Table_2_Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.XLSX

<p>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 to understand 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, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 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 indicates 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 cereal-based 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.</p

Table_1_Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.XLSX

<p>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 to understand 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, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 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 indicates 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 cereal-based 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.</p

Image_2_Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.TIF

<p>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 to understand 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, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 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 indicates 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 cereal-based 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.</p

Purification of ELPylated hemagglutinin (H5-ELP) from transgenic leaves and seeds by membrane-based ITC.

<p>ELPylated hemagglutinins from leaves (A) and seeds (B) were purified by the standard or improved mITC methods (C) described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099347#s2" target="_blank">Materials and Methods</a> section. Proteins in the raw plant extract (RE), in the supernatant after passage through a 0.2 µm cellulose acetate membrane (Sm) and in the eluent (Pm) were collected during the mITC purification process and separated by 10% SDS-PAGE. Recombinant proteins were then detected using Coomassie Brilliant Blue staining (left) or an anti-c-myc monoclonal antibody (right).</p

Localization of hemagglutinin-ELP fusions in tobacco seeds.

<p>A, B. Fluorescence microscopy.C, D. Electron microscopy.A, C. Endosperm. B, D. Embryo. Note the ELP bodies (arrowheads, A, B) and those that are loosely packed (arrowheads, C, D). Cell wall (cw), oil bodies (ob), protein storage vacuole (arrows), nucleus (n). Bars 50 µm (A, B), 1 µm (C, D).</p

Glycosylation profile of recombinant HAs from leaves and seeds.

<p>Man_{6, 7, 8}: Mannose _{6, 7, 8}; Gn: <i>N</i>-acetylglucosamine; X: Xylose; F: Fucose.</p

Localization of hemagglutinin-hydrophobin I fusions in tobacco seeds.

<p>A. Fluorescence microscopy. B, C. Electron microscopy. B. Endosperm. C. Embryo. Scarce hydrophobin bodies in the endosperm (arrowheads, A, B). Abundant hydrophobin bodies in the embryo cells (arrowheads, A, C). Hydrophobin bodies show non-uniform electron density (*, C). Endosperm (end), embryo (emb), protein storage vacuole (PSV), ribosomes (arrow). Bars 25 µm (A), 0.5 µm (B, C).</p

Immunofluorescence analysis of recombinant HAs in plant leaves.

<p>Leaves were fixed, embedded in PEG and sectioned. Recombinant HAs were immunodecorated with an anti-c-myc monoclonal antibody followed by incubation with secondary antibody (anti-mouse-IgG conjugated with AlexaFluor488) and counterstaining with DAPI. A. H5; B. H5-HFBI; C. H5-ELP; D. wild type. Bars represent 50 µm.</p

Western blot analysis of purified HAs treated/untreated with PNGase F.

<p>Purified HAs from leaves were deglycosylated using the commercial PNGase F enzyme described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099347#s2" target="_blank">Materials and Methods</a> section. PNGase F-treated and untreated proteins were then separated in 10% SDS-PAGE. Recombinant proteins were detected using an anti-c-myc monoclonal antibody. “−” and “+” indicate PNGase F-untreated and treated samples, respectively.</p

Influence of hydrophobin and elastin-like polypeptide on hemagglutinin accumulation in transgenic plants under the control of either the CaMV 35S promoter (A) or the seed-specific USP promoter (B).

<p>Each column shows the mean value of expression level of the transgenic plants, and the error bars indicate the standard deviation. TSP: total soluble protein.</p