Supplemental Data

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• Supplemental Figure 1 - Supplemental Figure 1. Effect of various treatments on auxin-induced IAA17-LUC degradation and DR5-GUS expression. (A) Effect of three AtRacs that positively regulate auxin-responsive gene expression (Tao et al., 2002) on IAA17-LUC activity in transfected tobacco protoplasts. (B)Upper panel, effect of GFP-IAA7 and IAA17-GFP on DR5-GUS expression in the absence and presence of exogenous auxin in transfected tobacco protoplasts. Lower panel, effect of IAA17 and IAA17-GFP on auxin-induced DR5-GUS expression in transfected Arabidopsis protoplasts. Results were qualitatively similar in both protoplast systems. C Effect of NAA, brassinosteroid (BR), gibberellic acid (GA) and abscisic acid (ABA) on IAA17-LUC activity in transfected Arabidopsis protoplasts.
• Supplemental Figure 2 - Supplemental Figure 2. Plasmid vectors. (A) Cloning region for the vector used for inserting chimeric genes used in this study. The vector used was derived from pBluescript. A CaMV35S promoter fragment and a Nos 3' end fragment flank several cloning sites for insertion of chimeric genes. Cloning region is not drawn to scale. (B) The dual plasmid system for Dex-inducible chimeric genes (Yanagasami et al., 2003). Plasmid 35S-LSVG-nosT carries the nuclear-targeted GR repressor that binds the target site on plasmid OctLexAop-35STATA(-72), which expressed the regulated gene, e.g. IAA17-GFP. Both plasmids were constructed on pUC backbone. 10 μg of plasmid DNA for each of the constructs were used for transfection. After overnight culture of transfected protoplasts, gene expression was induced by Dex (10 μM) for 4-5 hours, a period of time long enough for GFP signal to be easily observed. Routinely, about 65-75% of transfected protoplasts were GFP-positive at this point. Dex-containing medium was removed and replaced with Dex-free protoplast medium, with various supplements as indicated in the text.
• Supplemental Figure 3 - Supplemental Figure 3. Expression of IAA17-GFP and its induced phenotype in transformed Arabidopsis seedlings. About 20 35S-IAA17-GFP transformed plants were obtained; about half of them showed detectable IAA17-GFP in very young seedlings (3-5 days post germination) in restricted cells types. Fluorescence levels declined as seedlings developed, and became barely or undetectable in two weeks and older seedlings. (A) Typical IAA17-GFP fluorescence signal pattern in very young seedlings (a 4 days old seedling shown here). Cells along the main vascular system showed the most prominent IAA17-GFP accumulation. Some of the transgenic seedlings show very low levels of IAA17-GFP in cortical cells (see Fig.3). (B) A cotyledon from the seedling shown in (A). (C)10 days old seedlings. Relative to control (seedlings 1, 3), the primary root in transformed seedlings were observable shorter and more adventitious root development was apparent in some of the transformed seedlings (e.g. seedlings 2, 4). Seedlings 2, 4 were each from an independent transgenic parent. Plants 5-9 were from another transgenic parent, more moderate phenotype was observed. The observed root-related phenotype was similar to those reported for axr3 mutants (Leyser et al., 1996) and 35S-IAA17 transformed seedlings (Worley et al., 2000). (D,E) IAA17-GFP signal from the adventitious root region of seedling 2 (D), 9 (E). Fluorescent dots were from nuclei. (F,G) IAA17-GFP signal in the primary root of seedling 2 (F) and in a leaf from seedling 4 (G), whose leaves had more elongated petiole. IAA17-GFP signals were detected along the main vascular strands only. At this age, IAA17-GFP was in general observable in seedlings that showed discernable phenotypes but already not in those that appeared normal. (H,I,J) 25 days old wild type plant (H), a 35S-IAA17-GFP transformed plant with a more bushy morphology (I) and another with elongated petioles (J), two most prevalent phenotypes among plants that show appearances discernable from wild type plants.
• Supplemental Figure 4 - Supplemental Figure 4. Quantitative analysis of auxin-signaled and Rac GTPase-mediated recruitment of nucleoplasmic IAA17-GFP into nuclear PBs. This figure is based on the same data sets as those shown in Fig. 4, 6 and Supplemental Fig. 2C. It provides more refined categorization among the NPB-containing category II cells, which are further categorized here into 4 subclasses, IIa-IId (S.Fig. 2A). Protoplasts were transfected with Dex-IAA17-GFP (A,B,D-F) or Dex-IAA17(P88L)-GFP (C). After transfection, overnight culture, Dex induction and withdrawal, protoplasts were cultured in 0, 1 or 10 μM NAA for 30 minutes as indicated. When MG132 was used (B), it was added to the 1 μM NAA-treated sample 1 hour prior to Dex withdrawal and included for the remaining duration of the experiment. When other hormones were used, they were also added at the end of Dex induction. At the end of 30 minutes after Dex withdrawal, the remaining IAA17-GFP-positive cells were observed using the 100X objective so the details of the IAA17-GFP signal in the nucleus could be seen. (A) Images of representative category I and II cells. All images were taken by autoexposure and so do not reflect relative fluorescence intensity. They are shown at the same scale (scale bar in top panel = 5 μm applies to all images). As indicated in Fig. 4 of the text, category I cells were those with diffuse nuclear IAA17-GFP signal and their signals were consistently the strongest. Category II cells presented in Fig. 4 were comprised of 4 subclasses, IIa-IId, and they were in general considerably weaker in overall green fluorescence level than the category I cells. Categories IIa cells had 1-4, IIb had 5-10, and IIc had more than 10 IAA17-GFP NPB in a single focal plane. Category IId cells had very weak overall signal within the nucleus (as reflected by the relatively high level of background fluorescence seen in the images). For many of the II-d cells, structures within the nuclei were obvious but too weak to be imaged well; the slightly brighter ones often showed numerous extremely weak and small NPBs, as in the nuclei shown here. Category II-d cells presumably would be those where IAA17-GFP degradation had been extensive. While the level of IAA17-GFP expression might have some contribution, the heterogeneity in the number and size of NPBs seen in cells within a culture was most probably contributed by differences in individual cells? sensitivity to the treatments and that IAA17-GFP was at various stages of being recruited into NPBs and had undergone different extent of proteolysis in different cells. (B-F) The number of cells belonging to each of the 5 categories (I, IIa to IId) was recorded for each experiment. The reporter gene used for each experiment is indicated at the top of each figure. Hormone (NAA, BR and GA3) concentrations, co-expression from either 35S-NtRac1(CA), (DN), 35S-AtRac1(CA) or (DN) are as indicated in each figure. Data bars show the average from three independent experiments. 100% represent all IAA17-GFP cells scored. In each experiment, a total of at least 200 IAA17-GFP or IAA17(P88L)-GFP positive cells maintained under each specified conditions were observed. Where no error bar is shown, standard deviation was negligible. Cells were grouped into category I or IIa-d. (B) Auxin induced considerable decline in the level of category I cells and appearance of all category II subclasses. Among category II cells, IId cells were the most abundant in the presence of NAA and had the least overall fluorescence signal, suggesting many transfected cells had most of their IAA17-GFP degraded. The intermediate subclasses, II-a,b,c, were of variable fluorescent intensity. They probably represented cells showing different extent of IAA17-GFP translocation into NPBs and degradation. Treatment with MG132 (last data bar, light gray, in each set) suppressed the auxin effect, category I cells remained high even in the presence of NAA. (C) Auxin had little effect on the nucleoplasmic localization of proteolytically stable IAA17(P88L)-GFP. Category I cells remained high in the presence of NAA. (D) Co-expressing NtRac1(CA) or AtRac1(CA) induced decline in category I cells. Nucleoplasmic IAA17-GFP was recruited into NPBs in various category II subclasses even in the absence of auxin. (E)Co-expressing NtRac1(DN) and AtRac1(DN) suppressed the auxin-induced recruitment of nucleoplasmic IAA17-GFP into NPBs as category I cells were maintained at higher levels than in auxin-treated control cells not co-expressing DN forms of Rac GTPases. (F) Hormones that did not induce Aux/IAA protein instability (see Supplemental Fig.3; Zenser et al., 2003; Nemhauser et al., 2004) did not affect nucleoplasmic IAA17-GFP localization as category I cells remained high when protoplasts were cultured with BR and GA₃. (data for category II subclasses were not collected for ABA treatment).
• Supplemental Figure 5 - Supplemental Figure 5. AtRac1(CA) induces formation of IAA17-GFP-containing NBPs and their decline in the absence of exogenous auxin. An Arabidopsis protoplast cotransfected by Dex-IAA17-GFP and 35S-AtRac1(CA) observed after Dex treatment and maintained in auxin-free medium. The cell was among the population of protoplasts that still maintained a diffused nucleoplasmic signal (category I) but was noticeably on the verge of showing discernable NBPs. Condition for imaging was the same as those described in Figs. 5, 6.
• Supplemental Figure 6 - Supplemental Figure 6. Co-localization of GFP-labeled components of SCF^TIR1, selected components of CSN and 26S proteasome core particle (CP) with co-expressed IAA17-RFP. Arabidopsis protoplasts were transfected with combinations of 35S-expressed GFP-labeled proteins and IAA17-RFP as indicated in each of the panels. From left to right in each row are image taken in the green channel, red channel, and merged from green/red channels, respectively. Results were qualitatively similar to those shown in Fig. 7.
• Supplemental References