the lipoxygenase lsozymes in soybean [glyche max (1.) · plant physiol. (1995) 107: 535-543 the...

Plant Physiol. (1995) 107: 535-543

The Lipoxygenase lsozymes in Soybean [Glyche max (1.) Merr.] Leaves

Changes during Leaf Development, after Wounding, and following Reproductive Sink Removal

David M. Saravitz’ and James N. Siedow*

Developmental, Cell and Molecular Biology/Department of Botany, Duke University, Box 91 000, Durham, North Carolina 27708-1 O00

The levels of individual lipoxygenase isozymes in soybean [Clycine max (1.) Merr.] leaves were assessed during leaf development, after mechanical wounding, and in response to reproductive sink removal. Native isoelectric focusing followed by immunoblotting was employed to examine individual lipoxygenase isozymes. In leaves of all ages, two distinct classes of lipoxygenase isozymes were detected. One class of lipoxygenase isozymes had nearly neutral isoelectric points (pls) ranging from pH 6.8 to 7.2. The other class of lipoxygenase isozymes had acidic pls ranging from pH 4.7 to 5.6. During leaf development, all of the neutral lipoxygenase isozymes and most of the acidic isozymes were present in greatest abundance in the youngest leaves examined and declined in amount as leaf age increased. However, four acidic lipoxygenase isozymes (pl = 4.70, 4.80, 4.90, 4.95) were more abundant in intermediate- age leaves than in either the youngest or oldest leaves examined. Following mechanical wounding of leaves, these same four acidic isozymes also increased in abundance both locally and systemically in leaves from wounded plants. Unlike the specific effects of wounding on the lipoxygenase isozymes in leaves, reproductive sink removal stimulated a general increase in most of the acidic lipoxygenase isozymes in leaves.

Multiple forms or isozymes of the enzyme lipoxygenase have been detected in both animal and plant species (Vick and Zimmerman, 1987; Siedow, 1991; Yamamoto, 1992). Although multiple isozymes of lipoxygenase have been identified in many plant species (Christopher et al., 1970; Ida et al., 1983; Yamamoto and Tani, 1986; Mulliez et al., 1987; Domoney et al., 1990; Todd et al., 1990), the physiological role of any specific plant lipoxygenase isozyme has yet to be established. Because lipoxygenase is required in the biosynthesis of JA in plants (Vick and Zimmerman, 1987), one or more lipoxygenase isozymes present within a given plant species must be involved in JA biosynthesis. The jasmonates (JA and its naturally occurring, structural analogs) are known to influence a wide variety of physiological processes in plants (Parthier, 1991; Koda, 1992). JA has also been proposed to serve as a mediator of plant

Present address: Department of Genetics, North Carolina State

* Corresponding author; e-mail [email protected]; fax University, Box 7614, Raleigh, NC 27695-7614.

1-919-613-8177.

defense responses to wounding and pathogen attack (Farmer and Ryan, 1992; Gundlach et al., 1992; Kauss et al., 1992). Thus, lipoxygenase may be involved in a variety of physiological processes due to its role in the synthesis of JA. Other proposed physiological roles of lipoxygenase, unrelated to JA biosynthesis, include membrane degradation during hypersensitive resistance responses (Croft et al., 1990), production of fatty acid-derived, anti-microbial molecules (Ohta et al., 1990, 1991; Kato et al., 1992a; Croft et al., 1993), and the synthesis of ABA (Creelman et al., 1992). In addition, lipoxygenase has been proposed to serve as a storage protein in both seeds (Peterman and Siedow, 1985b; Siedow, 1991) and leaves (Tranbarger et al., 1991). Given the presence of multiple isozymes of lipoxygenase in plants, it is possible that individual lipoxygenase isozymes within a plant may have distinct physiological roles.

The soybean [Glycine max (L.) Merr.] lipoxygenase isozymes have been the most intensively studied among plant lipoxygenases. In mature soybean seeds, three to four lipoxygenase isozymes are present, and during seed germination, three additional isozymes appear in soybean cotyledons (Christopher et al., 1970, 1972; Kato et al., 1992b). In addition, multiple isozymes have been identified in soybean hypocotyls. One major and two to three minor lipoxygenase isozymes appear in soybean hypocotyl/radicle axes during germination that have more acidic pIs than the well-characterized isozymes found in mature soybean seeds (Park and Polacco, 1989). Multiple lipoxygenase isozymes also occur in soybean leaves. Grayburn et al. (1991) used chromatofocusing to separate three distinct peaks of lipoxygenase activity in extracts from soybean leaves. On the basis of physical and kinetic characteriza- tions of the three leaf lipoxygenases, Grayburn et al. (1991) concluded that the soybean leaf lipoxygenase isozymes are distinct from the isozymes found in seeds and that at least one of the leaf isozymes is different from the lipoxygenase isozymes found in hypocotyl/radicle axes. IEF followed by immunoblotting has been used to demonstrate that each of the three leaf lipoxygenase activity peaks separated by chromatofocusing contains severa1 distinct lipoxygenase isoforms (Grayburn et al., 1991).

Abbreviations: JA, jasmonic acid; TBST, 10 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween-20.

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536 Saravitz and Siedow Plant Physiol. Vol. 107, 1995

In soybean leaves, total lipoxygenase protein and activity have been reported to vary with respect to leaf size (Hildebrand et al., 1988; Altschuler et al., 1989; Grayburn et al., 1991). Because soybean leaves contain multiple lipoxygenase isozymes, variation in total lipoxygenase protein and activity during Ieaf development probably results from changes in individual lipoxygenase isozymes, but the relationship of individual lipoxygenase isozyme levels to leaf age has not been established.

Increases in host lipoxygenase activity and individual lipoxygenase isozymes after infection with bacterial and funga1 pathogens have been observed for severa1 plant species (Yamamoto and Tani, 1986; Keppler and Novacky, 1987; Croft et al., 1990; Kato et al., 1992b; Koch et al., 1992). Physical wounding also increases lipoxygenase expression in soybean leaves both locally and systemically (Hildebrand et al., 1989; Bell and Mullet, 1991). Increases in soybean lipoxygenase transcript levels have been detected in leaves within 8 h after wounding (Bell and Mullet, 1991). In Arabidopsis thaliana, differential expression of lipoxygenase genes was observed following wounding (Bell and Mullet, 1993). Local and systemic increases in AtLox2 transcript levels were detected in leaves following wounding, but AtLoxl transcript levels were unaffected by wounding (Bell and Mullet, 1993). To date, the effects of wounding on the levels of individual lipoxygenase isozymes in soybean or any other plant species have not been reported. Given the proposed role of lipoxygenase in the defense systems of plants against attacks by pests and pathogens (Farmer and Ryan, 1992; Gundlach et al., 1992; Kauss et al., 1992; Koch et al. 1992; Ryan, 1992; Croft et al., 1993), it is important to understand the effects of physical damage on the levels of individual lipoxygenase isozymes.

To understand more fully the role of lipoxygenase isozymes in soybean, the behavior of lipoxygenase isozymes in soybean leaves was examined during leaf development, after wounding, and in response to reproductive sink removal.

MATERIALS AND METHODS

Plant Materiais

For studies of leaf development, wounding, and reproductive sink removal, soybean [Glycine max (L.) Merr. cv Ransoml seeds were sown directly in a mixture containing equal parts (v/v/v) of soil, sand, and perlite. Plants were grown in the Duke University greenhouses under natural photoperiods. For wounding experiments, approximately I-month-old plants with mature, fully expanded first and second trifoliate leaves were used. Each leaflet of a first trifoliate leaf was wounded once at the beginning of the experiment with a hemostat. Wounds were centrally lo- cated in each leaflet and were perpendicular to and through the midrib. After harvest, leaves were immediately frozen with liquid nitrogen and stored at -80°C. Soybean cotyledon and hypocotyl/radicle axes originated from etiolated seedlings. Seedlings were grown in complete darkness for 3 d after imbibition at 28°C. Cotyledons and hypocotyl/radicle axes were excised from the seed-

lings and the plumules were discarded. Seedling tissues were immediately frozen with liquid nitrogen and stored at -80°C. Frozen plant tissues were powdered with a mortar and pestle that was precooled with liquid nitrogen. Pow- dered tissue samples were stored at -80°C for later use in preparation of tissue extracts.

In experiments to examine the effects of reproductive sink removal, soybean plants were grown as described above, and beginning at anthesis, flowers and flower buds were removed daily for a period of 2 weeks. Floral tissues were not removed from control plants and seed pods were allowed to develop normally. Leaves were harvested at anthesis and at 2 weeks postanthesis as described above.

Preparation of Tissue Extracts for IEF

Ice-cold 1.7-mL plastic microcentrifuge tubes were filled approximately three-fourths full with frozen, powdered plant tissue. While the tubes were held on ice, 300 p L of ice-cold extraction buffer (5.0 mM sodium phosphate, pH 7.0, 1.0% [w/vl polyvinylpolypyrrolidone) was added. After thawing on ice, each mixture was homogenized with a disposable pellet pestle while in the microcentrifuge tube (Kontes Scientific Glassware/Instruments, Vineland, NJ). AI1 subsequent operations were conducted at 4°C. After homogenization, the tubes were centrifuged for 20 min at 13,OOOg. Each supernatant was collected to a second tube and centrifuged again for 10 min at 13,0008. Each supernatant was collected to a new tube and centrifuged again for 10 min at 13,OOOg. The final supernatant was collected, divided into 50-pL aliquots, frozen with liquid nitrogen, and stored at -80°C for later use. Soluble protein concen- trations were determined as described by Lowry et al. (1951) using BSA as a standard.

IEF

Native IEF was performed on vertical polyacrylamide gels as described by Robertson et al. (1987) with the following modifications. For IEF in the range pH 4.5 to 6.5, a 1:l (v/v) mixture of pH 4 to 7 and pH 5 to 6 ampholytes was used. For IEF in the range pH 5.5 to 7.5, a 1:l (v/v) mixture of pH 5 to 8 and pH 6 to 7 ampholytes was used. A11 ampholytes were purchased from Serva Biochemicals (Westbury, NY). Prior to IEF, the frozen tissue extracts were thawed and immediately placed on ice. Each sample was mixed with one-half volume of an aqueous solution of 60% (v/v) glycerol and 2% (v/v) of each of the component ampholytes used in the gel preparation as described above. Focusing was conducted for 1 h at 200 V and 4 h at 500 V. During IEF, the gel temperature was maintained at 24°C. pI standards were purchased from Sigma.

lmmunoblot Analysis of lipoxygenase lsozymes

After IEF was completed, the gels were incubated at 4°C for 45 min in 250 mL of transfer buffer (25 mM Tris, 192 mM Gly, 0.1% [w/v] SDS, pH 8.5). Proteins from gels were then electrotransferred at 300 mA for 12 to 15 h at 4°C to nitrocellulose membranes (BA-S, Schleicher & Schuell). After protein transfer, the nitrocellulose filters were rinsed

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Soybean Leaf Lipoxygenase Isozymes 537

in distilled H2O and stained with Ponceau S to determineprotein transfer uniformity (Harlow and Lane, 1988). Stain-ing and all subsequent incubations of the nitrocellulosefilters were conducted at 25°C. After destaining in distilledH2O, the filters were incubated with agitation for 5 min inTEST, pH 8.0. This incubation was repeated twice withfresh TEST added each time. The filters were then incu-bated for 2 h in quench solution (5% [w/v] non-fat dry milkand 0.02% [w/v] sodium azide in TEST, pH 8.0). After twobrief rinses in TEST, the filters were incubated in TEST for15 min, followed by two additional washes of 5 min each.The filters were then incubated for 2 h with rabbit anti-soybean lipoxygenase-2 antibodies (Peterman and Siedow,1985a) diluted 1:2000 in quench solution. Following incu-bation with primary antibodies, the filters were rinsedtwice with TEST and washed once for 15 min and twice for5 min each in TEST. Goat anti-rabbit immunoglobulinsconjugated to horseradish peroxidase (Sigma) were diluted1:5000 in quench solution and incubated with the filters for1 h. The filters were then rinsed three times, washed oncefor 15 min and four times for 5 min in TEST. Bound,secondary antibodies were detected using the ECL chemi-luminescence system according to the manufacturer's di-rections (Amersham).

In-Gel Staining for Lipoxygenase Activity

After IEF was completed, the gels were rinsed in distilledH2O and stained for lipoxygenase activity by the method ofFunk et al. (1985) using the modifications employed byPark and Polacco (1989). Both linolenic and linoleic acidswere used as substrates in separate experiments. Gels wereincubated with gentle agitation in the presence of the re-action mixture for 2 to 3 h at 25°C. After staining wascompleted, the gels were rinsed several times with distilledH2O and incubated in distilled H2O with gentle agitationfor 15 min at 25°C. After the gels were photographed,activity-stained bands were excised and stored at —80°Cfor later use in SDS-PAGE.

SDS-PAGE and Immunoblotting

SDS-PAGE was performed on 82 X 60 X 1.5 mm slab gelsaccording to Laemmli (1970) using 12% (w/v) acrylamidein the resolving gel. Following electrophoresis, immuno-blotting was conducted as described above for lipoxyge-nase isozymes.

RESULTS

Developmental Patterns of Leaf Lipoxygenase Isozymes

Although multiple lipoxygenase isozymes have beendemonstrated to exist in soybean leaves (Grayburn et al.,1991), the relationship between the levels of individuallipoxygenase isozymes and leaf age has not been deter-mined. Developmental variation in total leaf lipoxygenaseprotein likely results from alterations in the levels of indi-vidual lipoxygenase isozymes. Therefore, an investigationof the developmental expression of the lipoxygenaseisozymes in soybean leaves was conducted.

First trifoliate leaves were harvested at four intervals (19,23, 26, and 31 d after planting) spanning the complete cycleof leaf development. The initial harvest was made whenthe middle leaflet of the first trifoliate leaves had an aver-age length of 16.8 mm. The final harvest consisted of ma-ture, fully expanded leaves having an average middle leaf-let length of 61.6 mm. Average leaf weights (in mg) in orderof increasing leaf age for the four sets of leaves were 46.3,199.3, 457.3, and 574.7, respectively.

To evaluate the levels of lipoxygenase isozymes, solubleprotein extracts were prepared from the leaf tissues, sub-jected to IEF on polyacrylamide gels, electrotransferred tonitrocellulose membranes, and immunoblotted using anti-soybean lipoxygenase-2 antibodies. After initial experi-ments using IEF gels with a pH gradient from 4.0 to 7.0(data not shown), it became clear that this pH gradientwould not resolve all of the lipoxygenase isozymes insoybean leaves. To resolve the leaf isozymes, each samplewas electrophoresed on two separate IEF gels optimizedfor more narrow pH ranges. Several lipoxygenase isozymeshaving pis between 6.8 and 7.2 were detected in leaves ofall ages (Fig. 1A). All the lipoxygenase isozymes in thisregion were most abundant in the youngest leaves exam-ined and declined in amount as leaf age increased. Inaddition to the lipoxygenase isozymes with neutral pis,lipoxygenase isozymes with more acidic pis were alsoobserved. The acidic lipoxygenases were observed to havepis ranging from 4.7 to 5.6. Assuming that the anti-lipoxy-genase-2 antibody employed has roughly equal avidity for

LEAF AGE-

1 2 3 4

7.2 -

6.8 -6.6 -

B LEAF AGE -

p| 1 2 3 4 H

5.9 - «">

5.1 -,

4.6 -

Figure 1. Immunoblot analysis of the developmental variation inlipoxygenase isozymes from first trifoliate soybean leaves. Lipoxyge-nase isozymes resolved between pH 6.0 and 7.5 (A) and between pH4.5 and 6.0 (B). Leaf extracts were prepared from first trifoliate leavesharvested 19 (lanes 1), 23 (lanes 2), 26 (lanes 3), and 31 (lanes 4) dafter planting. Leaf extracts containing 100 ^g of soluble proteinwere subjected to native IEF on polyacrylamide gels, and lipoxyge-nase isozymes were detected by immunoblotting using a polyclonalantibody prepared against soybean lipoxygenase-2. An extract from3-d-old, etiolated hypocotyI/radicle axes was focused in lane H.Arrowheads in B indicate the position of the minor acidic lipoxyge-nase isozymes. pis appear on the left. www.plantphysiol.orgon August 7, 2019 - Published by Downloaded from

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all the lipoxygenase isozymes, the relative abundance ofthe individual acidic isozymes is somewhat variable withinleaves of any age class (Fig. IB). At all leaf ages, the mostabundant isozymes have pis between 5.0 and 5.3. Many ofthe acidic isozymes were most abundant in the youngestleaves and declined with increasing leaf age. However,several minor lipoxygenase isozymes (pi = 4.70, 4.80, 4.90,4.95) were more abundant in intermediate-age leaves thanin either the youngest or oldest leaves (Fig. IB, arrow-heads). For comparison with previously identified lipoxy-genase isozymes, extracts prepared using hypocotyl/radi-cal axes from 3-d-old, etiolated seedlings were subjected toIEF. In hypocotyl/radicle axes, two lipoxygenase isozymeswith pis of 5.1 and 5.3 were observed that co-migrated withisozymes from leaves (Fig. IB). In addition, an abundantlipoxygenase isozyme (lipoxygenase-1) with a pi of 5.9 wasdetected in hypocotyl/radical axes but not in leaves of anyage (Fig. IB).

Effects of Wounding on Leaf Lipoxygenase Isozymes

Increases after wounding in the expression of at least twodifferent members of the soybean lipoxygenase gene fam-ily have been shown to occur at the transcriptional level(Bell and Mullet, 1991). To date, the effects of wounding onthe lipoxygenase isozymes in soybean leaves have not beenreported. Therefore, an investigation of the local and sys-temic effects of wounding on soybean leaf lipoxygenaseisozymes was undertaken. For wounding experiments, ap-proximately 1-month-old plants with mature, fully ex-panded first and second trifoliate leaves were used. At thestart of the experiment, each leaflet of the first trifoliateleaves was wounded once by a single crush with a hemo-stat. To examine the local response of lipoxygenase iso-zymes to wounding, first trifoliate leaves were harvestedfrom wounded and unwounded control plants at 12, 24,and 48 h after wounding. To examine the systemic re-sponse to wounding, second trifoliate leaves (unwounded)were harvested from plants with wounded first trifoliateleaves and from unwounded controls. Soluble protein ex-tracts were prepared from the leaf tissues and subjected toIEF followed by immunoblotting as described above.

Several lipoxygenase isozymes were found to increase inabundance in first trifoliate leaves after wounding (Fig. 2).No consistent changes in the levels of the neutral lipoxy-genase isozymes were observed following wounding (Fig.2A). Among the acidic, leaf lipoxygenase isozymes, how-ever, the four relatively minor isozymes having the lowestpis (4.70, 4.80, 4.90, 4.95) increased after wounding (Fig. 2B,arrowheads). In wounded leaves, the levels of all fouracidic isozymes were higher than in unwounded controlsat 12, 24, and 48 h after wounding. No changes wereobserved in the levels of the most abundant, acidic lipoxy-genase isozymes (pi = 5.0-5.3) after wounding (Fig. 2B).

In addition, systemic increases in the levels of specificlipoxygenase isozymes were detected in second trifoliateleaves following wounding (Fig. 3). Examination of theneutral soybean leaf isozymes present in second trifoliateleaves from both wounded and unwounded plants did notreveal any consistent increases in individual isozymes (Fig.

5.1

4.6 —

Figure 2. Immunoblot analysis of lipoxygenase isozymes in firsttrifoliate leaves from wounded ( + ) and unwounded (-) soybeanplants. Lipoxygenase isozymes resolved between pH 6.0 and 7.5 (A)and between pH 4.5 and 6.0 (B). For wounded plants, each leaflet ofthe first trifoliate leaves was wounded once with a hemostat at thestart of the experiment (h 0). Leaf extracts containing TOO ju,g ofsoluble protein were subjected to native IEF and immunoblotting asdescribed in Figure 1. Arrowheads indicate the location of lipoxyge-nase isozymes that increase after wounding, pis appear on the left.

3A). The four acidic lipoxygenase isozymes that increasedlocally after wounding (Fig. 2B) also increased in un-wounded second trifoliate leaves from wounded plants,relative to controls (Fig. 3B, arrowheads). At 12 h afterwounding, increased levels of the two least acidic isozymes(pi = 4.90 and 4.95) of the group of four were detected inleaves from wounded plants (Fig. 3B, upper two arrow-heads). At 24 h postwounding, higher levels were detectedin second trifoliate leaves from wounded plants for all fourof the acidic lipoxygenase isozymes, compared to similarleaves from unwounded plants (Fig. 3B, arrowheads). By48 h after wounding, only the two least acidic isozymes(pi = 4.90 and 4.95) of the group of four were detected inleaves from wounded plants (Fig. 3B). As was the case withthe wounded, first trifoliate leaves, changes were not ob-served in the levels of the most abundant, acidic lipoxyge-nase isozymes (pi = 5.0-5.3) after wounding (Fig. 3B).

In Figures 2B and 3B, the four acidic wound-responsivelipoxygenase isozymes are not present in any lanes con-taining extracts from unwounded leaves. However, lowlevels of material that cross-reacted with the anti-lipoxy-genase-2 antibody and co-migrated with the four acidicwound-responsive isozymes were routinely observedwhen the immunoblots were overexposed (data notshown).

Effects of Reproductive Sink Removal onLipoxygenase Isozymes in Leaves

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Soybean Leaf Lipoxygenase Isozymes 539

48

48

" HOURS: 0 12 24

pl_ " " " . - + - .

7.2 —

6.8 — "̂̂ ^

6.6 —

B HOURS: 0 12 24

fL5.9 -

5.1 -

4.6 -

Figure 3. Immunoblot analysis of lipoxygenase isozymes in secondtrifoliate leaves (unwounded) from wounded (+) and unwounded (-)soybean plants. Lipoxygenase isozymes resolved between pH 6.0and 7.5 (A) and between pH 4.5 and 6.0 (B). For wounded plants,each leaflet of the first trifoliate leaves was wounded once with ahemostat at the start of the experiment (h 0). Leaf extracts containing100 ^g of soluble protein were subjected to native IEF and immu-noblotting as described in Figure 1. Arrowheads indicate the locationof lipoxygenase isozymes that increase after wounding, pis appear onthe left.

(Tranbarger et al., 1991). The 94-kD protein and two lowermolecular mass glycoproteins of 27 and 29 kD are believedto be involved in the temporary storage of nitrogen and canbe induced to accumulate in soybean leaves following theremoval of reproductive sink tissues (Wittenbach, 1982;Franceschi et al., 1983; Tranbarger et al., 1991). Using poly-clonal, anti-soybean leaf lipoxygenase antibodies, the94-kD protein was found to accumulate primarily in thevacuoles of leaf paraveinal mesophyll cells after flowerremoval and has been termed the paraveinal mesophylllipoxygenase (Tranbarger et al., 1991). However, the spe-cific lipoxygenase isozyme (or isozymes) that correspondsto this vegetative storage protein lipoxygenase was notidentified by Tranbarger et al. (1991). Recently, Kato et al.(1993) demonstrated that at least one specific soybean li-poxygenase isozyme (L-4) is a component of the 94-kDvegetative storage protein described by Tranbarger et al.(1991).

To identify which soybean lipoxygenase isozymes con-stitute the 94-kD vegetative storage protein, an investiga-tion of the effect of reproductive sink removal on soybeanleaf lipoxygenase isozymes was conducted. Flowers andflower buds were removed daily from soybean plants be-ginning at anthesis (75 d after planting) and continuing fora period of 2 weeks. The floral tissues were left on controlplants and seed pods were allowed to develop normally.Leaves were collected at anthesis, and 2 weeks later fromboth sets of plants. Unlike leaves from 1-month-old plants(Figs. 2A and 3A), leaves from plants at anthesis and 2weeks postanthesis (with pods) lacked the neutral lipoxy-genase isozymes (Fig. 4A). However, a lipoxygenase

isozyme with a pi of 6.9 was observed in leaves fromdeflowered plants (Fig. 4A). This lipoxygenase isozymewas also detected in leaves from younger plants (Figs. 2Aand 3A). The daily removal of floral tissues was found toincrease the levels of most of the acidic lipoxygenaseisozymes in soybean leaves (Fig. 4B). Two of the four acidiclipoxygenase isozymes that increased after wounding(pi = 4.90 and 4.95) also increased in response to repro-ductive sink removal (Fig. 4B, arrowheads). The other twoacidic isozymes (pi = 4.70 and 4.80) that increased afterwounding were not detected in leaves from reproductiveplants (Fig. 4B).

Identification of Active Lipoxygenase Isozymes inSoybean Leaves

In the experiments described in the preceding sections,immunoblotting was used to assess the levels of leaf li-poxygenase isozymes. However, this procedure does notdetermine whether these lipoxygenase isozymes possessenzyme activity. Some of the lipoxygenase isoforms de-tected immunologically could be pi variants resulting fromdenaturation or partial degradation of native isozymes.Isozymic variation was probably not due to proteolyticdegradation, however, because all leaf extracts used wereanalyzed by immunoblotting following SDS-PAGE andfound to possess only a single molecular mass species thatco-migrated with purified soybean seed lipoxygenase-2(data not shown). To identify active lipoxygenase iso-zymes, IEF followed by in-gel activity staining has beenemployed previously (Verhue and Francke, 1972; Funk etal., 1985; Park and Polacco, 1989).

p, 1 2 3

7.2 —

6.8 —

6.6 —

Pi 1 2 3B

5.9 —

•44.6 —

Figure 4. Immunoblot analysis of lipoxygenase isozymes in trifoliateleaves from plants with and without reproductive sinks. Lipoxyge-nase isozymes resolved between pH 6.0 and 7.5 (A) and between pH4.5 and 6.0 (B). For plants lacking reproductive sinks, floral tissueswere removed daily beginning at anthesis. Leaves were harvested atanthesis (lanes 1) and 2 weeks later from plants with (lanes 2) andwithout reproductive sinks (lanes 3). Leaf extracts containing TOO /j.gof soluble protein were subjected to native IEF and immunoblottingas described in Figure 1. Arrowheads indicate the position of wound-inducible lipoxygenase isozymes. pis appear on the left.




Extracts were prepared from immature leaves (from 19 dafter planting) and from both unwounded and woundedmature leaves and focused as before, although the amountof soluble protein per lane was increased to 400 jug. Afterfocusing, the gels were stained for lipoxygenase activity atpH 7.0 using linolenic acid as the substrate. In general,more intense lipoxygenase activity staining was observedwith extracts from immature leaves. Six activity-stainedbands were observed with an extract from immature leavesin the same region of the IEF gels where the neutral lipoxy-genase isozymes were observed by immunoblotting (Fig.5A). These same bands were also observed in matureleaves, and no significant differences in the intensity of anyband were observed when wounded and unwoundedleaves were compared (data not shown).

In the region of the major acidic isozymes (pi = 5.0-5.3)detected by immunoblotting, strong lipoxygenase activitystaining was observed using extracts from both un-wounded and wounded leaves (Fig. 6, A and B, bands 2-4).In the regions that corresponded to the locations of the fouracidic, wound-inducible lipoxygenase isozymes identifiedby immunoblotting, weak staining was observed (Fig. 6, Aand B, bands 5-8). However, the activity staining in theseregions was more intense with an extract from woundedleaves (Fig. 6B, bands 5-8) than with an extract from un-wounded leaves (Fig. 6A, bands 5-8). In several indepen-dent experiments, low levels of activity staining in theregion corresponding to the four acidic, wound-induciblelipoxygenases were always detected when extracts fromwounded leaves were utilized. Unfortunately, these faintbands are not readily apparent following photographicreproduction (Fig. 6, A and B, bands 5-8). In other exper-iments, linoleic acid was used as the substrate, but theactivity staining of all bands was more rapid and intensewith linolenic acid as the substrate (data not shown).Lipoxygenase activity staining was also conducted at pH5.0 (100 mM citrate), 6.0 (100 mM Mes), 8.0 (100 mM Tris),

IB

Figure 5. Identification of active lipoxygenase isozymes with pisbetween pH 6.0 and 7.5. A, Leaf proteins (400 /ig) resolved on anative IEF gel and stained for lipoxygenase activity. A soluble proteinextract was prepared from immature soybean leaves (19 d afterplanting) and subjected to IEF on a polyacrylamide gel. B, Immuno-blot of activity-stained bands 1 to 6 from A. Activity-stained bandswere excised from the gel shown in A and subjected to denaturing gelelectrophoresis on 12% SDS-polyacrylamide gels. Lipoxygenaseisozymes were detected by immunoblotting using a polyclonal anti-body prepared against soybean lipoxygenase-2. Molecular massesappear on the left in B. Lane S in B contains soybean lipoxygenase-2.

1 2 3 4 5 6 7 8 S

Figure 6. Identification of active lipoxygenase isozymes with pisbetween pH 4.5 and 6.0. Leaf proteins (400 (xg) from unwounded (A)and wounded (B) mature first trifoliate leaves resolved by native IEFand stained for lipoxygenase activity. Soluble protein extracts wereprepared from unwounded and wounded soybean leaves (12 h afterwounding) and subjected to IEF. C, Immunoblot of activity-stainedbands 1 to 8 from B. Activity-stained bands were excised from the gelshown in B and subjected to SDS-PACE and immunoblotting asdescribed in Figure 5. Molecular masses appear on the left in C. LaneS in C contains soybean lipoxygenase-2.

and 9.0 (100 mM borate). For all activity-stained bands fromleaf extracts, the staining was most intense at pH 7.0 withlinolenic acid as the substrate, and no additional bandsappeared under any other conditions (data not shown).Varying the pH and/or substrate did not appear to affectdifferentially the activity staining of individual leaf lipoxy-genase isozymes (data not shown).

To be certain that the activity-stained bands representedlipoxygenase isozymes, the stained bands were excisedfrom IEF gels, denatured, and subjected to immunoblot-ting. All activity-stained bands contained protein thatcross-reacted with anti-lipoxygenase-2 and co-migratedwith purified soybean seed lipoxygenase-2 (Figs. 5B and6C). In some lanes (Fig. 6C, lanes 2-8), two bands of verysimilar molecular mass were observed after immunoblot-ting. This may represent some partial degradation of thelipoxygenase isozymes during the 3-h activity-stainingprocedure.

DISCUSSION

IEF on polyacrylamide gels followed by immunoblottinghas been utilized to resolve the lipoxygenase isozymes insoybean leaves and to examine the levels of the differentisozymes in soybean leaves during leaf development (Fig.1), after wounding (Figs. 2 and 3), and in response toreproductive sink removal (Fig. 4). As leaf age increased,most lipoxygenase isozymes decreased in relative abun-dance. However, the four most acidic isozymes (pi = 4.70,4.80, 4.90, 4.95) were more abundant in intermediate-ageleaves than in either the youngest or the oldest leavesexamined (Fig. IB, arrowheads). After wounding, thesesame four lipoxygenase isozymes were found to increaseboth in wounded leaves (Fig. 2B, arrowheads) and in un-wounded leaves from wounded plants (Fig. 3B, arrow-heads). The removal of reproductive sinks resulted in in-creased levels of most all of the acidic lipoxygenaseisozymes in soybean leaves (Fig. 4B). Finally, neither



Soybean Leaf Lipoxygenase lsozymes 541

wounding nor reproductive sink removal resulted in the appearance of any new lipoxygenase isozymes that were not detected in leaves from untreated plants.

To determine whether the leaf lipoxygenase isozymes revealed by immunoblotting have enzyme activity, leaf extracts were subjected to IEF on polyacrylamide gels followed by in-gel staining for lipoxygenase activity. Activity- stained bands were detected in the same regions of the IEF gels as the lipoxygenase isozymes detected immunologically (Figs. 5A and 6, A and B). Furthermore, when the activity-stained bands were excised from the gels and subjected to SDS-PAGE, a11 activity-stained bands contained material that cross-reacted with anti-lipoxygenase-2 antibodies and had molecular masses similar to soybean lipoxygenase-2 (Figs. 58 and 6C). Given that the wound- responsive, acidic isozymes are present in relatively minor amounts (Figs. 2B and 3B), it is not surprising that their activity staining was relatively weak (Fig. 6B, bands 5-8). In several similar experiments, low levels of activity staining were consistently detected in this same region of IEF gels when extracts from wounded leaves were analyzed (not shown). These results indicate that the lipoxygenase isozymes identified by immunoblotting following IEF are active lipoxygenases that do not result from degradation. In addition, the relative levels of lipoxygenase isozymes detected by immunoblotting correlate well with the relative intensity of bands detected by in-gel staining for lipoxygenase activity (Figs. lA, 2B, 5A, and 6, A and B). This correlation implies that the anti-lipoxygenase-2 antibodies utilized have roughly equal avidity for the individual lipoxygenase isozymes in soybean leaves.

The lipoxygenase isozymes found in soybean seeds have been characterized by IEF, and p1 values of 5.68,6.25, and 6.15 have been observed for lipoxygenases-1, -2, and -3, respectively (Christopher et al., 1972). In the experiments presented here, the soybean seed lipoxygenase isozymes were not detected in extracts from leaves under any cir- cumstance. In a previous investigation of lipoxygenase isozymes found in unfractionated leaf extracts, lipoxygenase isozymes corresponding to the seed isozymes were not observed, but after the leaf extracts were fractionated by chromatofocusing, a lipoxygenase isozyme with a pI nearly identical to that of lipoxygenase-1 from soybean seeds was detected by immunoblotting (Grayburn et al., 1991). Whether this isozyme is present but undetectable in unfractionated leaf extracts or is an artifact of the purification procedure is unclear. Park and Polacco (1989) have identified active lipoxygenase isozymes in hypocotyl/radicle axes with pIs more acidic than the three seed isozymes. Although they were not examined extensively in the current investigation, two lipoxygenase isozymes were found to be common between leaves and hypocotyl/radicle axes (Fig. 1B). The observation that soybean leaves and hypocotyls possess some common acidic lipoxygenase isozymes was reported previously (Grayburn et al., 1991).

Tranbarger et al. (1991) have identified the 94-kD vegetative storage protein as a lipoxygenase. Following the removal of reproductive sink tissues, lipoxygenase accu- mulates primarily in the vacuoles of paraveinal mesophyll

cells in soybean leaves (Tranbarger et al., 1991). Grimes et al. (1992) suggested that the paraveinal mesophyll lipoxygenase is most likely the product of a single member of the soybean lipoxygenase gene family. More recently, however, Grimes et al. (1993) have suggested that the paraveinal mesophyll lipoxygenase is probably composed of several lipoxygenase isoforms. Kato et al. (1993) have demonstrated that soybean lipoxygenase-4 is elevated in leaves of depodded plants relative to control leaves from podded plants. Because the predicted amino acid sequence for lipoxygenase-4 was not identical to the partial amino acid sequence of the paraveinal mesophyll lipoxygenase reported by Tranbarger et al. (1991), Kato et al. (1993) suggested that paraveinal mesophyll lipoxygenase may be composed of several closely related proteins.

To identify other lipoxygenase isozymes that respond to reproductive sink removal, the lipoxygenase isozymes in leaves of deflowered plants were compared to leaves from podded control plants. After daily removal of floral tissues for 2 weeks, higher levels of a11 acidic lipoxygenase isozymes were observed in leaves, relative to podded controls (Fig. 4B). An increase in the leve1 of a single neutra1 lipoxygenase isozyme (pI = 6.9) also occurred in leaves in response to reproductive sink removal (Fig. 4A). The results presented here clearly indicate that the accumulation of lipoxygenase in soybean leaves following reproductive sink removal involves increases in the levels of multiple, primarily acidic, lipoxygenase isozymes and suggest that the paraveinal mesophyll lipoxygenase is not a single lipoxygenase isozyme. In addition, both the results of Kato et al. (1993) and the current study cal1 into question the partial amino acid sequence reported for the paraveinal mesophyll lipoxygenase, which was obtained by sequenc- ing peptides derived from cyanogen bromide cleavage of a 94-kD protein resolved by SDS-PAGE (Tranbarger et al., 1991). Because the individual lipoxygenase isozymes present in soybean leaves were not resolved prior to cyanogen bromide cleavage, it is unlikely that the peptide sequence obtained was from a single lipoxygenase polypeptide.

Although reproductive sink removal may alter leaf nitrogen status, it is possible that some of the lipoxygenase isozymes that increase after flower removal responded to influences other than leaf nitrogen status. Hildebrand et al. (1989) demonstrated that repetitive wounding of soybean leaves for 7 or more days causes local and systemic increases in lipoxygenase protein and activity. Because the removal of reproductive tissues cannot be accomplished without wounding, some of the increases in leaf lipoxygenase isozymes after reproductive sink removal may be part of a systemic response to wounding. Two of the acidic lipoxygenase isozymes (pI = 4.90 and 4.95) that increase locally and systemically after wounding (Figs. 2B and 3B, upper two arrowheads) also increased following reproductive sink removal (Figs. 4B, arrowheads). In addition, ABA has been shown to increase in soybean leaves after reproductive sink removal (Setter et al., 1980), and ABA has been reported to elevate the expression of Arabidopsis lipoxygenase-1 mRNA (Melan et al., 1993). Additional studies must




be conducted to determine the effects of plant growth substances, such as ABA and JA, on the levels of lipoxygenase isozymes in soybean leaves.

The neutral lipoxygenase isozymes detected in leaves from young vegetative plants (about 30 d after planting) (Figs. 2A and 3A) were not detected in leaves from plants at anthesis (75 d after planting) or from podded plants 2 weeks after anthesis (Fig. 4A). Although the physiological roles of the neutral lipoxygenase isozymes in soybean leaves are unknown, this observation indicates that these lipoxygenases are not involved in the physiological processes that occur within the leaves of unwounded, reproductive plants.

Wounding stimulated increases both locally and systemically in the levels of the four most acidic lipoxygenase isozymes. In wounded leaves, increases in a11 four isozymes occurred within 12 h after wounding (Fig. 2B). In unwounded second trifoliate leaves from plants with wounded first trifoliate leaves, increases in only two of the four acidic lipoxygenase isozymes were observed at 12 h after wounding (Fig. 3B, upper two arrowheads), but by 24 h postwounding increased levels of a11 four isozymes were detected compared to controls (Fig. 3B, arrowheads). In addition, a11 four of the acidic, wound-responsive lipoxygenase isozymes were observed in intermediate-age leaves from vegetative plants (Fig. lB), and two (pI = 4.90 and 4.95) of the four isozymes were observed in mature leaves from reproductive plants (Fig. 4B). These observa- tions coupled with the wound inducibility of the four acidic lipoxygenase isozymes suggest that these isozymes may be important in physiological processes that are common to both wounded and unwounded plants. Alterna- tively, the physiological functions of these four minor lipoxygenase isozymes may be exclusively for plant defense. Their presence prior to wounding may be necessary to aid in plant defenses before any wound-induced gene expression can occur. Subsequent increases in these four acidic lipoxygenase isozymes may be part of an amplifica- tion of the initial response to physical damage. Although the results presented here clearly indicate which lipoxygenase isozymes increase in abundance following physical damage to leaves, the lack of increases in other lipoxygenase isozymes in leaves does not exclude their participa- tion in plant defense responses.

The presence of multiple isozymes will make the task of defining the physiological roles of plant lipoxygenase chal- lenging. However, given the proposed roles of lipoxygenase in plant defense against pests and pathogens (Farmer and Ryan, 1992; Gundlach et al., 1992; Kauss et al., 1992; Koch et al., 1992; Ryan, 1992; Croft et al., 1993), understand- ing the function of lipoxygenase is important. Defining both the expression of individual lipoxygenase genes and the physiological roles of the specific lipoxygenase isozymes that they encode may be useful in developing strat- egies to genetically engineer crop plants that have en- hanced resistance to pests and pathogens. To understand fully the physiological functions of lipoxygenase in plants, the function of individual lipoxygenase isozymes must ultimately be determined.

Received July 28, 1994; accepted November 3, 1994. Copyright Clearance Center: 0032-0889/95/ 107/0535/09.

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