δ-Tonoplast intrinsic protein defines unique plant vacuole functions

Abstract

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...

A Proteomics Dissection of Arabidopsis thaliana Vacuoles Isolated from Cell Culture

“…We thus demonstrated that aquaporins are well expressed on Arabidopsis cultured cells and showed that one of the most abundant proteins present on the tonoplast is the AtTIP2.1 (Table I). Previous studies argued that plant cells have the ability to generate and maintain separate vacuole organelles with each being marked by a different TIP subtype (5,6). For example, the fully differentiated cell types PSVs are marked by ␣-TIP plus ␦-TIP, and lytic vacuoles are marked by ␥-TIP.…”

“…PSVs contain vacuolar storage proteins to be used for anabolism during seedling growth. The function of the different vacuoles seems to be correlated with the presence of specific tonoplast intrinsic protein (TIP) isoforms (5,6). Current knowledge of the cellular traffic of higher eukaryotes indicates that vacuole biogenesis is closely related to the traffic of proteins resident in these compartments (7)(8)(9)(10).…”

Identification and Characterization of a Prevacuolar Compartment in Stigmas of Nicotiana alata

“…This compartment fractionates distinctly from both vacuole membrane markers, ␣ -TIP and ␥ -TIP. In some plant systems, different TIP proteins mark distinct vacuoles (Paris et al, 1996;Jauh et al, 1998); however, it has not been determined whether multiple types of vacuoles are present in stigma cells. The distinct fractionation pattern for Na-PI and ␣ -TIP, however, indicates that Na-PI is unlikely to travel within "dense vesicles" similar to those that transport storage proteins in developing pea cotyledons.…”

Isolation of a fruit ripening-related tonoplast aquaporin (GjTIP) gene from Gardenia jasminoides

“…TIP1 subfamily is preferentially associated with the large lytic vacuoles (Jauh et al 1998). Arabidopsis thaliana AtTIP1;1 was shown to accumulate in intravacuolar invaginations made of a double tonoplast membrane (Saito et al 2002), and expression of the AtTIP1;1 is associated with cell enlargement in Arabidopsis roots, hypocotyls, leaves, and flower stems (Ludevid et al 1992), the growth-promoting hormone gibberellic acid (GA3) would up-regulated the transcriptional level of AtTIP1;1 (Phillips and Huttly 1994).…”