Transcription factor WRKY23 assists auxin distribution patterns during Arabidopsis root development through local control on flavonol biosynthesis

Abstract

Gradients of the plant hormone auxin, which depend on its active intercellular transport, are crucial for the maintenance of root meristematic activity. This directional transport is largely orchestrated by a complex interaction of specific influx and efflux carriers that mediate the auxin flow into and out of cells, respectively. Besides these transport proteins, plant-specific polyphenolic compounds known as flavonols have been shown to act as endogenous regulators of auxin transport. However, only limited information is available on how flavonol synthesis is developmentally regulated. Using reduction-of-function and overexpression approaches in parallel, we demonstrate that the WRKY23 transcription factor is needed for proper root growth and development by stimulating the local biosynthesis of flavonols. The expression of WRKY23 itself is controlled by auxin through the Auxin Response Factor 7 (ARF7) and ARF19 transcriptional response pathway. Our results suggest a model in which WRKY23 is part of a transcriptional feedback loop of auxin on its own transport through local regulation of flavonol biosynthesis.

Fig. 1.

WRKY23 controls maintenance of the root stem cell niche. (A–C) WRKY23::WRKY23^RNAi plants showing a phenotypic variation from WT-looking plants (class I) (A) to mildly (class II) (B) and strongly (class III) (C) affected seedlings. (D) Schematic representation of the Arabidopsis root tip. Blue, QC; light pink, columella initials; pink, columella root cap; brown, lateral root cap. (E–G) Primary root tips of WT (E) (94.1%; n = 17), class II WRKY23::WRKY23^RNAi (F) (91.9%; n = 62), and 35S::WRKY23-SRDX (G) (96.7%; n = 30) seedlings. White arrowheads indicate extra divisions, and asterisks mark the rows of columella cells. (H) Ectopic expression of WRKY23 reduces root growth (Left, WT; Right, 35S::WRKY23). (I–L) Q0680 (I and J) and QC184 (K and L) marker analysis in WT (I and K) and 35S::WRKY23 (J and L) root tips. Arrowheads point toward columella initials.

Fig. 2.

WRKY23 is a negative regulator of auxin transport. (A–C) Auxin response in WT (A), WRKY23::WRKY23^RNAi (B), and 35S::WRKY23-SRDX (C) root tips visualized by short staining of DR5::GUS. Arrowheads indicate QC cells. (D and E) DR5::GUS-visualized auxin response during WT (D) and 35S::WRKY23-SRDX (E) lateral root initiation. Arrowheads denote the cell walls of asymmetrically divided pericycle cells. P, pericycle; En, endodermis; Co, cortex; Ep, epidermis. (F and G) Auxin response in WT (F) and 35S::WRKY23 (G) root tips visualized by long staining of DR5::GUS. Arrowheads indicate QC cells. (H) Auxin transport measurements showing an enhanced transport in WRKY23::WRKY23^RNAi and a reduced acropetal transport in 35S::WRKY23-GR roots on Dex treatment compared with the WT and DMSO control, respectively. *P < 0.05; **P < 0.01, Student t test.

Fig. 3.

Flavonol biosynthesis is regulated by WRKY23. (A) qRT-PCR expression analysis of TT4, TT5, TT6, and TT7 in WT, WRKY23::WRKY23^RNAi, RPS5A>>WRKY23, and mock-treated and Dex-treated 35S::WRKY23-GR seedlings. Error bars represent SD. (B and C) DPBA-stained WT (B) and 35S::WRKY23 (C) roots. (D and E) Extracted ion chromatograms (m/z = 447.09) showing the increased quercetin-3-O-rhamnoside (Q-3-O-R, green arrow) in 35S::WRKY23 roots (E) compared with WT C24 (D). (F and G) Base peak intensity chromatograms demonstrating the reduction in Q-R-G (red arrow) and rhamnetin-O-neohesperidoside (Rh, blue arrow) in WRKY23::WRKY23^RNAi roots compared with WT Col-0.

Fig. 4.

Local flavonol production is stimulated by WRKY23 in an ARF7/ARF19-dependent manner. (A) Auxin-induced WRKY23 expression is abolished in tir1-3 and arf7arf19 mutants. Black, mock-treated seedlings; gray, NAA-treated seedlings. Error bars represent SD. (B) qRT-PCR data showing auxin-induced expression of TT4 and TT7. Error bars represent SD. (C and D) DPBA-visualized flavonol accumulation induced by auxin (NAA) (D) compared with mock-treated roots (C). (E and F) Upon auxin treatment, WRKY23 expression is induced in the basal meristem, an area of strong flavonoid accumulation. Note that E and F are composite images. (G–J) Auxin-induced flavonol accumulation is not observed in the arf7arf19 background (G and H) or in WRKY23::WRKY23^RNAi roots (I and J). (K–M) Early root meristem defects typical for 35S::WRKY23 seedlings are rescued by the absence of flavonols in the tt4-8 mutant. (K) tt4-8. (L) 35S::WRKY23. (M) tt4-8 × 35S::WRKY23.