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 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 characterizations 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).
The levels of individual lipoxygenase isozymes in soybean [Clycine max (1.)Merr.] leaves were assessed during leaf development, after mechanical wounding, and i n response to reproductive sink removal. Native isoelectric focusing followed by immunoblotting was employed t o 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 p H 6.8 t o 7.2. The other class of lipoxygenase isozymes had acidic pls ranging from p H 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 intermediateage leaves than in either the youngest or oldest leaves examined. Following mechanical wounding of leaves, these same four acidic isozymes also increased i n 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 University, Box 7614, Raleigh, NC 27695-7614. * Corresponding author; e-mail
[email protected]; fax 1-919-613-8177.
Abbreviations: JA, jasmonic acid; TBST, 10 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween-20. 535
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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 A N D 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 located 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-
Plant Physiol. Vol. 107, 1995
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. Powdered 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 concentrations 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
Soybean Leaf Lipoxygenase Isozymes
537
in distilled H2O and stained with Ponceau S to determine protein transfer uniformity (Harlow and Lane, 1988). Staining and all subsequent incubations of the nitrocellulose filters were conducted at 25°C. After destaining in distilled H2O, the filters were incubated with agitation for 5 min in TEST, pH 8.0. This incubation was repeated twice with
First trifoliate leaves were harvested at four intervals (19, 23, 26, and 31 d after planting) spanning the complete cycle of leaf development. The initial harvest was made when the middle leaflet of the first trifoliate leaves had an average length of 16.8 mm. The final harvest consisted of mature, fully expanded leaves having an average middle leaf-
fresh TEST added each time. The filters were then incubated for 2 h in quench solution (5% [w/v] non-fat dry milk and 0.02% [w/v] sodium azide in TEST, pH 8.0). After two brief rinses in TEST, the filters were incubated in TEST for
let length of 61.6 mm. Average leaf weights (in mg) in order
15 min, followed by two additional washes of 5 min each. The filters were then incubated for 2 h with rabbit antisoybean lipoxygenase-2 antibodies (Peterman and Siedow, 1985a) diluted 1:2000 in quench solution. Following incubation with primary antibodies, the filters were rinsed twice with TEST and washed once for 15 min and twice for 5 min each in TEST. Goat anti-rabbit immunoglobulins conjugated to horseradish peroxidase (Sigma) were diluted 1:5000 in quench solution and incubated with the filters for 1 h. The filters were then rinsed three times, washed once
for 15 min and four times for 5 min in TEST. Bound, secondary antibodies were detected using the ECL chemiluminescence system according to the manufacturer's directions (Amersham). In-Gel Staining for Lipoxygenase Activity
After IEF was completed, the gels were rinsed in distilled H2O and stained for lipoxygenase activity by the method of
Funk et al. (1985) using the modifications employed by
of 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, soluble protein extracts were prepared from the leaf tissues, subjected to IEF on polyacrylamide gels, electrotransferred to nitrocellulose membranes, and immunoblotted using antisoybean lipoxygenase-2 antibodies. After initial experiments using IEF gels with a pH gradient from 4.0 to 7.0 (data not shown), it became clear that this pH gradient would not resolve all of the lipoxygenase isozymes in
soybean leaves. To resolve the leaf isozymes, each sample was electrophoresed on two separate IEF gels optimized for more narrow pH ranges. Several lipoxygenase isozymes having pis between 6.8 and 7.2 were detected in leaves of all ages (Fig. 1A). All the lipoxygenase isozymes in this region were most abundant in the youngest leaves examined and declined in amount as leaf age increased. In addition to the lipoxygenase isozymes with neutral pis, lipoxygenase isozymes with more acidic pis were also observed. The acidic lipoxygenases were observed to have pis ranging from 4.7 to 5.6. Assuming that the anti-lipoxy-
genase-2 antibody employed has roughly equal avidity for
Park and Polacco (1989). Both linolenic and linoleic acids
were used as substrates in separate experiments. Gels were incubated with gentle agitation in the presence of the reaction mixture for 2 to 3 h at 25°C. After staining was completed, the gels were rinsed several times with distilled H2O and incubated in distilled H2O with gentle agitation for 15 min at 25°C. After the gels were photographed, activity-stained bands were excised and stored at —80°C
LEAF AGE-
1
2
3
4
7.2 -
6.8 6.6 -
for later use in SDS-PAGE.
B SDS-PAGE and Immunoblotting
SDS-PAGE was performed on 82 X 60 X 1.5 mm slab gels according to Laemmli (1970) using 12% (w/v) acrylamide in the resolving gel. Following electrophoresis, immuno-
blotting was conducted as described above for lipoxygenase isozymes.
LEAF AGE p|
1
2
3
5.9 -
4
H
«">
5.1 -,
4.6 -
RESULTS
Developmental Patterns of Leaf Lipoxygenase Isozymes
Although multiple lipoxygenase isozymes have been demonstrated to exist in soybean leaves (Grayburn et al., 1991), the relationship between the levels of individual lipoxygenase isozymes and leaf age has not been determined. Developmental variation in total leaf lipoxygenase protein likely results from alterations in the levels of individual lipoxygenase isozymes. Therefore, an investigation of the developmental expression of the lipoxygenase isozymes in soybean leaves was conducted.
Figure 1. Immunoblot analysis of the developmental variation in lipoxygenase isozymes from first trifoliate soybean leaves. Lipoxygenase isozymes resolved between pH 6.0 and 7.5 (A) and between pH 4.5 and 6.0 (B). Leaf extracts were prepared from first trifoliate leaves harvested 19 (lanes 1), 23 (lanes 2), 26 (lanes 3), and 31 (lanes 4) d after planting. Leaf extracts containing 100 ^g of soluble protein were subjected to native IEF on polyacrylamide gels, and lipoxygenase isozymes were detected by immunoblotting using a polyclonal antibody prepared against soybean lipoxygenase-2. An extract from 3-d-old, etiolated hypocotyI/radicle axes was focused in lane H. Arrowheads in B indicate the position of the minor acidic lipoxygenase isozymes. pis appear on the left.
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Saravitz and Siedow
all the lipoxygenase isozymes, the relative abundance of the individual acidic isozymes is somewhat variable within leaves of any age class (Fig. IB). At all leaf ages, the most abundant isozymes have pis between 5.0 and 5.3. Many of the acidic isozymes were most abundant in the youngest leaves 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 than in either the youngest or oldest leaves (Fig. IB, arrowheads). For comparison with previously identified lipoxygenase isozymes, extracts prepared using hypocotyl/radical axes from 3-d-old, etiolated seedlings were subjected to IEF. In hypocotyl/radicle axes, two lipoxygenase isozymes with pis of 5.1 and 5.3 were observed that co-migrated with isozymes from leaves (Fig. IB). In addition, an abundant lipoxygenase isozyme (lipoxygenase-1) with a pi of 5.9 was detected in hypocotyl/radical axes but not in leaves of any age (Fig. IB). Effects of Wounding on Leaf Lipoxygenase Isozymes
Increases after wounding in the expression of at least two different members of the soybean lipoxygenase gene family have been shown to occur at the transcriptional level (Bell and Mullet, 1991). To date, the effects of wounding on the lipoxygenase isozymes in soybean leaves have not been
reported. Therefore, an investigation of the local and systemic effects of wounding on soybean leaf lipoxygenase isozymes was undertaken. For wounding experiments, approximately 1-month-old plants with mature, fully expanded first and second trifoliate leaves were used. At the start of the experiment, each leaflet of the first trifoliate leaves was wounded once by a single crush with a hemostat. To examine the local response of lipoxygenase iso-
Plant Physiol. Vol. 107, 1995
5.1
4.6 —
Figure 2. Immunoblot analysis of lipoxygenase isozymes in first trifoliate leaves from wounded ( + ) and unwounded (-) soybean plants. 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 of the first trifoliate leaves was wounded once with a hemostat at the start of the experiment (h 0). Leaf extracts containing TOO ju,g of soluble protein were subjected to native IEF and immunoblotting as described in Figure 1. Arrowheads indicate the location of lipoxygenase isozymes that increase after wounding, pis appear on the left.
from wounded and unwounded control plants at 12, 24, and 48 h after wounding. To examine the systemic response to wounding, second trifoliate leaves (unwounded)
3A). The four acidic lipoxygenase isozymes that increased locally after wounding (Fig. 2B) also increased in unwounded second trifoliate leaves from wounded plants, relative to controls (Fig. 3B, arrowheads). At 12 h after wounding, increased levels of the two least acidic isozymes (pi = 4.90 and 4.95) of the group of four were detected in leaves from wounded plants (Fig. 3B, upper two arrowheads). At 24 h postwounding, higher levels were detected in second trifoliate leaves from wounded plants for all four
were harvested from plants with wounded first trifoliate
of the acidic lipoxygenase isozymes, compared to similar
leaves and from unwounded controls. Soluble protein extracts were prepared from the leaf tissues and subjected to IEF followed by immunoblotting as described above. Several lipoxygenase isozymes were found to increase in abundance in first trifoliate leaves after wounding (Fig. 2). No consistent changes in the levels of the neutral lipoxy-
leaves from unwounded plants (Fig. 3B, arrowheads). By 48 h after wounding, only the two least acidic isozymes (pi = 4.90 and 4.95) of the group of four were detected in
zymes to wounding, first trifoliate leaves were harvested
genase isozymes were observed following wounding (Fig. 2A). Among the acidic, leaf lipoxygenase isozymes, however, the four relatively minor isozymes having the lowest pis (4.70, 4.80, 4.90, 4.95) increased after wounding (Fig. 2B,
arrowheads). In wounded leaves, the levels of all four acidic isozymes were higher than in unwounded controls at 12, 24, and 48 h after wounding. No changes were observed in the levels of the most abundant, acidic lipoxygenase isozymes (pi = 5.0-5.3) after wounding (Fig. 2B). In addition, systemic increases in the levels of specific lipoxygenase isozymes were detected in second trifoliate leaves following wounding (Fig. 3). Examination of the neutral soybean leaf isozymes present in second trifoliate leaves from both wounded and unwounded plants did not reveal any consistent increases in individual isozymes (Fig.
leaves from wounded plants (Fig. 3B). As was the case with
the wounded, first trifoliate leaves, changes were not observed in the levels of the most abundant, acidic lipoxygenase isozymes (pi = 5.0-5.3) after wounding (Fig. 3B). In Figures 2B and 3B, the four acidic wound-responsive lipoxygenase isozymes are not present in any lanes con-
taining extracts from unwounded leaves. However, low levels of material that cross-reacted with the anti-lipoxygenase-2 antibody and co-migrated with the four acidic wound-responsive isozymes were routinely observed when the immunoblots were overexposed (data not shown). Effects of Reproductive Sink Removal on Lipoxygenase Isozymes in Leaves
Recently, the 94-kD vegetative storage protein of soybean leaves was demonstrated to be a lipoxygenase
539
Soybean Leaf Lipoxygenase Isozymes
"
HOURS: pl_
0
12
""".-
+
24 -
48
.
7.2 — 6.8 —
^"^^
6.6 —
B
HOURS:
0
12
24
48
fL 5.9 -
5.1 -
4.6 -
Figure 3. Immunoblot analysis of lipoxygenase isozymes in second trifoliate leaves (unwounded) from wounded (+) and unwounded (-) soybean plants. 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 of the first trifoliate leaves was wounded once with a hemostat at the start of the experiment (h 0). Leaf extracts containing 100 ^g of soluble protein were subjected to native IEF and immunoblotting as described in Figure 1. Arrowheads indicate the location of lipoxygenase isozymes that increase after wounding, pis appear on the left.
(Tranbarger et al., 1991). The 94-kD protein and two lower molecular mass glycoproteins of 27 and 29 kD are believed to be involved in the temporary storage of nitrogen and can be induced to accumulate in soybean leaves following the removal of reproductive sink tissues (Wittenbach, 1982;
isozyme with a pi of 6.9 was observed in leaves from deflowered plants (Fig. 4A). This lipoxygenase isozyme was also detected in leaves from younger plants (Figs. 2A and 3A). The daily removal of floral tissues was found to increase the levels of most of the acidic lipoxygenase isozymes in soybean leaves (Fig. 4B). Two of the four acidic lipoxygenase isozymes that increased after wounding (pi = 4.90 and 4.95) also increased in response to reproductive sink removal (Fig. 4B, arrowheads). The other two acidic isozymes (pi = 4.70 and 4.80) that increased after wounding were not detected in leaves from reproductive plants (Fig. 4B). Identification of Active Lipoxygenase Isozymes in Soybean Leaves
In the experiments described in the preceding sections, immunoblotting was used to assess the levels of leaf lipoxygenase isozymes. However, this procedure does not determine whether these lipoxygenase isozymes possess enzyme activity. Some of the lipoxygenase isoforms detected immunologically could be pi variants resulting from denaturation or partial degradation of native isozymes. Isozymic variation was probably not due to proteolytic degradation, however, because all leaf extracts used were analyzed by immunoblotting following SDS-PAGE and found to possess only a single molecular mass species that co-migrated with purified soybean seed lipoxygenase-2 (data not shown). To identify active lipoxygenase isozymes, IEF followed by in-gel activity staining has been employed previously (Verhue and Francke, 1972; Funk et al., 1985; Park and Polacco, 1989).
Franceschi et al., 1983; Tranbarger et al., 1991). Using poly-
clonal, anti-soybean leaf lipoxygenase antibodies, the
p,
94-kD protein was found to accumulate primarily in the
vacuoles of leaf paraveinal mesophyll cells after flower removal and has been termed the paraveinal mesophyll lipoxygenase (Tranbarger et al., 1991). However, the specific lipoxygenase isozyme (or isozymes) that corresponds to this vegetative storage protein lipoxygenase was not identified by Tranbarger et al. (1991). Recently, Kato et al. (1993) demonstrated that at least one specific soybean lipoxygenase isozyme (L-4) is a component of the 94-kD vegetative storage protein described by Tranbarger et al. (1991). To identify which soybean lipoxygenase isozymes constitute the 94-kD vegetative storage protein, an investigation of the effect of reproductive sink removal on soybean
leaf lipoxygenase isozymes was conducted. Flowers and flower buds were removed daily from soybean plants beginning at anthesis (75 d after planting) and continuing for a period of 2 weeks. The floral tissues were left on control plants and seed pods were allowed to develop normally.
Leaves were collected at anthesis, and 2 weeks later from both sets of plants. Unlike leaves from 1-month-old plants (Figs. 2A and 3A), leaves from plants at anthesis and 2 weeks postanthesis (with pods) lacked the neutral lipoxygenase isozymes (Fig. 4A). However, a lipoxygenase
1
2
3
7.2 — 6.8 — 6.6 —
B Pi
1
2
3
5.9 —
•4 4.6 —
Figure 4. Immunoblot analysis of lipoxygenase isozymes in trifoliate leaves from plants with and without reproductive sinks. Lipoxygenase isozymes resolved between pH 6.0 and 7.5 (A) and between pH 4.5 and 6.0 (B). For plants lacking reproductive sinks, floral tissues were removed daily beginning at anthesis. Leaves were harvested at anthesis (lanes 1) and 2 weeks later from plants with (lanes 2) and without reproductive sinks (lanes 3). Leaf extracts containing TOO /j.g of soluble protein were subjected to native IEF and immunoblotting as described in Figure 1. Arrowheads indicate the position of woundinducible lipoxygenase isozymes. pis appear on the left.
540
Saravitz and Siedow
Extracts were prepared from immature leaves (from 19 d after planting) and from both unwounded and wounded mature leaves and focused as before, although the amount of soluble protein per lane was increased to 400 jug. After focusing, the gels were stained for lipoxygenase activity at pH 7.0 using linolenic acid as the substrate. In general, more intense lipoxygenase activity staining was observed with extracts from immature leaves. Six activity-stained bands were observed with an extract from immature leaves in the same region of the IEF gels where the neutral lipoxygenase isozymes were observed by immunoblotting (Fig. 5A). These same bands were also observed in mature leaves, and no significant differences in the intensity of any band were observed when wounded and unwounded
leaves were compared (data not shown). In the region of the major acidic isozymes (pi = 5.0-5.3) detected by immunoblotting, strong lipoxygenase activity staining was observed using extracts from both unwounded and wounded leaves (Fig. 6, A and B, bands 2-4). In the regions that corresponded to the locations of the four acidic, wound-inducible lipoxygenase isozymes identified by immunoblotting, weak staining was observed (Fig. 6, A and B, bands 5-8). However, the activity staining in these
regions was more intense with an extract from wounded leaves (Fig. 6B, bands 5-8) than with an extract from unwounded leaves (Fig. 6A, bands 5-8). In several independent experiments, low levels of activity staining in the region corresponding to the four acidic, wound-inducible lipoxygenases were always detected when extracts from wounded leaves were utilized. Unfortunately, these faint bands are not readily apparent following photographic reproduction (Fig. 6, A and B, bands 5-8). In other experiments, linoleic acid was used as the substrate, but the activity staining of all bands was more rapid and intense with linolenic acid as the substrate (data not shown).
Lipoxygenase activity staining was also conducted at pH 5.0 (100 mM citrate), 6.0 (100 mM Mes), 8.0 (100 mM Tris),
I B
Plant Physiol. Vol. 107, 1995
1
2
3
4
5
6
7
8
S
Figure 6. Identification of active lipoxygenase isozymes with pis between pH 4.5 and 6.0. Leaf proteins (400 (xg) from unwounded (A) and wounded (B) mature first trifoliate leaves resolved by native IEF and stained for lipoxygenase activity. Soluble protein extracts were prepared from unwounded and wounded soybean leaves (12 h after wounding) and subjected to IEF. C, Immunoblot of activity-stained bands 1 to 8 from B. Activity-stained bands were excised from the gel shown in B and subjected to SDS-PACE and immunoblotting as described in Figure 5. Molecular masses appear on the left in C. Lane S in C contains soybean lipoxygenase-2.
and 9.0 (100 mM borate). For all activity-stained bands from leaf extracts, the staining was most intense at pH 7.0 with linolenic acid as the substrate, and no additional bands appeared under any other conditions (data not shown).
Varying the pH and/or substrate did not appear to affect differentially the activity staining of individual leaf lipoxygenase isozymes (data not shown). To be certain that the activity-stained bands represented lipoxygenase isozymes, the stained bands were excised from IEF gels, denatured, and subjected to immunoblotting. All activity-stained bands contained protein that cross-reacted with anti-lipoxygenase-2 and co-migrated with purified soybean seed lipoxygenase-2 (Figs. 5B and
6C). In some lanes (Fig. 6C, lanes 2-8), two bands of very similar molecular mass were observed after immunoblotting. This may represent some partial degradation of the lipoxygenase isozymes during the 3-h activity-staining procedure. DISCUSSION
IEF on polyacrylamide gels followed by immunoblotting has been utilized to resolve the lipoxygenase isozymes in soybean leaves and to examine the levels of the different isozymes in soybean leaves during leaf development (Fig.
1), after wounding (Figs. 2 and 3), and in response to reproductive sink removal (Fig. 4). As leaf age increased, Figure 5. Identification of active lipoxygenase isozymes with pis between pH 6.0 and 7.5. A, Leaf proteins (400 /ig) resolved on a native IEF gel and stained for lipoxygenase activity. A soluble protein extract was prepared from immature soybean leaves (19 d after planting) and subjected to IEF on a polyacrylamide gel. B, Immunoblot of activity-stained bands 1 to 6 from A. Activity-stained bands were excised from the gel shown in A and subjected to denaturing gel electrophoresis on 12% SDS-polyacrylamide gels. Lipoxygenase isozymes were detected by immunoblotting using a polyclonal antibody prepared against soybean lipoxygenase-2. Molecular masses appear on the left in B. Lane S in B contains soybean lipoxygenase-2.
most lipoxygenase isozymes decreased in relative abundance. However, the four most acidic isozymes (pi = 4.70, 4.80, 4.90, 4.95) were more abundant in intermediate-age
leaves than in either the youngest or the oldest leaves examined (Fig. IB, arrowheads). After wounding, these same four lipoxygenase isozymes were found to increase
both in wounded leaves (Fig. 2B, arrowheads) and in unwounded leaves from wounded plants (Fig. 3B, arrowheads). The removal of reproductive sinks resulted in increased levels of most all of the acidic lipoxygenase isozymes in soybean leaves (Fig. 4B). Finally, neither
Soybean Leaf Lipoxygenase lsozymes 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. Activitystained 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 woundresponsive, 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 circumstance. 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 accumulates primarily in the vacuoles of paraveinal mesophyll
541
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 sequencing 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
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Saravitz and Siedow
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 observations 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. Alternatively, 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 amplification 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 participation in plant defense responses. The presence of multiple isozymes will make the task of defining the physiological roles of plant lipoxygenase challenging. 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),understanding 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 strategies to genetically engineer crop plants that have enhanced 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.
Plant Physiol. Vol. 107, 1995
Received July 28, 1994; accepted November 3, 1994. Copyright Clearance Center: 0032-0889/95/ 107/0535/09. LITERATURE ClTED
Altschuler M, Grayburn WS, Collins GB, Hildebrand DF (1989) Developmental expression of lipoxygenases in soybeans. Plant Sci 63: 151-158 Bell E, Mullet JE (1991) Lipoxygenase gene expression is modulated in plants by water deficit, wounding, and methyl jasmonate. Mo1 Gen Genet 230: 456-462 Bell E, Mullet JE (1991) Characterization of an Arabidopsis lipoxygenase gene responsive to methyl jasmonate and wounding. Plant Physiol 103: 1133-1 137 Christopher JP, Pistorius EK, Axelrod B (1970) Isolation of an isoenzyme of soybean lipoxygenase. Biochim Biophys Acta 198: 12-19 Christopher JP, Pistorius EK, Axelrod B (1972) Isolation of a third isoenzyme of soybean lipoxygenase. Biochim Biophys Acta 2 8 4 54-62 Creelman RA, Bell E, Mullet JE (1992) Involvement of a lipoxygenase-like enzyme in abscisic acid biosynthesis. Plant Physiol 99: 1258-1260 Croft KPC, Jiittner F, Slusarenko AJ (1993) Volatile products of the lipoxygenase pathway evolved from Phaseolus vulgaris (L.) leaves inoculated with Pseudomonas syringae pv phaseolicola. Plant Physiol 101: 13-24 Croft KPC, Voisey CR, Slusarenko AJ (1990) Mechanism of hypersensitive cell collapse: correlation of increased lipoxygenase activity with membrane damage in leaves of Phaseolus vulgaris (L.) inoculated with an avirulent race of Pseudomonas syringae pv. phaseolicola. Physiol Mo1 Plant Pathol 36: 49-62 Domoney C, Firmin JL, Sidebottom C, Ealing PM, Slabas A, Casey R (1990) Lipoxygenase heterogeneity in Pisum sativum. Planta 181: 3 5 4 3 Farmer EE, Ryan CA (1992) Octadecanoid precursors of jasmonic acid activate the synthesis of wound-inducible proteinase inhibitors. Plant C e l l 4 129-134 Franceschi VR, Wittenbach VA. Giaquinta RT (1983) Paraveinal mesophyll of soybean leaves in relation to assimilate transfer and compartmentation. 111. Immunohistochemical localization of specific glycopeptides in the vacuole after depodding. Plant Physiol72 586-589 Funk MO, Whitney MA, Hausknecht EC, OBrien EM (1985) Resolution of the isoenzymes of soybean lipoxygenase using isoelectric focusing and chromatofocusing. Ana1 Biochem 146 246-251 Grayburn WS, Schneider GR, Hamilton-Kemp TR, Bookjans G, Ali K, Hildebrand DF (1991) Soybean leaves contain multiple lipoxygenases. Plant Physiol 9 5 1214-1218 Grimes HD, Koetje DS, Franceschi VR (1992) Expression, activity, and cellular accumulation of methyl jasmonate-responsive lipoxygenase in soybean seedlings. Plant Physiol 100: 433443 Grimes HD, Tranbarger TJ, Franceschi VR (1993) Expression and accumulation patterns of nitrogen-responsive lipoxygenase in soybeans. Plant PhysiollO3: 457-466 Gundlach H, Miiller MJ, Kutchan TM, Zenk MH (1992)Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc Natl Acad Sci USA 8 9 2389-2393 Harlow E, Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Hildebrand DF, Hamilton-Kemp TR, Legg CS, Bookjans G (1988). Plant lipoxygenases: occurrence, properties and possible functions. Curr Top Plant Biochem Physiol 7: 201-219 Hildebrand DF, Rodriguez JG, Legg CS, Brown GC, Bookjans G (1989) The effects of wounding and mite infestation on soybean leaf lipoxygenase levels. Z Naturforsch 44c: 655-659 Ida S, Masaki Y, Morita Y (1983) The isolation of multiple forms and product specification of rice lipoxygenase. Agric Biol Chem 47: 637-641 Kato T, Maeda Y, Hirukawa T, Naimai T, Yoshioka N (1992a) Lipoxygenase activity increment in infected tomato leaves and
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Soybean Leaf Lipoxygenase lsozymes oxidation product of linolenic acid by its in vitro enzyme reaction. Biosci Biotechnol Biochem 56: 373-375 Kato T, Ohta H, Tanaka K, Shibata D (1992b) Appearance of new lipoxygenases in soybean cotyledons after germination and evidence for expression of a major new lipoxygenase gene. Plant Physiol98 324-330 Kato T, Shirano Y, Iwamoto H, Shibata D (1993) Soybean lipoxygenase L-4, a component of the 94-kilodalton storage protein in vegetative tissues: expression and accumulation in leaves induced by pod remova1 and by methyl jasmonate. Plant Cell Physiol 34: 1063-1072 Kauss H, Krause K, Jeblick W (1992) Methyl jasmonate conditions parsley suspension cells for increased elicitation of phenylpropanoid defense responses. Biochem Biophys Res Commun 189: 304-308 Keppler LD, Novacky A (1987) The initiation of membrane lipid peroxidation during bacteria-induced hypersensitive reactions. Physiol Mo1 Plant Biol 3 0 233-245 Koch E, Meier BM, Eiben H-G, Slusarenko A (1992) A lipoxygenase from leaves of tomato (Lycopersicon esculentum Mill.) is induced in response to plant pathogenic pseudomonads. Plant Physiol 99: 571-576 Koda Y (1992)The role of jasmonic acid and related compounds in the regulation of plant development. Int Rev Cytoll35: 155-199 Laemmli UK (1970)Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mo1 Biol 80: 575-599 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 193: 263-275 Melan MA, Dong X, Endara ME, Davis KR, Ausubel FM, Peterman TK (1993) An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate. Plant Physiol 101: 441450 Mulliez E, LeBlanc J-P, Girerd J-J, Riguad M, Chottard J-C (1987) 5-Lipoxygenase from potato tubers. Improved purification and physicochemical characteristics. Biochim Biophys Acta 916: 13-23 Ohta H, Shida K, Peng Y-L, Furusawa I, Shishiyama J, Aibara S, Morita Y (1990) The occurrence of lipid hydroperoxide-decomposing activities in rice and the relationship of such activities to the formation of antifungal substances. Plant Cell Physiol 31: 1117-1122 Ohta H, Shida K, Peng Y-L, Furusawa I, Shishiyama J, Aibara S, Morita Y (1991) A lipoxygenase pathway is activated in rice
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after infection with the rice blast fungus Magnaporthe grisea. Plant Physiol 97: 94-98 Park TK, Polacco JC (1989)Distinct lipoxygenase species appear in the hypocotyl/radicle of germinating soybean. Plant Physiol90: 285-290 Parthier b (1991)Jasmonates, new regulators of plant growth and development: many facts and few hypotheses on their actions. Bot Acta 104: 446-454 Peterman TK, Siedow JN (1985a) Immunological comparison of lipoxygenase isoenzyme-1 and -2 with soybean seedling lipoxygenases. Arch Biochem Biophys 3 8 476-483 Peterman TK, Siedow JN (198513)Behavior of lipoxygenase during establishment, senescence, and rejuvenation of soybean cotyledons. Plant Physiol 78: 690-695 Robertson EF, Dannelly HK, Malloy PJ, Reeves HC (1987) Rapid isoelectric focusing in a vertical polyacrylamide minigel system. Ana1 Biochem 167: 290-294 Ryan CA (1992) The search for the proteinase inhibitor-inducing factor, PIIF. Plant Mo1 Biol 19: 123-133 Setter TL, Brun WA, Brenner ML (1980) Effect of obstructed translocation on leaf abscisic acid, and associated stomatal closure and photosynthesis decline. Plant Physiol 64: 1111-1115 Siedow JN (1991) Plant lipoxygenase: structure and function. Annu Rev Plant Physiol Plant Mo1 Biol42 145-188 Todd JF, Paliyath G, Thompson JE (1990) Characteristics of a membrane-associated lipoxygenase in tomato fruit. Plant Physiol 94: 1225-1232 Tranbarger JL, Franceschi VR, Hildebrand DF, Grimes HD (1991) The soybean 94-kilodalton vegetative storage protein is a lipoxygenase that is localized in paraveinal mesophyll cell vacuoles. Plant Cell 3: 973-987 Verhue WM, Francke A (1972) The heterogeneity of soyabean lipoxygenase. Biochim Biophys Acta 285: 43-53 Vick BA, Zimmerman DC (1987) Oxidative systems for the modification of fatty acids: the lipoxygenase pathway. In PK Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol 9. Academic Press, New York, pp 53-90 Wittenbach VA (1982) Purification and characterization of a soybean leaf storage glycoprotein. Plant Physiol 73: 125-129 Yamamoto H, Tani T (1986)Possible involvement of lipoxygenase in the mechanism of resistance of oats to Puccina coronata avenae. J Phytopathol 116: 329-337 Yamamoto S (1992) Mammalian lipoxygenases: molecular studies and functions. Biochim Biophys Acta 1128: 117-131
