DEV025932 Supplementary Material

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• Supplemental Table S1 -

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• Supplemental Table S2 -

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• Supplemental Table S3 -

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• Supplemental Table S4 -

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• Supplemental Table S5 -

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• Supplemental Figure S1 -

  Fig. S1. Root phenotypes of wild-type and iaa18-1 seedlings. (A) Structure of root tip of 7-day-old wild-type and iaa18-1 seedlings. (B) Primary root growth. Five-day-old seedlings were transferred to plates supplemented with IAA (Indole-3-acetic acid) and grown for 3 days under long day conditions. Triangles, Ler; circles, iaa 18-1/IAA18 (n=20). Shown are both raw data (upper graph) and growth inhibition relative to root growth in the absence of exogenous IAA (lower graph). (C) Lateral root initiation from excised roots. Black bars, Ler; white bars, iaa18-1/IAA18 (n=40). Five-day-old dark-grown seedling roots were excised, transferred to plates supplemented with IAA and grown for 10 days under long day conditions. Error bars indicate s.d.

• Supplemental Figure S2 -

  Fig. S2. Recapitulation of iaa18-1 phenotypes in iaa18-1:GUS transgenic plants. iaa18-1:GUS T1 seedling phenotypes. (A) Seedling with curled leaves. (B) Monocot. (C) Rootless monocot. (D) Dicot with closed cotyledons. Table S3 lists frequencies of T1 seedlings obtained with various phenotypes.

• Supplemental Figure S3 -

  Fig. S3. Effect of iaa18-1 on PIN1:GFP^Col and P_DR5 gene expression in embryos. (A-J) Expression of PIN1:GFP fusion in wild-type (A,B,E,F,G) and iaa18-1 (C,D,H,I,J) embryos, viewed by confocal fluorescence microscopy. The PIN1:GFP fusion line in this figure is in the Columbia background (Benkova et al., 2003), and is distinct line from the one in the Ler background shown in Fig. 4. In our hands, the Columbia line produced a weaker and more variable signal in wild-type globular embryos, and was therefore used only to characterize iaa18-1 heart stage embryos. (A,B) Z-stack projection (A) and medial optical section (B) of wild-type heart stage embryo. (C,D) Z-stack projection (C) and medial optical section (D) of iaa18-1 heart stage embryo. The arrowhead in D indicates decreased fluorescence in the tier of provascular cells just beneath the apical meristem. Whereas 20 of 21 wild-type control embryos had PIN1:GFP expression in these cells, 17 of 35 embryos from a population segregating for iaa18-1 lacked this expression (i.e. about 65% of embryos carrying at least one iaa18-1 allele, assuming Mendelian segregation). Many of these embryos had apparently wild-type morphology (C,D), indicating that altered PIN1:GFP expression did not arise from aberrant cotyledon outgrowth. The tier of cells beneath the meristem express IAA18 (Fig. 3J,K,R), suggesting that IAA18-1 inhibits PIN1 expression there. (E-G) Z-stack projection (E), glancing epidermal optical section (F) and medial optical section (G) of wild-type late heart stage embryo. (H-J) Z-stack projection (H), glancing epidermal optical section (I) and medial optical section (J) of iaa18-1 late heart stage monocot embryo. Arrowhead in J indicates discontinuity in PIN1:GFP expression in the cotyledon provasculature. (K,L) P_DR5:GUS expression in wild-type (K) and iaa18-1 (L) late heart stage embryos. (M,N) P_DR5:GFP expression in wild-type (M) and iaa18-1 monocot (N) late heart stage embryos. Scale bar: 25 µm. Diffuse P_DR5:GFP expression was occasionally detected at the growing cotyledon edge of monocot embryos (N). P_DR5:GUS and P_DR5:GFP expression at the root pole of wild-type embryos (K,M) was usually also present in iaa18-1 embryos (L,N). Decreased or absent expression at the root pole of the iaa18-1 embryos shown occurred at a low frequency (<1%) similar to the frequency with which iaa18-1 embryos lacked a root pole. (O) Frequency of late heart stage embryos with indicated numbers of PIN1:GFP-expressing axis cell files at the midpoint of the central vascular cylinder in wild-type and iaa18-1/IAA18 segregating populations. For all fluorescent images, wild-type and mutant embryos were photographed in parallel using identical settings.

  Reference

  Benkova, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertova, D., Jurgens, G. and Friml, J. (2003). Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115, 591-602.

• Supplemental Figure S4 -

  Fig. S4. Q0990 UAS:iaa18-1 T1 seedlings. (A) Seedling with a single cotyledon and lacking a root. (B) Seedling with a trumpet-shaped cotyledon and lacking a root.

• Supplemental Figure S5 -

  Fig. S5. Quantitative real-time RT-PCR analyses of ARF gene expression in iaa18-1/IAA18 P_35S:ARF plant lines. Shown are calculated expression levels relative to those of non-transgenic plants ±s.e.m. Reactions were performed in triplicate, and expression levels were normalized to corresponding values for the UBQ10 gene. Note different scales on the y-axis for different genes. Data were analyzed using the software REST (Pfaffl et al., 2002) (http://gene-quantification.com/rest.html), which uses a non-parametric randomization method to calculate probabilities. Asterisks indicate lines for which the null hypothesis of equal expression to control for the indicated ARF gene had P<0.05. Line P_35S:ARF7-B was obtained from Thomas Berleth (Hardtke et al., 2004) and crossed with iaa18-1/IAA18. Other lines are Basta-resistant T2 progeny of self-pollinated primary transformants.

  References

  Hardtke, C. S., Ckurshumova, W., Vidaurre, D. P., Singh, S. A., Stamatiou, G., Tiwari, S. B., Hagen, G., Guilfoyle, T. J. and Berleth, T. (2004). Overlapping and non-redundant functions of the Arabidopsis auxin response factors MONOPTEROS and NONPHOTOTROPIC HYPOCOTYL 4. Development 131, 1089-1100.

  Pfaffl, M. W., Horgan, G. W. and Dempfle, L. (2002). Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30, e36.