Cathrine Lillo

PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1

When brassinosteroid levels are low, the GSK3-like kinase BIN2 phosphorylates and inactivates the BZR1 transcription factor to inhibit growth in plants. Brassinosteroid promotes growth by inducing dephosphorylation of BZR1, but the phosphatase that dephosphorylates BZR1 has remained unknown. Here, using tandem affinity purification, we identified protein phosphatase 2A (PP2A) as a BZR1-interacting protein. Genetic analyses demonstrated a positive role for PP2A in brassinosteroid signalling and BZR1 dephosphorylation. Members of the B’ regulatory subunits of PP2A directly interact with BZR1’s putative PEST domain containing the site of the bzr1-1D mutation. Interaction with and dephosphorylation by PP2A are enhanced by the bzr1-1D mutation, reduced by two intragenic bzr1-1D suppressor mutations, and abolished by deletion of the PEST domain. This study reveals a crucial function for PP2A in dephosphorylating and activating BZR1 and completes the set of core components of the brassinosteroid-signalling cascade from cell surface receptor kinase to gene regulation in the nucleus.

Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress

We examined eight putative consistently expressed genes-actin (ACT), beta-tubulin, elongation factor 1alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), ribosomal protein L2 (RPL2), ubiquitin (UBI), and a catalytic subunit of protein phosphatase 2A (PP2Acs)-for their potential as references for the normalization of gene expression in tomato leaves. Expression stability of candidate reference genes was tested during growth conditions of nitrogen (N) starvation, low temperature, and suboptimal light. The geNorm algorithm, using reciprocal cross-validation among a larger group of candidate references, was applied for this purpose. The widely used reference genes GAPDH and PGK were top ranked during light stress but poorly ranked during N and cold stress. In contrast, EF1 was top ranked during N and cold stress but poorly ranked during light stress. The novel references RPL2 and PP2Acs, as well as the traditional references ACT and UBI, appeared to be stably expressed when looking at the data set as a whole. No gene was identified that exhibited such a constant level of expression as to outperform the other candidates under all experimental conditions. Thus, the results highlight the need for normalizing gene expression in tomato using the geometric average of multiple carefully selected reference genes.

Nitrogen deficiency enhances expression of specific MYB and bHLH transcription factors and accumulation of end products in the flavonoid pathway

Expression of regulators of the flavonoid pathway was examined in Arabidopsisthaliana 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.

Identification of a Protein That Inhibits the Phosphorylated Form of Nitrate Reductase from Spinach (Spinacia oleracea) Leaves

The low-activity, phosphorylated form of nitrate reductase (NR) became activated during purification from spinach (Spinacia oleracea) leaves harvested in the dark. This activation resulted from its separation from an approximately 110-kd nitrate reductase inhibitor protein (NIP). Readdition of NIP inactivated the purified phosphorylated NR, but not the active dephosphorylated form of NR, indicating that the inactivation of NR requires its interaction with NIP as well as phosphorylation. Consistent with this hypothesis, NR that had been inactivated in vitro in the presence of NR kinase, ATP-Mg, and NIP could be reactivated either by dephosphorylation with protein phosphatase 2A or by dissociation of NIP from NR.

Differential expression of four Arabidopsis PAL genes; PAL1 and PAL2 have functional specialization in abiotic environmental-triggered flavonoid synthesis

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the first step in the phenylpropanoid pathway, and is considered an important regulation point between primary and secondary metabolism. In the present work we analyzed expression of the PAL genes in leaves of Arabidopsis thaliana rosette-stage plants in response to nitrogen depletion at temperatures ranging from 5 to 30 degrees C. Only PAL1 and PAL2 responded strongly to both environmental factors, nitrogen and temperature. Regardless of nitrogen treatments, PAL1 and 2 transcript levels increased at 5 and 10 degrees C. Averaged across all temperatures, nitrogen depletion led to a two-fold increase in PAL1 and PAL2 transcripts. PAL activity was correlated with PAL transcript levels (R=0.94). Accumulation of major soluble phenylpropanoids, sinapic acid esters and flavonoids, increased in response to lowering temperature. The flavonoids, kaempferols, quercetins and anthocyanins, showed significantly increased levels as a result of nitrogen depletion (two-, five- and six-fold increases, respectively) when averaged across all temperatures. PAL1, PAL2 and PAL4 have previously been shown to be related with tissue-specific lignin synthesis, and the present work shows that PAL1 and PAL2 also have functional specialization in abiotic environmental-triggered flavonoid synthesis.

Synergetic effects of nitrogen depletion, temperature, and light on the content of phenolic compounds and gene expression in leaves of tomato

Tomato plants (Solanum lycopersicum, cv. Suzanne) were subjected to complete nutrient solution or a solution without nitrogen (N), and placed at different temperatures and light conditions to test the effects of environment on flavonoids and caffeoyl derivatives and related gene expression. N depletion during 4-8days resulted in enhanced levels of flavonoids and caffeoyl derivatives. Anthocyanins showed pronounced increased levels when lowering the growth temperature from 24 degrees C to 18 degrees C or 12 degrees C. Flavonol levels increased when the light intensity was increased from 100 micromol m(-2) s(-1) PAR to 200 micromol m(-2) s(-1) PAR. Synergistic effects of the various environmental factors were observed. The increase in content of quercetin derivatives in response to low temperatures was only found under conditions of N depletion, and especially at the higher light intensity. Expression of structural genes in the phenylpropanoid and flavonoid pathways, PAL (phenylalanine ammonia lyase), CHS (chalcone synthase), F3H (flavanone 3-hydroxylase), and FLS (flavonol synthase) increased in response to N depletion, in agreement with a corresponding increase in flavonoid and caffeoyl content. Expression of these structural genes generally also increased in response to lower temperatures. As indicated through expression studies and correlation analysis, effects of N depletion were apparently mediated through the overall regulators of the pathway the MYB transcription factor ANT1 (ANTHOCYANIN 1) and SlJAF13 (a bHLH transcription factor orthologue of petunia JAF13 and maize RED genes). A PAL gene (PAL6) was identified, and correlation analysis was compatible with PAL6 being an actively expressed gene with function in flavonoid synthesis.

Is nitrate reductase a major player in the plant NO (nitric oxide) game

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.

Signalling cascades integrating light-enhanced nitrate metabolism

In higher plants, light is crucial for regulation of nitrate uptake, translocation and assimilation into organic compounds. Part of this metabolism is tightly coupled to photosynthesis because the enzymes involved, nitrite reductase and glutamate synthase, are localized to the chloroplasts and receive reducing power from photosynthetic electron transport. However, important enzymes in nitrate acquisition and reduction are localized to cellular compartments other than chloroplasts and are also up-regulated by light, i.e. transporters in cell and organellar membranes and nitrate reductase in the cytosol. This review describes the different light-dependent signalling cascades regulating nitrate metabolism at the transcriptional as well as post-transcriptional level, and how reactions in different compartments of the cell are co-ordinated. Essential players in this network are phytochrome and HY5 (long hypocotyls 5)/HYH (HY5 homologue)-dependent signalling pathways, the energy-related AMPK (AMP-activated protein kinase) protein kinase homologue SNRK1 (sucrose non-fermenting kinase 1-related kinase), chloroplastic thioredoxins and the prokaryotically originated PII protein. A complex light-dependent network of regulation emerges, which appears to be necessary for optimal nitrogen assimilation and for avoiding the accumulation of toxic intermediates and side products, such as nitrite and reactive oxygen compounds.

Temperature and nitrogen effects on regulators and products of the flavonoid pathway: experimental and kinetic model studies

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 degrees 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 degrees 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.

Posttranslational Regulation of Nitrate Reductase Strongly Affects the Levels of Free Amino Acids and Nitrate, whereas Transcriptional Regulation Has Only Minor Influence

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 15N 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.