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. 2021 Dec 17;34(3):1117–1143. doi: 10.1093/plcell/koab306

Figure 3.

Figure 3

GmACBP3 and GmACBP4 interact with type-1 LOXs. A, Expression and purification of GST-fusion proteins. GST:GmACBP3.1 and GST:GmACBP4.1 were expressed in E. coli and purified from soluble fractions on GSTrap HP affinity and HiTrap Q HP anion-exchange chromatography columns. GST alone was purified on a GSTrap HP affinity column and served as a control in pull-down assays. Arrowheads indicate target bands of 62-kD GST:ACBP3.1 (apparent: 70 kD), 63-kD GST:ACBP4.1 (apparent: 70 kD), and 28-kD GST. B, GST pull-down assays. Purified bait proteins were immobilized to glutathione agarose for capturing potential interactors from crude soybean soluble proteins. A silver-stained SDS-PAGE gel indicates prey proteins in eluates. Arrowheads indicate target bands of 62-kD GST:ACBP3.1 (apparent: 70 kD), 63-kD GST:ACBP4.1 (apparent: 70 kD), and 28-kD GST. C, LC–MS/MS identification of prey proteins from GST:GmACBP4.1 pull-down assays. Confident proteins were identified using a target–decoy approach with a reversed database, strict false-discovery rate of <1% at peptide and PSM levels. Peptides of seven type-1 LOXs were identified. D, Neighbor-joining phylogenetic tree of soybean and Arabidopsis LOX homologs. Bootstrap values with 2,000 repetitions (%) are given at respective nodes. The scale bar indicates the distance scale (substitutions per site). Identified LOXs from LC–MS/MS as listed in (C) are shown in red and blue. See Supplemental Files S1, S2. E, GmACBP3.1:EGFP and GmACBP4.1:EGFP colocalization with DsRed:VLXB in agroinfiltrated N. benthamiana leaf epidermal cells. Signals were colocalized at the ER cisternae (blue arrowheads) and ER tubules. Bars = 50 or 10 μm (insets). F, Subcellular localization of DsRed:VLXB and DsRed:AtLOX1 in transgenic Arabidopsis by confocal laser scanning microscopy. In petioles and roots of 2-week-old plants, signals were detected at the tubular ER and ER cisternae. In root hair cells of 7-day-old seedlings, DsRed-fusion proteins were colocalized with the ER-Tracker signals at the membrane of ER-derived vesicles (open arrowheads). Pearson’s correlation coefficient ®, Manders’ overlap coefficients M1 (fraction of ER-Tracker Green overlapping DsRed), and M2 (fraction of DsRed overlapping ER-Tracker Green) were computed from regions of interest of 400 × 100 pixels. Values represent the mean ± sem of 10 cells. Bars = 10 μm.