Planta

Planta (1986) 167:300-302

9 Springer-Verlag 1986

Short communication

Occurrence of diamine oxidase in the apoplast of pea epicotyls R. Federico and R. Angelini Dipartimento di Biologia Vegetale, Universitfi " L a Sapienza", P~~ Aldo Moro 5, 1-00185 Roma, Italia

Abstract. Most of the diamine oxidase (EC 1.4.3.6) present in pea (Pisum sativum L. cv. Rondo) epicotyls is found in the fluid obtained by centrifuging pea epicotyl sections previously infiltrated under vacuum with a buffer solution. No detectable amount of the cytoplasmic enzyme glucose-6-phosphate dehydrogenase is present in this fluid, showing that there is very little contamination by cell contents. Polyacrylamide-gel electrophoresis and specific-activity data indicate that diamine oxidase is the most plentiful protein in the extracellular solution obtained from pea epicotyl sections and that an active process is involved in the selective transfer of the enzyme outside the cell. The possible involvement of diamine oxidase in the supply of HzO2 to peroxidase-catalyzed reactions occurring inside the cell wall is discussed.

Key words: Diamine oxidase - Extracellular solution - Hydrogen peroxide production - Pisum (diamine oxidase).

Introduction

While much information is available on the localization of diamine oxidase (DAO) in animal tissues (Argento-Cerl] and Autuori 1984), data concerning the distribution of DAO in plant tissues and cells are at present very poor. Although DAO is reported to be mainly a soluble component of plant cells (Suzuki 1970), we have recently determined the presence of the enzyme in the cell walls of lentil seedlings by an immunohistochemical method (Federico et al. 1985 a). Abbreviations:

phosphate

DAO = diamine oxidase; Glc6P = glucose-6-

In addition, Kaur-Sawhney etal. (1981) reported that polyamine-oxidase activity appears to be localized in cell walls of oat leaves while Goldberg and Perdrizet (1984) observed that in differentiated cells of mung-bean hypocotyls, most of the polyamines substrates of amine oxidases were present in the cell wall. Terry and Bonner (1980) described a method for the isolation, from the apoplast of pea epicotyls, of a fluid containing considerable amounts of the soluble components of the cell wall with little cytoplasmic contamination. In this paper we report an investigation of the presence of DAO activity in the fluid removed from cell walls of pea epicotyl sections by this centrifugation technique. Material and methods o-Aminobenzaldehyde, 1,4-diaminobutane and N A D P + were purchased from Fluka, Buchs, Switzerland and glucose-6-phosphate (Glc6P) from Sigma, St. Louis, Mo., USA. All chemicals were of analytical grade. Seeds of Pisum sativum L. cv. Rondo were soaked for 2 h in aerated tap water and then grown in moistened vermiculite for 7 d in the dark at 27 ~ C. Epicotyl sections (2 cm long) were cut 0.5 cm below the apical hook in the dark using specially designed harvesting blades and placed in distilled water for 1 h at room temperature. All subsequent operations were performed with normal room illumination. Extracellular solutions were obtained essentially according to the method of Terry and Bonner (1980). Briefly, following the 1-h incubation, the sections were packed vertically in the barrel of a 20-ml plastic syringe and washed thoroughly with cold distilled water using a circulating pump. The packed epicotyl sections were successively infiltrated with the appropriate buffer under vacuum for 5 rain and then centrifuged at 1000 g for 7 min. The extraction procedure was repeated up to four times and the fluids obtained were used for enzymic tests and polyacrylamide-gel electrophoresis. The activity of DAO in the extracellular solutions and crude homogenate was determined according to the method of Holmsted et al. (1961) as previously described (Federico et al. 1985b). For the determination of Glc6P-dehydrogenase activity the method used was essentially that of Kornberg and Horecker

R. Federico and R. Angelini: Diamine oxidase in the apoplast of pea epicotyls (1955). Proteins were determined by the method of Lowry et al. (1951). Electrophoresis under non-denaturing condition was performed according to the method of Gabriel (1971); protein bands with DAO activity were detected by staining the gels in 0.1 M potassium phosphate, pH 7, containing 100 gg peroxidase, 1 mg benzidine and 17 mM putrescine (1,4-diaminobutane).

Table 1. Activity of DAO in pea epicotyl sections, extracellular fluid fractions and residual material. Pea epicotyl sections (70) were infiltrated with 10 mM potassium phosphate, pH 7, and extracellular-fluid fractions were obtained as described in Material and methods. Activity of Glc6P dehydrogenase in extracellular solutions was undetectable

Results and discussion

Table 1 shows DAO activity and protein content in crude homogenate, extracellular-fluid fractions and residual material obtained from pea epicotyl sections. The lack of Glc6P-dehydrogenase activity in the extracellular fluid demonstrates the absence of any contamination by the cytoplasmic contents of cells. Extracellular-fluid fractions contained less than 1% of the total protein and about 80% of the DAO activity present in the epicotyl sections. It is worth noting that the specific activity of DAO in the extracellular solution is very high and its value is comparable to that of the pure protein. Polyacrylamide-gel-electrophoresis data confirm these findings and only one main band was detected whether the gel was stained for protein or DAO activity. The results reported show that DAO is the main protein of the soluble components of the cell wall of pea epicotyls obtainable with the described extraction method. The v e r y high level of DAO in the extracellular fluid implies the action of a mechanism for the selective export of the protein, as was also reported for a soluble peroxidase which has been found in the intercellular fluid of tobacco leaves (Rathmell and Sequeira 1974). In order to obtain better insight into the interaction of DAO with the cell wall, we extracted the extracellular fluid with buffers at different ionic strengths. The enzyme seems to be loosely bound to the cell wall as it could be extracted even with low-ionic-strength buffer (10 mM potassium phosphate, pH 7). Increasing the salt concentration in the infiltration media did not affect the release of the protein. Conversely, a strong positive dependence of buffer ionic strength on the release of peroxidase activity was found (data not shown). Similar overall results were obtained with lentil seedlings. The reported occurrence of DAO in the apoplast of Leguminosae seedlings and previous findings on polyamine-oxidase localization in the cell wall of Graminae (Kaur-Sawhney et al. 1981) indicate that the occurrence of amino oxidases in the apoplast of fully expanded cells could be a general phenomenom and raise the question concerning the possible role of these enzymes within the cell wall. Extracellular amino oxidases could

301

Pea epicotyls

Protein Protein (rag. ml- 1) (mg. g- 1 FW)

DAO activity (U. g- 1 FW)

10

5.99

Extracellular fluid : Fraction I 0.16 Fraction II 0.14 Fraction III 0.12 Fraction IV 0.08 Residual material

9,95

20 0.032 0.028 0.024 0.016 19.9

1.3 1.2 1.1 0.7 1.2

DAO specific activity (U (mg protein)- 1) 0.30 30 31 31 34 0.06

easily have a role in regulating the level of polycationic polyamines in the apoplast where these compounds can influence the flux of important cations like Ca 2+ and K + (Kaur-Sawhney etal. 1981). Moreover, we suggest that catabolic products of polyamines oxidation, namely aminoaldehydes and H202, might have a special role within the cell wall. Both can act in protecting plants from pathogen infections and, furthermore, HzO2 is needed in several peroxidase-catalyzed reactions involving the peroxidatic coupling of extensin and lignin subunits. As far as H202 production is concerned, a very interesting theory has been suggested in which NADH-driven reversal of the general peroxidase reaction is supposed to provide the required hydrogen peroxide for ligniflcation and wall stiffening (Groos et al. 1977). According to this theory a possible role for a cell-wall-bound malate dehydrogenase has been identified in providing NADH for the reduction of Oz to H202. Although malate dehydrogenase can be detected in the cell wall compartment and the secretion of malate by the cell is well established, it is not clear if NAD § can actually be present within the cell wall (McNeil et al. 1984). The available evidence indicates that amino oxidases present in the apoplast may have a physiological role within the cell wall and can account for the H202 needed in the peroxidatic coupling of lignin subunits and in wall stiffening, at least in the species examined. The marked regulation of DAO activity by endogenous and environmental factors like hormones and light (Federico et al. 1985 b; Machohln and Minfi~ 1974) are likely to be related to this important function.

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R. Federico and R. Angelini: Diamine oxidase in the apoplast of pea epicotyls

Research work supported by CNR, Italy. Special grant I.P.R.A. - Sub-project 1 Paper N. 638.

References Argento-CertS, M.P., Autuori, F. (1984) Localization of diamine oxidase in animal tissues. In: Structure and function of amine oxidase, Mondovi, B., ed. C.R.C. Press, Boca Raton (in press) Federico, R., Angelini, R., Argento-Cerfi, M.P., Manes, F. (1985a) Immunohistochemical demonstration of lentil diamine oxidase. Cellul. Mol. Biol. 31, 171-174 Federico, R., Angelini, R., Cesta, A., Pini, C. (1985b) Determination of diamine oxidase in lentil seedlings by enzymic activity and immunoreactivity. Plant Physiol. 79, 62-64 Gabriel, O. (1971) Analitical disc gel electrophoresis. Methods Enzymol. 22, 565-578 Goldberg, R., Perdrizet, E. (1984) Ratio of free to bound polyamines during maturation in mung-bean hypoeotyl cells. Planta 161, 531-535 Gross, G.C., Janse, C., Elstner, E.F. (1977) Involvement of malate, monophenols and the superoxide radical in hydrogen peroxide formation by isolated cell walls from horseradish (Armoraeia lapathifolia Gilib.). Planta 136, 271-276 Holmsted, B., Larsson, L., Tham, R. (1961) Further studies on spectrophotometric method for the determination of amine oxidase activity. Biochim. Biophys. Acta 48, 182-186

Kaur-Sawhney, R., Flores, H.E., Galston, A.W. (1981) Polyamine oxidase in oat leaves: a cell wall-localized enzyme. Plant Physiol. 68, 494-498 Kornberg, A., Horecker, B.L. (1955) Glucose 6-phosphate dehydrogenase. Methods Enzymol. 1,323-327 Lowry, O.H., Rosebrough, N.J., Farr, L., Randall, R.J. (1951) Protein measurement with the Folin Phenol reagent. J. Biol. Chem. 193, 265-275 Machol~n, L., Minfif', J. (1974) The depression of pea diamine oxidase due to light and the verification of its participation in growth processes using competitive inhibitors. Biol. Plant. 16, 86-93 McNeil, M., Darvill, A.G., Fry, S.C., Albersheim, P. (1984) Structure and function of the primary cell walls of plants. Annu. Rev. Biochem. 53, 625-663 Rathmell, W.G., Sequeira, L. (1974) Soluble peroxidase in fluid from the intercellular spaces of tobacco leaves. Plant Physiol. 53, 317-318 Suzuki, Y. (1970) Intracellular distribution of amine oxidase in pea seedlings. Naturwissenschaften 57, 94 Terry, M.E., Bonner, B.A. (1980) An examination of centrifugation as a method of extracting an extracellular solution from peas, and its use for the study of indolacetic acidinduced growth. Plant Physiol. 66, 321-325

Received 3 August; accepted 9 September 1985

Occurrence of diamine oxidase in the apoplast of pea epicotyls.

Most of the diamine oxidase (EC 1.4.3.6) present in pea (Pisum sativum L. cv. Rondo) epicotyls is found in the fluid obtained by centrifuging pea epic...
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