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

Cereal 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. During development, protein trafficking and storage require an extensive reorganization of the endomembrane system. Consequently, endomembrane-modifying proteins will influence the final grain quality and yield. However, little is known about the molecular mechanism underlying endomembrane system remodeling during barley grain development. By using label-free quantitative proteomics profiling, we quantified 1,822 proteins across developing barley grains. Based on proteome annotation and a homology search, 94 proteins associated with the endomembrane system were identified that exhibited significant changes in abundance during grain development. Clustering analysis allowed characterization of three different development phases; notably, integration of proteomics data with in situ subcellular microscopic analyses showed a high abundance of cytoskeleton proteins associated with acidified PBs at the early development stages. Moreover, 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 PBs during barley endosperm development. Together our data identified promising targets to be genetically engineered to modulate seed storage protein accumulation that have a growing role in health and nutritional issues.

of total protein extracts of GP grains at 10 and 20 DAP using the polyclonal rabbit anti-SNF7. black asterisks -background band, magenta asterisks -positive signals from hetero-homodimers and different isoforms of HvSNF7. The amount of loaded sample is indicated in µg. Note the stage dependent signal of 10 and 12 DAP. Exposure time of the film was 10 minutes to visualize the weak bands below 25 kDa. After film development, the membrane was stained with Ponceau S. (d) Polyclonal rabbit anti-SNF7 confirms the intact fusion protein p6U::SNF7.1-mEosFP of total proteins extracts of this transgenic line. black asterisk -background band, magenta asterisk -positive signals from p6U::SNF7.1-mEosFP (49 kDa). In total, 22 µg protein was loaded. Note the strong signal from p6U::SNF7.1-mEosFP compared to the signal of GP. Exposure time of the film was 3 minutes. After film development, the membrane was stained with Ponceau S. Western blot is grouped of cropped lines from the same blot, indicated by the dividing lines. Semi-quantitative Western blot analyses with anti-eEF1a and anti-VSR1 of protein extracts of different development stages reveal a constant and an increasing signal, respectively. The signal intensities (peak area density) of the Western blots were quantified by ImageJ. Normalized LFQ intensities of identified HveEF1a (F2EG53) and HvVSR1 peptides (A0A287H7X6, A0A287NZS5, A0A287R1U7) at 6, 10, 12 and ≥ 20 DAP were averaged over three biological replicates. Bars represent standard deviation. (c) Hierarchical-Clustering-Analysis shows that the measured protein abundances were highly reproducible with an average Pearson's correlation coefficients of 0.96 between biological replicates.  (a) Toluidine blue staining of sections prepared at 6, 12 and ≥ 20 DAP. At 6 DAP, cellularization of the barley endosperm is finished and three aleurone cell layers are shown. Toluidine blue-stained compartments (arrows) were more abundant at ≥ 20 DAP compared to 12 and 6 DAP, confirming that the protein level of SSPs increase during development. Note the indicated tissues: aleurone, subaleurone and starchy endosperm. Bars = 100 µm. (b) Single, spherical PBs (asterisks) were observed by TEM in starchy endosperm cells engulfed by putative vacuolar membranes (arrows). Note vesicles (arrowheads) attached to the PBs. ER, endoplasmic reticulum. Bar = 0.5 µm. (c) Data-matrix heat map representing z-score values of 6, 10, 12 and ≥ 20 DAP. Heat map was prepared using Microsoft Excel. Scale: grey = smallest value; blue = 50% quantile; pink = highest value. Identified proteins involved in the accumulation of SSPs were highest at ≥ 20 DAP. Note that all identified SSPs could be found in cluster 3 except F2CYL7; Vicilin-like seed storage protein, that was grouped into cluster 2.

Sample preparation for proteomics analyses
Total proteins were extracted from approximately 20 barley grains (one ear) harvested at 6-8, 10, 12-18 and ≥ 20 DAP in three biological replicates. Stages were chosen as previously described 2 . Barley flowers were not emasculated, and DAP were counted from the first dehiscence of anthers. Extraction was performed following an adapted phenol-phase extraction protocol as described in 3 . Subsequently, proteins were re-suspended in urea buffer (8 M urea, 100 mM ammonium bicarbonate) to measure protein concentration with a Bradford Assay prior protein content normalization and trypsin digestion. Following overnight digestion, peptides were desalted using a C18 solid phase extraction (Agilent Technologies, Santa Clara, USA). After solid-phase extraction, the corresponding eluates were dried in a vacuum concentrator.

Nano-HPLC and Orbitrap Elite tune methods
Mass spectrometry (MS) was performed as previously described 4 . Peptide pellets were resolved at a protein concentration equivalent of 0.1 µg/µL. A total of 0.5 µg of the mixture was separated on an EASY-Spray PepMap RSLC 75 μm × 50 cm column (Thermo Fisher Scientific, Waltham, Massachusetts, USA). After elution using a 150 min linear gradient at a 300 nL/min flow rate generated with an UltiMate 3000 RSLCnano system, the peptides were measured with an LTQ-Orbitrap Elite (Thermo Fisher Scientific, Waltham, Massachusetts, USA) as described in 4 . Mass analyzer settings were as follows: ion transfer capillary temperature 275 °C, full scan range 350-1800 m/z, FTMS resolution 60,000. Each FTMS full scan was followed by up to twenty data-dependent (DDA) CID tandem mass spectra (MS/MS spectra) in the linear triple quadrupole (LTQ) mass analyzer. Dynamic exclusion was enabled using list size 500 m/z values with an exclusion width ±10 ppm for 60 s. Charge state screening was enabled and unassigned and +1 charged ions were excluded from MS/MS acquisitions. For injection control, automatic gain control for full scan acquisition in the Orbitrap was set to 1 x 106 ion population, and the maximum injection time (max IT) was set to 200 ms. Multistage activation was enabled with neural losses of 24.49, 32.66, 48.999, 97.97, 195.94, and 293.91 Da for the 10 most intense precursor ions. Prediction of ion injection time was enabled, and the trap was set to gather 5 x 103 ions for up to 100 ms. Orbitrap online calibration using internal lock mass calibration on m/z 371.10123 from polydimethylcyclosiloxane was used.

Western blots
To confirm the MS results, we have used the same extraction protocol for SDS Page and the Western blots as for the MS analysis. For SDS page, between five and seven wild type (GP) seeds of 6 and 10 DAP, and three seeds of 10, 12 and ≥ 20 DAP were taken for protein extraction. After protein extraction, we measured the quantity of protein with a Bradford assay. 15 ug total protein amount were loaded of each sample and the gel was stained for 30 min by Coomassie and destained overnight. A total of 2 µg of PageRuler Plus Prestained Protein Ladder (#26 619, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was loaded on a 10% acrylamide gel. For the analysis of the transgenic line p6U::SNF7.1-mEosFP, five transgenic and wild type (GP) grains were extracted by PBS buffer pH 6. After protein extraction, we measured the quantity of protein with a Bradford assay. Western blots were performed as previously described 4 . For the analyses of the specificity of anti-V-ATPase, proteins were extracted at 6 DAP as described in 2 and 4 µl was loaded. A total of 2 µg of PageRuler Plus Prestained Protein Ladder (#26 619, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 20 µg of the samples were loaded on a 10% acrylamide gel. Antibodies were diluted as following: polyclonal rabbit anti-V-ATPase antibody (#AS 07 213, Agrisera, Vännäs, Sweden), dilution 1:5000; actin (#AS13 2640, Agrisera, Vännäs, Sweden), 1:5000; tubulin alpha (#AS10 680, Agrisera, Vännäs, Sweden), 1:1000 polyclonal rabbit anti-SNF7 antibody (kindly provided by 1 , 1/1000; and Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey) (#NA934VS, GE Healthcare, Illinois, Chicago, United States), 1/10,000. The ECL prime Western blotting detection reagent (#RPN2232, GE Healthcare, Illinois, Chicago, United States) was used for development.

Phenotyping of barley grains
The weight was measured each time of five GP seeds of three biological replicates at 6, 10, 12 and ≥ 20 DAP. Data was visualized by a boxplot.
Besides bioinformatic analyses, the specificity of the primers was tested by PCR on the cDNAs. At least three biological replicates were used, and three technical replicates were performed for RT-qPCR. For the normalization studies of ESCRT transcripts, we used the following reference genes as described in 2 : ARF (ADP-ribosylation factor), FBPA (fructose-bisphosphate aldolase), and SAM (S-adenosyl-Lmethionine) for whole grains. Normalization was calculated as described in 2,6 . For statistical analyses we performed a Student's t-test (two-tailed distribution, two-sample unequal variance (heteroscedastic)) using the Microsoft Excel software program.