The processes by which soluble proteins are sorted from the secretory pathway to the vacuolar compartments in plant cells are poorly understood. In contrast to receptormediated sorting of lysosomal proteins in mammalian cells, where the sorting determinant is a Man-6-P residue added to Asn-linked oligosaccharides (Kornfeld, 1992), plant vacuolar sorting is determined by sequences within the polypeptides themselves (Bednarek and Raikhel, 1991;Matsuoka and Nakamura, 1991;Neuhaus et al., 1991;Saalbach et al., 1991;Holwerda et al., 1992). A similar strategy is used in yeast, where the tetrapeptide QRPL within the . '

We identify new organelles associated with the vacuolar system in plant cells. These organelles are defined biochemically by their internal content of three integral membrane proteins: a chimeric reporter protein that moves there directly from the ER; a specific tonoplast intrinsic protein; and a novel receptor-like RING-H2 protein that traffics through the Golgi apparatus. Highly conserved homologues of the latter are expressed in animal cells. In a developmentally regulated manner, the organelles are taken up into vacuoles where, in seed protein storage vacuoles, they form a membrane-containing crystalloid. The uptake and preservation of the contents of these organelles in vacuoles represents a unique mechanism for compartmentalization of protein and lipid for storage.

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SummaryCaleosin is a Ca 2þ -binding oil-body surface protein. To assess its role in the degradation of oil-bodies, two independent insertion mutants lacking caleosin were studied. Both mutants demonstrated significant delay of breakdown of the 20:1 storage lipid at 48 and 60 h of germination. Additionally, although germination rates for seeds were not affected by the mutations, mutant seedlings grew more slowly than wild type when measured at 48 h of germination, a defect that was corrected with continued growth for 72 and 96 h in the light. After 48 h of germination, wild-type central vacuoles had smooth contours and demonstrated internalization of oil bodies and of membrane containing a-and d-tonoplast intrinsic proteins (TIPs), markers for protein storage vacuoles. In contrast, mutant central vacuoles had distorted limiting membranes displaying domains with clumps of the two TIPs, and they contained fewer oil bodies. Thus, during germination caleosin plays a role in the degradation of storage lipid in oil bodies. Its role involves both the normal modification of storage vacuole membrane and the interaction of oil bodies with vacuoles. The results indicate that interaction of oil bodies with vacuoles is one mechanism that contributes to the degradation of storage lipid.

Prevacuolar compartments (PVCs) are membrane-bound organelles that mediate protein traffic between Golgi and vacuoles in the plant secretory pathway. Here we identify and define organelles as the lytic prevacuolar compartments in pea and tobacco cells using confocal immunofluorescence. We use five different antibodies specific for a vacuolar sorting receptor (VSR) BP-80 and its homologs to detect the location of VSR proteins. In addition, we use well-established Golgi-markers to identify Golgi organelles. We further compare VSR-labeled organelles to Golgi organelles so that the relative proportion of VSR proteins in Golgi vs. PVCs can be quantitated. More than 90% of the BP-80-marked organelles are separate from Golgi organelles; thus, BP-80 and its homologs are predominantly concentrated on the lytic PVCs. Additionally, organelles marked by anti-AtPep12p (AtSYP21p) and anti-AtELP antibodies are also largely separate from Golgi apparatus, whereas VSR and AtPep12p (AtSYP21p) were largely colocalized. We have thus demonstrated in plant cells that VSR proteins are predominantly present in the lytic PVCs and have provided additional markers for defining plant PVCs using confocal immunofluorescence. Additionally, our approach will provide a rapid comparison between markers to quantitate protein distribution among various organelles.

The mechanisms by which cis-acting hormone response elements affect transcription is unclear. In this study, we demonstrated that a second "coupling element," identified as O2S, must be present to allow a single copy of either the gibberellin response element (GARE) or the abscisic acid response element (ABRE) to mediate their hormonal effects in the barley Amy32b alpha-amylase gene promoter. The interactive effects of the O2S and the GARE are constrained positionally and spatially; thus, together they form a gibberellin response complex (GARC). The absolute requirement of the O2S for function of the ABRE demonstrates that these together form an abscisic acid response complex (ABRC). A second copy of the GARE can substitute for the O2S in the GARC, but only in one orientation. By expressing the GARC-containing and ABRC-containing promoters in developing aleurone tissue, we showed that hormonal effects prevent alpha-amylase gene expression during the second half of grain development, but other mechanisms suppress expression earlier. Our results suggest that the specific sequence serving as a coupling element in a given gene promoter will greatly affect where and when the GARE or ABRE will be able to regulate transcription.

The Amy32b gene is a representative member of a closely related family of a-amylase genes expressed under hormonal control in aleurone layers of barley grains. Transcription of this gene is induced by gibberellin (GA) and suppressed by abscisic acid. In this study, we functionally defined the promoter elements of the Amy32b gene that govern the developmental and hormonal control of its expression in aleurone. Two functionally distinct yet physically associated elements are essential: a gibberellin response element mediates regulation by GA and abscisic acid, and an Opaque-2 binding sequence (02s) is thought to interact with a barley homolog of the maize endosperm-specific transcriptional regulator Opaque-2. An additional element CCTTTT, which with the 0 2 s forms part of a canonical =endosperm box," is important in modulating the absolute leve1 of expression of the Amy32b promoter, as is another separate, highly consenred element TATCCATGCAGTG.

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