Genome-wide patterns of carbon and nitrogen regulation of gene expression validate the combined carbon and nitrogen (CN)-signaling hypothesis in plants

BACKGROUND: Carbon and nitrogen are two signals that influence plant growth and development. It is known that carbon- and nitrogen-signaling pathways influence one another to affect gene expression, but little is known about which genes are regulated by interactions between carbon and nitrogen signaling or the mechanisms by which the different pathways interact. RESULTS: Microarray analysis was used to study global changes in mRNA levels due to carbon and nitrogen in Arabidopsis thaliana. An informatic analysis using InterAct Class enabled us to classify genes on the basis of their responses to carbon or nitrogen treatments. This analysis provides in vivo evidence supporting the hypothesis that plants have a carbon/nitrogen (CN)-sensing/regulatory mechanism, as we have identified over 300 genes whose response to combined CN treatment is different from that expected from expression values due to carbon and nitrogen treatments separately. Metabolism, energy and protein synthesis were found to be significantly affected by interactions between carbon and nitrogen signaling. Identified putative cis-acting regulatory elements involved in mediating CN-responsive gene expression suggest multiple mechanisms for CN responsiveness. One mechanism invokes the existence of a single CN-responsive cis element, while another invokes the existence of cis elements that promote nitrogen-responsive gene expression only when present in combination with a carbon-responsive cis element. CONCLUSION: This study has allowed us to identify genes and processes regulated by interactions between carbon and nitrogen signaling and take a first step in uncovering how carbon- and nitrogen-signaling pathways interact to regulate transcription

Qualitative network models and genome-wide expression data define carbon/nitrogen-responsive molecular machines in Arabidopsis

BACKGROUND: Carbon (C) and nitrogen (N) metabolites can regulate gene expression in Arabidopsis thaliana. Here, we use multinetwork analysis of microarray data to identify molecular networks regulated by C and N in the Arabidopsis root system. RESULTS: We used the Arabidopsis whole genome Affymetrix gene chip to explore global gene expression responses in plants exposed transiently to a matrix of C and N treatments. We used ANOVA analysis to define quantitative models of regulation for all detected genes. Our results suggest that about half of the Arabidopsis transcriptome is regulated by C, N or CN interactions. We found ample evidence for interactions between C and N that include genes involved in metabolic pathways, protein degradation and auxin signaling. To provide a global, yet detailed, view of how the cell molecular network is adjusted in response to the CN treatments, we constructed a qualitative multinetwork model of the Arabidopsis metabolic and regulatory molecular network, including 6,176 genes, 1,459 metabolites and 230,900 interactions among them. We integrated the quantitative models of CN gene regulation with the wiring diagram in the multinetwork, and identified specific interacting genes in biological modules that respond to C, N or CN treatments. CONCLUSION: Our results indicate that CN regulation occurs at multiple levels, including potential post-transcriptional control by microRNAs. The network analysis of our systematic dataset of CN treatments indicates that CN sensing is a mechanism that coordinates the global and coordinated regulation of specific sets of molecular machines in the plant cell

NRT2.1 phosphorylation prevents root high affinity nitrate uptake activity in Arabidopsis thaliana

In Arabidopsis thaliana, NRT2.1 codes for a main component of the root nitrate high-affinity transport system. Previous studies revealed that post-translational regulation of NRT2.1 plays an important role in the control of root nitrate uptake and that one mechanism could correspond to NRT2.1 C-terminus processing. To further investigate this hypothesis, we produced transgenic plants with truncated forms of NRT2.1. It revealed an essential sequence for NRT2.1 activity, located between the residues 494-513. Using a phospho-proteomic approach, we found that this sequence contains one phosphorylation site, at serine 501, which can inactivate NRT2.1 function when mimicking the constitutive phosphorylation of this residue in transgenic plants. This phenotype could neither be explained by changes in abundance of NRT2.1 and NAR2.1, a partner protein of NRT2.1, nor by a lack of interaction between these two proteins. Finally, the relative level of serine 501 phosphorylation was found to be modulated by nitrate in wildtype plants. Altogether, these observations allowed us to propose a model for a new and essential mechanism for the regulation of NRT2.1 activity

A Systems Approach Uncovers Restrictions for Signal Interactions Regulating Genome-wide Responses to Nutritional Cues in Arabidopsis

As sessile organisms, plants must cope with multiple and combined variations of signals in their environment. However, very few reports have studied the genome-wide effects of systematic signal combinations on gene expression. Here, we evaluate a high level of signal integration, by modeling genome-wide expression patterns under a factorial combination of carbon (C), light (L), and nitrogen (N) as binary factors in two organs (O), roots and leaves. Signal management is different between C, N, and L and in shoots and roots. For example, L is the major factor controlling gene expression in leaves. However, in roots there is no obvious prominent signal, and signal interaction is stronger. The major signal interaction events detected genome wide in Arabidopsis roots are deciphered and summarized in a comprehensive conceptual model. Surprisingly, global analysis of gene expression in response to C, N, L, and O revealed that the number of genes controlled by a signal is proportional to the magnitude of the gene expression changes elicited by the signal. These results uncovered a strong constraining structure in plant cell signaling pathways, which prompted us to propose the existence of a “code” of signal integration

Regulation of root nitrate uptake in Arabidopsis thaliana using NRT2.1 as a target

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Régulation du transport racinaire de nitrate chez Arabidopsis thaliana

Chez Arabidopsis NRT2.1 code pour le principal transporteur racinaire de nitrate à forte affinité (HATS). Sa régulation a été étudiée principalement au niveau transcriptionnel ce qui a permis de montrer une forte corrélation entre l’expression de NRT2.1 et l’activité du HATS en réponse à des traitements carbone et azote. Cependant, malgré le rôle central de NRT2.1 dans la nutrition des plantes peu de choses sont connues concernant les mécanismes moléculaires impliqués dans sa régulation. Au cours des dernières années nous avons combiné différentes approches pour étudier le contrôle de l’expression de NRT2.1 par la signalisation carbone et la régulation de NRT2.1 au niveau de la protéine. Nous avons montré que NRT2.1 est induit par la lumière et les sucres à travers le fonctionnement de la voie oxidative des pentoses phosphates (OPPP). De plus, en combinant des approches d’immunologie à l’utilisation de lignées transgéniques exprimant un transgène fonctionnel 35S::NRT2.1 dans le mutant atnrt2.1, nous avons pu montrer l’existence de mécanismes de régulation post-transcriptionnelle. Plus récemment, nous avons également démarré une approche de biologie des systèmes en collaboration avec le laboratoire de Rodrigo Gutierrez au Chili et un groupe de physiciens de l’université Montpellier 2. Pour ce travail, NRT2.1 est utilisé comme gène cible en réponse à des combinaisons de traitements entre la lumière, le carbone et l’azote pour décrypter et modéliser les réseaux de gènes impliqués dans le contrôle du transport de nitrate. L’objectif à terme de ces approches est d’utiliser les informations obtenues grâce à NRT2.1, comme point de départ pour étudier l’impact des mécanismes de régulation identifiés sur les autres acteurs du transport et du métabolisme azoté et obtenir ainsi une vision globale de la régulation de la nutrition azotée des plantes

Regulation of root NO3- uptake in Arabidopsis thaliana using NRT2.1 as a target

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Post-transcriptional regulation of the root nitrate uptake transporter NRT2.1 in Arabidopsis thaliana

Post-transcriptional regulation of the root nitrate uptake transporter NRT2.1 in Arabidopsis thaliana. 2nd International Symposium on the Nitrogen Nutrition of Plant

Signal interactions in the regulation of root nitrate uptake.

International audienceIn most aerobic soils, nitrate (NO3(-)) is the main nitrogen source for plants and is often limiting for plant growth and development. To adapt to a changing environment, plants have developed complex regulatory mechanisms that involve short and long-range signalling pathways in response to both NO3(-) availability in the soil and other physiological processes like growth or nitrogen (N) and carbon (C) metabolisms. Over the past decade, transcriptomic approaches largely contributed to the identification of molecular elements involved in these regulatory mechanisms, especially at the level of root NO3(-)uptake. Most strikingly, the data obtained revealed the high level of interaction between N and both hormone and C signalling pathways, suggesting a strong dependence on growth, development, and C metabolism to adapt root NO3(-) uptake to both external NO3(-) availability and the N status of the plant. However, the signalling mechanisms involved in the cross-talk between N, C, and hormones for the regulation of root NO3(-) uptake remain largely obscure. The aim of this review is to discuss the recent advances concerning the regulatory pathways controlling NO3(-) uptake in response to N signalling, hormones, and C in the model plant Arabidopsis thaliana. Then, to further characterize the level of interaction between these signalling pathways we built on publicly available transcriptome data to determine how hormones and C treatments modify the gene network connecting root NO3(-) transporters and their regulators

Glucose Elevates NITRATE TRANSPORTER2.1 Protein Levels and Nitrate Transport Activity Independently of Its HEXOKINASE1-Mediated Stimulation of NITRATE TRANSPORTER2.1 Expression.

International audienceMineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth