Brefeldin A (BFA) causes a block in the secretory system of eukaryotic cells by inhibiting vesicle formation at the Golgi apparatus. Although this toxin has been used in many studies, its effects on plant cells are still shrouded in controversy. We have reinvestigated the early responses of plant cells to BFA with novel tools, namely, tobacco Bright Yellow 2 (BY-2) suspension-cultured cells expressing an in vivo green fluorescent protein–Golgi marker, electron microscopy of high-pressure frozen/freeze-substituted cells, and antisera against Atγ-COP, a component of COPI coats, and AtArf1, the GTPase necessary for COPI coat assembly. The first effect of 10 μg/mL BFA on BY-2 cells was to induce in <5 min the complete loss of vesicle-forming Atγ-COP from Golgi cisternae. During the subsequent 15 to 20 min, this block in Golgi-based vesicle formation led to a series of sequential changes in Golgi architecture, the loss of distinct Golgi stacks, and the formation of an endoplasmic reticulum (ER)–Golgi hybrid compartment with stacked domains. These secondary effects appear to depend in part on stabilizing intercisternal filaments and include the continued maturation of cis- and medial cisternae into trans-Golgi cisternae, as predicted by the cisternal progression model, the shedding of trans-Golgi network cisternae, the fusion of individual Golgi cisternae with the ER, and the formation of large ER-Golgi hybrid stacks. Prolonged exposure of the BY-2 cells to BFA led to the transformation of the ER-Golgi hybrid compartment into a sponge-like structure that does not resemble normal ER. Thus, although the initial effects of BFA on plant cells are the same as those described for mammalian cells, the secondary and tertiary effects have drastically different morphological manifestations. These results indicate that, despite a number of similarities in the trafficking machinery with other eukaryotes, there are fundamental differences in the functional architecture and properties of the plant Golgi apparatus that are the cause for the unique responses of the plant secretory pathway to BFA.

A method was designed for in vivo observation of sieve element/companion complexes by using confocal laser scanning microscopy. A leaf attached to an intact fava bean plant was mounted upside down on the stage of a confocal microscope. Two shallow paradermal cortical cuts were made in the major vein. The basal cortical window allowed us to observe the phloem intact. The apical window at 3 cm from the site of observation was used to apply phloem-mobile fluorochromes, which identified living sieve elements at the observation site. In intact sieve tubes, the sieve plates did not present a barrier to mass flow, because the translocation of fluorochromes appeared to be unhindered. Two major occlusion mechanisms were distinguished. In response to intense laser light, the parietal proteins detached from the plasma membrane and formed a network of minute strands and clustered material that aggregated and pressed against the sieve plate. In response to mechanical damage, the evenly distributed P plastids exploded, giving rise to the formation of a massive plug against the sieve plate. In case of mechanical damage, the parietal proteins transformed into elastic threads (strands) that extended throughout the sieve element lumen. Our observations cover the phenomena encountered in previous microscopic and electron microscopic studies and provide a temporal disentanglement of the events giving rise to the confusing mass of structures observed thus far.

Developing pea cotyledons contain functionally different vacuoles, a protein storage vacuole and a lytic vacuole. Lumenal as well as membrane proteins of the protein storage vacuole exit the Golgi apparatus in dense vesicles rather than in clathrin-coated vesicles (CCVs). Although the sorting receptor for vacuolar hydrolases BP-80 is present in CCVs, it is not detectable in dense vesicles. To localize these different vacuolar sorting events in the Golgi, we have compared the distribution of vacuolar storage proteins and of α-TIP, a membrane protein of the protein storage vacuole, with the distribution of the vacuolar sorting receptor BP-80 across the Golgi stack. Analysis of immunogold labeling from cryosections and from high pressure frozen samples has revealed a steep gradient in the distribution of the storage proteins within the Golgi stack. Intense labeling for storage proteins was registered for the cis-cisternae, contrasting with very low labeling for these antigens in the trans-cisternae. The distribution of BP-80 was the reverse, showing a peak in the trans-Golgi network with very low labeling of the cis-cisternae. These results indicate a spatial separation of different vacuolar sorting events in the Golgi apparatus of developing pea cotyledons.