Intracellular localization of a lipid transfer protein in Vigna unguiculata seeds

PHYSIOLOGIA PLANTARUM 122: 328–336. 2004 Printed in Denmark – all rights reserved

doi: 10.1111/j.1399-3054.2004.00413.x Copyright # Physiologia Plantarum 2004

Intracellular localization of a lipid transfer protein in Vigna unguiculata seeds Andre´ de O. Carvalhoa, Carlos Eduardo de S. Teodoroa, Maura Da Cunhab, Anna L. Okorokova-Fac¸anhaa, Lev A. Okorokova, Ka´tia V. S. Fernandesc and Valdirene M. Gomesa,* a

Laborato´rio de Fisiologia e Bioquı´mica de Microrganismos, Centro de Biocieˆncias e Biotecnologia, Universidade Estadual do Norte Fluminense, Avenida Alberto Lamego 2000, 28013–600, Campos dos Goytacazes, RJ, Brazil b Laborato´rio de Biologia Celular e Tecidual, Centro de Biocieˆncias e Biotecnologia, Universidade Estadual do Norte Fluminense, Avenida Alberto Lamego 2000, 28013–600, Campos dos Goytacazes, RJ, Brazil c Laborato´rio de Quı´mica e Func¸a˜o de Proteı´nas e Peptı´deos, Universidade Estadual do Norte Fluminense, Avenida Alberto Lamego 2000, 28013–600, Campos dos Goytacazes, RJ, Brazil *Corresponding author, e-mail: [email protected] Received 25 May 2004; revised 13 July 2004

Lipid transfer proteins (LTP) facilitate transfer of lipids between membranes in vitro. Up to now, they have been found to be localized basically in the plant cell wall and in compartments linked to lipid metabolism, such as glyoxysomes. Accordingly, LTP are considered to be involved in the plant defence against pathogen microbes and lipid metabolism. We herein show, by immunoelectron microscopy, that besides the cell wall, LTP are localized in the lumen of organelles which we suggest to be the protein storage vacuoles, as well as in vesicles similar to the lipid-containing ones and in the extracellular space of Vigna unguiculata

seeds. To further characterize these organelles, we performed subcellular fractionation of membranes isolated from imbibed seeds on a sucrose-density gradient. The analysis of these fractions revealed that the lightest membrane vesicles, derived probably from PSV, contain LTP, a-TIP and K1 independent PPiase, but not g -TIP and K1 stimulated PPiase. The presence of LTP and vicilins (typical storage protein) in the lumen of these vesicles was confirmed by immunoelectron microscopy. Taken together, the data suggest that the intracellular LTP in the V. unguiculata seeds are localized in protein storage vacuoles and in lipid-containing vesicles.

Introduction The idea that a membrane biogenesis requires the transfer of phospholipids from the site of their synthesis to different cellular membranes led to the discovery of proteins that facilitate the exchange of lipids between membranes, in vitro (Kader 1975, Arondel and Kader 1990). These-non-specific lipid transfer proteins (LTP) share several common properties: the ability to bind fatty acids and their derivatives, molecular masses of 9–10 kDa, high isoelectric points and the presence of eight cysteine residues engaged in four disulphide bridges (Kader 1996). The ability of LTP to bind hydrophobic molecules (Tsuboi et al. 1992, Sodamo et al. 1997) agrees well with structural studies which have shown that LTP

possess four a-helices stabilized by disulphide bounds forming an internal hydrophobic cavity which runs through the protein molecule. It has been also shown that this cavity is able to accommodate hydrophobic molecules of different sizes (Lerche et al. 1997, Lee et al. 1998, Zachowski et al. 1998). The finding of a putative signal peptide in the LTP sequences suggested that the protein could be targeted to a specific intracellular compartment and/or secreted (Vignols et al. 1994). In the latter case, the participation of LTP in lipid metabolism, including the lipid transfer between intracellular membranes, was not obvious, raising the possibility of the existence of at least two populations

Abbreviations – DTT, dithiothreitol; LTP, lipid transfer protein; MOPS, 3-[N-morpholino] propane-sulphonic acid; PMSF, phenylmethylsufonylfluoride; PPiase, vacuolar H1 translocating inorganic pyrophosphatase; PSV, protein storage vacuole; PVPP, polyvinylpolypyrrolidone; TIP, tonoplast intrinsic protein.

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of LTP, an intracellular one and an extracellular another, with different functions. The participation of LTP in the b-oxidation of fatty acids was correlated to the intracellular form since it was localized in the glyoxysome matrix of Ricinus comunis seeds (Tsuboi et al. 1992). LTP were additionally found outside plant cells, mainly in the cell wall (Tchang et al. 1988, Arondel et al. 1991, Sterk et al. 1991, Thoma et al. 1994, Pyee et al. 1994). LTP extracted from leaves of Arabidopsis, spinach, barley, maize and sugar beet and from seeds of wheat, cowpea, radish, sunflowers and pearl millet have been demonstrated to inhibit phytopathogens, such as Pseudomonas solanacearum, Clavibacter michiganensis, Fusarium solani, Rhizoctonia solani, Trichoderma viride and Cercospora beticola (Terras et al. 1991, Molina et al. 1993, Segura et al. 1993, Dubreil et al. 1998, Kristensen et al. 2000, Regent and De la Canal 2000, Carvalho et al. 2001, Velazhahan et al. 2001). Although the precise mechanism of the antimicrobial activity of LTP is not yet known, it has been proposed that they may act as plasma membrane permeabilizing agents (Kader 1996). Other plant proteins exhibiting antimicrobial activities such as defensins and vicilins, are also thought to permeabilize the plasma membrane (Gomes et al. 1998a, Thevissen et al. 1999). We previously demonstrated, by means of immunofluorescence microscopy, that LTP from cowpea seeds were localized in the cell wall of both cotyledons and embryonic axes cells and organelles which we assumed were protein storage vacuoles (PSV) (Carvalho et al. 2001). The present work further examines the LTP localization in these seeds using immunoelectron microscopy and the LTP distribution in membrane fractions isolated from imbibed cowpea seeds and separated on a sucrose density gradient.

(1979). Nitrocellulose membranes were blocked with PBS [0.1 M sodium phosphate and 0.15 M NaCl (pH 7.3)] containing 1% BSA (w/v) for 1 h, incubated overnight with antiLTP:PBS 1 BSA (1 : 2000, v/v) or anti-a-TIP:PBS 1 BSA (1 : 15 000, v/v) or anti-g-TIP:PBS 1 BSA (1 : 7500, v/v), washed 10 times for 5 min and incubated for 1 h with goat peroxidase-conjugated antirabbit IgG:PBS 1 BSA (1 : 2000, v/v). Immunoblots were developed using a chemiluminescence kit (ECL reagent; Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. Antibodies Rabbit antiserum raised against a-TIP (Johnson et al. 1989) and g-TIP (Marty-Mazars et al. 1995) were kindly provided by Professor F. Marty (Laboratoire de PhytoBiologie Cellulaire, Universite´ de Bourgogne, France). Rabbit antiserum raised against a purified LTP from V. unguiculata seeds was prepared as described by Carvalho et al. (2001) and rabbit antiserum raised against vicilin from V. unguiculata seeds, cultivar Pitiu´ba, was prepared as described by Gomes et al. (1998b). Immunolocalization of LTP in V. unguiculata seeds by microscopy

SDS-tricine-gel electrophoresis was performed according to the method of Schagger and Von Jagow (1987). SDS-PAGE was carried out according to the method of Laemmli (1970).

For immunocytochemical analysis, cowpea (EPACE-10) quiescent seeds or seeds imbibed for 4 h, in water, were fixed for 2 h in 0.05 M cacodylate buffer containing 0.1% glutaraldehyde (v/v) and 4% paraformaldehyde (v/v) (pH 7.0), rinsed three times in 0.05 M cacodylate buffer (pH 7.0), dehydrated in solutions of increasing concentrations of methanol [30–90% (v/v)] and processed for LR Gold embedding. Ultrathin sections (400 nm) were laid on nickel slit grids and submitted to immunogold labelling. A solution containing PBS 1 BSA [0.1 M sodium phosphate contaning 0.15 M NaCl and 1% BSA (w/v) (pH 7.3)] was used for all rinsing steps and for dilution of the reagents. The sections were immunolabelled by immersing grids in drops (40 ml) of solutions in the following sequence: (a) PBS 1 BSA, 30 min; (b) pre-immune serum in PBS 1 BSA, 20 min; (c) anti-LTP serum in PBS 1 BSA [1 : 50 (v/v)], 2 h; (d) 10 changes of PBS 1 BSA, 10 min each; (e) goat antirabbit IgG conjugated with 10 nm colloidal gold in PBS 1 BSA [1 : 300 (v/v)], 2 h; (f) 10 changes of PBS 1 BSA, 10 min; (g) two changes of PBS, 10 min each; (h) two changes of deionized water, 10 min each. The nickel grids were stained with uranyl acetate and lead citrate and examined under a Zeiss 900 (Carl Zeiss, Germany) transmission electron microscope. Control sections were prepared by replacing the primary antiserum with pre-immune serum. All above procedures were performed at room temperature.

Western blotting

Membrane fractionation

After electrophoresis, proteins were electrotransferred to nitrocellulose membranes, as described by Towbin et al.

Subcellular fractions of cowpea seeds were prepared as described by Okorokov and Lehle (1998) with some

Materials and methods Plant material Cowpea (Vigna unguiculata (L) Walp) seeds of the EPACE-10 cultivar were supplied by the ‘Centro de Cieˆncias Agra´rias’ of the Universidade Federal do Ceara´, Fortaleza, Brazil. Protein determination Protein content was determined as described by Bradford (1976) using BSA as protein standard. Gel electrophoresis

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modifications. Initially, 80 g, fresh weight, of 4-h-soaked cowpea seeds were peeled and homogenized in ice-cold homogenizing buffer [12.5% sucrose (w/v), 100 mM TrisHCl, 0.5% PVPP (w/v), 1 mM DTT, 1 mM benzamidine and 1 mM PMSF (pH 7.4)] using pre-cooled mortar and pestle. The homogenate was strained through four layers of cheesecloth and then centrifuged at 2500 g for 10 min. The resulting supernatant was centrifuged at 110 000 g for 45 min. Total membrane vesicles were re-suspended in ice-cold buffer containing 12.5% sucrose (w/v), 5% glycerol (v/v), 25 mM MOPS-KOH, 2 mM DTT, 1 mM benzamidine, 1 mM PMSF (pH 7.2) and a cocktail of protease inhibitors (consisting of antipain, aprotinin, leupeptin and pepstatin, 1 mg ml 1 each). The suspension containing total membrane vesicles was layered on a discontinuous sucrose gradient [56, 52, 48, 45%, 42, 39, 36, 33, 30, 25 and 20% (w/w) sucrose]. After centrifugation at 110 000 g for 2 h and 45 min, membrane fractions (approximately 50 fractions with 300 ml each) were collected and stored at 70 C. Immunolocalization of LTP in subcellular fractions of cowpea seeds by microscopy After analysis of subcellular fractions by Western blotting, the 45, 47 and 49 fractions were chosen for immunocytochemical analysis. Aliquots (250 ml) were fixed for 2 h in a 0.05-M cacodylate buffer containing 0.1% glutaraldehyde (v/v) and 4% paraformaldehyde (v/v) (pH 7.0), rinsed three times in 0.05 M cacodylate buffer (pH 7.0), dehydrated in solutions of increasing concentrations of methanol [30–90% (v/v)] and processed for LR Gold embedding. Between each change of solutions the fractions were submitted to 5 min centrifugation of 16 000 g at room temperature. After this procedure, fractions were treated as described for immunolocalization of LTP in V. unguiculata seeds. Co-localization of LTP and vicilin in seeds and subcellular fractions of V. unguiculata seeds For this procedure, fractions were treated as described for immunolocalization of LTP in V. unguiculata seeds, except for the following differences. Sections were immunolabelled by immersing grids in drops (40 ml) of the following solutions: (a) PBS 1 BSA, 30 min; (b) preimmune serum in PBS 1 BSA, 20 min; (c) antivicilin serum in PBS 1 BSA [1 : 500 (v/v)], 2 h; (d) 10 changes of PBS 1 BSA, 10 min each; (e) goat antirabbit IgG conjugated with 10 nm colloidal gold in PBS 1 BSA [1 : 300 (v/v)], 2 h; (f) 10 changes of PBS 1 BSA, 10 min each; (g) PBS 1 BSA for 18 h; (h) anti-LTP serum in PBS 1 BSA [1 : 50 (v/v)], 2 h; (i) 10 changes of PBS 1 BSA; (j) goat antirabbit IgG conjugated with 5 nm colloidal gold in PBS 1 BSA [1 : 300 (v/v)], 2 h; (k) 10 changes of PBS 1 BSA, 10 min each; (l) two changes of PBS, 10 min each; (m) two changes of deionized water, 10 min each. Final procedures were performed as previously described. 330

Catalase determination For catalase (EC 1.11.1.6; hydrogen-peroxide: hydrogenperoxide oxireductase) determination, 10 ml of each gradient fraction were incubated with 10 ml of 15% (v/v) hydrogen peroxide in 0.05 M phosphate buffer (pH 7.4). Substrate consumption was monitored at 240 nm on a spectrophotometer every 5 s for 5 min. The resulting data were calculated as DA240 for the first 3 min (Beers and Sizer 1952). PPiase, P-ATPase and V-ATPase determination For determination of PPiase (EC 3.6.1.1; vacuolar H1 translocating inorganic pyrophosphatase) activity, 30 ml aliquots of membrane vesicles were incubated with 1 ml of a solution containing 30 mM MOPS-imidazol, 200 mM ammonium molybdate, 100 mM KCl, 100 mM sorbitol, 2.5 mM MgSO4 and 1 mM potassium pyrophosphate (pH 7.2) for 1 h at 30 C. After incubation, the reaction was stopped by the addition of 0.5% ammonium molybdate (w/v), 0.5% SDS (w/v) and 2% H2SO4 (v/v) and the release of Pi was measured (Fiske and Subbarow 1925) on a Shimadzu UV-1203 spectrophotometer (Shimadzu Corporation, Kyote, Japan). For determination of ATPase activity, 60 ml aliquots of membrane vesicles were incubated with 300 ml of a solution containing 30 mM MOPS-imidazole, 200 mM ammonium molybdate, 100 mM KCl, 100 mM sorbitol, 2.5 mM MgSO4 and 1.6 mM ATP (pH 7.2), in the presence of 100 mM NaNO3 (a V-ATPase inhibitor) (EC 3.6.1.3; adenosinetriphosphatase) or in the presence of 200 mM sodium orthovanadate (a P-ATPase inhibitor) (EC 3.6.3.6; H1-exporting ATPase) for 1 h at 30 C. After incubation, the volume was adjusted to 1 ml with water and the reaction was terminated by the addition of 0.5% ammonium molybdate (w/v), 0.5% SDS (w/v) and 2% H2SO4 (v/v). The release of Pi was measured on a Shimadzu UV-1203 (Fiske and Subbarow 1925).

Results and discussion Localization of LTP in cowpea seed The analysis of ultrathin sections from imbibed cowpea seeds, treated with the anti-LTP antibodies, revealed immunolabelling in extracellular space (Fig. 1B), cell wall (Fig. 1D) and intracellular organelles (Fig. 2B and C). The major localization of LTP in plant cell walls was previously reported by different groups and is widely accepted (Kader 1996). Intracellular localization of LTP in glyoxysomes of castor bean (Tsuboi et al. 1992) and within aleurone cells of Triticum aestivum seeds (Dubreil et al. 1998) has been previously reported. The immunolabelled intracellular organelles were morphologically similar to PSV detected in other plants (Mollenhauer and Totten 1971, Mollenhauer et al. 1978, Marty 1997). As treatment of ultrathin sections with OsO4 was omitted to facilitate subsequent procedures of immunolocalization, the usual osmiophilic reactions were absent, Physiol. Plant. 122, 2004

Fig. 1. Immunolocalization of LTP in cotyledons sections from cowpea seeds by transmission electron microscopy using an anti-LTP serum followed by treatment with 10 nm colloidal goldconjugated secondary antibody. The immunolabelling can be observed in extra-cellular space (B) and in the cell wall (B and D). A and C correspond to control sections in which the primary antiserum was replaced by pre-immune serum. CW, cell wall; ?, extracellular space. Bars: A and B, 350 nm; C, 150 nm; and D, 200 nm.

PSV-like organelles did not show the strong osmiophility that is commonly observed after treatment with this substance. In these conditions, LTP immunolabelling can be clearly seen in the organelle lumen (Fig. 2B and C). It is noteworthy that the presence of LTP was additionally detected in small vesicles (Fig. 1B and D), which were grouped along the plasma membrane and displayed aspects similar to the lipid-containing vesicles (Mollenhauer and Totten 1971, Mollenhauer et al. 1978). Thus, as judged from the immunoelectron microscopy, our data indicate a dual intracellular and extracellular localization of LTP in V. unguiculata seeds. In order to verify if the immunolabelled organelles were PSV, we analysed the distribution and localization of vicilin in cowpea cotyledons. Vicilins, also known as 7S globulins, are classical storage proteins of legume seeds (Shewry et al. 1995, Sales et al. 2000) and are located in storage organelles or PSV (Derbyshire et al. 1976). To support our idea that LTP was localized in PSV-like organelles, LTP and vicilin were immunolocalized in the same ultrathin sections. The analysis revealed that LTP (labelled with 5 nm colloidal gold particles) and vicilin (labelled with 10 nm colloidal gold particles) were co-localized in the same organelle (Fig. 2C). The result supports our suggestion that these organelles are in the fact PSV. There were no major differences between the localization of LTP in imbibed and quiescent seeds (data not shown). An Physiol. Plant. 122, 2004

Fig. 2. Immunolocalization of LTP in cotyledons sections from cowpea seeds by transmission electron microscopy using an anti-LTP serum followed by treatment with 10 nm (B) and 5 nm (C) colloidal gold-conjugated secondary antibody and using antivicilin serum followed by treatment with 10 nm (C only) colloidal gold-conjugated secondary antibody. The immunolabelling of LTP was observed in the lumen of a vesicle (B) and indicated by arrows in C. Vicilin was used as a PSV marker and its labelling could be seen in the lumen of seed vesicles (C). This figure also shown the immunolabelling of LTP was co-localized with the immunolabelling of vicilin, suggesting that these two proteins share the same compartment in cowpea seeds. A correspond to control sections in which the primary antiserum was replaced by pre-immune serum. Bars: A and C, 200 nm; B, 150 nm.

evaluation of LTP distribution in different cell compartments showed that they were mainly localized in extracellular space/cell walls, being also present in PSV and those lipid-containing-like small vesicles. 331

TM

S

Fig. 3. Localization of LTP in both membrane-free and membraneassociated fractions. The homogenate obtained from imbibed cowpea seeds was centrifuged at 110 000 g as described in ‘Materials and methods’. The aliquots (200 mg) of the resulting supernatant (S) and pellet (total membranes, TM) were resolved by SDS-PAGE and immunoblotted with anti-LTP antibodies.

Distribution of LTP in subcellular fractions of cowpea seeds To further characterize the cellular compartments containing LTP we fractionated a cell homogenate from imbibed cowpea seeds on a discontinuous sucrose density gradient. Total membranes and a supernatant obtained after sedimentation were shown to contain LTP, with a major concentration of the protein being found in the membrane-free fraction (Fig. 3). This finding is in agreement with the microscopic analysis which showed the predominant localization of LTP in cell walls and extracellular spaces. We assume therefore that the presence of LTP on membrane-free fraction is mainly due to extracellular LTP, and may also be partly derived from the lumen of vesicles and PSV as a result of their disruption during the membrane preparation. Immunoblotting analysis of membrane fractions demonstrated that LTP was concentrated in fractions 44–49 which migrated at 22.2–18% sucrose. The immunoreactivity of LTP was unequal in those fractions and exhibited a peak with a maximum in fractions 47 and 48 (Fig. 4A).

To further characterize membrane vesicles enriched with LTP, we probed the membrane fractions for the distribution of tonoplast intrinsic proteins (TIP), such as a-TIP and g-TIP or aquaporins which function as water channels (Chrispeels and Maurel 1994, MartyMazars et al. 1995, Maurel 1997). It is widely accepted that antibodies raised against a-TIP recognize specifically PSV from vegetative cells (Maurel 1997) whereas those raised against g-TIP label mainly lytic vacuoles (Ho¨fte et al. 1992, Marty-Mazars et al. 1995). The combined use of these two antibodies would be helpful to immunochemically define two distinct populations of vacuolar membranes (Paris et al. 1996). Surprisingly, a-TIP was found in all membrane fractions, thus showing no specific localization (Fig. 4B). The g-TIP was absent in the heavy and light membrane fractions (fractions 1–5 and 37–49, respectively) and the strongest immunoresponse was found in fractions 7–21 with a slight decrease in the following fractions (Fig. 4C). Membrane fractions 44–49, which were enriched with LTP and demonstrated the presence of a-TIP, displayed no signal for g-TIP (Fig. 4). This indicates that vesicles enriched with LTP were not derived from lytic vacuoles, but very likely from PSV. Wide distribution of a-TIP in all membrane fractions is of particular interest and despite having already reported for Pisum sativum cotyledons (Hoh et al. 1995), the distribution of two main aquaporins in membranes of imbibed V. unguiculata seeds can be taken as an indication that probably all membranes of the secretory pathway possess at least one or even both aquaporins during the first stages of seed germination. We decided to measure the activities of various marker enzymes to verify the efficiency of the employed membrane separation method that had previously been

Fractions 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 44

45

46

47

48

49

A 56

1

50

3

5

7

46.8

9

11 13

43.6

15 17 19 21 23 25

32.4

27 29

24.2

20.4

31 33 35 37 39 41 43

45

18

47

49 TM S

B C 56

50

46.8

37.4

31.2

24.2

18

Sucrose (%) Fig. 4. Distribution of LTP (A), a-TIP (B) and g-TIP (C) in membrane fractions isolated from cowpea seeds. Total membranes were isolated and fractionated on a sucrose density gradient as described in ‘Materials and methods’. The aliquots (20 ml) of membrane fractions were separated by SDS-PAGE. Fractions were than analysed by immunoblotting using polyclonal anti-LTP, anti-a-TIP and anti-g-TIP antibodies. TM, total membranes; S, supernatant obtained after sedimentation of total membranes.

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60 50

40

40 30 30 20 20 10

10

0

0 900 PPi hydrolysis (nmol Pi.60 min–1 /30 µl)

B

Catalase (∆240 nm) and sucrose (%)

50

4000 800 700 3000

600 500

2000

400 300

1000

200

Protein content (µg.ml–1)

ATP hydrolysis (nmol Pi.30 min–1 /30 µl)

A

100 0

0 0

5

10

15

20

25

30

35

40

45

50

Fractions Fig. 5. (A) Distribution of P-type H1-ATPase (–&–), V-type H1-ATPase (–*–), sucrose concentration (–*–) and catalase (–~–) in the membrane fractions isolated from cowpea seeds. (B) H1 PPiase activity (–*–) and protein content (–&–) in the membrane fractions. Membrane fractions were obtained after separation of total membranes isolated from imbibed cowpea seeds on a sucrose density gradient and used for determination of the orthovanadate and nitrate-sensitive ATP hydrolysis, hydrogen peroxide consumption and K1-stimulated PPi hydrolysis as described in ‘Materials and methods’. Note the presence of two peaks of PPiase in fractions 7–25 and fractions 44–50. The second peak comigrates with LTP in fractions 44–49 (see Fig. 4).

shown to be efficient in the case of yeast membranes (Okorokov and Lehle 1998, Okorokov et al. 2001) and membranes isolated from the Ricinus communis stem and V. unguiculata hypocotyls (Okorokov LA; personal comm.). The determination of the V type H1-ATPase activity (Fig. 5A) revealed an efficient separation of membranes comparable with that of membranes from pea cotyledons (Hoh et al. 1995). Similar results were obtained when P type H1-ATPase activity was determined (Fig. 5A), demonstrating that the major activities of V and P type H1-ATPases are found in denser membrane vesicles. Measurement of the membrane bound PPiase activity performed in the presence of the osmotic stabilizer sorbitol revealed two main peaks of K1-stimulated PPiase exactly in the same heavy membrane fractions which were enriched with both P and V type H1-ATPases (Fig. 5B). An additional peak of PPiase activity was Physiol. Plant. 122, 2004

found in the lightest membrane vesicles (fractions 43–50, Fig. 5B), although it did not show significant stimulation by 100 mM K1. By this property, this enzyme is different from the one found in dense membranes (Fig. 4) and is similar to Arabidopsis APV2p isoform of PPiase (Drozdowicz et al. 2000). We further measured catalase activity in membrane fractions to determine whether membrane vesicles derived from glyoxysomes were enriched with LTP, as reported previously for R. communis seeds (Tsuboi et al. 1992). The main peak of catalase activity was found in the heavy membrane fractions 14 to 18, which did not contain LTP (Fig. 5A). An additional peak of catalase activity was found in the lightest membrane fractions (Fig. 5A) enriched with LTP (fractions 44–49, 22.2–18% sucrose) (Fig. 4A). The analysis of catalase activity and immunolabelling of LTP in the membrane fractions 43–49 revealed the partly separated peaks of those 333

Fig. 6. Immunolocalization of LTP in membrane fractions isolated from cowpea seeds by transmission electron microscopy using an anti-LTP serum followed by treatment with 10 nm (A to C) and 5 nm (F) colloidal gold-conjugated secondary antibody and using an antivicilin serum followed by treatment with 10 nm (E and F). The immunolabelling of LTP can be observed mainly in the lumen of the vesicles in the fraction 45 (B and C) and in the fraction 47 (F), as indicated by arrows. Vicilin was used as a marker of membranes derived from PSV and its labelling could be seen in the lumen of vesicles of 47 fraction (F). A and D correspond to control sections in which the primary antiserum was replaced by pre-immune serum. Bars: A and B, 400 nm; C, D and E, 250 nm; F, 150 nm.

proteins with corresponding maximums in fractions 46 and 48. This fact may be taken as an indication that catalase and LTP are probably associated with different populations of light membranes. It is worthy of note that the main peak of catalase activity co-migrated with a peak of ATPase and PPiase which was distinct from other neighbouring peak of ATPase and PPiase (fractions 6–10) (Fig. 5). This is another good piece of evidence of the efficiency of our membrane separation conditions. The light membrane fractions were also analysed by immunoelectron microscopy to confirm whether LTP are associated with membrane vesicles. Indeed, as shown in Fig. 6B, C and E, LTP were detected mainly inside of vesicles. As can be also shown by Fig. 6F, vicilins (labelled with 10 nm colloidal gold particles) were co-localized in vesicles of fraction 45 together with LTP (labelled with 5 nm colloidal gold particles). The data corroborate our biochemical analysis and suggest that these fractions are enriched with membranes derived from PSV-like cellular protein storage compartments. 334

The localization of LTP in distinct compartments of the plant cell and their possible involvement in different processes such as lipid transfer and plant defence raise the possibility of existence of at least two different isoforms of LTP. We suggest that one of the important functions of the extracellular LTP isoform in V. unguiculata seeds is defence against phytopathogens. This isoform would be preferentially localized in the cell wall and extracellular space of the cotyledon cortex (Fig. 1), which are sites of primary contact with a pathogen. We have also demonstrated that LTP were released during the imbibition of cowpea seeds (Diz et al. 2003). Further investigations are needed to elucidate a role for LTP in PSV, but it should be noted that vicilins, 2S albumins and lectins which are classical storage proteins, also possess antimicrobial activities (Terras et al. 1991, Gomes et al. 1998a, Freire et al. 2002) and are also released during the imbibition of cowpea seeds (Gomes VM; personal comm.). In conclusion, our results provide evidence for intracellular localization of LTP in the PSV and lipid containing vesicles. They also point to a dynamic Physiol. Plant. 122, 2004

redistribution of TIPs between the secretory pathway organelles during a transition of the seed membranes to the vegetative membranes in the course of the germination process. Acknowledgements – This study is a part of the MSc degree thesis of A.O.C., carried out at the Universidade Estadual do Norte Fluminense. We acknowledge the financial support of the Brazilian agencies CNPq, FAPERJ, CAPES and FINEP, of the Universidade Estadual do Norte Fluminense supporting body FENORTE and the International Foundation for Science (IFS), Stockholm, Sweden, through a grant to C/2806–1. We are grateful to B.R. Ferreira for the preparation of samples for microscopy, M.A. da Silva Carvalho for photography and to M. T. Gobo and F.A.Q. Ribeiro for technical assistance. We are grateful to Dr F. Marty for the constructive discussion of our data on the electron microscopy analysis as well as for providing the antibodies against a-TIP and g-TIP.

References Arondel V, Kader JC (1990) Lipid transfer in plants. Experimentia 46: 579–585 Arondel V, Tchang F, Baillet B, Vignols F, Grellet F, Delseny M, Kader JC, Puigdomenech P (1991) Multiple mRNA coding for phospholipid-transfer protein from Zea mays arise from alternative splicing. Gene 99: 133–136 Beers RF, Sizer IW (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195: 133–140 Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 Carvalho AO, Machado OLT, Da Cunha M, Santos IS, Gomes VM (2001) Antimicrobial peptides and immunolocalization of a LTP in Vigna unguiculata seeds. Plant Physiol Biochem 39: 137–146 Chrispeels MJ, Maurel C (1994) Aquaporins: the molecular basis of facilitated water movement through living plant cell? J Cell Biol 138: 45–54 Derbyshire E, Wright DJ, Boulter D (1976) Legumin and vicilin storage proteins of legume seeds. Phytochemistry 15: 3–24 Diz MSS, Carvalho AO, Gomes VM (2003) Purification and molecular mass determination of a lipid transfer protein exuded from Vigna unguiculata seeds. Braz J Plant Physiol 15: 171–175 Drozdowicz YM, Kissinger JC, Rea PA (2000) AVP2, a sequencedivergent, K1-insensitive H1-translocating inorganic pyrophosphatase from Arabidopsis. Plant Physiol 123: 353–362 Dubreil L, Gaborit T, Bouchet B, Gallant DJ, Broekaert WF, Quillien L, Marion D (1998) Spatial and temporal distribution of the major isoforms of puroindolines (puroindolines-a and puroindolines-b) and non specific lipid transfer protein (ns-LTP1e1) of Triticum aestivum seeds. Relationships with their in vitro antifungal properties. Plant Sci 138: 121–135 Fiske CH, Subbarow Y (1925) The colorimetric determination of phosphorus. J Biol Chem 66: 378–400 Freire MGM, Gomes VM, Corsin R, Simone SG, Nonelo JC, Marangoni S, Macedo MLR (2002) Inhibition of fungus growth by a novel lectin from Tarlisia esculenta plant seeds. Plant Physiol Biochem 40: 61–68 Gomes VM, Okorokov LA, Rose TL, Fernandes KVS, XavierFilho J (1998a) Ultrastructure and immunolabelling of Vigna unguiculata vicilins (7S storage proteins) associated to fungi cells. Plant Sci 138: 81–89 Gomes VM, Okorokov LA, Rose TL, Fernandes KVS, XavierFilho J (1998b) Vicilins (7S globulins) inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. Biochim Biophys Acta 1379: 207–216 Ho¨fte H, Hubbard L, Reizer J, Ludevid D, Herman EM, Chrispeels MJ (1992) Vegetative and seed-specific forms of tonoplast intrinsic protein in the vacuolar membrane of Arabidopsis thaliana. Plant Physiol 99: 561–570 Hoh B, Hinz G, Jeong BK, Robinson DG (1995) Protein storage vacuoles form de novo during pea cotyledon development. J Cell Sci 108: 299–310 Physiol. Plant. 122, 2004

Johnson KD, Herman EM, Chrispeels MJ (1989) An abundant, highly conserved tonoplast protein in seeds. Plant Physiol 91: 1006–1013 Kader JC (1975) Proteins and the intracellular exchange of lipids. Stimulation of phospholipid exchange between mitochondria and microsomal fractions by proteins isolated from potato tuber. Biochim Biophys Acta 380: 31–44 Kader JC (1996) Lipid-transfer proteins in plants. Annu Rev Plant Physiol Plant Mol Biol 47: 627–654 Kristensen AK, Brunstedt J, Nielsen KK, Roepstorff P, Mikkelsen JD (2000) Characterization of a new antifungal non-specific lipid transfer protein (nsLTP) from sugar beet leaves. Plant Sci 155: 31–40 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 Lee JY, Min K, Cha H, Shin DH, Hwang KY, Shu SW (1998) Rice non-specific lipid transfer protein: The 1.6 A˚ crystal structure in the unliganded state reveals a small hydrophobic cavity. J Mol Biol 276: 437–448 Lerche MH, Kragelund BB, Bech LM, Poulsen FM (1997) Barley lipid-transfer protein complexed with palmitoyl CoA: The structure reveals a hydrophobic binding site that can expand to fit both large and small lipid-like ligands. Structure 5: 291–306 Marty F (1997) The biogenesis of vacuoles: insights from microscopy. Adv Plant Res 25: 1–42 Marty-Mazars D, Cle`mencet MC, Dozolme P, Marty F (1995) Antibodies to the tonoplast from the storage parenchyma cells of beetroot recognize a major intrinsic protein related to TIPs. Eur J Cell Biol 66: 406–118 Maurel C (1997) Aquaporins and water permeability of plant membranes. Annu Rev Plant Physiol Plant Mol Biol 48: 399–429 Molina A, Segura A, Gracı´ a-Olmedo F (1993) Lipid transfer protein (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens. FEBS Lett 316: 119–122 Mollenhauer HH, Morre´ DJ, Jelsema CL (1978) Lamellar bodies as intermediates in endoplasmic reticulum biogenesis in maize (Zea mays L.) embryo, bean (Phaseolus vulgaris L.) cotyledon and pea (Pisum stivum L.) cotyledon. Bot Gaz 139: 1–10 Mollenhauer HH, Totten C (1971) Studies on seeds. I. Fixation of seeds. J Cell Biol 48: 387–394 Okorokov AL, Lehle L (1998) Ca21-ATPases of Saccharomyces cerevisiae: diversity and possible role in protein sorting. FEMS Microbiol Lett 162: 83–91 Okorokov LA, Silva FE, Okorokova-Fac¸anha AL (2001) Ca21 and H1 homeostasis in fission yeast: a role of Ca21/H1 exchange and distinct V-H1-ATPases of the secretory pathway organelles. FEBS Lett 505: 321–324 Paris N, Stanley CM, Jones RL, Rogers JC (1996) Plant cells contain two functionally distinct vacuolar compartments. Cell 85: 563–572 Pyee J, Yu H, Kolattukudy PE (1994) Identification of a lipid transfer protein as the major protein in the surface wax of broccoli (Brassica oleracea) leaves. Arch Biochem Biophys 311: 460–468 Regente MC, de la Canal L (2000) Purification, characterization and antifungal properties of a lipid transfer protein from sunflower (Heliantus annuns) seeds. Physiol Plant 110: 158–163 Sales MP, Gerhardt IR, Grossi-d, e-Sa´ MF, Xavier-Filho J (2000) Do legume storage proteins play a role in defending seeds against bruchuids? Plant Physiol 124: 515–522 Scha¨gger H, Von Jagow G (1987) Tricine-sodium dodecylsulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368–379 Segura A, Moreno M, Gracı´ a-Olmedo F (1993) Purification and antipathogenic activity of lipid transfer proteins (LTPs) from the leaves of Arabidopsis and spinach. FEBS Lett 332: 243–246 Shewry PR, Napier JA, Tatham AS (1995) Seed storage proteins: structure and biosynthesis. Plant Cell 7: 945–956 Sodamo P, Caille A, Sy D, Person G, Mairon D, Ptak M (1997) 1H NRM and fluorescence studies of the complexation of DMPG by wheat non-specific lipid transfer protein. Global fold of the complex. FEBS Lett 416: 130–134 Sterk P, Booij H, Schellekens GA, Van Kammen A, De Vries SC (1991) Cell-specific expression of the carrot EP2 lipid transfer protein gene. Plant Cell 3: 907–921

335

Tchang F, This P, Stiefel V, Arondel V, Morch MD, Pages M, Puigdomench P, Grellet F, Delseny M, Bouillon P, Huet FC, Guerbette F, Beauvais-Cante F, Duranton H, Pernollet JC, Kader JC (1988) Phospholipids transfer proteins: full length cDNA and aminoacid sequence in maize. J Biol Chem 263: 16849–16855 Terras FRG, Schoofs HME, de Bolle MFC, van Leuven F, Rees SB, Vanderlyden J, Cammue BPA, Broekaert WF (1991) Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 267: 15301–15309 Thevissen K, Terras FRG, Broekaert WF (1999) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl Environ Microbiol 65: 5451–5458 Thoma S, Hecht U, Kippers A, Botella J, De Vries S, Somerville C (1994) Tissue-specific expression of a gene encoding a cell wall-localized lipid transfer protein from Arabidopsis. Plant Physiol 105: 35–45

Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 Tsuboi S, Osafune T, Tsugek IR, Nishimura M, Yamada M (1992) Nonspecific lipid transfer protein in castor bean cotyledons cells: subcellular localization and a possible role in lipid metabolism. J Biochem 111: 500–508 Velazhahan R, Radhajeyalakshmi R, Thangavelu R, Muthukrishnan S (2001) An antifungal protein purified from pearl millet seeds shows sequence homology to lipid transfer proteins. Biol Plantarum 44: 417–421 Vignols F, Lund G, Pammi S, Tre`mousaygue D, Grellet F, Kader JC, Puigdome`nech P, Delseny M (1994) Characterization of a rice gene coding for a lipid transfer protein. Gene 142: 265–270 Zachowski A, Guerbette F, Grosbois M, Jolliot-Croquin A, Kader JC (1998) Characterization of acyl binding by a plant lipid-transfer protein. Eur J Biochem 257: 443–448

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