Plant cell vacuoles may have storage or lytic functions, but biochemical markers specific for the tonoplasts of functionally distinct vacuoles are poorly defined. Here, we use antipeptide antibodies specific for the tonoplast intrinsic proteins ␣ -TIP, ␥ -TIP, and ␦ -TIP in confocal immunofluorescence experiments to test the hypothesis that different TIP isoforms may define different vacuole functions. Organelles labeled with these antibodies were also labeled with antipyrophosphatase antibodies, demonstrating that regardless of their size, they had the expected characteristics of vacuoles. Our results demonstrate that the storage vacuole tonoplast contains ␦ -TIP, protein storage vacuoles containing seed-type storage proteins are marked by ␣ -and ␦ -or ␣ -and ␦ -plus ␥ -TIP, whereas vacuoles storing vegetative storage proteins and pigments are marked by ␦ -TIP alone or ␦ -plus ␥ -TIP. In contrast, those marked by ␥ -TIP alone have characteristics of lytic vacuoles, and results from other researchers indicate that ␣ -TIP alone is a marker for autophagic vacuoles. In root tips, relatively undifferentiated cells that contain vacuoles labeled separately for each of the three TIPs have been identified. These results argue that plant cells have the ability to generate and maintain three separate vacuole organelles, with each being marked by a different TIP, and that the functional diversity of the vacuolar system may be generated from different combinations of the three basic types.

In lily, adhesion of the pollen tube to the trans-

Prevacuolar compartments (PVCs) are membrane-bound organelles that mediate protein traffic between Golgi and vacuoles in the plant secretory pathway. Here we identify and define organelles as the lytic prevacuolar compartments in pea and tobacco cells using confocal immunofluorescence. We use five different antibodies specific for a vacuolar sorting receptor (VSR) BP-80 and its homologs to detect the location of VSR proteins. In addition, we use well-established Golgi-markers to identify Golgi organelles. We further compare VSR-labeled organelles to Golgi organelles so that the relative proportion of VSR proteins in Golgi vs. PVCs can be quantitated. More than 90% of the BP-80-marked organelles are separate from Golgi organelles; thus, BP-80 and its homologs are predominantly concentrated on the lytic PVCs. Additionally, organelles marked by anti-AtPep12p (AtSYP21p) and anti-AtELP antibodies are also largely separate from Golgi apparatus, whereas VSR and AtPep12p (AtSYP21p) were largely colocalized. We have thus demonstrated in plant cells that VSR proteins are predominantly present in the lytic PVCs and have provided additional markers for defining plant PVCs using confocal immunofluorescence. Additionally, our approach will provide a rapid comparison between markers to quantitate protein distribution among various organelles.

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (beta-D-glucosyl)3 Yariv phenyl-glycoside [(beta-D-Glc)3] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(beta-D-Glc)3 complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall.

Plant cell vacuoles may have either storage or degradative functions. Vegetative storage proteins (VSPs) are synthesized in response to wounding and to developmental switches that affect carbon and nitrogen sinks. Here we show that VSPs are stored in a unique type of vacuole that is derived from degradative central vacuoles coincident with insertion of a new tonoplast intrinsic protein (TIP), ␦-TIP, into their membranes. This finding demonstrates a tight coupling between the presence of ␦-TIP and acquisition of a specialized storage function and indicates that TIP isoforms may determine vacuole identity.Uniquely, in contrast to yeast or mammalian cells, plant cells may contain separate vacuoles with protein storage and digestive functions (1, 2). It is not known how functionally distinct vacuoles are generated or maintained. Plant vacuole tonoplast membranes contain abundant tonoplast intrinsic proteins (TIPs) that may function as aquaporins (3), but the quantities present seem to be far in excess of what is required for water transport (4). Protein storage vacuoles (PSVs), containing seed-type storage proteins, are marked by the presence of ␣-TIP, and lytic or degradative vacuoles (LVs) are marked by the presence of ␥-TIP (1, 2). These observations indicate that a specific TIP isoform correlates with a specific vacuole function (5). A further test of this hypothesis would require demonstration that a third functionally distinct vacuole carried a different TIP isoform. Here we define a third functionally distinct vacuole in plant cells and demonstrate that it is marked specifically by ␦-TIP. MATERIALS AND METHODSAntibodies and Immunocytochemistry. Anti-␣-TIP protein antiserum (6) and VM23 antiserum to purified ␥-TIP protein from radish root (7) were generously provided by M. Chrispeels (6) and M. Maeshima (7), respectively. Synthetic peptides were synthesized and antisera to the peptides coupled to keyhole limpet hemocyanin were generated by Quality Controlled Biochemicals, Hopkinton, MA. For antibody purification, the peptides were coupled via an amino-terminal Cys residue to SulfoLink agarose (Pierce) according to the manufacturer's instructions, and peptide-specific antibodies were affinity-purified as described previously (8) for use in all procedures. Antisera to proteinase inhibitor I (Inh I) and II (Inh II) have been described previously (9-11). Fluorochrometagged secondary antibodies were purchased from Jackson ImmunoResearch. Plant tissues were fixed in either formaldehyde͞acetic acid͞ethyl alcohol or 3.7% paraformaldehyde, and paraffin-embedded sections were prepared as described (12). After removal of paraffin and rehydration, the sections were blocked as described (2). The double-label protocol to identify two different rabbit antibodies separately with immunofluorescence has been described (2). Briefly, the first primary antibody was completely covered by incubation with an excess of anti-rabbit F(abЈ) 2 fragment coupled to lissamine rhodamine before incubating with the second primary antibo...