an examination of the peroxidases from lupinus albus l. hypocotyls

Planta (1994)194:311-317 P l a ~ t ~

�9 Springer-Verlag 1994

An examination of the peroxidases from Lupinus albus L. hypocotyls Philip Jackson 1, C~ndido P.P. Ricardo m

11nstituto de Tecnologia, Quimica e Biol6gica, Apartado 127, 2780 Oeiras, Portugal 2 Departamento de Bot~nica e Engenharia Biol6gica, Instituto Superior de Agronomia, 1399 Lisboa Codex, Portugal

Received: 2 July 1993 / Accepted: 9 March 1994

Abstract. Peroxidases (EC 1.11.1.7) from hypocotyls of Lupinus albus L. cv. Rio Maior have been characterised using one- and two-dimensional, native electrophoretic techniques. Data are presented showing the complexity in charge and molecular size or shape of these peroxidases. We report the finding of a new acidic peroxidase and several new basic peroxidases in these hypocotyls, and of their stability to treatments considered to break ligand- induced variants and conformational variants derived from differences in polypeptide folding. Densitometric data demonstrate that these new peroxidases contribute up to 60% of the total peroxidase activity in hypocotyls. Studies of intercellular fluid, cell-wall and soluble fractions, with assays of purity were conducted in an attempt to define the subcellular locations of these additional peroxidases. The acidic form (pI 4.1) is greatly enriched in soluble fractions, three of the basic peroxidases (pls 9.5, 9.7 and > 9.7) are strongly associated to the cell wall, ad a minor, basic component (pI 9.7) is enriched in the intercellular fluid. Individual peroxidase activities with the substrates coniferyl alcohol, ferulic acid or indole acetic acid were compared by densitometric analysis of zymograms with those for guaiacol, and notable differences between these peroxidases in their capacity to oxidise indole acetic acid in vitro were identified. The possible functions of these peroxidases in vivo and their implica- tions to current understanding of peroxidases in L. albus are discussed.

Key words: Cell wall - Conformer (peroxidase) - Indole- 3-acetic acid (oxidases) - Leguminosae - Lupinus (hypocotyl) - Peroxidase (acidic, basic)

Abbreviations: APAGE = anionic polyacrylamide gel electrophoresis; CA = coniferyl alcohol; CPAGE = cationic polyacrylamide gel electrophoresis; IEF = isoelectric focusin; NEIEF = non-equilibrated isoelectric focusing; 2D=two dimensional; pI=isoelectric point; RCPAGE=reversed current polyacrylamide gel electrophoresis

Correspondence to: P. Jackson; FAX: 351(1)4428766

Introduction

Peroxidases (EC 1.11.1.7.) are ubiquitous in plants, and perform important biochemical functions such as lignification (Milder 1980), suberisation (Espelie et al..1986), cell-wall rigidification through formation of diphenolic cross-links between pectin (Fry 1979, 1982a, b) or ex- tensin (Fry 1986), and IAA oxidation (Mato and Vietez 1986). These enzymes have been repeatedly suggested to play important roles in differentiation (Mato and Vietez 1986; Mato et al. 1988; Joersbso et al. 1989; Rao et al. 1990; Cordewener et al. 1991).

One task facing investigators in the field of peroxidases is defining the reasons for the extreme diversity of these enzymes. Within the plant kingdom, the peroxidase families are numerous, each supposedly performing a different biological function, although the precise function in vivo is often unclear (Welinder 1991). In many of the plants studied to date, peroxidase is present in multiple forms. Some of these, such as in horseradish, show suffi- cient sequence dissimilarity to be classified as members of separate families (Welinder 1991). Other peroxidases have been shown to be distinct molecular species with high sequence similarities. Variation can also arise within a single plant, however, through heterogeneity in post- translational modifications such as glycosylation or C- terminal processing (Welinder 1991). The significance of these slighter differences to biological function, if any, is unknown (Welinder 1991).

With this large natural diversity among peroxidases, it is essential that in the analysis of their heterogeneity in any plant material, distinction of individual isoperoxidases be based on differences in their molecular make-up. The native systems utilised for preservation of biological activity are especially prone to charge variants, either ligand-induced or "true" conformational variation (Ep- stein and Shechter 1968), giving rise to a banding complexity which is not a true measure of heterogeneity. These "conformers" must be recognised before any valid examination can be made.

312 P. Jackson and C.P.P. Ricardo: Peroxidases from Lupinus hypocotyls

Helpful in fo rmat ion can also be produced by the iden- t ification of the subcel lular locat ion of indiv idual peroxidases, since growing knowledge of peroxidat ic act ion in situ can provide suggest ions as to the par t icular funct ion of the perioxidase in vivo. Suppor t for that biological funct ion can also be ob ta ined from the caut ious interpre- ta t ion of peroxidase activity in zymograms with a variety of substrates.

Fol lowing our recent repor t of peroxidases in Lupinus albus roots (Jackson and Ricardo 1992) our interest tu rned to the further character isa t ion of peroxidases in hypocotyl sections. For this purpose we have utilised cat ionic polyacrylamide gels (CPAGE), C P A G E with reversed current (RCPAGE) , isoelectric focusing (IEF), non -equ i l i b r ium isoelectric focusing (NEIEF) , C P A G E in con junc t ion with I E F (2-D-CPAGE) , an ionic P A G E (APAGE) , and A P A G E in con junc t ion with IEF (2D- APAGE) .

The part ial charac ter i sa t ion of L. albus hypocotyl peroxidases has been reported by Barcel6 et al. (1987, 1989a) using similar mater ia l and condi t ions. F rom their careful studies it was conc luded that the peroxidase activity in hypocotyls is due to three subsets of isoenzymes: an acid pair, A~ and A2 which are ionical ly associated to the cell wall and in the intercel lular fluid (Barcel6 et al. 1988), a cytosolic basic pair, B 1 and B 2 (Barcel6 et al. 1989a), and a further basic group, B 3 and B 4 which are ionically associated to tonoplas t ic m e m b r a n e s in young seedlings (Barcel6 et al. 199 !). We here report the presence of other peroxidases in L. albus hypocotyls and present results that suggest that these add i t iona l isoperoxidases are not conformat iona l isomers. We also investigate the cellular locat ion of these peroxidases and discuss their possible funct ion in vivo.

Materials and methods

Plant material and extraction procedures. Seeds of Lupinus albus L. cv. Rio Major were obtained from the Departmento de Botfinica e Engenharia Biol6gica, Instituto Superior de Agronomia, Lisbon, Portugal. Hypocotyls were taken from 10-d-old plantlets grown in sand under a photoperiod of 16/8 h of light/dark (160 gmol quan- ta.m 2 s ~), and watered every second day.

Tissues were homogenised in 50 mM Tris-HCl (pH 7.2), containing 5 mM phenylmethylsulfanyl fluoride. The homogenate was filtered through a 200-gm net and centrifuged at 4500.g to yield a soluble fraction and a crude cell-wall fraction. The soluble fraction was then clarified by centrifugation at 120000.g. The crude cell- wall fraction was resuspended in 1% Triton X-100 before centrifugation at 4500.g followed by washing of the pellet three times in 50 mM Tris-HCl buffer (pH 7.2). The cell-wall preparation was then extracted with l M KC1 for 10 min. Cell-wall debris was removed by centrifugation at 4500-g and the supernatant desalted on PD-10 columns (Pharmacia, Uppsala, Sweden) equilibrated in 50 mM Tris- HCI (pH 7.2). Both soluble and cell-wall extracts were concentrated in Diaflow| using PY10 membranes (Amicon, Danvers, Ma., USA).

Extraction of intercellular fluid by vacuum-infiltration was performed as reported by others (Barcel6 et al. 1989b). Assays of purity of the cell-wall fraction and intercellular fluid employed the cytosolic marker enzyme glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as described by Douce et al. (1987). Guaiacol oxidase (EC 1.11.1.7) was assayed as before (Jackson and Ricardo 1992) except that 6 A47 o was measured instead of 6 A450. Conversion of A4v o to kats was achieved by defining 1 nkat as the amount of protein that produces 1 nmol-s l of product and using a e.47o = 26.6.103. M- ~. cm- ~ for tetraguaiacol (Ferrer et al. 1991 a).

Electrophoresis. Cationic PAGE (CPAGE) was performed as previously reported (Jackson and Ricardo 1992). Reverse current PAGE (RCPAGE) was performed as for CPAGE except that the poles were reversed. Anionic PAGE (APAGE) was performed with 1.5 mm, 7.5% T, 3.0% C gels buffered with 0.1 M glycine adjusted to pH 8.9 with Tris, containing 3.8 mM NAN3, and solidified with 600 gl of 10% ammonium persulphate and 120 gl N,N,N',N' te- tramethylethylene diamine (TEMED; BioRad, Hercules, Calif., USA) per 100 ml of gel solution. The parameters utilised for electrophoresis were a constant current of 50 mA over 10 h, with cooling at 4 ~ C. Isoelectric focusing (IEF) was performed horizontally in 2-mm-thick polyacrylamide gels (5% T, 3% C), containing 5% 3.5- 10.0 ampholites, or where more basic ranges were required, 2.5% 6 9 and 2.5% 9-11 ampholites. Gels were solidified with 170gl 10% ammonium persulphate and 25 pl TEMED per 60 ml gel solution. Focusing was achieved after 1.5 h withthe constant power of 1 W.cm 1 width of gel, and on a cooling plate at 4 ~ C. The non- equilibrated IEF (NEIEF) was performed as for IEF with the time reduced to 1.2 h. Two-dimensional (2D)-electrophoresis was performed by the combination of IEF or NEIEF as the first dimension, with CPAGE or APAGE as the second. First-dimension strips of 0.5 cm in width were incubated for 10 min with 10% (v/v) glycerol in the appropriate electrophoresis buffers before embedding in 5% T, 3% C polyacrylamide. Where 2D-CPAGE was performed, gels were overlaid with 1 mg.ml I cytochrome c in 5% (v/v) glycerol.

Zymograms were developed using either guaiacol, coniferyl alcohol, ferulic acid (Barcel6 et al. 1987) or IAA (Gove and Hoyle 1975) as substrates.

Densitometry. Gels were photographed using Polaroid 665 film and one-dimensional densitometry was performed on the negatives. Variation in the signal caused by constriction or spreading of the sample lanes was compensated for by measurement of the lane width and subsequent normalisation of th signal strength. Baselines were constructed from traces obtained from empty sample wells. In addition, where necessary, significant valley-to-valley baselines were also utilised. Isolated peaks or groups were quantified by their total area. Overlapping peaks within groups were quantified by the division of group areas into their symmetrical Gaussian components. Each sample lane was scanned three times and the traces averaged after component analysis. The values presented here are the average of the analysis of at least four different samples. Conver- sion of values obtained from densitometric analysis to gkat.g FW. was achieved by the appropriate division of the previously measured, total activities of each fraction.

Results

The diversity of peroxidases in L. albus. Compar i son of peroxidase actitivies from the fractions studied (Table 1) demons t ra ted that in L. albus, as in m a n y of the plants studied to date, the major i ty of peroxidase activity is ionically ligated to the cell wall (Barcel6 et al. 1987). After exhaustive extract ion with saline buffer, the digest ion of cell walls with commercial prepara t ions of cellulase and pectinase released insignificant addi t iona l activity. Init ial zymograms produced from I E F gels (3.5 10.0) showed the presence of an acidic and a basic group of peroxidases in bo th the cell-wall and cytosolic fractions (Fig. 1 A). In order to search for addi t ional peroxidases in hypocotyls, several electrophoretic systems were used. For acidic peroxidases, R C P A G E and A P A G E with a variety of time and current settings were utilized. No further acidic peroxidases other than those seen in I E F gels were found in either the cell-wall or soluble fractions. For basic peroxidases however, I EF gels (7.0-10.0) part ial ly resolved the highly basic group of the cell walls and revealed an addi-

P. Jackson and C.P.P. Ricardo: Peroxidases from Lupinus hypocotyls 313

Table 1. Peroxidases of the cell-wail and cytosolic fractions of L. albus hypocotyls (see Fig. 1) and their contributions to the total peroxidase activity

Peroxidase pI Activity (nkat. g- 1 FW)

Cell Wall B8 >9.7 12,5_+26.3 B7 9.7 0.2_+ 3.1 B6 9.7 8.2+ 6.9 B5 9.3 2,5 _ 18.9 A2 5.2 10.9 + 17.4 A2 4.7 4.6_+ 27.0

Total 38.9

Cytosol B2 8.8 6.6 + 2.6 B1 8.3 4.3 _+ 12.8 A0 4.2 4.9_+11.1

Total - 15.8

Fig. 1 A, B. Isoelectric focusing (A) and NEIEF (B) of cell-wall and soluble peroxidases. Peroxidase activity (6.3 nkat) was applied to all sample lanes in both types of electrophoresis. B*, Unresolved basic peroxidases. The label for B7 marks the position of this faint band invisible on the photograph; ew, cell-wall peroxidases; s, soluble peroxidases; +, anode; - , cathode

tional basic peroxidase in the soluble fraction. These gels enabled the assignment of isoelectric points (pIs) to the alkaline soluble peroxidase and two of the basic peroxidases from the cell wall (Table 1). Better resolution was obtained using NEIEF with the run time reduced to 0.8 of that required to achieve equilibrium, by which method the basic group could be resolved into three distinct bands (Fig. I B). Densitometry of these zymograms demonstrated a higher activity of the cell-wall basic group as compared with that seen in equilibrium IEF, indicating that in the latter, some of the basic peroxidases were lost to the cathode as previously reported for other basic proteins (O'Farrel et al. 1977). This was also true for the minor basic peroxidase of the soluble fraction which could not be detected in equilibrium IEF. The N E I E F with even less time (0.5 and 0.7 of that required to achieve equilibrium), resolved no further peroxidases with higher mobilities in either the cell-wall or soluble fractions.

So, the majority of peroxidase activity in these hypocotyls is represented by a minor alkaline peroxidase and two acidic and two basic groups of distinct pI. The highly basic group of the cell wall, and both the acid group and the minor basic band of the soluble fraction, are apparently additional to those previously reported in L. albus hypocotyls by Barce6 and co-workers (Barcel6 et al. 1987, 1989a, 1991) who utilised etiolated seedlings of cv. Multolupa. However, using this cultivar and etiolated plantlets of cv. Rio Maior, we still find all the additional peroxidases. The zymograms produced were similar to that shown in Fig. 1 (data not shown).

The highly basic group in the cell well is composed of three peroxidases which comprise 47-64% of the total peroxidase activity in this fraction. Following the nomen- clature proposed by Barcel6 et al. (1987, 1989a, 1991), we term these peroxidases B 5 (pI 9.3), B 6 (pI 9.7) and B 8 (pI > 9.7) (Fig. 1). The minor basic peroxidase in the soluble fraction of pI 9.7 we refer to as B 7. The acidic group of the soluble fraction (Fig. 1 A) which composes 32% of the activity in this fraction, we refer to as A 0. The pI's of these peroxidases and their contributions to the overall peroxidase activity in hypocotyls are shown in Table 1.

Further heterogeneity resulting from differences in shape and/or size of these charge groups was searched for using 2D-CPAGE and 2D-APAGE. The relevant zymograms are shown in Figs. 2A-D. What was found was that A2, A 1 (Fig. 2A), B 6 (Fig. 2B) and 8 7 (Fig. 2C) resolved to single points, indicating that these peroxidases are most likely homogeneous in molecular charge and form. In the soluble fraction, B~ and B 2 resolved close together in both APAGE and CPAGE, so that it is not clear if these peroxidases are homogeneous. However, definite further heterogeneity was seen in Bs, Bs (Fig. 2 B) and Ao (Fig. 2D) which each resolved into two components in the second dimension, suggesting that these bands in IEF gels are composed of two peroxidases which differ in molecular size or form.

Artefacts in peroxidase banding. We subjected cell-wall and soluble fractions to methods reported to break or avoid conformational variation. Fractions were subjected to chromatography in Sephadex G-25 equilibrated with 2 M CaC12 (Barcel6 et al. 1987), or extracts were


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Fig. 3. Denaturation/renaturation of peroxidases by heat (70 ~ C). Solid circles, cell-wall-derived peroxidases; open squares, soluble peroxidases

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Fig. 2A-D. Two dimensional electrophoresis of cell-wall and soluble peroxidases. A 2D-APAGE and B 2D-CPAGE of cell-wall peroxidases. C 2D-CPAGE of soluble peroxidases. Arrow marks the position of By. D 2D-APAGE of soluble peroxidases. Arrow marks the position of A o. +, anode; - , cathode; m, marker lane of appropriate peroxidase sample applied with 6.3 nkat. All first di- mensions were prepared from NEIEF of peroxidase samples applied at 25.1 nkat peroxidase activity.cm-~ width of gel utilized

made with bovine serum albumin (BSA) at 1% (w/v) as a phenol-binding agent. Additionally, denaturation of these isoenzymes by heating to 70~ and renaturation by rapid cooling (0 ~ C) was performed. Interestingly, soluble peroxidases show greater resistance to heat destruc- tion of the heme unit as compared to cell-wall-derived peroxidases (Fig. 3). By selection of the times at which 35-50% of the peroxidases were irreversibly denatured, it was assumed that a large proportion of the apoprotein has undergone polypeptide unfolding (Milder 1980). The results in Fig. 4 show that no treatment could identify the additional peroxidases as conformers. This apparent stability leads us to suggest that B 5, B6, B7, Bs, and A0 are distinct molecular species, and that their differences in electrophoretic mobility must therefore be related to differences in their primary structure or to the level of glycosylation.

Subcel lular location o fperoxidases . Figure 1 shows clearly that each peroxidase was selectively enriched in either the cell-wall or soluble fractions. In the cell-wall fraction, densitometric analysis showed that the major peroxidase of the soluble fraction, B2, contributed as little as 0.5% of the total activity. Low levels of contamination of the cell- wall fractions with soluble peroxidases is also indicated by a low glucose-6-phosphate dehydrogenase activity (0.12 % of cytosolic activity).

B5 ,,.m ~ ~ ~,,.-, B 2

Fig. 4. Assay for the presence of conformational isoperoxidases using NEIEF. The label for B 7 marks the position of this faint band. cw, cell-wall-derived peroxidases; s, soluble peroxidases. Lanes 1, control; lanes 2, samples extracted in the presence of 1% (w/v) BSA; lanes 3, samples subject to column chromatography in Sephadex G-25 equilibrated in 2 M CaC12; lanes 4, samples reversibly denatured by heating to 70 ~ C

In the soluble fraction, although the majority of peroxidase activity is represented by B2 and A0, minor amounts of A2 (see Fig. 1A), Bs, B6 and By are present (see Fig. 5, lane 5). Peroxidases can be released into the cytosolic fraction upon tissue homogenisation (Barcel6 et al. 1989b). In order to investigate the strength of association of peroxidases to the cell wall, we have infiltrated hypocotyls with buffers of low ionic (50 mM Tris-HC1, pH 7.2) or high ionic strength (1 M KC1 in 50 mM Tris- HC1, pH 7.2). Measurements of the cytosolic marker enzyme glucose-6-phosphate dehydrogenase in infiltrates indicated insignificant contamination with cytoplasmic proteins (cytosolic contribution to total peroxidases of the intercellular fluid was 0-3.3%). Infiltrates with 1 M KC1 contained much higher peroxidase activity (240 pkat. g - 1 FW) than those obtained with 50 mM Tris (75 pkat .g l FW), indicating that the majority of peroxidase activity in the extracellular matrix is ionically ligated to the cell wall. The fraction extracted with 1 M KC1 was enriched in Bs, B6, and BT, but with low levels of B1 and B2 (Fig. 5), which suggests that the three former peroxidase are residents of the extracellular space, and con- firms the location of the latter two as cytoplasmic, where they are abundant. By solubilising peroxidases of the extracellular space by infiltration with low- or high-ionic-


Fig. 6. Substrate utilization of peroxidases. Non-equilibrated IEF was the electrophoretic method utilized. Lanes 1, 3, 5, 7 are cell- wall-derived peroxidases. Lanes 2, 4, 6, 8 are cytosolic peroxidases. G, guaiacol; IAA, indole-3-acetic acid; F, ferulate; CA, coniferyl alcohol

Fig. 5. Cationic PAGE of cytosolic samples and infiltrates from hypocotyls subject to vacuum infiltration. Lane 1 (3.2 nkat), 1 M KCL infiltrate; lanes 2 (3.2 nkat) and 4 (6.3 nkat), cytosolic samples from hypocotyls infiltrated with 1 M KCL; lane 3, cytosolic samples from hypocotyls infiltrated with 50 mM Tris-HC1 (pH 7.2); lane 5, cytosolic samples from control hypocotyls, lane 6, cell-wall-derived peroxidases extracted with l M KC1

strength buffers, and homogenising tissues without prior removal of the infiltrate, quantitative differences were easily detected in the cytosolic fractions (Fig. 5). That B 6 and B~ are strongly ligated to the cell wall is shown by their enrichment in the soluble fraction from tissues infiltrated with high-ionic-strength buffer. However, after infiltration with either high- or low-ionic-strength buffer, the level of B 7 in the cytosol was similar to that in control soluble extracts. Since B7 is enriched in the intercellular fluid, this leads us to suggest that this peroxidase is the most loosely bound to the cell wall at neutral pH. Of particular interest is the apparent difficulty in solubilising B 8 by infiltration, whereas it is readily released from purified cell walls after a brief incubation in 1 M KC1. In conclusion, it appears that Ao, B 1 and B 2 reside in the soluble fraction in vivo, that AI, A2 and B 7 a re the most loosely associated to the cell wall, and that B 5, B 6 and B 8 are strongly, and ionically ligated to the extracellular matrix.

Substrate utilisation o f peroxidases. We have chosen the substrates guaiacol, ferulic acid coniferyl alcohol (CA) and IAA for analysis of substrate utilisation by the several isoenzymes. A typical sample gel is depicted in Fig. 6. Zymograms were analysed after revelation was complete, and differences between the isoenzymes were expressed by comparing their product accumulation with the various substrates to that with guaiacol. A qualitative distinction between substrate selectivity amongst the various peroxidases was not apparent, as also observed with other plant sources (Goldberg et al. 1991). A casual in- spection of the gel shows that for CA and ferulic acid the product accumulation was roughly proportional to that of tetraguaiacol, indicating no detectable differences in substrate utilisation between the isoenzymes (Fig. 6). The ratio of IAA-oxidase to guaiacol-oxidase activity shows that A 2 and B 8 of the cell wall, and B 2 of the cytosolic fraction are clearly the more capable of decarboxylating IAA (Table 2). However, the correlate of the accumulated products, tetraguaiacol against indole-3-methanol, was

Table 2. Comparative activities of guaiacol oxidase and IAA oxidase of cell-wall- and cytosol-derived peroxidases of L. albus hypocotyls. N.D., not determined

Peroxidase Ratio IAAox/Guaiac ox.

Cell Wall B8 0.159+0.032 B7 N.D. B6 0.075 + 0.011 B5 0.020___0.012 A2 0.152___0.004 A1 0.055 _+ 0.022

Cytosol B2 0.133 _ 0.009 B1 0.040_+0.012 A0 0.035_+0.016

positive with a reasonable coefficient (0.7-0.9), suggesting at first that there is a low level of selectivity for substrate utilisation. In order to ascertain whether the deviations from this gradient were significant, similar analyses were made of zymograms produced with cytosolic extracts using a range of peroxidase activities (1.1-28.3 gkat). It was found that highly linear correlates of product accumulation could be obtained with all isoenzymes using either guaiacol or IAA as substrates. The ratios calculated for individual isoenzymes were highly independent of the quantity applied and had low degrees of variation. Addi- tionally, results obtained for four distinct cytosolic extracts, each extracted from separate material, showed highly conserved orders of ratios, namely, Bz>B 1 > A 0. These results lead us to conclude that the ratios observed in this way are credible as definitions of differences of substrate selectivity in these isoenzymes in vitro. The results are summarised in Table 2.

Discussion

Of the 12 peroxidases we have detected in L. albus hypocotyls, we find A 2 and B 2 are present with similar pIs to those previously reported by Barcel6 et al. (1987, 1989 a). The basic peroxidases (B 3 and B4), reported to be associated to tonoplastic membranes in 5-d-old seedlings (Barcel6 et al. 1991), could not be detected in our extracts from older plants. Of the eight new peroxidases we find in hypocotyls, five have been previously demonstrated by


CPAGE as important components in root cell walls (Jackson and Ricardo 1992). The reason for our different results may lie within the electrophoretic techniques utilised. To our knowledge, with the exception of the characterisation of two highly basic peroxidases from tonoplastic membranes (Barcel6 et al. 1991), tube gel IEF was employed across a pH gradient of 4-9, where all of the additional peroxidases described here would either be close to the limits of the pH gradient or outside it. In the case of A o, its acidity would necessitate its resolution proximal to the anode. For the additional basic peroxidases, we have demonstrated that in equilibrium IEF they are largely lost to the cathode and could only be properly resolved using NEIEF or CPAGE techniques.

It seems unlikely that the additional peroxidases are artefacts caused by manipulation of the extracts since complete denaturation would disable their detection by the methods utilised here, and partial degradation, such as by proteases, would lead to smears in gels, which was not seen. The binding of phenols to proteins might pro- duce conformers (Srivastava and Van Huystee 1977), as might differences in polypeptide folding (Epstein and Shechter 1968). In this report we have presented evidence that none of the additional peroxidases are conformers produced from differences in polypeptide folding or lig- and binding. Therefore, the differences in electrophoretic behaviour between these peroxidases must be due to differences in their primary structure or in the level of their glycosylation (Milder 1980).

The heterogeneity seen in Bs, B5 and A0 seems to be principally caused by differences in molecular form and/ or weight. Possible charge variants are A~ and B~, both of which are minor components of, and deviate in charge slightly from their possible counterparts A 2 and B 2 respectively, which is a sign of sub-banding. Similarly, B 5 might be related to eiher B 8 or B 6. However, all of these peroxidases varied in charge only slightly from either an acid or a basic pI. It seems that this minor variation might be caused by heterogeneity in glycosylation or C- terminal processing, but that the larger differences in charge between the acid and basic group of peroxidases of both the cell wall and cytosol can be more credibly related to larger differences in the primary sequence. We therefore propose that in L. albus, there are at least two additional subsets of peroxidases to consider, a soluble acid form and a cell-wall-bound basic form.

The histological and cytochemical location of peroxidases, and the IAA-oxidase system in L. albus has come under considerable attention (Barcel6 et al. 1989 b, 1990; Ferrer et al. 1990b). However, because peroxidase activity was attributed to only a few of the existing peroxidase species, in the light of additional peroxidases, other inter- pretations are possible.

The isoform B 2 is considered a cytoplasmic peroxidase (Barcel6 et al. 1989b) that could function as an IAA oxidase (Ferrer et al. 1991 a). We have found both A 0 and B 2 in non-particulate fractions; possibly, they occupy different zones of the hypocotyl. However, B 2 distribution has been studied along the hypocotyl axis and shown to closely follow total peroxidase activity (Ferrer et al. 1991 a), so it is also possible that A0 follows B2 distribu-

tion, since variation in the ratio of the abundant A o (32% of total soluble activity) to B 2 would largely alter the ratio of B 2 to total activity, which was not seen. Our studies of substrate utilisation have shown that B 2 has the highest capacity, and A 0 the lowest capacity of all L. albus peroxidases for IAA oxidation in vitro. This sup- ports the role of B 2 as the cytoplasmic IAA oxidase, as proposed by others, and indicates that A 0 is likely to perform other functions which remain to be clarified. These results also support the general observation that basic peroxidases are those responsible for 1AA decar- boxylation in plants (Gaspar et al. 1991).

A~ and A 2 have previously been shown to be bound to the cell wall or soluble in the intercellular fluid depending on the pH (Barcel6 et al. 1989c). The additional extracellular peroxidases differ in their degree of association to the cell wall. By is the most soluble in the intercellular fluid, and during extraction B s showed a different behaviour from that of the other ionically ligated peroxidases B 5 and B 6 in that it could not be released into the intercellular fluid. If B 8 is associated to cell walls of cells residing in compact areas of the hypocotyl, solubilisation into the infiltrate would be difficult. This does not find support in histochemical studies however, where the majority of cell-wall peroxidase activity is found associated to juvenile phloem and xylem elements (Barcel6 et al. 1989c).

While the presence of positively charged residues is apparently not the only pre-requisite for peroxidase binding to cell walls [Ros et al. (1986) found negligible binding of B~ and B2 to cell walls over the pH range 4.2 7.2, and we find B7 most freely soluble in the intercellular fluid], a stronger binding of the highly alkaline B 8 to cell walls might also explain the results. However, since B 8 is readily solubilised from purified cell walls simultaneously with the other cell-wall peroxidases by a short incubation with 1 M KC1, dissociation of the ionic link- age was apparently not the limiting factor in infiltration experiments. Therefore, it appears that the diffusion of this peroxidase through the cell-wall matrix is restricted by some physical barrier. An association of B 8 to cell-wall components proximal to the plasmalemma, or a large molecular size might explain these results.

In a proposed model of peroxidase activity in developing lupin hypocotyls, an oxidative/peroxidative cycle of peroxidases (Halliwell 1978) occurs with the oxidation of IAA and the peroxidation of the lignin precursor, CA (Ferrer et al. 1990b). This mechanism is inactive in juvenile and expanding cells despite the presence of peroxidases (Barcel6 et al. 1988, 1989c) and IAA (Sfinchez- Bravo et al. 1986) due to inhibitory isoflavones (Ferrer et al. 1990a, 1991b). This model is attractive since it de- scribes a means by which the developing hypocotyl can achieve, and restrict, lignification to cells undergoing differentiation. However, this model was based on biochemical studies performed with whole cell-wall extracts, but has been attributed to only Aland A 2 (Ferrer et al. 1990b) without considering the contribution of the basic peroxidases.

Although the distribution of these basic peroxidases along the hypocotyl axis is not known, the total cell-wall


activity has been largely placed in juvenile phloem and xylem elements (Barcel6 et al. 1989c). The high activity represented by the basic peroxidases (up to 60% of total cell wall activity) suggests that they are major con- stituents of at least some of these tissues. This implies that H202 produced in the oxidative cycle with IAA, could be utilized by more than one perox.idase type in the peroxidative cycle. Variation of the peroxidase components in cell walls might therefore result in differences in their net products. We have found some evidence for different ca- pacities for either CA or IAA oxidation in vitro. Al- though we cannot say these isoenzymes degrade these substrates in vivo, in light of the above biochemical model it would seem that cell walls with an enriched content of B5 and B 6 would more readily oxidise CA at the expense of IAA. Cell walls enriched in B8, on the other hand, would have higher IAA-oxidase acitivities. This peroxidase could compete for H202 produced in the oxidative cycle and simultaneously decarboxylate IAA in a peroxidative cycle. Such a reaction would both slow the build-up of H~O2 necessary for the initiation of CA oxidation and diminish the local IAA concentration. To- gether, these effects would results in a reduction of CA oxidation, and maintain cell walls in a juvenile state.

In conclusion, for the clarification of the role of peroxidases in the growth and development of hypocotyls, it is necessary to include the additional peroxidases found here. For their detection, electrophoretic techniques such as NEIEF would be suitable. Similarly, determination of the roles and metabolism of individual peroxidases in both cytosol and cell walls from the various hypocotyl zones will have to await their purification.

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an examination of the peroxidases from lupinus albus l. hypocotyls

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