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 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.

PANOMICS meets germplasm

“…This suggests that differences in protein profiles can provide a useful tool for examining more complex traits and identify novel protein marker for crop improvement. In recent studies, we have combined shotgun proteomics and cell biological studies of barley seeds to unravel the spatio‐temporal expression and subcellular localization of hordoindoline across development in barley endosperm (Ibl et al , 2018; Shabrangy et al , 2018).…”

Protein sorting into protein bodies during barley endosperm development is putatively regulated by cytoskeleton members, MVBs and the HvSNF7s

“…Histological and immunofluorescence studies. At least three randomly selected GP grains were harvested at 6, 12 and ≥20 DAP and fixed, embedded and sectioned as described in 15,16 . The 1.5 µm sections on glass slides were stained with toluidine blue (0.1%) for 30 s at 80 °C on a hot plate and rinsed with distilled water.…”

“…The 1.5 µm sections on glass slides were stained with toluidine blue (0.1%) for 30 s at 80 °C on a hot plate and rinsed with distilled water. Immunofluorescence microscopy of developing barley grains was performed as described by 16 using: polyclonal rabbit anti-V-ATPase antibody (#AS 07 213, Agrisera, Vännäs, Sweden, raised against Arabidopsis thaliana At4g11150, specific for higher plants including Hordeum vulgare), dilution 1:100; polyclonal rabbit anti-actin antibody (#AS 13 2640, Agrisera, Vännäs, Sweden, raised against Arabidopsis thaliana actin-1/-2/-3/-4/-5/-7/-8/-11 and-12, specific for higher plants including Hordeum vulgare), dilution 1:50; polyclonal rabbit anti-tubulin-α antibody (#AS 10 680, Agrisera, Vännäs, Sweden, raised against Arabidopsis thaliana tubulin alpha-1/-2/-4/-5/-6-chain, specific for higher plants including Hordeum vulgare, dilution 1:50); polyclonal rabbit anti-VSR1 antibody, www.nature.com/scientificreports www.nature.com/scientificreports/ OsO 4 for one and a half hours at room temperature followed by washing with buffer. The chemically fixed samples underwent dehydration in an ethanol series (30%, 50%,70%, 95% ethanol for 10 min each, and two times 100% ethanol).…”

“…Data processing and protein identification. Sample preparation for proteomics analyses and Mass spectrometry (MS) was performed as previously described in 16 and in the supplementary material. Raw files were processed with MaxQuant 1.5 (http://www.maxquant.org) and the Andromeda search algorithm [18][19][20] on the barley UniProt database (http://uniprot.org).…”

“…Raw files were processed with MaxQuant 1.5 (http://www.maxquant.org) and the Andromeda search algorithm [18][19][20] on the barley UniProt database (http://uniprot.org). Peptide identification was performed as previously described 16 . Label-free quantification was done at the MS1 level with at least two peptides per protein.…”