Plant Physiol.

(1992) 98, 324-330

0032-0889/92/98/0324/07/$01 .00/0

Received for publication May 3, 1991 Accepted August 5, 1991

Appearance of New Lipoxygenases in Soybean Cotyledons after Germination and Evidence for Expression of a Major New Lipoxygenase Gene
Tomohiko Kato, Hiroyuki Ohtal, Kunisuke Tanaka, and Daisuke Shibata* Mitsui Plant Biotechnology Research Institute, TCI-A 10, Sengen 2-1-6, Tsukuba, Ibaraki 305, Japan (T.K., H.O., D.S.); and Department of Biochemistry, College of Agricultural Chemistry, Kyoto Prefectural University, Shimogamo, Kyoto 606, Japan (K. T.)
ABSTRACT
The appearance and subsequent disappearance of lipoxygenase activity at pH 6.8 in germinated cotyledons of soybean (Glycine max [L.]) was shown using a variant soybean cultivar (Kanto 101) that lacks the two lipoxygenase isozymes, L-2 and L-3, that are present in dry seeds of a normal soybean cultivar (Enrei). Three new lipoxygenases, designated lipoxygenase L-4, L-5, and L-6, were purified using anionic or cationic ion exchange chromatography. The major lipoxygenase in 5-day-old cotyledons of the variant soybean was lipoxygenase L-4. Lipoxygenases L-5 and L-6 preferentially produced 13(S)-hydroperoxy-9(Z),11(E)octadecadienoic acid (13S-HPOD) as a reaction product of linoleic acid, whereas lipoxygenase L-4 produced both 13S-HPOD and 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid. All three isozymes have pH optima of 6.5, no activity at pH 9.0, and preferred linolenic acid to linoleic acid as a substrate. Partial amino acid sequencing of lipoxygenase L-4 showed that this isozyme shares amino acid sequence homology with lipoxygenases L-1, L-2, and L-3 but is not identical to any of them. This indicates that a new lipoxygenase, L-4, is expressed in

cotyledons.

Lipoxygenases are enzymes that use molecular oxygen to produce hydroperoxides from fatty acids that have a cis,cispentadiene structure. These enzymes are widely distributed in nature. A number of lipoxygenases have been enzymatically characterized, particularly soybean lipoxygenase L- 1 (1). Recently, the soybean cDNAs encoding lipoxygenases L-1 (16), L-2 (17), and L-3 (21), and the soybean genomic DNAs encoding lipoxygenases L-3 (21) and SC514 (15), have been cloned. Lipoxygenases are involved in a number of diverse aspects of plant physiology, including growth and development, pest resistance, senescence, and wounding responses (see refs. 3, 5, and 8 for recent reviews). For example, jasmonic acid, one product of a lipoxygenase pathway in plant cells (20), has been proposed as a regulator of gene expression (18), and Ohta et al. (1 1) suggested that lipoxygenases in rice leaves are involved in the defense mechanism against rice blast
' Present address: Department of Biological Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Yokohama 227, Japan.

disease. Thus, although the biochemistry of lipoxygenases has been studied extensively, the role of these enzymes in plant metabolism remains rather speculative. Mature soybean seeds contain high levels of lipoxygenases L-1, L-2, and L-3. The activity of these lipoxygenases were measured in cotyledons after germination and the activity of the L-l isozyme at pH 9.0 was shown to disappear by day 9 after germination (6, 14). Hildebrand and Hymowitz (6) found that lipoxygenase activity that was measured at pH 7.0 was maximal between days 4 and 7 of germination, whereas Peterman and Siedow (14) showed that the pH 6.8 activity in soybean cotyledons continued to decrease in the early stages after germination. The discrepancy in these results may be due to different conditions of plant growth, because the former study was conducted in dark-grown cotyledons, whereas the latter work was done on cotyledons grown in the light (14). These researchers associated the activity at pH 6.8 with lipoxygenases L-2 and L-3; however, no evidence was given to prove that these two enzymes were the only ones responsible for this activity. We isolated three different cDNAs from a library made from germinated soybean cotyledons (15). One of the cDNAs, SC514 (15), and the other cDNAs (our unpublished data) shared nucleotide sequence homology with the genes encoding L-1, L-2, and L-3, but are not identical to these genes (15). These findings suggested that new, previously unidentified lipoxygenase genes are expressed in the cotyledons, presumably after germination. We purified the lipoxygenases in germinating cotyledons and studied their enzymatic properties to understand the physiological role of these enzymes. We report on the appearance of three new lipoxygenases in soybean cotyledons after germination and their enzymatic characteristics. Amino acid sequencing of one of these proteins indicates that it is expressed from a different gene than those previously identified.
MATERIALS AND METHODS Plant Materials Soybean seeds (Glycine max [L.]) of a lipoxygenase-minus cultivar, Kanto 101, were kindly provided by Dr. K. Kitamura, National Agriculture Research Center, Tsukuba, Ibaraki, Japan. The dry seeds were sterilized by immersion in 0.1 % benomyl[methyl-(butylcarbamoyl)-2-benzoimidazole324

LIPOXYGENASES FROM GERMINATED COTYLEDONS OF SOYBEAN

325

carbamate] (BENLATE, Du Pont) for 6 h at room temperature, then grown in vermiculite at 28C for 9 d under a photoperiod of 16 h light/8 h dark.
Enzyme Assay

extract (12 mL) was then desalted on a Sephadex G-25 column (2.7 x 23.5 cm) equilibrated with buffer A: 10 mM sodium

Lipoxygenase activity was measured by the method of Grossman and Zakut (4) with the minor modification of using an oxygen electrode. The reaction mixture (3.0 mL) contained 7.5 mM linoleic acid and 0.25% (w/v) Tween 20 in 0.1 M sodium phosphate (pH 6.8) or 0.1 M Tris/Cl (pH 9.0). The activity (1 kat) is defined as the quantity of enzyme catalyzing the consumption of 1 mol 02/s at 30C. The specific activities of the enzyme fractions were calculated based on the amount of protein in the fraction. Protein was estimated by the Bradford method (2) using bovine plasma gamma globulin as a standard.
Extraction and Purification of Enzymes

Extraction and purification of the soybean lipoxygenases were carried out according to the methods of Ohta et al. for purifying rice lipoxygenases (9) with several modifications. Germinated cotyledons (5 g) were homogenized with 25 mL of 50 mm sodium phosphate and 1.5% (w/v) Triton X-100 (pH 6.8) using a Polytron homogenizer and centrifuged at 18,000g for 15 min. The supernatant was used as a crude extract and assayed for lipoxygenase activity (Fig. 1). Crude extracts from 5-d-old cotyledons were further purified by adding Amberlite XDA-2 (5 g) to remove Triton X-100. This

phosphate, 10% (w/v) glycerol, 0.1 mm EDTA, and 0.1 % (w/ v) Tween 20 (pH 6.8). The crude extract was clarified by passing it through either a DEAE- or a CM-Toyopearl column. This was necessary to obtain good resolution of the lipoxygenase activities, which were finally separated on either a Mono Q or a Mono S column. The desalted extract was applied to a DEAE-Toyopearl column (1.0 x 11 cm) equilibrated with buffer A, then washed with buffer A. The enzyme activities bound to the DEAEToyopearl column were eluted with 100 mm sodium phosphate, 10% (w/v) glycerol, 0.1 mm EDTA, 0.1% (w/v) Tween 20 (pH 6.8). The eluate (16 mL) was concentrated to 8 mL using a Centriprep 30 (Amicon), then desalted on a Sephadex G-25 column (2.7 x 23.5 cm) with buffer A. The desalted solution was applied to a Mono Q HR5/5 column equilibrated with buffer A and the enzyme activities were eluted with a linear gradient of NaCl, 0 to 0.2 M, in buffer A. Lipoxygenase activity not bound to the DEAE-Toyopearl column was prepared from 20 g of cotyledons using a larger scale procedure. This preparation was adjusted to pH 6.2 by adding 0.1 N HCI, then applied to a CM-Toyopearl column (1.0 x 11 cm) equilibrated with buffer B: 10 mm sodium malonate, 10% (w/v) glycerol, 0.1 mM EDTA, 0.1% Tween 20 (pH 6.2). The lipoxygenase activity was eluted with 0.5 M NaCl in buffer B. After desalting the extract with buffer B, it was applied to a Mono S HR5/5 column equilibrated with buffer B. Enzyme activities were eluted with a linear gradient

C
0

4'

LL.

Days after planting

Figure 1. Changes in lipoxygenase activities and fresh weight in soybean cotyledons after germination. A normal soybean cultivar, Enrei (0), and a variant line, Kanto 101 (0), which lacks both lipoxygenases L-2 and L-3 in dry seeds, were grown at 28C under 16 h light/8 h dark. Activities at pH 9.0 (a) and 6.8 (b) in cotyledons were measured using linoleic acid as the substrate. Fresh weights were shown in c. Five pairs of cotyledons were analyzed for each time point. The points are the mean values of three different experiments and the bars denote SD; where absent, SD limits are within the symbol dimensions.

326

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Plant Physiol. Vol. 98, 1992

of NaCl, 0 to 0.25 M, in buffer B. The enzyme fractions were stored at -20C. Lipoxygenases L-1, L-2, and L-3, isolated from ungerminated seeds of cv Enrei, with wild-type levels of lipoxygenases L-1, L-2, and L-3, were also placed on a Mono Q column to determine their elution positions from this column under the same conditions as those used to separate lipoxygenases L-4, L-5, and L-6.
Determination of Regiospecificity for Hydroperoxidation Fractions containing 1.7 nkat of lipoxygenase activity were added to 1 mL of 2 mm linoleic acid and 0.25% (w/v) Tween 20 in 0.1 M sodium phosphate (pH 6.8) and incubated at 4C for 1 h with vigorous mixing. The reaction products were extracted using chloroform and then subjected to HPLC. HPLC was performed on a silica column (6 x 150 mm, YMCPack A-01 2 S-5 120A) using hexane:isopopanol:acetic acid (97.3:2.5:0.2, v/v) as a solvent. The retention times of the reaction products were compared with those of authentic standards.
Amino Acid Sequencing The lipoxygenase L-4 fraction was desalted using a Centricon 30 (Amicon) with deionized water and then lyophilized. The enzyme (200 ,ug) was dissolved in 100 ML of 8 M urea, 0.1 M Tris/Cl (pH 8.0), then diluted with 300 uL of 0.1 M Tris/Cl (pH 8.0). The protein was incubated with 2 MAg of

lysylendopeptidase (Wako Pure Chemical Industries Ltd, Osaka, Japan) at 37C for 6 h. Complete digestion was verified by SDS-PAGE. The digested mixture was lyophilized, then dissolved in 100 ML of 0.1% trifluoroacetate. The peptides were resolved on reverse-phase HPLC using an ODS column (4.6 x 250 mm, Biofine RPC-SC1 8, Japan Spectroscopic Co., Ltd., Tokyo, Japan) equilibrated with 0.1% trifluoroacetate. The peptides were eluted with linear gradients of acetonitrile in trifluoroacetate (0-20% for the first 5 min, then 20-80% for another 60 min) at a flow rate of 1 mL/min and monitored at 220 nm. The separated peptides were lyophilized and then subjected to automated amino acid sequencing on an ABI sequenator.
RESULTS Changes in Lipoxygenase Activities in Cotyledons after Germination Lipoxygenase activities in soybean cotyledons were measured at pH 6.8 and pH 9.0 until 9 d postgermination in two soybean cultivars (Fig. 1); Enrei is a normal cultivar with respect to lipoxygenase, and Kanto 101 is a cultivar that is missing some of the lipoxygenase activities present in Enrei. Enrei has three lipoxygenase activities, L-1, L-2, and L-3, in dry seeds (Fig. 2a). Kanto 101 contains lipoxygenase L-1, active at pH 9.0, but completely lacks lipoxygenases L-2 and L-3, both of which are active at pH 6.8 in dry seeds (7) (Fig. 2c). We took advantage of the absence of these lipoxygenase

0.68a

L-2

L-3

L-1

1.0
0.8

0..6

0-6E
-O.

E
O. vw m x

:0.40
0-.-~~~~~~~~~~~.
b
L-4 L-2 L-3 L-5 L-1

00
X 0
Lo

0.2 8

>,0..8
4 ._

.6L
.4 ,'~~~~~~~~~~.

.0.6 0 O

0.

0.44
0.2
rl

0,
-

o 10 20 30 40 50 Fraction number (0.5ml/tube)

10 20 30 40 50 Fraction number (0.5ml/tube)

Figure 2. Mono 0 column chromatography of cotyledon lipoxygenases. Lipoxygenases were extracted from ungerminated (a, c) or 5-d-old (b, d) cotyledons of the normal soybean, Enrei (a, b), or the variant line, Kanto 101 (c, d). Lipoxygenases L-1, L-2, L-3, L-4, and L-5 were separated on the Mono Q column using a linear gradient of NaCI in the buffer (- - -). The lipoxygenase activity was measured at pH 6.8 (0) or 9.0 (0). The activity toward arachidonic acid was also measured for the L-2 and L-3 fractions of a (data not shown). (- - - -) Absorbance at 280 nm.

LIPOXYGENASES FROM GERMINATED COTYLEDONS OF SOYBEAN

327

activities in the Kanto 101 line and identified lipoxygenase activities that are active at pH 6.8 and are either new activities or modified forms of the lipoxygenases present in dry seeds. Figure 1 shows lipoxygenase activities in germinated cotyledons of both of these cultivars at pH 6.8 and 9.0. The fresh weight of the cotyledons of both cultivars increased approximately twofold during the 9 d after germination; however, the weight of Kanto 101 cotyledons was approximately 30% less than that of Enrei cotyledons throughout this period. The activity at pH 9.0 in cotyledons from both cultivars decreased continually until day 9 after germination (Fig. la), indicating disappearance of the L-l isozyme in both cases. This result is consistent with previously described work (6, 14). The normal cultivar, Enrei, showed high levels of lipoxygenase activity at pH 6.8 in ungerminated seeds, due to three isozymes, L-1, L-2, and L-3. Ungerminated seeds of the variant cultivar, Kanto 101, also contained the pH 6.8 activity at a much lower level, which was due to low-level activity of the L-l isozyme at pH 6.8. This activity in the normal cultivar decreased rapidly in the first 24 h after germination, increased again until 3 d postgermination, then by day 9 it declined to a level that was 13% of the activity found in ungerminated seeds. In the variant cultivar, the pH 6.8 activity increased until day 5 postgermination and then declined dramatically in activity (Fig. lb). These increases and decreases in lipoxygenase activity in both types of cotyledons after germination are consistent with the results of Hildebrand and Hymowitz (6), although they used a normal soybean cultivar and a cultivar missing the L-1 isozyme. As shown below, the appearance of the pH 6.8 activity was due to new lipoxygenases in both cases.
Purification of Lipoxygenases on Ion Exchange Columns
To characterize the lipoxygenases found in germinating cotyledons of both soybean cultivars, we purified lipoxygenases extracted from 5-d-old cotyledons using either Mono Q or Mono S ion exchange column chromatography (Figs. 2 and 3). We identified three new lipoxygenases that are different from L-1, L-2, and L-3, as indicated on the chromatograms. A water-soluble extract from the cotyledons was desalted and applied to a DEAE-Toyopearl column. Nine percent of the total pH 6.8 lipoxygenase activity from the normal cultivar and 25% of that from the variant cultivar did not bind to the DEAE column, whereas all of the pH 9.0 activity from both cultivars bound to this column. As this unbound activity was observed even when smaller amounts of extract were applied to this column, it is unlikely that the failure to bind was due to an overloaded column. Lipoxygenase activity bound to the DEAE-Toyopearl column was eluted with 100 mm sodium phosphate (pH 6.8). The eluate was desalted, then subjected to Mono Q column chromatography. Three lipoxygenases, L- 1, L-2, and L-3, were seen in the ungerminated cotyledons of the normal cultivar (Fig. 2a). The order of elution for L-2 and L-3 on the Mono Q column was the opposite of that observed in a previous experiment using a DEAE-Sephadex A-50 column (1). We assigned the pH 6.8 activities using arachidonic acid as a

E
_0.

0c
~~~O.5
0

~~~~~~025 0.25C
-

~~~~~~~~~~~~~0
c

30 20 10 Fraction number

(0.5m1/tube)

40

50

Figure 3. Mono S column chromatography of 5-d-old cotyledon lipoxygenases of the variant soybean line, Kanto 101. Lipoxygenases that did not bind to the DEAE-Toyopearl column (see "Materials and Methods") were placed on a Mono S column and then eluted with a linear NaCI gradient ( - ). The lipoxygenase activity was measured at pH 6.8 (0). (-- - -) Absorbance at 280 nm.

substrate, for which the activity of L-2 is markedly increased relative to L-3 (1). Chromatography of extracts from 5-d-old cotyledons of the normal cultivar showed decreased amounts of the L-1, L-2, and L-3 activities and two new lipoxygenase activities (Fig. 2b). The variant cultivar showed two new lipoxygenase activities as well as the L- 1 activity (all in the same position as those in the normal cultivar) in chromatograms of extracts from 5-d-old cotyledons (Fig. 2d). Ungerminated seeds of this cultivar contained lipoxygenase L-1 (Fig. 2c). The two new lipoxygenase activities were designated lipoxygenase L-4 and L-5 according to the elution positions in an increasing salt gradient. The activities that did not bind the DEAE-Toyopearl column were applied to a CM-Toyopearl column and subsequently eluted with buffered 0.5 M NaCl. The eluted fraction was desalted, then applied to a Mono S column. One major peak of activity, designated lipoxygenase L-6, was identified on this chromatogram (Fig. 3). Purification of the lipoxygenases from 5-d-old cotyledons of the variant cultivar is summarized in Table I. The activity of lipoxygenase L-4 consists of approximately 66% of the activity of the new lipoxygenases after purification. Lipoxygenases L-4 and L-6 were purified almost to homogeneity by these chromatographic procedures, whereas the lipoxygenase L-5 fraction still contained a number of polypeptides, identified by SDS-PAGE (Fig. 4).

Characterization of New Lipoxygenases

We characterized the three new lipoxygenases, purified from 5-d-old cotyledons of the variant cultivar, using linoleic acid as a substrate. They all had pH optima at 6.5 and none of them were active at pH 9.0. The position of hydroperoxidation on the substrate (regiospecificity) were analyzed using HPLC (Fig. 5). Lipoxygenases L-5 and L-6 preferentially

328

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Plant Physiol. Vol. 98, 1992

Table I. Purification of Lipoxygenases from 5-d-old Cotyledons (5

g) of the Variant Soybean Line, Kanto 101, Which Lacks Both

Lipoxygenase L-2 and L-3 in its Dry Seeds
Purification Step

Protein Activity Specific Activity mg pkat pkat/mg

L-1 L-2 L-3 L-4 L-5 L-6

97.468.0-

411 21.6 Crude extract 277 13.4 Amberlite treatment 294 8.63 Desalting with Sephadex G-25 DEAE-Toyopearl chromatography 59.0 6.40 Bound fraction 30.0 2.20 Unbound fraction DEAE-Toyopearl bound fraction Mono Q chromatography 2.78 1.28 Lipoxygenase L-4 0.87 0.52 Lipoxygenase L-5 DEAE-Toyopearl unbound fraction' CM-Toyopearl chromatography 30.3 2.87 Mono S chromatography 1.12 1.47 Lipoxygenase L-6 a The unbound fraction obtained from a larger scale from 20 g of cotyledons.

0.053 0.048 0.030

0.108 0.073 0.461 0.172

43.00.095

1.310 purification

29.0Figure 4. SDS-PAGE of lipoxygenase L-4, L-5, and L-6 purified from 5-d-old cotyledons of the variant soybean line, Kanto 101. Purified lipoxygenases L-4 and L-6 (1 ,g each) and 5 ug of a fraction containing lipoxygenase L-5 were electrophoresed. For comparing the molecular masses of the new isozymes, lipoxygenase L-1, L-2, and L-3 were also electrophoresed. (The L-3 preparation contains contaminants less than 55 kD.) Molecular masses are indicated on the left.

produced 13S-HPOD2 to 9S-HPOD at a ratio of 85:15, whereas lipoxygenase L-4 produced both 1 3S-HPOD and 9SHPOD at a ratio of 46:54. The regiospecificity of lipoxygenases L-5 and L-6 resembled that of L-1 (1), suggesting that these two enzymes belong to a new class of soybean lipoxygenases. Lipoxygenase L-4 showed similar regiospecificity to L-2 and L-3 (1), but it differed from both of them in primary structure, as shown below. The relative activities of these enzymes using linoleic acid and linolenic acid as substrates were determined and compared with those of L-1, L-2, and L-3 (Table II). All lipoxygenase except L-1 showed a preference in activity for linolenic acid.
Lipoxygenase L-4 Is a Newly Expressed Activity To determine whether lipoxygenase L-4 was synthesized de novo or derived from modification of a preexisting lipoxygenase, we partially determined the amino acid sequence of the protein purified from 5-d-old cotyledons of the variant cultivar. The protein was digested completely at the lysyl bonds using lysylendopeptidase. The resulting peptides were separated by reverse-phase HPLC (Fig. 6), then selected peptides were micro-sequenced. The amino acid sequences obtained were compared with those of soybean lipoxygenases L-1 (16), L-2 (17), L-3 (21), and the gene product of a soybean lipoxygenase gene SC5 14, which is expressed in germinated cotyledons (15) (Fig. 7). This comparison shows that the amino acid sequences do not match any of the previously identified lipoxygenase sequences, but share homology with them, indicating that L-4 was synthesized from a gene that is distinct from the genes encoding these four previously identified lipoxygenases.
2 Abbreviations: 13S-HPOD, 13(S)hydroperoxy-9(Z), 1 l(E)-octadecadienoic acid; 9S-HPOD, 9(S)-hydroperoxy-10(E), 12(Z)-octadecadienoic acid; pIs, isoelectric points.

a
E
C

b
C13

C13

09 C13

N
a

.0
0

.0
.4

JC9
_

A C9
_ _ _ _ _

2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 Retention time (minutes)

Figure 5. Regiospecificity of three new lipoxygenases on linoleic acid. HPLC separation of the products of lipoxygenase L-4 (a), L-5 (b), and L-6 (c) activities using linoleic acid as a substrate. Arrows indicate the elution positions of 1 3S-HPOD (C13) and 9S-HPOD (C9) that were identified using authentic standards.

LIPOXYGENASES FROM GERMINATED COTYLEDONS OF SOYBEAN

#13 L-1 L-2 L-3 SC514
LFILDHHDYLIPYLR ::M: :Y::IFM: :V: ::M: :Y: :VFM: :I: : :L:G:: :PIM::::
::::

329
#15
L-1

??VQEYV?FYY
(622-632)
TW:::: : PL:: TW:::: :SL:: TW:H: :FL:: TW:H: :SL::
DPSAPHGVRLLIEDY
:T:.. .: .

Table IL. Substrate Specificity of Soybean Lipoxygenases Activities of lipoxygenases L-4, L-5, L-6, L-1, L-2, and L-3 with linoleic and linolenic acids were measured at pH 6.8 (or pH 9.0 for the L-1 isozyme). The relative activities of these enzymes with these fatty acids are presented.
Substrate Specificity
Fatty acid L4 L-5 L-6 L-1 L-2 L-3

(404-418) (433-447) (424-438) (430-444)

:Y():AF::: :T

L-2 (651-661) L-3 (642-652) SC514 (649-659) #20

L-1 L-2 L-3 SC514

#16
(338-347) L-2 (367-376) L-3 (356-365) SC514 (362-371)
L-1

SA?MTDEEFA

(593-607) (622-636) (613-627) (620-634)

(453-467) (481-495) (452-466) (479-493)

:L: .ot.

#24

ITLPTLGAGEQAYDV
TS::::::: :S:FNI VS::::::: :S:FNI

Linoleicacid Linolenic acid

100 293

100 261

relative activity (%/) 100 100 100 311 88 300

L-1 (69-83) L-2 (99-113) L-3 (87-101) SC514 (95-109)

TS:::::::QS:FKI P:::::::RQD:FSI
ETLIDLTVIEILS
0:::: :S:::::: ... ::::::..

#25 L-1 L-2 L-3 SC514

P?PQGE?YGPVSEVY
:HSA:DLSAA: :Q:V :H:A:DLS:A: :0:I :H:: :DQS:AF:Q:F :H:D:DNL: :E:I:V

100 447

#27 (742-754) (771-783) L-3 (762-774) SC514 (769-781)

L-1 L-2

DISCUSSION Here we describe the identification and characterization of new types of lipoxygenases in soybean cotyledons. These activities appear between 3 and 5 d after germination and almost disappear by 9 d postgermination. We also showed that the activities of lipoxygenases L- 1, L-2, and L-3 decline after germination in the cotyledons. Previous work (6, 14) referred to an activity at pH 6.8 in the cotyledons after germination as being due only to lipoxygenases L-2 and/or L-3. In this study, we took advantage of a variant soybean cultivar (Kanto 101) that lacks both lipoxygenase L-2 and L3 to help distinguish the new activities from those that have already been identified or a modified form of those lipoxygenases. It is unlikely that the new lipoxygenases found in the variant cultivar are variant forms of the L-2 and L-3 isozymes because they were also found in the normal soybean cultivar, Enrei (Fig. 2). Although the new lipoxygenases found in both the normal and variant cultivars have the same elution positions on the Mono Q column (Fig. 2), we cannot exclude the possibility that they differ from each other, particularly in amino acid sequence, because these cultivars are not isogenic. Our northern blotting experiments using a cDNA clone (SC50 1) encod-

Figure 7. Comparison of the amino acid sequences of lipoxygenase L-4 with known soybean lipoxygenase sequences. The partial amino acid sequences of proteolytic peptides of lipoxygenase L-4 were compared with those of soybean lipoxygenase L-1 (16), L-2 (17), L3 (21), and SC514 (15). The numbered peptides correspond to those labeled in Figure 6. Unidentified amino acid residues are indicated by a question mark (?). The position of each peptide (from the amino terminus of its respective lipoxygenase) that was used for this comparson is indicated in parentheses. Amino acid residues identical to those in lipoxygenase LA are indicated by a colon (:).

gene was

20

27

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c
0

15

13

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4.

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0
a

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.0

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.--

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.-

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20 0
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10 15 20 25 30 35 40 45 50 55 60 65 Time (minutes)

Figure 6. Separation of peptides obtained by proteolytic digestion of lipoxygenase L-4 by HPLC. Purified lipoxygenase L4 (200 9g) was completely digested using lysylendopeptidase (2 jg). The resulting peptides were separated by HPLC using an ODS column with a linear gradient of acetonitrile. The numbered peptides were subjected to amino acid sequencing (see Fig. 7).

ing lipoxygenase L-4 (see below) as a probe showed that the expressed in cotyledons of the normal cultivar, Enrei, after germination (our unpublished data). This indicates that lipoxygenase L-4 is synthesized in the cotyledons of both cultivars. Vernooy-Gerritsen et al. (19) showed the distribution of lipoxygenase L-1 and L-2 in germinated cotyledons using antibodies. Lipoxygenase L- 1 and L-2 were initially localized in the storage tissues of etiolated soybean cotyledons during the first days after germination, but later appeared in the epidermal and vascular tissues of the developing cotyledons, hypocotyls, and primary leaves. This observation coincides with our results, that is, the disappearance of preexisting lipoxygenases L-1, L-2, and L-3 and the appearance of new lipoxygenases in cotyledons after germination. It is likely that the lipoxygenases that appear in the epidermal and vascular tissues are the new lipoxygenases identified in this study. Preliminary experiments with antilipoxygenase L- 1 antibody (provided by Dr. B. Axelrod) showed cross-reactivity with the new lipoxygenases we purified. A number of investigators have observed changes in lipoxygenase activity during the early stages of seedling growth in other plants, suggesting that lipoxygenases have some as yet unknown function during seed germination (8). It is interesting to note that the fresh weight of the cotyledons increased after day 3 at the same time that the new lipoxygenases appeared (Fig. 1), although the dry weight and protein in the cotyledons decreased rapidly during the first 11 d after germination (14). It is possible that the new lipoxygenases have a function in the developmental process. Park and Polacco (12) demonstrated the appearance of new lipoxygenases in germinated seedlings of soybean using electrofocusing gels. The lipoxygenases they found showed much lower pls than those of L- 1, L-2, and L-3. As lipoxygenase L4 and L-5 were eluted with lower concentrations of salt than

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KATO ET AL.

Plant Physiol. Vol. 98, 1992

L- 1 on the Mono Q column and lipoxygenase L-6 did not bind to the column, indicating their pIs are higher than that of L- 1, it seems that the enzymes we have identified differ from those found by Park and Polacco in hypocotyl/radical sections. To know whether lipoxygenases that have lower pls exist in 5-d-old cotyledons or not, we washed the DEAEToyopearl column with buffered 1 M NaCl after eluting the lipoxygenases we identified in this study. However, less than 1% of the total activity applied to the column was detected (data not shown). We did not characterize this activity further. The fate of a free fatty acid through a lipoxygenase pathway is decided by the position on the fatty acid that is hydroperoxidized and subsequent metabolism of the hydroperoxide moiety, by enzymes such as hydroperoxide lyase, hydroperoxide dehydrase, hydroperoxide isomerase, and hydroperoxide peroxygenase (3). Future research should take account of the regiospecificity of newly identified lipoxygenases to help elucidate their physiological functions. Lipoxygenase L-5 and L-6 preferentially introduced molecular oxygen at carbon 13 on linoleic acid, whereas lipoxygenase L4 attaches 02 at either carbon 13 or 9 (Fig. 5). We did not examine the regiospecificities of the new lipoxygenases for linolenic acid, which is another substrate in plant cells. It should be noted that 1 3-hydroxylinolenic acid is a precursor of jasmonic acid (20), which may be involved in regulating soybean vegetative storage protein genes (18). Amino acid sequencing of lipoxygenase L-4 showed that the isozyme has a distinct amino acid sequence from known lipoxygenases, indicating that this is a novel gene product. We recently isolated three lipoxygenase clones from a cDNA library made from germinated cotyledons of soybean (15). We are currently investigating a soybean lipoxygenase gene, SC50 1, which was isolated using one of these three cDNAs as a probe. The amino acid sequences of lipoxygenase L-4 that we analyzed in this study were found in amino acid sequences deduced from the genomic nucleotide sequence of the SC501 gene (our unpublished data), indicating that the gene encodes lipoxygenase L-4. Expression of the gene in cotyledons, hypocotyls, roots, and leaves was shown using a cDNA clone, pTK18 (13), for the SC501 gene by Park and Polacco (personal communication) and indicated that expression of the lipoxygenase gene is transcribed in such tissues. Appearance of lipoxygenase L-4 in germinated cotyledons seems likely to be a result of de novo synthesis of the protein. In this study, we showed continuous decreases in lipoxygenases L- 1, L-2, and L-3 and the appearance of new lipoxygenases. These results differ from expression of rice lipoxygenases after germination. Rice lipoxygenases L-2 and L-3, synthesized during seed maturation, are also synthesized after germination in the seeds (10). This suggests different mechanisms of gene expression of lipoxygenases in dicot and monocot seeds. We isolated cDNAs from germinated soybean cotyledons (15) and rice seedlings (our manuscript in preparation) and are currently analyzing their gene expression in various tissues. Analysis of expression of the genes encoding the lipoxygenases that appear in germinated cotyledons are ongoing in our laboratory and will be helpful in understanding the physiological role of these enzymes.

ACKNOWLEDGMENTS We thank Dr. Keisuke Kitamura for the gift of seeds of the lipoxygenase variant cultivar (Kanto 101). We wish to express our thanks to Drs. Tae K. Park and Joseph C. Polacco for the opportunity to see the nucleotide sequence of pTK18 in advance of publication. We also thank Dr. Bernard Axelrod for the gift of antilipoxygenase L- I antibody. We thank Yumiko Shirano for drawing figures.
LITERATURE CITED Axelrod B, Cheesbrough TM, Laakso S (1981) Lipoxygenase from soybeans. Methods Enzymol 71: 441-451 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 Gardner HW (1991) Recent investigations into the lipoxygenase pathway of plants. Biochim Biophys Acta 1084: 221-239 Grossman S, Zakut R (1979) Determination of the activity of lipoxygenase (lipoxidase). Methods Biochem Anal 25: 303-329 Hildebrand DF (1989) Lipoxygenases. Physiol Plant 76:249-253 Hildebrand DF, Hymowitz T (1983) Lipoxygenase activities in developing and germinating soybean seeds with and without lipoxygenase-1. Bot Gaz 144: 212-216 Kitamura K, Kumagai T, Kikuchi A (1985) Inheritance of lipoxygenase-2 and genetic relationships among genes for lipoxygenase- 1, -2, -3 isozymes in soybean seeds. Japan J Breed 35: 413-420 Mack AJ, Peterman TK, Siedow JN (1987) Lipoxygenase isozymes in higher plants: biochemical properties and physiological role. Curr Top Bio Med Res 13: 127-154 Ohta H, Ida S, Mikami B, Morita Y (1986) Purification and characterization of rice lipoxygenase component 3 from embryos. Agric Biol Chem 50: 3165-3171 Ohta H, Ida S, Mikami B, Morita Y (1986) Changes in lipoxygenase components of rice seedlings during germination. Plant Cell Physiol 27: 911-918 Ohta H, Shida K, Peng Y-L, Furusawa I, Shishiyama J, Aibara S, Morita Y (1990) The occurrence of lipid hydroperoxidedecomposing activities in rice and the relationship of such activities to the formation of antifungal substances. Plant Cell Physiol 31: 1117-1122 Park TK, Polacco JC (1989) Distinct lipoxygenase species appear in the hypocotyl/radicle of germinating soybean. Plant Physiol 90: 285-290 Park TK, Polacco JC (1990) Lipoxygenase isozymes synthesized during soybean germination: analysis of cDNA clones (abstract No. 5). Plant Physiol 93: S-2 Peterman TK, Siedow JN (1985) Behavior of lipoxygenase during establishment, senescence, and rejuvenation of soybean cotyledons. Plant Physiol 78: 690-695 Shibata D, Kato T, Tanaka K (1991) Nucleotide sequences of a soybean lipoxygenase gene and the short intergenic region between an upstream lipoxygenase gene. Plant Mol Biol 16: 353-359 Shibata D, Steczko J, Dixon JE, Hermodson M, Yazdanparast R, Axelrod B (1987) Primary structure of soybean lipoxygenase-l. J Biol Chem 262: 10080-10085 Shibata D, Steczko J, Dixon JE, Andrews PC, Hermodson M, Axelrod B (1988) Primary structure of soybean lipoxygenase L-2. J Biol Chem 263: 6816-6821 Staswick PE (1990) Novel regulation of vegetative storage protein genes. Plant Cell 2: 1-6 Vernooy-Gerritsen M, Bos ALM, Veldink GA, Vliegenthart JFG (1983) Localization of lipoxygenases 1 and 2 in germinating soybean seeds by an indirect immunofluorescence technique. Plant Physiol 73: 262-267 Vick BA, Zimmerman DC (1984) Biosynthesis of jasmonic acid by several plant species. Plant Physiol 75: 458-461 Yenofsky RL, Fine M, Liu C (1988) Isolation and characterization of a soybean (Glycine max) lipoxygenase-3 gene. Mol Gen Genet 211: 215-222

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