PIN1, PIN2, and PIN4 Immunolocalization in Roots of 5-d Flavonoid Mutant Seedlings.
Confocal images of PIN immunohistochemical localizations. Asterisks indicate the position of quiescent center cells in PIN4 localization images. PIN2 and PIN4 confocal images have been false-colored; color is not as a result of autofluorescence. Bars in (A) to (K), (P) to (S), and (X) to (AA) = 25 μM; (L) to (O), (T) to (W), and (BB) to (EE) = 10 μM.
(A) to (E) PIN1 localization (green) in pin1 (A), Ler (B), tt4 (C), tt7 (D), and tt3 (E).
(F) to (J) PIN2 localization (red) in pin2 (F), Ler (G), tt4 (H), tt7 (I), and tt3 (J).
(K) to (O) PIN4 localization (blue) in pin4 (K), Ler (L), tt4 (M), tt7 (N), and tt3 (O).
(P) to (S) PIN1 localization in seedlings treated with 10 μM TIBA in Ler (P), tt4 (Q), tt7 (R), and tt3 (S).
(T) to (W) PIN4 localization in seedlings treated with 10 μM TIBA in Ler (T), tt4 (U), tt7 (V), and tt3 (W).
(X) to (AA) PIN1 localization in seedlings treated with 1 μM NPA in Ler (X), tt4 (Y), tt7 (Z), and tt3 (AA).
(BB) to (EE) PIN4 localization in seedlings treated with 1 μM NPA in Ler (BB), tt4 (CC), tt7 (DD), and tt3 (EE).

Altered Auxin Transport in 4.5-d Flavonoid Mutant Seedlings.
Auxin transport from shoot apex to R-S TZ and root tips of 4.5-d seedlings. Altogether 20 nM naringenin (N) was coapplied with radiolabeled auxin in Ler plus N and tt4 plus N treatments. The mean Ler wild-type values were 2,933.3 ± 137.6 disintegrations per minute (DPM) at the R-S TZ and 655.8 ± 162.5 DPM at the root tip. Values shown are means ± sd for two independent experiments. The experiment was replicated with identical results. *, Significance was determined by ANOVA followed by Tukey post hoc test; P < 0.05 compared with untreated wild type.

Flavonoid–Auxin Interactions in Roots.
(A) to (J) Confocal images.
(K) to (P) Epifluorescence images.
(A) to (F) Five-day tt4 seedlings treated with exogenous flavonoids. PIN1 immunolocalization (green) in tt4 seedlings treated with 1 nM naringenin (N) in (A), 10 nM kaempferol (K) in (B), and 1 nM quercetin (Q) in (C). PIN4 localization (blue) in tt4 seedlings treated with 1 nM naringenin (N) in (D), 10 nM kaempferol (K) in (E), and 1 nM quercetin (Q) in (F). Asterisk is above quiescent center cells in PIN4 localization images.
(G) and (J) PIN1 localization in 5-d root tips treated with BFA and after flavonol washout.
(G) PIN1 is observed in BFA bodies after BFA treatment in tt4.
(H) PIN1 signal is diffuse in addition to polar localization in the vascular tissue after BFA washout in tt4.
(I) PIN1 signal is observed at the plasma membrane after BFA washout with flavonols in wild-type root tips. Inset, PIN1 signal remains in BFA bodies in a nonflavonol-containing region of the root tip.
(J) PIN1 remains in BFA bodies after flavonol washout of BFA in tt4.
(K) to (P) Flavonoid fluorescence in 5-d Ler seedlings stained with DPBA.
(K) Kaempferol fluorescence is observed at the plasma membrane in the proximal elongation zone of 4.75-d untreated Ler. This is the region above the brightly fluorescing naringenin chalcone area (Murphy et al., 2000; Peer et al., 2001).
(L) Kaempferol fluorescence is observed in pericycle, columella initials, and columella tiers 1, 2, and 3; kaempferol and quercetin fluorescence is observed in the root cap in 4.75-d untreated Ler. Arrows point to kaempferol fluorescent columella initials and tier 3/story 3 columella cells.
(M) Quercetin aggregates are observed in the proximal elongation zone of 4.75-d Ler seedlings treated with a droplet of IAA placed at shoot apex and stained 5 h after IAA application (as for auxin transport assays).
(N) Root tip of 4.75-d Ler plus IAA droplet placed at shoot apex and stained 5 h after application (as for auxin transport assays). Arrow points to tier 3/story 3 columella (C3/S3) cells with quercetin fluorescence after IAA treatment.
(O) Proximal elongation zone of 5-d Ler transferred to NPA at 3 d (long-term global NPA treatment).
(P) Root tip of 5-d Ler transferred to NPA at 3 d (long-term global NPA treatment).
Bars in (A) to (C) = 25 μM; (D) to (J) = 10 μM; and (K) to (P) = 25 μM.