Plant Physiol. (1993) 102: 1027-1031

Tissue Printing as a To01 for Observing lmmunological and Protein Profiles in Young and Mature Celery Petioles

Rosannah Taylor*, Gordon Inamine', and James D. Anderson

Weed Science Laboratory, Beltsville Agriculture Research Center West, Agricultura1 Research Service, United States Department of Agriculture, Beltsville, Maryland 20705

Tissue printing onto membranes such as nitrocellulose is a tech- nique employed to study the localization of proteins, nucleic acids, and soluble metabolites from freshly cut tissue slices. We probed tissue prints of young and mature celery (Apium graveolens) petioles with antibodies raised against two proteins, spinach ribu- lose-1,5-bisphosphate carboxylase and tomato fruit catalase. The purposes of this study were to determine if these proteins are developmentally regulated and to determine if the patterns and intensities of cross-reactivity of antibodies on tissue blots corre- sponded only to the presence of specific epitopes or was related to the amount of protein present in any given area on the tissue prints. Different and distinct cross-reactivity patterns were ob- served with each of the two antibodies used. Tissue prints from young and mature tissues also showed differences in antibody cross-reactivity. Comparison of Coomassie blue staining patterns with antibody reactivity patterns showed that there is little relationship between tissue protein concentration and antibody reactivity.

Most studies conceming regulation of protein levels involve the use of complex tissues or organs. Data obtained from such studies are extremely valuable but do not necessarily indicate in which cells or tissues the changes are occumng. Determining the amount and distribution of proteins in spe- cific tissues or cells could yield valuable insights into cell development. Tissue printing is a relatively new technique that is being used to study cell-specific localization of mac- romolecules (Cassab et al., 1985; Cassab and Vamer, 1987; Spruce et al., 1987; Cassab et al., 1988; Cassab and Vamer, 1989; McClure and Guilfoyle, 1989; Vamer and Taylor, 1989; Lagrimini, 1991; Ye and Vamer, 1991; Bailey et al., 1992; Taylor, 1992b). The technique was described by Cassab and Vamer (1987) and consists of transfemng cellular materials from the freshly cut surface of tissues to nitrocellulose mem- branes.

We sought to develop this method in a more quantitative way, i.e. measuring the amount of material transferred from one cell type or tissue. Quantitative interpretation in antibody reactivity on tissue prints is difficult if one tries to explain differences in the degree of cross-reactivity without knowing the amount of protein transferred from each tissue; specific signal might simply reflect the bulk transfer of protein from regions of high cell density and yield no information on the

Present address: The Public Health Research Institute, Depart- ment of Microbiology, 455 First Avenue, New York, NY 10016.

* Corresponding author; fax 1-301-504-6491. 1027

concentration of target protein relative to total protein. To address this problem, we have examined the reactions of two antibodies to tissue blots of celery (Apium graveolens) cross- sections representing Y (inner) or M (outer) petiole tissues. We compared the cross-reactivity of the two antibodies with the relative amount of protein transferred to the blots within cell types and tissues.

MATERIALS AND METHODS

Plant Tissue

Celery petioles (Apium graveolens L.) were purchased from local markets. The petioles (Fig. 1A) selected represent two developmental stages: Y (nongreen petioles from the center of the stalk) and more M (green extemal petioles). The sections printed of both Y and M tissues were taken from the central portion of each petiole used. The arrows show this area only in the most M petiole.

Tissue Printing

Freehand cross-sections of approximately 1- to 3-mm thickness were made with a razor blade from celery petioles (Vamer and Taylor, 1989; Taylor, 1992a, 1992b). The sections were gently blotted onto filter paper prior to printing. Sections were then carefully placed onto a piece of NC membrane. A piece of smooth paper was placed over the section to protect the membrane from fingerprints while applying pressure with one or two fingers for 10 to 30 s. Similar freehand cross- sections were stained with toluidine blue for observation of anatomy (Fig. 18).

Tissue-Print Western Analysis

Tissue-print westem blots were previously described (Cas- sab and Vamer, 1987, 1989). Antibodies used to locate pro- teins on tissue prints of developing celery petioles were against (a) RuBP (Inamine et al., 1985) or (b) CTL (Inamine and Baker, 1989). Alkaline phosphatase-conjugated goat anti-rabbit antibodies were used as the secondary antibodies. Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-in-

Abbreviations: Cb, collenchyma bundles; CTL, tomato fruit cata- lase; E, epidermis; Fc, fasicular cambium; M, mature; NC, nitrocel- lulose; Ph, phloem; Pp, pith parenchyma; RUBI', spinach ribulose- 1,5-bisphosphate carboxylase; Xy, xylem; Y, young.

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1028 Taylor et al. Plant Physiol. Vol. 102, 1993

doxyl phosphate, p-toluidine salt (Promega Corp.)2 were usedas substrates for color development. All micrographs weretaken either under a Zeiss SV8 stereomicroscope equippedwith a MC 100 camera or with a 35-mm Nikon camera. Allphotographic films were from Eastman Kodak.

Electrophoresis and Immunoblotting

Protein extracts for SDS-PAGE (Schagger and Von Jagow,1987) were prepared by homogenizing tissue in ice-cold 50mM Tris-HCl (pH 6.8) buffer containing 10 mM thiourea. Thehomogenate was centrifuged at ll,000g for 10 min, and thecleared supernatant was collected. Protein was determinedaccording to Bradford (1976). The following proteins weresubjected to SDS-PAGE: RuBP, CTL, and extracts from Yand M petiole tissues. Protein staining was performed withCoomassie brilliant blue R-250. Proteins from the gels wereelectrotransferred to NC membranes (Towbin et al., 1986).Immunoblots for substrate binding were detected as de-scribed above.

Figure 1. Celery tissue used for this study. The sections printed ofboth Y and M tissues were taken from the central portion of eachpetiole (A). The arrows show this area only in the most M petiole.Dark-field illumination of a freehand cross-section that was stainedwith toluidine blue for anatomical observation (B). Magnificationbar = 1 mm.

Total Protein Profiles

Freehand cross-sections of celery petioles were prepared,gently blotted, and printed directly onto NC membranes.Several protein-staining procedures were tried (e.g. Coo-

2 The mention of specific instruments, trade names, or manufac-turers is for the purpose of identification and does not imply anyendorsement by the United States government.

massie blue, Indian ink, colloidal gold, and napthol blueblack). Coomassie blue was adopted because of its ease ofuse and better reproducibility. The membranes were gentlyagitated for several minutes in a filtered solution of 0.1%Coomassie blue in 20% aqueous methanol containing 7%acetic acid at room temperature. The membrane was removedand washed with distilled H2O and destained in 50% meth-anol containing 7% acetic acid with gentle agitation. Initial

Y M

RuBP

CTL

...<*•--....̂ -E

Fc-

PP Ph

VPh

Cb

Xy— Ph

Xy

Figure 2. Antibody reactivity profiles of celery petiole tissue blots. Cross-sections of Y and M celery petioles were printedonto NC and the blots challenged with antibodies against RuBP (A-C) and CTL (D-F). Profiles on the left (A and D)represent prints from Y, or inside, petioles and profiles B and E represent prints from M, or outside, petioles. Profiles onthe far right (C and F) represent prints from M petioles enlarged to show detail. Magnification bars = 1 mm.

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Protein Binding on Nitrocellulose 1029

Table I. Summary of analysis of Y and M celery petiole tissue printsTissue prints were probed with antibodies raised against RuBP and CTL, whereas total protein was

visualized with Coomassie blue.Antibodies Total Protein

Tissue RuBP CTLM

M M

ECbPhFcXyPp

' The symbols +, ++, +++, ++++, and — represent little, moderate, intense, most intense, or noapparent cross-reactivity with antibodies or Coomassie blue staining, respectively.

problems with NC solubility were solved by lowering themethanol content of both the staining and destaining solu-tions. After destaining, the membranes were placed betweenabsorbent paper and allowed to dry.

RESULTS

Specific Antibody Profiles in Celery Petioles

The results of the two specific polyclonal antibodies usedto locate proteins on tissue blots of celery petioles (Fig. 2) aresummarized in Table I. Antibodies raised against RuBP (Fig.2, A-C) showed strong cross-reactivity with epidermal cells,the Fc of the vascular tissue, and the Pp. Cb were devoid ofproducts that are cross-reactive with RuBP antibodies. Theseantibodies reacted similarly in both Y and M tissues exceptfor the E and Fc, which were more distinct in the more Mtissue, and the Xy, which was more pronounced in the Ytissue. Antibodies raised against CTL (Fig. 2, D-F) cross-

reacted strongly with the Cb and Ph, moderately with thePp, little with the E, and not at all with the Xy and Fc. Therewas little difference in the degree of cross-reactivity betweenY and M tissues.

SDS-PAGE and Western Blots

Coomassie blue staining of RuBP and CTL antigens, aswell as extracts from Y and M celery petioles, are presentedin Figure 3A (lanes 1-4, respectively). Immunological analysisof polypeptides (Fig. 3B) showed that RuBP (lanes 5-7)and CTL (lanes 8-10) antibodies reacted with their respec-tive antigens and did not cross-react with many otherpolypeptides.

Coomassie Blue Staining of Tissue Blots

An important aspect of this research was to determine therelative amount of protein transferred to NC membranes and

1 2 3

RuBP CTL Y

Figure 3. SDS-PAGE analysis of polypeptides(A) and antigens (B) in extracts from Y and Mcelery petioles. The following amounts of pro-tein 0*g) were added: Lanes 1, 5 /jg; 2, 4 Mg; 3,4, 6, 7, 9, 10, 10 Mg; 5, 0.25 ^g; and 8, 2 Mg. A,Coomassie blue staining of RUBP, CTL, andextracts from Y and M petioles (lanes 1-4). B,Immunoblots of the proteins in A, reacted withspecific antibodies against RUBP (lanes 5-7)and CTL (lanes 8-10).

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1030 Taylor et al. Plant Physiol. Vol. 102, 1993

B

Pp

...;. -,y' "x;

'*&•£#£$/

K'4

P E

' '•'•'.;• •"••.'JjJ-v5:_--:.*'.r;v^

Figure 4. Tissue prints of Y (A) and M (B) celery petioles on NIC stained with Coomassie blue. C, Print from M petioleenlarged to show detail. Magnifications, A, 4X; B, 2x; C, 15x.

to relate that information to the type of tissue being examined(Table I). Coomassie blue stained the Ph, Cb, and Fc in-tensely, the Pp and Xy moderately, and the E only a little inboth tissue types. However, tissue prints of the Y petioles(Fig. 4, A and C) seemed to be more intensely stained thanthese same tissues in more M petioles (Fig. 4B).

DISCUSSION

The purpose of this study was to determine if the patternof cross-reactivity of antibodies on tissue blots actually relatesto the presence of specific epitopes or to the amount ofprotein present in any given tissue. Two different cross-reactivity patterns observed with the antibodies used in tissueblotting are summarized in Table I. The differences were bothgreater in intensity and more in number between the anti-bodies used than between the Y and M tissues used. Some-what surprisingly, neither of the antibody patterns coincidedwith the pattern of total protein staining. Tissues that exhib-ited high protein content did not show greater binding ofboth antibodies. Thus, the pattern one gets with antibodiesshowing good specificity is not related to the amount ofprotein transferred, but rather to specific epitopes present inparticular tissues.

We used several antibody preparations in this study. Onlyresults with those antisera that cross-reacted with their spe-cific antigens as well as with proteins of the correct mol wtblotted from SDS-polyacrylamide gels of tissue extracts arereported. Discrete, immunoreactive proteins from SDS-PAGEblots of tissue extracts were taken to be the source of signalon the tissue prints. One antiserum failed to react with theblotted, SDS-solubilized proteins of celery petioles and waseliminated from further characterization, despite the fact thatit produced tissue-specific patterns on tissue prints. Otherantibodies not used gave good signals on tissue prints butdid not show good specificity with the antigen and/or tissueextracts. Our criteria for identifying true recognition of anti-gens on tissue prints called for corresponding recognition ofSDS-solubilized proteins electroblotted from polyacrylamideslab gels.

ACKNOWLEDGMENTS

We are indebted to Paul Snyder for technical assistance. We alsoacknowledge Dr. Joe Varner of Washington University in St. Louis,MO, for his stimulating discussions and ideas. The authors extendthanks to H. Dave Clark for assistance with the photography.

Received March 4, 1993; accepted April 7, 1993.Copyright Clearance Center: 0032-0889/93/102/1027/05.

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