Abstract
Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination.
Highlights
Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation
Most of the studies on the roles of vacuolar sorting receptor (VSR) in plants have been focused on vegetative cells and developing seeds, but little is known about the possible roles of VSRs during seed germination
As a first step to study the functional roles of VSRs during seed germination, we study the subcellular localization of VSR proteins via both confocal immunofluorescence and immunogold electron microscopy (EM) with VSRat-1 antibodies in germinating mung bean seeds
Summary
Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. Both storage proteins and proteases are transported to the same PSVs during seed development, but proteases remain inactive due to the presence of inhibition factors, which are removed upon seed germination and lead to activation of stored proteases and subsequent protein degradation within PSVs (Muntz et al, 2001). Transport of soluble proteins to lytic vacuoles in plant cells is a receptor-mediated process involving a protein family termed vacuolar sorting receptors (VSRs; Neuhaus and Rogers, 1998; Neuhaus and Paris, 2005). Research to date indicates that most VSRs are concentrated on PVCs (Li et al, 2002; daSilva et al, 2005; Miao et al, 2006), even though a proteomic analysis points to the possibility of VSR isoforms located to the endoplasmic reticulum (ER) and/or Golgi apparatus in Arabidopsis (Arabidopsis thaliana) cultured cells (Dunkley et al, 2006)
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