Extended Data Fig. 9: The inverse relationship of NO and ROS and nitrosation of BrFER1 and RBOHs in B. rapa stigmas.
From: Stigma receptors control intraspecies and interspecies barriers in Brassicaceae
a, b, Intraspecific CP pollen (2nd row), but not SI and B. oleracea (UI) pollen, induced NO in B. rapa stigmas. c–e, Scavenging NO increased ROS (c) and RBOH enzymatic activity of UP B.rapa stigmas (d), and inhibited the growth of CP pollen tubes in B. rapa stigmas (e). f–h, GSNO treatment increased NO (f), reduced ROS (f) and RBOH enzymatic activity of UP B. rapa stigmas (g), and promoted the growth of SI or B. oleracea pollen in mature or B. vulgaris polen in bud-stage B. rapa stigmas (h). i, BrFER1(KD), not BrFER1 (ED), was nitrosated by GSNO. See also Fig. 4f. j, k, LC-MS spectrum showing the nitrosated cysteine residues in BrFER1 protein. l, FER amino acid sequence alignment showing the nitrosated cysteines, Cys730 and Cys752 (*) are evolutionarily conserved. m, Pull-down assay shows GSNO treatment induced quantitative inhibition of MBP-BrFER1(KD) with GST-BrROP2 complex. See also Fig. 4h. n, The nitrosomimetic mutation, Cys730W, reduced the amount of BrFER1 that was pulled down by GFP-BrROP2. o, Phylogenetic analysis of BrRBOHs and AtRBOHD. BrRBOHD1 and BrRBOHD2 are closer to AtRBOHD than other BrRBOHs. p–r, RBOHD was nitrosated by NO. In vitro nitrosation of BrRBOHD1 by GSNO (p); CP-induced nitrosation of GFP-AtRBOHD protein from A. thaliana stigmas (q), and LC–MS spectra showing nitrosation of Cys891 of GST-BrRBOHD1 (CT, C-terminal) (r). Nitrosated amino acid residues were labelled with TMT. Proteins were representative of more than three independent preparations. s, t, GST-BrRBOHD2 (CT) (s) and GST-BrRBOHF (CT) (t) proteins were nitrosated in vitro. The protein samples were derived from the same experiment and the blots were processed in parallel (i, m, n, p, q, s, t). For gel source data, see Supplementary Fig. 1. Scale bars, 500 μm. Line chart (b): average ± SD. Average relative NO intensities from three biological replicates of stigmas shown in a (two tailed t-test, n = 3). Data bars (d, g): Average activity of ROS producing enzymes from three technical replicates of one stigma sample containing 100 stigmas (two tailed t-test, n = 3). Data bar (m, n): average ± SD. Average relative intensities from three biological replicates of stigmas in (a) and blots in Fig 4h and in (n) (two tailed t-test, n = 3). Box plots (c, e, f, h): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of stigmas. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results unless otherwise specified.
