Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks - PubMed (original) (raw)

Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks

Daniel R Lewis et al. Plant Physiol. 2011 May.

Abstract

Auxin and ethylene are key regulators of plant growth and development, and thus the transcriptional networks that mediate responses to these hormones have been the subject of intense research. This study dissected the hormonal cross talk regulating the synthesis of flavonols and examined their impact on root growth and development. We analyzed the effects of auxin and an ethylene precursor on roots of wild-type and hormone-insensitive Arabidopsis (Arabidopsis thaliana) mutants at the transcript, protein, and metabolite levels at high spatial and temporal resolution. Indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) differentially increased flavonol pathway transcripts and flavonol accumulation, altering the relative abundance of quercetin and kaempferol. The IAA, but not ACC, response is lost in the transport inhibitor response1 (tir1) auxin receptor mutant, while ACC responses, but not IAA responses, are lost in ethylene insensitive2 (ein2) and ethylene resistant1 (etr1) ethylene signaling mutants. A kinetic analysis identified increases in transcripts encoding the transcriptional regulators MYB12, Transparent Testa Glabra1, and Production of Anthocyanin Pigment after hormone treatments, which preceded increases in transcripts encoding flavonoid biosynthetic enzymes. In addition, myb12 mutants were insensitive to the effects of auxin and ethylene on flavonol metabolism. The equivalent phenotypes for transparent testa4 (tt4), which makes no flavonols, and tt7, which makes kaempferol but not quercetin, showed that quercetin derivatives are the inhibitors of basipetal root auxin transport, gravitropism, and elongation growth. Collectively, these experiments demonstrate that auxin and ethylene regulate flavonol biosynthesis through distinct signaling networks involving TIR1 and EIN2/ETR1, respectively, both of which converge on MYB12. This study also provides new evidence that quercetin is the flavonol that modulates basipetal auxin transport.

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Figures

Figure 1.

Figure 1.

The flavonoid biosynthetic pathway. Intermediate compounds and enzymes in the pathway are shown with mutant alleles represented in brackets. The structures of naringenin and quercetin are shown, and downstream products are represented at the bottom of the diagram. Enzymes, mutants, and metabolites central to this study are shown in boldface. This figure is modified from Buer and Muday (2004).

Figure 2.

Figure 2.

IAA and ACC enhance the expression of genes encoding flavonol pathway enzymes. The abundance of CHS, FLS, and F3′H transcripts relative to actin and normalized to the untreated wild type (WT) 6 h after treatment with 1 μ

m

IAA and ACC is shown in the wild type, tir1, ein2, etr1, and myb12. The basal level and fold induction of CHS (A), FLS (B), and F3′H (C) transcript abundance after treatment with IAA and ACC are shown in all genotypes. Mean and

se

of three biological replicates are shown. * Significant difference (P < 0.05) between the mutant and the wild type within a treatment; # significant difference between treated and untreated controls within a genotype as determined by Student’s t test (P < 0.05).

Figure 3.

Figure 3.

Determination and validation of LSCM settings for kaempferol and quercetin measurement. A, Graph of the emission spectra for kaempferol (K), quercetin (Q), and isorhamnetin (IR) acquired with tt7-2 roots as well as isorhamnetin- and quercetin-fed tt4-2 roots, with the wavelength range used for K-DPBA collection shaded in green and the Q-DPBA collection window in gold. B, Confocal micrographs of 6-d-old DPBA-stained wild-type, tt4-2, tt7-2, and tt4-2 primary roots 6 h after treatment with 10 μ

m

quercetin illustrate the specificity of these settings for detecting only K-DPBA in green or Q-DPBA in yellow. Bar = 100 μm.

Figure 4.

Figure 4.

IAA and ACC increase flavonoid accumulation in primary roots and alter the distribution of these metabolites. A, Representative images of primary roots stained with DPBA 8 h after mock treatment or exposure to 1 μ

m

IAA or 1 μ

m

ACC. K-DPBA- and Q-DPBA-specific signals were collected, pseudocolored, and displayed as individual channels and an overlay. The right column shows higher magnification images that were used for quantification in the area defined by the red box, which contains seven cells. Mock-, IAA-, and ACC-treated roots are shown in rows one to three, respectively. Bar in left three columns = 100 μm; bar in right column = 50 μm. B, Comparison of DPBA and FIE-MS analyses of flavonoid accumulation in primary root tips in the elongation zone (DPBA) or in 5-mm root tips containing the elongation zone and an additional 4 mm (FIE-MS) after treatment with IAA and ACC. The two methods show a similar increase in flavonol accumulation 12 h after hormone treatment. # Significant difference between treated and untreated controls within a metabolite as determined by Student’s t test (P < 0.05). C, Six-day-old DPBA-stained wild-type, tir1-1, and ein2-5 primary roots. Representative images are from three separate analyses, each of which consisted of more than five micrographs each per genotype taken with identical gain and laser intensity. Gain settings were increased relative to A to better visualize DPBA staining in tir1-1 and ein2-5. Bar = 100 μm.

Figure 5.

Figure 5.

In Arabidopsis roots, IAA and ACC differentially induce gene expression, which predicts metabolite accumulation kinetics. A and B, Kinetic analysis of CHS, F3′H, and FLS expression via qRT-PCR after treatment with 1 μ

m

IAA (A) and ACC (B). Abundance of the mRNA from three genes relative to actin is represented as fold increase over untreated controls. C and D, Q-DPBA- and K-DPBA-specific fluorescence in primary roots stained with DPBA was monitored over time in response to IAA (C) and ACC (D). E and F, Kinetic analysis of IAA-induced (E) and ACC-induced (F) changes in CHS, MYB12, TTG1, and PAP1 expression determined from RNA samples used in A and B. Average and

se

of normalized gene expression from three pools of individuals are shown in A, B, E, and F; average and

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of DPBA fluorescence from three independent trials containing eight individual roots each are shown in C and D.

Figure 6.

Figure 6.

CHSpro:CHS-GFP is induced in the elongation zone of primary roots in response to IAA and ACC. A, Representative images of CHSpro:CHS-GFP in primary roots of 6-d-old seedlings counterstained with propidium iodide (shown in white) at 8 h after the indicated treatment. A digitally reconstructed maximum projection is shown from a z-stack of micrographs, as is a cross-section showing a single optical slice from the center of the root. Representative images are shown from two trials in which 20 total micrographs were taken. Bar = 100 μm. B, Comparison of CHSpro:CHS-GFP fluorescence, CHS transcript, and flavonol accumulation during a time course after IAA treatment. C, Comparison of CHSpro:CHS-GFP fluorescence, CHS transcript, and flavonol accumulation during a time course after ACC treatment. For CHSpro:CHS-GFP, the average and

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are shown from two separate experiments, quantitatively imaging six roots per time point. The transcript data from Figure 5 is included for comparison. To reflect the total flavonol pool, the Q-DPBA and K-DPBA fluorescence from Figure 5 are combined.

Figure 7.

Figure 7.

Auxin and ethylene responsiveness of PAP1 and TTG1 in roots requires TIR1, EIN2, and MYB12. A, The basal level and fold induction of MYB12, PAP1, and TTG1 transcript abundance 6 h after treatment with IAA and ACC is shown in all genotypes. TIR1 and EIN2 are necessary for auxin- and ethylene-dependent gene expression changes, respectively. All data are normalized to untreated wild-type (WT) expression levels. B, Mutations in MYB12 prevent the induction of PAP1 and TTG1 transcript changes after IAA and ACC treatment. Mean and

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of three biological replicates are shown. * Significant difference (P < 0.05) between the mutant and the wild type within a treatment; # significant difference between treated and untreated controls within a genotype as determined by Student’s t test (P < 0.05).

Figure 8.

Figure 8.

tt4-2 and tt7-2 exhibit altered auxin transport and signaling and dependent processes. A, Basipetal [3H]IAA transport in the wild type (wt), tt4-2, and tt7-2 is shown. Average and

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of three independent trials of eight individuals are shown. B, Morphometric analysis was used to quantify the tip angle of roots after gravitropic stimulation. The wild type, tt4-2, and tt7-2 in the presence and absence of 1 μ

m

ACC are shown (mean and

se

of more than seven individual roots). Data from tt4-2 and tt7-2 as well as these genotypes treated with ACC are overlapping, and in some cases, difficult to visualize. C, Growth inhibition by 1 μ

m

ACC in the wild type, tt4-2, and tt7-2. The tt4-2 growth responses in both the presence and absence of ACC are masked by overlapping tt7-2 growth responses. Mean and

se

are shown for three independent trials, each containing six individuals. D, Comparison of the wild type (WT) and tt4-2 expressing the DR5:vYFP auxin-responsive promoter 12 h after treatment. tt4-2 exhibits reduced ACC induction of DR5 expression, especially in the cortical cell layer (white boxes) and in the lateral root cap (arrows), where the ACC effect on DR5 activation is most apparent. Representative images from two independent trials of eight individuals are shown. Bar = 100 μm.

Figure 9.

Figure 9.

Order of signaling events in hormone-induced flavonol metabolism. A combination of auxin- and ethylene-specific and MYB-dependent transcriptional control may explain the observed data. TIR1- and ARF-dependent auxin signaling activates the auxin-responsive elements of the pathway genes shown, which include F3′H. In parallel, MYB12 is activated, leading to further up-regulation of the same pathway genes and PAP1 and TTG1 through their MYB-responsive elements. The line between MYB12 and PAP1 and TTG1 is dotted based on the complexity of interpreting these mutant data, as described in the text. EIN2- and ETR1-dependent ethylene signaling also increases the expression of a subset of pathway genes, excluding F3′H, through the activation of ethylene-responsive elements. This auxin-specific regulation of F3′H may explain the observed differences in the Q/K ratio after treatment with IAA and ACC. [See online article for color version of this figure.]

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