The content of flavonoids increases in response to nitrogen and phosphorus depletion in plants. Manipulation of these macronutrients may therefore be used to control the levels of desirable compounds and improve plant quality. Key enzymes in the shikimate pathway, which feeds precursors into the flavonoid pathway, are regulated post-translationally by feedback from aromatic amino acids, and possibly by redox control through photosynthesis. Use of microarrays for global transcript analysis in Arabidopsis has revealed that transcript levels are less influenced by mineral nutrients in the shikimate pathway compared with the flavonoid pathway. The responses in the shikimate pathway appear complex, whereas in the flavonoid pathway, a single gene often responds similarly to mineral depletion, high light intensity and sucrose. MYB [production of anthocyanin pigment 1 (PAP1)/production of anthocyanin pigment 2 (PAP2)] and bHLH [GLABRA3 (GL3)] transcription factors are important for the nutrient depletion response. PAP1/2 stimulate gross activation of the flavonoid pathway, and different investigations support merging signal transduction chains for various abiotic treatments on PAP1/2. Flavonol synthase is not part of the PAP1/2 regulon, and expression is mainly enhanced by high light intensity and sucrose, not mineral depletion. Nevertheless, both cyanidin and flavonol derivatives increase in response to nitrogen depletion. Kaempferols are the dominating flavonols in Arabidopsis leaves under normal cultivation conditions, but quercetin accumulation can be triggered by nitrogen depletion in combination with other abiotic factors.

Expression of regulators of the flavonoid pathway was examined in Arabidopsis thaliana wild type and pap1D plants, the latter being a T-DNA activation-tagged line over-expressing the PAP1/MYB75 gene which is a positive regulator of the pathway. Anthocyanin accumulation was induced in plants grown in soil, on agar plates, and hydroponics by withdrawing nitrogen from the growth medium. The agar-grown seedlings and rosette stage plants in hydroponics were further explored, and showed that nitrogen deficiency resulted in the accumulation of not only anthocyanins, but also flavonols. The examination of transcript levels showed that the general flavonoid pathway regulators PAP1 and PAP2 were up-regulated in response to nitrogen deficiency in wild type as well as pap1D plants. Interestingly, PAP2 responded much stronger to nitrogen deficiency than PAP1, 200- and 6-fold increase in transcript levels, respectively, for wild-type seedlings. In rosette leaves the increase was 900-fold for PAP2 and 6-fold for PAP1. At least three different bHLH domain transcription factors promote anthocyanin synthesis, and transcripts for one of these, i.e. GL3 were found to be sixfold enhanced by nitrogen deficiency in rosette leaves. The MYB12 transcription factor, known to regulate flavonol synthesis, was slightly induced by nitrogen deficiency in seedlings. In conclusion, four out of eight regulators involved in the flavonoid pathway showed an enhanced expression from 2 to 1,000 times in response to nitrogen deficiency. Together with MYB factors, especially PAP2, GL3 appears to be the BHLH partner for anthocyanin accumulation in response to nitrogen deficiency.

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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.