Tissue Printing as a Tool for Observing Immunological and Protein Profiles in Young and Mature Celery Petioles

Abstract

Tissue printing onto membranes such as nitrocellulose is a technique 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 ribulose-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 corresponded 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 observed 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 specific 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 macromolecules (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 membranes.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 MATERIALS AND METHODS Plant TissueCelery 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 PrintingFreehand cross-sections of approximately 1-to 3-mm thickness were made with a razor blade from celery petioles (Vamer and Taylor, 1989;T...

Histochemical Demonstration and Localization of H2O2 in Organs of Higher Plants by Tissue Printing on Nitrocellulose Paper

“…Tissue prints on nitrocellulose paper that can be developed with specific reagents for visualizing certain RNAs or proteins (enzymes) provide a simple, ingenious tool for the detection and histological localization of these macromolecules (McClure and Guilfoyle, 1989;Bailey et al, 1991;Lagrimini, 1991;Taylor et al, 1993). This technique, which was introduced simultaneously by Cassab and Vamer (1987) and Spruce et al (1987), has an inherent potential for a large variety of additional applications (Reid et al, 1992).…”

Tomato prosystemin promoter confers wound-inducible, vascular bundle-specific expression of the β-glucuronidase gene in transgenic tomato plants

“…Tissue-print experiments were carried out as described by Taylor et al (1993). Petioles, petiolules and main stems were excised with a razor blade from three-week-old wildtype tomato plants.…”

Detection of ‘Candidatus Liberibacter asiaticus’ in citrus by concurrent tissue print‐based qPCR and immunoassay

“…In contrast, immunotissue printing is a simple and inexpensive technique and also preserves the cellular locations of macromolecules, such as proteins, enzymes, soluble metabolites and nucleic acids (Cassab & Varner, ; Taylor et al ., ). The method is widely used in both field and laboratory for the detection of other important phloem‐limited pathogens of citrus such as citrus tristeza virus (CTV; Garnsey et al ., ) and Spiroplasma citri (Shi et al ., ).…”