Plant Cell Reports

Plant Cell Reports (1996) t5:620-626

9 Springer-Verlag1996

Immunolocalization of lipoxygenase in pea (Pisum sativum L.) carpels Manuel Rodriguez-Concepci6n, Maria Dolores G6mez, and Jos~-Pio Beltrfin Instituto de Biologia Molecular y Celular de Plantas, C.S.I.C.-Universidad Polit6cnica de Valencia, Camino Vera 14, 46022 Valencia, Spain Received /2 July 1995/Revised version received 27 September 1995 - Communicated by E. W. Weiler

Summary Polyclonal antibodies against a part of pea (Pisum sativum L.) LOXG protein have been raised to study the pattern of distribution of related lipoxygenases in pea carpels. The antiserum recognized three lipoxygenase polypeptides in carpels. One of them became undetectable 24 hours after fruit development induction, suggesting that it may correspond to the protein derived from loxg cDNA. Immunolocalization experiments showed that lipoxygenase protein was present only in pod tissues: it was abundant in the mesocarp and, from the day of anthesis, in the endocarp layers. Lipoxygenase distribution is regulated throughout development. The association of lipoxygenase with cells in which processes of expansion and growth will potentially take place support a role in pod growth and development.


DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; IgG, immunoglobulin G; GA3, gibberellic acid; LOX, lipoxygenase, PAGE, polyacrylamide gel; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; Tris, 2-amino-2hydroxymethyl-1,3-propanediol.

Introduction Lipoxygenases (LOXs, linoleate:oxygen reductase, EC are non-heme iron-containing enzymes which catalyze the oxygenation of polyunsaturated fatty acids with a cis, cis-l,4-pentadiene system (linoleic and linolenic acids in plants) to produce lipid hydroperoxides (Siedow 1991). They are commercially important due to their involvement in flavor and odor formation in seeds, fruits and vegetables, through the production of hexanal and other compounds. The conversion of the fatty acid hydroperoxide products through the lipoxygenase pathway generates molecules such as jasmonic acid and traumatin, which mediate various physiological and pathological processes (Hildebrand 1989). In addition, a LOX reaction has been implicated in the production of abscisic acid via

Correspondence to: J.-P. Beltrfin

violaxanthin (Creelman et al. 1992). LOXs are ubiquitous and have been described to exist in many tissues of higher plants and animals (Siedow 1991). There is evidence for roles of LOXs in plant growth and development, stress responses and senescence, either through the production of those regulatory molecules or by some other activity (Siedow 1991, Hildebrand 1989). The multiple functions ascribed to LOXs are consistent with the variety of forms or isozymes which are often present in plants. In pea (Pisum sativum L.), several LOXs have been reported (Domoney et al. 1990, Domoney et al. I991). Seed LOX2 and LOX3 are first detected more than two weeks after anthesis and accumulate with seed fresh weight (Domoney et al. 1990). Other pea LOX species are detected in flowers, stems and roots. Water stress induces the expression of loxP1 in stems and leaves (Bell and Mullet 1991). Nevertheless, no clear role has been established for any of these LOXs. We have previously reported the isolation from pea carpels of the cDNA loxg, which corresponds to a novel LOX gene (Rodrfguez-Concepci6n and Beltr~in 1995). Expression of loxg was also found in young organs such as apical leaves and seedlings, but it was specially high in flowers. In all these organs, the natural pattern of development involves loxg down-regulation. The patterns of loxg expression suggest that this LOX has to do with the growth of the tissues in which it is expressed (Rodffguez-Concepci6n and Beltrfin 1995). We have raised antibodies against a fragment of LOXG protein to study the dis~ibution of related LOX polypeptides in carpels and young fruits in order to throw new light on the physiological function. Fruit set and development was induced by treating unpollinated carpels with gibberellic acid (GA3) to control the time of induction and better determine early changes in the development of fruit tissues. GA3 treatment gives rise to parthenocarpic, seedless fi'uits, which are identical to those produced by pollination and fertilization (Garcfa-Martinez and Carbonell 1980, Garcfa-Marffnez et al. i987, Vercher et al. 1984). The antibody showed immunological reactivity with LOXs and revealed an association of LOX with pod cell growth and expansion.


Materials and methods

Plant material Pea (Pisum sativum L., cv. Alaska) plants were grown as described (Rodriguez-Concepci6n and Beltr~n 1995). Flowers from the first and second flowering nodes were emasculated two days before anthesis (day -2, taking anthesis as day 0) and at the beginning of day +2, 20 gl of gibberellic acid (GA 3, Fluka) 0.3 mM in 0.1% (v/v) Tween 80 were applied directly to the carpel. Control carpels were untreated or treated with 0.1% (v/v) Tween 80. After the indicated times, carpels were harvested, and immediately either frozen or fixed for further experiments.

Expression of a Ioxgfragment in E. coli A 1149 bp EcoRI-HindlII fragment encoding b2EH, a polypeptide of 383 amino acid residues (43 kD), was obtained from loxg cDNA clone pU/91b2 (Rodrfguez-Concepci6n and Belmin 1995) and subcloned in frame in the expression vector pRSET-B (Invitrogen). The plasmid confers resistance to ampicillin andtpermits the cloning of foreign DNA in frame with phage:T7 gene 10 modified (which encodes a protein of 45 amino acid residues and 5 kD), under the control of the T7 promoter. The construct pRSET-B/b2EH was used to transform Escherichia coli K38 cells. This strain was previously transformed with the plasmid pGP1-2 (Tabor and Richardson 1985), which confers resistance to kanamicin and contains the gene 1 encoding T7 RNA polymerase under the control of a promoter inducible by heat-shock. A colony of K38 cells transformed with both plasmids was cultured at 30~ overnight in 10 ml of LB medium (2% [w/v] tryptone, 0.5% [w/v] yeast extract, 1% [w/v] NaC1, pH 7) containing 75 gg/ml ampicillin and 50 gg/ml kanamicin. The heat-shock necessary to produce T7 RNA polymerase and the overproduction of the fusion protein were carried out as described (Edwards et al. 1995). A control experiment was performed with cells containing pRSET-B without an insert.

Visualization and purification of the fusion protein The analysis of synthesized proteins was performed from preparations of bacterial protein extracts. After induction, the cells were pelleted by centrifugation at 5000xg for 10 rain and resuspended in 1 ml of 0.1 M DTT, 20% (v/v) glycerol, 4% (w/v) SDS, 80 mM Tris-HC1 pH 6.8. The resulting extracts were boiled for 5 rain, cooled down, and sonicated 5 x 15 s. After centrifugation in a microfuge at 12000 rpm for 5 min, supernatant was selected for protein analysis. Samples of I0 gl were analyzed in 10% polyacrylamide gels (SDS-PAGE; Laemmli 1970) with the Miniprotean system (Bio-Rad). The proteins were visualized by staining the gels with a solution of 0.1% (w/v) Coomassie Brilliant Blue R-250 (Merck) in 46% (v/v) methanol, 8% (v/v) acetic acid for at least 1 hour and subsequent destaining in 30% methanol, 10% acetic acid. For purification of the fusion protein, preparative SDS-PAGE electrophoreses were performed. After electrophoresis, gels were stained with 0.25 M KC1 in an ice bath and the band corresponding to the recombinant protein was excised. The strip was washed twice in 5 mM D T r and equilibrated for 1 hour in elution buffer (2% [w/v] SDS, 0.4 M NH4HCO3, 5% [v/v] l]-mercaptoethanol) prior to electroelution.

Antibody production Polyclonal antibodies against the fragment b2EH of LOXG protein were raised in New Zealand white rabbits injected with alliquots of 150 gg purified fusion protein as described (Domingo et al. 1994). After 4 injections, immune serum was collected and dialyzed overnight against Tris-buffered saline (TBS: 0.9% [w/v] NaC1, 20 mM Tris, pH 8.2).

Protein analysis Carpel protein extracts were prepared by homogenization of frozen samples in liquid nitrogen. The extraction buffer contained 0.1 M sodium phosphate pH 7.5,2 mM DTY, 1 mM EDTA and 0.1% (v/v) Triton X-100. The homogenates were clarified by centrifugation at 12000xg for 60 min. Supernatant was used for total protein quantification with the method of Bradford (t976) using bovine serum albumin (BSA) as standard. Aliquots of 3 gg total protein were boiled in Laemmli (1970) sample buffer for 5 rain and separated by SDS-PAGE for Western analysis. After electrotransference to Immobilon-P PVDF membranes (Millipore), the blots were blocked in TBS-t (TBS, 2% [w/v] powdered nonfat dry milk and 0.05% [v/v] Tween-20) for 1 h. The same buffer was used for the incubation with the antiserum diluted 1:2000 for 1 h. The membranes were washed three times in TBS-T (same as TBS-t but 0.5% [v/v] Tween-20) prior to incubation with the secondary antibody and immunodetection using tile ECL system (Amersham).

lmmunolocalization Pea carpels were harvested from the plant, cut transversely and immediately fixed as described (Domingo et al. 1994). Sections (2 gm thick) were cut and incubations and washes were performed as described (Domingo et al. 1994) using an anti-b2EH dilution of 1:200, and an anti-rabbit IgG gold conjugate (10 nm, Sigma) diluted 1:40 as a secondary antibody in combination with a silver enhancement kit (IntenSE, Amersham). Control sections were incubated with preimmune serum. Sections were stained with 0.5% safranin O for 30 rain, and a Nikon Diaphot microscope with an episcopic fluorescence attachment (IGS filter) was used for sample visualization and photography.


Antibody production. A fragment of 1.15 kb (b2EH) was obtained after the digestion o f loxg c D N A ( R o d r i g u e z - C o n c e p c i 6 n and Beltr~n 1995) with EcoRI and HindIII and cloned in frame in the expression vector pRSET-B (figure 1). The construct pRSET-B/b2EH encodes a 428 amino acid fusion protein (383 residues from b2EH and 45 from the ORF of the plasmid) with a predicted molecular mass o f 48 kD, and this was the size of the polypeptide overproduced by cultures of E. coli cells transformed with pRSET-B/b2EH. The control cells transformed with pRSET-B without an insert synthesize a 5 kD protein which is not detected in the 10% polyacrylamide gels (figure 1). The fusion protein was purified by electroelution and used for polyclonal antibody production in rabbits. I m m u n e serum diluted 1:2000 reacted with b2EH protein but also with soybean seed LOX1, indicating that anti-LOX antibodies had been generated (figure 1).

Immunoblot analysis of LOX polypeptides in pea carpels. Protein extracts from pea carpels were electrophoresed, blotted and probed with the anti-b2EH serum. Unpollinated carpels either induced or not to become growing fruits with G A 3 were tested for L O X protein content with the antiserum diluted 1:2000 (figure 2). In untreated ovaries, the antibodies recognized three polypeptides of ca 97 kD: a (the minor band), b and e, a being the largest and c the smallest. One of them, c, progressively disappeared in carpels treated with GA3 and it was undetectable 24 hours


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Figure 1. Strategy to produce antibodies against the polypeptide fragment b2EH from pea LOXG protein. An EcoRI-HindIII fragment from loxg cDNA clone pU/91b2 was cloned in frame in the multiple cloning site (MCS) of the expression vector pRSET-B, under the control of the T7 RNA polymerase promoter (PT7). Shadow boxes in loxg and b2EH cDNA sequences indicate coding regions and dark areas represent highly conserved regions in all LOXs. After induction of T7 RNA polymerase production, protein extracts from E. coli K38 cells transformed with either pRSET-B (pB) or pRSET-B/b2EH (pB/b2EH) were electrophoresed in SDS-PAGE together with molecular mass markers (P)(box 1). The arrow shows the position of the expressed recombinant fusion protein b2EH, which was afterwards purified by electroelution. In box 2, a Coomassiestained SDS-PAGE of pB/b2EH protein extract (left) and the purified fusion protein b2EH (right) is shown. This fusion protein was injected in rabbit for polyclonal antibodies production. The antiserum was used in Western analyses of 0.5 gg purified fusion protein b2EH (W2) and 0.5 gg soybean LOX1 (Wl). Size standards (5 gg) were used as a control of the specificity of the anti-LOX serum (W1). P1 is the same as W l but Coomassie-stained.

623 after the application of the hormone. The polypeptides were visible in the gels stained with Coomassie Brilliant Blue: three bands in untreated carpels and only two (a and b) 24 hours after GA3-treatment (figure 2).

was detected from day 0 in the cells of the transition layer and the epidermis of the endocarp (figures 4B and 4C). Control incubations with preimmune serum (figure 4D) and with the antiserum previously incubated with an excess of b2EH protein (not shown) did not show any specific signal. The distribution shown in carpels at day 0, which coincides with the localization of loxg mRNAs (Rodrfguez-Concepci6n and Beltr~in 1995), was observed up to day +3 in untreated carpels. In carpels induced to fruit set with GA 3 the amount of LOX seemed to be lower. In these young parthenocarpic fruits, LOX polypeptides were detected in all the endocarp layers, including the cells of the middle zone (figure 4F). The same distribution was found in young pollinated fruits (data not shown). Discussion

Figure 2. Immunoblot analysis of pea carpel protein extracts with the anti-b2EH serum. Carpels from emasculated flowers were either treated (+) or not (-) with GA 3 at day +2 and collected after 24 hours. Samples of 3 lag total protein were loaded in SDS-PAGE. Some lanes were stained with Coomassie (P) and the rest were used for Western analysis (W) with anti-b2EH serum diluted 1:2000. Arrows a, b and c, and lines between boxes, indicate the polypeptides recognized by the antibody. The position and mass of size standards is indicated in the left.

Immunolocalization of LOX in pea carpels Samples of carpels of different developmental stages were embedded in 2-hYdroxyethyl methacrylate. Transverse sections were incubated with the anti-b2EH serum diluted 1:200. Immunostaining with the LOX antibody was detected as bright spots appearing after epifluorescence illumination. To better determine the position of the antibody reaction signal in the tissue, sections were stained with safranin. Figure 3A shows a safranin-stained transverse section of a pea carpel at day -2 (two days before anthesis). At this stage of development LOX polypeptides were found only in the mesocarp cells of the pod (figure 3). Virtually no signal was detected in the ovule (figure 3B), and LOX was also absent in the exocarp and the endocarp layers (figure 3D). The distribution of LOX polypeptides was not only tissue-specific but also cell-specific. In the mesocarp, the signal was stronger in the cells adjacent to the endocarp (figure 3D) and specially around vascular bundles (figure 3E). In addition, some parenchyma cells in the vascular bundle sheath showed high amounts of LOX whereas it was absent in adjacent cells with identical morphology (arrows in figure 3E). An identical pattern of LOX distribution was found in carpels from day -2 to day +3, except that LOX appeared in the endocarp cells as they developed (figure 4). LOX

Polyclonal antibodies against the fragment b2EH of pea LOXG protein have been raised to study the distribution of LOX in pea carpels and young fruits. We selected this fragment because its sequence showed high predicted antigenic index and surface probability, and so probably being, a priori, efficient as an antigen. Although only part of two of the four regions which are highly conserved in plant and animal LOXs were present in the b2EH amino acid sequence (figure 1), it was expected that the anti-b2EH serum recognized other LOX polypeptides besides LOXG. The LOXG amino acid sequence is more than 70% similar to the rest of plant LOXs reported, and the b2EH fragment also shows high degrees of similarity (more than 75%) with other LOXs. For instance, the corresponding regions of pea seed LOX2 and LOX3 are, respectively, 77.8% and 84.5% similar to b2EH. Plant LOXs have a molecular mass in the vicinity of 95 kD (Siedow 1991), which corresponds to the size of the polypeptides recognized by the antiserum in pea protein extracts. In addition, the antib2EH antibody cross-reacted with soybean LOX1 (figure 1), suggesting that it was specific for LOX. The anti-LOX serum was used to determine LOX polypeptide content of pea carpels. Western analysis of protein extracts showed that three LOX species were recognized in carpels. One of the two major polypeptides (which we have called c) becomes undetectable after fruit set and development induction (figure 2). The disappearance of polypeptide c 24 hours after the application of GA3 to unpollinated carpels would explain the lower LOX activity measured in these carpels compared to that in non-induced ones (Rodrfguez-Concepci6n and Beltr~n 1995). The size of polypeptide c and its pattern of disappearance in carpels induced to keep growing suggest that it may correspond to the polypeptide whose sequence was deduced from loxg cDNA (Rodrfguez-Concepci6n and Belmin 1995). Therefore, changes in loxg expression seem to be physiologically relevant during the early stages of fruit development, as they are correlated with changes in the amount of LOX protein and activity. The anti-LOX serum also reacted with two other polypeptides in pea carpels (a and b) which may be responsible for the remaining activity measured in GA3-treated carpels. The physiological role of LOXs remains unclear, but they have been implicated in processes of growth and


Figure 3. Immunolocalization of LOX in pea carpels at day -2. A: Cross-section of the ovule region of the carpel (xl0). Boxes indicate magnified areas (x40) shown in B, C, D and E. B: Region of insertion of the ovule. C and D: the same area of pod wall. E: main vascular bundles. Tissue was stained with safranin for visualization (A and C). Immunostaining with LOX antiserum diluted 1:200 was detected by illumination with epipolarized light (D). A combination of both illuminations is shown in B and E. en, endocarp ex, exocarp; f, funicle; m, mesocarp; ov, ovule; v, vascular bundles. Arrows in E indicate cells of similar morphology with and without signal of immunostaining.


Figure 4. Immunolocalization of LOX in cross-sections of pea carpels. Pictures (x40) correspond to regions of the ovule and adjacent pericarp of unpollinated and untreated carpels at day 0 (A to D) or treated at day +2 with GA 3 and collected 24 hours later (E and F). A: safranin-stained carpel at day +2. B: equivalent section as A after incubation with anti-b2EH serum diluted 1:200 and illumination with epipolarized light. C: a combination of A and B. D: cross-section similar to C but incubated with preimmune serum as a control. E: safranin-stained GA3-treated carpel. F: equivalent section as E after incubation with anti-b2EH serum diluted 1:200 and illumination with epipolarized light, a, transition layer; b, pre-sclerenchyma layer; c, middle zone; d, inner epidermis; m, mesocarp; or, ovule.

626 development, senescence, and stress responses (Siedow 1991, Hildebrand 1989). Despite a decrease in LOX is found soon after the induction of fruit set and development, the analysis of the distribution of LOX polypeptides in pea carpels suggests that LOX is associated with growth processes. During the final steps of carpel maturation and early stages of fruit development, the most important changes take place in the endocarp and the mesocarp layers of the pod (Vercher et al. 1984), which are the tissues where reaction with the anti-b2EH antibody is detected (figures 3 and 4). However, from day -2 to day +3 LOX polypeptides were virtually absent in the ovules, which begin their growth phase much later in fruit development (Pate and Flinn 1977). LOX polypeptides are found in specific cells of the pod in certain steps of their development, once they conclude their first stage of cell division and begin to expand. At day -2, no LOX is detected in the endocarp, where cell division and differentiation is actively taking place (Vercher and Carbonell 1991). In contrast, in the mesocarp cells, which are completely differentiated and growing (Vercher et al. 1987), LOX polypeptides are present, predominantly in the region closer to the endocarp (figure 3). From day 0 to day +2, three cell layers become distinguishable in the endocarp (Vercher et al. 1987): the "transition layer" lining the mesocarp, the middle zone (composed by 2-4 "strata" of cells) and the inner layer or inner epidermis, lining the ovarian cavity (figure 4). At day 0, the transition layer and the epidermis showed a strong reaction with the anti-LOX serum (figure 4B and 4C). In these layers no mitoses are observed and the cells begin to elongate and grow. The cells of the middle zone, which are still dividing (Vercher and Carbonell 1991), showed no immunostaining with the anti-LOX serum. By day +3, the ceils of the middle zone of stimulated carpels (either pollinated or treated with GA3) increase their size and this coincides with the appearance of LOX in such cells of the endocarp (figure 4F). Therefore, LOX seems to be primarily associated with cell expansion and growth. A similar pattern of LOX distribution has been reported in soybean primary leaves, growing organs ontogenically related to carpels (Gillaspy et al. 1993). LOXs occur in vacuolating parenchyma cells but not in non-differentiated ceils of primary leaves (Vernooy-Gerritsen et al. 1984). In all the stages studied in pea carpels, LOX is specially abundant in the cells surrounding the vascular bundles of the mesocarp. Similarly, in soybean mature leaves, the majority of the protein cross-reactive with a LOX antibody was localized in the bundle sheath and the cells of the paraveinal mesophyll immediately surrounding the bundle sheath (Tranbarger et al. 1991). Localization of LOX in soybean seedlings is also associated with the vascular cylinder after germination (Vernooy-Gerritsen et al. 1983). These authors describe the presence of LOX in cells in which will take place processes involving drastic changes in their metabolism, growth and intracellular organization, similarly to those described during pea carpel maturation and fruit set and early development. Data from immunolocalization experiments in pea carpels support a role for LOX in pod growth and development, and are consistent with the fact that loxg is mainly expressed at

the beginning of the stage of active growth of these and other tissues potentially committed to keep growing. The anti-LOX serum used in this work is a tool to identify and analyze other LOX species besides loxg involved in fruit development, and it will be useful to study LOX regulation throughout the plant.

Acknowledgements. We thank Dr Luis Cafias for critical reading of the manuscript. We also wish to thank Dr C. Martin and Dr. J. Martfnez-Garcfa (John Innes Institute, Norwich, U.K.) for their gift of plasmids and bacterial strains, and A. Montoya (U.P.V., Valencia, Spain) for his help with the techniques of antibody production. The collaboration of R. Martfnez-Pardo and A. Villar in the greenhouse and the technical assistance from A. Ptrez are gratefully acknowledged. This work was supported by DGICYT (grants PB90-0133 and PB93-0140) and EEC-Bridge (BIOT-CT90017l). MRC and MDG received fellowships from the Spanish Ministerio de Educaci6n y Ciencia. References Bell E, Mullet JE (1991) Mol. Gen. Genet. 230: 456-462. Bradford MM (1976) Anal. Biochem. 72: 248-254. Creelman RA, Bell E, Mullet JE (1992) Plant Physiol. 99: 1258-1260. Domingo C, G6mez MD, Cafias L, Hernfindez-Yago J, Conejero V, Vera P (1994) Plant Cell 6: 1035-1047. Domoney C, Firmin JL, Sidebottom C, Ealing PM, Slabas A, Casey R. (1990) Planta 181: 35-43. Domoney C, Casey R, Turner L, Ellis N (1991) Theor. Appl. Genet. 81: 800-805. Edwards A, Marshall J, Sidebottom C, Visser RGF, Smith AM, Martin C (1995) Plant J. 8: 283-294. Garcfa-Martfnez JL, Carbonell J (1980) Planta 147: 451-456. Garcfa-Martfnez JL, Sponsel VM, Gaskin P (1987) Planta 170: 130-137. Gillaspy G, Ben-David H, Gruissem W (1993) Plant Cell 5: 1439-1451. Hildebrand DF (1989) Physiol. Plant. 76: 249-253. Laemmli UK (1970) Nature 227: 680-685. Pate JS, Flinn AM (1977) In: Sutcliffe JF, Pate JS (eds) The physiology of the garden pea. Chapter 15. Academic Press, London, pp. 431-468. Roddguez-Concepci6n M, Beltr~in JP (1995) Plant Mol. Biol. 27: 887-899. Siedow JN (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 145-188. Tabor S, Richardson CC (1985) Proc. Natl. Acad. Sci. USA 82: 1074-1078. Tranbarger TJ, Franceschi VR, Hildebrand DF, Grimes HD (1991) Plant Cell 3: 973-987. Vercher Y, Molowny A, L6pez C, Garcfa-Martfnez JL, Carbonell J (1984) Plant Sci. Lett. 36: 87-91. Vercher Y, Molowny A, Carbonell J (1987) Physiol. Plant. 71. 302-308. Vercher Y, Carbonell J (1991) Physiol. Plant. 81: 518-526. Vernooy-Gerritsen M, Bos ALM, Veldink GA, Vliegenthart JFG (1983) Plant Physiol. 73: 262-267. Vernooy-Gerritsen M, Leunissen JLM, Veldink GA, Vliegenthart JFG (1984) Plant Physiol. 76: 1070-1079.

Immunolocalization of lipoxygenase in pea (Pisum sativum L.) carpels.

Polyclonal antibodies against a part of pea (Pisum sativum L.) LOXG protein have been raised to study the pattern of distribution of related lipoxygen...
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