Supplemental Data

Files in this Data Supplement:

• Supplemental Table 1
• Supplemental Figure 1 - Supplemental Figure 1. Regulation of PGP4 and PGP21 gene expressions by chemical stresses. Untreated control (Cont.). 14 day seedlings in liquid MS medium were treated for 24 hr with 10 μM indole-3-acetic acid (IAA), 10 μM 1-naphthaleneacetic acid (NAA), 10 μM 2, 4- dichlorophenoxyacetic acid (2, 4-D), 10 μM 6-benzyladenine (BA), 10 μM kinetin (kin.). Seedlings were frozen in liquid nitrogen. RNA extraction was performed using RNeasy Plant Mini Kit (Qiagen). Semi-quantitative RT-PCR was performed using cDNA templates prepared using SuperScript^TM III RNase H^- reverse transcriptase (Invitogen Corp.). Numbers indicate the cycles of PCR. Amplified DNA fragments were stained ethidium bromide. As an internal control, the gene coding for actin was chosen.
• Supplemental Figure 2 - Supplemental Figure 2. Complementation of pgp4-1. (A) Lateral root phenotype complementation of pgp4-1. Bar, 1 cm. (B) PGP4OX lines and pgp4-1 complementation. Bar, 1 cm. (C) Root basipetal auxin transport complementation of pgp4-1. Transport was not statistically different from WT.
• Supplemental Figure 3 - Supplemental Figure 3. MPIN2 localization in pgp4-1 and PGP4OX. Confocal images showing immunohistochemical localization of PIN2 in 5-d seedlings. (A), (E), (I). 20x images of PIN2 localization in WT, PGP4OX, and pgp4-1, respectively. (B), (F), (J). 60x images of PIN2 localization in WT, PGP4OX, and pgp4-1, respectively. (C), (G), (K). DIC images of WT, PGP4OX, and pgp4-1, respectively. (D), (H), (L). Overlays of confocal and DIC images of WT, PGP4OX, and pgp4-1, respectively. (M). 100x image showing normal PIN2 localization in epidermal and cortical layers of pgp4-1. Size bars: A, E, I, 200 μm; B-D, F-H, J-L, 150 μm; M, 40μm.
• Supplemental Figure 4 - Supplemental Figure 4. PIN1 localization in pgp4-1and PGP4OX. (A) PIN1 localization in wild type root tips. (B) PIN1 localization in PGP4OX root tips. (C) PIN1 localization in pgp4-1 root tips. Size bar, 50μm.
• Supplemental Figure 5 - Supplemental Figure 5. Flavonol accumulations in pgp4. Although flavonols act inhibit auxin transport at the root and shoot apices, they also accumulate at sites of (Murphy et al., 2000; Brown et al., 2001; Peer et al., 2001; 2004). Observations of Arabidopsis tissues responding to exogenous application of auxin and AEIs indicate that flavonols are sensitive indicators of localized auxin accumulations as flavonoid localization and speciation are altered by local auxin concentrations (Peer et al., 2004; Buer and Muday, 2004). We compared the flavonoid accumulation pattern in pgp4-1 with wild-type using diphenyl boronic acid (DPBA) staining. Consistent with decreased root basipetal auxin transport, cortical tissues in upper roots of pgp4-1 exhibited decreased accumulations of quercetin (Fig. S5C, D), and root elongation zone cells that are quercetin-deficient in wild-type accumulated intracellular quercetin aggregations (Fig. 5E, F) similar to those seen after localized application of auxin to wild-type (Peer et al., 2004). Quercetin accumulation at the cotyledonary node was not observed in pgp4-1 seedlings (Fig. S5A, B). (A) Cotyledonary nodes of Col-0 wild-type (WT) seedlings accumulate quercetin (golden fluorescence). (B) Cotyledonary nodes of pgp4-1 seedlings do not accumulate quercetin; however, quercetin accumulates in the petioles. Red is chlorophyll autofluorescence (C) WT seedlings accumulate quercetin in the upper root. (D) Quercetin accumulation was restricted to a smaller region in the upper root of pgp4-1 seedlings than in WT. Bar, 100 μm.