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.

HY5 and HYH are bZIP transcription factors well known to be involved in photomorphogenesis and light signalling. Loss-of-function mutants of HY5 and HYH revealed that these genes are essential for induction of a key enzyme in nitrogen assimilation, nitrate reductase (EC 1.7.1.1). In Arabidopsis thaliana seedlings nitrate reductase was expressed under low irradiance far-red or red light at the same level as under higher irradiance, photosynthetic active, white light. However, high NR expression at low light levels occurred only in the presence of sucrose in the growth medium. Sucrose did not promote expression in darkness. Whereas HY5 was necessary for high nitrate reductase expression in far-red light, HYH was important in red light. COP1 is known to promote degradation of HY5 and HYH, and in the cop1 mutant, nitrate reductase activity was relatively high also in darkness. PhyA and PhyB mutants were tested, and confirmed the phytochrome dependency for far-red and red light induction of nitrate reductase in seedlings. In rosette leaves of 3-week-old green plants the daily increase in nitrate reductase expression in response to light-on was abolished in the hyh and hy5 hyh double mutant. The hy5 hyh double mutant had lower nitrate reductase activity than any of the single mutants in photosynthetic active light in both seedlings and rosette leaves.

Nitric oxide (NO) is a diffusible, very reactive gas that is involved in the regulation of many processes in plants. Several enzymatic sources of NO production have been identified in recent years. Nitrate reductase (NR) is one of them and it has been shown that this well-known plant protein, apart from its role in nitrate reduction and assimilation, can also catalyse the reduction of nitrite to NO. This reaction can produce large amounts of NO, or at least more than is needed for signalling, as some escape of NO to the outside medium can be detected after NR activation. A role for NO and NR in stomata functioning in response to abscisic acid has also been proposed. The question that remains is whether this NR-derived NO is a signalling molecule or the mere product of an enzymatic side reaction like the products generated by the oxygenase activity of RuBisCO.

Diurnal variations in nitrate reductase (NR) activity and nitrogen metabolites were examined in wild-type Nicotiana plumbaginifolia and transformants with various degrees of NR deregulation. In the C1 line, NR was only deregulated at the transcriptional level by placing the NR gene under the control of the cauliflower mosaic virus 35S RNA promoter. In the Del8 and S521D lines, NR was additionally deregulated at the posttranslational level either by a deletion mutation in the N-terminal domain or by a mutation of the regulatory phosphorylation site (serine-521). Posttranslational regulation was essential for pronounced diurnal variations in NR activity. Low nitrate content was related to deregulation of NR, whereas the level of total free amino acids was much higher in plants with fully deregulated NR. Abolishing transcriptional and posttranslational regulation (S521D plants) resulted in an increase of glutamine and asparagine by a factor of 9 and 14, respectively, compared with wild type, whereas abolishing transcriptional regulation (C1 plants) only resulted in increases of glutamine and asparagine by factors ,2. Among the minor amino acids, isoleucine and threonine, in particular, showed enhanced levels in S521D. Nitrate uptake rates were the same in S521D and wild type as determined with 15 N feeding. Deregulation of NR appears to set the level of certain amino acids, whereas diurnal variations were still determined by light/dark. Generally, deregulation of NR at the transcriptional level did not have much influence on metabolite levels, but additional deregulation at the posttranslational level resulted in profound changes of nitrogen metabolite levels.