Fig. 2: OsASTOL1 encodes a chloroplast-localized O-acetylserine(thiol)lyase (OAS-TL).
From: A molecular switch in sulfur metabolism to reduce arsenic and enrich selenium in rice grain
a Subcellular localization of OsASTOL1. Rice protoplasts were expressed with the eYFP (top), OsASTOL1-eYFP (middle), or Osastol1-eYFP (bottom) driven by the cauliflower mosaic virus 35S promoter. Left to right: YFP fluorescence, chlorophyll autofluorescence, bright-field images, and merged images. Scale bars, 10 µm. At least five protoplasts were investigated with similar results to the images shown in a. b Validation of OsASTOL1/ OsASTOL1S189N protein (lower band) levels in 3-week-old WT, astol1(+/−), and astol1(+/+) using immunoblot analysis. OsASTOL1/ OsASTOL1S189N protein levels are detected by AtOAS-TL A1 antibody (anti-OAS-TL A) and Rubisco large subunit is used as a loading control. The experiment was repeated two times and similar results were obtained. c Total OAS-TL enzyme activity in whole soluble protein extracts of the shoots of 3-week-old WT, astol1(+/−), and astol1(+/+). Data are shown as means ± s.d., n = 4 biological replicates; each biological replicate represents an individual plant. d Expression of recombinant mature OAS-TL proteins in E. coli. Proteins were separated on SDS-PAGE and visualized by Coomassie Brilliant Blue (CBB) staining (upper) and by immunoblot with anti-His antibody (bottom). M, marker. The experiment was repeated two times and similar results were obtained. e In vitro OAS-TL enzyme activity of purified mature OsASTOL1 and AtOAS-TL A and their corresponding mutant proteins. Data are shown as means ± s.d., n = 3 technical replicates. WT, wild type. astol1(+/−), astol1 heterozygote. astol1(+/+), astol1 homozygote. Asterisks in c, e indicate significant difference by two-sided Student’s t test: **P < 0.01, ***P < 0.001.
