Cell layer-specific distribution of transiently expressed barley ESCRT-III component HvVPS60 in developing barley endosperm

Hilscher, Julia; Kapusi, Eszter; Stöger, Eva; Ibl, Verena

doi:10.1007/s00709-015-0798-1

Cited by 7 publications

(12 citation statements)

References 88 publications

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“…Recently, the overexpressed ESCRT-III-associated component HvVPS60 was shown to be involved in protein targeting in developing barley endosperm (Hilscher et al, 2016). Here, seven of the identified ESCRT proteins were most expressed at development stage I and II, indicating a possible involvement in MVB body formation.…”

Section: How Do Escrts and Mvbs Contribute To Pb Formation?mentioning

confidence: 77%

“…The backbone of all vectors (MKK4_SPYCE and MPK3_SPYNE, kindly provided by Dr. Andrea Pitzschke) used in this study contain a p35S promoter (Cauliflower Mosaic virus 35S promoter), 5´UTR (untranslated region from tobacco etch virus), our genes of interest (Hvsnf7.1), C-terminal or N-terminal sequence of YFP (SPYCE or SPYNE, t35 (Cauliflower Mosaic virus 35S terminator), HA-tag (Human influenza hemagglutinin) or c-MYC-tag, and a kanamycin antibiotic resistance sequence. HvSNF7.1 (according to HvSNF7a.1 described in (Hilscher et al, 2016) was cloned into the vector pCR2.1 (#K200001, Thermo Fisher Scientific, Massachusetts, USA) using HvSNF7.1_NcoI-F and HvSNF7.1_NotI-R as primers, digested by NcoI and NotI and inserted into previously digested MKK4_SPYCE and MPK3_SPYNE, respectively. To obtain a pSPYCE vector without insert for control reactions, plasmids were cut (NcoI/NotI), blunted using Klenow fragment and religated.…”

Section: Cloning Of Constructsmentioning

confidence: 99%

“…p6U::SNF7.1-mEosFP. To obtain HvSNF7-mEosFP driven by the barley hordein D promoter (p6U) and ended by the nopaline synthase terminator, HvSNF7.1-mEosFP (previously described in (Hilscher et al, 2016) was ligated in the following into the vector p6U_pHordeinD (kindly provided by Eszter Kapusi, unpublished) using MluI. The p6U vector backbone originates from DNA Cloning Service e.K.…”

Section: Cloning Of Constructsmentioning

confidence: 99%

“…In barley, microscopic analyses revealed that spherical PSVs, which are stable in the aleurone during development, underwent a dynamic rearrangement including fusion, rupture and degeneration in the subaleurone and starchy endosperm (Cameron-Mills, 1980;Hilscher et al, 2016). Between 8 and 12 DAP, PSVs reduced their size and even degenerated in the starchy endosperm .…”

mentioning

confidence: 99%

“…Recent bioinformatic, proteomic and RT-qPCR analyses shed the first light on proteins possibly involved in the rearrangement of the endomembrane system in barley endosperm (Hilscher et al, 2016;Roustan et al, 2018;Shabrangy et al, 2018): ESCRT-III proteins were identified by a bioinformatic assay (Hilscher et al, 2016) and localization studies of recombinantly expressed HvSNF7a, HvVPS24 and HvVPS60a ESCRT III components in barley revealed different localization of these proteins within the endosperm (Hilscher et al, 2016). It was further suggested that the steady-state association of ESCRT-III may be influenced by cell layer-specific protein deposition or trafficking and remodeling of the endomembrane system in the endosperm (Hilscher et al, 2016). Additionally, the ER was identified to be most abundant in the starchy endosperm and ER rearrangements were characterized, including relocalization of HvPDIL1-1 during development (Roustan et al, 2018).…”

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confidence: 99%

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Cytoskeleton members, MVBs and the ESCRT-III HvSNF7s are putative key players for protein sorting into protein bodies during barley endosperm development

Roustan

Hilscher

Weidinger

et al. 2019

Preprint

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Twitter: @VerenaIbl m: 4; f: 7 Abbreviations AMSH1, associated molecule with the SH3 domain of Stam1; A. AbstractCereal endosperm is a short-lived tissue adapted for nutrient storage, containing specialized organelles, such as protein bodies (PBs) and protein storage vacuoles (PSVs), for the accumulation of storage proteins. PBs can be used as efficient biotechnological systems to produce high yields of stable recombinant proteins. During development, protein trafficking and storage require an extensive reorganization of the endomembrane system. Consequently, endomembrane-modifying proteins will influence the final grain quality, yield and recombinant protein production. Barley, a cereal crop of worldwide importance for the brewing industry, animal feed and to a lesser extent, human nutrition, has been identified as promising candidate for recombinant protein production. However, little is known about the molecular mechanism underlying endomembrane system remodeling during barley grain development. By using in vivo label-free quantitative proteomics profiling, we quantified 1,822 proteins across developing barley grains. Based on proteome annotation and a homology search, 95 proteins associated with the endomembrane system were identified, and 83 of these exhibited significant changes in abundance during grain development. Clustering analysis allowed characterization of three different development stages; notably, integration of proteomics data with in situ subcellular microscopic analyses showed a high abundance of cytoskeleton proteins associated with acidified protein bodies at the early development stages. Endosomal sorting complex required for transport (ESCRT)-related proteins and their transcripts are most abundant at early and mid-development. Specifically, multivesicular bodies (MVBs), and the ESCRT-III HvSNF7 proteins are associated with protein bodies (PBs) during barley endosperm development. Taken together, our proteomics results specifically identified members of the cytoskeleton, MVBs, and ESCRT as putative key players for protein sorting into PBs during barley endosperm development. These results present a comprehensive overview of proteins involved in the rearrangement of the endomembrane system during barley early grain development and will provide the basis for future work on engineering the endomembrane system to optimize nutrient content and to produce high yields of recombinant proteins.

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Section: How Do Escrts and Mvbs Contribute To Pb Formation?mentioning

confidence: 77%

Section: Cloning Of Constructsmentioning

confidence: 99%

Section: Cloning Of Constructsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Cytoskeleton members, MVBs and the ESCRT-III HvSNF7s are putative key players for protein sorting into protein bodies during barley endosperm development

Roustan

Hilscher

Weidinger

et al. 2019

Preprint

Self Cite

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Immunolocalization of hordein synthesis and transport in developing barley endosperm

Tanner,

van de Meene,

Bacic

2024

Plant Direct

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The spatial accumulation of hordeins in the developing endosperm of barley grains was examined by immunofluorescence microscopy (immunolight microscopy [iLM]) and immunoelectron microscopy (iEM) to establish the timing and subcellular pattern of hordein synthesis and deposition. The pattern seen for hordeins was compared to other abundant grain proteins, such as serpin Z4 and lipid transfer protein 1 (LTP1). Hordein accumulates throughout grain development, from 6 to 37 days post‐anthesis (DPA). In contrast, serpin Z4 was present at 6 DPA, but the greatest synthesis and accumulation occurred during the middle of seed development, from 15 to 30 DPA. LTP1 accumulated later in seed development, from 15 to 30 DPA. Hordeins accumulated within the lumen of the endoplasmic reticulum (ER), were exocytosed from the ER membrane, and accumulated in protein bodies, which then fused either with the protein storage vacuoles or with other protein bodies, which also later fused with the protein storage vacuoles. iEM showed hordein, and LTP1 appeared not to traverse the Golgi apparatus (GA). Hordein, LTP1, and serpin Z4 colocalized to the same protein bodies and were co‐transported to the protein storage vacuole in the same protein bodies. It is likely that this represents a general transport mechanism common to storage proteins in developing grains.

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ESCRTing in cereals: still a long way to go

Ibl

2019

Sci. China Life Sci.

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Cell layer-specific distribution of transiently expressed barley ESCRT-III component HvVPS60 in developing barley endosperm

Cited by 7 publications

References 88 publications

Cytoskeleton members, MVBs and the ESCRT-III HvSNF7s are putative key players for protein sorting into protein bodies during barley endosperm development

Cytoskeleton members, MVBs and the ESCRT-III HvSNF7s are putative key players for protein sorting into protein bodies during barley endosperm development

Immunolocalization of hordein synthesis and transport in developing barley endosperm

ESCRTing in cereals: still a long way to go

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