SUPPLEMENTAL METHODS

Real-time Quantitative RT-PCR Analysis

1. Analysis of Arabidopsis GID1s[p1] mRNA Levels during Development

Total RNA was isolated from of the following samples, with three independent biological replicates: (1) dry seed, (2) seed imbibed on wet 3MM grade 1 Whatman paper for 24h, (3) seedlings grown for 3d on vertical 1x MS plates (containing Gamborg B5 vitamins, 1% sucrose, 2.5 mM MES, 0.7% gelrite) (n=180 plants), (4) shoot and (5) root of seedlings grown on vertical MS plates for 7 d (n=45 plants), (6) to (10) tissues were harvested from plants grown on soil (n=10 plants): (6) whole shoot (rosette) of plants at 11 d on soil, (7) all leave, (8) vegetative stem and (9) inflorescence of plants at 24 d, (10) inflorescence stem, (11) inflorescence (from youngest open flower upward) and (12) all siliques of plants at 31d. Seeds for seedling and plant material were first stratified at 4˚C for 3d. Seeds on paper and seedlings on plates were grown at 22˚C under continuous light, plants on soil were grown under a 16 h light-23˚C/8 h dark-18˚C regime. RNA was extracted from 100 mg tissue using an RNeasy plant RNA isolation kit with on-column DNase treatment (Qiagen), except for the dry seed and imbibed seed samples, for which a modified hot-borate method was used (Toorop et al., 2005), followed by an RNA clean-up using the RNeasy plant RNA isolation kit. One g total RNA was treated with Turbo DNA-free kit (Ambion) and used as a template to synthesize cDNA using the SuperScript III Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen). PCR reactions were performed on a ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using Platinum SYBR Green qPCR SuperMix-UDG reagents (Invitrogen), according to the manufacturers specification, with the cDNA equivalent of 17.5 ng RNA in a 25 L reaction volume. Reactions were performed in duplicate and the absence of genomic DNA and primer dimers was confirmed by analysis of RT-minus and water control samples and by examination of dissociation curves. Results from three reference genes were used to calculate a geNorm Normalization Factor (Vandesompele et al., 2002), using primer efficiencies determined by analysis of the amplification curves with LinReg software (Ramakers et al., 2003). Stability of the endogenous control genes across samples was indicated by the low pair-wise variation between the resulting Normalization Factor before and after inclusion of the third most stable gene (V2/3=0.118, which is below the recommended cut-off value of 0.150). Standard curves of the GID1 genes were prepared using PCR fragments as the template. For calculation of the absolute ratios, the number of GID1 cDNA templates per sample was normalized against the corresponding geNorm Normalization Factor and results from the three biological replicates were averaged. The primers for GID1s (GID1a/b/c F/R) were designed using Primer Express v.2.0 (Applied Biosystems) or GENOPLANTE SPADS software ( Primers for the reference genes At1g13320, At4g34270 and At2g28390 have been described (Czechowski et al., 2005).

2. Analysis of GA3ox1 and GID1s mRNA Levels in ga1 and DELLA Mutants and in Response to GA Treatment

8-day-old ga1-3 seedlings that were grown vertically on MS agar plates were sprayed with water or 2 M GA4 using Preval Sprayer (Precision Valve Corporation, Yonkers, NY) and incubated for 15 min, 30 min, 1h or 3 hr before harvesting. For Ler, rga-24 ga1-3, rga-24 gai-t6 ga1-3 and rga-17, 8-day-old seedlings that were grown vertically on MS agar plates were sprayed with water and incubated for 1 hr before harvesting. The seeds of the rga-17line were produced from hemizygous parents because homozygous plants are sterile (Dill et al., 2001). For this line, WT seedlings were discarded and a mixture of hemizygous and homozygous rga-17 seedlings were harvested. Total shoot RNA was isolated and DNase-treated for each line, and cDNA was synthesized using 1 g of total RNA as described (Tyler et al., 2004), except that the DNA-free Kit from Ambion (Austin, TX) was used for DNase treatment. cDNAs were then purified using Qiaquick PCR purification kit (Qiagen, Valencia, CA), and eluted with 50 L elution buffer, and then with 150 L H2O. The levels of GA3ox1 and the Arabidopsis GID1 transcripts in each sample were quantified using 2 L of purified cDNA by real-time PCR using gene-specific primers, the Light Cycler GastStart DNA Master SYBR Green I Kit and a Roche Light Cycler as described (Tyler et al., 2004). The housekeeping gene GAPC whose expression is not affected by GA treatment (Dill et al., 2004) was used as a control to normalize all samples. The GAPC and GA3ox1 primers (Table 1S) were made previously (Dill et al., 2004; Mitchum et al., 2006). The primers forGID1a, GID1b and GID1c are listed in Table 1S, and all qPCR reactions were performed using 3 mM MgCl2 with an annealing temperature of 55°C. One-way ANOVA analyses were performed with LSD multiple comparison tests at an alpha level of 0.01,using the Statistical Package for the Social Sciences (SPSS) Version 10 (Chicago, IL).

3. Analysis of GA3ox1 mRNA Levels in the gid1 Mutants and in Response to GA

The gid1 mutants and wild-type Col-0 were grown on 1 x MS plates for 10 days and whole seedlings were harvested. Total RNA isolation, DNase treatment, reverse transcription and qPCR were performed as described for the developmental series. At1g13320, At4g33380 and At5g25760 were used as endogenous control genes for normalization. Stable expression of these genes across samples was confirmed as described for the developmental series (V2/3= 0.120). Primer efficiencies were determined from the amplification curves (see above) and relative quantities were calculated using qBase software (v1.3.4, Primers for At5g25760 were designed using GENOPLANTE SPADS software and are listed in Table 1S. Primers for At4g33380 have been described (Czechowski et al., 2005).

REFERENCES

Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., and Scheible, W.R. (2005). Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5-17.

Dill, A., Jung, H.-S., and Sun, T.-p. (2001). The DELLA motif is essential for gibberellin-induced degradation of RGA. Proc. Natl. Acad. Sci. USA 98, 14162-14167.

Dill, A., Thomas, S.G., Hu, J., Steber, C.M., and Sun, T.-p. (2004). The Arabidopsis F-box protein SLEEPY1 targets GA signaling repressors for GA-induced degradation. Plant Cell 16, 1392-1405.

Mitchum, M.G., Yamaguchi, S., Hanada, A., Kuwahara, A., Yoshioka, Y., Kato, T., Tabata, S., Kamiya, Y., and Sun, T.P. (2006). Distinct and overlapping roles of two gibberellin 3-oxidases in Arabidopsis development. Plant J. 45, 804-818.

Ramakers, C., Ruijter, J.M., Deprez, R.H., and Moorman, A.F. (2003). Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339, 62-66.

Toorop, P.E., Barroco, R.M., Engler, G., Groot, S.P., and Hilhorst, H.W. (2005). Differentially expressed genes associated with dormancy or germination of Arabidopsis thaliana seeds. Planta 221, 637-647.

Tyler, L., Thomas, S.G., Hu, J., Dill, A., Alonso, J.M., Ecker, J.R., and Sun, T.-p. (2004). DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis. Plant Physiol. 135, 1008-1019.

Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, 34.31-34.11.

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