Dataset Information

Using RT-qPCR, proteomics and microscopy to unravel the spatio-temporal expression and subcellular localization of hordoindolines across development in barley endosperm

ABSTRACT: Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1 and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness, is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required tounderstand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four ((6 days after pollination (dap), 10 dap, 12 dap and ≥ 20 dap)) as well as from aleurone, subaleurone and starchy endosperm at two (12 dap and ≥ 20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥ 20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicats a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the high end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Hordeum Vulgare (barley)

TISSUE(S): Aleurone Layer, Endosperm

SUBMITTER: Valentin Roustan

LAB HEAD: Verena Ibl

PROVIDER: PXD009708 | Pride | 2018-07-02

REPOSITORIES: Pride

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Publications

Microscopic and Proteomic Analysis of Dissected Developing Barley Endosperm Layers Reveals the Starchy Endosperm as Prominent Storage Tissue for ER-Derived Hordeins Alongside the Accumulation of Barley Protein Disulfide Isomerase (HvPDIL1-1).

Roustan Valentin V Roustan Pierre-Jean PJ Weidinger Marieluise M Reipert Siegfried S Kapusi Eszter E Shabrangy Azita A Stoger Eva E Weckwerth Wolfram W Ibl Verena V

Frontiers in plant science 20180910

Barley (<i>Hordeum vulgare</i>) is one of the major food sources for humans and forage sources for animal livestock. The average grain protein content (GPC) of barley ranges between 8 and 12%. Barley hordeins (i.e., prolamins) account for more than 50% of GPC in mature seeds and are important for both grain and flour quality. Barley endosperm is structured into three distinct cell layers: the starchy endosperm, which acts essentially as storage tissue for starch; the subaleurone, which is charac ...[more]

PMID: 30250475

Publication: 1/2

Similar Datasets

Project description:Barley (Hordeum vulgare) is one of the major food sources for humans and forage source for animal livestock. Barley endosperm is structured into three distinct cell layers: the starchy endosperm, which acts essentially as storage tissue for starch, the subaleurone, which is characterized by a high accumulation of endoplasmic reticulum (ER)-derived seed storage proteins (SSP) and finally the aleurone beside the seed coat with a prominent role during seed germination. Prolamins account for more than 50% of the total protein amount in mature seeds. Together with other seed storage proteins (SSPs) they are important for both grain quality and flour quality. Prolamins are synthesized on the rough ER, translocated into the ER lumen and accumulate in distinct, ER-derived protein bodies (PBs) that are most abundant in the SE. PB formation is regulated by the protein disulfide isomerase (PDI) that is involved in the disulfide transfer pathway. Here, we used laser microdisection (LMD) to characterize spatio-temporal molecular and morphological differences of the ER during barley endosperm development. We revealed by nanoLC-MS/MS proteomic analyses performed on whole seeds and collected tissues at different seed development stages that the protein level of the protein disulfide isomerase HvPDIL1-1 is spatio-temporally regulated in developing barley endosperm. Our microscopic studies showed that HvPDIL1-1 preferentially accumulates in SE, especially at 12 days after pollination (dap). HvPDIL1-1 re-localized from PBs to the protein matrix at the periphery of starch granules along grain filling process. Detailed analysis of SE proteome dynamics identified clusters of proteins with similar expression pattern as HvPDIL1-1, which were analysed in a protein-protein network. It revealed a strong functional interconnection between transcription and translation, protein folding and amino acid synthesis with sucrose and starch metabolism. Our data indicate a role of HvPDIL1-1 in the coordination of protein synthesis and prolamins deposition during grain filling processes in developing barley endosperm. These results are discussed in relation to the putative role of HvPDIL1-1 for cereal food end-product quality and recombinant protein production in cereal seeds.

Project description:Abstract Barley is a cereal crop of worldwide importance for brewing industry, animal and in lesser extent humannutrition. Grain development is a tightly controlled process which predominantly contributes to the final grain quality. To understand molecular changes occurring in the early seed development stage up to the initial storage phase, proteome profiling of developing barley seed at 8, 10 18 and 20 days after flowering was analyzed. In vivo label free quantification approach allowed the quantification of more than 1200 proteins across the different stages. Confirmation of the reproducibility of the developmental data obtained in our proteomics analysis was performed using western-blot technics to validate the specific pattern of selected proteins and comparison with previously published data. Multi- and uni-variates statistics were used to unravel the biochemical dynamics underlying the grain filling process. Seed storage proteins (SSPs) were found to accumulate during the grain filling process suggesting a higher activity of the endoplasmic reticulum playing a major role in protein synthesis. This hypothesis was supported by a spatio-temporal analysis of the ER marker protein-disulfide-isomerase (PDI) at both transcript and protein level coupled with microscopic study of the ER dynamics. Results indicates that ER is predominantly active in the starchy endosperm up to 18 DAP and is subsequently degraded while the cells undergo program cell death. The central role of the ER was further strengthening by the analysis of all ER related proteins. Similarly, the pattern of vacuolar H+ ATPases suggested a decrease of the vacuolar acidity, microscopic analysis of the endosperm unravel a concomitant decrease of both vacuolar size and acidity associated with an abundance switch in different class of vacuolar annotated proteins (eg: Sucrose related metabolism proteins). Observed endomembrane system dynamics were supported by specific abundance changes of ESCRT related proteins such as VPS4 and SNF7 or VPS5 a protein associated to the retromer complex. Collectively, our results provide insight into endomembrane molecular processes taking place during barley early grain development, additionally the proteome dataset provide a comprehensive overview of a wide range of metabolic pathway which will be helpful for the cereal community.

Project description:Heat stress occurrence during endosperm development and seed filling forms chalky portion in the limited zone of starchy endosperm of rice grains. In this study, isolation of aleurone, dorsal, central and lateral tissues of developing endosperm by laser-microdissection (LM) coupled with gene expression analysis of 44K microarray was performed to identify key regulatory genes involved in the formation of milky-white (MW) and white-back (WB) chalky grains during heat stress. Gene regulatory network analysis classified the genes changed under heat stress into five modules. the modules of genes changed in developing starchy endosperm corresponding to MW and WB portion under heat stress suggested different regulatory genes involved in each type of grain chalk. The most distinct expression pattern was observed in a M1 and M2 modules where most of the small heat shock proteins and cellular organisation genes being upregulated under heat stress in dorsal aleurone cells and dorsal starchy endosperm zones The histological observation supported the significant increase in cell number and size of dorsal aleurone cells in WB grains. With regard to the central zone of starchy endosperm, preferential downregulation of high molecular weight heat shock proteins (HMW HSPs) including a prominent member encoding for endoplasmic reticulum (ER) chaperones by heat stress were observed, while expression of starch-biosynthesis genes remained unaffected. Characterization of transgenic plants supressing endosperm lumenal binding protein (BiP1), an ER chaperone preferentially downregulated at MW portion under heat stress, showed an evidence of forming the chalky grains without disturbing the expression of starch-biosynthesis genes. The present LM-based comprehensive expression analyses provides novel inferences that HMW HSPs play important role in controlling the redox, nitrogen and amino aicd metabolism in endosperm leading to the formation of MW and WB chalky grains.Keywords ; developing endosperm, gene expression, grain chalkiness, heat stress, laser-microdissection, Oryza sativa.

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