Specific Compartmentalization of Peroxidase Isoenzymes in Relation

Chapter 7

Downloaded by NORTH CAROLINA STATE UNIV on September 28, 2012 | http://pubs.acs.org Publication Date: August 13, 1998 | doi: 10.1021/bk-1998-0697.ch007

Specific Compartmentalization of Peroxidase Isoenzymes in Relation to Lignin Biosynthesis in the Plant Cell A. Ros Barceló, M . Morales, and M. A. Pedreño Department of Plant Biology, University of Murcia, E-30100 Murcia, Spain

The compartmentalization of peroxidase isoenzymes related to lignin biosynthesis was studied in the cell walls and the cell wall-free spaces of several woody and non-woody plant species. The results illustrate a specific compartmentalization of peroxidase isoenzymes in cell walls compared with that found in vacuoles. Cell wall peroxidase isoenzymes may be classified into three main groups: one with acidic pI (APrx), and two with a basic pI (BPrx LpI and BPrx HpI). While basic peroxidases are constitutively expressed, acidic peroxidases are normally developmentally regulated. This developmental regulation and the greater efficacy in the oxidation of coniferyl alcohol shown by acidic peroxidases suggest that this isoenzyme group plays a key role in lignin biosynthesis, although some contribution to this process by basic peroxidases cannot be ruled out. Peroxidase (EC 1.11.1.7) is the enzyme responsible for the linking and cross-linking of monolignols during the biosynthesis of lignins in the plant cell wall. As may be expected from its specific role in lignin biosynthesis, peroxidase is mainly located in the cell wall, although other subcellular localizations have been reported. Over the last few years, considerable information has been accumulated about the subcellular localization of peroxidase isoenzymes (7). However, it remains unclear whether the findings concerning isoenzyme localization in a few plants can be generalized and rationalized. Combined histochemical, cytochemical and biochemical studies are necessary to rationalize some concepts which are only just beginning to be understood. To cast light on this question, we have studied peroxidase isoenzyme localization in lupin (Lupinus albus) (2-4), grapevine (Vitis vinifera) (5, 6), pepper (Capsicum annuum) (7, 8), lettuce (Lactuca sativa) (9), catharanthus (Catharanthus roseus) (10), oats (Avena sativa) (Rojas, M.C.; Ros Barcelo, A., unpublished data) and aleppo pine (Pinus halepensis) (Ros Barcelo, A . , unpublished data). Electron microscope cytochemistry, vacuum infiltration and subcellular fractionation, together with protoplast and vacuole isolation techniques, show that in all the studied plant materials both basic (BPrx, pi > 7.0) and acidic (APrx, pi < 7.0) peroxidase isoenzymes are located in the cell wall free spaces, probably in equilibrium with those bound to the cell 84

©1998 American Chemical Society

In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

85 walls, while basic peroxidase isoenzymes of high pi (BPrx Hpl, pi > 9.2) are the only isoenzymes located in vacuoles (Table I).


Table I.

Compartmentalization of Peroxidase Isoenzyme Groups in the Plant Cell APrx

BPrx L p l

BPrx Hpl

reference

Lupinus albus

cw

nd

CW, V

2,3,4

Vitis vinifera

cw

cw

CW, V

5,6

Capsicum annuum

cw

cw

CW, V

7,8

Lactuca sativa

a

a

CW, V

9

Catharanthus roseus

a

a

CW, V

10

Avena sativa

cw

cw

CW, V ?

np

Pinus halepensis

cw

cw

CW, V ?

np

cw: cell wall; v: vacuole; nd: not determined; a: absent; np: not published; v?: vacuolar localization not studied. The BPrx Hpl Isoenzyme Group: a Distinctive Peroxidase Isoenzyme Group Located in Vacuoles and Cell Walls The spent medium in which a plant cell culture has grown may be considered to be a large free intercellular space forming a continuum with the plant cell wall (77). For this reason, suspension cultured cells are useful for studying the specific compartmentalization of peroxidase isoenzymes in the cell wall once the cell wall free space that constitutes the spent medium has been isolated by non-disruptive techniques. In this simple experimental system, peroxidase isoenzymes isolated from grapevine cell cultures may be classified into three main groups: APrx (acidic peroxidases), BPrx L p l (basic peroxidases of low pi, 7.0 < pi < 9.2), and BPrx Hpl (basic peroxidases of high pi, pi > 9.2). In grapevine cell cultures, APrx and BPrx L p l are largely located in the cell walls, as can be expected from the fact that these isoenzymes are present exclusively in the spent medium in which the cells were cultured (6, 12), while BPrx Hpl are located in both cell walls (5, 6, 12) and vacuoles (5, 6, 13). In fact, through protoplast and vacuole isolation techniques, it was found that the grapevine peroxidase isoenzyme group BPrx Hpl was the only group present in protoplast and vacuole preparations. Taking oc-mannosidase as a vacuolar marker, 87% of the protoplast BPrx Hpl was located in the vacuolar fraction (Table II). Similarly, when using grapevine anthocyanins as vacuolar markers, the yield of vacuolar BPrx Hpl was found to be 110% of the starting protoplast fraction (Table II). These results suggest that almost all, if not all, the protoplast BPrx Hpl was located in the vacuolar fractions.


86


Table II. Comparison between Compartmentalization of the BPrx Hpl Isoenzyme Group in Vacuoles and Protoplasts protoplast

vacuolar marker

vacuoles

reference

Lupinus albus

-

oc-mannosidase

93%

3

Vitis vinifera

100%

oc-mannosidase

87%

6

100%

anthocyanins

110%

6 7 9

Capsicum annuum

100%

oc-mannosidase

164%

Lactuca sativa

100%

oc-mannosidase

28%

These results confirm those previously obtained using Lupinus albus hypocotyls (2-4). In this case, the cell wall localization of peroxidase isoenzymes was studied by vacuum infiltration of tissue sections with buffers, while the vacuolar localization was studied by purification of vacuolar membranes through 20-40% sucrose gradients. Using this experimental approach, only the peroxidase isoenzyme group BPrx Hpl was located in cell walls and vacuole fractions (Figure 1). As was the case for the grapevine peroxidase isoenzyme group BPrx Hpl, lupin peroxidase isoenzyme group BPrx H p l was recovered with a yield of 93% in the vacuole fraction (Table II). Similar results (Table II) were found in Capsicum annuum (7, 8), lettuce (Lactuca sativa) (9) and catharanthus (Catharanthus roseus) (10). Electron microscope cytochemical studies, using 3,3'-diaminobenzidine as a hydrogen donor, revealed that, in Lupinus albus, the vacuolar peroxidase activity, i.e. the peroxidase isoenzyme group (BPrx Hpl), was mainly located at the internal face of the tonoplast membrane (3), a localization pattern similar to that found in suspension cultured grapevine cells (5, 13) and Catharanthus leaves (14). Molecular Heterogeneity of the Peroxidase Isoenzyme Group BPrx Hpl The fact that the peroxidase isoenzyme group BPrx Hpl is located in both the cell walls and vacuoles (Table I) suggests that this isoenzyme group follows the two secretion routes found in plant cells: (1) that where the subcellular target is the cell wall and (2) that where the subcellular target is the vacuole. The double compartmentalization of this peroxidase isoenzyme group in the cell wall and vacuoles is not surprising since similar results have been obtained with their homologous isoenzymes in other plant species, such as Nicotiana tabacum (15) and Lupinus polyphyllus (16). The existence of two subcellular target compartments for this peroxidase isoenzyme group in plant cells raises a question about the molecular heterogeneity of this, apparently single, hemoprotein isoenzyme. The molecular heterogeneity of peroxidase isoenzymes is easily studied by non-equilibrium IEF (NEIEF), a tool which makes it possible to detect isomers differing slightly in either molecular weight or isoelectric point. Since peroxidases are monomeric glycoproteins, this heterogeneity frequently arises from the different patterns of glycosylation, a feature common to all basic peroxidases (17, 18). On this basis, we may suspect that the peroxidase isoenzyme group BPrx Hpl will be composed of at least two isomers, probably differing in their glycosylation patterns, as would be expected for an isoenzyme group that is secreted simultaneously into the cell wall and the vacuole. As was anticipated above, NEIEF of the Lupinus albus peroxidase isoenzyme group BPrx Hpl reveals the presence of two isoenzymes (de Pinto, M.C.; Ros Barcelo,


87 A., /. Plant Physiol, in press), previously known as B and B peroxidase isoenzymes (3, 4). A similar heterogeneity (i.e. the presence of at least two isoenzymes) for this peroxidase isoenzyme group was detected in lettuce (Lactuca sativa) (79), Catharanthus roseus (10), and strawberries (Fragaria ananassa) (Lopez-Serrano, M . ; Ros Barcelo, A., unpublished data). Surprisingly, the two isozymes were simultaneously located in the cell walls and vacuoles in the case of Lupinus albus hypocotyls (3, 4). Target sequences (signal peptides) on the primary structure of these isoenzymes remain to be elucidated. 3

4


Physical State of Plant Peroxidase Isoenzymes in the Cell Wall The presence of the complete set of peroxidase isoenzymes in the spent medium of cultured cells suggests that peroxidase isoenzymes move freely through the cell wall barrier, the cell wall network having pores of sufficient size to permit their free diffusion from the plasma membrane to the periplasmic free space. This is not surprising since cell wall pores admit the free diffusion of proteins as large as 60 kDa (20), and plant peroxidase isoenzymes have an average molecular weight of 43 kDa. Likewise, this suggests that most of the plant peroxidase isoenzymes located in the cell walls are found in the cell wall free spaces as soluble forms. This does not preclude, however, the possibility that peroxidases may also be bound to the cell walls, linked to the polygalacturonate anionic groups of pectins particularly (27). In order to discriminate which peroxidase isoenzymes are bound to cell walls and which are free in the cell wall free spaces, vacuum infiltration of intact plant tissues with buffers is a useful tool. When this technique was used with oat coleoptiles, it was found that both the APrx and BPrx L p l isoenzyme groups were easily extracted from the cell wall free spaces with buffers of low ionic strength (Figure 2, lane a), suggesting that they occur unbound in the cell wall free spaces, while the BPrx Hpl isoenzyme group was only extracted when CaCl was added to the buffer (Figure 2, lane c). This is due to the fact that calcium competes with peroxidases for the anionic groups of the plant cell walls (27), releasing peroxidases from the bound to the soluble state. Peroxidases from plant cell walls may be classified, according to the dependence on pH of their binding to cell walls, as being A - , N - or B-type. Type A peroxidases (generally those that are acidic in nature and therefore belong to the APrx isoenzyme group) bind to cell walls only at acidic pHs (27), precisely at those pH values where they are charged positively (pH values lower than their pi). Type B peroxidases (those that are basic in nature and generally belong to the BPrx Hpl isoenzyme group) bind to cell walls within the entire range of physiological pH values (4), since they are charged positively throughout this pH range. Type N peroxidases (generally belonging to the BPrx L p l isoenzyme group) do not bind to cell walls at any pH value. Since it is known that the binding of peroxidases to cell walls is typically electrostatic in nature (27), N-type peroxidases must experience some steric hindrance (probably caused by the polysaccharide chains on the hemoprotein surface) that prevents binding to the cell wall surface. 2

Phenol Oxidase (Laccase-like) Isoenzyme Group BPrx Hpl

Activities

of

the

Plant

Peroxidase

It has long been known that plant peroxidases can oxidize some phenols in the absence of exogenous H 0 . This is due to the ability of plant peroxidases to show an 'oxidase-like activity', in which the peroxidase intermediate compound III appears to play a key role in the catalytic activity of the enzyme (22, 23). Compound IE is formed in the presence of a suitable reducing agent (RO) according to the reaction scheme: 2

2

+3

+2

peroxidase(Fe ) + RO" -> peroxidase(Fe ) + RO* +2

peroxidase(Fe ) + 0 -» compound III 2


(1) (2)

88

3.5

-

6.0

Pi


8.0

10.0

b

a

Figure 1. Isoelectric focusing in 3.5-10 pH gradients of cell wall (lane a) and vacuolar (lane b) lupin hypocotyl peroxidase isoenzymes. Peroxidase isoenzymes were stained with 1.0 mM 4-methoxy-alphanaphthol in the presence of 0.5 mM H 0 . Arrowheads indicate basic peroxidase isoenzymes with high isoelectric point (BPrx Hpl). 2

3-5

2

-

•

• 6-0

Pi 8.0

t f t

%

10-0

a

c

b

d

Figure 2. Isoelectric focusing in 3.5-10 pH gradients of peroxidase isoenzymes extracted from oat coleoptile cell walls by vacuum infiltration with 10 mM Tris-HCl buffer (pH 7.5), in the absence (lane a) and presence (lane c) of 25 mM CaCl . Peroxidase isoenzyme pattern in the tissues after vacuum infiltration with 10 mM Tris-HCl buffer (pH 7.5), in the absence (lane b) and presence (lane d) of 25 mM CaCl . Peroxidase isoenzymes were stained with 1.0 mM 4-methoxy-alphanaphthol in the presence of 0.5 mM H 0 . Arrowheads indicate basic peroxidase isoenzymes with high isoelectric point (BPrx Hpl). 2

2

2

2


89 +3

Thus, in the presence of 0 and a suitable reducing agent for Fe (RO), and in the absence of hydrogen peroxide, the catalytic cycle of peroxidase is drawn towards compound III, which is generally considered to be a resonance hybrid between F e - 0 and Fe - O2 • The importance of compound III is that it is broken down (22) in the presence of a phenol (PhOH) giving: 2

+2

2

+3

+3


compound III + PhOH -» peroxidase(Fe ) + PhO' + H O2

(3)

and thus generating the hydrogen peroxide which later may be used in the 'normal' catalytic cycle of the enzyme; the latter, of course, passes through compound I and compound II intermediates (21). Consequently, if a particular peroxidase isoenzyme can be reduced by a phenol (PhOH), the oxidation of the phenol by this peroxidase isoenzyme would be expected to take place with concomitant reduction of 0 . Cell wall localized peroxidase isoenzymes may show phenol oxidase activity (24, 25) and, due to this behavior, they have sometimes been confused with laccase isoenzymes. The phenol oxidase activity of certain cell wall peroxidase isoenzymes is inhibited by the addition of catalase and may easily be monitored by vacuum infiltration followed by staining with a phenol in the absence of H 0 , once these isoenzymes have been separated, e.g. by isoelectric focusing. When this was done with cell wall peroxidase isoenzymes extracted by vacuum infiltration of stems and needles (Figure 3) from aleppo pine (P. halepensis), it was found that only the BPrx Hpl isoenzyme group showed phenol oxidase (laccase-like) activity. These results are not surprising since Chabanet et al. (25) found similar results with cationic peroxidase isoenzymes from Vigna radiata (mung bean) hypocotyls. Furthermore, this differential phenol oxidase (laccase-like) activity of the peroxidase isoenzyme group BPrx Hpl is to be expected since it is generally accepted that basic peroxidases are potentially more easily reduced (see equation 1) than acidic peroxidases (26). These results point to the special catalytic properties of the BPrx H p l isoenzyme group, and its ability to show phenol-oxidase (laccase-like) activity. Despite this special phenol-oxidizing behavior, when these peroxidase isoenzymes were purified from C. roseus leaves (Sottomayor, M . ; Lopez Serrano, M . ; Di Cosmo, F.; Salema, R.; Ros Barcelo, A., unpublished data), they exhibited a Soret band at 404 nm, and two visible bands at 501 nm and 633 nm, typical of high-spin ferric heme peroxidases. 2

2

Kinetic Behavior of the BPrx Oxidation of Coniferyl Alcohol

2

Hpl Isoenzyme

Group

during

the

Because the BPrx Hpl isoenzyme group is located in the cell walls and vacuoles (Table I), a study of their ability to oxidize coniferyl alcohol appears to be of special significance. The fact that the oxidation/reduction potential of coniferyl alcohol is as high as 420 mV at pH 6.0 (27) suggests that it is incapable of reducing peroxidase according to equation 1. For this reason, the oxidation of coniferyl alcohol by peroxidase may only be expected to occur, in the absence of any other reducing agent, through the peroxidative cycle. In this respect, Hayashi and Yamazaki (28) determined redox potentials of about 950 mV for compound I/compound II and compound II/ferric couples at slightly acidic pH values, which suggests that, although coniferyl alcohol appears to be incapable of reducing ferriperoxidase to ferroperoxidase, it is a good substrate for both compound I and compound II reduction. Oxidation of coniferyl alcohol by the basic peroxidase isoenzyme group BPrx H p l from grapevines follows Michaelis-Menten type kinetics with inhibition by substrate at higher concentrations (Figure 4). To calculate the microscopic rate coefficients (k and k ) for the oxidation of coniferyl alcohol by this basic peroxidase isoenzyme group, the rates of oxidation of coniferyl alcohol were treated, at each concentration of coniferyl alcohol and H 0 , according to the generally accepted mechanism for the peroxidase cycle (29): x

3

2

2


90

3.5

-

6-0

Pi


8-0

10-0

a

b

Figure 3. Isoelectric focusing in 3.5-10 pH gradients of peroxidase isoenzymes extracted from aleppo pine needle cell walls by vacuum infiltration with 0.1 M acetate buffer (pH 6.0) in the presence of 50 mM CaCl , stained with 1.0 mM 4-methoxy-alpha-naphthol in the presence (lane a) and absence (lane b) of 0.5 mM H 0 . Arrowheads indicate basic peroxidase isoenzymes with high isoelectric point (BPrx Hpl) showing laccase-like activity. 2

2

2

0.3

o 0.0 J 0

1

1

1

2 4 [ H 0 ] (mM) 2

2

Figure 4. Dependence of the oxidation rate of coniferyl alcohol catalyzed by the basic peroxidase isoenzyme B of grapevines (an isoenzyme belonging to the BPrx Hpl isoenzyme group) on H 0 concentration in a reaction medium containing 0.1 M Tris-acetate buffer (pH 5.0), [25 p M (•), 50 p M ( A ) and 100 p M (•)] coniferyl alcohol and enzyme. Bars represent standard errors (n = 3). 5

2

2


91 P +H 0 2

^

2

compound I + H 0

compound I + ArOH

—> compound II + ArO* k 3

compound H + ArOH

(5)

> P + ArO*

(6)

where ArOH is coniferyl alcohol and compound I and compound II are the key intermediates in the peroxidase cycle. Assuming that k » k , as is the case for most peroxidases (29), the steady-state rate equation may be written as: 2


(4)

2

3

2[E]k [ArOH][H Q ]

v

3

2

2

(k /k )[ArOH] + [ H 0 ] 3

1

2

2

from which the dependence of v on [H 0 ] may be written as: 2

v

2

= « B + [H 0 ] 2

(8)

2

where A = 2 [E] k [ArOH] and B = (k /k,) [ArOH]. Double-reciprocal plots (1/v vs. l/[ H 0 ]) (Figure 5) of the data shown in Figure 4 allow us to calculate A and B values for each coniferyl alcohol concentration. Plotting A versus B values gives a straight line (A = 2 [E] k, B , Figure 6), which confirms the proposed mechanism (Equations 4-6), and from its slope it is possible to calculate the k value. Similarly starting again from Equation 7, the dependence of v on [ArOH] may be written as: 3

3

2

2

x

A[ArOH] B + [ArOH] where A = 2 [E] k, [H 0 ] and B = (k,/k ) [H 0 ]. Double reciprocal plots (1/v vs. l/[ArOH]) allow us to calculate A and B values for each H 0 concentration. Plotting A versus B values gives a straight line (A = 2 [E] k B , Figure 7), which further confirms the mechanism proposed, and from its slope it is possible to calculate the k value. From the slopes of the straight lines plotted in Figures 6 and 7, it is therefore possible to determine the values of k and k . The values obtained from the steady-state kinetic data were k (compound I formation constant) = 10.4 pM" s" and k (compound II reduction constant) = 8.7 pM" s" . Thus, the oxidation of coniferyl alcohol by peroxidases from the BPrx H p l isoenzyme group follows the accepted model for peroxidase oxidation, in which compound I and compound II appear as the main intermediates in the catalytic cycle (Equations 4-6). This was supported by the good fits to a straight line which passed through the coordinate origin when the parameters A vs B were plotted for varying H 0 (Figure 6) and coniferyl alcohol (Figure 7) concentrations. 2

2

3

2

2

2

2

3

3

{

3

1

{

2

1

3

1

1

2

Catalytic Properties of Acidic and Basic Peroxidase Isoenzymes during the Oxidation of Coniferyl Alcohol From the results shown in Table I, the plant cell wall contains both acidic and basic peroxidase isoenzymes. To date, it has been difficult to decide which of these two isoenzyme groups is responsible for lignin biosynthesis in the cell wall, and it even


1/[H 0 ]


2

8

Figure 5. Double reciprocal plots of data shown in Figure 4.

0.3

CA 0.2

0.1

0.0 £ 0.00

> 0.08

1

0.04

1

B 1

Figure 6. Plots of parameters A (nmol s" ) versus B (mM) obtained by varying H 0 at three coniferyl alcohol concentrations [25 p M (1), 50 p M (2) and 100 p M (3)]. From the slope of the straight line (2[E]k,), k, was calculated to be 10.4 p M ' V . 2

2

1

1.2

A

0.6

0.0

0.2

0.4

B

0.0

1

Figure 7. Plots of parameters A (nmol s" ) versus B (mM) obtained by varying coniferyl alcohol at three H 0 concentrations [0.25 m M (1), 0.5 mM (2) and 2 mM (3)]. From the slope of the straight line (2[E]k ), k was calculated to be = 8.7 p M ' V . 2

2

3

1


3

93 seems possible that both peroxidase isoenzyme groups may be involved. Mader et al. (30) suggested that basic peroxidases generate the H 0 which is later used by acidic peroxidases in phenolic coupling. However, both basic and acidic peroxidases are only temporary residents in the plant cell wall and, in most cases, they are not present simultaneously. Furthermore, although it is well established that acidic peroxidases play a central role in the lignification of the vascular bundles in stems (31, 32), this does not appear true in other cases where basic peroxidases are clearly and selectively associated with the lignification of vascular structures (33-36). To cast some light on this controversy, it is interesting to compare the catalytic activities of acidic and basic peroxidase isoenzymes during the oxidation of coniferyl alcohol. An examination of the macroscopic apparent Michaelis constants ( K ) shows that acidic peroxidase isoenzymes have a greater affinity for coniferyl alcohol (Table III), while basic peroxidases have a greater affinity for H 0 (Table IV). 2

2

obs


M

2

2

Table III. Observed K Values for Coniferyl Alcohol during Its Oxidation at pH 5.0 by Acidic and Basic Peroxidase Isoenzymes with H 0 M

2

isoenzyme

K

o b s

[H 0 ] mM

source

reference

10

0.33

Lupinus

37

BPrx Hpl

176

0.25

Vitis

-

BPrx Hpl

266

0.50

Vitis

-

BPrx Hpl

369

2.00

Vitis

-

M

APrx

pM

2

2

2

A similar conclusion may be reached when comparing the microscopic catalytic rate coefficients for compound I formation (kj) and compound II reduction (k ). In this case, the reactivity of ferriperoxidase with H 0 [whose magnitude is evaluated through the coefficient for compound I formation (kj] is greater for basic peroxidases than for acidic peroxidases (Table V), while the reactivity of compound II toward coniferyl alcohol [whose magnitude is evaluated through the coefficient for compound II reduction (k )] is greater for acidic peroxidases than for basic peroxidases (Table V). 3

2

2

3

Table IV. Observed K Values for H 0 during Its Reduction at pH 5.0 by Acidic and Basic Peroxidase Isoenzymes with Coniferyl Alcohol M

isoenzyme APrx

K

o b s M

pM

2

2

[coniferyl alcohol] mM

source

160

0.100

Lupinus

BPrx Hpl

38

0.025

Vitis

BPrx Hpl

67

0.050

Vitis

BPrx Hpl

88

0.100

Vitis

reference


37

94 In any case, cell wall acidic peroxidases show greater intrinsic efficacy 2k,[H 0 ]

b s =

2

2

=

2

k

3

(k /k [H 0 l


1

3

2

2

than basic peroxidases for coniferyl alcohol oxidation (see Table V), suggesting that they are evolutionarily better adapted than basic peroxidases for the in situ oxidation of coniferyl alcohol to lignins. Nevertheless, this redundancy in biological function shown by the different peroxidase isoenzyme groups, underlined by both their differing reactivities toward coniferyl alcohol and their specific localization in the several domains of the primary cell wall and secondary thickening domains (32), could explain why lignins are not uniformly deposited throughout the plant cell wall and why they are intrinsically so heterogeneous. Table V. k and k Values at 25°C for the Oxidation of Coniferyl Alcohol by Acidic and Basic Peroxidase Isoenzymes t

isoenzyme

3

k, p M V

1

k pMV 3

1

pH

source

reference

1.4

16.0

4.50

Armoracia

38

BPrx L p l

12.0

2.8

4.50

Armoracia

38

BPrx L p l

6.7

2.4

3.96

Hordeum

39

BPrx Hpl

10.4

8.7

5.00

Vitis

-

APrx

Acknowledgments This work has been partially supported by grants from the CICYT (Spain), Projects # ALI 93/573 and ALI 95/1018. The authors thank Dr. M.C. Rojas (Departamento de Quimica, Universidad de Chile) for permitting us to use unpublished data. Literature Cited 1. Pedreño, M . A.; Bernal, M . A.; Calderón, A . A.; Ferrer, M . A . ; López-Serrano, M . ; Merino de Cáceres, F.; Muñoz, R.; Ros Barceló, A . In Plant Peroxidases: Biochemistry and Physiology; Welinder, K . G., Rasmussen, S. K . , Penel, C., Greppin, H., Eds.; University of Geneva: Geneva, 1993; pp 307-314. 2. Ros Barceló, A.; Munoz, R.; Sabater, F. Plant Sci. 1989, 63, 31. 3. Ros Barceló, A . ; Ferrer, M . A . ; García-Florenciano, E.; Muñoz, R. Bot. Acta 1991, 104, 272. 4. Ferrer, M . A . ; Pedreño, M . A . ; Ros Barceló, A.; Muñoz, R. J. Plant Physiol. 1992, 139, 611. 5. García-Florenciano, E.; Calderón, A . A . ; Pedreño, M . A . ; Muñoz, R.; Ros Barceló, A. Plant Growth Regul. 1991, 10, 125. 6. Calderón, A . A . ; García-Florenciano, E.; Pedreño, M . A . ; Muñoz, R.; Ros Barceló, A . Z. Naturforsch. 1992, 47c, 215. 7. Bernal, M . A . ; Pedreño, M . A . ; Calderón, A . A.; Muñoz, R.; Ros Barceló, A . ; Merino de Cáceres, F. Ann. Bot. 1993, 72, 415.


95

1989,


1994,

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