expression, activity, andcellular accumulation methyl ...organs, however, mostof the methyl...

Plant Physiol. (1992) 100, 433-4430032-0889/92/100/0433/11 /$01 .00/0

Received for publication December 24, 1991Accepted April 17, 1992

Expression, Activity, and Cellular Accumulation of Methylasmonate-Responsive Lipoxygenase in Soybean Seedlings'

Howard D. Grimes*, David S. Koetje, and Vincent R. Franceschi

Department of Botany, Washington State University, Pullman, Washington 99164-4238

ABSTRACT

Exposure of soybean (Glycine max) seedlings to low levels ofatmospheric methyl jasmonate induced the expression and accu-mulation of one or more lipoxygenase(s) in the primary leaves,hypocotyls, epicotyls, and cotyledons. In the primary leaf, the majorsite of lipoxygenase accumulation in response to methyl jasmonatewas in the vacuoles of paraveinal mesophyll cells. In the otherorgans, however, most of the methyl jasmonate-responsive lipox-ygenase(s) were associated with both the epidermal and corticalcells and were present in both vacuoles and plastids. In plastids,the methyl jasmonate-responsive lipoxygenase was sequesteredinto protein inclusion bodies; no lipoxygenase was evident in eitherthe thylakoids or the stroma. Both spectrophotometric measure-ment of conjugated diene formation and thin layer chromatographyof lipoxygenase product formation indicated that methyl jasmonatecaused an increase in the amount of lipoxygenase activity. Electronmicroscopy of the methyl jasmonate-responsive lipoxygenase pro-tein in the vacuoles showed that it was arranged into a stellate,paracrystalline structure in various cell types other than the para-veinal mesophyll cells. The paracrystals appeared to be composedof tubular elements of between 5 and 8 nm in diameter, were ofvariable length, and were observed in most cell types of theseedling organs.

Jasmonic acid and its methyl ester, methyl jasmonate, arerapidly emerging as strong candidates for intracellular orintercellular messengers in higher plants. When applied di-rectly to plants or suspension cultures, they produce a numberof phytohormone-like effects (1, 19, 26, 31). The case forthese compounds functioning as messengers was strength-ened by the recent demonstration that exceptionally lowlevels (approximately iO-' M maximum) of atmosphericmethyl jasmonate would stimulate the expression of proteaseinhibitor proteins in tomato leaves (3) and the accumulationof VSPs2 in soybeans (7).

Jasmonic acid is structurally similar to eicosanoids such asthe prostaglandins present in a wide range of animal species

l This research was partially supported by the U.S. Department ofAgriculture (grant No. 91-37305-6707). Instrumentation for themicroscopy and image analysis was purchased with National ScienceFoundation grant (DIR9016138).

2Abbreviations: VSP, vegetative storage protein; 13(s)HPOTrE,13(s) hydroperoxy linolenic acid; 9(s)HPOTrE, 9(s) hydroperoxy lin-olenic acid; pvmLOX, paraveinal mesophyll lipoxygenase; PVM,paraveinal mesophyll; TTBS + BSA, 10 mm Tris, 500 mm NaCl, 0.3%Tween 20, 1% (w/v) BSA.

(27). In mammals, eicosanoids are synthesized following therelease of arachidonic acid into the cytoplasm and functionas intracellular stress messengers (27). Analogously, plantcells are able to utilize linolenic acid as a substrate for thelipoxygenase-dependent synthesis of jasmonic acid (30). Al-though it has yet to be definitively established that jasmonicacid or methyl jasmonate are intracellular messengers in plantsystems, the results of Farmer and Ryan (3) suggest that theymay be involved in a wound-responsive signal cascade.The expression and accumulation of soybean VSPs is reg-

ulated by jasmonic acid and methyl jasmonate (1, 7, 10, 14,22, 23). The VSPs consist of three proteins of 27, 29, and 94kD (vsp27 or vspa, vsp29 or vsp#, and vsp94, respectively),which accumulate during vegetative growth in a specializedcell layer termed the PVM layer (5, 6, 8, 11, 13). The PVMlayer apparently serves as a direct conduit from the palisadeparenchyma to the phloem (4-6, 8). VSP transcripts and theirproteins respond to the immediate need to store nitrogen oramino acids (22, 34) and to the removal of sink tissue or pods(20, 25, 32, 33). The VSPs decrease in abundance during seeddevelopment (21, 34). Apparently, the VSPs have been re-cruited for temporary storage of nitrogen during vegetativegrowth, and this nitrogen is later contributed to the devel-oping seed.

In soybean (Glycine max) seedlings, atmospheric methyljasmonate induces the accumulation of all three VSPs, in-cluding vsp94, in the shoot tips, primary leaves, stems, andcotyledons (7). These organs normally express the VSPs atvery low levels, and, of these organs, only the primary leafhas PVM cells. In response to atmospheric methyl jasmonate,vspa accumulated in primary leaves and expanding firsttrifoliolates, whereas vsp(# preferentially accumulated in thecotyledons (7). These results suggest that methyl jasmonatemay alter the source-sink relationships by shifting the priorityof nitrogen utilization or increasing the rate of nitrogenassimilation and amino acid storage. Hence, methyl jasmon-ate appears to affect nitrogen partitioning in soybean plants.We recently demonstrated that the 94-kD VSP present inmature soybean leaves is actually a member of the lipoxy-genase gene family (25). Because of the strong localization ofvsp94 in the PVM vacuoles, we termed this protein pvmLOX.These observations raise the obvious question of whether

the 94-kD protein induced by methyl jasmonate in seedlingsis also a lipoxygenase. This question takes on added impor-tance when one considers that in the seedling only theprimary leaves have a PVM layer (13). We have demonstratedthat methyl jasmonate induces the expression of the 94-kDprotein in all above-ground organs of the soybean seedling

433

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Plant Physiol. Vol. 100, 1992

(7). If the 94-kD protein that is induced by methyl jasmonatein seedlings is a lipoxygenase, then it becomes necessary todetermine with which cell types, and in which subcellularcompartments, the methyl jasmonate-responsive lipoxygen-ase is associated and to compare this pattern of expressionand accumulation with that of the nitrogen-responsive lipox-

ygenase present in mature soybean leaves. A last significantissue concerns whether the methyl jasmonate-responsive 94-kD protein, if it is a lipoxygenase, is an active lipoxygenase.

This question is especially relevent in terms of the evidenceindicating that jasmonic acid and methyl jasmonate are syn-

thesized via a lipoxygenase-dependent pathway (30).

MATERIALS AND METHODS

Plant Material and Growth Conditions

Soybean (Glycine max Merr. cv Wye) seeds were plantedin potting compost in 3-inch pots and grown in a controlledenvironment chamber with 360 to 400 ,mol m-2 s-' PPFDat canopy height, 16 h photoperiod and 24/180C day/nighttemperatures.

Methyl Jasmonate Treatments

Methyl jasmonate was prepared by diluting with 100%ethanol at a ratio of 1:9 (methyl jasmonate:ethanol). Themethyl jasmonate was obtained from Bedoukian ResearchCo. (Danbury, CT) and consisted of 90.6% 1R,2R-enantiomerand 8.1% 1RF,2S-enantiomer according to the manufacturer.Controls consisted of 100% ethanol.Four seeds were sown in 3-inch pots and allowed to

germinate in the growth chamber until the cotyledons were

above ground (approximately 5 d after sowing). Just after thecotyledons emerged from the soil, the seedlings were exposedto methyl jasmonate vapor by placing methyl jasmonate ontothe tip of a cotton swab anchored in a central location in thepot but not directly contacting the plants. The plants were

placed inside an air-tight plexiglass box for the remainder ofthe experiment; these boxes were placed inside the growthchamber. The final concentration of diluted methyl jasmonatewas 120 ,L/L of box volume, with the controls receiving 120,uL/L of ethanol. At the initiation of our experiments, themaximum amount of atmospheric methyl jasmonate (assum-ing a fully saturated atmosphere and 240C) has been calcu-lated to be about 2.4 x 1i0' mol/L of air. Exposure to methyljasmonate was for 5 d, unless specified otherwise in the text.

Protein Extraction and Electrophoresis

Samples from various organs were weighed and quick-frozen in liquid nitrogen and stored at -800C. Tissue was

ground to a powder in liquid nitrogen with a chilled mortarand pestle and total soluble protein was extracted with 2volumes/g of tissue of homogenization buffer, which con-

sisted of 25 mm N-tris [hydroxymethyl] methylglycine (Tri-cine, pH 7.5), 1 mi EDTA, 10 mm ,B-mercaptoethanol, and1% insoluble polyvinylpolypyrrolidone. Protease inhibitorswere added to the homogenate in the following concentra-tions: 1 ,lM leupeptin, 1 ,UM pepstatin, and 100 ,ug mL-1 PMSF.The homogenate was centrifuged at 10,OOOg for 15 min at

40C, and the supernatant was filtered through Miracloth andassayed for protein with the Bio-Rad Protein Assay reagentaccording to the manufacturer's instructions (Bio-Rad Labo-ratories). Extracts were mixed 1:1 (v/v) with 2x Laemmlisample buffer (12), heated at 900C for 5 min, and centrifugedat 13,000g for 5 min, and the supernatants were loaded ontoSDS-PAGE gels on an equal protein basis. Proteins wereresolved according to Laemmli (12) except that a 7.5 to 15%acrylamide gradient was used with an accompanying 7.5 to15% glycerol gradient. Gels were stained with Coomassiebrilliant blue G-250.

Immunoblotting and Immunostaining

Soluble protein extracts resolved by SDS-PAGE were elec-troblotted to nitrocellulose according to Towbin et al. (24) at100 mA for 12 h followed by 200 mA for 1 h. The blot wasblocked for 2 to 3 h with 10% nonfat Carnation instant milkin Tris-buffered saline buffer (20 mm Tris, 500 mm NaCl, pH7.5) with the pH readjusted to 7.5. After blocking, the blotswere incubated with soybean leaf lipoxygenase antiserum(1:5000 dilution) in the blocking solution. This antibody wasprepared against isoelectric-focused soybean leaf lipoxygen-ase peak 3 (9) and was kindly supplied by Dr. David Hilde-brand (University of Kentucky). Secondary antibody was goatanti-rabbit immunoglobulin G-peroxidase (Pierce ChemicalCo.) in the blocking solution at a concentration of 1:5000 for1 h. Color was developed by immersing the blot in 60 mg of4-chloro-1.-napthol (first dissolved in 20 mL of cold metha-nol) and 60 ,uL of H202 in 100 mL of Tris-buffered saline.

RNA Isolation and Northern Analysis

RNA was extracted from seedling tissues according toChomczynski and Sacchi (2). Tissue was ground in liquid N2to a powder in a mortar and pestle. All subsequent volumesare for 1.0 g fresh weight of tissue. A denaturing solutioncontaining 4 M guanidine thiocyanate, 25 mm sodium citrate(pH 7.0), 0.5% Sarkosyl, and 0.1 M /3-mercaptoethanol wasadded to the powder. The resulting slurry was transferred toa Corex tube containing 10 mL of phenol (pH 8.0) and 2 mLof chloroform. The phenol/chloroform aqueous phase wasthoroughly mixed and incubated on ice for 15 min. Sampleswere centrifuged at 15,000g for 20 min at 40C. The aqueousphase was collected and precipitated with an equal volumeof isopropanol for 1 h at -200C. Precipitated nucleic acidwas pelleted at 10,OOOg for 20 min at 40C and resuspendedin 500 IuL of denaturing solution. The resuspended nucleicacids were extracted twice with an equal volume of phenol(pH 8.0):chloroform (1:1). The final aqueous phase was col-lected in a 1.5-mL microcentrifuge tube and precipitated withan equal volume of isopropanol for 1 h at -200C. The 1.5-mL microcentrifuge tubes were centrifuged at maximumspeed in a microcentrifuge at 40C for 10 min, the pellets wererinsed with 80% ethanol, and were dried in a Speed-Vacuntil dry. The final pellets were resuspended in diethylpyro-carbonate-treated water for spectrophotometric analysis.

Total RNA (10 jig) was resolved on formaldehyde gelsaccording to Sambrook et al. (18) and transferred to Gene-screen Plus (DuPont) membranes via capillary action (18).

434 GRIMES ET AL.



METHYL JASMONATE-RESPONSIVE LIPOXYGENASE

Hybridization to probe DNA (using 10-15 ng of probe/mLof hybridization solution) was performed according to themanufacturer's recommendations. The probe used was a 1.4-kb EcoRI fragment of pTK1 1 (containing a cDNA to a hypo-cotyl/radicle-associated lipoxygenase, ref. 15). After hybrid-ization overnight at 650C, the membranes were washed twicefor 5 min each with 2x SSC at room temperature, twice for30 min each with 2x SSC and 1% SDS at 650C, and twicefor 30 min each with O.lX SSC at room temperature. Mem-branes were then air-dried and subjected to autoradiography.

Lipoxygenase Activity Assays

Soluble protein was extracted from primary leaves andshoot apices as described above except that 1 volume ofhomogenization buffer/g fresh weight of tissue was used.Extracts were analyzed immediately or frozen in liquid N2and stored at -800C. Two independent assays were used.The first was a spectrophotometric assay that measured theformation of conjugated dienes at A235 using linolenic acid asthe substrate at pH 6.8. All assay conditions were as describedby Graybum et al. (9). The second assay analyzed lipoxygen-ase product formation using TLC. Assay conditions were 40mM Tris (pH 8.7), 1 mM CaCl2, 0.1% Triton X-100, 0.5 mmlinolenic acid, and approximately 50 ,uCi of [14C]linolenicacid. Final volume for the assays was 200 ,uL, and 2 usL ofextract was added to initiate the reaction. The reaction wasallowed to proceed for 5 min at 300C and then was stoppedwith 360 ,uL of diethyl ether:methanol:1 M citric acid (30:4:1),vortexed, and centrifuged for 1 min at 13,000g. The upperphase was collected and evaporated to near dryness in aSpeed Vac. The volume was adjusted to 20 uL with ethylacetate, and the entire volume was spotted onto TLC plates.Chromatography was done for approximately 1 h usingchloroform:methanol:acetic acid (70:30:1). Standards were['4C]linolenic acid and [`4C]13(s)HPOTrE, an initial productof lipoxygenase action on linolenic acid, and were obtainedfrom Cayman Chemical Co. (Ann Arbor, MI).

Tissue Preparation and Immunolocalization

Soybean tissue was gently sliced into pieces approximately2 mm2 and placed immediately into a vessel containing afreshly prepared fixative with the following components: 2%(v/v) paraformaldehyde, 1.25% (v/v) glutaraldehyde, 50 mmPipes, pH 7.2, and 2 mm CaCl2. Tissue was allowed to fix for6 h at room temperature with gentle agitation before beingrinsed with 50 mm Pipes buffer (pH 7.2) three times for 15min and dehydrated with an ethanol series. Tissue wasinfiltrated with L.R. White resin and polymerized in gelatincapsules at 600C for 24 h. Sections (1 ,um) were cut and driedonto gelatin-coated slides.

Sections were blocked with TTBS + BSA for 60 min, thenincubated in soybean leaf lipoxygenase antiserum (1:150dilution) at room temperature for 3 h. Nonimmune serumwas used at the same dilution. Sections were then washedfour times for 5 min with the diluted blocking solution beforebeing incubated with protein A-conjugated gold (15 nm,Janssen, 1:50 in Tris-buffered saline) for 2 h. The sectionswere rinsed in the blocking solution three times for 5 min,

then with TTBS + BSA three times for 5 min, followed byTTBS for 5 min. Sections were rinsed in water for 30 to 60 sand allowed to air dry and were then enhanced with silver(IntenseM, Amersham) twice for 5 min. Sections were thenstained with 1% Safranin at room temperature and coveredwith immersion oil and a coverslip for light microscopeexamination.

Light microscopy was performed using a Leitz Aristoplanmicroscope. The Bioquont Meg IV image analysis programswere used for background correction and quantification ofsilver grains within cells. Quantification was as described inTranbarger et al. (25).

Transmission EM Immunocytochemistry

Leaf samples were those used for light microscopy immu-nolocalization. Thin sections were picked up onto nickel gridsand incubated in TTBS + BSA for 1 h at room temperatureto block nonspecific protein-binding sites. The grids wereincubated for 4 h in soybean leaf lipoxygenase antiserum ata dilution of 1:100 with TTBS + BSA. They were rinsed fourtimes by immersion (5 min) in TTBS + BSA and then incu-bated for 1 h in protein A-gold (15 nm, Janssen) diluted 1:50with TTBS + BSA. After extensive washes by immersion inTTBS + BSA, TTBS, and then water, the sections werepoststained with uranyl acetate, potassium permanganate,and lead citrate. Sections were examined and photographedwith a Hitachi H-300 transmission electron microscope.

RESULTS

Atmospheric Methyl Jasmonate Induces the Expression andAccumulation of a Lipoxygenase in Soybean Seedlings

In previous work, we demonstrated that atmosphericmethyl jasmonate induced the accumulation of the VSPs,including vsp94, in soybean seedlings (7). We subsequentlydemonstrated, in fully expanded trifoliolates from adult soy-beans, that vsp94 was a member of the lipoxygenase genefamily, was localized in the vacuoles of PVM cells, and wasregulated by nitrogen status (25). Hence, the question wasraised as to whether the 94-kD VSP induced by methyljasmonate in seedlings was also a member of the lipoxygenasegene family.

This question was addressed by first examining the expres-sion and accumulation of the 94-kD VSP in various organsof soybean seedlings. Figure 1A shows a northern assay usingtotal RNA probed with an EcoRI fragment from a plasmid(pTK1 1) containing a cDNA to a lipoxygenase expressed insoybean seedling stems (hypocotyls and epicotyls), primaryleaf, and cotyledons. This cDNA is to a highly conservedbase sequence in the lipoxygenase gene family. The resultsclearly demonstrate that a higher level of lipoxygenasemRNA accumulates in response to methyl jasmonate (Fig.1A). Very high levels of lipoxygenase mRNA are found incotyledons, primary leaves, and stem tissue after exposure tomethyl jasmonate, although it appears that the cotyledonsmay be the most responsive tissue to methyl jasmonate(Fig. 1A).Two possibilities exist to explain the increase in lipoxygen-

ase mRNA levels after exposure to methyl jasmonate. First,

435




ACotyledon Prim. Least Stemr 1 r M.. M

6-0- i*

BCB o nt roI Methyt Jasmonate

Figure 1. Northern analysis of lipoxygenase mRNA response tomethyl jasmonate exposure. A, Comparison of lipoxygenase mRNAlevels in various organs after a 5-d exposure to atmospheric methyljasmonate (MJ) or ethanol (C). B, Time course of lipoxygenasemRNA induction upon exposure to atmospheric methyl jasmonate.In B, total RNA was isolated from entire seedlings for analysis. Inboth A and B, 15 ,g of total RNA was electrophoresed. The size ofthe transcript was 2.6 kb.

the methyl jasmonate may stabilize the lipoxygenase mRNAduring seedling growth from normal degradation. This pos-sibility is a concern because lipoxygenase activity and proteinlevel is known to decrease in cotyledonary tissue as the tissueages (16, 17). Second, the methyl jasmonate may induce theexpression of lipoxygenase transcript. To differentiate be-tween these two possibilities, a time-course experiment was

performed. Seeds were germinated and the time course was

initiated after the cotyledons were above ground. Time zero

was prior to any treatment or enclosure of the seedlings inthe plexiglass boxes. Seedlings were placed into the plexiglassboxes and whole seedlings were removed after 1, 3, and 5 dof exposure to ethanol (control) or methyl jasmonate. TotalRNA was isolated from entire seedlings and was subjectedto northern analysis (Fig. 1B). This experiment demonstratesthat methyl jasmonate induces the expression of lipoxygenasetranscript.

Because the expression of lipoxygenase was induced bymethyl jasmonate treatment, we wanted to determine howmuch lipoxygenase protein accumulated in response tomethyl jasmonate. Figure 2A shows that vsp94 accumulates

in the cotyledons, shoot tip plus first expanding trifoliolateleaf, epicotyl, and the hypocotyl region. The other two VSPs,vsp-a and vsp-f, also accumulate in these organs (Fig. 2A).In Figure 2A, vsp-a is referred to as 'A' and vsp-3 as 'B.' Awestern assay was performed using a lipoxygenase anti-serum. The results of this assay are shown in Figure 2B andindicate that the methyl jasmonate-responsive 94-kD proteinis cross-reactive with the lipoxygenase antiserum. This anti-serum is quite specific for 94-kD lipoxygenase (25) and,hence, only a portion of the immunoblot is shown in Figure2B. Densitometric measurements of these bands indicate thatlipoxygenase expression is stimulated approximately 3.1-foldin the cotyledon, 2.5-fold in the shoot tip, and 2.4-fold instem tissue (epicotyl and hypoctyl). This accumulation cor-relates with the increases observed in the lipoxygenasemRNA levels (Fig. 1A). These results show that methyljasmonate causes the accumulation of lipoxygenase in soy-bean seedlings.

Atmospheric Methyl Jasmonate Causes an Increase in theAmount of Lipoxygenase Activity in Primary Leaves andShoot Apices

Because methyl jasmonate induced the expression andaccumulation of a lipoxygenase in various organs in thesoybean seedling, it was of interest to determine if this methyljasmonate-responsive lipoxygenase caused an increase in li-poxygenase activity. This issue is important in light of evi-dence indicating that jasmonic acid and methyl jasmonateare probably synthesized via a lipoxygenase-dependent path-way (30). To address this problem, two independent assayswere performed to measure lipoxygenase activity before andafter exposure to methyl jasmonate.The first assay measures lipoxygenase product

[13(s)HPOTrE] formation using TLC. Figure 3A presents theresults of control and methyl jasmonate-treated extracts ex-amined by this method. It is clear from this figure that methyljasmonate induced more 13(s)HPOTrE formation when sam-ples were loaded on an equal leaf volume basis. The proteincontents of these two samples were very close (88 ug/juL forthe control and 92 ,ug/,gL for methyl jasmonate-treated).Hence, the lipoxygenase activity is increased when expressedas either total activity per leaf area or as specific activity. Thesecond product visible just under the 13(s)HPOTrE is prob-ably another lipoxygenase product [9(s)HPOTrE], but this hasnot been confirmed at this time.A second assay was also used to quantitate the increase in

lipoxygenase activity after methyl jasmonate treatment. Thisassay measured conjugated diene formation at 235 nm usinglinolenic acid as a substrate, and the results are presented inFigure 3B. Both of these assay methods indicate that methyljasmonate causes an increase in lipoxygenase activity.

Atmospheric Methyl jasmonate-Responsive LipoxygenaseIs Found in Several Types of Cells in Soybean Seedlings

In mature soybean leaves, the vsp94/pvmLOX is localizedprimarily in the vacuoles of bundle sheath and PVM cells.The PVM cell layer, however, is found only in primary leavesand trifoliolates in soybeans; it is not present in the hypocotyl,

436 GRIMES ET AL.




A Control MethyI Jasmonate

C/ 1 D.O ,O m QL >9 O m CL >C) U) I2J I (L c) 11 I

ji -1_M jill~~~~~~~~~~~il_I'j"4::44:..:.:;.~~~~~~~~~~~~~~~~~~~' 4i.

e I.

in the hypocotyl (epidermis, cortex, pith, vascular, paren-chyma) but was most abundant in the epidermal and corticalcells. The epidermal and cortical cells in the hypocotyl ac-counted for about 90% of the total lipoxygenase present inthis organ (data not shown). Comparison of Figure 5, A andB demonstrates that lipoxygenase protein levels increase sig-nificantly after exposure to methyl jasmonate.

Figure 6 shows lipoxygenase protein immunolocalizationbefore (A) and after (B) methyl jasmonate treatment in coty-ledonary tissue. In the cotyledons after methyl jasmonateexposure, lipoxygenase was concentrated in the vacuoles ofthe epidermal cells as large protein aggregates (Fig. 6B). Mostof the remainder of the cotyledon was made up of chloren-chymatous palisade cells with no noticeable protein labeling.

In all methyl jasmonate-exposed seedling tissues, with theexception of the PVM layer, the lipoxygenase protein oftenappeared as clumps composed of large branched or spikedclusters. This is in contrast with our earlier observations ofthe pvmLOX in fully expanded leaves, where it appears as aflocculent mass distributed fairly uniformly throughout thevacuoles (25). It is also evident from these immunocytochem-ical studies that methyl jasmonate consistently causes lipox-

94 -_. -- -_0_ _ p_

A LOX Product Formation

Figure 2. SDS-PAGE and immunoblot analysis of soybean seedlingorgans before and after a 5-d exposure to atmospheric methyljasmonate. A, SDS-PAGE gel of soluble proteins extracted fromvarious organs in control and atmospheric methyl jasmonate-ex-posed seedlings. Primary leaf includes the first expanding primaryleaf as well as the shoot apex. The markers point to the 94 kDlipoxygenase, vspa (A, 27 kD) and vsp, (B, 29 kD). B, Immunoblotof A using a leaf lipoxygenase antiserum. Only the cross-reactiveregion of the blot is shown.

epicotyl, or the cotyledon. Because of this, it was determinedwhether the methyl jasmonate-responsive lipoxygenase was

expressed in all cell types or whether it was specificallyassociated with a subset of cell types in seedlings. Immuno-localization experiments were performed on thick-sectionedmaterial using the lipoxygenase antibody.

Figure 4 shows the location of lipoxygenase within primaryleaves before and after exposure to atmospheric methyl jas-monate. Preimmune controls (Fig. 4A) were unlabeled inprimary leaf and all other organs. In the primary leaf, lipox-ygenase is found predominantly in the PVM cell layers andthe associated bundle sheath (Fig. 4B). The primary leafcharacteristically has two PVM layers (13), and both showaccumulation of the pvmLOX. It is also present in the epi-dermis but to a much lesser extent than in the PVM cells. Bycomparing Figure 4, B and C, it is apparent that methyljasmonate causes a significant increase in the level of lipox-

ygenase protein accumulation in bundle sheath, PVM, andepidermal cells.

Figure 5 shows lipoxygenase accumulation before (A) andafter (B) exposure to methyl jasmonate in hypocotyl tissue.Lipoxygenase was found in the vacuoles of most cell types

B LOX Activity (nmol/ min / mg)

Control

14.1(± 0.2)

MJ-Treated

17.5(± 0.7)

Figure 3. Lipoxygenase activity after exposure to methyl jasmonate.A, Two microliters of primary leaf and shoot apex extract was

incubated with [14C]linolenic acid. The amount of 1 3(s)HPOTrE andputative 9(s)HPOTrE produced is shown in controls and methyljasmonate-treated seedlings. B, Results of a spectrophotometricassay measuring conjugated diene formation in controls and methyljasmonate-treated seedlings (±SE).

-6

05co

70

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0

E

05

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437




A"

4..

B

A~~~~~~~~~~~~~~~

Figure 4. Immunolocalization of lipoxygenase in primary leaves. A,

Pre-immune control of primary leaf. B, Control primary leaf of a 5-

d-old seedling (5 d after cotyledon emergence) immunostained

with lipoxygenase antiserum. Control consisted of exposing the

seedlings to ethanol. C, Methyl jasmonate-treated primary leaf of a

5-d-old seedling immunostained with lipoxygenase antiserum.

ygenase protein to accumulate in various tissues and celltypes in the soybean seedling and are thus methyl jasmonate-responsive lipoxygenases.Image analysis techniques were used to quantify the dis-

tribution of the methyl jasmonate-responsive lipoxygenasein various cell types of different organs. The silver grains inthe micrographs were quantitated by converting individualpixels that lie above a specific gray level to 'marked' pixels(25). Figure 7 shows the data from quantifying the relativeamount of silver grains present in different cell types invarious organs. These data demonstrate that the PVM layerin primary leaves is a significant site of methyl jasmonate-responsive lipoxygenase accumulation. Epidermal and corti-cal cells in cotyledons and hypocotyls, respectively, alsocontain large amounts of methyl jasmonate-responsive lipox-ygenase. These data, coupled with the densitometry on theimmunoblot in Figure 1B, indicate that the hypocotyl andcotyledons are a major site for accumulation of methyl jas-monate-responsive lipoxygenase in soybean seedlings.

Ultrastructural Studies Indicate that the Methyl jasmonate-Responsive Lipoxygenase in Hypocotyls and Cotyledons IsPresent as a Paracrystalline Structure

The pvmLOX in the vacuoles of PVM and bundle sheathcells of primary leaves and trifoliolates appears as a flocculentmaterial (25). In contrast with this, pvmLOX deposits in thevacuoles of the hypocotyl and cotyledon tissues are highlyaggregated in discrete clumps. Because of these observationsat the light microscope level, we performed transmission EManalysis of thin-sectioned material to probe the structuralnature of the methyl jasmonate-responsive lipoxygenases.

Figure 8A shows a preimmune control for the lipoxygenaseantibody in the hypocotyl. The protein cluster appears as aconglomeration of elongate and amorphous elements. Figure8B shows the stellate, paracrystalline arrangement that theseprotein bodies' often acquire. The inset in Figure 8B showsan enlargement of part of the paracrystal. Distinct tubularelements of variable length appear to be arranged as packetsof tubules. The surrounding material seems to be amorphous,but this material may actually consist of tubules cut tangen-tially and in cross-section. One observation consistent withthis interpretation is the abundance of circular profiles inthese areas, which may be individual tubules. These tubularelements appear to be highly regular and to have an outerdiameter of about 5 to 8 nm. As is clearly evidenced in Figure7B, both of these regions are strongly labeled with the lipox-ygenase antibody, indicating that the methyl jasmonate-responsive lipoxygenase(s) is organized in this manner.The subepidermal cells and some cortical cells of the hy-

pocotyl have plastids with thylakoids and protein inclusions(Fig. 8C). The lipoxygenase antibody cross-reacted specifi-cally with the protein inclusions of the plastids, whereas nolabeling was observed in the stroma or associated with thy-lakoids (Fig. 8D). These protein inclusions did not label whenantibody to vspa/3 was used (data not shown).The methyl jasmonate-responsive lipoxygenase protein

was present in the epidermal cell vacuoles of cotyledons andalso demonstrated a paracrystalline structure (Fig. 9A). Athigher magnification, the tubular elements can be visualized

GRIMES ET AL.438




A-: 8 f,*+~~~~~

ft .N

I;

N.

VI'

-tsi;I. o. a+ t '. JeerL a.w USA ' a.+ 5., .,.

At .) t

_,#... _ . iAt\

*.Figure 5. Immunolocalization of lipoxygenase in hypocotyl tissue.A, Control hypocotyl of a 5-d-old seedling immunostained withlipoxygenase antiserum. B, Methyl jasmonate-treated hypocotyl ofa 5-d-old seedling immunostained with lipoxygenase antiserum.

in the cotyledon protein clusters (Fig. 9B). These regions oftubular elements contained methyl jasmonate-responsive li-poxygenase, as indicated by the distribution of labeling alongthem (Fig. 9B).

DISCUSSION

The results presented here demonstrate that low levels ofatmospheric methyl jasmonate induce the expression, accu-

mulation, and activity of lipoxygenase in soybean seedlings.Upon exposure to atmospheric methyl jasmonate, lipoxygen-ase was fairly uniformly stimulated in shoot tips, primaryleaves, epicotyls, hypocotyls, and cotyledons. The regulationof this increase is at the transcriptional level since methyljasmonate induces the expression of a lipoxygenase mRNAin intact seedlings. Previous work in our laboratory hasindicated that these responses are due to atmospheric methyljasmonate and not to increased ethylene levels in the cham-bers (7). Collectively, these results strongly indicate that thereis a methyl jasmonate-responsive lipoxygenase present in

Figure 6. Immunolocalization of lipoxygenase in cotyledonary tis-sue. A, Control cotyledon of a 5-d-old seedling immunostainedwith lipoxygenase antiserum. B, Methyl jasmonate-treated cotyle-don of a 5-d-old seedling immunostained with lipoxygenase anti-serum.

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Figure 7. Quantification of lipoxygenase in different cell types andorgans in methyl jasmonate-exposed seedlings. Data were quanti-tated on an individual cell basis. Error bars indicate the SE ofmeasurement, and at least 20 cells were quantitated for each celltype.

439

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Figure 8. Transmission electron micrographs of hypocotyl tissue in 5-d-old seedlings. All of the panels in this figure show tissue sections aftera 5-d exposure to atmospheric methyl jasmonate. A, Vacuolar region of hypocotyl tissue stained with preimmune serum (x19,900; bar = 0.5,gin). B, Vacuolar region of hypocotyl tissue stained with lipoxygenase antiserum. Note the paracrystalline structure (X1O,500; bar = 1 gin).Inset shows an enlargement (x74,000) of the paracrystalline structure. C, Section showing cell wall, plastid, and vacuole after staining withlipoxygenase antiserum. Note that there is reactive material in all of these subcellular compartments (XI 3,800; bar = 1 gin). D, Plastid presentin the hypocotyl after methyl jasmonate exposure stained with lipoxygenase antiserum. The plastid shows a significant amount of lipoxygenasepresent inside of a protein inclusion body (x49,500; bar = 0.2 gi). V, Vacuole; P, plastic; CW, cell wall.

GRIMES ET AL.440




Figure 9. Transmission electron micrographs of cotyledonary tissueafter a 5-d exposure to atmospheric methyl jasmonate showing theparacrystalline nature of the methyl jasmonate-responsive lipoxy-genase. A, A region of the cotyledonary vacuole immunostainedwith the lipoxygenase antiserum (x1 7,800; bar = 0.5 jm). B, Highermagnification (x74,000) of vacuolar region immunostained with thelipoxygenase antiserum showing the tubular arrangement of theseparacrystalline structures (bar = 2 Jim).

soybean seedlings. This is an intriguing observation consid-ering that (a) jasmonic acid and methyl jasmonate are poten-tially derived from a lipoxygenase-dependent pathway (30)and (b) methyl jasmonate is a strong candidate as an intra-or intercellular regulator of VSP gene expression and nitrogenpartitioning (7, 22).The potential activity of this methyl jasmonate-responsive

lipoxygenase is a difficult area to assess. In our in vitroexperiments, we have been able to demonstrate that lipoxy-genase activity is stimulated somewhat (approximately 25%)after methyl jasmonate exposure. This increase in activitycorrelates with, but does not closely parallel, the increase thatwe observe in transcript level or lipoxygenase protein level.The issue of lipoxygenase activity is further complicated bythe fact that the major site of accumulation for the methyljasmonate-responsive lipoxygenase(s) is in the vacuole. Ourin vitro assay conditions may not closely simulate the in vivoconditions. At this time, then, all we can conclude is that themethyl jasmonate-responsive lipoxygenase(s) is potentiallyactive. We are currently assaying for in vivo lipoxygenaseproduct formation to resolve this uncertainty.

In mature soybean leaves, the nitrogen-responsive lipoxy-genase is primarily associated with the PVM layer, and wehave named this protein the pvmLOX to reflect this fact (25).Although a small amount of VSP, including the lipoxygenase,is expressed in epidermal cells, quantitative immunochemis-try indicates that this amount is at least two orders of mag-nitude less than the level associated with the PVM layer (25).

In seedlings, however, only the primary leaf contains PVMcells, where they exist as a double layer rather than the singlelayer present in mature trifoliolate leaves (13). Becausemethyl jasmonate induced the expression of a lipoxygenasein organs that do not contain a PVM layer, we wanted todetermine where in these organs the lipoxygenase was ac-cumulating. The results of immunochemical localization ex-periments indicated that several different types of cells havematerial that is cross-reactive with the lipoxygenase anti-serum. In the primary leaf, both layers of the PVM showhigh amounts of the lipoxygenase, and the epidermal cellsalso contain a small amount of lipoxygenase protein. Palisadeparenchyma and spongy mesophyll do not contain detecta-ble amounts of lipoxygenase. This pattern is very similar tothe pattern seen in mature soybean leaves after removal ofthe sink tissue (25). In hypocotyl tissue, small amounts of thelipoxygenase were found in most cell types including theepidermis, cortex, pith, and vascular parenchyma, but it wasmost abundant in the epidermis and underlying cortex re-gions. Cambial cells did not appear to have any label asso-ciated with them. In cotyledonary tissue, the lipoxygenasewas primarily associated with epidermal cells. The remainingchlorenchymatous palisade cells had no detectable label.Huang et al. (10) presented evidence that in mature soy-

bean leaves from depodded plants, methyl jasmonate causedan increase in vspa/3 levels in many cell types. Apparently,the methyl jasmonate eliminated the normal cell-specificexpression of vspa/f3 and caused these proteins to be ex-pressed in numerous cell types (10). In seedlings, we alsoobserve that methyl jasmonate-responsive lipoxygenase isfound in numerous cell types. We are currently performing adevelopmental study of lipoxygenase localization to deter-

441




mine if the expression pattern is altered by methyl jasmonateexposure or if the methyl jasmonate-responsive lipoxygenaseis regulated differently than vspa/f3.

Ultrastructural studies were initiated to determine the sub-cellular localization of the methyl jasmonate-responsive li-poxygenase in these various cell types and to investigate thestructural nature of the methyl jasmonate-responsive lipox-ygenase. In our earlier studies with mature leaves, thepvmLOX was shown to accumulate within the cell vacuoles(25). Although cytoplasmic labeling was observed, we con-sider most of this to be due to synthesis and transport of thevacuolar lipoxygenase from the cytoplasm to the vacuoles(25). In seedling primary leaves and cotyledons, the majorityof the methyl jasmonate-responsive lipoxygenase was asso-ciated with the vacuoles. In the hypocotyl, however, othercellular compartments, such as the cytosol and plastids, con-tained appreciable levels of the methyl jasmonate-responsivelipoxygenase. Specifically, the subepidermal and cortical cellshave plastids with both thylakoids and protein inclusions.The lipoxygenase antibody specifically cross-reacted with theprotein inclusions but not with the stroma or thylakoids.Vernooy-Gerritsen et al. (28, 29) have examined the subcel-lular distribution of soybean seed lipoxygenases and suggestthat during germination they are initially present in thestorage tissues of etiolated cotyledons and later appear in thecytoplasm and protein bodies. Our results, on the other hand,show that the vast majority of lipoxygenase that cross-reactswith our antiserum is present in the vacuoles, both beforeand after exposure to methyl jasmonate. This difference isprobably due to the fact that our seedling tissue (5-d-old)was older than theirs (1-d-old). In our data, we detect someactivity associated with the cell wall in the hypocotyls, butthis is probably due to the presence of lignin in this tissueand antiserum binding nonspecifically to the lignin fraction.The morphology of the vacuolar-deposited methyl jasmon-

ate-responsive seedling lipoxygenase is very different thanthe morphology of these proteins in mature leaves. ThepvmLOX is present in the vacuoles of mature leaf PVM cellsas a flocculant material. In seedlings, the proteins are presentas paracrystalline structures in the vacuoles. When sectionsare taken through these paracrystals, tubular elements inboth tangential sections and cross sections are observed. Thematerial in cross-section appears to be composed of circularprofiles representing individual tubules, and the diameter ofthese tubules is between 5 and 8 nm. Lipoxygenase antiserumstrongly cross-reacts with the tubular elements that comprisethe paracrystals. This paracrystalline arrangement and tubu-lar substructure of the methyl jasmonate-responsive lipoxy-genase is unprecedented for any vacuolar protein of whichwe are aware.From our immunocytochemical and ultrastructural studies,

it would appear that methyl jasmonate is inducing the expres-sion and accumulation of more than one member of thelipoxygenase gene family. In the primary leaves, atmosphericmethyl jasmonate clearly induces the pvmLOX as well as alipoxygenase present in the epidermal cells. All of the VSPs,including the pvmLOX, are present primarily in the PVMlayer and, to a much lesser extent, in the epidermal cells.

Hence, this lipoxygenase is likely the product of a singlegene that is expressed in two cell types. In other tissues,

however, numerous types of cells express a lipoxygenase inresponse to methyl jasmonate exposure. In hypocotyls, wefound distinct evidence indicating that methyl jasmonateinduced the expression of a lipoxygenase that was associatedwith protein inclusion bodies in the plastids present in epi-dermal and cortex cells. These same cells also contained avacuolar lipoxygenase. Because it is difficult to imagine asingle gene product being targeted to both vacuoles andplastids, we hypothesize that in the hypocotyl, methyl jas-monate induces the expression of at least two lipoxygenasegenes. Determination of the exact number of lipoxygenasegenes that respond to methyl jasmonate will have to awaitthe availability of gene-specific probes to the members of thelipoxygenase gene family.

In summary, we have demonstrated that low levels ofatmospheric methyl jasmonate induce the expression andaccumulation of lipoxygenase(s) in various cell types of soy-bean seedlings. This increase in the lipoxygenase protein levelis correlated with an increase in the amount of lipoxygenaseactivity after exposure to methyl jasmonate. Ultrastructuralstudies indicate that most of the methyl jasmonate-responsivelipoxygenases are associated with vacuoles where the lipox-ygenases are present as paracrystalline structures of appar-ently tubular elements.

ACKNOWLEDGMENTS

We thank Tae Kyung Park and Dr. Joseph Polacco (BiochemistryDepartment, University of Missouri-Columbia) for the lipoxygenasecDNA (pTK11) and Dr. David Hildebrand (Department of Agron-omy, University of Kentucky) for the lipoxygenase antiserum. Theassistance of Dr. Ted Farmer with the lipoxygenase TLC assay isgratefully acknowledged. Lastly, we thank Dr. Chris Davitt and thestaff of the Electron Microscopy Center for their assistance.

LITERATURE CITED

1. Anderson JM, Spilatro SR, Klauer SF, Franceschi VR (1989)Jasmonic acid-dependent increases in the level of vegetativestorage protein in soybean. Plant Sci 62: 45-50

2. Chomczynski P, Sacchi N (1987) Single-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 162: 156-159

3. Farmer EE, Ryan CA (1991) Interplant communication: airbomemethyl jasmonate induces synthesis of proteinase inhibitors inplant leaves. Proc Natl Acad Sci USA 87: 7713-7716

4. Franceschi VR, Giaquinta RT (1983) The paraveinal mesophyllof soybean leaves in relation to assimilate transfer and com-partmentation. I. Ultrastructure and histochemistry duringvegetative development. Planta 157: 411-421

5. Franceschi VR, Giaquinta RT (1983) The paraveinal mesophyllof soybean leaves in relation to assimilate transfer and com-partmentation. II. Structural, metabolic and compartmentalchanges during reproductive growth. Planta 157: 422-431

6. Franceschi VR, Giaquinta RT (1983) Specialized cellular ar-rangements in legume leaves in relation to assimilate transportand compartmentation. comparison of the paraveinal meso-phyll. Planta 159: 415-422

7. Franceschi VR, Grimes HD (1991) Low levels of atmosphericmethyl jasmonate induce the accumulation of soybean vege-tative storage proteins and anthocyanins. Proc Natl Acad SciUSA 88: 6745-6749

8. Franceschi VR, Wittenbach VA, Giaquinta RT (1983) Para-veinal mesophyll of soybean leaves in relation to assimilatetransfer and compartmentation. III. Immunohistochemical lo-calization of specific glycopeptides in the vacuole after depod-ding. Plant Physiol 72: 586-589

442 GRIMES ET AL.




9. Grayburn WS, Schneider GR, Hamilton-Kemp TR, BookjansG, Ali K, Hildebrand DF (1991) Soybean leaves containmultiple lipoxygenases. Plant Physiol 95: 1214-1218

10. Huang JF, Bantroch DJ, Greenwood JS, Staswick PE (1991)Methyl jasmonate treatment eliminates cell-specific expressionof vegetative storage protein genes in soybean leaves. PlantPhysiol 97: 1512-1520

11. Klauer SF, Franceschi VR, Ku MSB (1991) Protein compositionsof mesophyll and paraveinal mesophyll of soybean leaves atvarious developmental stages. Plant Physiol 97: 1306-1316

12. Laemmli UK (1970) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227:680-685

13. Liljebjelke KA, Franceschi VR (1991) Differentiation of meso-phyll and paraveinal mesopyll in soybean leaf. Bot Gaz 152:34-41

14. Mason HS, Mullet JE (1990) Expression of two soybean vege-tative storage protein genes during development and in re-sponse to water deficit, wounding, and jasmonic acid. PlantCell 2: 569-579

15. Park TK, Polacco JC (1989) Distinct lipoxygenase species appearin the hypocotyl/radicle of germinating soybean. Plant Physiol90: 285-290

16. Peterman TK, Siedow JN (1985) Behavior of lipoxygenase dur-ing establishment, senescence, and rejuvenation of soybeancotyledons. Plant Physiol 78: 690-695

17. Peterman TK, Siedow JN (1985) Immunological comparison oflipoxygenase isozymes-1 and -2 with soybean seedling lipox-ygenase. Arch Biochem Biophys 238: 476-483

18. Sambrook J, Fritsch EG, Maniatis T (1989) Molecular Cloning:A Laboratory Manual. Ed 2. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY

19. Saniewski M, Czapski J (1989) Stimulatory effect of methyljasmonate on polyphenol oxidase and peroxidase activities,and starch degradation in scales of tulip bulbs. Bull PolishAcad Sci 37: 49-53

20. Staswick PE (1989) Developmental regulation and the influenceof plant sinks on vegetative storage protein gene expression insoybean leaves. Plant Physiol 89: 309-315

21. Staswick PE (1989) Preferential loss of an abundant storageprotein from soybean pods during seed development. PlantPhysiol 90: 1252-1255

22. Staswick PE (1990) Novel regulation of vegetative storage pro-tein genes. Plant Cell 2: 1-6

23. Staswick PE, Huang JF, Rhee Y (1991) Nitrogen and methyljasmonate induction of soybean vegetative storage proteingenes. Plant Physiol 96: 130-136

24. Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transferof proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354

25. Tranbarger T, Franceschi VR, Hildebrand DF, Grimes HD(1991) The soybean 94-kilodalton vegetative storage proteinis a lipoxygenase that is localized in paraveinal mesophyll cellvacuoles. Plant Cell 3: 973-987

26. Uedo J, Kato J (1980) Isolation and identification of a senescence-promoting substance from wormwood (Artemesia absinthiumL.). Plant Physiol 66: 246-249

27. Van Kuijk FJGM, Sevanian A, Handelman GJ, Dratz EA(1987) A new role for phospholipase A2: protection of mem-branes from lipid damage. Trends Biochem Sci 12: 31-34

28. Vernooy-Gerritsen M, Bos ALM, Veldink GA, VliegenthartJFG (1983) Localization of lipoxygenase-1 and -2 in germinat-ing soybean seeds by an indirect immunofluorescence tech-nique. Plant Physiol 72: 262-267

29. Vernooy-Gerritsen M, Leunissen JLM, Veldink GA (1984)Intracellular localization of lipoxygenases-1 and -2 in germi-nating soybean seeds by indirect labelling with protein A-colloidal gold complexes. Plant Physiol 76: 1070-1079

30. Vick BA, Zimmerman DC (1984) Biosynthesis of jasmonic acidby several plant species. Plant Physiol 75: 458-461

31. Weidhase RA, Kramell H, Lehmann J, Liebisch H, Lerbs W,Parthier V (1987) Methyl jasmonate-induced changes in thepolypeptide pattem of senescing barley leaf segments. PlantSci 51: 177-186

32. Wittenbach VA (1983) Effect of pod removal on leaf photosyn-thesis and soluble protein composition of field-grown soy-beans. Plant Physiol 73: 121-124

33. Wittenbach VA (1983) Purification and characterization of asoybean leaf storage glycoprotein. Plant Physiol 73: 125-129

34. Wittenbach VA, Franceschi VR, Giaquinta RT (1984) Soybeanleaf storage proteins. Curr Topics Plant Biochem Physiol 3:19-30

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expression, activity, andcellular accumulation methyl ...organs, however, mostof the methyl...

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