Secretory proteins and extracellular glycans are transported to the extracellular space during cell growth. These materials are carried in secretory vesicles generated at the trans-Golgi network (TGN). Analysis of the mammalian post-Golgi secretory pathway demonstrated the movement of separated secretory vesicles in the cell. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco (Nicotiana tabacum) BY-2 cell as a model cell, we characterized the transport machinery in plant cells. A combination of analyses, including electron microscopy of quick-frozen cells and four-dimensional analysis of cells expressing fluorescent-tagged SCAMP2, enabled the identification of a clustered structure of secretory vesicles generated from TGN that moves in the cell and eventually fuses with plasma membrane. This structure was termed the secretory vesicle cluster (SVC). The SVC was also found in Arabidopsis thaliana and rice (Oryza sativa) cells and moved to the cell plate in dividing tobacco cells. Thus, the SVC is a motile structure involved in mass transport from the Golgi to the plasma membrane and cell plate in plant cells.

The well-characterized secretory glycoprotein, rice (Oryza sativa) a-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal-dependent manner. INTRODUCTIONCereal a-amylases (EC 3.2.1.1) are typical secretory proteins found in many plants. In germinating cereal seeds, these enzyme molecules are biosynthesized and secreted from the secretory tissues, the scutellar epithelium and the aleurone, to the starchy endosperm, which has undergone programmed cell death. Numerous a-amylase isoforms have been identified in cereals, but the predominant a-amylase isoform I-1 (AmyI-1) in rice (Oryza sativa) is a unique glycoprotein that bears N-linked oligosaccharide side chains (Hayashi et al., 1990;Terashima et al., 1994). The biosynthesis and secretion of AmyI-1 have been extensively investigated: mRNA translation on endoplasmic reticulum (ER) membrane-bound ribosomes, signal sequence-dependent translocation of the ER, core glycosylation in the ER lumen, vesicular transport to the Golgi apparatus, oligosaccharide modification to the complex type, and exocytosis all proceed according to the canonical secretory mechanism (Palade, 1975;Blobel, 1980;Kornfeld and Kornfeld, 1985).Protein targeting into plastids is an essential cellular event for maintaining plant function and plant life. Plastids, including chloroplasts in green leaves and amyloplasts in starchy cells, contain the genetic machinery required to synthesize their own proteins, although most plastidial proteins are encoded in the nuclear DNA. Nuclear-encoded plastidial...

Life on earth relies upon photosynthesis, which consumes carbon dioxide and generates oxygen and carbohydrates. Photosynthesis is sustained by a dynamic environment within the plant cell involving numerous organelles with cytoplasmic streaming. Physiological studies of chloroplasts, mitochondria and peroxisomes show that these organelles actively communicate during photorespiration, a process by which by-products produced by photosynthesis are salvaged. Nevertheless, the mechanisms enabling efficient exchange of metabolites have not been clearly defined. We found that peroxisomes along chloroplasts changed shape from spherical to elliptical and their interaction area increased during photorespiration. We applied a recent femtosecond laser technology to analyse adhesion between the organelles inside palisade mesophyll cells of Arabidopsis leaves and succeeded in estimating their physical interactions under different environmental conditions. This is the first application of this estimation method within living cells. Our findings suggest that photosynthetic-dependent interactions play a critical role in ensuring efficient metabolite flow during photorespiration.

A nucleotide pyrophosphatase/phosphodiesterase (NPP) activity that catalyzes the hydrolytic breakdown of ADP-glucose (ADPG) has been shown to occur in the plastidial compartment of both mono-and dicotyledonous plants. To learn more about this enzyme, we purified two NPPs from rice (Oryza sativa) and barley (Hordeum vulgare) seedlings. Both enzymes are glycosylated, since they bind to concanavalin A, stain with periodic acid-Schiff reagent, and are digested by Endo-H. A complete rice NPP cDNA, designated as NPP1, was isolated, characterized, and overexpressed in transgenic plants displaying high ADPG hydrolytic activity. Databank searches revealed that NPP1 belongs to a functionally divergent group of plant nucleotide hydrolases. NPP1 contains numerous N-glycosylation sites and a cleavable hydrophobic signal sequence that does not match with the N-terminal part of the mature protein. Both immunocytochemical analyses and confocal fluorescence microscopy of rice cells expressing NPP1 fused with green fluorescent protein (GFP) revealed that NPP1-GFP occurs in the plastidial compartment. Brefeldin A treatment of NPP1-GFP-expressing cells prevented NPP1-GFP accumulation in the chloroplasts. Endo-H digestibility studies revealed that both NPP1 and NPP1-GFP in the chloroplast are glycosylated. Collectively, these data demonstrate the trafficking of glycosylated proteins from the endoplasmic reticulum-Golgi system to the chloroplast in higher plants.

SummaryHigh temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism-related genes. Here we report the involvement of a starch-hydrolyzing enzyme, a-amylase, in high temperature-triggered grain chalkiness. In developing seeds, high temperature induced the expression of a-amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as a-amylase activity, while it decreased an a-amylase-repressing plant hormone, ABA, suggesting starch to be degraded by a-amylase in developing grains under elevated temperature. Furthermore, RNAi-mediated suppression of a-amylase genes in ripening seeds resulted in fewer chalky grains under high-temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of a-amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.