Flavonoids redirect PIN-mediated polar auxin fluxes during root gravitropic responses

Abstract

The rate, polarity, and symmetry of the flow of the plant hormone auxin are determined by the polar cellular localization of PIN-FORMED (PIN) auxin efflux carriers. Flavonoids, a class of secondary plant metabolites, have been suspected to modulate auxin transport and tropic responses. Nevertheless, the identity of specific flavonoid compounds involved and their molecular function and targets in vivo are essentially unknown. Here we show that the root elongation zone of agravitropic pin2/eir1/wav6/agr1 has an altered pattern and amount of flavonol glycosides. Application of nanomolar concentrations of flavonols to pin2 roots is sufficient to partially restore root gravitropism. By employing a quantitative cell biological approach, we demonstrate that flavonoids partially restore the formation of lateral auxin gradients in the absence of PIN2. Chemical complementation by flavonoids correlates with an asymmetric distribution of the PIN1 protein. pin2 complementation probably does not result from inhibition of auxin efflux, as supply of the auxin transport inhibitor N-1-naphthylphthalamic acid failed to restore pin2 gravitropism. We propose that flavonoids promote asymmetric PIN shifts during gravity stimulation, thus redirecting basipetal auxin streams necessary for root bending.

FIGURE 1.

Defects in basipetal auxin transport are associated with altered root flavonoid accumulation. A-D, flavonoid accumulation in the entire root and root elongation zone of wild type (Col Wt; A and C) and pin2 (B and D). Accumulation of flavonoids in control and 2-h gravity-stimulated roots visualized in situ using DPBA (yellow fluorescence) as described under “Experimental Procedures.” The arrows indicate the direction of the gravity vector relative to the root. Bar, 100 μm. E, total amount of flavonoid derivatives detected in the entire root and RT-EZ of wild type and pin2. F, gravity-induced root phenolic compound accumulation normalized to phenolic compound accumulation in vertical control. Values represent mean ± S.E. (n = 2-5 replicates); ^*, significantly different from the wild type (Student's t test, p < 0.05). G, representative sum of extracted ion chromatograms [M - H]^- of flavonoid derivatives found in wild type and pin2 RT-EZ analyzed by HPLC-ESI-MS. Significantly altered compounds are indicated by arrows. Peak numbers correspond to flavonoid derivates listed in Table 1. Note the 10 times lower intensity scale for pin2 root elongation zone in comparison to wild type.

FIGURE 2.

Exogenous flavonols partially rescue the agravitropic response of pin2 by restoring asymmetric auxin gradients. A, gravity responses of wild-type (Col Wt) and pin2 (eir1-4) roots 24 h after reorientation of 90° to horizontal in the presence of the indicated auxin transport inhibitors. Each gravity-stimulated root was assigned to one of 12, 30° sectors (see schematic). The length of each bar represents the mean percentages ± S.D. of seedlings showing the same direction of root growth of at least three independent experiments; numbers correspond to the mean (±S.D.) percent occurrence of 90 and 60° bending (sum of 90° and 60° sectors). B, time series of wild-type (Col Wt) and pin2 (eir1-4) root curvature after reorientation of 90° to horizontal in the presence and absence of 100 nm quercetin analyzed as described in Ref. 24). Data are mean ± S.E. (n = 4 with 50 seedlings per allele and treatment). C, expression of the auxin-reporter construct DR5_rev-GFP in wild type (Col Wt) and pin2 (eir1-4) root tips was assessed prior to, and after 2 h gravity stimulation on control (top row), quercetin (middle row), and kaempferol-treated roots (bottom row). White asterisks indicate more pronounced DR5-GFP expression at the lower side of gravistimulated roots suggesting enhanced basipetal auxin reflux. The gravity vector relative to the root tip is indicated by an arrow. QC, quiescent center; QI, columella initials; col, mature columella cells; dLRC, distal lateral root cap; pLRC, proximal lateral root cap. Bar, 75 μm.

FIGURE 3.

Exogenous NPA does not rescue the agravitropic response of pin2. A, gravity responses of wild-type (Col Wt) and pin2 (eir1-4) roots 24 h after reorientation of 90° to horizontal in the presence of the indicated concentrations of NPA. Each gravity-stimulated root was assigned to one of 12, 30° sectors (see Fig. 2). The length of each bar represents the mean percentages ± S.D. of seedlings showing the same direction of root growth of at least three independent experiments; numbers correspond to the mean (±S.D.) percent occurrence of 90 and 60° bending (sum of 90 and 60° sectors). B, mean percentages of plants showing 90 and 60° bending (sum of 90 and 60° sectors) ± S.D. of three independent experiments.

FIGURE 4.

Flavonoid-dependent rescue of pin2 agravitropic phenotype requires PIN1 and is correlated with asymmetric PIN1 distribution across gravity-stimulated tissues. A, gravity responses of pin2 pin3 pin7 and pin1 pin2 (pin1 eir1) roots after 24 h in the presence of quercetin. The length of each bar represents the mean percentages ± S.D. of seedlings showing the same direction of root growth of at least three independent experiments; numbers correspond to the mean (±S.D.) percent occurrence of 60 and 90° bending (sum of 60 and 90° sectors). B, whole-mount in situ immunolocalization of PIN1 protein (red) in 5-day pin2 (eir1-4; ii and iv) and wild-type (Col Wt; i and iii) vertical seedlings transferred on media supplemented with 100 nm quercetin. Gravity vector is indicated by an arrow. White arrows indicate PIN1 protein apical localization in the epidermis and basal localization in the cortex cells of pin2 root tip. Note that the appearance of slightly different PIN1 signals in the pin2 epidermis and cortex (ii-iv) do not reflect unequal expression but are the result of unequal background intensities due to scattered light. v, vascular bundle; en, endodermis; c, cortex; e, epidermis. Bar, 30 μm. C, whole mount in situ immunolocalization of PIN1 protein in pin2 after 2 h of gravity stimulation; gravity vector is indicated by an arrow. 4-Day pin2 seedlings were transferred on media supplemented with 100 nm quercetin or the solvent (control). Red, PIN1; green, DR5_rev-GFP expression. White arrows indicate more pronounced PIN1 proteins levels at the lower or upper side of the gravity-stimulated root tip. Bar, 30 μm. Percentages indicate relative occurrence of asymmetric or symmetric PIN1 distributions with asymmetric DR5-GFP signals; the total number of analyzed roots that showed simultaneous clear DR5-GFP and PIN1 signals was 47.