assays/2_2-FAME-analysis/README.md

+
+## 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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assays/2_2-FAME-analysis/protocols/FAME-analysis.md

+# 2.2 Fatty acid methylester (FAME) analysis
+
+The ricinoleic acid content in the castor bean endosperm was determined using gas chromatography-mass spectrometry (GC- MS)-based fatty acid methyl ester (FAME) method (Hielscher et al., 2017). Endosperm tissue was dissected from dry seeds, 24h- imbibed seeds and 1- to 6-day old dark-grown seedlings by removing the embryo and roots with the blunt end of a scalpel blade, weighed and shock frozen in liquid nitrogen.
+FAME analyses were generated by acid catalyzed methyl-ester formation using methanolic HCl as described by Hielscher et al. (2017). 20 mg endosperm was incubated at 90°C for one hour in 1 ml 3 N methanolic-HCl containing heptadecanoic acid (C17:0) as internal standard. The subsequent extraction of fatty acids from the sample was performed with 1 ml n-hexane and 1 ml 1% (w/v) sodium chloride. Samples were centrifuged at 2,000 g for five minutes. The resulting upper hexane phase was transferred into GC vials. The FAME extracts (1:100 diluted) were analyzed by GC Agilent 7890A gas chromatograph coupled to Waters GCT-TOF Premier mass spectrometer equipped with a Gerstel MPS2XL auto sampler.
+Quantification and identification of the detected fatty acids was carried out with QuantLynx and MassLynx, respectively. Peaks were integrated and the resulting areas used for determination of relative changes in the abundance of the castor oil-specific ricinoleic acid. As the Ricinus endosperm started to gain fresh weight during seedling development due to water uptake, the fatty acid quantities were normalized to the fresh weight of an average endosperm. All samples were analyzed in three biological replicates.
+
+
+- Hielscher, B., Charton, L., Mettler-Altmann, T., and Linka, N. (2017). Analysis of peroxisomal b-oxidation during storage oil mobilization in Arabidopsis thaliana Seedlings. Methods Mol. Biol. 1595, 291–304. doi: 10.1007/978-1-4939-6937-1_27
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assays/2_3-OrganellesFromEndosperm/README.md

+
+
+![fpls-14-1182105-g003.jpg](dataset/fpls-14-1182105-g003.jpg)
+
+**Figure 3** 
+
+Isolation of organelles from etiolated castor bean seedlings. Four fractions of the sucrose density step gradient after centrifugation were taken from the gradient for various analyses at the interface 30% - 44% (w/w) sucrose solution (F1), 44% - 48% (w/w) sucrose solution (F2), 48% - 49% (w/w) sucrose solution (F3), and 50% - 54% (w/w) sucrose solution (F4).
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assays/2_3-OrganellesFromEndosperm/protocols/OrganellesFromEndosperm.md

+# 2.3 Preparation of organelles from castor bean endosperm
+
+The isolation of castor bean endosperm was performed according to Cooper and Beevers (1969) and Beevers and Breidenbach (1974). The protocols were modified by using the grinding buffer as described by Reumann et al. (2007). 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 flask (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) were carefully collected, pooled, and stored at -80°C for further experiments.
+
+- Beevers, H., and Breidenbach, R. W. (1974). “Glyoxysomes,” in Methods in enzymology, Vol 16 Biomembranes, Part A. Eds. S. Fleciher and L. packer (New York London: Academic Press), 565–571.
+- Cooper, T. G., and Beevers, H. (1969). Mitochondria and glyoxysomes from castor bean endosperm. J. Biol. Chem. 244, 3507–3513. doi: 10.1016/S0021-9258(18)83401-9
+- Reumann, S., Babujee, L., Ma, C., Wienkoop, S., Siemsen, T., Antonicelli, G. E., et al. (2007). Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms. Plant Cell 19, 3170–3193. doi: 10.1105/tpc.107.050989
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assays/2_4-EnzymeActivityOrganellarMarkers/README.md

+![fpls-14-1182105-t001.jpg](dataset\fpls-14-1182105-t001.jpg)
+
+**Table 1**
+
+Distribution of marker enzyme activities between the fractions.
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assays/2_4-EnzymeActivityOrganellarMarkers/protocols/EnzymeActivityOrganellarMarkers.md

+# 2.4 Measurements of enzyme activity of organellar marker enzymes
+
+The distribution of peroxisomes, mitochondria, and plastids within the four organellar fractions were examined using enzymatic marker proteins. All enzyme assays were performed photospectrometrically in a plate reader (SynergyH1, BioTek) at room temperature. For each sample three technical replicates were measured. Total protein concentration of the fractions was determined using the Pierce BCA protein assay kit (ThermoFisher Scientific). Enzyme activity was expressed as units per mg total protein. The activities of the following marker enzymes were analyzed: Catalase for peroxisomes (Breidenbach et al., 1968), fumarase for mitochondria (Nishimura and Beevers, 1981), and phosphoglycerate dehydrogenase for plastids (Benstein et al., 2013).
+
+- Breidenbach, R. W., Kahn, A., and Beevers, H. (1968). Characterization of glyoxysomes from castor bean endosperm. Plant Physiol. 43, 705–713. doi: 10.1104/ pp.43.5.705
+- Nishimura, M., and Beevers, H. (1981). Isoenzymes of sugar phosphate metabolism in endosperm of germinating castor beans. Plant Physiol. 67, 1255–1258. doi: 10.1104/ pp.67.6.1255
+- Benstein, R. M., Ludewig, K., Wulfert, S., Wittek, S., Gigolashvili, T., Frerigmann, H., et al. (2013). Arabidopsis phosphoglycerate dehydrogenase1 of the phosphoserine pathway is essential for development and required for ammonium assimilation and tryptophan biosynthesis. Plant Cell 25, 5011–5029. doi: 10.1105/tpc.113.118992
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