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  • ceplas/Wrobel-2023-CastorBeanEndospermProteome
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# meeting notes
## 11.04.2022
### orig idea
- new transporter in peroxisome membrane
- proteome sequence all organelle fractions
- confirm peroxisomal location
### special issue
- cooperation multiple organelles in cell
- degradation of ricinus storage amongst organelles (- cytosol missing)
- Special issue “Photosynthetic and photorespiratory organelles: metabolism, dynamics, and signaling” Frontiers of Plant Science
### sfb
- associate fractions with marker proteins (based on publications)
- project workflow
### TODO: upload data to PRIDE
- swate template
-
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# Publication
downloaded from https://doi.org/10.3389/fpls.2023.1182105
## Citation
Wrobel TJ, Brilhaus D, Stefanski A, Stühler K, Weber APM, Linka N. Mapping the castor bean endosperm proteome revealed a metabolic interaction between plastid, mitochondria, and peroxisomes to optimize seedling growth. Front Plant Sci. 2023 Oct 6;14:1182105. doi: 10.3389/fpls.2023.1182105. PMID: 37868318; PMCID: PMC10588648.
## License
Copyright © 2023 Wrobel, Brilhaus, Stefanski, Stühler, Weber and Linka
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
## Supplementary material
https://www.frontiersin.org/articles/10.3389/fpls.2023.1182105/full#supplementary-material
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# Plant growth conditions
Dry seeds of *Ricinus communis var. zanzibariensis* were surface-sterilized in a solution of 0.1% (w/v) 8-quinolinol in water for ten minutes and soaked in running tap water over night for seed imbibition. The imbibed seeds were placed on moist vermiculite and incubated at 30°C in the dark {Beevers:1974ug}.
# Preparation of organelles from castor bean endosperm
The isolation of castor bean endosperm was performed according to Cooper & Beevers (1969) and Beevers & Breidenbach (1974) {Cooper:1969tz} {Beevers:1974ug}. The protocols were modified by using the grinding buffer as described by Reumann et al. (2007) {Reumann:2007wi}. All steps were carried out on ice in a cold room (4°C) unless indicated otherwise. 30 g of endosperm tissue from 5-day old dark-grown Ricinus seedlings was harvested by removing the yellow cotyledons using the blunt side of a scalpel blade. The resulting endosperm was chopped using an onion chopper in 60 mL grinding buffer (170 mM Tricine pH X (KOH), 1 M Sucrose, 1% (w/v) BSA, 10 mM KCl, 1 mM MgCl2, 2 mM EDTA, 0,5 % (w/v) PVP-40, and 5 mM DTT). The suspension was further homogenized using mortar and pestle. The homogenate was filtered through four layers cheesecloth. The crude extract was centrifuged at 1,200 g for 10 minutes to remove cell debris. The supernatant was carefully decanted into a new flasks (approx. 40 mL). To separate the organelles, 6 mL of the obtained extract was loaded onto the top of a discontinuous sucrose gradient prepared in 20 mM Tricine-KOH (pH 7.5) and 1 mM EDTA. The density gradient consists of the following sucrose steps (from top to bottom): 5 ml 30% (w/w) sucrose, 3 ml 44% (w/w) sucrose, 5 ml 48% (w/w) sucrose, 5 ml 49% (w/w) sucrose, 1 ml 50% (w/w) sucrose, 2 ml 54% (w/w) sucrose, and 2 ml 60% (w/w) sucrose. The organelles were separated by ultracentrifugation at 105,026 g using a swing-out rotor for 3 hours. Four visible bands at the interface 30% − 44% (w/w) sucrose solution (fraction 1), 44% − 48% (w/w) sucrose solution (fraction 2), 48% − 49% (w/w) sucrose solution (fraction 3), and 50% − 54% (w/w) sucrose solution (fraction 4) was carefully collected, pooled and stored at -80°C for further experiments.
# Isolation of organellar membranes
Membranes were isolated from the collected organellar fractions (F1-4) as described by Fujiki et al. (1982) and Reumann et al. (1995) with modifications {Fujiki:1982gt} {Reumann:1995wh}. The obtained fractions were slowly diluted with hypo-osmotic buffer (20 mM HEPES-KOH, pH 6.8 and 0.8 mM MgCl2) and incubated on ice for 30 minutes to osmotically disrupt the organelles. The suspension was subjected to 10 freeze/thaw cycles. (freezing in liquid nitrogen, thawing at room temperature) to lyse efficiently the organelles. After each thaw cycle the sample was homogenized by vortexing. Membranes were sedimented by centrifugation at 100,000 g at 4°C for 1 h and washed in 100 mM sodium carbonate (pH 11.5) to remove membrane-associated proteins. A second centrifugation step (100,000 g at 4°C for 1 h) was performed to harvest the organellar membranes. The membrane pellet was resuspended in 20 mM HEPES-KOH, pH 6.8 and 0.8 mM MgCl2 and stored at -80°C for further experiments. Concentration of the membrane proteins were determined using Pierce BCA protein assay kit (ThermoFisher Scientific).
# Proteome acquisition via mass spectrometry analysis
Total proteins and the enriched membrane proteins of the organellar fractions (F1-4) isolated from Ricinus endosperm tissue were analyzed by mass spectrometry (MS). Therefore, protein samples were loaded on an SDS-polyacrylamide gel, concentrated in the stacking gel, silver stained according to MS-compatible protocol, reduced, alkylated and digested with trypsin. Peptides were extracted from the gel with 0.1% trifluoroacetic acid and subjected to liquid chromatography. For peptide separation over a 140 minutes LC-gradient an Ultimate 3000 Rapid Separation liquid chromatography system (Dionex; ThermoFisher Scientific) equipped with an Acclaim PepMap 100 C18 column (75 μm inner diameter x 50 cm length x 2 mm particle size from ThermoFisher Scientific) was used. Mass spectrometry was carried out on an Obitrap Elite high-resolution instrument (ThermoFisher Scientific) operated in positive mode and equipped with a Nano electrospray ionization source. Capillary temperature was set to 275°C and source voltage to 1.5 kV. Survey scans were conducted in the orbitrap analyzer at a mass to charge (m/z) ranging from 350-1700 and a resolution of 60,000 (at 400 m/z). The target value for the automatic gain control was 1,000,000 and the maximum fill time 200 ms. The 20 most intense doubly and triply charged peptide ions (minimal signal intensity 500) were isolated, transferred to the linear ion trap (LTQ) part of the instrument and fragmented using collision induced dissociation (CID). Peptide fragments were analyzed using a maximal fill time of 200 ms and automatic gain control target value of 100,000. The available mass range was 200- 2000 m/z at a resolution of 5400 (at 400 m/z). Two fragment spectra were summed up and already fragmented ions excluded from fragmentation for 45 seconds.
# Computational MS data analysis
For peptide and protein identification the acquired MS spectra were analyzed using the MaxQuant version 1.3.0.5 (MPI for Biochemistry, Planegg, Germany) with default parameters {Cox:2008ir}. Quantification was performed using the unlabeled quantification option of MaxQuant. The identified spectra were matched against the Ricinus proteome using the peptide search engine Andromeda {Cox:2011eg}. Only proteins containing at least two unique peptides and a minimum of three valid values in at least one group were quantified. A full list of all identified peptides from the proteome experiment is presented in supplemental Table S1. The raw files and identified spectra were submitted to ProteomeXChange (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository and can be accessed under the identifier PXDXXXXXX.
All identified Ricinus proteins were analyzed by bidirectional BLAST against the Arabidopsis proteome {Altschul:1990vt}. Organelle distribution within the collected fractions was assayed using a set of marker proteins. Proteins were assigned as organelle markers if the experimental localization of their Arabidopsis homologues in the SUBA 4.0 database {Hooper:2017cf} corresponded with their sequence-based localization prediction in Ricinus. We predicted protein localization to peroxisomes, mitochondria, and plastids manually and using the publicly available tools PPero, PredPlantPTS1, and TargetP (Emanuelsson et al. 2000; Reumann et al., 2012; Wang et al. 2017).
## FAME-analysis
![fpls-14-1182105-g002.jpg](dataset/fpls-14-1182105-g002.jpg)
**Figure 2**
Storage oil mobilization in castor bean endosperm. (A) Relative fresh weight of the endosperm in dry seeds (-1d), imbibed seeds (0d) and 1- to 7-day old dark-grown castor bean seedlings (1-7d) as percentage of fresh weight of the whole plant. Data represent arithmetic means ± SD of 3 biological replicates. (B) Relative ricinoleic acid content of the endosperm from seeds and seedlings. Samples were normalized to the average fresh weight of on endosperm and expressed as percentages of their initial quantities determined in dry seeds (-1d). Error bars show standard deviations of the means of at least three biological replicates.
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