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The flavonoid pathway is known to be up-regulated by different environmental stress factors. Down-regulation of the pathway is much less studied and is emphasized in the present work. Flavonoid accumulation was induced by exposing plants for 1 week to nitrogen depletion at 10°C, giving high levels of anthocyanins and 3-glucoside-7-rhamnosides, 3,7-di-rhamnosides and 3-rutinoside-7-rhamnosides of kaempferol and quercetin. Flavonol accumulation as influenced by temperatures and nitrogen supply was not related to the glycosylation patterns but to the classification as quercetin and kaempferol. When nitrogen was re-supplied, transcripts for main regulators of the pathway, PAP1/GL3 and PAP2/MYB12, fell to less than 1 and 0.1% of initial values, respectively, during 24 h in the 15-30°C temperature range. Anthocyanins showed a half-life of approximately 1 d, while the degradation of flavonols was much slower. Interestingly, the initial fluxes of anthocyanin and flavonol degradations were found to be temperature-independent. A kinetic model for the flavonoid pathway was constructed. In order to get the observed concentration-temperature profiles as well as the temperature compensation in the flavonoid degradation flux, the model predicts that the flavonoid pathway shows an increased temperature sensitivity at the end of the pathway, where the up-regulation by PAP/GL3 has been found to be largest.

The bHLH transcription factors EGL3 (ENHANCER OF GLABRA3) and its close homologue GL3 (GLABRA3) are important regulators of the anthocyanin pathway in Arabidopsis thaliana, and together with TTG1 (a WD40 repeat protein) and MYB transcription factors regulate specific genes in the pathway. In response to nitrogen depletion, the MYB genes PAP1/PAP2 (production of anthocyanin pigment 1/2) and GL3 are strongly induced, and anthocyanin synthesis is activated in seedlings and rosette stage plants. In this study we show that anthocyanins accumulate in both wild type and egl3, but not in gl3 loss-of-function mutants when depleted of nitrogen. Several structural genes of flavonoid metabolism including CHS (chalcone synthase), FLS1 (flavonol synthase 1) and ANS (anthocyanidin synthase) were induced in response to nitrogen depletion in wild type as well as in the egl3 and gl3 mutants. Strikingly, in the gl3 mutant DFR (dihydroflavonol-4-reductase) transcript level was only 2% of the levels in wild type or egl3 mutant. Hence, low expression of DFR appears to be the bottleneck preventing anthocyanin synthesis in the gl3 mutant. The specific effect on DFR, but not ANS is compatible with involvement of the MYBL2 inhibitor.

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