The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 m nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism.

A nitrate reductase (NR)-null mutant of Arabidopsis was constructed that had a deletion of the major NR gene NIA2 and an insertion in the NIA1 NR gene. This mutant had no detectable NR activity and could not use nitrate as the sole nitrogen source. Starch mobilization was not induced by nitrate in this mutant but was induced by ammonium, indicating that nitrate was not the signal for this process. Microarray analysis of gene expression revealed that 595 genes responded to nitrate (5 mm nitrate for 2 h) in both wild-type and mutant plants. This group of genes was overrepresented most significantly in the functional categories of energy, metabolism, and glycolysis and gluconeogenesis. Because the nitrate response of these genes was NR independent, nitrate and not a downstream metabolite served as the signal. The microarray analysis also revealed that shoots can be as responsive to nitrate as roots, yet there was substantial organ specificity to the nitrate response.

Nitrate regulatory mutants (nrg) of Arabidopsis (Arabidopsis thaliana) were sought using a genetic screen that employed a nitrate-inducible promoter fused to the yellow fluorescent protein marker gene YFP. A mutation was identified that impaired nitrate induction, and it was localized to the nitrate regulatory gene NLP7, demonstrating the validity of this screen. A second, independent mutation (nrg1) mapped to a region containing the NRT1.1 (CHL1) nitrate transporter gene on chromosome 1. Sequence analysis of NRT1.1 in the mutant revealed a nonsense mutation that truncated the NRT1.1 protein at amino acid 301. The nrg1 mutation disrupted nitrate regulation of several endogenous genes as induction of three nitrate-responsive genes (NIA1, NiR, and NRT2.1) was dramatically reduced in roots of the mutant after 2-h treatment using nitrate concentrations from 0.25 to 20 mM. Another nrt1.1 mutant (deletion mutant chl1-5) showed a similar phenotype. The loss of nitrate induction in the two nrt1.1 mutants (nrg1 and chl1-5) was not explained by reduced nitrate uptake and was reversed by nitrogen deprivation. Microarray analysis showed that nitrate induction of 111 genes was reduced and of three genes increased 2-fold or more in the nrg1 mutant. Genes involved in nitrate assimilation, energy metabolism, and pentose-phosphate pathway were most affected. These results strongly support the model that NRT1.1 acts as a nitrate regulator or sensor in Arabidopsis.

Nitrate serves as a potent signal to control gene expression in plants and algae, but little is known about the signaling role of nitrite, the direct product of nitrate reduction. Analysis of several nitrate-induced genes showed that nitrite increases mRNA levels as rapidly as nitrate in nitrogen-starved Arabidopsis (Arabidopsis thaliana) roots. Both nitrite and nitrate induction are apparent at concentrations as low as 100 nM. The response at low nitrite concentrations was not due to contaminating nitrate, which was present at ,1% of the nitrite concentration. High levels of ammonium (20 mM) in the growth medium suppressed induction of several genes by nitrate, but had varied effects on the nitrite response. Transcriptome analysis using 250 or 5 mM nitrate or nitrite showed that over one-half of the nitrate-induced genes, which included genes involved in nitrate and ammonium assimilation, energy production, and carbon and nitrogen metabolism responded equivalently to nitrite; however, the nitrite response was more robust and there were many genes that responded specifically to nitrite. Thus, nitrite can serve as a signal as well as if not better than nitrate.

To accommodate fluctuating nutrient levels in the soil, plants modulate their metabolism and root development via signaling mechanisms that rapidly reprogram the plant transcriptome. In the case of nitrate, over 1,000 genes are induced or repressed within minutes of nitrate exposure. To identify cis-regulatory elements that mediate these responses, an enhancer screen was performed in transgenic Arabidopsis (Arabidopsis thaliana) plants. A 1.8-kb promoter fragment from the nitrate reductase gene NIA1 was identified that acts as a nitrate enhancer when fused to a 35S minimal promoter. Enhancer activity was localized to a 180-bp fragment, and this activity could be enhanced by the addition of a 131-bp fragment from the nitrite reductase promoter. A promoter construct containing the 180-and 131-bp fragments was also induced by nitrite and repressed by ammonium, indicating that it was responsive to multiple nitrogen signals. To identify specific regulatory elements within the 180-bp NIA1 fragment, a transient expression system using agroinfiltration of Nicotiana benthamiana was developed. Deletion analysis identified three elements corresponding to predicted binding motifs for homeodomain/E-box, Myb, and Alfin1 transcription factors. A fully active promoter showing nitrate and nitrite enhancer activity equivalent to that of the wild-type 180-bp fragment could be built from these three elements if the spacing between the homeodomain/E-box and Myb-Alfin1 sites was equivalent to that of the native promoter. These findings were validated in transgenic Arabidopsis plants and identify a cisregulatory module containing three elements that comprise a nitrate enhancer in the NIA1 promoter.

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Abstract. Cyclin D1 is a significant regulator of the G1-to S-phase transition and is often aberrant in human tumors of various origins. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms by which they contribute to neoplasia remaind to be elucidated. We previously identified a cyclin D1 mutation (Δ266-295) in esophageal cancer with deleted codons from 266 to 295 of wild-type cyclin D1, the critical COOH-terminal regulatory sequences necessary for cyclin D1 nuclear export. In the present study, this cancer-derived cyclin D1-Δ266-295 was shown to be a constitutively nuclear cyclin D1 protein with a significantly increased oncogenic potential. Moreover, the cancer-derived cyclin D1-Δ266-295 mutant was found to retain its ability to bind to and activate CDK4, which in turn phosphorylates and inactivates the pRb protein and promotes cell cycle progression. In comparison to wild-type cyclin D1a, D1-Δ266-295 exhibited enforced nuclear accumulation. In addition, the transient transfection and ectopic expression of this nuclear localized D1-Δ266-295 up-regulated endogenous Notch1 expression, indicating that the mutant retained its ability as a transcriptional regulator. Furthermore, data from the flow cytometry assay showed that D1-Δ266-295 fractionally increased >4N cell accumulation, and further analysis suggested the retriggering of DNA replication relevant to its inhibition of Cdt1 proteolysis. Therefore, the inappropriate nuclear localization of this cyclin D1 mutant may interfere with DNA replication in cultured cells, thereby contributing to genomic instability. IntroductionCyclin D1 is a significant regulator of cell cycle progression in numerous cell types. Cyclin D1 elicits its pro-proliferative function early in the G1 phase, as it is capable of activating cyclin-dependent kinases (CDKs) 4 or 6. Active CDK4/6-cyclin D1 complexes phosphorylate and inactivate the retinoblastoma protein (Rb), which is critical for modulating G1-to S-phase progression, and in this manner promote cell proliferation (1-3). In addition to its well-established cell cycle roles, cyclin D1 is involved in CDK-independent function in transcription by acting as a molecular bridge between DNA-bound transcription factors and chromatin-modifying enzymes (4-6).Cyclin D1 expression is regulated mainly by extracellular mitogenic and oncogenic signals, allowing cyclin D1 to serve as a mediator of growth factor signaling and cell cycle progression (7). It is therefore unsurprising that cyclin D1 is often deregulated in tumors of various origins (8). The overexpression of cyclin D1 caused by gene amplification is observed in several carcinomas, including those of the esophagus, head and neck, breast and colon (9-16). Notably, unlike strong oncogenes, such as Ras, the overexpression of cyclin D1 alone is not capable of transforming immortalized murine fibroblasts in vitro (17). Furthermore, whereas the overexpression of cyclin D1 is considered to be the initial genetic trigger in mantle cell lymphoma...