Anionic Peroxidase from the Seed Coat of Glycine max' by ltq93779

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									Plant Physiol. (1991) 96, 214-220                                                                  Received for publication November 6, 1990
0032-0889/91/96/0214/07/$01 .00/0                                                                                  Accepted January 18, 1991



      Purification and Developmental Analysis of the Major
     Anionic Peroxidase from the Seed Coat of Glycine max'
                                      Jeffrey W. Gillikin and John S. Graham*
           Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403

                           ABSTRACT                                        is an abundant protein within the seed coat, represents the
   We show that the majority of peroxidase activity in soybean             dominant peroxidase isozyme, and accumulates during the
(Glycine max var Williams 82) seeds is localized to the seed coat.         maturation of this tissue.
A single isozyme is responsible for this activity and has been
purified to electrophoretic homogeneity by successive chroma-                               MATERIALS AND METHODS
tography on DEAE Sepharose Fast Flow, concanavalin A-Sepha-
rose, and Sephadex G-75. The peroxidase exhibits a pl of 4.1, an           Plant Material
apparent molecular mass of 37 kilodaltons, and has properties
characteristic of a glycoprotein. The enzyme begins to accumu-                Seeds (Glycine max var Williams 82) used for the purifi-
late approximately 21 days after anthesis and continues to do so           cation of peroxidase were obtained from Midwood, Inc.,
throughout the maturation of the seed coat where it can represent          Bowling Green, OH. Soybean plants were grown in potting
at least 5% of the soluble protein in dry seed coats. Due to its           soil under glasshouse conditions.
localization in the seed, we propose that this isozyme may play
a role in the hardening of the seed coat.                                  Enzyme Assay and Protein Determination
                                                                              Peroxidase activity toward guaiacol was quantitated by
                                                                           following the rate of conversion of this substrate to tetraguaia-
                                                                           col at 470 nm. The reaction mixture contained 8 mm guaiacol,
   Peroxidases (donor: H202 oxidoreductase; EC 1.1 1.1.7), a               0.5 mM H202, 50 mm potassium acetate (pH 5.5), and was
ubiquitous class of plant proteins, are enzymes whose primary              run at 25TC. A second assay of seed coat peroxidase activity
function is to oxidize a variety of hydrogen donors at the                 employed the use of 4-chloro-I-napthol as the electron donor.
expense of hydrogen peroxide. Peroxidase can also use molec-
                                                                           Nitrocellulose imprints and IEF2 gels were placed in a solution
ular oxygen as an electron acceptor in oxidasic reactions (4).             containing 8.5 mm 4-chloro-I-napthol, 10 mm Na2HPO4 (pH
This class of enzyme has been extensively studied during this              7.2), 75 mM NaCl, and 3.3 mM H202.
century resulting in considerable knowledge about the chem-                   Protein concentration was determined by the Bio-Rad assay
istry and biochemistry of the molecule (9).                                system (1) according to the manufacturer's procedures. BSA
   Peroxidase has been implicated in a variety of physiological            was used as the standard.
processes in plants. The literature abounds with reports of
peroxidase involvement in lignin biosynthesis (10), extensin               Enzyme Purification
polymerization (8), auxin metabolism (9), disease resistance                  Seed coats were obtained by soaking whole seeds in distilled
(1 1, 20), wound healing (6), and the response to air pollutant            water for approximately 5 min or until the coats became
stress (17). Considering the vast number of publications in                wrinkled. The coats were then removed from the seed and
this field, the physiological function of individual members               homogenized with a Tekmar Tissuemizer in extraction buffer
within this class of enzymes is only partially understood. The             (25 mm KH2PO4 [pH 7.5]) at a ratio of 10 mL extraction
lack of knowledge is due, in part, to the complexity of per-               buffer per gram tissue. All purification steps were performed
oxidase isozymes expressed within a given plant tissue. The                at 40C. The homogenate was filtered through two layers of
presence of multiple isozymes, each of which may be capable                Miracloth and the filtrate centrifuged (12,000g, 10 min). The
of utilizing a variety of cellular substrates, makes the assign-           supernatant fluid was applied to a DEAE Sepharose Fast Flow
ment of specific biological functions to individual isoforms               (Sigma Chemicals) column (2.5 x 20 cm) previously equili-
difficult.                                                                 brated with extraction buffer. Peroxidase activity was eluted
   Our laboratory is interested in studying the physiological              with a linear salt gradient from 0 to 300 mM KC1 in extraction
function of peroxidase isozymes in the soybean plant. To this              buffer. Fractions containing peroxidase activity were made
end, we have chosen to isolate individual isozymes as a                    l x in Con A-Sepharose (Sigma Chemicals) binding buffer by
prerequisite to the assignment of in vivo function. This com-              addition of one-fourth volume of a 5x stock solution (250
munication reports on the isolation to homogeneity of a
soybean seed peroxidase activity. We show that the enzyme                    2Abbreviations: IEF, isoelectric focusing; Con A-Sepharose, con-
                                                                           canavalin A-Sepharose; NMWL, nominal molecular weight limit;
   ' Supported by Ohio Board of Regents.                                   TMFS, trifluoromethanesulfonic acid; daa, days after anthesis.
                                                                     214
                              PURIFICATION OF MAJOR PEROXIDASE FROM SOYBEAN SEED COAT                                           215


mM Tris-HCl [pH 6.8], 2.5 M NaCl, 25 mM MnCl2, and 25              was allowed to proceed for 15 s. The filters were immediately
mM CaCl2). This was then applied to a Con A-Sepharose              transferred to a 65TC oven and allowed to dry for 10 min.
column (2.5 x 20 cm) previously equilibrated with lx binding
buffer. Peroxidase activity retained on the column was eluted      Chemical Deglycosylation of SP4.1
with a linear gradient from 0 to 1 M methyl a-D-mannopyr-
anoside in 2.5 M NaCl, 50 mM Tris-HCl (pH 6.8), and detected          Five hundred micrograms of purified SP4. 1 was deglyco-
as described above. The activity was concentrated to 5 mL          sylated by the TFMS method previously described by Edge et
using an Omegacell (Pharmacia), 10,000 NMWL ultrafiltra-           al. (5). Following deglycosylation, the sample was dialysed
tion system, and loaded directly onto a Sephadex G-75 (Phar-       overnight against 2 mm pyridine (pH 5.5), followed by dialysis
macia) column (2.5 x 120 cm) equilibrated in water. Fractions      against several changes of distilled H20 for 1 d. The sample
containing peroxidase activity were pooled and lyophilized.        was then lyophilized to dryness and taken up in electropho-
                                                                   resis loading buffer and analyzed by denaturing gel
Determination of Seed Coat Peroxidase Isoelectric Point            electrophoresis.
   The pI of the seed coat isozyme was determined by analyt-
ical flatbed isoelectric focusing polyacrylamide gels containing   Antibody Production
ampholines in the pH range of 3.5 to 9.5 (LKB, Bromma,               Antibodies to SP4.1 were raised in a rabbit with three
Sweden). The gel was loaded with a seed coat extract in one        subcutaneous injections of the purified peroxidase (200 jig/
lane and was mock loaded with H20 in an adjacent lane. The         injection) at 2 week intervals. Partial purification of poly-
gel was subjected to electrophoresis for 1.5 h at 0.125 W/cm2      clonal antibodies was accomplished by ammonium sulfate
at 10°C (14). The lanes were then separated and the one            precipitation followed by DEAE chromatography according
containing the seed coat extract was stained with 4-chloro- 1-     to the methods of Livingston (15). The preparation was
napthol solution to determine the distance the peroxidase          adjusted to the original serum volume with 1O mM Na2HPO4
activity migrated relative to the anode. The mock loaded lane      (pH 6.8). Western blot analysis was performed using a 1: 1500
was sliced into 0.25 cm pieces. Gel slices were then added to      dilution of the partially purified primary antibody.
2 mL of H20 and incubated at room temperature overnight
with shaking. The pH of the solution containing each gel slice     SDS-PAGE and Western Blot
was determined and plotted as a function of distance from
the anode. By comparing the distance from the anode that             Denaturing gel electrophoresis (SDS-PAGE) was performed
the activity migrated to the pH of the gel at that position, the   as described by Laemmli (1 3) and silver staining was carried
pI of the enzyme was ascertained.                                  out according to the methods employed by Morrissey (16).
                                                                   Molecular mass markers used for gels were bovine serum
Determination of Peroxidase Isozyme Tissue-Specific                albumin (66 kD), egg albumin (45 kD), glyceraldehyde-3-
Activity                                                           phosphate dehydrogenase (36 kD), carbonic anhydrase (29
                                                                   kD), trypsinogen (24 kD), trypsin inhibitor (20.1 kD), and a-
   Determination of isozyme activity levels in specific tissues    lactalbumin (14.2 kD). Immunoblots were performed as de-
was accomplished by densitometric tracing of peroxidase            scribed by Towbin et al. (21).
activity-stained gels. The cotyledons and seed coats were
homogenized with a Tekmar Tissuemizer in distilled H20 at
a ratio of 10 mL per g of tissue. Samples were then centrifuged                            RESULTS
(12,000g, 10 min). Samples were electrophoresed on analyti-        Localization of Peroxidase Activity Within the
cal IEF gels as described above. Gels were then placed in 4-       Soybean Plant
chloro- 1 -napthol solution and allowed to develop for 60 s
followed by a 5 min wash in distilled H20. Densitometric               Tissue imprinting experiments reveal that the majority of
tracings of the activity stained gels were performed at 582 nm     the peroxidase activity in soybean seeds directed toward 4-
using a Shimadzu CS-930 Dual Wavelength TLC Scanner.               chloro- l-napthol is localized to the seed coat (Fig. lA). Analy-
Units of activity are arbitrary values calculated by measuring     sis of peroxidase activity (guaiacol) in dissected immature
peak areas of peroxidase activity in the IEF gel. The specific     seeds shows that the activity in the seed coat is 99-fold higher
activity of the SP4. 1 seed coat peroxidase is defined as units/   than observed in the cotyledon on a dry weight basis (data
mg of soluble protein applied to IEF gels.                         not shown). Seed coats do show other minor electrophoretic
                                                                   forms on analytical IEF gels other than the major seed per-
Localization of Peroxidase Activity                                oxidase. However, the detection of these minor species (<5%)
                                                                   requires 50 times the amount of protein loaded in Figure l B.
  Visualization of peroxidase activity in immature seeds was       The isoelectric point (pI) of the peroxidase was determined
accomplished by tissue imprinting (2). Nitrocellulose filters      using analytical IEF gels and was shown to be approximately
were soaked in 4-chloro-1-napthol solution (see above) and         4. 1. The major seed coat peroxidase isozyme will be hence-
then lightly blotted to remove excess substrate. Immature          forth referred to as SP4. 1. The observation that the greatest
soybean seeds were halved with a razor blade and allowed to        concentration of SP4. 1 activity is localized to the seed coat
dry for 1 min before blotting onto the moist filter. Seed halves   and is the dominant isozyme suggests that this tissue be used
were then pressed firmly onto the filter and color development     in the purification of the enzyme.
216                                                           GILLIKIN AND GRAHAM                                Plant Physiol. Vol. 96, 1991

                                                                           Accumulation of SP4.1 in Maturing Seed Coats
                                                          B
                                                                              Before attempting the isolation of SP4. 1, we determined
                                                                           the stage of seed coat maturation at which the enzyme reached
                                                                           its highest specific activity. Figure 2 shows a general increase
                                                                           in the total peroxidase specific activity as the seed coat ma-
                                                                           tures. Peroxidase specific activity begins to increase 21 to 28
                                                                           daa and continues throughout the maturation of the seed
                      C                                                    coat. Commercially obtained dry seed actually show higher
                                                                           peroxidase specific activity (1.2x) than that observed in 49
                                                                           daa seeds still attached to the plant.
                                                                              The increase in SP4. 1 specific activity during the develop-
                                                                           ment of the seed coat correlates with the accumulation of a
                                                                           polypeptide with a molecular mass of 37 kD (Fig. 3). Figure
                                                                           3 also shows that the complexity of the pattern of soluble seed
                                                                           coat polypeptides decreases dramatically during the matura-
                                                                           tion of the coat. Taken together, these data suggest that the
                                                                           increase in peroxidase specific activity is due, in part, to the
                                                                           accumulation of SP4. 1. The increase in specific activity may
                                                                           also be due to a general increase in the degradation of other
                                                                           soluble proteins during seed coat maturation (cf. lanes 2 and
                                                                     4.4   7, Fig. 3). Indeed, SP4. 1 activity remains high in seeds at least
                                                                           3 years after harvest suggesting that this activity is stable.
Figure 1. Localization and identification of peroxidase activity in a      Thus, we isolated the peroxidase activity from mature seed
developing soybean seed. (A) Nitrocellulose print of a soybean seed        coats to determine if the 37 kD polypeptide is SP4. 1.
35 daa. The seed was halved and pressed onto nitrocellulose im-
pregnated with 4-chloro-1-napthol solution (see "Materials and Meth-
ods." C, cotyledons; SC, seed coat. (B) Analytical isoelectric focusing
gel of a soluble protein extract (2 9g) of seed coat tissue stained with
4-chloro-1-napthol solution. The pH of the anodic and cathodic ends
of the gel are shown in the right margin.



                                                                                                 17.



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-9
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       E
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       E
       2
       E
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 a) 0
 ._
    P.-
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                   10          20          30          40          50

                             Days After Anthesis
Figure 2. Specific activity of SP4.1 in the developing soybean seed        Figure 3. Silver-stained denaturing polyacrylamide gel of soybean
coat. Seed coats were harvested at weekly intervals beginning 14           seed coat soluble protein. Samples are identical to those used in
daa. Protein extracts were assayed for peroxidase activity using           Figure 2. Lanes 1 to 6 represent 5 ,9g of extracts from seed coats
guaiacol and protein determinations were performed as described in         14, 21, 28, 35, 42, and 49 daa, respectively. Lane 7 is a seed coat
"Materials and Methods." Bars indicate ± SE (n = 3).                       extract from commercially obtained seeds. The position of molecular
                                                                           mass markers (kilodaltons) are shown in the left margin.
                                  PURIFICATION OF MAJOR PEROXIDASE FROM SOYBEAN SEED COAT                                                           217


Enzyme Purification                                                        Table I. Purification of SP4. 1 from Seed Coats of Glycine max
   Purification of SP4.1 required 10 g of seed coats (wet                       Procedure       Total   Total      Specific    Recovery Purification
weight). Figure 4A shows the elution profile of seed coat                                      Protein Activity    Activity
homogenates from a DEAE Sepharose Fast Flow ion exchange                                         mg      units
                                                                                                                  units mg-1
                                                                                                                                  %         -fold
column. The activity eluted from DEAE Sepharose Fast Flow                                                           protein
at 0.10 M KCl and was 5.5-fold purified after this step (see               Crude extract   45.0         9,960         221        100         1.0
Table I). The pooled peroxidase activity was applied to Con                DEAE Sepharose   2.9         3,690       1,212         37         5.5
A-Sepharose and eluted at 0.75 to 1 M methyl a-D-manno-                    Con A-Sepharose 0.25         1,442       5,768         15        26.1
pyranoside (see "Materials and Methods") resulting in a 26.1-              G-75 Sephadex    0.15        1,352       9,013         14        40.8
fold purification (see Table I). The final step employed a
Sephadex G75 gel filtration column (Fig. 4B), resulting in a
40.8-fold purification, a recovery of 14% (Table I), and a                 its affinity for Con A; this was confirmed by treatment with
Reinheitzahl value of 2.8. Due to the abundance of this                    TFMS. TFMS treatment resulted in a shift in the molecular
protein in the crude extract (Fig. 3, lane 7) a 40-fold purifi-            mass of the polypeptide from 37 kD to 30 kD, thus the
cation is sufficient to purify the enzyme.                                 modifications represent a significant portion of the mass of
   Analysis of the material from the Sephadex G75 column                   this protein (Fig. 5). The polypeptide also stains with Schiff's
on both silver stained denaturing gels (Fig. 5A) and on ana-               Reagent (24) on SDS-PAGE (data not shown).
lytical IEF gels (Fig. 6A) demonstrate the presence of a single
polypeptide. Glycosylation of purified SP4. 1 is suggested by              Accumulation of SP4.1 during Maturation of the
                                                                           Seed Coat
                                                                             Polyclonal antibodies generated against the purified per-
                    A                                                      oxidase were used as additional confirmation that the 37 kD
                                                       *150                polypeptide accumulating during seed coat maturation is
                                                                           SP4. 1. Western analysis shows an increase of a single poly-
  E                                                                 E      peptide with a molecular mass of 37 kD beginning approxi-

 0
                                                          100       .I
 co
 Cm
                                                        50          >
                                                                                              A                                B
  c;                                                            I
                                                                     50
                                                                    ._




                0       20 40 60 80 1 00
                         Fraction No.
                    B
                                                       *120
                                                       -100                        37-*
E                                                                    in
                                                        80          ._

                                                                                                                    30-
                                                       *60          -

CO
o
cm                                                      40          *5
                                                                    ._



                                                        20          :
                0           20          40           60
                          Fraction No.
Figure 4. Fractionation of seed coat peroxidase. A, DEAE Sepharose
Fast Flow chromatography of crude seed coat extract. Absorbance
at 280 nm (El); peroxidase activity (*). The linear salt gradient from 0   Figure 5. Analysis of SP4.1 purity and extent of glycosylation. A,
to 300 mm KCI began at tube 34; 8 mL fractions were collected. B,          Silver-stained SDS-PAGE of Sephadex G75 purified SP4.1 (10 lAg);
Sephadex G75 gel filtration chromatography of post-Con A-Sepha-            B, silver-stained SDS-PAGE of Sephadex G75 purified SP4.1 (5 ,g)
rose peroxidase activity. Absorbance at 280 nm (E); peroxidase             after treatment with TFMS. The position of relevant molecular mass
activity (*). Eight milliliter fractions were collected.                   markers (kilodaltons) are shown in the left margin of each lane.
218                                                          GILLIKIN AND GRAHAM                                  Plant Physiol. Vol. 96, 1991


                                    A      B                                                         DISCUSSION
                                                                              The partial purification of the predominant peroxidase
                          /.
                                                                           activity within soybean seeds has been previously reported by
                   ....




                                                                           Sessa and Anderson (18) using whole seeds as the source of
                                                                           enzyme. Their preparation contained one-major anionic per-
                                                                           oxidase activity and at least two minor activities along with
                                                                           several other contaminating polypeptides. The study was con-
                                                                           cerned with the peroxidatic properties of the mixture and the
                                                                           possible involvement of peroxidase in generating off-flavors
                                                                           from endogenous seed constituents (18). A closer examination
                                                                           of the seed shows that the preponderance of the peroxidase
                                                                           activity is localized within the testa or seed coat (Fig. 1, Table
                                                                           II). By employing seed coats as the source of enzyme, we
                                                                           are able to purify the major peroxidatic activity within the
                                                                           seed to homogeneity by standard biochemical separation
                               I.                                          techniques.
                                                                              The relative ease in isolation of this isozyme is due to the
                                                                           fact that mature soybean seed coats do not contain a complex
                                                                           assortment of soluble proteins. Moreover, the anionic perox-




Figure 6. Analysis of purified SP4.1 by analytical isoelectric focusing.
A, Silver-stained analytical isoelectric focusing gel of purified SP4.1    fb 6   -




(5 ug); B, activity stained analytical isoelectric focusing of purified
SP4.1 (10 ng). The pH of the anodic and cathodic ends of the gel are
shown in the left margin.



mately 28 daa (Fig. 7). This result correlates with the appear-
ance of the 37 kD polypeptide observed with SDS-PAGE (Fig.
3) and the increase in the peroxidase specific activity (Fig. 2)
during maturation of the seed coat.

Specific Activity of SP4.1 in Soybean Tissues
  The presence of SP4. 1 was determined in other regions of
the soybean plant to ascertain the abundance of this isozyme.
The seed pod contained the highest specific activity of SP4. 1
in tissues other than the seed coat, but the activity was only
4% of the level in seed coats (Table II). Other tissues examined
had specific activities of SP4. 1 less than 1% of mature seed
coats. These tissues contain multiple anionic and cationic
forms of peroxidase activities. We were interested in confirm-
ing the IEF data with Western blot analysis, but found that
the technique was not useful because multiple bands were
observed in these tissues. The banding pattern was probably
due to the presence of similar carbohydrate moieties shared
by SP4. 1 and other soybean proteins. In contrast, seed coat               Figure 7. Western blot of SDS-PAGE gel of soybean seed coat
tissue yields only a single band by Western analysis. We have              soluble protein. Samples are identical to those used in Figure 2.
observed as many as 20 different isozymes among soybean                    Lanes 1 to 6 represent 5 Ag of extracts from seed coats 14, 21, 28,
plant tissues using analytical isoelectric focusing (data not              35, 42, and 49 daa, respectively. Lane 7 is a seed coat extract from
shown). No tissue, except the seed coat, exhibits a peroxidase             commercially obtained seeds. The position of molecular mass mark-
isozyme pattern where one form predominates.                               ers are shown in the left margin.
                                  PURIFICATION OF MAJOR PEROXIDASE FROM SOYBEAN SEED COAT                                                        219


Table II. Specific Activity of SP4. 1 from Various Soybean Plant            suggest that there is sufficient activity present during early
Tissues                                                                     seed coat development to catalyze this reaction. As mentioned
   Soluble protein extracts were electrophoresed on an analytical IEF       previously, the increase in specific activity of SP4. 1 at latter
gel (pH 3.5-9.5), and stained with 4-chloro-1 -napthol (see "Materials      stages of seed development appears to be due to the inherent
and Methods"). Gels were washed in distilled H20 for 5 min before           stability of this protein relative to other seed coat proteins (see
obtaining densitometric tracings of the peroxidase activity. Units of       Fig. 3).
activity are arbitrary values which are calculated by measuring the            The lignification of the seed coat may also require the
peak area of the SP4.1 position on analytical IEF gels. Specific activity   presence of SP4. 1. The polymerization of cinnamyl alcohols
is reported as units/mg soluble protein.                                    is widely accepted as being a peroxidase catalyzed reaction
                    Tissue                   Specific Activity              (10). The hardening of the seed coat during later stages of
             Mature seed coat                    20,620                     maturation most certainly requires the presence of peroxidase
             Seed coat 35 daa                    15,620                     activity. This may explain the abundance of this isozyme in
             Cotyledon 35 daa                      <100                     mature soybean seed coats.
             Pod                                     807                       The suberized tissue of the mature seed coat represents a
             Leaf                                  <1 00                    diffusion barrier sealing the seed coat from the environment
             Stem                                  <1 00                    (12). Previous studies have implicated an anionic peroxidase
                                                                            in the deposition of the aliphatic and aromatic components
                                                                            of suberin (19). Although we have not yet measured the
                                                                            hydroxyproline or lignin content in developing seed coat
idase represents a significant portion of the total soluble                 tissue, we do have preliminary transmission electron micro-
protein complement (Fig. 3, lane 7) within the seed coat.                   graphs showing the deposition of this peroxidase within the
Based on our purification data (Table I) it appears that the                cell walls of both hour glass and palisade cells of the mature
seed coat tissue of soybean seeds may represent one of the                  soybean seed coat. Additional experimentation is in progress
richest sources of a single peroxidase isozyme yet described in             to elucidate the physiological function of this abundant seed
plants (23).                                                                coat protein.
  The results presented in this report demonstrate that the
accumulation of a 37 kD polypeptide begins approximately                                            ACKNOWLEDGMENTS
21 to 28 daa within the seed coat tissue and coincides with
the increase in peroxidase specific activity observed during                   The authors wish to acknowledge Joe Baker and Barbara Randall
                                                                            for their valuable technical assistance. We are also grateful to George
the same time frame (cf Figs. 2 and 3). We purified the major               S. Bullerjahn for his critical review of the manuscript.
peroxidase activity to homogeneity and generated polyclonal
antibodies to the polypeptide. The identity of the 37 kD                                              LITERATURE CITED
polypeptide was shown to be SP4. 1 by Western analysis. This
molecular mass is consistent with peroxidases reported from                  1.   Bradford MM (1976) A rapid and sensitive method for quanti-
other plant sources (7). We believe that this activity represents                   tation of microgram quantities of protein utilizing the principle
                                                                                    of protein-dye binding. Anal Biochem 72: 248-254
the major isozyme studied by Sessa and Anderson based on                     2.   Cassab GI, Varner JE (1987) Immunocytolocalization of exten-
similarity in pH optima, heat stability, and chromatographic                        sin in developing soybean seed coats by immunogold-silver
behavior. Partial amino acid sequence information (data not                         staining and by tissue printing on nitrocellulose paper. J Cell
shown) has been obtained from SP4. 1 and shows considerable                         Biol 105: 2581-2588
amino acid identity with previously reported peroxidase                      3.   Cooper JB, Varner JE (1983) Insolubilization of hydroxyproline-
                                                                                    rich cell wall glycoprotein in aerated carrot slices. Biochem
sequences.                                                                          Biophys Res Commun 112: 161-167
   The major function of the mature seed coat is to provide a                4.   Dunford HB, Stillman JS (1976) On the function and mechanism
barrier to protect the enclosed embryo. We have not yet                             of action of peroxidase. Coord Chem Rev 19: 187-251
assessed the physiological function of this isozyme in seed                  5.   Edge AS, Faltynek CR, Hof L, Reichert LE, Weber P (1981)
                                                                                    Deglycosylation of glycoproteins by trifluoromethanesulfonic
coat biogenesis. The involvement of peroxidase has been                             acid. Anal Biochem 118: 13 1-137
implicated in several processes operative with the seed coats.               6.   Espelie KE, Franceschi VR, Kolattukudy PE (1986) Immuno-
These processes are extensin polymerization, lignification,                         cytochemical localization and time course of appearance of an
and/or suberization. During the maturation process the ac-                          anionic peroxidase associated with suberization in wound-
cumulation and deposition of extensin polymers within the                           healing potato tuber tissue. Plant Physiol 81: 487-492
                                                                             7.   Espelie KE, Kolattukudy PE (1985) Purification and character-
cell wall of soybean seed coat has recently been documented                         ization of an abscisic acid-inducible anionic peroxidase asso-
(2). The accumulation of extensin begins approximately 16                           ciated with suberization in potato. Arch Biochem Biophys 240:
daa and exists in an insoluble form in the mature seed coat.                        539-545
It is thought that the deposition of extensin within cell walls              8.   Fry SC (1987) Formation of isodityrosine by peroxidase iso-
                                                                                    zymes. J Exp Bot 38: 853-862
serves a structural function in the testa (22). Peroxidase is                9.   Gaspar T, Penel C, Thorpe T, Greppin H (1982) Peroxidases
suspected to be involved in the formation of isodityrosine                          1970-1980. A survey of their biochemical and physiologic
during extensin polymerization (3, 8). The time course of                           roles in higher plants. University of Geneva Press, Geneva,
extensin and SP4.1 accumulation are very similar in devel-                          Switzerland
                                                                            10.   Gross GG (1977) Biosynthesis of lignin and related monomers.
oping seed coats, suggesting that this peroxidase may be                            Recent Adv Phytochem 11: 141-184
involved in the polymerization of extensin. Although the                    11.   Hammerschmidt R, Lamport DTA, Muldoon EP (1984) Cell wall
specific activity of SP4. 1 is greatest in dry seed coat tissue, we                 hydroxyproline enhancement and lignin deposition as an early
220                                                        GILLIKIN AND GRAHAM                                  Plant Physiol. Vol. 96, 1991

        event in the resistance of cucumber to Cladosporium cucumer-    19. S"mons PC, Kolattukudy PE, Bienfait HF (1985) Iron deficiency
        inum. Physiol Plant Pathol 24: 43-47                                  decreases suberization in bean roots through a decrease in
12.   Kolattukudy PE (1984) Biochemistry and function of cutin and            suberin-specific peroxidase activity. Plant Physiol 78: 115-120
        suberin. Can J Bot 62: 2918-2933                                20. Smith JA, Hammerschmidt R (1988) Comparative study of acid
13.   Laemmli UK (1970) Cleavage of structural proteins during the            peroxidases associated with induced resistance in cucumber,
        assembly of the head of the bacteriophage T4. Nature 227:             muskmelon, and watermelon. Physiol Mol Plant Pathol 33:
        680-685                                                               255-261
14.   Lagrimini LM, Rothstein S (1987) Tissue specificity of tobacco    21. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer
        peroxidase isozymes and their induction by wounding and               of proteins from polyacrylamide gels to nitrocellulose sheets:
        tobacco mosaic virus infection. Plant Physiol 84: 438-442             procedure and applications. Proc Natl Acad Sci USA 76:4350-
15.   Livingston DM (1974) Immunoaffinity chromatography of pro-              4354
        teins. Methods Enzymol 34: 723-729                              22. Van Etten CH, Miller RW, Earle FR, Wolff IA, Jones Q (1961)
16.   Morrissey JH (1982) Silver stain for proteins in polyacrylamide         Hydroxyproline content of seed meals and distribution of the
        gels: a modified procedure with enhanced uniform sensitivity.         amino acid in the kernel, seed coat, and pericarp. J Agric Food
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