, with the accumulation of tetraguaiacol monitored at 470 nm .
K„
isoenzymes in distal and proximal tissues may also be significant . In this regard, the major staining cationic iscenzyme in the pulvinus may be important . As an IAA oxidase, it can be part of a mechanism by which the concentration of endogenous auxin is reduced in tissue distal to the zone, so enhancing the stimulatory effect of ethylene .
tissues . Amongst these, only two anionic isoenzymes have
In terms of total peroxidase activity, increases were
been identified as being induced preferentially in the
most pronounced in the abscission zone . This is evident
abscission zone, and one (designated A7, Fig . 2B) is also
especially in the ionically bound fraction indicating that
evident on the pulvinus surface immediately adjacent to
of those proteins present in this fraction, peroxidase is a
the abscission zone (suggesting that it is secreted) .
major component .
Peroxidase in bean leaf abscission These data also support the concept that during cell separation the activity of peroxidase (and certain isoenzymes in particular) is induced, in part by an increase in activity (cf. total enzyme units), but also by a change in the concentration of other proteins (cf . specific activity) . There is evidence to support this on consideration of the kinetic induction of activity in the abscission zone . Here, a significant increase in V.., was observed, while the K,,, for guaiacol did not alter . These observations are consistent with the view that the induction involves an increase in total activity (mediated through two specific isoenzymes in particular) and not by the induction of new or existing isoenzymes with altered substrate affinity . In sum, the approach used in this study-to identify, for the first time, isoenzyme changes specific to the abscission zone (by comparing changes with those observed in distal and proximal tissue)-highlights which are the key isoenzymes to purify and investigate further in order to elucidate the role of this enzyme either directly in the cell separation process, or in regulating post-abscission events. EXPERIMENTAL
Plant material . Phaseolus vulgaris var . Masterpiece (Asmer Seeds Ltd, Leicester, U.K.) was grown in a glasshouse in Levingtons Universal compost (Fison, Suffolk, U.K .). The plants were illuminated with 400 Watt mercury vapour lamps to maintain a minimum day length of 14 hr, and supplementary heating was provided to ensure a minimum temp . of 15° . The first leaf pair was harvested for use in experiments when fully expanded (usually 12-15 days) . Preparation and extraction of plant tissue . To extract tissue at 0 hr, sections were excised as 2-mm-thick slices from the middle of the distal pulvinus, while petiole sections were excised, again as 2-mm-thick slices, ca 3 mm from the zone . Tissue containing predominantly abscission zone cells was obtained by nicking the junction between the distal pulvinus and the petiole, where after the application of gentle pressure, separation occurs and the zone can be sliced from the petiole side as a thin sheet of cells of (less than) 1 mm in thickness . To obtain ethylene-treated tissue, abscission explants were excised to include the distal leaf petiole :pulvinus abscission zone, the pulvinus and a 15 mm length of the petiole [30] . Explants were incubated in an atm . of 10 p11'' ethylene, or in air (with 0.4 M mercuric perchlorate), for 16 hr at 25°, and after brief aeration, incubated for a further 24 hr in ethylene or air, as appropriate. After this time the pulvinus had sepd from the petiole. To obtain ethylene-treated tissue, ca 2 mm-thick sections were excised from the middle of the senescent pulvinus and from a point along the petiole, 3 mm from the zone. The abscission zone was excised from the petiole as a thin (ca 1 mm thick) sheet of cells . Excised plant tissue (both 0 and 36 hr) was homogenized in H20 containing 10 mM 2-mercaptoethanol and 0.5 mM PMSF (I g fr . wt : 3 ml extraction soln) . Insoluble polyvinylpyrrolidone (PVPP) was added to give a final
571
concn of 2% (w/v) and the extraction continued for 15 min on ice . The slurry was centrifuged at 600 g for 15 min, the supernatant decanted (designated the soluble fr.), and the pellet washed with the same soln and centrifuged a further 12 times. After the final wash, the pellet was resuspended in 50 mM NaPi buffer, pH 7 .4, containing I M NaCl and extracted for I hr at 25° . After centrifugation at 600 g, the supernatant was decanted (designated the ionically bound fr.), the pellet re-extracted with the same buffer, centrifuged and the supernatant pooled with the ionically bound fr . Finally the pellet was resuspended in a soln of 3 M LiCl, extracted for 1 hr at 25°, centrifuged and the supernatant pooled with the ionically bound extracts . These soluble and ionically bound extracts were either : (i) pooled and dialysed against several changes of H .0 for 24 hr at 4°, frozen and lyophilized for isoenzyme determination using gel electrophoresis ; or (ii) dialysed separately against several changes of 50 mM NaPi buffer, pH 7 .4, for 18 hr, at 4° for enzyme assays . Peroxidase assay. Peroxidase activity was determined using guaiacol (Sigma, Poole, U.K .) as substrate. The appropriate extract was dialysed in 50 mM NaPi, pH 6 (assay buffer), the reaction initiated by the addition of 100-pl aliquots to 3 ml of assay buffer containing a final concn of 25 mM guaiacol and i mM H 2 02. Accumulation of tetraguaiacol was measured at 470 nm, where E470 nm=26.6 cm"' mM - ' [31] . To determine the K„ for guaiacol of peroxidase in abscission zone, a range of substrate conens from 1 to 25 mM was used, with H 20 2 used at a constant concn of I mM in each reaction . Isoenzyme determination. Peroxidase isoenzymes were resolved in 12 .5% polyacrylamide gels using the buffer systems described for basic isoenzymes [32], and for acidic isoenzymes [33] . Lyophilized tissue extracts were resuspended in H20 for 2 hr at 25°, centrifuged briefly, the supernatant made I % (v/v) with glycerol and ca 100-200,ug (total protein) loaded as appropriate . Gel electrophoresis using both buffer systems was performed at 25 mA for 4-6 hr and the sepd isoenzymes visualized with 0 .01% (w/v) 3', 3'-diaminobenzidine, 0.03% (v/v) H 2 0 2 in 50 mM Tris-HCI, pH 7 .5. To visualize isoenzymes sepd using the buffer system of ref. [32), preincubation in 50 mM Tris-HCI, pH 7 .5, for 10-15 min, at 25°, was necessary before the addition of substrate . Protein content in extracts was determined using the method of ref. [34] with bovine serum gamma globulin (BSGG) as standard . REFERENCES
1 . Gaspar, T ., Penel, C ., Thorpe, T . and Greppin, H . (1982) in Peroxidases, a Survey and their Biochemical and Physiological Roles in Higher Plants. University of Geneva Press, Geneva, Switzerland . 2. Van Huystee, R . B . (1986) A. Rev. Plant Physiol. 38, 205. 3. Mader, M . and Fussl, R . (1982) Plant Physiol . 70, 1132.
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4 . Goldberg, R ., Le, T. and Catesson, A.-M. (1985) J. Exp. Bot. 36, 503. 5. Imberty, A., Goldberg, R . and Catesson, A.-M . (1985) Planta 164, 221 . 6 . Lagrimini, L . M ., Burkhart, W ., Moyer, M . and Rothstein, S . (1987) Proc. Natn. Acad. Sci . U.S.A. 84, 7542. 7. Lamport, D. T. A. (1986) in Molecular Aspects of Plant Peroxidases (Greppin, H., Penel, C . and Gaspar, T ., eds), pp . 199-208 . University of Geneva Press, Geneva, Switzerland . 8. Espelie, K . E. and Kolattukudy, P . E. (1985) Arch. Biochem. Biophys. 240, 539. 9. Espelie, K . E., Franceschi, V . R . and Kolattukudy, P. E. (1986) Plant Physiol. 81, 487. 10. Mottley, C . and Mason, R . P. (1986) J . Biol. Chem . 261, 16860. 11 . Osswald, W. F., Schutz, W . and Elstner, E . F. (1988) Plant Physiol. 86, 1310. 12. Pressey, R . (1990) Plant Physiol. 93, 798 . 13. Metodiewa, D ., de Melo, M . P., Escobar, J . A., Cilento, G . and Dunford, H . B. (1992) Arch. Biochem. Biophys. 2%, 27. 14. Park, R . D. and Park, C. K. (1987) Plant Physiol. 84, 826. 15. Sexton, R. and Roberts, J . A. (1982) A . Rev . Plant Physiol. 33, 133 . 16. Poovaiah, B . W . and Rasmussen, H . P. (1973) Plant Physiol. 52, 263 . 17. Hall, J. L. and Sexton, R . (1974) Ann. Bot. 38, 855.
18. Webster, B. D., Dunlap, T . W. and Craig, M . E. (1976) Am. J. Bot . 63, 759. 19. Poovaiah, B. W ., Rasmussen, H. P. and Bukovac, M . J. (1973) J. Am. Soc . Hort. Sci. 98, 16. 20. Henry, E. W . and Jensen, T. E. (1973) J. Cell Sci. 13, 591 . 21 . Henry, E. W. (1975) Caryologia 28, 477 . 22. Henry, E. W. (1979) Cytologia 44, 135. 23. Henry, E . W ., Valdovinos, J . G . and Jensen, T. E. (1974) Plant Physiol. 54, 192. 24. Gaspar, T., Goren, R ., Huberman, M . and Dubucq, M . (1978) Plant Cell Environ . 1, 225 . 25. Morgan, P . W. and Fowler, J. L. (1972) Plant Cell Physiol. 13, 727 . 26. Imaseki, H . (1970) Plant Physiol. 46, 172. 27. Birecka, H ., Catalfamo, J . and Urban, P. (1976) Plant Physiol. 57, 74. 28. Abeles, F . B., Dunn, L . J., Morgens, P., Callahan, A ., Dinterman, R. E. and Schmidt, J. (1988) Plant Physiol. 87, 609. 29. del Campillo, E . and Lewis, L. N. (1992) Plant Physiol. 98, 955. 30. Wright, M . and Osborne, D. J. (1974) Planta 120, 163 . 31 . Chance, B . and Maehly, A. C. (1955) Meth. Enzymol . 2,764. 32. Davis, B . (1964) Ann. N. Y. Acad. Sci . 125, 404 . 33. Reisfeld, R . A., Lewis, U. J. and Williams, D . W. (1962) Nature 195, 281 . 34. Bradford, M . M . (1955) Analyt . Biochem . 72, 248.
Phytochem stry, Vol. 35, No. 3, pp. 567-572, 1994 Copynght Q 1994 Elsevier Science Ltd Printed in Great Britain . All rights reserved
0031-9422/94 $6 .00+0.00
PEROXIDASES IN THE SEPARATION ZONE DURING ETHYLENE-INDUCED BEAN LEAF ABSCISSION MICHAEL T . MCMANUS •
Department of Biochemistry, Royal Holloway and Bedford New College, University of London, Surrey TW20 OEX, U .K. (Received in revised form 22 June 1993)
Key Word Index-Phaseolus vulgaris; Leguminosae; leaf abscission; peroxidase isoenzymes .
Abstract-To determine if peroxidase activity is induced specifically in the abscission zone during ethylene-induced cell separation, the spectrum of isoenzymes and activity of the enzyme in extracts from the distal pulvinus, the pulvinus : petiole abscission zone and the petiole of Phaseolus vulgaris leaves were compared . For the cationic isoenzymes, the onset of ethylene-induced cell separation correlated with an increase in the expression of two isoenzymes that are present in both the pulvinus and the abscission zone . In terms of the anionic isoenzymes, two isoenzymes were induced at the time of ethylene-induced cell separation . Both of these are expressed preferentially in the zone, while another was induced predominantly in the petiole . No peroxidase isoenzymes could be detected that were induced specifically in the abscission zone at the time of pulvinus separation . When total enzyme activity was compared, ethylene treatment increased the measurable activity in all tissues, although the highest was at the zone at the time of cell separation . When specific activity was compared, these differences were not as marked. Together, these results show that while peroxidase activity is induced at the abscission zone during ethylene-induced cell separation, changes in specific isoenzymes are observed both in the zone and in neighbouring (non-abscission) tissue . These changes in the spectrum of isoenzymes observed in neighbouring tissue, therefore, should not be ignored when postulating a role for peroxidase in mediating cell-cell separation at the abscission zone .
INTRODUCTION
Peroxidases (donor: hydrogen peroxide oxidoreductase, EC 1 .11 .1 .7) have been implicated in the regulation of a number of biochemical and physiological processes in higher plants [1, 2]. Of these, the role of specific isoenzymes in regulating the terminal steps of lignification, or as indole-3-acetic acid (IAA) oxidases has received considerable attention . Anionic isoenzymes of peroxidase have been implicated in the terminal steps of lignification by mediating the polymerization of substituted cinnamyl alcohols [3-6] . These isoenzymes have also been implicated in the crosslinking of extension monomers and feruloylated polysaccharides in cell walls [7], and in wound healing through suberization [8, 9] . Cationic isoenzymes are implicated as IAA oxidases. While the molecular mechanism of IAA oxidation by horse-radish peroxidase, in vitro, has been established [10-13], the significance of IAA catabolism by peroxidase, in vivo, is still unclear, particularly since the enzyme can also oxidize IAA-amino acid conjugates [14]. A number of studies have now been published concerning the correlation of peroxidase activity with leaf abscis*Present address : Plant Molecular Genetics Laboratory, Grasslands Research Centre, AgResearch, Private Bag 11008, Palmerston North, New Zealand .
sion, although as yet, there is little consensus on the specific role of this enzyme in the mechanism of cell-cell separation [15] . Using histochemical staining procedures, intense activity of the enzyme has been localized in the wall and middle lamella region of cells at the separation layer in bean leaves [16-18], the lower (pedicel :fruit) zone in sweet cherry [19] and in tobacco flower pedicels [20-22] . As well as studies on the specific cellular localization of peroxidase activity at cell separation, changes in the isoenzyme profile of the enzyme at the onset of abscission in the zone have also been documented . In most tissues examined so far, only quantitative changes in the isoenzyme profile are observed [16, 23, 24], although qualitative changes have been recorded during leaf and cotyledon abscission in cotton [25]. In general though, these isoenzyme studies have only concentrated on changes specifically in the abscission zone in an attempt to link the enzyme with the cell separation process . However, it is known that ethylene can induce peroxidase activity in a number of other tissues, including the leaves and storage roots of sweet potato [26, 27], and in senescent cucumber cotyledons [28]. The identification of abscission zone-specific peroxidase isoenzymes is of interest because other ethylene-induced proteins (e.g. B-1,4-glucanase and chitinase) have been identified in bean leaf abscission zones, and their function in mediating cell--cell separation considered [29]. In order to implicate peroxidase in this process, a comparison of 567
568
M . T . MCMANUS
ethylene-induced isoenzymes in both the abscission zone and in neighbouring tissue is necessary . In this study, the profile of peroxidase isoenzymes has been monitored during ethylene-induced cell separation at the distal leaf pulvinus :petiole abscission zone in Phaseolus vulgaris L., to determine whether there are any that are abscission cell-specific . Further, the induction of enzyme activity in these tissues has also been investigated, in order to determine if any increase in total peroxidase activity during abscission is confined precisely to the zone. RESULTS
Changes in the spectrum of peroxidase isoenzymes during ethylene-induced cell separation
Cell extracts from the pulvinus, abscission zone and petiole were prepared at the time of explant excision (0 hr) and after 36 hr in ethylene (the time of pulvinus separation). Using diaminobenzidine as substrate, the peroxidase isoenzyme profile was determined for each extract . On separation of the cationic isoenzymes, no qualitative differences could be discerned between the extracts at the time of excision (Fig. IA) and at pulvinus separation (Fig. 1B). However, the concentration of two of the prevalent isoenzymes (C1, C2) increased with the onset of cell separation in both the pulvinus and the abscission zone . With separation of anionic isoenzymes, several changes in the staining intensity of the existing isoenzymes could be identified with the onset of cell separation (Fig . 2). Of these, two isoenzymes in particular, A3 and A7 (Fig . 2B, lane f), have increased in concentration, most notably in
the abscission zone, although A3 has increased to a lesser degree in the pulvinus (Fig . 2B, lane d). Isoenzyme A7 can also be detected on the pulvinus surface immediately adjacent to the abscission zone, suggesting that it is secreted (Fig . 2B, lane e). One isoenzyme, A5, has increased in the petiole (Fig . 2B, lane g) during the onset of cell separation, while isoenzyme A l has decreased in intensity in the pulvinus after 36 hr in ethylene (cf. Fig. 2, lane a with d) . In common with changes observed in the cationic isoenzyme profile during cell separation, no qualitative changes in anionic iscenzymes were observed (A7 is present in 0 hr zone, albeit at very low concentrations) . Overall, these results indicate that during the onset of ethylene-induced cell separation at the distal pulvinus :petiole abscission zone in P. vulgaris, the use of diaminobenzidine as substrate does reveal that quantitative changes in the peroxidase isoenzyme profile occur, but these are not restricted solely to the zone. Changes in peroxidase activity during ethylene-induced cell separation
Using guaiacol (o-methoxyphenol) as substrate, total and specific peroxidase activity in the abscission zone and surrounding tissue was measured at 0 hr and after ethylene treatment to induce pulvinus separation (36 hr) . For these experiments, it was convenient to divide each tissue extract into two fractions, a water-soluble fraction and an ionically bound (high salt-soluble) cell wall fraction . In terms of total activity (expressed as enzyme units), an increase was observed with the onset of cell separation in the soluble and ionically bound fractions from all three tissues. However, activity was highest in both abscission
C1
C2
Fig. 1. Separation of cationic isoenzymes. Extracts were subjected to polyacrylamide gel electrophoresis using the method of Reisfeld, Lewis and Williams [33] and the separated peroxidase isoenzymes visualized using 3',3'-diaminobenzidine . A. Extracts from tissues at 0 hr, a = leaf pulvinus, b = abscission zone, c = petiole . B . Extracts from tissue after 36 hr in ethylene ; d = leaf pulvinus, e = abscission zone, 1= petiole .
Peroxidase in bean leaf abscission
569
a b c
-A1 -A2 -A3 - A4 -A -A - A7 - A8 Fig. 2 . Separation of anionic isoenzymes . Extracts were subjected to polyacrylamide gel electrophoresis using the method of Davis [32] . Visualization of separated peroxidase isoenzymes was achieved using 3',3'-diaminobenzidine . A. Extracts from tissues at 0 hr, a =leaf pulvinus, b = abscission zone, c = petiole . B. Extracts from tissue after 36 hr in ethylene ; d = leaf pulvinus, a=leaf pulvinus surface adjacent to the abscission zone, f= abscission zone, g = petiole .
zone fractions when compared with neighbouring tissue (Table 1) . Of the two abscission fractions, activity is greater in the ionically bound preparation . In terms of specific activity, the highest activity in any tissue at the time of cell separation (36 hr) was again detected in the abscission zone. This occurs both in the soluble and ionically bound fractions, although the difference between the abscission zone and neighbouring tissue is now not as marked as with comparisons made for total activity . This is particularly so for the soluble fraction, and presumably reflects an increase in the relative protein content of the abscission zone after ethylene treatment, particularly with the induction of cell wall hydrolases . These results confirm that during ethylene-induced cell separation, peroxidase activity is highest in the abscission zone, but the enzyme is also induced in neighbouring tissues. Kinetic parameters of peroxidase induction
The increase in enzyme activity in the abscission zone with the onset of cell separation was investigated in terms of two kinetic parameters, V., and K . for the substrate guaiacol (Table 2) . The induction of enzyme activity is accompanied by the significant increase in VV for the PHYTO 35 :3-C
enzyme at 36 hr in both fractions, when compared with 0 hr. However, the K . for guaiacol has not decreased significantly over this time-course . These results suggest that for this substrate at least, the observed increase in peroxidase activity at the abscission zone is mediated essentially through an overall increase in enzyme synthesis and not through any significant change in the specificity for the substrate . DISCUSSION
This study has sought to identify isoenzymes of peroxidase which are induced specifically in the distal pulvinus :petiole abscission zone of bean leaves at the onset of ethylene-induced cell separation. Cytochemical staining [16, 17] has identified peroxidase as preferentially expressed in the abscission zone . Poovaiah and Rasmussen [16] also identified specific anionic isoenzymes that were induced (or decreased) in the abscission zone and compared these with changes 2 cm below the zone. The results presented here show that, in common with observations made by other workers, the concentration of several isoenzymes in the abscission zone does increase with ethylene-induced cell separation, but many of these changes also occur in neighbouring
570
M. T. MCMANUS
Table 1 . Peroxidase activity in various tissue extracts from bean leaf abscission explants at the time of excision (0 hr, A) and after incubation for 36 hr in ethylene (B)
Time of assay (hr) A
0 hr
B
36 hr
Specific activity (nkat mg - ' protein)
Extract
Enzyme unit (nkat)
Soluble Pulvinus Abscission zone Petiole
0.19±0.005* 0.15±0 .005 0.09±0 .002
0.31±0.070 0.43±0 .100 0.03±0.003
lonically bound Pulvinus Abscission zone Petiole
0.41+0 .005 0.22+0 .003 0.04±0 .001
3.96+0.040 2.47+_0.036 0.91 ±0.017
Soluble Pulvinus Abscission zone Petiole
0.17+0 .003 0.63+0 .020 0.23±0 .004
1 .24±0 .009 1 .52±0.042 0.78±0.013
lonically bound Pulvinus Abscission zone Petiole
0.19±0 .011 1 .42±0 .100 0.15±0 .010
5.67±0.298 15.12±0 .622 3.65+0.244
Soluble Pulvinus Abscission zone Petiole
0.18±0 .005 0 .20±0 .007 0.12+0 .005
0.30±0.050 0.60±0.040 0.10±0.005
lonically bound Pulvinus Abscission zone Petiole
0.35±0 .055 0.40±0 .070 0.08 ±0.005
3.70±0.050 3 .50±0.120 1 .42 ±0.060
*Values are+S .E.M. Peroxidase activity was determined using constant substrate concentrations of 25 mM guaiacol and I mM H202 in 50 mM phosphate buffer, pH 6 at 25°, with the accumulation of tetraguaiacol monitored at 470 nm . Table 2 . K,. and V., for the substrate guaiacol of peroxidase extracted from the abscission zone at the time of explant excision (0 hr) and at cell separation (36 hr)
These observations, therefore, suggest which isoenzymes, of those induced during separation of the pulvinus, may have either a more direct involvement in the cell separation process, or in post-abscission events .
Time of assay (hr) Ohr 36 hr
The two anionic isoenzymes identified here could be Extract
K,,, (mM)
V., (nkat)
Soluble lonically bound Soluble lonically bound
8.75+ _ 0.65* 8.60+1 .20 7.20± 1 .10 7.30±2 .10
0.23+_0.024 0.24±0 .025 1 .05±0 .075 1 .85±0 .115
involved simply in post-abscission lignification and suberization reactions, or may have a more direct role (e .g . the detoxification of lipid peroxides) which may result from modifications to cell membranes during cell separation . The observed changes in the concentration of specific
*Values are + S.E.M. and V., determinations for the substrate guaiacol were performed at 25' in the presence of a constant concentration of 1 mM H 2 0 2 in 50 mM phosphate buffer, pH 6, with the accumulation of tetraguaiacol monitored at 470 nm .
K„
isoenzymes in distal and proximal tissues may also be significant . In this regard, the major staining cationic iscenzyme in the pulvinus may be important . As an IAA oxidase, it can be part of a mechanism by which the concentration of endogenous auxin is reduced in tissue distal to the zone, so enhancing the stimulatory effect of ethylene .
tissues . Amongst these, only two anionic isoenzymes have
In terms of total peroxidase activity, increases were
been identified as being induced preferentially in the
most pronounced in the abscission zone . This is evident
abscission zone, and one (designated A7, Fig . 2B) is also
especially in the ionically bound fraction indicating that
evident on the pulvinus surface immediately adjacent to
of those proteins present in this fraction, peroxidase is a
the abscission zone (suggesting that it is secreted) .
major component .
Peroxidase in bean leaf abscission These data also support the concept that during cell separation the activity of peroxidase (and certain isoenzymes in particular) is induced, in part by an increase in activity (cf. total enzyme units), but also by a change in the concentration of other proteins (cf . specific activity) . There is evidence to support this on consideration of the kinetic induction of activity in the abscission zone . Here, a significant increase in V.., was observed, while the K,,, for guaiacol did not alter . These observations are consistent with the view that the induction involves an increase in total activity (mediated through two specific isoenzymes in particular) and not by the induction of new or existing isoenzymes with altered substrate affinity . In sum, the approach used in this study-to identify, for the first time, isoenzyme changes specific to the abscission zone (by comparing changes with those observed in distal and proximal tissue)-highlights which are the key isoenzymes to purify and investigate further in order to elucidate the role of this enzyme either directly in the cell separation process, or in regulating post-abscission events. EXPERIMENTAL
Plant material . Phaseolus vulgaris var . Masterpiece (Asmer Seeds Ltd, Leicester, U.K.) was grown in a glasshouse in Levingtons Universal compost (Fison, Suffolk, U.K .). The plants were illuminated with 400 Watt mercury vapour lamps to maintain a minimum day length of 14 hr, and supplementary heating was provided to ensure a minimum temp . of 15° . The first leaf pair was harvested for use in experiments when fully expanded (usually 12-15 days) . Preparation and extraction of plant tissue . To extract tissue at 0 hr, sections were excised as 2-mm-thick slices from the middle of the distal pulvinus, while petiole sections were excised, again as 2-mm-thick slices, ca 3 mm from the zone . Tissue containing predominantly abscission zone cells was obtained by nicking the junction between the distal pulvinus and the petiole, where after the application of gentle pressure, separation occurs and the zone can be sliced from the petiole side as a thin sheet of cells of (less than) 1 mm in thickness . To obtain ethylene-treated tissue, abscission explants were excised to include the distal leaf petiole :pulvinus abscission zone, the pulvinus and a 15 mm length of the petiole [30] . Explants were incubated in an atm . of 10 p11'' ethylene, or in air (with 0.4 M mercuric perchlorate), for 16 hr at 25°, and after brief aeration, incubated for a further 24 hr in ethylene or air, as appropriate. After this time the pulvinus had sepd from the petiole. To obtain ethylene-treated tissue, ca 2 mm-thick sections were excised from the middle of the senescent pulvinus and from a point along the petiole, 3 mm from the zone. The abscission zone was excised from the petiole as a thin (ca 1 mm thick) sheet of cells . Excised plant tissue (both 0 and 36 hr) was homogenized in H20 containing 10 mM 2-mercaptoethanol and 0.5 mM PMSF (I g fr . wt : 3 ml extraction soln) . Insoluble polyvinylpyrrolidone (PVPP) was added to give a final
571
concn of 2% (w/v) and the extraction continued for 15 min on ice . The slurry was centrifuged at 600 g for 15 min, the supernatant decanted (designated the soluble fr.), and the pellet washed with the same soln and centrifuged a further 12 times. After the final wash, the pellet was resuspended in 50 mM NaPi buffer, pH 7 .4, containing I M NaCl and extracted for I hr at 25° . After centrifugation at 600 g, the supernatant was decanted (designated the ionically bound fr.), the pellet re-extracted with the same buffer, centrifuged and the supernatant pooled with the ionically bound fr . Finally the pellet was resuspended in a soln of 3 M LiCl, extracted for 1 hr at 25°, centrifuged and the supernatant pooled with the ionically bound extracts . These soluble and ionically bound extracts were either : (i) pooled and dialysed against several changes of H .0 for 24 hr at 4°, frozen and lyophilized for isoenzyme determination using gel electrophoresis ; or (ii) dialysed separately against several changes of 50 mM NaPi buffer, pH 7 .4, for 18 hr, at 4° for enzyme assays . Peroxidase assay. Peroxidase activity was determined using guaiacol (Sigma, Poole, U.K .) as substrate. The appropriate extract was dialysed in 50 mM NaPi, pH 6 (assay buffer), the reaction initiated by the addition of 100-pl aliquots to 3 ml of assay buffer containing a final concn of 25 mM guaiacol and i mM H 2 02. Accumulation of tetraguaiacol was measured at 470 nm, where E470 nm=26.6 cm"' mM - ' [31] . To determine the K„ for guaiacol of peroxidase in abscission zone, a range of substrate conens from 1 to 25 mM was used, with H 20 2 used at a constant concn of I mM in each reaction . Isoenzyme determination. Peroxidase isoenzymes were resolved in 12 .5% polyacrylamide gels using the buffer systems described for basic isoenzymes [32], and for acidic isoenzymes [33] . Lyophilized tissue extracts were resuspended in H20 for 2 hr at 25°, centrifuged briefly, the supernatant made I % (v/v) with glycerol and ca 100-200,ug (total protein) loaded as appropriate . Gel electrophoresis using both buffer systems was performed at 25 mA for 4-6 hr and the sepd isoenzymes visualized with 0 .01% (w/v) 3', 3'-diaminobenzidine, 0.03% (v/v) H 2 0 2 in 50 mM Tris-HCI, pH 7 .5. To visualize isoenzymes sepd using the buffer system of ref. [32), preincubation in 50 mM Tris-HCI, pH 7 .5, for 10-15 min, at 25°, was necessary before the addition of substrate . Protein content in extracts was determined using the method of ref. [34] with bovine serum gamma globulin (BSGG) as standard . REFERENCES
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