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Figure S1. Derivatization of IPyA and IAAld by cysteamine.

(a) MS/MS spectrum of IAAld-TAZ after derivatization of IAAld bycysteamine solution. The product ion scan was measured underoptimized MS conditions (see Experimental procedures) with acollision energy of 15 eV. (b) MS/MS spectrum of IPyA-TAZafter derivatization of IPyA by cysteamine solution. The production scan was measured under optimized MS conditions with acollision energy of 20 eV. (c) The pH effect on derivatizationrecovery of IAAld (black squares) and IPyA (white squares) wastested with 0.25 M cysteamine solutions at pH 4.0–10.0(adjusted by 1M HCl or 25% NH4OH). Before LC-MS/MSanalysis, 10 pmol of both analytes were incubated for 1 hourand evaporated to dryness. The optimal pH for the derivatizationreaction was found to be pH 8.0. (d) Improving the sensitivityof IPyA determination after derivatization with cysteamine comparedto derivatization using hydroxylamine according to the previouslypublished method (Tam and Normanly, 1998). 20 mg ofArabidopsis seedling material (Col-0) was extracted, halvedbefore derivatization and purified by the SPE method described inExperimental procedures. After derivatization, the oxime forms ofIPyA and 2H4-IPyA were detected using the MRMtransitions 219.2 > 130.1 and223.2 > 133.1, respectively, and the thiazolidineforms were measured under optimized MS conditions (see Table 2).(e) Representative chromatograms of Arabidopsis extractsderivatized by 0.25 M solution of cysteamine (pH 8.0) underoptimized conditions. Stable isotope-labelled internal standards,10 pmol of [2H4]-IPyA and[13C6]-IAAld, were added during extraction of10 mg Arabidopsis seedlings (Col-0) and the extractswere purified by the protocol described in  Experimentalprocedures.

References

Tam, Y.Y. and Normanly, J. (1998) Determinationof indole-3-pyruvic acid levels in Arabidopsis thaliana bygas chromatography–selected ion monitoring-mass spectrometry.J. Chrom. A, 800, 101-108.

Figure S2. Test of buffer extraction capacity and plant matrix effects on IAA recovery.

(a) Test of extraction capacity of the sodium-phosphate buffer.Different amounts of plant tissue was extracted and purified on C8(black squares) and HLB (white squares) SPE columns. The extractionprocedure is linear up to 100 mg of plant material andindependent on the stationary phase of the SPE columns. (b) Test ofthe plant matrix effect on the recovery of the[13C6]-IAA internal standard added afterpurification of different amounts of plant tissue on silica based(C8, black squares, solid line) and polymer based (Oasis HLB, whitesquares, dotted line) solid phase extraction columns.

Figure S3. Test of different loading conditions on the recovery of IAA metabolites after purification by HLB solid phase extraction.

Different loading conditions (pH 2.7, black bars; pH 7.0, white bars) were used for purification of IAA metabolites on HLB columns, and the recovery was calculated.

Figure S4. Test of different extraction and purification protocols for IAA and IBA quantification and IBA spiking experiments of Arabidopsis extracts.

(a-d). Comparison of three different extraction methods andthree solid phase extraction protocols for the recovery of IAA andIBA. Comparison of recoveries from three different extractionbuffers (80% methanol, white bars; 65% isopropanol/0.2M-imidazolebuffer (pH 7.0), grey bars; sodium-phosphate buffer (pH 7.0), blackbars) and three SPE protocols (C8, C18 and MAX columns) forpurification of IAA (a,c) and IBA (b,d). Samples (10 pmol of IAAand IBA in mixture) were purified without (a,b) and with plantmatrix (20 mg of plant material) (c,d).

(e-f). Different concentrations of IBA (0, 0.1, 1 and 10 pmol)were added to Arabidopsis extracts (20 mg fresh weight),extracted with 50 mM Na-phosphate buffer (pH 7.0) containing0.025% DEDTCA and purified by the mixed-mode anion exchangesorbent. To some of the extracts, 1 pmol[13C8,15N1]-IBAinternal standard was also added.

Figure S5. Representative MRM chromatograms of20 mg samples from wild-type (Col-0; a) and mutant(sur1-3; b) plants.

Figure S6. Multivariate modelling of IAA metabolite profiles.

Principal component analysis plots show the correlation between observations (samples, black dots) with the variables (metabolites, red squares). Components are 1 and 2, which in each plot contain 60-90 % of the total variation of the data. The closer the dots are to the variables, the stronger the correlation. (a) Mutant line shoots versus mutant line roots –35S:YUC1/sur1-3/sur2-1(R/S); (b) Col-0 versustwo ecotypes (Ler, Ws), shoots (S); (c) Col-0 versus two ecotypes(Ler, Ws), roots (R).

Figure S7. IAA metabolite profiles in theestradiol inducible lines pMDC7:TAA1 (TAA1ox), pMDC7:YUC6(YUC6ox) and the empty vector wild-type controlpER8.

IAA, the IAA precursors (ANT, TRP, TRA, IAOx, IAM, IPyA, IAN, IAAld), conjugates (IAAsp and IAGlu) and the catabolite oxIAA were quantified in root (white bars) and shoot (black bars) tissues. Samples were analyzed in four independent biological replicates, and error bars represent the SD. Asterisks indicate statistically significant difference between Col-0 and other ecotypes and mutant lines (shoot and root tissues) at 95 % confidence level in a one-way ANOVA analysis.

Table S1. Auxin metabolite profiling of Arabidopsis wildtype and mutant lines.

Levels of IAA metabolites in 10 DAG Arabidopsis seedlings of the ecotypes Col-0, Ws and Ler, the mutant lines 35S:YUC1,sur1-3 and sur2-1 and the estradiol inducible linespMDC7:TAA1 And pMDC7:YUC6 (pER8 is the empty vector wildtype control). 20mg FW samples of root and shoot tissues were extracted in sodium phosphate buffer, partially derivatized by cysteamine, purified by one step SPE and quantified using LC-MRM-MS. IAEt, IBA, IAAla, IAGly, IALeu, IAPhe, IATrp and IAVal were not detected.

Data S1. Preparation of stable isotope-labelled internal standards.

(a) Synthesis of [indole-2H4]-IPyA and (b)synthesis of [indole-2H5]-IAOx.

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