DEV006759 Supplementary Material

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• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. Northern blot analysis of AMP1 gene expression in T-DNA insertion mutants. Total RNA was extracted from 7-day-old seedlings and 15 μg of RNA was loaded in each lane.

• Supplemental Figure S2 -

  Fig. S2. The effect of clv2 on mp inflorescences. Scanning electron micrographs of inflorescence meristems of wild type (A), mpG92 (B,C), clv2-1 (D) and mpG92 clv2-1 (E,F) double mutants. Single clv2-1 mutants have enlarged meristems (D) relative to wild type (A). (E,F) mpG92 clv2-1 double mutants form pin-shaped inflorescences devoid of floral organs (F) or mutant flowers consisting only of carpel-like structures (E), similar to mp single mutants (B,C). Representative phenotypes are shown. Scale bars: 100 μm in A,D; 350 μm in B; 240 μm in C; 600 μm in E; 300 μm in F.

• Supplemental Figure S3 -

  Fig. S3. Transgenic complementation of amp1 and mp. (A) amp1-1 mutants display an altered phyllotaxis and an increased rate of leaf initiation. (B) Restoration of the normal near 180° angle in amp1-1 plants carrying the AMP1::AMP1-GFP transgene. (C-F) Restoration of primary root formation and vascular complexity in MP::MP-GUS; mpG12 (D,F). Scale bars: 5 mm in A,B; 1 mm in C,D.

• Supplemental Figure S4 -

  Fig. S4. Subcellular localization of MP and AMP1. (A,B) CLS micrographs showing AMP1-GFP expression in the primary root tip in all cell types including the stele, endodermis and cortex. Localization of AMP1-GFP surrounds the nuclei. (C) Cleared whole-mount preparations, DIC optics, showing predominantly nuclear localization of MP::MP-GUS in the stele near the tip. To the right is an enlargement of the red boxed area showing nuclear expression in a single cell. (D) Membrane fractionation on sucrose density gradient. Ribosome dissociation in the absence of Mg²⁺ leads to redistribution of ER-associated proteins such as SNO-GFP but not of plasma membrane marker Lti6b-GFP. Note that AMP1-GFP co-fractionates with SNO-GFP. (E) Immunodetection of AMP1-GFP (left) and 35S::GFP (right) from 5 DAG seedlings; lanes 1 and 2 express the transgenes (AMP1-GFP and 35S::GFP, respectively), whereas lanes 3 do not express the transgene. All seedlings were grown on MS medium; those for lanes 2 were exposed to 5 μg/μl tunicamycin for 24 hours. Upper panel, rubisco band in Coomassie-stained loading control; lower panel, immunodetection by GFP antibody at approximately 104 kDa (left) and 27 kDa (right). Scale bars: 50 μm in A,C; 20 μm in B.

• Supplemental Figure S5 -

  Fig. S5. Expression patterns of AMP1 and MP. Expression patterns of AMP1::AMP1-GFP (A-C) and MP::MP-GUS (D-F) functional fusion proteins. (A,D) Expression in SAM and young leaf primordia at 3 DAG. MP-GUS expression becomes confined to areas from which procambial cells are recruited. (B,C,E,F) Nearly congruent expression of AMP1-GFP and MP-GUS in lateral root primordia (B,E) and emerging lateral roots (C,F). Sample size observed was between 30-50 seedlings for each transgene. (A-C) CLS microscopy; (D-F) DIC optics. Scale bars: 50 μm.