Gloria M. Coruzzi

Cell-specific nitrogen responses mediate developmental plasticity

The organs of multicellular species consist of cell types that must function together to perform specific tasks. One critical organ function is responding to internal or external change. Some cell-specific responses to changes in environmental conditions are known, but the scale of cell-specific responses within an entire organ as it perceives an environmental flux has not been well characterized in plants or any other multicellular organism. Here, we use cellular profiling of five Arabidopsis root cell types in response to an influx of a critical resource, nitrogen, to uncover a vast and predominantly cell-specific response. We show that cell-specific profiling increases sensitivity several-fold, revealing highly localized regulation of transcripts that were largely hidden from previous global analyses. The cell-specific data revealed responses that suggested a coordinated developmental response in distinct cell types or tissues. One example is the cell-specific regulation of a transcriptional circuit that we showed mediates lateral root outgrowth in response to nitrogen via microRNA167, linking small RNAs to nitrogen responses. Together, these results reveal a previously cryptic component of cell-specific responses to nitrogen. Thus, the results make an important advance in our understanding of how multicellular organisms cope with environmental change at the cell level.

Tissue-specific and light-regulated expression of a pea nuclear gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase.

We have examined the expression of a member of the multigene family encoding the small subunit (rbcS) of ribulose‐1,5‐bisphosphate carboxylase in various tissues of pea. The rbcS gene, pPS‐2.4, was characterized by DNA sequence analysis and 5′ and 3′ end mapping of its mRNA transcript. rbcS polypeptides were shown to be differentially present in various tissues of light‐ and dark‐grown plants. Northern analysis shows that compared with green leaves, the level of rbcS mRNA is reduced to approximately 50% in pericarps, 8% in petals and seeds, and 1‐3% in etiolated leaves, stems, and roots. 5′ S1 nuclease mapping of total rbcS mRNA was used to quantitate the relative amount of pPS‐2.4 gene‐specific transcripts in each tissue. pPS‐2.4 mRNA accounts for approximately 30‐35% of total rbcS transcripts in green leaves, but only 5‐10% in pericarps, 15‐20% in seeds, and is below detection in petals and etiolated leaves. We conclude that the pPS‐2.4 gene is expressed in a tissue‐specific, light‐regulated fashion and that transcriptional controls of individual rbcS genes vary.

Nitrate-responsive miR393/AFB3 regulatory module controls root system architecture in Arabidopsis thaliana

One of the most striking examples of plant developmental plasticity to changing environmental conditions is the modulation of root system architecture (RSA) in response to nitrate supply. Despite the fundamental and applied significance of understanding this process, the molecular mechanisms behind nitrate-regulated changes in developmental programs are still largely unknown. Small RNAs (sRNAs) have emerged as master regulators of gene expression in plants and other organisms. To evaluate the role of sRNAs in the nitrate response, we sequenced sRNAs from control and nitrate-treated Arabidopsis seedlings using the 454 sequencing technology. miR393 was induced by nitrate in these experiments. miR393 targets transcripts that code for a basic helix-loop-helix (bHLH) transcription factor and for the auxin receptors TIR1, AFB1, AFB2, and AFB3. However, only AFB3 was regulated by nitrate in roots under our experimental conditions. Analysis of the expression of this miR393/AFB3 module, revealed an incoherent feed-forward mechanism that is induced by nitrate and repressed by N metabolites generated by nitrate reduction and assimilation. To understand the functional role of this N-regulatory module for plant development, we analyzed the RSA response to nitrate in AFB3 insertional mutant plants and in miR393 overexpressors. RSA analysis in these plants revealed that both primary and lateral root growth responses to nitrate were altered. Interestingly, regulation of RSA by nitrate was specifically mediated by AFB3, indicating that miR393/AFB3 is a unique N-responsive module that controls root system architecture in response to external and internal N availability in Arabidopsis.

Genomic Analysis of the Nitrate Response Using a Nitrate Reductase-Null Mutant of Arabidopsis

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.

Glutamate-receptor genes in plants.

In animal brains, ionotropic glutamate receptors (GluRs) function as glutamate-activated ion channels in rapid synaptic transmission. We have now discovered that genes encoding putative ionotropic GluRs exist in plants, and we present preliminary evidence for their involvement in light-signal transduction. It may be that signalling between cells by excitatory amino acids in animal brains evolved from a primitive signalling mechanism that existed before the divergence of plants and animals. Our findings also help to explain why neuroactive compounds made by plants work on receptors in human brains.

Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1

Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. Phase response curve analysis shows that distinct N-metabolites can advance or delay the CCA1 phase. Regulation of CCA1 by organic N signals may represent a novel input mechanism for N-nutrients to affect plant circadian clock function.

Use of Arabidopsis Mutants and Genes To Study Amide Amino Acid Biosynthesis

Studies of enzymes involved in nitrogen assimilation in higher plants have an impact on both basic and applied plant research. First, basic research in this area should uncover the mechanisms by which plants regulate genes involved in a metabolic pathway. Second, because nitrogen is a rate-limiting element in plant growth (Hageman and Lambert, 1988), it may be possible to increase the yield or improve the quality of crop plants by the molecular or genetic manipulation of genes involved in nitrogen assimilation. Research on nitrogen assimilation into amino acids has been complicated by the fact that some of these reactions are catalyzed by multiple isoenzymes located in distinct subcellular compartments. With traditional biochemical approaches, it has been impossible to sort out the function of each isoenzyme in plant nitrogen metabolism. The discovery that genes for chloroplastic and cytosolic isoenzymes of glutamine synthetase (GS) are expressed in distinct cell types (Edwards et al., 1990; Carvalhoet al., 1992; Kamachi et al., 1992)suggeststhat traditional biochemical studies, which begin with tissue disruption, artificially mix isoenzymes that may not coexist in the same cell type in vivo. Thus, in vitro biochemical methods commonly used to define the rate-limiting enzyme in a pathway in unicellular microorganisms may lead to erroneous interpretations when employed to study plant metabolic pathways. An alternative way to define the in vivo function of a particular isoenzyme or to define a rate-limiting enzyme in a pathway is by mutant analysis, as shown by studies of Escherichia coli and yeast. Plant mutants defective in particular isoenzymes of GS or ferredoxin-dependent glutamate synthase (Fd-GOGAT) have been identified in screens for photorespiratory mutants in Arabidopsis and barley (Somerville and Ogren, 1980,1982; Wallsgrove et al., 1987). More recently, Arabidopsis mutants with alterations in the activity of additional enzymes of nitrogen assimilation have been identified using a screening method that does not depend on a growth phenotype (Schultz and Coruzzi, 1995). The in vivo role of the mutated isoenzyme

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

BackgroundCarbon (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.ResultsWe 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.ConclusionOur 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.

A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants

Members of the plant NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family display protein sequence homology with the SLC15/PepT/PTR/POT family of peptide transporters in animals. In comparison to their animal and bacterial counterparts, these plant proteins transport a wide variety of substrates: nitrate, peptides, amino acids, dicarboxylates, glucosinolates, IAA, and ABA. The phylogenetic relationship of the members of the NRT1/PTR family in 31 fully sequenced plant genomes allowed the identification of unambiguous clades, defining eight subfamilies. The phylogenetic tree was used to determine a unified nomenclature of this family named NPF, for NRT1/PTR FAMILY. We propose that the members should be named accordingly: NPFX.Y, where X denotes the subfamily and Y the individual member within the species.

Nitrate signaling: adaptation to fluctuating environments

Nitrate (NO(3)(-)) is a key nutrient as well as a signaling molecule that impacts both metabolism and development of plants. Understanding the complexity of the regulatory networks that control nitrate uptake, metabolism, and associated responses has the potential to provide solutions that address the major issues of nitrate pollution and toxicity that threaten agricultural and ecological sustainability and human health. Recently, major advances have been made in cataloguing the nitrate transcriptome and in identifying key components that mediate nitrate signaling. In this perspective, we describe the genes involved in nitrate regulation and how they influence nitrate transport and assimilation, and we discuss the role of systems biology approaches in elucidating the gene networks involved in NO(3)(-) signaling adaptation to fluctuating environments.