Monoclonal antibodies against phloem P‐protein from plant tissue cultures. I. Microscopy and biochemical analysis

Abstract

Most research involving phloem proteins is done with phloem exudates, which are not easily obtained from many plants. We report here on the use of tissue cultures to study phloem proteins. Monoclonal antibodies against the filamentous phloem protein, P‐protein, were made by injecting mice with a phloem‐enriched fraction isolated from Streptanthus tortuosus callus grown on a medium that stimulates the differentiation of xylem and phloem (phloem[+] cultures). Monoclonal antibodies specific for P‐protein were identified by incubating free‐hand stem sections of S. tortuosus in hybridoma supernatants, then in a goat anti‐mouse antibody conjugated to fluorescein isothiocyanate (FITC), and observing the FITC under an epifluorescence microscope. Antibodies specific for P‐protein in stem sections were used to probe nitrocellulose blots of polyacrylamide gels separating proteins isolated from both phloem(+) and phloem(‐) tissue cultures. Immunoblots were incubated overnight in hybridoma supernatants followed by a secondary antibody conjugated to alkaline phosphatase. Three monoclonal antibodies—RS21, RS22, and RS23—bound to an 89‐kD band in the phloem(+) lanes but failed to bind to any proteins in the phloem(—) lanes. In leaf sections of Arabidopsis thaliana processed by freeze‐substitution, a mixture of RS21 and RS22 bound to the P‐protein filaments in sieve elements, but not to any proteins in adjacent cells. A control antibody specific for tubulin did not bind to the P‐protein filaments.

An Early Nodulin-Like Protein Accumulates in the Sieve Element Plasma Membrane of Arabidopsis

“…Phloem (1) tissue cultures of California shield leaf (Streptanthus tortuosus) were grown as previously described by Toth et al (1994). Cultures were maintained in the dark at room temperature on a Murashige and Skoog medium containing 2% Suc, 0.9% agarose, 4.65 mM kinetin, and 0.54 mM naphthalene acetic acid.…”

“…A sieve element-enriched fraction was isolated from the phloem-induced tissue cultures of California shield leaf as previously described by Sjölund (1990) and Toth et al (1994). The primary inoculation of 12-week-old BALB/c mice with sieve elementenriched fractions mixed with Freund's complete adjuvant (1:1) was followed by two sary inoculations with Freund's incomplete adjuvant at 3-week intervals.…”

“…Spleen cells from mice showing a strong immunological response during prescreening of the sera were fused with myeloma cells to produce the mab. Sieve element-specific antibodies were identified using immunofluorescence microscopy of free-hand sections of California shield leaf plants as described by Toth et al (1994). Monoclonal cell lines were established from hybridomas producing the sieve element-specific RS6 antibodies.…”

The Phloem Sieve Element: A River Runs through It.

“…As expected, the monoclonal antibodies recognize phloem-specific antigens not only in callus sieve elements but also in the sieve elements of intact plants. The antigens, including P-protein and a sieve element-specific form of P-amylase, are detected on immunoblots of total proteins from phloem (+) cultures but are not detected on immunoblots of proteins from phloem (-) cultures (Tóth and Sjolund, 1994;Tóth et al, 1994;Wang et ai., 199.9, demonstrating the differential expression of phloemspecific genes in this culture system.…”

Transformation of the Collateral Vascular Bundles into Amphivasal Vascular Bundles in an Arabidopsis Mutant1

“…The corresponding genes were shown to be specifically induced during xylogenesis in the plant (Demura and Fukuda, 1994;Ye and Varner, 1994). In terms of phloem differentiation, several genes have been characterized, such as those encoding ␤-amylase (Wang et al, 1995), P-protein (Tó th et al, 1994) and Arabidopsis H ϩ -ATPase isoform 3 (Dewitt and Sussman, 1995). The proteins are specifically present in either sieve elements (␤-amylase and P-protein) or companion cells (Arabidopsis H ϩ -ATPase isoform 3).…”