Isolation, characterization and immunolocalization of a novel, modular tomato arabinogalactan‐protein corresponding to the LeAGP‐1 gene

Abstract

SummaryArabinogalactan-proteins (AGPs) are a family of hydroxyproline-rich glycoproteins implicated to function in plant growth and development. This report focuses on a novel, modular AGP found in tomato, LeAGP-1, which was predicted by DNA cloning and herein verified at the protein level as a major AGP component. LeAGP-1 was isolated from tomato suspension-cultured cells and verified to be an AGP by precipitation with (β-D-galactosyl) 3 Yariv phenylglycoside and by amino acid composition analysis. Furthermore, LeAGP-1 was determined to correspond to LeAGP-1 clones based on three criteria: (1) amino acid composition identity, (2) amino acid sequence identity, and (3) specific immunoreactivity of glycosylated and deglycosylated LeAGP-1 with an antibody developed against the highly basic subdomain predicted from LeAGP-1 clones. The antibody was also used to immunolocalize LeAGP-1 in tomato to the cell surface of suspension-cultured cells, maturing metaxylem elements in young internodes and petioles, and stylar transmitting tissue cells. At the subcellular level, LeAGP-1 immunolocalized to the cell walls of these particular cells as well as to intercellular spaces between stylar transmitting tissue cells. LeAGP-1 now emerges as one of the most comprehensively studied AGPs in terms of (1) characterization at the genomic DNA, cDNA and protein levels, (2) known organ-specific and developmentally regulated mRNA expression patterns, (3) development of an antibody against a unique, peptide subdomain which specifically recognizes LeAGP-1 in its glycosylated and deglycosylated states, and (4) immunolocalization of a

Arabinogalactan-proteins: structure, expression and function

“…Thus, the model that emerges is that AGPs containing a GPI anchor are associated with the plasma membrane and are cleaved so as to release the AGP to and beyond the cell wall. Immunolocalizations as well as biochemical isolations of identical, or nearly identical AGPs (family members), in multiple surface locations (e. g. cell wall and culture media; plasma membrane, cell wall and culture media) provide evidence for multiple surface locations for a given AGP and are consistent with the above processing model for GPI-anchored AGPs [5, 10,77,78]. Nonetheless, the possibility of exclusively targeting certain AGPs to specific cell surface locations cannot be excluded, and the contribution of particular sequence determinants in the carbohydrate or protein moieties to such a process remains to be investigated.…”

“…In contrast, few AGP antibodies are directed against the core protein, which is typically extensively glycosylated. Two notable exceptions are the PAP antibody, which has reactivity directed against a putatively unglycosylated, Lys-rich region of a tomato AGP known as LeAGP-1, and an antibody which has reactivity directed against a bacterially expressed tobacco TTS core protein (table 1) [10,58]. These antibodies have proven useful in localizing the respective expression of an individual AGP core protein (i.e.…”

“…Clearly, the carbohydrate moieties of AGPs contain a reservoir of structural information that could serve as potential chemical signals consistent with oligosaccharide signaling in plants as championed by Peter Albersheim [114]. In addition, limited regions of AGP core proteins may also be accessible, as appears to be the case for at least some AGPs such as LeAGP-1, and may also provide chemical signaling information [10]. Many scenarios or models can be envisioned whereby AGPs serve as cellsignaling and/or cell adhesion molecules involved in various aspects of growth and development already mentioned above.…”

“…In these sequences, relatively few Pro are not hydroxylated, and the occurrence of Hyp-Ala and Ala-Hyp dipeptide repeats is common. References for these data are as follows: carrot [3], ryegrass [4], rose [5], maize [6], pear (Pc1) [7], pear (Pc2) [8], tobacco [9] and tomato [10].…”

Functional Identification of a Hydroxyproline-O-galactosyltransferase Specific for Arabinogalactan Protein Biosynthesis in Arabidopsis

“…AGPs were extracted from WT, galt2-1, and galt2-2 plants as described by Schultz et al (33). Specifically, 5 g of aerial tissue from 14-day-old plants were used for each line to obtain ␤-Yariv-precipitable AGPs, which were quantified spectrophotometrically as described by Gao et al (34).…”