MAX1, a regulator of the flavonoid pathway, controls vegetative axillary bud outgrowth in Arabidopsis

Abstract

We show that MAX1, a specific repressor of vegetative axillary bud outgrowth in Arabidopsis, acts a positive regulator of the flavonoid pathway, including 11 structural genes and the transcription factor An2. Repression of bud outgrowth requires MAX1-dependent flavonoid gene expression. As the flavonoidless state leads to lateral outgrowth in Arabidopsis, our data suggest that a flavonoid-based mechanism regulates axillary bud outgrowth and that this mechanism is under the control of MAX1. Flavonoid gene expression results in the diminished expression of auxin transporters in the bud and stem, and this, in turn, decreases the rate of polar auxin transport. We speculate that MAX1 could repress axillary bud outgrowth via regulating flavonoid-dependent auxin retention in the bud and underlying stem. Because MAX1 is implicated in synthesis of the carotenoid-derived branch regulator(s) from the root, it likely links long-distance signaling with local control of bud outgrowth.

Fig. 1.

MAX1 is a positive regulator of the flavonoid pathway in the late vegetative stage plant. (A) RNA blot analysis of flavonoid pathway genes in late vegetative stage trunk. The plant genotypes are wt, loss-of-function mutant max1 (max1-3), and gain-of-function transgenic (Txs). Plants were grown under normal conditions (control) or exposed to light stress (≈160 μE cm^-2·s^-1) for 2 days. Flavonoid pathway genes, with locus tags in parentheses, are as follows: CHS, chalcone synthase (At5g13930); CHI, chalcone isomerase (At5g05270); F3H, flavanone 3-hydroxylase (At3g51240); DFR, dihydroflavonol 4-reductase (At5g42800); FLS, flavonol synthase (At5g08640); F3′H, flavonoid 3′ hydroxylase (At5g07990); ANS, anthocyanidin synthase (At4g22870); UFGT, UDP glucose:flavonoid 3-O-glucosyltransferase (At5g17050); RT, UDP rhamnose-anthocyanindin-3-glucoside rhamnosyltransferase (At4g27570); AAC, anthocyanin 5-aromatic acyl-transferase (At1g03495); GST, glutatione S-transferase (At5g17220); An2, anthocyanin 2 (At1g56650). (B) A semiquantitative RT-PCR analysis of An2/PAP1 expression in late-vegetative-stage trunk using 0.5, 1.0, and 2.0 μl of the reverse transciption reaction as template; actin-1 is the internal control. (C) Light stress response under continuous illumination after 9 days. Longitudinal sections of rosette region stem of wt (Upper Left and Lower Left) and max1-3 (Upper Right); repressed axillary buds are marked by arrowheads. (Lower Center)wtand max1-3 leaves. (Lower Right) Extractable anthocyanins from uppermost rosette leaves (500 mg of fresh weight). (Scale bar, 1 mm.) (D) Expression profiles of flavonoid pathway genes in the dissected trunk of late-vegetative-stage plants. Semiquantitative RT-PCR was performed as described for B. (Ea-Ed) Flavonoid staining. Cross sections of the vegetative rosette stem were stained with diphenylboric acid-2-aminoethyl ester and viewed through a FITC filter. Flavonols (kempferol and quercetin) stain orange, naringenin stains yellow, sinapate esters are green, and chlorophyll autofluorescence is red. White arrows indicate axillary buds before (a-c) and during bud outgrowth (d). (F) Expression of flavonoid pathway genes in axillary buds (<0.5 mm). Semiquantitative RT-PCR was used as described for D. (G) Chemical complementation of max1-3 using exogenous application of flavonoid pathway intermediates: naringenin, kempferol, and quercetin. Axillary buds and rosette stem were treated daily for 2 weeks, starting 7 days before bolting. Plants were grown for another week before documentation. (H) A model for MAX1-regulated flavonoid gene expression in the late-vegetative-stage plant. Genes that require MAX1 for normal level expression are red. An2 controls at least four genes of the pathway (15), as shown by dotted lines. Pathway intermediates used in the chemical complementation of max1-3 are highlighted.

Fig. 2.

Polar auxin transport and expression of auxin transporter and flavonoid genes in the inflorescence stem. (A) Comparison of [³H]IAA transport in the rosette proximal inflorescence stem segment of wt, loss-of-function (max1-3), and gain-of function (Txs) plants without (upper bar graph) and with NPA (lower bar graph). Segment 6 is the trunk. Data are the average ± SE of three segments. (B) An RNA blot analysis of spatial expression of auxin transporter and flavonoid genes in seed-setting-stage stem. Ethidium bromide-stained gel is shown below as a loading control. (Upper) Stem segments included the trunk, which encompassing the rosette stem with the axillary buds and base of petioles (Rs), the lower stem (Ls), the middle stem (Ms), and the upper stem (Us). The auxin transporter genes, with locus tags in parentheses are AUX1 (At2g21050), LAX1 (At 5g01240), PIN1 (At1g73590), PIN3 (At1g70940), PIN4 (At2g01420), PIN5 (At1g23080), PIN6 (At1g77110), and PIN8 (At5g16530).

Fig. 3.

Expression profiles of auxin transporter genes in the axillary bud and rosette stem of late-vegetative-stage plants. Semiquantitative RT-PCR was performed as described for Fig. 1D.