Supplemental Data: Figures Direct isolation of flavonoids from plants using ultra-small anatase TiO2 nanoparticles Jasmina Kurepa, Ryo Nakabayashi, Tatjana Paunesku, Makoto Suzuki, Kazuki Saito, Gayle E. Woloschak and Jan A. Smalle Figure S1 List and nomenclature of cyanidin derivatives and flavonoid glycosides detected in Col-0 plants grown on MS/2 media with 4% sucrose. Figure S2 UPLC-PDA chromatograms of aqueous acid methanol extracts of Col-0 plants grown on 1% or 4% sucrose media. Figure S3 Figure S4 Nanoharvesting flavonoids from rosettes and roots. Effects of nanoparticle concentration and incubation temperature on nanoharvesting yield. Figure S5 Figure S6 Figure S7 Effects of pH on nanoharvesting. Efficiency of nanoharvesting at different pH values. Representative ion chromatograms of total plant extracts and anthocyanins released from nanoparticle coronas. Cellular damage and superoxide radical production in leaves treated with nanoparticles. Figure S8 1 Supplementary Figure S1. List and nomenclature of cyanidin derivatives and flavonoid glycosides detected in Col-0 plants grown on MS/2 media with 4% sucrose. Total flavonoids were isolated and analyzed using LC-ESI-Q-TOF-MS/MS as described (Matsuda et al. 2009). (a) Isolated antocyanin derivatives (A1-A11) A1: cyanidin 3-O-[2-O-(-D-xylopyranosyl)--D-glucopyranoside]-5-O--D-glucopyranoside A2: cyanidin glucopyranoside] 3-O-[2-O-(-D-xylopyranosyl)--D-glucopyranoside]-5-O-[6-O-(malonyl)--D- A3: cyanidin 3-O-[2-O-(-D-xylopyranosyl)-6-O-(E-p-coumaroyl)--D-glucopyranoside]-5-O--Dglucopyranoside A4: cyanidin 3-O-[2-O-(2-O-(E-sinapoyl)--D-xylopyranosyl)--D-glucopyranoside-5-O--Dglucopyranoside A5: cyanidin 3-O-[2-O-(-D-xylopyranosyl) -6-O-(E-p-coumaroyl)--D-glucopyranoside]-5-O-[6O-(malonyl)--D-glucopyranoside] A6: cyanidin 3-O-[2-O-(-D-xylopyranosyl) -6-O-(4-O-(-D-glucopyranosyl)-E-p-coumaroyl)--Dglucopyranoside]-5-O--D-glucopyranoside 2 A7: cyanidin 3-O-[2-O-(2-O-(E-sinapoyl)--D-xylopyranosyl)-6-O-(4-O-E-p-coumaroyl)--Dglucopyranoside]-5-O--D-glucopyranoside A8: cyanidin 3-O-[2-O--D-xylopyranosyl-6-O-(4-O-E-p-coumaroyl)--D-glucopyranoside]-5-O[6-O-(malonyl)--D-glucopyranoside] A9: cyanidin-3-O-[2-O-(2-O-(E-sinapoyl)--D-xylopyranosyl)-6-O-(4-O-E-p-coumaroyl)--Dglucopyranoside]-5-O-[6-O-(malonyl)--D-glucopyranoside] A10: cyanidin 3-O-[2-O-(2-O-(E-sinapoyl)--D-xylopyranosyl)-6-O-(4-O-(-D-glucopyranosyl)-Ep-coumaroyl)--D-glucopyranoside]-5-O--D-glucopyranoside A11: cyanidin 3-O-[2-O-(2-O-(E-sinapoyl--D-xylopyranosyl)-6-O-(4-O-(-D-glucopyranosyl)-(Ep-coumaroyl)--D-glucopyranoside)-5-O-[6-O-(malonyl)--D-glucopyranoside] (b) Isolated flavonols (f1-f8). f1: kaempferol 3-O--L-rhamnopyranoside-7-O--L-rhamnopyranoside f2: kaempferol 3-O--D-glucopyranoside-7-O--L-rhamnopyranoside f3: kaempferol rhamnopyranoside 3-O--L-rhamnopyranosyl (1 → 2)--D-glucopyranoside-7-O--L- f5: quercetin 3-O--D-glucopyranoside-7-O--L-rhamnopyranoside f6: quercetin 3-O--L-rhamnopyranoside-7-O--L-rhamnopyranoside f8: quercetin 3-O--L-rhamnopyranosyl (1→2)--D-glucopyranoside-7-O--L-rhamnopyranoside 3 Supplementary Figure S2. UPLC-PDA chromatograms of aqueous acid methanol extracts of Col-0 plants grown on 1% or 4% sucrose media. (a) Accumulation of anthocyanins in plants grown on 4% sucrose media. Anthocyanins were extracted by aqueous acid methanol followed by chloroform partitioning (Kubasek et al. 1992). Data are mean ± standard error of mean of three measurements. (b) Peak labels correspond to compounds shown in Supplementary Figure 2. s1 and s2 are sinapoyl derivatives, and s2i is an isomer of s2 (Yonekura-Sakakibara et al. 2008). The analyses show that in addition to anthocyanin species such as A11 (cyanidin 3-O-[2-O-(2O-(E-sinapoyl--D-xylopyranosyl)-6-O-(4-O-(-D-glucopyranosyl)-(E-p-coumaroyl)--Dglucopyranoside)-5-O-[6-O-(malonyl)--D-glucopyranoside]), sucrose led to an increase in flavonols such as f3 and f1 (kaempferol 3-O--L-rhamnopyranosyl (1→2)--D-glucopyranoside7-O--L-rhamnopyranoside and kaempferol 3-O--L-rhamnopyranoside-7-O--Lrhamnopyranoside, respetively) and sinapoyl derivates. The zoomed chromatogram and relative abundances of flavonols in 1% and 4% sucrose-treated plants are shown in the Figure 3. 4 Supplementary Figure S3. Nanoharvesting flavonoids from rosettes and roots. Methods: Col-0 plants grown on MS/2 media with 1% sucrose for three weeks were dissected, and an equal mass (~20 mg) of shoot or root tissue was mixed with TiO 2 nanoparticles (pH 5.0, 76.7 mg/L). Harvesting was done for 24 hours at 22°C in the dark. Plant tissues were removed from the tubes prior to photography. Nanoconjugates (NC) where pelleted for 1 min at 1000 g, resuspended in a 10 mM phosphate buffer (pH 5.7), vortexed and sonicated for 1 min. 1 µl of NC suspension was used to determine the absorption spectra using NanoDrop 2000. (a) Nanoharvesting from shoots and roots. (b) The UV-Vis light absorption spectra of nanoconjugates isolated from rosettes and roots. NP, phosphorylated TiO2 nanoparticles. Conclusions: The most prominent flavonoid species in roots are quercetin and kaempherol derivates (Yonekura-Sakakibara et al. 2008). Both quercetin and kaempherol upon conjugation to anatase nanoparticles form yellow-orange nanoconjugates (Figure 1). The UV-Vis absorption spectrum of root NCs has a distinct peak at 420 nm, which corresponds to the conjugated flavonols, and the spectrum of shoot NC has a peak at 600 nm that corresponds to the conjugated cyanidin derivatives. 5 Supplementary Figure S4. Effects of nanoparticle concentration and incubation temperature on nanoharvesting yield. (a) and (b) Equal amounts of leaf tissue (~20 mg) from plants grown on 4% sucrose were incubated in 10 mM sodium phosphate buffer (pH 6.0) containing the denoted concentration of ultra-small TiO2 nanoparticles (NP). After 4 hours of co-incubation at 22°C in the dark, the tissues were removed, the incubation vessels were photographed (a). The nanoconjugates were pelleted (1 min, 1000 g, room temperature) and resuspended in 10 mM sodium phosphate buffer (pH 6.0). One µl was used to determine the absorption at 590 nm (peak absorption for anthocyanin-TiO2 nanoconjugates (b). (c) Equal amounts of leaf tissue (~20 mg) from plants grown on 4% sucrose were incubated in a nanoparticle suspension in the dark at 4°C or 24°C for 12 hours. The leaf tissues were removed prior to photography. Supplementary Figure S5. Effects of pH on nanoharvesting. Methods: An equal mass (~10 mg) of true leaves from plants grown on MS/2 with 4% sucrose or of roots from plants grown on vertically positioned MS/2 plates with 1% sucrose was incubated in a 500 µl of TiO2 nanoparticle suspension in 10 mM Na phosphate buffer (final mass concentration 76.7 mg/L) for 4 hours in the dark at 22°C. The pH of the nanoparticle suspension was adjusted with HCl or NaOH. 6 (a) pH dependence of nanoharvesting from leaves. The highest yield of anthocyanin nanoconjugates (NCs) was observed at the pH 5–6 range. A similar trend was observed when the pH of the nanoparticle suspensions was adjusted by H2PO4 or KOH (not shown). SN, supernatant. (b) Nanoconjugates shown in (a) were pelleted by centrifugation, and resuspended in a 0.2 V of 10 mM Na phosphate buffer at pH 6. These suspensions were used for the quantifications by UV-Vis spectroscopy shown in Figure S6. (c) pH dependence of nanoharvesting from roots. Similar to the nanoharvesting efficiency from leaves, the maximal yield of nanoparticles with plant metabolite corona was obtained within the pH 5-6 range. Supplementary Figure S6. Efficiency of nanoharvesting at different pH values. Methods: After nanoharvesting, the source plant tissue was saved, briefly washed in a 10 mM sodium phosphate buffer (pH 6.0), and used for anthocyanin extraction by the acid aqueous methanol method (Kubasek et al. 1992). Nanoconjugates (shown in Figure S6) were pelleted by centrifugation (1 min at 1000 g) and the pellet was resuspended in 100 µl of a 10 mM sodium phosphate buffer (pH 6.0). The supernatant was also saved for further analyses. The acid aqueous methanol extracts of the source plant tissue, supernatants of the nanoharvesting reactions and nanoconjugates suspensions in phosphate buffer were used for spectral analyses using NanoDrop 2000. 7 (a) Comparison of the UV-Vis spectra of nanoconjugates (NC) isolated at different pH values (red line) and control, phosphorylated TiO2 nanoparticles (NP, blue line). The representative UVVis spectra are shown, and the arrowhead pointing at 590 nm (a peak unique for nanoparticles with anthocyanin corona) is added for clarity. (b) Relative levels of anthocyanins (measured at 530 nm, A530) and anthocyanin nanoconjugates (measured at 590 nm, A590). The absorption was measured in the coincubation solutions separated from nanoconjugates by centrifugation (supernatant), nanoconjugates (pellet) and in the acid methanol extracts of the plants used for nanoharvesting (residual). Data are shown as mean ± SD (n=9) and are expressed in relative units (RU). 8 Supplementary Figure S7. Representative ion chromatograms of total plant extracts and anthocyanins released from in vivo-formed nanoconjugates (NC). The identities of cyanidin glycosides (A) are listed in Supplemental Figure 2. Arrows point to the peaks with the denoted m/z ratios. 9 Supplementary Figure S8. Cellular damage and superoxide radical production in leaves treated with nanoparticles. (a) Axenically grown seedlings were incubated with a 76.7 mg/L nanoparticle suspension for 4 hours, rinsed in sterile water and stained with the vital stain SYTOX Green (Molecular Probes, http://www.lifetechnologies.com) essentially as described (Truernit and Haseloff 2008). In brief, seedlings were incubated in 0.2 μM SYTOX Green solution (pH 7) in the dark for 20 min and then rinsed in distilled water. Seedlings were mounted in Fluoro-Gel solution (Electron Microscopy Sciences, http://www.emsdiasum.com) and analyzed using an Olympus BX51 fluorescent microscope. Representative images (scale bar = 100 µm) are shown. (b) The number of nuclei per leaf area (mm2) was quantified using ImageJ software. Data are an average ± SD of 5 counts. (c) Superoxide radical (O2.-) detection. Plants were grown and treated with nanoparticles as described in (a). The nitro blue tetrazolium (NBT) staining of leaves was done as described (Kurepa et al. 2010). 10 References Kubasek, W.L., Shirley, B.W., McKillop, A., Goodman, H.M., Briggs, W. and Ausubel, F.M. (1992) Regulation of flavonoid biosynthetic genes in germinating Arabidopsis seedlings. Plant Cell, 4, 1229-1236. Kurepa, J., Paunesku, T., Vogt, S., Arora, H., Rabatic, B.M., Lu, J., Wanzer, M.B., Woloschak, G.E. and Smalle, J.A. (2010) Uptake and distribution of ultrasmall anatase TiO2 alizarin red S nanoconjugates in Arabidopsis thaliana. Nano Lett, 10, 2296-2302. Matsuda, F., Yonekura-Sakakibara, K., Niida, R., Kuromori, T., Shinozaki, K. and Saito, K. (2009) MS/MS spectral tag-based annotation of non-targeted profile of plant secondary metabolites. Plant J, 57, 555-577. Truernit, E. and Haseloff, J. (2008) A simple way to identify non-viable cells within living plant tissue using confocal microscopy. Plant Methods, 4, 15. Yonekura-Sakakibara, K., Tohge, T., Matsuda, F., Nakabayashi, R., Takayama, H., Niida, R., Watanabe-Takahashi, A., Inoue, E. and Saito, K. (2008) Comprehensive flavonol profiling and transcriptome coexpression analysis leading to decoding gene-metabolite correlations in Arabidopsis. Plant Cell, 20, 2160-2176. 11