Plant Physiol. (1998) 116: 923-933

Specific Soybean Lipoxygenases Localize to Discrete Subcellular Compartments and Their mRNAs Are Differentially Regulated by Source-Sink Status¹

Lowry C. Stephenson, Thomas W. Bunker, Wesley E. Dubbs, and Howard D. Grimes^*

Department of Genetics and Cell Biology (L.C.S., H.D.G.), and Department of Botany (T.W.B., W.E.D., H.D.G.), Washington State University, Pullman, Washington 99164-4238

	ABSTRACT

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Members of the lipoxygenase multigene family, found widely in eukaryotes, have been proposed to function in nitrogen partitioning and storage in plants. Lipoxygenase gene responses to source-sink manipulations in mature soybean (Glycine max [L.] Merr.) leaves were examined using gene-specific riboprobes to the five vegetative lipoxygenases (vlxA-vlxE). Steady-state levels of all vlx mRNAs responded strongly to sink limitation, but specific transcripts exhibited differential patterns of response as well. During reproductive sink limitation, vlxA and vlxB messages accumulated to high levels, whereas vlxC and vlxD transcript levels were modest. Immunolocalization using peptide-specific antibodies demonstrated that under control conditions, VLXB was present in the cytosol of the paraveinal mesophyll and with pod removal accumulated additionally in the bundle-sheath and adjacent cells. With sink limitation VLXD accumulated to apparent high levels in the vacuoles of the same cells. Segregation of gene products at the cellular and subcellular levels may thus permit complex patterns of differential regulation within the same cell type. Specific lipoxygenase isoforms may have a role in short-term nitrogen storage (VLXC/D), whereas others may simultaneously function in assimilate partitioning as active enzymes (VLXA/B).

	INTRODUCTION

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The lipoxygenases are a family of enzymes that are widespread in higher plants and animals. Catalyzing the hydroperoxidation of specific pentadiene moieties in long-chain fatty acids, lipoxygenase activity has been correlated with diverse processes in development, maturation, and senescence in plants (for review, see Siedow, 1991). Lipoxygenases have also been implicated in responses to wounding (Creelman et al., 1992; Farmer and Ryan, 1992; Peña-Cortés et al., 1993; Bell et al., 1995) and in plant defense and pathogen resistance (for review, see Siedow, 1991; Rosahl, 1996). The plant lipoxygenases have an enzymatic role in the biosynthesis of signaling molecules and regulatory compounds such as the jasmonates and hexenals (Vick and Zimmerman, 1987; Hildebrand, 1989; Gardner, 1991; Song and Brash, 1991).

Lipoxygenases in soybean (Glycine max [L.] Merr.) function in nitrogen and assimilate partitioning (Tranbarger et al., 1991; Grimes et al., 1993; Bunker et al., 1995), mechanisms evolved by plants to temporarily store and subsequently remobilize nutrients to meet specific needs (Dalling, 1985; Zapata et al., 1987; Peoples and Dalling, 1988). The levels of gene transcript and protein accumulation of one or more lipoxygenases in mature soybean generally increase in response to increasing levels of available nitrogen (Grimes et al., 1993). Lipoxygenase genes are regulated in response to plant nitrogen status in both tissue-specific and developmentally specific patterns (Tranbarger et al., 1991; Grimes et al., 1993). Removal of developing pods, a strong assimilate sink, causes a reallocation of nitrogen and other assimilates to lipoxygenases (Tranbarger et al., 1991; Grimes et al., 1993; Bunker et al., 1995) and to VSPA and VSPB, well-characterized as VSPs in the leaves of these "sink-regulated" plants (Franceschi et al., 1983; Wittenbach, 1983b; Staswick, 1988; Klauer et al., 1991).

Lipoxygenases (Tranbarger et al., 1991) and VSPs (Franceschi and Giaquinta, 1983a, 1983b, 1983c; Franceschi et al., 1983) accumulate transiently in the specialized cells of the PVM, a single cell layer that interconnects the veins of the leaf with the palisade parenchyma and spongy mesophyll (Fisher, 1967). The cells of the PVM are hypothesized to act as an intermediary for temporary storage and mobilization of nutrients among photosynthetic source tissues, the vasculature of the plant, and, ultimately, the reproductive sink organs, the developing pods, or regions of organogenesis during vegetative growth (Franceschi and Giaquinta, 1983a; Franceschi et al., 1983; Everard et al., 1990a, 1990b). Thus, legumes have developed structural and molecular strategies to modulate the transient supply of nitrogen to meet changing metabolic demands.

Multiple gene copies within a species have been proposed as a means for sophisticated organ-, tissue-, or cell-specific regulation to respond to specific developmental or other internal or external stimuli (Eiben and Slusarenko, 1994; Harper et al., 1994; Palmgren, 1994; Zimmer et al., 1996). Lipoxygenases in soybean are examples of such a multigene family (Bunker et al., 1995); they are very highly conserved and consist of at least eight genes. The three seed-storage lipoxygenases (L-1, L-2, and L-3) and their genes have been well characterized, but the gene products appear to be active only at the earliest stage of germination (for review, see Siedow, 1991). The cDNAs of five VLX genes, termed vlxA to vlxE, have been cloned (for derivation and terminology, see Bunker et al., 1995). Differential regulation of the several VLX isoforms may account for the diverse functions attributed to the lipoxygenase family.

The objective of the research reported in this paper was to investigate the relationship between multiple lipoxygenase genes and nitrogen partitioning in soybean, by characterizing quantitative gene response and protein subcellular localization of specific vegetative members of the lipoxygenase multigene family in mature plants subject to source-sink manipulations. To accomplish this goal, a sensitive, gene-specific RNase-protection assay was developed (Bunker et al., 1995), and antisera were raised against synthetic peptides that represent unique regions of the five VLX isoforms. The ability to quantitate relative levels of steady-state vlx mRNAs by phosphor imaging has allowed documentation of complex regulatory aspects that were previously not possible. The results of these experiments suggest that specific lipoxygenase proteins may function in short-term, inactive nitrogen storage in leaves, whereas one or more may function in assimilate partitioning as active enzymes. Specific lipoxygenase genes respond differentially within the same cell type. These experiments provide a rigorous characterization of the responses of all five vegetative members of the lipoxygenase multigene family to sink limitation in soybean, and provide a case study of integrated regulation in such a highly conserved family.

	MATERIALS AND METHODS

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Seeds of unnodulated soybean (Glycine max [L.] Merr. cv Wye) were planted in potting compost in 1-gallon pots and grown in controlled-environment growth chambers with a minimum light intensity of 360 to 400 µmol photons m² s¹, a daylength of 16 h, and a day/night temperature regime of 25/18°C. Plants were fertilized with 500 mL of Peter's Professional and, on alternate fertilization dates, Peter's Excell nutrient solutions, both prepared at 4 g L¹ concentration (Grace-Sierra Horticulture Products Co., Allentown, PA). Plants were fertilized once per week at 2 to 4 weeks of age and twice per week thereafter. At each time point from each plant in a treatment, three leaflets were selected for collection from fully expanded axial trifoliate leaves in such a way that developmental effects of leaf age were mitigated. One leaflet was randomly selected from the younger leaves, one from the older leaves, and one from the middle of the stem. Collected plant material was immediately frozen in liquid nitrogen and stored at 80°C.

RNA Analysis

The 596-bp lox7-DomI fragment was isolated from a depodded soybean cDNA library by PCR amplification, using the gene-specific forward primer 5-CACAAGCTTAAGAAGTAGCAAAGATGTTTGG-3 (determined from the published sequence of the lox7 cDNA; Saravitz and Siedow, 1996) and the loxA/B/7-domain I reverse primer 5-CAACTGCAGTTAGTTGGCAAAGAAAATGCGATC-3. Gels from RNase-protection assays were exposed on x-ray film for visualization by autoradiography, then exposed on a phosphor plate for 2 h and immediately scanned for computer analysis with a phosphor imager (model 445SI, Molecular Dynamics, Sunnyvale, CA). Bands representing full-length protected riboprobes were quantitated using ImageQuant 4.1 software, following the manufacturer's protocols (Molecular Dynamics). The integrated phosphor signal volume scanned was directly proportional to radioactivity in each protected band and thus proportional to the number of gene-specific transcripts present. To allow direct comparison of results among riboprobes and experiments, the specific activity for each riboprobe was normalized to an arbitrary standard specific activity (2.0 × 10⁵ cpm fmol¹). The ratio of standard to calculated specific activity was then used to adjust each scanned phosphor signal value. The data presented in this report are these normalized measurements.

Preparation of Peptide-Specific Antibodies

All chosen peptides were close to the N terminus. Peptides were synthesized commercially: the VLXD-specific peptide was from Chiron Mimotopes (San Diego, CA), and the remainder were from Princeton Biomolecules (Columbus, OH). Peptides were prepared with C-terminal cysteines, and were N-terminal acetylated and C-terminal amidated.

Peptides were conjugated with Inject Maleimide activated keyhole limpet hemocyanin carrier protein (Pierce) in a 1:1 (w/w) ratio via sulfhydryl linkage. Peptide was injected into New Zealand white rabbits at 0.05 to 0.25 mg mL¹ per injection set using the monophosphoryl lipid A plus trehalose dicarynomycolate plus cell wall skeleton adjuvant system (Sigma). Rabbits were boosted at 4 and 8 weeks. Final serum collection was at 10 weeks. Serum was rimmed, coagulated, spun twice at 3500g, and stored with the addition of Tris, pH 8.0, to 0.25% and 1% thimerosal to 0.01%.

Peptide affinity-purification columns were prepared using chromatography columns (Poly-Prep, Bio-Rad) and coupling gel (Sulfo-Link, Pierce). Four milligrams of peptide was conjugated with 2 mL of coupling gel following the manufacturer's protocol. Columns were stored in 0.05% NaN₃ in column buffer. Antisera were affinity purified as follows: Columns were equilibrated with column buffer (10 mm Tris/1 mm EDTA, pH 7.5). Two milliliters of serum was diluted to 20 mL and passed through the column three times (once for anti-VLXD). Columns were then washed with 20 mL of column buffer and 20 mL of 500 mm NaCl in column buffer. Antibody was eluted with 5 mL of 0.1 m Gly, pH 2.4, and 0.8-mL fractions were collected into 0.2 mL of 1 m Tris, pH 9.0. Columns were then washed with 20 mm Tris, pH 8.8 (10 mm Tris for anti-VLXD), until equilibrated at pH 8.5. Antibody was further eluted with 5 mL of 100 mm triethylamine, pH 11.5, and fractions were collected as above into 1 m Tris, pH 7.0. Columns were washed with column buffer and stored as above. Protein in the antisera fractions was quantitated using a protein assay system following the manufacturer's microassay protocol (Bio-Rad).

Specificities of these antisera have not been tested against purified proteins. Because specific, unique peptides were synthesized for antibody preparation, the probability that an antibody would cross-react with other lipoxygenase isozymes was very low. In particular, the peptide sequences recognized by anti-VLXA/B were highly dissimilar from those that anti-VLXC/D recognize, specificities strongly supported by the unique patterns of immunolocalization and differential regulation for VLXB and VLXD reported in this manuscript. However, we cannot eliminate the possibility, although remote, that anti-VLXA cross-reacts with VLXB or that anti-VLXC cross-reacts with VLXD.

Tissue Preparation, Immunolocalization, and Transmission Electron Microscopy Immunocytochemistry

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	RESULTS

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Response of VLX Leaf mRNAs to Reproductive Sink Removal in Soybean

A composite autoradiograph from a representative experiment was assembled (Fig. 1a), and radiolabeled data from two independent experiments were combined and quantitated (Fig. 1, b and c). The increase of the transcript level for each VLX gene and vspB after 6 weeks of daily pod removal (above the values for the pod-bearing controls at the same age) demonstrated that transcript levels of all VLX genes in leaves responded coordinately to long-term pod removal with at least a 6-fold increase over 6 weeks (Fig. 1b). The vlx message in control plants bearing normally developing pods declined over the same time period (Fig. 1b). The vlxA transcript accumulated to the highest level during daily reproductive sink removal (Fig. 1c), although vlxC mRNA increased its message level 35-fold over that of the pod-bearing controls (Fig. 1b). However, levels of transcript for vlxA and vlxB were already at high base levels at week 1 compared with vlxC and vlxD (Fig. 1c), and these isoforms accumulated 2- to 4-fold greater levels of transcript in leaves over 6 weeks of treatment than that for vlxC/D (Fig. 1c). Response of vspB transcript level to pod removal was 99-fold above the control level (Fig. 1b), but the total transcript that accumulated was equivalent to that for vlxA.

View larger version (77K): [in this window] [in a new window]   Figure 1. Quantitative analysis of vlx and vsp steady-state mRNA levels in sink-regulated soybean leaves from plants with pods removed daily for 6 weeks. a, Compilation of autoradiographs from a representative RNase-protection analysis. Pod removal began 1 week after anthesis with plants 9 weeks old. Selected trifoliate leaflets were harvested beginning at the time of first pod removal (Week 1) and at weekly intervals thereafter (Weeks 2-6). Total RNA was extracted and mRNA analyzed using 5 µg of total RNA and 2 fmol 32P-labeled antisense riboprobe specific to 5 domains of vlx genes A through E and to vspB. Hybridization products were then separated on polyacrylamide gels. Only the portions of the gels representing full- length riboprobes protected by specific mRNAs are shown. b, Quantitation of vlxA through vlxE and vspB steady-state transcript from phosphor-image analysis of the 32P label in full-length protected bands from independent RNase-protection assays. The scale varies for each riboprobe. Error bars show sd (n = 2). c, Display of data described in Figure 1b, but at a common vertical scale to demonstrate comparative base levels of transcript accumulation and subsequent responses.

Response of VLX Leaf mRNAs to Removal of Shoot Tips in Soybean

A composite autoradiograph from a representative experiment was assembled (Fig. 2a) and radiolabeled data from two independent experiments were combined and quantitated (Fig. 2, b and c). The increase of transcript level in leaves for each VLX gene and vspB after 16 d of shoot-tip removal (measured from the untreated controls at 16 d, data not shown) demonstrated that, as with daily pod removal, transcript levels of all VLXs in leaves responded coordinately, showing at least a 2-fold increase, whereas vlxD increased its transcript level 20-fold over that of the control plant at the same age (Fig. 2b). The vlxC transcripts also increased to nearly the same level as those of vlxD after 16 d of tip removal, both about 2- to 4-fold above the levels of vlxA and vlxB mRNAs (Fig. 2c). It is interesting that the background levels of vlxA and vlxB steady-state mRNAs in leaves from 30-d-old plants were less than one-half of those for vlxA and vlxB in leaves from 60-d-old plants (compare scales between Figs. 1b and 2b), reflecting a strong developmental difference. In control plants with tips allowed to develop normally, the transcript levels of the vlx and vsp genes remained constant or increased slightly (data not shown). The level of vspB message in treated plants was very high, responding to treatment 20-fold over that of the control plants (Fig. 1b). The responses of the vlxC and vlxD steady-state transcript levels to vegetative sink removal (Fig. 2, b and c) were therefore in contrast to those in long-term reproductive sink removal (Fig. 1, b and c), where vlxA and vlxB transcripts responded most strongly to sink limitation. Thus, it appears that vlxA/B and vlxC/D may play differing roles in nitrogen storage or partitioning in leaves, and are under developmental control as well. Cessation of vegetative sink limitation and resumption of shoot-tip growth reduced the message accumulation of all of the genes dramatically (Fig. 2, a and b). These data confirm the direct correlation between sink limitation and accumulation of VLX gene transcript in mature leaves.

View larger version (71K): [in this window] [in a new window]   Figure 2. Quantitative analysis of vlx and vsp steady-state mRNA levels in soybean leaves from plants with shoot tips removed daily for 16 d, then with shoots allowed to regrow for 2 weeks. a, Compilation of autoradiographs from a representative RNase-protection analysis. Plants were 4 weeks old at first treatment. Terminal and axial shoots bearing leaves less than or equal to one-eighth-expanded were excised. Leaf samples were collected at the time of first tip removal (0 d), then subsequently at 4, 8, 12, and 16 d after tip removal. Beginning at 16 d, plants were allowed to regrow their shoots. Further collections were made at 1 and 2 weeks after regrowth began (23 and 30 d, respectively). Tissue collection and subsequent analysis were identical to that described for Figure 1. b, Quantitation of vlxA-E and vspB steady-state transcript levels from phosphor-image analysis of the 32P label in full-length protected bands from independent RNase-protection assays. The scale varies for each riboprobe. Error bars show sd (n = 2). c, Display of data described in Figure 2b, but at a common vertical scale to demonstrate comparative base levels of transcript accumulation and subsequent responses.

Immunocytolocalization Demonstrates That VLXB Protein Accumulates in the Cytosol and VLXD Protein Accumulates in the Vacuoles of Cells of the Leaf PVM and Bundle Sheath in Soybean Plants Subjected to Reproductive Sink Removal

Generating isoform-specific antisera to VLXA through VLXE has proven difficult because of the high level of sequence conservation among these proteins. The regions of highest variability exist in the amino-terminal -barrel domain and, consequently, regions of 12 to 16 amino acids were selected in this domain for the generation of anti-peptide antisera. Specific antisera to VLXA were obtained through VLXE using these methods, but only the antisera to VLXB, VLXD, and VLXE are of sufficient avidity for immunocytochemistry. Here we focus on VLXB and VLXD immunolocalization; the data for VLXE will be published separately.

Leaf cross-sections incubated with anti-VLXB antibody were examined with bright-field (Fig. 3, a, c, and e) and epipolarization (Fig. 3, b, d, and f) light microscopy. VLXB from control plants was found localized primarily in the cytosol of cells of the PVM, the single cell layer here seen highlighted by epipolarized light reflected off of anti-VLXB-decorated silver grains (Fig. 3, b and d). In leaves of plants subjected to sink regulation by removal of developing pods, the level of VLXB accumulation in the PVM increased slightly (Fig. 3f), but this protein was found additionally in the bundle-sheath cells and to a lesser extent in the cells surrounding these tissues (Fig. 3f), suggesting a role for this enzyme in assimilate partitioning. The VLXB protein remained localized in the cytosol with sink regulation (Fig. 3f). Sections were also incubated with VLXB preimmune serum, but no staining was observed (data not shown). Transmission electron microscopy confirmed the cytosolic localization of VLXB (Fig. 4a). Anti-VLXB-decorated immunogold particles were observed primarily in the cytosol from both depodded plants (Fig. 4a) and control plants (data not shown). Density of particles within the cytosol of individual cells increased only slightly with source-sink manipulation, indicating that most of the increase in vlxB mRNA levels observed after pod removal (Fig. 1) resulted in VLXB accumulation in the bundle-sheath cells.

View larger version (137K): [in this window] [in a new window]   Figure 3. Light microscopy immunolocalization of VLXB protein in mature soybean leaves after 4 weeks of continuous pod removal. Mature leaves were collected at 13 weeks of age, fixed, and sectioned. Sections were incubated with the peptide-specific antibody, then with protein A-conjugated gold, before silver enhancement and histochemical staining. a and b, Bright-field and epipolarization images, respectively, of leaf cross-section from untreated control plant incubated with anti-VLXB antibody. The protein under these nonsink-limited conditions can be seen localized to the cytosol of the PVM cells. Scale bar = 40 µm. c and d, Bright-field and epipolarization images, respectively, of a vascular bundle and adjacent PVM and bundle-sheath cells shown at higher magnification from untreated control plant incubated with anti-VLXB antibody. Scale bar = 25 µm. e and f, Bright-field and epipolarization images, respectively, of leaf cross-section from a plant with pods removed continuously for 4 weeks and incubated with anti-VLXB antibody. VLXB protein slightly increases its accumulation in the PVM under these sink-regulated conditions, but additional accumulation occurs in the bundle-sheath cells surrounding the vein and to a lesser extent in adjacent cells. VLXB protein remains localized in the cytosol. Scale bar = 40 µm.

View larger version (75K): [in this window] [in a new window]   Figure 4. Transmission electron microscopy immunocytolocalization of VLXB and VLXD protein in mature soybean leaves after 4 weeks of continuous pod removal. a, Portion of a leaf cross-section from a plant with pods removed daily for 4 weeks and incubated with anti-VLXB antibody. Tissue sections are identical to those described for Figure 3. Sections were mounted onto nickel grids, incubated with peptide-specific antibody and protein A-gold conjugate, and poststained. Immunogold particles are localized primarily in the cytosol in this cell from a sink-regulated plant. Scale bar = 200 nm. b, Portion of a leaf cross-section from a plant with pods removed daily for 4 weeks and incubated with anti-VLXD antiserum. Tissue sections are identical to those described for Figure 5 and were prepared as in a. Immunogold particles are dense in the vacuole and localize strongly to electron-dense flocculent material in this cell from a sink-regulated plant. Minimal localization is apparent elsewhere. Scale bar = 200 nm.

Leaf cross-sections incubated with anti-VLXD antibody were examined with bright-field (Fig. 5, a, c, and e) and epipolarization (Fig. 5, b, d, and f) light microscopy. Accumulation of VLXD protein was minimal in leaves from control plants (Fig. 5b). In contrast, VLXD protein accumulated to apparent high concentration in the vacuoles of the PVM and bundle-sheath cells in response to daily pod removal (Fig. 5, d and f). A lower level of accumulation was noted in cells adjoining these tissues. Leaf sections were also incubated with VLXD preimmune serum, but no staining was observed (data not shown). Transmission electron microscopy confirmed the vacuolar localization of VLXD (Fig. 4b). Anti-VLXD-decorated immunogold particles were readily visible in vacuoles in tissues prepared from plants with pods removed (Fig. 4b), here seen associated with electron-dense material. Little or no staining was observed in the cytosol, nor was any observed in cellular organelles such as the ER, Golgi bodies, or chloroplasts (data not shown).

View larger version (117K): [in this window] [in a new window]   Figure 5. Light microscopy immunolocalization of VLXD protein in mature soybean leaves after 4 weeks of continuous pod removal. Sections were prepared as described for Figure 3. a and b, Bright-field and epipolarization images, respectively, of leaf cross-section from untreated control plant incubated with anti-VLXD antibodies. The protein under these nonsink-limited conditions can be seen localized at very low levels in the vacuoles of the PVM and bundle-sheath cells. Scale bar = 25 µm. c and d, Bright-field and epipolarization images, respectively, of leaf cross-section from a plant with pods removed continuously for 4 weeks and incubated with anti-VLXD antibodies. VLXD protein accumulates in the vacuoles of the PVM and bundle-sheath cells and, to a lesser extent, in adjacent cells under these sink-regulated conditions. Scale bar = 30 µm. e and f, Bright-field and epipolarization images, respectively, of a vascular bundle and surrounding bundle-sheath cells shown at higher magnification from a plant with pods removed continuously for 4 weeks and incubated with anti-VLXD antibodies. VLXD protein accumulates in the vacuoles of the PVM and bundle-sheath cells under these sink-regulated conditions. Scale bar = 25 µm.

Both cellular and subcellular immunolocalization studies of mature leaves demonstrate that VLXB and VLXD accumulate differentially in response to reproductive sink limitation in soybean. VLXB is present in the cytosol of PVM cells but, under sink-regulated conditions, accumulates additionally in the bundle sheath and surrounding cells. VLXD protein is found in the vacuoles of the PVM and bundle-sheath cells at very low levels, but accumulates to apparent high concentrations in response to sink limitation.

	DISCUSSION

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A quantitative analysis of molecular regulation of specific VLX mRNA levels in response to source-sink manipulations in soybean was performed and the cellular and subcellular localization of specific VLX gene products was determined. Because of the complexity of vlx mRNA accumulation patterns observed in a preliminary study (Bunker et al., 1995), and because of the cloning of a fifth VLX cDNA (vlxE/lox 7; Saravitz and Siedow, 1996), it was imperative to obtain quantitative data documenting the responses of the soybean lipoxygenase multigene family response to sink limitation. Only with quantitative data for each individual vlx mRNA is it possible to determine the potential roles of these genes and their products in nitrogen metabolism and plant function. Furthermore, while one or more lipoxygenases accumulate to very high levels in sink-regulated leaves (Tranbarger et al., 1991), there is a coordinate increase in lipoxygenase activity (Grimes et al., 1992), thus indicating a possible bifunctionalitystorage and lipid peroxidationwithin this multigene family. To investigate this bifunctionality and to further characterize the regulation of this multigene family, gene-specific riboprobes and a sensitive RNase-protection protocol (Bunker et al., 1995) were developed to distinguish between the highly conserved mRNA sequences. Additionally, peptide-specific antibodies were generated to visualize the cellular and subcellular localization of the specific gene products.

Steady-state levels of five VLX mRNAs were examined in leaves of mature soybean with pods removed daily, and in leaves of 4- to 6-week-old soybean with terminal and axial shoot tips removed daily. It is interesting that steady-state transcript levels of all VLX genes rose severalfold, suggesting a global regulation of the multigene family by these sink-limitation treatments. However, specific vlx isoforms demonstrated significant differential message accumulation beyond the general coordinate response. Transcripts of vlxA and vlxB accumulated to much higher levels than those for vlxC and vlxD during pod-removal treatments, whereas vlxC and vlxD transcripts accumulated to greater levels when developing shoot tips were removed. These data suggest differential functions for the specific gene products.

Cellular and subcellular localization of VLX gene products was examined in mature leaves by light and transmission electron microscopy. Previous immunolocalization studies (Vernooy-Gerritsen et al., 1983, 1984) had shown differential patterns of seed lipoxygenase accumulation during early seed germination. Here, using peptide-specific antibodies, we demonstrated that VLXB protein accumulated only in the cytosol of the PVM cells in plants with pods developing normally. The level of VLXB protein accumulation in the PVM did not appear to change significantly with sink limitation (4 weeks of pod removal), but was observed to accumulate additionally in the cytosol of bundle-sheath cells after daily pod removal. Immunolocalization studies showed that VLXD protein accumulated only in the cells of the PVM and bundle sheath and, in contrast to VLXB, only within the vacuole. VLXD protein level was very low in plants with pods developing normally, but accumulated to apparent high concentrations in plants with pods removed daily for 4 weeks. VLXD remained localized to the vacuoles under these treatment conditions.

The responses of the five VLX genes in soybean to sink manipulation provide a case study of coordinated gene regulation within a highly conserved multigene family of a single plant species. Differences of molecular regulation of specific gene transcripts, and cellular and subcellular localization of protein products, suggest that at least three lipoxygenase genes respond differentially to sink limitation and may provide a first level of regulation in cell function. The specific vacuolar localization of VLXD protein in the PVM and its accumulation in response to reproductive sink limitation suggest that this protein functions as a temporary storage protein. Biochemical studies of soybean leaf lipoxygenases demonstrate that they possess the pH optima and other characteristics of active cytosolic enzymes (Grayburn et al., 1991). Sequestration of VLXD in the typical low-pH environs of vacuoles (Boller and Wiemken, 1986) may render the VLXD isoform inactive, enabling it to function as an inert storage form (Wink, 1993; Staswick, 1994). Localization of VLXD in the cells of the PVM, a specialized cell layer involved in assimilate partitioning and storage (Franceschi and Giaquinta, 1983a; Franceschi et al., 1983; Everard et al., 1990a, 1990b), provides a further indication that VLXD is involved in the temporary storage of nitrogen in vegetative organs.

In contrast, immunolocalization data suggest that VLXB protein may function as an active enzyme. It was observed to be strictly cytosolic, and levels of protein within individual cells did not appear to vary significantly with sink- limitation treatments. However, 4 weeks of daily pod removal caused accumulation of protein in the bundle-sheath cells, in addition to the PVM, where it was found constitutively. These data are consistent with an active role for VLXB in nitrogen or assimilate partitioning in response to reproductive sink limitation. Thus, two specific VLX gene products may have completely different functions in a single cell type in response to sink limitation. Our data suggest that VLXB may function as an active enzyme in assimilate partitioning, whereas VLXD may function as a vacuolar storage protein.

In contrast to VLXB and VLXD localization to tissues specifically associated with nitrogen partitioning and storage, VLXE protein associated with thylakoid membranes of chloroplasts from diverse cell types, and the vlxE gene transcript was especially responsive to wound treatments. Because response and accumulation patterns suggest that the VLXE isozyme may be functionally and structurally distinct, and because additional studies are in progress, these data will be reported elsewhere (A.M. Fischer, L.C. Stephenson, and H.D. Grimes, unpublished data), but they suggest yet a third distinct cellular function for a soybean VLX. Bunker et al. (1995) previously demonstrated that removal of pods in sink-limitation treatments did not provoke a response from the wound-signaling pathway, suggesting that expression patterns displayed in the experiments reported in this manuscript were not due to wounding. Effects of shoot-tip removal on the wound-response pathway were not specifically tested.

How a lipoxygenase gene product might function in the PVM to mediate assimilate partitioning is unknown at this time. However, VLXB localization in the cytosol of the PVM suggests that it may have an important function in this specialized cell layer. Everard et al. (1990a) have shown that oxygen-consumption rates in the PVM are approximately 3-fold higher than in the palisade parenchyma cells, and it now becomes a distinct possibility that oxygen consumption by lipoxygenase is responsible for this. One lipoxygenase studied in mammals functions in reticulocyte maturation by remodeling membranes through peroxidation and subsequent breakdown of the membrane (Kuhn et al., 1990). Maccarrone et al. (1994) propose that soybean lipoxygenase-2, a seed lipoxygenase, can differentially oxygenate specific soybean membranes. A similar enzymatic role is conceivable for VLXB in the PVM and bundle-sheath cells, with the isozyme functioning in maintenance or dissolution of vacuolar or other membrane systems. Such an isozyme would remain cytosolic and enzymatically active in this scenario, as evidence has suggested for VLXB. Alternatively, VLXB may function in initiation of a signal transduction cascade to regulate partitioning. Several studies have implicated components of the octadecanoid pathway in the regulation of assimilate partitioning during seed development (Lopez et al., 1987; Wilen et al., 1991; Simpson and Gardner, 1995). It is clear that the biochemical activities of these lipoxygenase isoforms must now be investigated.

Molecular-regulation data from sink-limitation experiments show that levels of vlxB transcript were high in leaves of mature plants at 1 week postanthesis ("Week 1"), twice the levels observed in the 4-week-old plants, and 2-fold higher in the mature plants than the levels of vlxC and vlxD message. Because mature soybean normally undergoes translocation of assimilates from temporary storage to reproductive organs in the weeks after anthesis, the correlative high accumulation of vlxB message at this developmental stage is additional evidence that VLXB may be functioning as an active enzyme involved in assimilate partitioning. The vlxA message levels accumulate to significantly higher levels and respond to sink manipulation treatments in the same patterns as vlxB transcript. Furthermore, vlxA and vlxB nucleotide sequences are very similar, sharing 94% identity at the deduced amino acid level. Thus, we predict that VLXA will be found in the cytosol. Studies of molecular regulation demonstrate that vlxC and vlxD accumulate similar levels of transcript in both pod- and shoot-tip-removal treatments. Additionally, these two genes have closely related sequences, sharing 87% identity at the deduced amino acid level. The C-terminal coding region of the vlxC gene sits less than 1 kb upstream of the vlxD coding region, presumably the result of the duplication event that characterizes the soybean genome (Zhu et al., 1994; Shoemaker et al., 1996). It was observed that very few of these duplications in soybean are silent (Zhu et al., 1994). These data are consistent with a common role for vlxC and vlxD, encoding gene products that function as vacuolar-storage proteins. Development of antibodies to VLXA and VLXC, with sufficient avidity for immunocytochemistry, will be needed to verify subcellular localization of these isozymes.

The patterns of steady-state mRNA accumulation in leaves for vlxC and vlxD in both reproductive- and vegetative-sink limitation closely parallel each other. The fact that VLXD protein also accumulates to high levels after pod removal, whereas vlxD transcript levels accumulate only modestly, is evidence for posttranscriptional regulation. Molecular-regulation data for vlxC and vlxD transcript levels and vacuolar localization for VLXD protein thus support roles for these genes and their products in nitrogen storage and partitioning. However, molecular regulation of the lipoxygenase multigene family is complex, suggesting that additional levels of cellular regulation may exist as well. Although multiple members of the family occur within the same cell type, segregation at the cellular and subcellular level may permit complex patterns of differential regulation. These levels of developmental and spatial regulation may permit members of the lipoxygenase multigene family to have multiple roles in a complex function, e.g. processing assimilates through the PVM. Thus, by a specific function of the gene product or by multiple functions controlled by alternative regulation or localization, members of a multiple gene family such as the lipoxygenases may respond to diverse internal and external signals and yield integrated responses in plant growth and physiology.

In this report the responses to assimilate sink limitation of the five vegetative members of a highly conserved multigene family, the soybean VLXs, were quantitatively examined in mature leaves. The data strongly suggest that specific isoforms participate in temporary storage and partitioning of nitrogen and other assimilates through the specialized leaf PVM tissue. VLXD, and perhaps VLXC, may function as relatively inert vacuolar-storage proteins. VLXB, and perhaps VLXA, may function as active cytosolic enzymes in assimilate partitioning. Regulation is tightly coordinated between the gene family members and responses are specific to treatment, isoform, tissue, developmental stage, and organelle. Multiple isoforms may function simultaneously within a single cell type, and regulation of the members of this multigene family is highly coordinated.

	FOOTNOTES

   Received August 27, 1997; accepted November 14, 1997.

	ABBREVIATIONS

Abbreviations: PVM, paraveinal mesophyll. VLX, vegetative lipoxygenase. VSP, vegetative storage protein.

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