Localization of myo-inositol-1-phosphate synthase to the endosperm in developing seeds of Arabidopsis.

Expression and localization of myo-inositol-1-phosphate synthase (MIPS) in developing seeds of Arabidopsis thaliana was investigated. MIPS is an essential enzyme for production of inositol and inositol phosphates via its circularization of glucose-6-phosphate as the initial step. myo-inositol-6-phosphate (InsP(6) or phytic acid) is the predominant form of phosphorus found in seeds and accumulates as a consequence of MIPS action. Three MIPS genes have been identified in Arabidopsis, all of which were expressed not only in siliques but in both leaves and roots. Immunoelectron microscopy using a MIPS antibody showed that MIPS localizes to the cytosol primarily in the endosperm during seed development and not in the embryo. This is consistent with results obtained using fluorescent microscopy and western blot analysis that showed a similar pattern of localization. However, InsP(6), which is the final product of inositol phosphate metabolism, was present mainly in the embryo. This suggests that a complex interaction between the endosperm and embryo occurs during the synthesis and subsequent accumulation of InsP(6) in developing seeds of Arabidopsis.

Vacuolar processing enzyme: an executor of plant cell death.

Apoptotic cell death in animals is regulated by cysteine proteinases called caspases. Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE deficiency prevents cell death during hypersensitive response and cell death of limited cell layers at the early stage of embryogenesis. Because plants do not have macrophages, dying cells must degrade their materials by themselves. VPE plays an essential role in the regulation of the lytic system of plants during the processes of defense and development. VPE is localized in the vacuoles, unlike animal caspases, which are localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.

A vacuolar processing enzyme, deltaVPE, is involved in seed coat formation at the early stage of seed development.

Vacuolar processing enzyme (VPE) is a Cys proteinase responsible for the maturation of vacuolar proteins. Arabidopsis thaliana deltaVPE, which was recently found in the database, was specifically and transiently expressed in two cell layers of the seed coat (ii2 and ii3) at an early stage of seed development. At this stage, cell death accompanying cell shrinkage occurs in the ii2 layer followed by cell death in the ii3 layer. In a deltaVPE-deficient mutant, cell death of the two layers of the seed coat was delayed. Immunocytochemical analysis localized deltaVPE to electron-dense structures inside and outside the walls of seed coat cells that undergo cell death. Interestingly, deltaVPE in the precipitate fraction from young siliques exhibits caspase-1-like activity, which has been detected in various types of plant cell death. Our results suggest that, at the early stage of seed development, deltaVPE is involved in cell death of limited cell layers, the purpose of which is to form a seed coat.

Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana.

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.