Plant

Planta (1988) 175:259-269

9 Springer-Verlag 1988

Biochemical differentiation in the tobacco flower probed with monoclonal antibodies PhiIlip T. Evans, Brian L. Holaway, and Russell L. Malmberg Botany Department, University of Georgia, Athens, GA 30602, USA Abstract. We have isolated a series of monoclonal antibodies that react to antigens in flowers of Nicotiana tabacum L. (tobacco) displaying specificity or preferentiality in their cell and tissue distributions. We immunized mice with extracts from tobacco flowers and then screened the hybridomas by enzyme-linked immunosorbent assay (ELISA) against extracts from leaves, sepals, petals, stamens and pistils; twenty five were chosen from the total screened. The antigens detected by about half of the antibodies were periodate-sensitive, implying that the epitopes were carbohydrate. Competition ELISA assays were used to determine if any antibodies were reacting to the same epitopes. Western blot analysis showed that while some antibodies reacted to specific bands, the bulk either failed to react or reacted to multiple bands, consistent with a glyco-conjugate nature for many of the antigens. Analysis of the spatial pattern of antigen distribution within tobacco flowers by immunolocalization showed that some antibodies recognized epitopes that were limited to very specific cells and tissues. We used the immunolocalization technique to analyze a mutant with stigmoid anthers: an antibody recognizing a pistil transmitting-tract antigen also reacted to cells in stigmoid anthers. Our results with this antibody set imply that biochemical differentiation within the tobacco flower includes celland tissue-specific glyco-moeities, and also that similarities, at the biochemical level, exist between a normal floral organ and the abnormal organ in a phenotype with a developmental switch. Key words: Flower differentiation - Monoclonal antibodies - Mutant, developmental - Nicotiana (flower differentiation). Abbreviations: ELISA=enzyme-linked immunosorbent assay;

Fg = immunoglobulin; kDa = kilodalton

Introduction

Flowers are among the most complex structures made during higher-plant development, and the study of the transition to flowering from the vegetative state has been one of the great continuing themes of plant physiology. In this paper we examine events that occur much later than floral induction and initiation, and focus instead on the differentiation of the floral parts, sepals, petals, stamens, pistils, that occurs after the vegetative meristem has become committed to floral development. Nicotiana tabacum L. (tobacco) has been one of the model systems frequently used for studies on floral induction (Watterkeyn etal. 1965; Lang etal. 1977; Chailakhyan and Lozhnikova 1985; Singer and McDaniel 1987) and differentiation (Hicks and Sussex 1971; McHughen 1980) including at the nucleic-acid level (Kamalay and Goldberg 1980). Tobacco has large flowers complete with all major organs, and flowers can be formed in culture directly from certain types of explants (Tran Thanh Van 1973). In previous research, we isolated tobacco mutants that had flowers with abnormal phenotypes, including apparent shifts in floral organs among the whorls (Malmberg et al. 1985), so that a variety of examples of developmental switches among the organs were available for study as well. In order better to understand floral differentiation in tobacco, we have developed monoclonal antibodies as probes for various cells and tissues within a tobacco flower. These antibodies allowed us to characterize some of the kinds of biochemical differentiation that occur between tissues within the flower, and also allowed us to study a floral morphology mutant in more detail.

260

Materials and methods Plant material. Normal and mutant tobacco plants (Nicotiana tabaeum L. cv. Xanthi) harvested for protein preparations were greenhouse-grown under natural lighting conditions. The selection and phenotype of the mutant, Mgr27, is described in Malmberg et al. (1985). Preparation of tissue extracts. The sources of extracts were whole flowers ranging in development from approx. 1 mm length to 1-d pre-anthesis, and stamens and pistils from preanthesis flowers of mixed sizes. The various materials were ground with mortar and pestle at 4 ~ C. Preparations 1, 2, and 3 of the text and Table 1 were extracted in 100 m M 4-(2-hydroxyethyl)-piperazineethanesulfonic acid (Hepes), 5 mM oc-dithiothreitol (DTT), 0.1% sodium dodecyl sulfate (SDS), pH 7.4, then heated in boiling water for 2 min, filtered through Miracloth (Calbiochem, San Diego, Cal., USA), and centrifuged at 3 000.g for 10 rain. Preparations 1 and 2 were precipitated overnight in nine volumes of acetone, each, at - 2 0 ~ C, and resuspended in Hepes, DTT minus SDS. Preparation 3 was precipitated at 4 ~ with ammonium sulfate, using 0-50% and 50-100% cuts, which were then dialyzed overnight. Preparation 4 was ground in 25 m M Hepes, 5 mM phenylmethyl sulfonyl fluoride (PMSF), 3 r a M ethylendiamine tetraacetic acid (EDTA), 3 m M ethylene glycol-bis(fl-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 10 m M DTT, 0.25 M sucrose, pH 7.4, filtered through Miracloth, and centrifuged at 1 500 "g for 10 rain. The pellet was discarded and the supernatant centrifuged at 100000-g for 1 h. The supernatant was precipitated in nine volumes of acetone at - 20 ~ C overnight, then resuspended in 10 m M 2-amino-2(hydroxymethyl)-l,3-propanediol (Tris), 25 mM NaC1, 1 m M EDTA, pH 7.4. Preparation 5 was the 100000.g pellet from preparation 4, resuspended in 10 m M Tris, 25 m M NaC1, 1 mM EDTA, pH 7.4, and used without further solubilization. Organ-specific extracts for the enzyme-linked immunosorbent assay (ELISA) reactions were prepared as follows. Preanthesis flowers larger than 5 mm in length were dissected and the parts grouped into sepals, petals, stamens and pistils. Young, fully expanded leaves of healthy appearance were also taken as a fifth category. Dissected parts were kept at 0-4 ~ C in grinding buffer (25 m M Hepes, 5 m M PMSF, 3 m M EDTA, 3 mM EGTA, 10 m M DTT, 0.25 M sucrose, pH 7.4) until grinding in a pre-chilled mortar and pestle followed by homogenization with a Tekmar Ultra-Turrax homogenizer (Tekmar Co., Cincinnati, Oh., USA). The extract was centrifuged for 10 rain at 1500.g. The supernatant was saved and centrifuged at 100000-g for i h. The pellet was resuspended in 2% (3-[(3cholamidopropyl)dimethylammonio] 1-propanesulfonate (Chaps), 10 m M Tris, 25 m M NaC1, 1 mM EDTA, pH 7.4, and this preparation was used in ELISAs as the organ-specific microsomal fraction. The 100000.g supernatant was precipitated overnight with nine volumes of acetone at - 20 ~ C, resuspended in 10 m Tris, 25 m M NaC1, I m M EDTA, pH 7.4, and used in ELISAs as the organ-specific soluble fraction. Protein concentrations in the extracts were estimated by the method of Bradford (1976) using bovine serum albumen (BSA) as a standard. Immunizations. Balb/C mice (Dominion Laboratories, Dublin, Va., USA) were injected with 100 ~tl of extract at a protein concentration of approx. 1 mg/ml. Initial immunizations were in complete Freund's adjuvant and subsequent injections with incomplete adjuvant were made at three- to four-week intervals, the last being 3 d prior to the spleen harvest and fusion. Hybri-

P.T. Evans et al. : Biochemical differentiation in the tobacco flower domas were prepared by fusion to the SP2/O-Ag14 myeloma cell line (obtained from Dr. Roger Kennett, University of Pennsylvania School of Medicine, Philadelphia, Pa., USA) using standard techniques (Kohler and Milstein 1976).

Enzyme-linked immunosorbent assays. These reactions were performed using minor modifications of standard protocols (Cordonnier et al. 1983). Each well was coated with 100 gl of antigen (50 ~tl/ml in 50 m M sodium carbonate, pH 9.6) and adsorbed onto 96-well microtiter plates overnight at 4 ~ C. Plates were washed three times with ELISA wash buffer (20 m M Tris, 0.9% NaC1, 0.1% BSA, pH 7.2), then blocked with 3% BSA in wash buffer for 1 h. The plates were washed again and hybridoma supernatants were added for a 2-h incubation at room temperature. Following three rinses with wash buffer, wells were incubated for 2 h at room temperature with a goat anti-tutus e immunoglobulin G (IgG)-alkaline phosphatase conjugate (Sigma Chemical Co., St. Louis, Mo., USA) at 1 : 500 dilution. Following three rinses with wash buffer, 100 gl of 1 mg/ml p-nitrophenyl phosphate in 10% diethylamine, pH 9.8, was added to each well. After 30 rain, 100 gl per well of 3 m N a O H was added to stop the reactions; absorbance at 400 nm was measured. Isotyping of antibodies. Isotyping was performed by ELISA using polyclonal anti-isotype antibodies obtained from Southern Biotechnology Associates (distributed by Fisher Scientific, Pittsburgh, Pa., USA). Goat anti-mouse immunoglobulins at 100 ~tg/ml were adsorbed onto 96-well microtiter plates for 2 h at room temperature. Wells were then blocked with 3% BSA for ~ h, rinsed with ELISA wash buffer, and 100 gl of hybridoma supernatant was added per well. Two hours later the wells were rinsed three times with ELISA buffer and goat antimouse isotypes conjugated to alkaline phosphatase were added at 0.2 gg/ml and incubated for an additional 2 h. Following three rinses with ELISA wash buffer, the plates were developed with p-nitrophenyl phosphate and handled as with the other ELISAs. Periodate sensitivity of antibody reactions. For the periodatesensitivity test (Woodward et al. 1985; Hahn et al. 1987), antigen at 50 ~tg/ml and 100 txl per well was adsorbed onto 96-well microtiter plates overnight at 4 ~ C. The plates were then rinsed three times with 50 mM sodium acetate, pH 4.5, and were incubated in acetate buffer (SAB) at 25~ for l h in the dark. For periodate treatment, wells were treated in the same way except for the addition of fresh periodic acid (0.1-20 raM) in SAB. All wells were then blocked with 1% glycine in SAB for 1 h followed by three rinses with ELISA wash buffer. The wells were next blocked in 3% BSA and subsequently handled according to the standard ELISA protocol. Competition and sandwich assays. Antibodies were purified using a rabbit anti-mouse affinity column. Hybridoma supernatant was brought to 50% saturation with ammonium sulfate; the precipitate was collected, resuspended in and dialyzed against Tris-saline buffer (TBS). The concentrated crude antibody was then passed over the affinity column, and eluted with a low-pH glycine buffer in a standard manner (Goding 1983, chapter 4). This was followed by precipitation with ammonium sulfate at 50% saturation and resuspension in TBS. Antibodies to be biotinylated were adjusted to a concentration of 1.5 mg/ml and dialyzed against 2 1 of 0.1 M sodium-carbonate, 0.15 M sodium-chloride, pH 8.0, buffer in a micro-dialysis chamber. Following dialysis, 20 ~tl of a 25-mg/ml solution of biotinyl-e-

P.T. Evans et al. : Biochemical differentiation in the tobacco flower aminocaproic acid N-hydroxysuccinamide ester (Calbiochem, San Diego, Cal., USA) in 100% dimethyl sulfoxide (DMSO) was added per 1 mg of protein. After a 2-h incubation at room temperature, antibodies were again dialyzed against N a H C O 3 - N a C 1 buffer at 4 ~ C. For competitive ELISAs (Pratt et al. 1986), antigen at 10 gg/ml in 50 mM NaHCO~ buffer pH 9.6 was bound to microtiter plates overnight at 4 ~ C. The plates were then washed with TBS and blocked with 1% BSA for 1 h at room temperature. All subsequent washes were performed with 0.05% Tween20 (polyoxyethylenesorbitan monolaurate) added to the TBS and all subsequent operations were at room temperature with washes between each step through the addition of substrate solution. Blocking antibody was added at 40 gg/ml and given 2 h to bind. Biotinylated antibodies diluted in 0.1% BSA were added at 2.5-40 gg/ml, depending upon the antibody, and incubated for 2 h. The next step was the addition of streptavidin (Calbiochem) at 1 gg/ml in 0.1% BSA for 2 h followed by a 2-h incubation in biotin-x-alkaline phosphatase (Calbiochem). Substrate was added and development handled as indicated for other ELISA procedures. For sandwich ELISAs (Pratt et al. 1986), purified antibodies at 5 gg/ml in 0.1 M NaHCO3-0.15 M NaC1, pH 8.0, buffer were bound to microtiter plates overnight at 4 ~ C. The plates were washed with TBS, blocked at room temperature for 1 h with 1% BSA, washed in TBS plus 0.05% Tween 20, and then incubated with antigen at 25 gg/ml in 0.1% BSA for 3 h at 4 ~ C. All subsequent incubations, until addition of substrate, were for 2 h at room temperature and washes were performed between steps with TBS-Tween 20. Biotinylated second antibodies were added at concentrations varying from 2.5 to 30 gg/ml in 0.1% BSA. This step was followed by streptavidin, biotin-x-alkaline phosphatase and substrate as described for the competition ELISA.

Western blotting. Protein samples for Western blots were prepared as follows. Tobacco flower buds of mixed ages from recently initiated to i d pre-authesis were ground to a fine powder under liquid nitrogen_ Grinding buffer (0.1 M Tris, pH 7.4, 5 m M EDTA, 20raM fl-mercaptoethanol, 1 mM PMSF, 0.1% SDS, 10 gg/ml leupeptin) was added (2 ml per I g of tissue), followed by further grinding with mortar and pestle at 4 ~ C. The homogenate was incubated at 37~ for 2 rain, chilled on ice for ~ 3 rain, filtered through Miracloth, and centrifuged at 4 ~ C, 12000.g for 15 min. The supernatant was taken and ammonium sulfate was added to 90% saturation at 4 ~ C. The precipitate was centrifuged at 12000.g, 4 ~ C for 15 rain. The pellet was resuspended in grinding buffer followed by an overnight dialysis at 4 ~ C against 20 m M Hepes, pH 7.4, 5 mM fl-mercaptoethanol, 0.1 mM E D T A 50 m M NaC1. Protein concentrations were determined by the method of Bradford (1976) using BSA as a standard. Protein samples were solubilized in buffer containing (final concentrations) 0.13 M Tris, pH 6.8, 5% glycerol, 1% SDS, 0.36 M fl-mercaptoethanol, 0.001% bromophenol blue. The samples were boiled in this mixture for 2 rain and then resolved, using the system of Laemmli (1970), on 7-15% polyacrylamide slab gels. The sample was applied across the entire surface of the stacking gel (one lane was reserved for molecular-weight markers). Five micrograms of protein were loaded per I mm 2 of sample-well surface area. The proteins were transferred from the gel to nitrocellulose sheets (BA85; Schleicher and Schuell, Keene, Cal., USA) using a Trans-Blot Cell (BioRad, Richmond, Cal.). The transfer was done in 25 m M Tris, pH 8.3, 192 mM glycine, 20% methanol at 70 V for 3 h. To visualize the transferred proteins, vertical strips were stained with 0.1% amido black 10B (Calbiochem), 45% methanol 10% acetic acid

261 f o r 2 rain, and destained in 2% acetic acid, 90% methanol. The rest of the blot was air-dried and cut into strips for testing with antibodies. Nitrocellulose strips were blocked overnight in 5% BSATBS (10 mM Tris, pH 7.4, 0.15 M NaC1), 0.05% Tween-20 and 4 ~ C and then incubated for 60 rain at room temperature with primary antibody diluted in 3% BSA-TBS, 0.05% Tween-20. Purified antibodies (see above) were used at 2 gg/ml. Appropriate monoclonal control antibodies were run in parallel at the same concentration. In some experiments, spent hybridoma supernatants were used instead of purified antibody solutions. Unused tissue-culture medium was run as a control. The results were identical in either case. Following incubation with primary antibody, strips were washed once in TBS, 0.05% Tween-20 for 10 min and twice in TBS for 10 min each. Strips were incubated at room temperature for 60 min with goat anti-mouse IgG coupled to alkaline phosphatase diluted 1:1500 in 3% BSA-TBS, 0.05% Tween-20. The strips were washed as above, incubated for 5 rain in 0.1 M Tris, pH 9.0, 0.5 mM MgC12, and transferred to the same buffer plus 0.1 mM 5-bromo-4chloro-3-indoylphosphate and 0.1 mM nitrobluetetrazolium; a purple color indicates antibody binding.

Immunoloealizations. Flowers were longitudinally slit and then fixed and cryoprotected according to Pratt et al. (1986) with minor modifications. Paraformaldehyde at 5% was used for the fixation and 0.5% paraformaldehyde was included in the initial cryoprotection step. Specimens were mounted on a microtome chuck with Histo-Prep (Fisher) and frozen at - 2 0 ~ C. Sections, 8 gm thick, were transferred to glass slides coated with 0.4% gelatin + 0.04% C r K ( S O 4 ) 2 9 12 H 2 0 and heated to 45~ for 30 rain. Samples for plastic sections were fixed as above, dehydrated in ethanol, and embedded in glycol methacrylate (Immunobed; Polysciences, Warrington, Pa., USA) according to the protocol provided. Sections (3-4 gm) were mounted on glass slides, allowed to air-dry, and treated with ethanol for 2 min before proceeding with immunolocalizations. Sections on slides were washed for 10 min in 0.01 M sodium phosphate, 0.15 M NaCI, pH 7.2 (PBS) to remove the Histo-Prep and hydrate the sections. All washes and incubation steps were done at room temperature. The sections were covered with normal goat serum (S-2007; Sigma) for 30 rain, followed by monoclonal antibody in PBS-3% BSA for 2 h. Monoclonal antibodies were purified from hybridoma medium with an anti-mouse IgG-agarose affinity column (Sigma). Purified antibody was assayed by absorbance at 280 nm and resuspended at 0.1 mg/ml in PBS-3% BSA. Stocks were diluted from 2 to 10 gg/ml for experiments. Control sections were treated with purified myeloma immunoglobulin (Sigma) of an appropriate subclass at a concentration equal to that of the experimental antibody. In some experiments sections were treated with spent hybridoma medium instead of purified antibody. In these experiments control slides were treated with unused hybridoma medium. The results of these experiments were indistinguishable from those where purified antibodies and control myeloma immunoglobulins were used. Sections were washed for 10 rain in PBS-0.05% Triton X-100 (octylphenoxy polyethoxyethanol) and twice for 10 min each in PBS. The sections were then covered with normal goat serum for 30 min followed by incubation for 2 h in goat anti-mouse alkaline phosphatase (A-4656; Sigma) diluted 1:300 in PBS-3% BSA. Sections were washed for 10 min in PBS-0.1% Triton X-100, twice for 10 rain each in PBS, and incubated for 30-40 min in 0.1 M Tris, pH 9.0, 0.5 mM MgC12, 0.1 mM 5-bromo-4chloro-3-indoylphosphate (B-8503 ; Sigma)+ 0.1 m M nitro blue tetrazolium (N-6876, Sigma); a purple precipitate forms at the site of localization.

262

Results

Isolation of antibodies. We isolated monoclonal antibodies by injecting various crude homogenates of tobacco flowers into mice and screening the resuiting hybridomas for antibodies that would demonstrate specificity for floral organs, tissues or cells. The types o f extracts injected were from: (1) whole pre-anthesis flowers of mixed ages; (2) stamens and pistils from pre-anthesis flowers of mixed ages; (3) whole pre-anthesis flowers of mixed ages, of which ammonium-sulfate precipitates were collected; (4) whole pre-anthesis flowers of mixed ages, o f which a 100000.g supernatant was collected; (5) whole pre-anthesis flowers of mixed ages, of which a 100 000.g pellet was collected. Hybridomas were isolated by standard techniques (Kohler and Milstein 1975), then screened by ELISA against sodium-dodecyl-sulfate-solubilized and acetone-precipitated extracts, one from whole flowers and the other from leaves. Positives were screened a second time against identically prepared extracts of leaves, sepals, petals, stamens and pistils, adjusted to equal protein concentrations. Those showing differential reactivity were retained for cloning and further testing; several exhibiting very uniform reactivity were retained as controls. The results of seven immunizations using five different immunogens are summarized in Table 1. Of 31 antibody lines retained for expansion, 29 were identified by ELISA as displaying some tissue- or cell-type specificity. The patterns of reactivity of 25 lines to extracts from various floral organs as judged by ELISA reaction are shown in Fig. 1. Two antibody lines 9F10 and 5D8, react very evenly across all extracts and are useful positive controls. Several others, including 3El0, 6G6, 8G1, 8D12 and STO1, have limited or complex patterns of specificity as determined by ELISA. One line, 2F10, is highly specific for stamen extracts, while 13 of the 25 lines primarily recognize pistil extracts. This may reflect either immunodominance of some pistil-specific components in the extracts, or it may indicate that pistils exhibit a much more distinctive biochemical differentiation than do other tissues. The differential reactivity of these antibodies may be the result either of changes in abundance of an antigen from tissue to tissue, or of changes in presentation of a given epitope on an antigen that is uniformly distributed across floral tissues. The antigens recognized by these antibodies could be a variety of types of molecules, ranging from proteins to carbohydrates to lipids, and various combinations of these. The antibody set can

P.T. Evans et al. : Biochemical differentiation in the tobacco flower Table 1. Results of immunizations of mice with various extracts from tobacco flowers

Immunogena

No. of spleens

No. of hybrid-

No. of initial

No. of clones

harvested o m a s positives retained screened (1) Whole flower, 1 acetone ppt. (2) Anther-pistil, 2 acetone ppt. (3) Whole flower, 1 100000.g pellet (4) Whole flower, 1 100000.g supern. (5) Whole flower, 2

33

5

5

520

85

14

158

5

l

421

65

4

607

27

7

1739

187

31

(N[-I4)SO 4 ppt. Totals

7

ppt. = precipitate; supern. = supernatant

therefore be used as a measure of the types of biochemical differentiation that occur within a tobacco flower. We have characterized the antigens by testing for carbohydrate epitopes, and by examining microsomal fractions isolated from the individual floral tissues.

The epitope recognized by many lines of monoclonal antibodies is periodate-sensitive. Some of the major constituents of plant cells are carbohydrates, either as oligosaccharides, or bound to other molecules as glycoproteins or glycolipids. W o o d w a r d et al. (1985) and Hahn et al. (1987) have demonstrated that periodate oxidation of extracts can be used to destroy the antigenicity of some carbohydrates. Using this method in ELISA reactions, we have tested whether or not the epitopes recognized by these monoclonal antibodies are likely to be carbohydrate in nature (Table 2). F o r 12 lines, the periodate reaction eliminated the antigenicity, for I0 it did not, and for others the test was not conclusive. This implies that the epitopes recognized by about half of the antibodies are probably carbohydrate in nature. Among the 13 antibody lines specifically reacting to pistil, fully 11 had periodate-sensitive epitopes. Anderson et al. (1984) found that approx. 50% of the total hybridomas from mice immunized with crude Nicotiana stigma and style extracts, and giving positive ELISA reactions with those extracts, produce antibodies against arabinogalactan proteins. Our results with antibodies selected for tissue or cell specificities are thus consistent with

P.T. Evans et al. : Biochemical differentiation in the tobacco flower

1o0%

4F3

6F 12

7H 12

2E 10

6F4

1 ICI

8CI0

?R i i

5E3

NST i

6F9

4G6

3F8

3EIO

4H6

8GI

9EI

81312

9F10

5D8

1 1C2

2F 10

8B 1

STO 1

6G6

LSPRC

LSPRC

LSPRC

LSPRC

LSPRC

-

100%

-

10o%

-

I 0 0 ~

-

I00%

263

-

the notion of immunodominance of these carbohydrates. If the 11 periodate-sensitive antibodies that are pistil-specific are subtracted from the total number of periodate-sensitive antibodies reported in Table 2, it appears that this immunodominance may be less pervasive in other tissues. Other reports of monoclonal antibodies to Nicotiana include antibodies against membranes from microsomal and protoplast preparations (Hahn et al. 1987; Norman etal. 1986) which also recognize carbohydrate epitopes and may be indicative of

Fig. 1. Summary of ELISA reactions of the various monoclonal antibody lines with crude extracts from tissues of tobacco flowers (L = leaf extract, S = s e p a l extract, P = p e t a l extract, A = stamen extract, C = pistil extract). All values are averages of absorbance quantitated on an automated reader and have been corrected to correspond to a 0 100% scale (0% = absorbance of a control blank, 100% = absorbance of strongest reaction with each antibody). Since bybridoma supernatants were used, it is not valid to compare antigen abundance between lines

a high degree of protein glycosylation on plant plasmalemmas.

Analysis of microsomal extracts from flowers. All 25 antibodies were tested against both soluble and microsomal antigen sources to help discriminate the epitopes recognized by individual lines. Twelve of the lines had measurable reactions with the microsomaI extracts, as displayed in Fig. 2. Some pistil-preferential antibodies that appear similar to each other simply on the basis of Fig, 1 can be

264

P.T. Evans et al. : Biochemical differentiation in the tobacco flower

Table 2. Summary of typing and of periodate sensitivity of monoclonal antibodies against tobacco flower extracts Periodate

Ig subclass

No.

Antibodies

Sensitive

IgG1 IgG2b IgG3 IgM

3 1 1 6

7Al 1 ; 3F8; 3El0 8G1 7H12 4F3; 6F12; 6F4; l l C 1 ; 8C10; NSY1 2El0

Insensitive

N o t classified

100% -

07.

100%

-

0~ lOO%

0%

IgM-IgG3

l

IgG1

6

IgG2b IgM

1 4

6F9; 4G8; 9 E l ; 8D12; 11C2; M G T I 5E3 6G6; 9F10; 5D8; 4H6

IgM IgG3

3 ]

STOI ; NSTI ; 2FI0 8B1

8CI0

11C1

7H12

2EI0

d A_l.l Lllh 3EI0

5D8

8D12

9FIO

1 1C2

5E3

4F3

6F 12

L S PAC

L S PAC

L S PAC

L SPAC

2. Summary of ELISA reactions of the various monoclonal antibody lines with microsomal extracts from tissues of tobacco flowers (L = leaf extract, S = sepal extract, P = petal extract, A = stamen extract, C = pistil extract). Otherwise as Fig. 1 Fig.

separated into classes by comparing periodate sensitivities and Fig. 2 histogram profiles. One class, represented by 4F3 and 6F12, has enhanced reactivity with leaf in the microsomal assay and is periodate-sensitive. Four other lines, 7H12, 11C1, 2E10 and 8C10, largely retain specificity in both the soluble and microsomal tests, with 8C10 and 11C1 being very similar to one another and 7H12 resembling 2E10. Two antibodies, 4G6 and 6F9, whose reactions are not periodate-sensitive, and which both resulted from the anther-pistil immunization, have virtually identical histogram profiles with soluble extracts and both fail to react with microsomal extracts.

Competition and sandwich ELISA assays. The results of the microsomal-extract experiment are a start to answer the question if antibodies with similar tissue ELISA reactivities represent independent isolations of antibodies derived from single immunodominant epitopes (Pratt et al. 1986). This question can be more directly addressed in two ways: (1) a competition ELISA assay can be used to ask if two antibodies bind to precisely the same epitope; (2) a sandwich ELISA assay can help distinguish if two antibodies bind to different epitopes, but on the same antigen. The antibodies are purified, and then one member of each pair to be tested is biotinylated; this allows subsequent detection of the presence of the biotinylated antibody with streptavidin. The streptavidin is in turn detected by a biotin-alkaline phosphatase conjugate. In the epitope-competition experiments, ELISA plates are coated with antigen which is reacted with the first antibody; a second biotinylated antibody is then added. If binding of the first antibody protects the epitope from the second antibody, there will be no detection of the biotin. In the sandwich assay, the first antibody is used to coat the ELISA plate, then antigen is added, followed by biotinylated second antibody. In this case, the biotin will be detected if the antibodies recognize different epitopes on the same antigen, or if the antigen has two or more identical epitopes on the same molecule. The results of these experiments are shown in Fig. 3 A (competition) and B (sandwich). The tissue-specific histogram profiles have to be carefully compared with the sandwich, and competition ELISAs to evaluate the relatedness of a pair of antibodies. Some antibodies may react to overlapping but slightly different epitopes, there may be multiple epitopes (identical or different) on a single antigen, and steric hindrance could occur during mixed-antibody experiments in a manner that is not dependent upon the epitope. Comparisons of the competitive and sandwich ELISA results show a pattern of relatedness similar to the groupings from the histogram profiles o f Fig. 1. All periodate-sensitive antibodies that are included here and that reacted well under these conditions show considerable cross-reactivity, especially on sandwich ELISAs. Antibodies 4E3, 6F12, 7H12, 8C10 and 2E10 appear very similar in competitive tests; all reacted with the microsomal extracts, and all reacted most strongly with pistil extracts. One other antibody, 9E1, also appears to be related to this group on the basis of both sandwich and competitive ELISAs; however, i t does not react with microsomal extracts, has a dif-

P.T. Evans et al. : Biochemical differentiation in the tobacco flower

265

(A) COMPETITION ELISA First Antlbody

Second Antibody - B i o t i n y l a t e d

3E10 6G6 3E10 6G6 8G1 4F3 2F10 466 7All 6F12 5D8 9E1 7H1~ 8D12

|

8C10 6F9 2E10

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Second ~nttbody - B t o t t n y l a t e d

Antibody 3E10 6G6 3E10

8S1

4F3 2F10

4G6 7All 6F12

II

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ferent profile of reactivities with soluble extracts, and is not periodate-sensitive. On the basis of sandwich ELISAs, two other antibodies may be related to this group, 3E10 and 8G1. Other groupings that may be taken from the competition tests include 7 A l l with 4G6 and 3E10 with 8C10, the former result being consistant with the other tests but the latter one being unexpected on the basis of the organ-specific histogram profiles.

Western-blot analysis. Twenty-one of the antibodies were analyzed with Western blots of tobacco floral protein; a representative sample of the results is shown in Fig. 4. Several antibodies recognized proteins of different sizes, resulting in a " s m e a r " on the blot; both 8C10 (lane 1) and 3E10 (lane 2) yielded a smear o f high-molecularweight material centered at 140 kilodaltons (kDa) and a broader smear of lower-molecular-weight material centered at 63 kDa. It is unlikely that

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II

II

Fig. 3A, B. Results of competition and sandwich ELISA reactions against combined extracts of pistils and anthers of tobacco flowers to determine if pairs of antibodies are binding to the same epitope, or to the same antigen but at different epitopes. A Competition ELISA: II = strong blocking, little ELISA reaction ( < 3 2 % of control); ~ = m e d i u m blocking, medium ELISA reaction (33-66% of control); iii = w e a k blocking, strong ELISA reaction (67-90% of control) ; blank = no blocking, full ELISA reaction. B Sandwich ELISA: I = recognition of same antigen, strong ELISA reaction ( > 6 7 % of control; I = p a r t i a l recognition., medium ELISA reaction (33-66% of control); iii = weak recognition, weak ELISA reaction (10-32% of control); blank = no recognition, no ELISA reaction

these two antibodies recognize the same molecule as the antigen-distribution profiles corresponding to the two antibodies are different. Antibody 11C2 (lane 3) also produces a high-molecular-weight smear pattern on a Western blot and additionally recognizes material larger than t80 kDa. Antibody 6F12 (lane 4) recognizes a continuous band of material between 140 kDa and 55 kDa. In particular, 6F12 recognizes material in the size range 107 to 125 kDa whereas 8C10, 3EI0 or 11C2 do not. Antibodies 8C10, 3El0, 11C2 and 6F12 may recognize epitopes on proteins that are extensively and variably modified through glycosylation or phosphorylation or some combination of both, resulting in complex smear patterns. Antibody 4H6 (lane 5) recognizes a polypeptide that is approx. 140 kDa. There is a very small amount of smearing above and below the main band of interest, indicating this protein may be modified to a limited extent.

266

P.T. Evans et al. : Biochemical differentiation in the tobacco flower

where either primary or secondary antibodies were omitted as controls.

Fig. 4. Western blot analysis of proteins from tobacco flowers. The protein displayed on nitrocellulose was reacted with monoclonal antibodies. Lane 1, antibody 8C10; lane 2, 3El0; lane 3 11C2; lane4, 6F12; lane5, 4H6; lane6, MGT-1; lane 7, 8B1; lane 8, IgGzb control antibody

Nine of the 21 antibodies tested do not react to flower protein that has been transferred to nitrocellulose; lane 7 is an example. It could be that the antigens in question are not protein and as such would either fail to migrate through the polyacrylamide gel or bind to the nitrocellulose. It is also possible that the denaturation of proteins prior to and during sodium dodecyl sulfate polyacrylamide gel electrophoresis is irreversible in some cases, resulting in loss of epitope recognition by some antibodies. Antibody MGT-1 is a control antibody that reacts with many tissues; we used it in these Western blots because it gives a very simple banding pattern. In Fig. 4 (lane 6), MGT-I gives a band of 51 kDa and a band of lesser intensity at 45 kDa. This control helps us to show that the multiple bands seen in lanes 1-4 are not some artifact of the protein preparation or immunoblot procedure. Control antibodies of the same isotypes as the experimental antibodies were included in the experiments. These controls did not react with the immunoblots and yielded blank lanes (e.g. lane 8) indistinguishable from the results of experiments

Localization of the antigens on floral sections. In order to examine the tissue and cell distributions of the epitopes recognized by some of the antibodies further, we developed an immunolocalization protocol. Most antibodies react with more than one part of the flower, in a manner that is consistent with the patterns produced by the ELISA reactions. Antibody 11C2 recognizes an antigen present in most tobacco flower tissues (Fig. 5 a, b), and we have used it as a control to check the integrity of flower tissue after fixation and freezesectioning, processes that can reduce recognition of an epitope by an antibody. In contrast to 11C2, some of the antibodies reacted almost exclusively with specific cells of the tobacco flower. Monoclonal 8BI recognizes an antigen that is localized in the tobacco anther. The antigen is found inside the anther in discrete patches of cells that are external to the locules (Fig. 5c, d) and is also found in a small group of cells on the periphery of the anther (not shown). The 8BI antigen is barely detectable in other types of flower tissue. Antibody 5E3 recognizes antigen most strongly in the transmitting tissue of the pistil; the antigen is present in lower abundance in the anther and in vascular cells of sepals. It is barely detectable in petals. Analysis of a stigmoid-anther mutant. Previously we recovered a series of tobacco plants that had abnormal flowers with rearrangements in differentiation resembling the homeotic and heterochronic mutants of animals (Malmberg et al. 1985). The abnormal phenotypes were classified and named on the basis of their visual appearance. This left open the question of the extent of tissue transformation and homology of the abnormal tissues to various parts in a normal flower. Mgr27 is a mutant plant line that forms stigrnoid anthers; the external appearance o f the bulk o f the stamens is nearly normal, but the anther is smaller than in the wild-type, and the tip of the anther has a green cap that looks like a stigma. Two of our antibodies were particularly useful in characterizing the stigmoid anthers of the mutant. As described above and shown in Fig. 5, monoclonal line 5E3 recognizes an antigen present in the transmitting tissue of the stigma of normal tobacco flowers, and line 8B1 recognizes discrete patches of cells within the anther. When the stigmoid anthers from Mgr27 plants were sectioned and probed with 5E3 or 8BI antibody, the 5E3

P.T. Evans et al. : Biochemical differentiation in the tobacco flower

267

Fig. 5a-h. Immunolocalization with selected antibodies to tobacco flower sections, a Longitudinal frozen section of a flower treated with 11C2 antibody ( x 15). b Negative control section ( x ]5). e Transverse frozen section of anther treated with 8B] antibody ( x 80). d Negative control section ( x 80). e Longitudinal plastic section of normal stigma treated with 5E3 antibody ( x 16). f Negative control section ( x 16). g Longitudinal plastic section of stigmoid anther treated with 5E3 antibody ( x 16). h Negative control section ( x 16). A purple precipitate forms at the site of antibody localization

antibody recognized cells in the stigma portion of the stigmoid anther that appear to be the equivalent of those in the transmitting tissue of normal tobacco flowers (Fig. 5g, h); 8B1 reacted to a few dispersed cells within the anther portion of the stigmoid anther (not shown). This demonstrates at a biochemical level that Mgr27 plants produce anthers that are a mixture of anther and pistil tissue, and hence that they really are stigmoid anthers. Discussion Few of the antibodies we were able to prepare are totally specific to an antigen of any one organ, as judged by ELISA, although there are strong organ preferences. Even though protein differences among floral parts have been demonstrated by

others (Marushige and Marushige 1962; Barber and Steward 1968; Sawhney et al. 1985) and our screening procedures included no intentional bias for carbohydrate epitopes, the antibodies with the greatest differential reactivity among extracts are those targeted against carbohydrate moieties. This may indicate a general role for glycosylation in plant differentiation. It has been suggested that arabinogalactan-proteins (AGPs) may be involved in expression of identity (Clarke et al. 1979) and differences in arabinogalactans or AGPs among plant organs of several species have been reported (Gleeson and Clarke/980; van Holst and Clarke/986; Gell et al. 1986; Tsumuraya et al. 1988). It is possible, though as yet undetermined, that some of the carbohydrate epitopes recognized by the anti2 bodies ,described in this paper are present in AGPs. The prevalence of non-protein antigens in plant

268

tissues also indicates that the Western blotting technique may be only of limited utility in analysis of some antibodies derived from plant extracts. Some of the antibodies may represent independent isolations from a single immunodominant compound or group of related compounds; an example of this are antibodies 7H12 and 2E10. The isolation and characterization of cell- and tissue-specific genes through differential hybridization of nucleic acids are certainly important steps in increasing our understanding of plant differentiation and development (Smith et al. 1987), but molecules of fundamental importance other than polypeptides may be overlooked, and these could be detected though hybridoma technology. However, because of low abundance in the tissues, either method may miss compounds of interrest. In addition, hybridoma techniques may not detect compounds of low antigenicity or those masked by immunodominance of others. To remove problems of immunodominance, immunodepletion through affinity chromatography or treatment of immunogen with trifluoromethanesulfonic acid for deglycosylation has been attempted elsewhere (Bradley et al. 1988). Other techniques such as careful staging of material for extractions, partial purification from crude extracts, enzymatic deglycosylation of proteins, and micro-innuoculation techniques combined with improved screening procedures may help to recover antibodies to antigens of interest. Plant organs are mixtures of similar and in many cases probably analogous tissues; differential reactivity of antibodies in ELISA reactions might reflect an unequal distribution of tissues or cell types among the floral organs. This would imply that some of the antibodies have a degree of specificity for a particular cell type or tissue within an organ, a fact borne out by the immunocytological staining reported here. It should also be noted that flowers of virtually all ages prior to anthesis were included in our extracts. Since the differential reactivities of the monoclonal antibodies may change with changes during development, our results represent a crude average over many developmental stages. In the past, plant cell types have been largely distinguished by morphological criteria aided by histochemistry. It may be possible, using monoclonal antibodies, to classify cells further based on their physiological or developmental states. This work has been supported by National Science Foundation (NSF) grants DMB-85-44021, DCB-85-00172, a McKnight Foundation Training Grant in Plant Biology, and by an NSF

P.T. Evans et al. : Biochemical differentiation in the tobacco flower postdoctoral fellowship to B.L.H. The hybridoma facility of the Botany Department was supported by NSF grant PCM-8315882. The authors thank Mary Sledge, Wilrna Lingle and Debra Rose for their help, and Natasha Raikhel (Michigan State University, East Lancing, USA), Richard Cyr, Cindy Evans, Michael Hahn and Lee Pratt for useful discussions and comments. R.L.M. also wishes to thank Susan Hockfield and Ron McKay for their help in the early stages of this work.

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Received 8 January; accepted 9 March 1988