a homolog of the substrate adhesion molecule vitronectin

The Plant Cell, Vol. 3, 629-635, June 1991 O 1991 American Society of Plant Physiologists

A Homolog of the Substrate Adhesion Molecule Vitronectin Occurs in Four Species of Flowering Plants

Luraynne C. Sanders, Co-Shine Wang, Linda L. Walling, and Elizabeth M. Lord'

Department of Botany and Plant Sciences, University of California, Riverside, California 92521

The extracellular matrix (ECM) has been implicated in the primary developmental processes of many organisms. A family of secretory adhesive glycoproteins called substrate adhesion molecules (SAMs) is believed to confer these dynamic capabilities to the ECM in animals. In this paper, we report the existence of SAM-like genes and gene products in flowering plants. Hybridizations with a human vitronectin cDNA probe and genomic DNA from broad bean, soybean, and tomato revealed vitronectin-like sequences. Human vitronectin antibodies cross-react with a 55-kilodalton protein in leaf and root protein extracts from lily, broad bean, soybean, and tomato. In addition, immunocytochemical staining of frozen sections of lily leaf and broad bean gynoecium demonstrated that vitronectin- like proteins were localized to the ECM on the cell surface, with the most intense labeling residing in the transmitting tract of broad bean gynoecium.

INTRODUCTION

Cells of all organisms produce products that are secreted into the environment to form an extracellular matrix (ECM). In plants, the ECM that encompasses each cell is referred to as the cell wall (Lamport and Catt, 1981). The structural role of the ECM, functioning in support and anchorage, has long been recognized in both plants and animals (Darvill et al., 1980; Hay, 1981). A more recent view of the ECM suggests it has a more dynamic role in growth and development (Adair and Mecham, 1990; Roberts, 1990). The ECM in animal cells has been shown to play an active role in developmental processes such as cellular polarity, differentiation, cell division, cell death, and cell migration (Hay, 1981 ; Hynes, 1981). These events are directed by a variety of adhesion molecules (Edelman, 1988). Substrate adhesion molecules (SAMs) are a family of adhesive glycoproteins that are secreted into the ECM and interact with the cell by way of a class of plasma membrane receptors known as integrins (Hynes, 1987). Many SAMs have been described and localized in animal tissues; they include fibronectin, laminin, vitronectin, and cytotactin (Edelman, 1988). The SAMs that have been implicated in facilitating cell spreading and/or cell migration during embryogenesis are vitronectin and fibronectin (Hayman et al., 1983; Dufour et al., 1988). In vivo experiments have demonstrated the importance of fibronectin in cell migration during embryogenesis. lnert latex particles placed within the neural crest region of a chicken embryo trans- located on the ECM in a pattern mimicking cell migration (Bronner-Fraser, 1982). If these particles were coated with

' To whom correspondence should be addressed

fibronectin, however, translocation did not occur (Bronner-Fraser, 1985). In vitro experiments showed that cells and latex particles migrate preferentially on sub- strates containing fibronectin (Newman et al., 1985). The more recently discovered vitronectin appears to be more active biologically with regard to cell attachment, cell spreading, and growth than fibronectin (Hayman et al., 1985; Underwood and Bennett, 1989).

It has been suggested that a process similar to cell migration may be operating in plants (Sanders and Lord, 1989). During pollination, a pollen tube extends through the transmitting tract of the gynoecium, physically moving its own protoplasm as well as two sperm cells to the embryo sac in the ovule. The in vivo journey of pollen tubes occurs within a specialized ECM called the stylar matrix, which is secreted by the cells of the style (Knox, 1984). Based on data demonstrating active movement of latex particles along the stylar ECM in three species of flowering plants, we have suggested that the style actively facilitates pollen tube extension by way of a biochemical recognition-adhesion system (Sanders and Lord, 1989). This hypothesis suggests that a pollen tube tip can be considered as analogous to a migrating cell, which leaves a trai1 of cell wall behind. The hypothesis also implies the presence of SAMs and their receptors in plants. Recently, Schindler et al. (1989), using a vitronectin receptor mono- clonal antibody, detected proteins similar to the 0-subunit of the human vitronectin receptor (an integrin) in cultured soybean cells. This implies the presence of integrin-like protein in plants. Here, we describe what may be a plant SAM. Using human vitronectin cDNA probes and human

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BFigure 1. Detection of a 55-kD Plant Protein by Human Vitronectin Antiserum.

Lanes 1 to 7 are protein extractions from plant tissue: lanes 1, lily leaf; lanes 2, lily root; lanes 3, broad bean leaf; lanes 4, broad beanroot; lanes 5, soybean leaf; lanes 6, soybean root; lanes 7, tomato leaf. The lanes labeled VN contain 100 ng of purified human vitronectin.(A) Protein blot incubated with human vitronectin antiserum.(B) Protein blot incubated with nonimmune serum.

vitronectin antibodies, we were able to detect vitronectin-like genes and proteins in four species of flowering plants.Fluorescent-labeled anti-human vitronectin antibody local-ized these proteins to the inner wall in sections of leaf,root, and gynoecial tissues.

RESULTS

Proteins were extracted from the leaves and roots of lily,broad bean, soybean, and tomato, fractionated on 13%SDS-polyacrylamide gels, and transferred to nitrocellulose.Figures 1A and 1B show protein blots that were incubatedwith human vitronectin antiserum or nonimmune serum. Inall four species and in both organs, a single 55-kD cross-reactive band was resolved strongly (Figure 1A). Minorprotein bands were observed upon longer development.These proteins are likely to have a decreased affinity forthe human vitronectin antibodies or may represent non-specific binding from the antiserum. The blot incubated innonimmune serum detected no cross-reactive molecules(Figure 1 B). To investigate immunological relatedness ofhuman vitronectin and the 55-kD protein from plants, weused purified human vitronectin to block the immunologicalreaction in immunoblots, as shown in Figure 2. Immuno-globulins (Ig) were purified from the human vitronectin

antiserum and their molar concentrations determined. Par-tial blots were then incubated in either the purified IgG,used as a control (Figure 2A), in purified IgG preincubatedin a 10 M excess of human vitronectin (Figure 2B), or inpurified IgG preincubated in a 10 M excess of BSA, usedas an additional control (data not shown). Reduction of theimmunological reaction occurred only when the purifiedIgG was preincubated with human vitronectin (Figure 2B).The protein bands affected were the 75-kD and 65-kDhuman vitronectin, which were eliminated, and the 55-kDproteins from extracts of broad bean leaf and root, whichwere reduced strikingly when compared with the control(Figure 2). The protein band observed at the bottom of theblots was not affected by the treatment and thereforeprobably represents background from other antibodies inthe antiserum, i.e., not a specific reaction between thevitronectin antibodies and a plant protein. The blot incu-bated in the purified IgG preincubated with BSA wasidentical to the control (data not shown).

To demonstrate further immunological relatedness be-tween human vitronectin and the 55-kD protein in plants,we used monospecific antibodies purified from either humanvitronectin or the 55-kD protein from lily and broad beanroots. The monospecific anti-human vitronectin antibodyrecognized a major band at 55 kD in all three plant speciesand both organs, as shown in Figure 3A. A few minor bandswere recognized in all five lanes. Conversely, the monospe-cific anti-55-kD root antibody was immunologically reactive

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BFigure 2. Reduction of Immuno-Cross-Reactivity in the Presenceof Human Vitronectin.

Lanes 1 and 2 are protein extractions from broad bean leaf androot tissue, respectively. The lanes labeled VN contain 100 ng ofpurified human Vitronectin.(A) Protein blot incubated with purified IgG from human Vitronectinantiserum.(B) Protein blot incubated with purified IgG from human Vitronectinantiserum preincubated with purified human Vitronectin.

with both the 65-kD and 75-kD human Vitronectin proteins,as well as the 55-kD protein from leaf extracts of lily, broadbean, and soybean, as shown in Figure 3B. Only one minorband was recognized in lane 3, broad bean leaf proteins,and most likely represents a breakdown product of the 55-kD protein. Identical blots, when incubated with monospe-cific antibodies made from nonimmune serum instead ofhuman Vitronectin antiserum, detected no proteins (datanot shown).

Localization of vitronectin-like proteins by immunofluo-rescence on frozen sections of lily leaves and broad beangynoecium is shown in Figure 4. Slides were incubatedwith human Vitronectin antiserum or, as controls, incu-bated in nonimmune serum or secondary antibody only.Frozen sections of lily leaf incubated with human vitronec-tin antiserum revealed a ubiquitous vitronectin-like proteinin the tissue. The fluorescence appeared in patches in theinnermost part of the wall of all cells in the leaf (Figure 4B).Cross-sections of broad bean gynoecial tissue had a sim-ilar pattern of fluorescence distribution in all cells, exceptthe transmitting tract, in which the fluorescence was moreintense and continuous (Figure 4E). Frozen sections of leafor gynoecium incubated with rabbit nonimmune serum(Figures 4C and 4F) or secondary antibody only (data notshown) showed limited fluorescence, which was slightlymore than autofluorescence (data not shown). Root tissueof lily was also frozen, sectioned, and incubated in thehuman Vitronectin antiserum as described above. Thesetissues revealed a similar distribution of the vitronectin-likeproteins (data not shown). Frozen sections of broad bean

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Lanes 1 to 5 are protein extractions from plant tissue: lanes 1, lilyleaf; lanes 2, lily root; lanes 3, broad bean leaf; lanes 4, broadbean root; lanes 5, soybean leaf. The lanes labeled VN contain150 ng (A) or 3 ng (B) of purified human Vitronectin.(A) Protein blot incubated with monospecific antibodies purifiedfrom human Vitronectin.(B) Protein blot incubated with monospecific antibodies, purifiedfrom the 55-kD protein of lily and broad bean root.

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Figure 4. Localization of a Vitronectin-like Protein on FrozenSections.

(A) to (C) Lily leaf cross-sections.(D) to (F) Broad bean gynoecium cross-sections.(A) Section stained with toluidine blue O; x400.(B) Section incubated with human vitronectin antiserum; x400.(C) Section incubated with nonimmune serum; x400.(D) Section of gynoecium stained with toluidine blue 0; boxedarea indicates transmitting tract; x50.(E) Section of transmitting tract incubated with human vitronectinantiserum; x495.(F) Section of transmitting tract incubated with nonimmune serum;X495.E, epidermis; M, mesophyll; C, cuticle. White arrows indicateareas of immunoreactivity.

tissue were incubated with anti-human fibronectin anti-body, but results were inconclusive.

To determine whether plant vitronectin-like sequencescould be detected using a heterologous vitronectin cDNAprobe, genomic DMA blots were hybridized with a 32P-labeled human vitronectin cDNA probe. Ten microgramsof genomic DMA from the leaves of lily, broad bean,soybean, and tomato were digested, fractionated on 1%agarose gels, and transferred to a nylon membrane. Sev-eral DMA fragments in broad bean, soybean, and tomatowere visualized with the human vitronectin probe, asshown in Figure 5. These bands persisted when the mem-brane was washed under stringent conditions. We wereunable to detect any vitronectin-like sequences in lily usingthe procedure described above. Similar experiments usingfibronectin cDNA probes were negative.

DISCUSSION

In three of the plant species examined, DNA gel blotanalysis showed several DNA fragments hybridized withhuman vitronectin probe. These data suggest that a familyof genes with similarity to human vitronectin may exist inthese plant genomes. Even though no vitronectin cross-hybridizing DNA fragments were visualized with the lilyDNA, the immunoblots (Figures 1A and 3) and immunoflu-orescence studies (Figure 4B) indicated that a vitronectin-like product exists in this species. The fact that the lilygenome is 10 to 50 times larger than the other threespecies examined may explain why strong hybridizationsignals were not observed (Bennett and Smith, 1976).Immunoblot analysis using the human vitronectin anti-serum revealed one major band at 55 kD in four plantspecies and in different organs. The immunoreactivity ofthis protein was greatly reduced in the presence of humanvitronectin. Furthermore, monospecific anti-human vitro-nectin antibody cross-reacted with the 55-kD protein. Mon-ospecific anti-55-kD-root antibody cross-reacted with the55-kD protein in leaves of three species and the 65-kDand 75-kD human vitronectin proteins. These data dem-onstrated that the 55-kD proteins from these four plantspecies are immunologically related to human vitronectin.It is not surprising that a higher amount of the humanvitronectin protein was needed to achieve a noticeableimmunological reaction with the monospecific anti-55-kDroot antibody because it is unlikely that epitope conser-vation is very high between plant and human vitronectin.Although the molecular weight of the vitronectin-like pro-tein in plants differs from that of human vitronectin, themolecular weight of animal vitronectins examined rangesfrom 56,000 to 80,000 (Kitagaki-Ogawa et al., 1990). Theplant protein fits into the lower end of this scale. Frozensections of lily and broad bean tissues incubated withhuman vitronectin antiserum localized the vitronectin-like

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Figure 5. Detection of Vitronectin-like Sequences Using DNAHybridization Gel Blot Analysis.

Lanes 1 to 4 represent genomic DNA from lily, broad bean,soybean, and tomato, respectively. All lanes were hybridized witha human 32P-labeled vitronectin cDNA probe. Two picograms ofhuman vitronectin cDNA served as a positive control (VN). Theposition of the 1-kb DNA ladder (Bethesda Research Laboratories)markers are indicated. B, BamHI; Ha, Haelll; Hi, Hindlll; P, Pvull.

proteins to what we believe is the cell surface. Vitronectinis a secretory protein in animals and this is likely to be thecase in plants. Sequencing of the vitronectin-like genesand gene products in plants is necessary to determine thedegree of similarity between plant and human vitronectin.Although we detected no fibronectin cross-hybridizing se-quences in plant genomic DNA, the possibility remains thatother SAMs occur in plants. Together, the genomic DNAblot, immunoblot, and immunofluorescence studies arestrong evidence that a molecule similar to human vitronec-tin exists in plants.

The fact that a SAM-like molecule, which has beenimplicated in facilitating cell spreading and/or migration inanimal systems, occurs in plants lends credence to theproposal that pollen tube extension is a special case of

cell migration in plants (Sanders and Lord, 1989). Immu-nolocalization with the human vitronectin antiserumshowed a strong reaction on the transmitting tract cellsurfaces that function to support pollen tubes (Figure 4E).Based on these results and previous work (Sanders andLord, 1989), as well as the extensive literature on pollina-tion, we propose that compatible pollen tube extension inthe gynoecium occurs in a manner similar to cell migrationin animal systems. A model reported by Thiery's group(Dufour et al., 1988) explains the role of SAMs in stationarycell adherence as well as the transitory adherence thatoccurs during cell motility. In the stationary cell, the bindingaffinity of the SAM for its integrin is strong and the micro-filaments of the cell bind to the integrin indirectly, forminga tight connection with the cytoskeleton and creating focaladhesions (Burridge et al., 1988). In the motile cell, theaffinity between the SAM and its integrin is weak and onlytransitory attachments occur, with the microfilamentsforming a loose arrangement. Recent literature on thecytoskeleton of the pollen tube shows a loose arrange-ment of microfilaments at the growing tip and a moreorganized cytoskeleton further back from the tip (Lancelleet al., 1987; Tiwari and Polito, 1988; Heslop-Harrison andHeslop-Harrison, 1989). In this region of stationary adher-ence, structures resembling focal adhesions occur, butonly in pollen tubes grown in vivo, not in those grown invitro (Pierson et al., 1986).

Detection of the vitronectin-like protein in all tissuesexamined suggests that it may have a more basic function,perhaps in linking the cytoskeleton to the cell wall. In animalcells, SAMs function as links between the cytoskeletonand the ECM by way of the transmembrane integrin (Bissellet al., 1982; Burridge et al., 1988; Dufour et al., 1988).Green's (1986) work on the biophysics of organogenesisin plants suggests a connection between the cytoskeletonand the microfibrils of cellulose in the wall. We proposethat, in plants, a SAM such as vitronectin may provide thislink by way of an integrin-like protein in the plasma mem-brane (Schindler et al., 1989). SAMs, like the one describedhere, may provide the ECM of plants with a dynamicscaffolding that can function in development, communica-tion, and, in a specialized case, cell movement duringpollination.

METHODS

Immunoblot Analysis

In this study, we used tissue from lily (LJIium longiflorum), broadbean (We/a faba), soybean (Glycine max), and tomato (Lycoper-sicon esculentum).

Leaf or root tissue was immersed in liquid nitrogen and groundin a mortar with pestle. Proteins were extracted by transferringsamples directly into boiling SDS (12% w/v), 200 mM Tris, 100mM DTT, pH 8.4 (Smith and Fisher, 1984) using 4 volumes for

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leaf tissue and 1.5 volumes for root tissue. The samples were centrifuged at 15,0009 for 5 min. The supernatant was collected; aliquots (15 pL to 30 pL for leaf tissue and 35 pL for root tissue) from each sample and 100 ng of purified human vitronectin (D. Cheresh, Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, CA; Telios Pharmaceuticals, Inc., San Diego, CA) were fractionated on 13% SDS polyacrylamide gels (Laemmli, 1970). The gels were either stained with 0.2% Coomassie Brilliant Blue R or electroblotted onto nitrocellulose. Molecular weight markers were purchased from Bio-Rad. Blots were blocked with 3% gelatin in 0.1% Tween 20, Tris-buffered saline (0.1% TTBS); 20 mM Tris, 500 mM NaCI, pH 7.5 (TBS). Blots were incubated for 1 hr with rabbit anti-human vitronectin serum (Telios Pharma- ceuticals, Inc.) at a dilution of 1 :500, or incubated for 1 hr in rabbit nonimmune serum at a dilution of 1500. lmmunoblots were washed three times in 0.1% TTBS, incubated in a secondary antibody (goat anti-rabbit IgG alkaline phosphatase conjugate; Bio-Rad) for 1 hr at a dilution of 1 :3000. Blots were then washed three times in 0.1% TTBS and two times in TBS and incubated with color development solution (Bio-Rad).

Purification of IgG from the human vitronectin antiserum was achieved by using an Econo-Pac Protein A Kit (Bio-Rad) per the manufacturer's instructions. The molar concentration of 1 mL of purified IgG was determined by using a Beckman DU-6 spectro- photometer (AzBo)., Aliquots of the purified IgG, diluted 1 :1, were preincubated with a 1 O M excess of purified human vitronectin or BSA (Sigma) for 15 min. Three partia1 blots, prepared as described above, were incubated for 1 hr either in the purified IgG diluted 1 :1, or with purified IgG preincubated with purified human vitronectin or BSA, used as a control.

Monospecific antibodies were purified from human vitronectin and a mixture of the 55-kD proteins from lily and broad bean roots. The 55-kD proteins were excised from a blot on which 500 pg of total proteins from the roots were fractionated and transferred. Forty micrograms of purified human vitronectin was excised from a blot. The excised proteins were separately incubated in either the human vitronectin antiserum or rabbit nonimmune serum (used as a control) at a dilution of 1:50. The monospecific antibodies were eluted by a low pH buffer and neutralized by 1 M sodium phosphate buffer (pH 7.7) as described by Smith and Fisher (1 984). lmmunoblots were prepared as described above with the following modifications: proteins were extracted by using a phenol extraction method as described by Hurkman and Tanaka (1 986), proteins of each sample were quantitated by the Byadford protein assay (Bradford, 1976), and 50 pg of total protein from each sample was fractionated and transferred. The amount of purified human vitronectin transferred to the immunoblots varied with the monospecific antibody in which the blot was incubated: 150 ng for blots incubated in the monospecific anti-human vitronectin antibody and 3 pg for blots incubated in the monospecific anti-55-kD root antibody. Blots were incubated in the monospecific antibodies overnight at 4OC.

and collected on slides subbed with 0.5% gelatin and 0.005% chrome alum (Pappas, 1971). Sections were blocked with 0.1% TTBS, incubated for 1 hr in either human vitronectin antiserum at a dilution of 1 :300 or, as a control, rabbit nonimmune serum at a dilution 1:300 for 1 hr. Slides were rinsed three times in 0.1% TTBS, then incubated in the secondary antibody (goat anti-rabbit IgG biotinylated; ICN lmmuno Biological, Irvine, CA) for 1 hr. Some slides were blocked and incubated in the secondary antibody only, as an additional control. All slides were then washed three times in 0.1% TTBS, incubated for 30 min in a fluorescein isothiocyanate-avidin conjugate (ICN lmmuno Biological) diluted 1 :200, and rinsed three times in 0.1% TTBS and twice in TBS. Sections were viewed with a Zeiss fluorescence microscope equipped with Zeiss filter set 487716 and Ealing interference filter 35-5347, a 560 nm shortwave pass filter used to block chloroplast autofluorescence, or Omega qualitative analysis filter set for fluorescein isothiocyanate. For light micrographs, cryosections were stained with 0.5% aqueous toluidine blue O (Sigma). Kodak 241 5 Tech- nical Pan film was used for light micrographs and Kodak Tri-X for fluorescence micrographs.

DNA Gel Blot Analysis

Genomic DNA was extracted using the procedure of Dellaporta et al. (1 983), purified by CsCl density gradient centrifugation. Ten micrograms of genomic DNA was digested with restriction en- zymes and fractionated on 1% agarose gels and transferred to a zeta-probe membrane (Bio-Rad) under conditions suggested by the manufacturer. The membrane was hybridized with a 3zP-labeled human vitronectin cDNA (Telios Pharmaceuticals, Inc.). Hybridization was as described previously (Walling et al., 1988) with the following modifications: hybridization buffer contained 40% formamide, and the blot was incubated at 33°C. The membrane was washed twice for 20 min in 0.1 times standard saline citrate (149 mM NaCI, 15 mM Na citrate) and 0.1% SDS at 50°C. The blot was exposed to Kodak XARS film with one screen for 48 hr.

ACKNOWLEDGMENTS

We thank Drs. Eugene Nothnagel, Carl Ware, Timothy Close, and David Cheresh for discussion and advice. We are grateful to Dr. David Cheresh for the generous gift of human vitronectin. This research was supported by National Science Foundation Grant PCM 88-18554 to E.M.L.

Received April 1, 1991 ; accepted April 17, 1991

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