Three-dimensional analysis of syncytial-type cell plates during endosperm cellularization visualized by high resolution electron tomography.

The three-dimensional architecture of syncytial-type cell plates in the endosperm of Arabidopsis has been analyzed at approximately 6-nm resolution by means of dual-axis high-voltage electron tomography of high-pressure frozen/freeze-substituted samples. Mini-phragmoplasts consisting of microtubule clusters assemble between sister and nonsister nuclei. Most Golgi-derived vesicles appear connected to these microtubules by two molecules that resemble kinesin-like motor proteins. These vesicles fuse with each other to form hourglass-shaped intermediates, which become wide (approximately 45 nm in diameter) tubules, the building blocks of wide tubular networks. New mini-phragmoplasts also are generated de novo around the margins of expanding wide tubular networks, giving rise to new foci of cell plate growth, which later become integrated into the main cell plate. Spiral-shaped rings of the dynamin-like protein ADL1A constrict but do not fission the wide tubules at irregular intervals. These rings appear to maintain the tubular geometry of the network. The wide tubular network matures into a convoluted fenestrated sheet in a process that involves increases of 45 and 130% in relative membrane surface area and volume, respectively. The proportionally larger increase in volume appears to reflect callose synthesis. Upon fusion with the parental plasma membrane, the convoluted fenestrated sheet is transformed into a planar fenestrated sheet. This transformation involves clathrin-coated vesicles that reduce the relative membrane surface area and volume by approximately 70%. A ribosome-excluding matrix encompasses the cell plate membranes from the fusion of the first vesicles until the onset of the planar fenestrated sheet formation. We postulate that this matrix contains the molecules that mediate cell plate assembly.

The arabidopsis cell plate-associated dynamin-like protein, ADL1Ap, is required for multiple stages of plant growth and development.

Dynamin and dynamin-like proteins are GTP-binding proteins involved in vesicle trafficking. In soybean, a 68-kD dynamin-like protein called phragmoplastin has been shown to be associated with the cell plate in dividing cells (Gu and Verma, 1996). Five ADL1 genes encoding dynamin-like proteins related to phragmoplastin have been identified in the completed Arabidopsis genome. Here we report that ADL1Ap is associated with punctate subcellular structures and with the cell plate in dividing cells. To assess the function of ADL1Ap we utilized a reverse genetic approach to isolate three separate Arabidopsis mutant lines containing T-DNA insertions in ADL1A. Homozygous adl1A seeds were shriveled and mutant seedlings arrested soon after germination, producing only two leaf primordia and severely stunted roots. Immunoblotting revealed that ADL1Ap expression was not detectable in the mutants. Despite the loss of ADL1Ap, the mutants did not display any defects in cytokinesis, and growth of the mutant seedlings could be rescued in tissue culture by the addition of sucrose. Although these sucrose-rescued plants displayed normal vegetative growth and flowered, they set very few seeds. Thus, ADL1Ap is critical for several stages of plant development, including embryogenesis, seedling development, and reproduction. We discuss the putative role of ADL1Ap in vesicular trafficking, cytokinesis, and other aspects of plant growth.

COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes.

COPII vesicle formation requires only three coat assembly subunits: Sar1p, Sec13/31p, and Sec23/24p. PI 4-phosphate or PI 4,5-bisphosphate is required for the binding of these proteins to liposomes. The GTP-bound form of Sar1p recruits Sec23/24p to the liposomes as well as to the ER membranes, and this Sar1p-Sec23/24p complex is required for the binding of Sec13/31p. Ultrastructural analysis shows that the binding of COPII coat proteins to liposomes results in coated patches, coated buds, and coated vesicles of 50-90 nm in diameter. Budding proceeds without rupture of the donor liposome or vesicle product. These observations suggest that the assembly of the COPII coat on the ER occurs by a sequential binding of coat proteins to specific lipids and that this assembly promotes the budding of COPII-coated vesicles.

Protein transport within the plant cell endomembrane system: an update.

The secretory pathway plays a central role in plant development and morphogenesis. Storage protein deposition, plant cell division and the expansion of the plasma membrane and extracellular matrix all require the synthesis and trafficking of membranes, proteins and polysaccharides through this network of organelles. Increasing evidence demonstrates that the plant secretory pathway is more complex than previously appreciated and that its formation and maintenance are guided/regulated by many different mechanisms.

Traffic COPs and the formation of vesicle coats.

Forward and retrograde trafficking of secretory proteins between the endoplasmic reticulum and the Golgi apparatus is driven by two biochemically distinct vesicle coats, COPI and COPII. Assembly of the coats on their target membranes is thought to provide the driving force for membrane deformation and the selective packaging of cargo and targeting molecules into nascent transport vesicles. This review describes our current knowledge on these issues and discusses how the two coats may be differentially targeted and assembled to achieve protein sorting and transport within the early secretory pathway.

COPI- and COPII-coated vesicles bud directly from the endoplasmic reticulum in yeast.

The cytosolic yeast proteins Sec13p-Sec31p, Sec23p-Sec24p, and the small GTP-binding protein Sar1p generate protein transport vesicles by forming the membrane coat termed COPII. We demonstrate by thin section and immunoelectron microscopy that purified COPII components form transport vesicles directly from the outer membrane of isolated yeast nuclei. Another set of yeast cytosolic proteins, coatomer and Arf1p (COPI), also form coated buds and vesicles from the nuclear envelope. Formation of COPI-coated, but not COPII-coated, buds and vesicles on the nuclear envelope is inhibited by the fungal metabolite brefeldin A. The two vesicle populations are distinct. However, both vesicle types are devoid of endoplasmic reticulum (ER) resident proteins, and each contains targeting proteins necessary for docking at the Golgi complex. Our data suggest that COPI and COPII mediate separate vesicular transport pathways from the ER.

A small GTP-binding protein from Arabidopsis thaliana functionally complements the yeast YPT6 null mutant.

A clone designated A.t.RAB6 encoding a small GTP-binding protein was isolated from a cDNA library of Arabidopsis thaliana leaf tissue. The predicted amino acid sequence was highly homologous to the mammalian and yeast counterparts, H.Rab6 and Ryh1/Ypt6, respectively. Lesser homology was found between the predicted Arabidopsis protein sequence and two small GTP-binding proteins isolated from plant species (44% homology to Zea mays Ypt1 and 43% homology to Nicotiana tabacum Rab5). Conserved stretches in the deduced amino acid sequence of A.t.Rab6 include four regions involved in GTP-binding, an effector region, and C-terminal cysteine residues required for prenylation and subsequent membrane attachment. Northern blot analysis demonstrated that A.t.Rab6 mRNA was expressed in root, leaf, stem, and flower tissues from A. thaliana with the highest levels present in roots. Escherichia coli produced histidine-tagged A.t.Rab6 protein-bound GTP, whereas a mutation in one of the guanine nucleotide-binding sites (asparagine122 to isoleucine) rendered it incapable of binding GTP. Functionally, the A.t.RAB6 gene was able to complement the temperature-sensitive phenotype of the YPT6 null mutant in yeast. The isolation of this gene will aid in the dissection of the machinery involved in soluble protein sorting at the trans-Golgi network of plants.

Determination of the functional elements within the vacuolar targeting signal of barley lectin.

We have previously demonstrated that the carboxyl-terminal propeptide of barley lectin is both necessary and sufficient for protein sorting to the plant vacuole. Specific mutations were constructed to determine which amino acid residues or secondary structural determinants of the carboxyl-terminal propeptide affect proper protein sorting. We have found that no consensus sequence or common structural determinants are required for proper sorting of barley lectin to the vacuole. However, our analysis demonstrated the importance of hydrophobic residues in vacuolar targeting. In addition, at least three exposed amino acid residues are necessary for efficient sorting. Sorting was disrupted by the addition of two glycine residues at the carboxyl-terminal end of the targeting signal or by the translocation of the glycan to the carboxy terminus of the propeptide. These results suggest that some components of the sorting apparatus interact with the carboxy terminus of the propeptide.

The barley lectin carboxyl-terminal propeptide is a vacuolar protein sorting determinant in plants.

We have previously shown that the 15-amino acid carboxyl-terminal propeptide of probarley lectin is necessary for the proper sorting of this protein to the plant vacuole. A mutant form of the protein lacking the carboxyl-terminal propeptide is secreted. To test whether the carboxyl-terminal propeptide is the vacuole sorting determinant of probarley lectin, we examined in transgenic tobacco the processing and sorting of a series of fusion proteins containing the secreted protein, cucumber chitinase, and regions of probarley lectin. Pulse-labeling experiments demonstrated that the fusion proteins were properly translocated through the tobacco secretory system and that cucumber chitinase and cucumber chitinase fusion proteins lacking the carboxyl-terminal propeptide were secreted. The cucumber chitinase fusion protein containing the carboxyl-terminal propeptide was properly processed and sorted to the vacuole in transgenic tobacco as confirmed by organelle fractionation and electron microscopy immunocytochemistry. Therefore, the barley lectin carboxyl-terminal propeptide is both necessary and sufficient for protein sorting to the plant vacuole.

A carboxyl-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco.

Barley lectin is synthesized as a preproprotein with a glycosylated carboxyl-terminal propeptide (CTPP) that is removed before or concomitant with deposition of the mature protein in vacuoles. Expression of a cDNA clone encoding barley lectin in transformed tobacco plants results in the correct processing, maturation, and accumulation of active barley lectin in vacuoles [Wilkins, T.A., Bednarek, S.Y., and Raikhel, N.V. (1990). Plant Cell 2, 301-313]. The glycan of the propeptide is not essential for vacuolar sorting, but may influence the rate of post-translational processing [Wilkins, T.A., Bednarek, S.Y., and Raikhel, N.V. (1990). Plant Cell 2, 301-313]. To investigate the functional role of the CTPP in processing, assembly, and sorting of barley lectin to vacuoles, a mutant barley lectin cDNA clone lacking the 15-amino acid CTPP was prepared. The CTPP deletion mutant of barley lectin was expressed in tobacco protoplasts, suspension-cultured cells, and transgenic plants. In all three systems, the wild-type barley lectin was sorted to vacuoles, whereas the mutant barley lectin was secreted to the incubation media. Therefore, we conclude that the carboxyl-terminal domain of the barley lectin proprotein is necessary for the efficient sorting of this protein to plant cell vacuoles.

Role of propeptide glycan in post-translational processing and transport of barley lectin to vacuoles in transgenic tobacco.

Mature barley lectin is a dimeric protein composed of two identical 18-kilodalton polypeptides. The subunits of barley lectin are initially synthesized as glycosylated proproteins, which are post-translationally processed to the mature protein preceding or concomitant with deposition of barley lectin in vacuoles. To investigate the functional role of the glycan in processing and intracellular transport of barley lectin to vacuoles, the sole N-linked glycosylation site residing within the COOH-terminal propeptide of barley lectin was altered by site-directed mutagenesis. cDNA clones encoding wild-type (wt) or glycosylation-minus (gly-) barley lectin preproproteins were placed under the transcriptional control of the cauliflower mosaic virus 35S promoter and introduced into Nicotiana tabacum cv Wisconsin 38. Barley lectin synthesized from both the wt and gly- constructs was processed and correctly targeted to vacuoles of tobacco leaves. Localization of barley lectin in vacuoles processed from the nonglycosylated gly- proprotein indicated that the high-mannose glycan of the barley lectin proprotein was not essential for targeting barley lectin to vacuoles. However, pulse-chase labeling experiments demonstrated that the glycosylated wt proprotein and the nonglycosylated gly- proprotein were differentially processed to the mature protein and transported from the Golgi complex at different rates. These results implicate an indirect functional role for the glycan in post-translational processing and transport of barley lectin to vacuoles.

Isolation and characterization of an ipt gene from the Ti plasmid Bo542.

A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes.

Cell-type-specific expression of a wheat-germ agglutinin gene in embryos and young seedlings of Triticum aestivum.

We have investigated the levels of cell-specific expression of wheat-germ agglutinin (WGA) during the development of embryos and in 3-d-old seedlings. Southern blot analysis of genomic DNA derived from hexaploid and diploid wheat (Triticum) species indicates that the isolated cDNA (complementary DNA) clone is specific for isolectin B (WGA-B). Specific accumulation of transcript for WGA-B was determined by RNA blot analysis and in-situ hybridization. The WGA-B mRNA increased tenfold during embryogenesis between 10 and 40 d post-anthesis and then declined again as the seed reached maturity. The root tips of 3-d-old seedlings contained approximately threefold more WGA-B mRNA than mature seeds. In-situ hybridization experiments showed that during embryogenesis, WGA-B mRNA was present only in the epidermal layers of the radicle and the coleorhiza, while in 3-d-old seedlings it was found in the root-cap cells. To analyze de-novo transcription of WGA-B mRNA a new variation of in-situ hybridization was developed. RNAs from developing embryos and seedlings were labeled with [(3)H]uridine and then hybridized in-situ with unlabeled antisense and sense transcripts. These experiments demonstrated that accumulation of WGA-B mRNA in embryos and 3-d-old seedlings resulted, at least partially, from de-novo transcription. All cells containing WGA-B mRNA also contained WGA, as shown by immunocytochemistry.