The tonoplast was proposed as a default destination of membrane-bound proteins without specific targeting signals. To investigate the nature of this targeting, we created type I fusion proteins with green fluorescent protein followed by the transmembrane domain of the human lysosomal protein LAMP1. We varied the length of the transmembrane domain from 23 to either 20 or 17 amino acids by deletion within the hydrophobic domain. The resulting chimeras, called TM23, TM20, and TM17, were expressed either transiently or stably in tobacco. TM23 clearly accumulated in the plasmalemma, as confirmed by immunoelectron microscopy. In contrast, TM17 clearly was retained in the endoplasmic reticulum, and TM20 accumulated in small mobile structures. The nature of the TM20-labeled compartments was investigated by coexpression with a marker localized mainly in the Golgi apparatus, AtERD2, fused to a yellow fluorescent protein. The strict colocalization of both fluorescent proteins indicated that TM20 accumulated in the Golgi apparatus. To further test the default destination of type I membrane proteins, green fluorescent protein was fused to the 19-amino acid transmembrane domain of the plant vacuolar sorting receptor BP-80. The resulting chimera also accumulated in the Golgi instead of in post-Golgi compartments, where native BP-80 localized. Additionally, when the transmembrane domain of BP-80 was lengthened to 22 amino acids, the reporter escaped the Golgi and accumulated in the plasma membrane. Thus, the tonoplast apparently is not a favored default destination for type I membrane proteins in plants. Moreover, the target membrane where the chimera concentrates is not unique and depends at least in part on the length of the membrane-spanning domain. INTRODUCTIONThe sorting of integral proteins in plants is not well understood. Nevertheless, it is accepted that peptidic signals exposed in the cytosol are responsible for targeting to the correct subcellular location in plant cells. This signal-mediated sorting is opposed to a default transport that is believed to happen when no signal is present on a protein.Although the default destination within the secretory pathway for a soluble protein is secretion, the default membrane is unclear. The most informative results about the location where membrane proteins would accumulate by default were provided by a study of ␣ -TIP (Höfte and Chrispeels, 1992). In this experiment, the last 48 amino acids of ␣ -TIP, which contains the sixth transmembrane domain, were sufficient to target a reporter protein to the tonoplast. Because the deletion of the cytosolic C-terminal 15 amino acids from the ␣ -TIP sequence did not prevent the tonoplast accumulation of the truncated protein, the authors indirectly deduced a role for the sixth membrane-spanning domain. Either this ␣ -TIP transmembrane domain would be sufficient for vacuolar location or the tonoplast would be the default destination for membrane proteins.More recently, the same sixth transmembrane domain of ␣ -TIP was used in a chimeric c...

Tobacco contains different isoforms of chitinase (EC 3.2.1.14), a hydrolase thought to be involved in the defense against pathogens. Deduced amino acid sequences for putatively vacuolar, basic chitinases differ from the homologous extracellular, acidic isoforms by the presence of a C-terminal extension. To examine the role of this C-terminal extension in protein sorting, Nicotiana silvestris plants were stably transformed with chimeric genes coding for tobacco basic chitinase A with and without the seven C-terminal amino acids. In plants expressing unmodified chitinase A, the enzyme activity was low in the intercellular wash fluid but high in protoplasts and isolated vacuoles. In contrast, in plants expressing mutant chitinase lacking the C terminus, the activity

The endochitinases (E.C. 3.2.1.14, chitinase) are a structurally diverse group of enzymes believed to be important in the biochemical defense of plants against potential pathogens. The gene for a chitinase of Nicotiana tabacum L. cv. Havana 425 has been cloned and sequenced. The major transcription start is 11 bp upstream of the ATG codon and 28 bp downstream of the TATA box. The gene contains two introns and encodes a basic chitinase of 329 amino acids with a 23 amino acid N-terminal signal peptide followed by a 43 amino acid, cysteine-rich domain, which is linked by a hinge region to the main structure of the enzyme. This gene appears to be expressed because the exons are identical to the coding sequence of a cDNA which was isolated. Comparison of chitinase amino acid sequences from different plants indicates there are at least three classes of these enzymes: class I, basic chitinases with an N-terminal cysteine-rich domain and a highly conserved main structure; class II, chitinases similar to the main structure of class I chitinases but lacking the cysteine-rich domain; and, class III, chitinases with conserved sequences different from those of the class I and II enzymes. The sequences encoding the cysteine-rich domain in class I chitinases are flanked by 9-10 bp imperfect direct repeats suggesting that these domains arose from a common ancestral gene and were introduced into genes for class I enzymes by transposition events.

Tobacco glucan endo-1,3-fi-glucosidase (13-1,3-glucanase; 1,3-13-D-glucan glucanohydrolase; EC 3.2.1.39) exhibits complex hormonal and developmental regulation and is induced when plants are infected with pathogens. We determined the primary structure of this enzyme from the nucleotide sequence of five partial cDNA clones and the amino acid sequence of five peptides covering a total of 70 residues.,3-1,3-Glucanase is produced as a 359-residue preproenzyme

beta-Aminobutyric acid (BABA) was used to induce resistance in grapevine (Vitis vinifera) against downy mildew (Plasmopara viticola). This led to a strong reduction of mycelial growth and sporulation in the susceptible cv. Chasselas. Comparing different inducers, the best protection was achieved with BABA followed by jasmonic acid (JA), whereas benzo (1,2,3)-thiadiazole-7-carbothionic acid-S-methyl ester (a salicylic acid [SA] analog) and abscisic acid (ABA) treatment did not increase the resistance significantly. Marker genes for the SA and JA pathways showed potentiated expression patterns in BABA-treated plants following infection. The callose synthesis inhibitor 2-deoxy-D-glucose partially suppressed BABA- and JA-induced resistance against P viticola in Chasselas. Application of the phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid and the lipoxygenase (LOX) inhibitor 5, 8, 11, 14-eicosatetraynoic acid (ETYA) also led to a reduction of BABA-induced resistance (BABA-IR), suggesting that callose deposition as well as defense mechanisms depending on phenylpropanoids and the JA pathways all contribute to BABA-IR. The similar phenotype of BABA- and JA-induced resistance, the potentiated expression pattern of JA-regulated genes (LOX-9 and PR-4) following BABA treatment, and the suppression of BABA-IR with ETYA suggest an involvement of the JA pathway in BABA-IR of grapevine leading to a primed deposition of callose and lignin around the infection sites.

An individual plant cell may contain at least two functionally and structurally distinct types of vacuoles: protein storage vacuoles and lytic vacuoles. Presumably a cell that stores proteins in vacuoles must maintain these separate compartments to prevent exposure of the storage proteins to an acidified environment with active hydrolytic enzymes where they would be degraded. Thus, the organization of the secretory pathway in plant cells, which includes the vacuoles, has a fascinating complexity not anticipated from the extensive genetic and biochemical studies of the secretory pathway in yeast. Plant cells must generate the membranes to form two separate types of tonoplast, maintain them as separate organelles, and direct soluble proteins from the secretory flow specifically to one or the other via separate vesicular pathways. Individual soluble and membrane proteins must be recognized and sorted into one or the other pathway by distinct, specific mechanisms. Here we review the emerging picture of how separate plant vacuoles are organized structurally and how proteins are recognized and sorted to each type.

SummaryThe green fluorescent protein (GFP) from Aequorea victoria can be detected in living plant cells after transient transformation of protoplasts. Expression of the GFP can be used to monitor protein trafficking in a mixed cell population and also to study the different function and importance of organelles in different cell types. We developed a vacuolar form of GFP that was obtained by replacing the C-terminal endoplasmic reticulum (ER)-retention motif of mGFP5-ER by the vacuolar targeting peptide of tobacco chitinase A. The vacuolar GFP was transported and accumulated in the vacuole as expected. However, we found two patterns of GFP accumulation after prolonged incubation (18-24 h) depending on the cell type. Most chloroplastrich protoplasts had a fluorescent large central vacuole. In contrast, most chloroplast-poor protoplasts accumulated the GFP in one smaller vacuole but not in the large central vacuole, which was visible under a light microscope in the same cell. This differential accumulation reflected the existence of two different vacuolar compartments as described recently by immunolocalization of several vacuolar markers. We were able to characterize the vacuolar compartment to which GFP is specifically targeted as non-acidic, since it did not accumulate neutral red while acidic vacuoles did not accumulate GFP.

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion.