Abstract
The significance of the endosomal sorting complexes required for transport (ESCRT)-III in cereal endosperm has been shown by the identification of the recessive mutant supernumerary aleurone layer1 (SAL1) in maize. ESCRT-III is indispensable in the final membrane fission step during biogenesis of multivesicular bodies (MVBs), responsible for protein sorting to vacuoles and to the cell surface. Here, we annotated barley ESCRT-III members in the (model) crop Hordeum vulgare and show that all identified members are expressed in developing barley endosperm. We used fluorescently tagged core ESCRT-III members HvSNF7a/CHMP4 and HvVPS24/CHMP3 and the associated ESCRT-III component HvVPS60a/CHMP5 for transient localization studies in barley endosperm. In vivo confocal microscopic analyses show that the localization of recombinantly expressed HvSNF7a, HvVPS24 and HvVPS60a differs within barley endosperm. Whereas HvSNF7a induces large agglomerations, HvVPS24 shows mainly cytosolic localization in aleurone and subaleurone. In contrast, HvVPS60a localizes strongly at the plasma membrane in aleurone. In subaleurone, HvVPS60a was found to a lesser extent at the plasma membrane and at vacuolar membranes. These results indicate that the steady-state association of ESCRT-III may be influenced by cell layer-specific protein deposition or trafficking and remodelling of the endomembrane system in endosperm. We show that sorting of an artificially mono-ubiquitinated Arabidopsis plasma membrane protein is inhibited by HvVPS60a in aleurone. The involvement of HvVPS60a in different cell layer-specific trafficking pathways, reflected by localization of HvVPS60a at the plasma membrane in aleurone and at the PSV membrane in subaleurone, is discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00709-015-0798-1) contains supplementary material, which is available to authorized users.
Highlights
In cereals, the endosperm supports the germinating seedling by providing stored nitrogen, carbon and minerals
endosomal sorting complexes required for transport (ESCRT)-III members constitute a family of small coiled coil proteins which are divided into the two subfamilies SNF7VPS20-VPS60 and VPS2-VPS24-VPS46; the number of paralogs for each subunit member varies between organisms (Howard et al 2001; Leung et al 2008; Winter and Hauser 2006)
ESCRT-III is expressed in developing barley endosperm
Summary
The endosperm supports the germinating seedling by providing stored nitrogen, carbon and minerals. SE cells accumulate starch and storage proteins (SP) which are mobilized during seed germination via the release of hydrolytic enzymes by the aleurone layer (Olsen 2004). Both SE cells and aleurone cells contain protein storage vacuoles (PSVs). Seed storage proteins (SSPs) reach their final destination by two main routes: soluble albumins and globulins travel through ER and Golgi to PSVs, whereas most prolamins accumulate in ER-derived protein bodies (PBs). SSP transport routes depend on the cereal species, endosperm tissue layer and developmental timepoint (Ibl and Stoger 2012; Zheng and Wang 2014), and some PBs are deposited in the PSV after bypassing the Golgi. The massive and complex SSP transport in the endosperm is accompanied by extensive reorganization of the endomembrane system during development (Hoh et al 1995; Ibl and Stoger 2012; Wang et al 2010)
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