Long-Distance Signaling in bypass1 Mutants: Bioassay Development Reveals the bps Signal to Be a Metabolite  Emma Adhikari, Dong-Keun Lee, Patrick Giavalisco, Leslie E. Sieburth  Molecular Plant  Volume 6, Issue 1, Pages 164-173 (January 2013) DOI: 10.1093/mp/sss129 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Growth Arrest of bps1 Mutants Is Associated with Altered Cell Division.(A) Seven-day-old wild-type (Col-0) and bps1-2 seedlings.(B) Expression pattern of pCYCB1;1::GUS in 4-day-old wild-type (Col) and bps1-2 mutant leaf primordia (top) and roots (bottom).(C) Relative expression of the cell cycle genes expressed in G2/M, S, and G1 phases of the cell cycle. Solid bars represent wild-type and open bars represent bps1. Error bars show SEM. Size bars: 2mm in (A) and 50µm in (B). Molecular Plant 2013 6, 164-173DOI: (10.1093/mp/sss129) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 The bps Signal Causes Reduced Wild-Type Leaf Cell Division whether Transmitted through Grafts or Applied through Extracts, But It Does Not Activate the pARR5::GUS Cytokinin Reporter.(A) Transient micrografts with 4-day-old wild-type (WT) pCYCB1;1::GUS scion coupled to WT or bps1-1 rootstocks. Arrows point to the agarose plug.(B) Expression of pCYCB1;1::GUS in WT leaf primordia 24 h after micrograft coupling.(C) Box and whisker plots of pCYCB1;1::GUS-stained cells in WT leaf primordia following transient micrografts (n = 32 for each micrograft couple). Boxes delineate the data points falling between 25% and 75%, the line bisecting the box shows the median, and the whiskers indicate the highest and lowest data point.(D) Test of crude extracts on WT leaf cell division. Strategy for extracts addition is to the left. Box plots show pCYCB1;1::GUS-stained cells in WT leaf primordia treated with water or extracts (n = 21 for each sample).(E) pARR5::GUS expression in WT leaf primordia 24 h after micrograft coupling to WT or bps1 rootstocks; positive controls used 1 µM cytokinin (BAP and Kinetin) supplied in the agarose plug (n = 16, each treatment). Results with significant differences are labeled with letters a and b (Mann-Whitney U-test; a = P < 0.005 and b = P < 0.05). Size bars: 1.0 mm in (A) and 50 µm in (B, E). Molecular Plant 2013 6, 164-173DOI: (10.1093/mp/sss129) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Wild-Type (WT) Root Meristem Cell Division Is Sensitive to the bps Signal.(A) WT pCYCB1;1::GUS-stained roots treated with WT or bps1 extracts. (B) Numbers of pCYCB1;1::GUS-stained cells in WT roots treated with water or extracts. The letter ‘a’ represents a statistical significance P < 0.005 (Mann-Whitney U-test). Size bars: 50 µm. Molecular Plant 2013 6, 164-173DOI: (10.1093/mp/sss129) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 The bps Signal in Crude Extract Disrupts the Columella Cells in Wild-Type (WT) Roots.(A) Five-day-old WT and bps1 with QC:46::GUS marker, GUS and lugol-stained.(B) GUS and lugol-stained WT roots, with QC:46::GUS marker, treated with WT or bps1 extracts. Size bars: 50 µm. Molecular Plant 2013 6, 164-173DOI: (10.1093/mp/sss129) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Partial Characterization of the bps Signal.(A) The time-course sensitivity of the wild-type (WT) root meristem to the bps signal (N = 14 for each sample).(B) WT (L.er) and bps1-1 extracts were diluted to different strengths according to the fresh weight of the seedlings (N = 12 for each sample).(C) Tests for sensitivity to heat, RNaseA and ProteinaseK of the bps signal (N = 20 for each sample). Results that are significantly different from the control samples are labeled with the letters a and b (Mann-Whitney U-test; a = P < 0.005 and b = P < 0.05). Molecular Plant 2013 6, 164-173DOI: (10.1093/mp/sss129) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions