INTRODUCTION

TOP INTRODUCTION REGULATION OF THE NITRATE... INSIGHTS INTO ENZYME STRUCTURE... THE NO3 TRANSPORT SYSTEM... LONG-RANGE SIGNALING IN PLANTS "OME"-OPHOBIA? CONCLUDING REMARKS

Nitrate, an ion more accustomed to the subterranean darkness of the soil, found its place in the sun for four days in July 2002 when it was the focus of the Fifth International Symposium on Nitrate Assimilation: Molecular and Genetic Aspects (NAMGA) at the University of Córdoba in Spain. The highly successful series of NAMGA meetings was initiated in 1982 by Andreas Müller and Ralf Mendel with the aim of bringing together scientists from diverse backgrounds working on NO₃ assimilation in plants, fungi, and bacteria. An attractive feature of almost 50 presentations was that their topics ranged from transcriptional regulation to cofactor biosynthesis and protein structure. Also included were brief overviews of developments in processes that compete with NO₃ assimilation, namely denitrification and NO₃ reduction to NH₃ by anaerobic, fermentative bacteria.

	REGULATION OF THE NITRATE ASSIMILATORY PATHWAY

TOP INTRODUCTION REGULATION OF THE NITRATE... INSIGHTS INTO ENZYME STRUCTURE... THE NO3 TRANSPORT SYSTEM... LONG-RANGE SIGNALING IN PLANTS "OME"-OPHOBIA? CONCLUDING REMARKS

Some of the most significant advances reported in Córdoba were concerned with how NO₃ assimilation is regulated. The different facets of this problem covered questions about how the presence of NO₃ is detected; how the NO₃ signal is communicated to the genome; how NO₃ assimilation is regulated by the general N status of the cell; how NH₄⁺ assimilation is regulated; and how N assimilation is regulated in response to the availability of carbon.

Transcription Factors and Signals

A common feature that links the NO₃ assimilatory pathway in plants, bacteria, and fungi is its induction by NO₃ and its feedback regulation by the products of NO₃ assimilation. The model for how this is achieved was developed in fungi and involves a combination of pathway-specific regulators that respond to NO₃ and global regulators that regulate NO₃ assimilation (and other metabolic pathways) in response to the general N status of the cell. In Emericella (Aspergillus) nidulans, the pathway-specific transcription factor is NirA, a member of the GAL4 Zn²⁺ binuclear cluster family, whereas the global regulator is AreA, a GATA transcription factor. The interactions between these transcription factors and their binding sites within the bidirectional promoter that regulates the niaD and niiA genes (for nitrate reductase [NR] and nitrite reductase, respectively) are the subject of a collaboration between the groups of Scazzocchio (Université Paris Sud, Paris, France) and Strauss (University of Agricultural Sciences, Vienna). The emerging impression from this work is that the distinctions between the roles of NirA and AreA in NO₃ induction and feedback repression are not as clear-cut as traditionally thought. Studies with novel gain-of-function mutants in areA and nirA have shown that AreA is also capable of a NO₃ sensing role, even in the absence of NirA. Scazzocchio presented genetic and biochemical evidence that the NirA and AreA proteins interact in vivo and in vitro. Berger (University of Agricultural Sciences, Vienna), using a NirA::GFP fusion expressed in fungal hyphae, observed that import of NirA into the nucleus is NO₃ dependent, but it seems that binding to DNA additionally requires the function of AreA.

Another long-standing idea about how NO₃ assimilation is regulated was placed under the microscope in a study reported by Galván (University of Córdoba). Pioneering work by David Cove and Claudio Scazzocchio led to a model in which the product of the niaD gene (NR) was able to repress its own transcription and that of the other NO₃ assimilatory genes, a classic case of autoregulation. Since then, the discovery of pathway-specific and global transcription factors (such as NirA and AreA) has shifted the attention to more conventional mechanisms of transcriptional control. What is the role, then, for NR in transcriptional regulation? The idea has gradually emerged that the phenomenon of "autoregulation" is in some way linked to NO₃ transport. The exciting question, convincingly answered at this meeting, was by what mechanism does NO₃ transport interact with the transcriptional apparatus to regulate NO₃ assimilation? The question was addressed by Galván and her colleagues using the unicellular alga Chlamydomonas reinhardtii, in which NR-deficient mutants show the overexpression of NO₃assimilatory genes that is characteristic of autoregulation. With the help of mutants defective in different NO₃ transport systems, they were able to demonstrate that NO₃ sensing occurs intracellularly so that the role of the NO₃ transport system is to deliver the NO₃ to the place where it is sensed. System I, the most efficient of the multiple NO₃ uptake systems in this alga, is effective at NO₃ uptake even at the submicromolar concentrations found in media unsupplemented with NO₃. If NR is inactive (such as in an NR mutant), the NO₃ will accumulate in the cell and will hyperinduce the NO₃ assimilatory pathway.

These results are very satisfying in their potential to explain apparent autoregulatory phenomena associated with NR mutants in other species. However, intriguing new results reported by Lepetit (Institut National de la Recherche Agronomique, Montpellier, France) suggest that all may not be so simple. Lepetit and colleagues studied the regulation of NO₃ assimilatory genes in Arabidopsis mutants defective for one or both of the two NR genes (NIA1 and NIA2). In the nia1nia2 double mutant (and in the nia2 single mutant), the expression of some NO₃ assimilatory genes was up-regulated in a manner reminiscent of autoregulation. The overexpressed genes were NIA1 and the AtNRT1.1 NO₃ transporter gene, whereas other genes of the pathway were unaffected. They were able to rule out the most obvious possibilities that the mechanism was related to superinduction by accumulated NO₃ or to diminished feedback repression by reduced N metabolites. They concluded that the most likely explanation was negative regulation of NIA1 and AtNRT1.1 by NO₂ (or perhaps NO), which was relieved in the NR mutants. Feedback regulation by NO₂ can be seen as a mechanism for preventing accumulation of toxic NO₂ in the roots under conditions that inhibit NR (such as anoxia during flooding).

Additional evidence that NO₃ and NO₂ are not interchangeable in their signaling effects came from studies on gene regulation in two diverse bacterial species. Gunsalus (University of California, Los Angeles) reported that the Escherichia coli NarX NO₃ sensor protein (a transmembrane His kinase) recognizes NO₃ and NO₂, but has an affinity for the former that is over 100-fold greater than for the latter. Omata (University of Nagoya, Japan) reported that in the cyanobacterium Synechococcus sp PCC7942, the inductive signal for the NO₃ assimilatory pathway comes not from NO₃, but from NO₂, generated intracellularly (by nitrite reductase) or supplied externally.

The cyanobacterium Synechococcus has one of the simplest regulatory systems, where the pathway-specific regulator is a LysR-type transcription factor (NtcB) and the global N regulator is a cAMP receptor protein-type transcription factor (NtcA). In most species, the metabolite(s) that are monitored by the cell to detect changes in N status have not been rigorously identified. However, using an in vitro transcription assay, Omata demonstrated that 2-oxoglutarate is required for transcription from NtcA-dependent promoters in Synechococcus. Flores (University of Seville, Spain) reported the engineering of a strain of Synechococcus sp. that can absorb 2-oxoglutarate from the medium and demonstrated that supplying 2-oxoglutarate to this strain stimulated expression of NH₄⁺-repressible genes. Therefore, "ammonium repression" in this species appears to be a consequence of the depletion of the 2-oxoglutarate pool when sufficient NH₄⁺ is available.

There was a notable absence at the meeting of information about transcription factors that regulate NO₃ assimilation in higher plants. For reasons that are unclear, forward genetic approaches of the kind that have been so successful in fungi have singularly failed to identify similar kinds of NO₃ regulatory mutants in plants. Selection for resistance to chlorate (a NO₃ analog) has been the method of choice for identifying NO₃ assimilatory mutants in plants as well as fungi. Cheng (University of Iowa) reported on progress with the analysis of a novel chlorate-resistant Arabidopsis mutant (cr88), which was isolated in a screen for mutants with abnormal regulation of NR. The cr88 mutant has around 50% of the wild-type levels of NR activity, but it also shows defects in photomorphogenesis and in the light-induction of a subset of light-regulated genes (including NIA2). Map-based cloning of CR88 has now revealed it to encode a Heat Shock Protein 90 chaperone protein that is localized to the stromal compartment of the chloroplast. Possible functions suggested for CR88 include a role in protein import into the stroma or in the maturation of a subset of stromal proteins. These results highlight the complexity of the mechanisms regulating NIA gene expression in plants and may help to explain why true NO₃ regulatory mutants have proved so elusive.

Reverse genetics seems likely to prove a more successful route to NO₃ regulatory loci in plants, but the lack of any clear plant orthologs of AreA or NirA has thwarted one obvious line of attack. However, there are hopes that Chlamydomonas may turn out to be a more useful stepping stone to NO₃ regulatory genes in higher plants. With this in mind, Galván (University of Córdoba) reported studies on the Chlamydomonas NIT2 gene, a putative positive regulator of NO₃ assimilation whose expression is repressed by NH₄⁺ and has now been found to require intracellular NO₃ for its function. The NIT2 gene encodes a 1,196-amino acid protein with no obvious conserved motifs, but with Gln-rich regions typical of some classes of transcription factor.

Beyond Transcription

Transcriptional control is by no means the whole story when it comes to regulation of the NO₃ assimilatory pathway. Caddick (University of Liverpool) reported that in E. nidulans, a number of structural and regulatory genes in the pathway (including areA, niaD, and niiA) are regulated by a mechanism involving sequence-specific degradation of their mRNAs. The signal for degradation appears to be Gln rather than NH₄⁺, and in the case of areA, the signaling mechanism has been shown to require a 218-nucleotide sequence within the 3'-untranslated region. To add further complexity to the picture, a separate mechanism seems to be involved in stabilizing the niaD and niiA transcripts in the presence of NO₂ or NO₃.

Florencio (University of Seville) reported that the activity of one isoform of Gln synthetase in Synechocystis is regulated by protein-protein interactions with two inactivating factors (IF7 and IF17) that are homologous polypeptides encoded by the gifA and gifB genes, respectively. Both gif genes are regulated by NtcA, and their expression is strongest in the presence of NH₄⁺ when Gln synthase is inactivated. Reactivation of Gln synthetase is mediated by a protease that degrades at least IF7 and probably IF17.

Siverio (University of La Laguna, Tenerife) reported evidence that the YNT1 NO₃ transporter in the yeast Hansenula polymorpha (Pichia angusta) is posttranslationally modified in response to changes in N status, indicating the possible existence of a mechanism for rapid modulation of NO₃ influx.

NO Signaling Here?

NO is an important second messenger in animals, and evidence that it has a similar role in diverse signaling pathways in plants is accumulating. Kaiser (University of Würzburg) reported data indicating that NR is a major contributor to NO production in plants. However, there was also evidence for significant proportion of NO biosynthesis that was not NR dependent; for example, plants cultivated in the absence of NO₃ and with no detectable NR activity still displayed a low level of NO emission. All attempts to demonstrate a role for NO synthase (NOS), the NO-generating enzyme so intensively studied in mammalian systems, proved unsuccessful. The apparent absence of NOS enzyme activity in plants is in line with the failure so far to detect sequences homologous to NOS in the plant genomes.

Stöhr (University of Darmstadt) described the properties of an NO-generating enzyme, NO₂:NO reductase, which is present in the plasma membrane fraction of roots. In combination with a plasma membrane-bound form of NR, this could provide a pathway for the enzymic conversion of NO₃ to NO at the cell surface. Stöhr suggested that the apoplastic NO produced by this pathway might serve as a second messenger at low external NO₃ concentrations or in defense from pathogens.

	INSIGHTS INTO ENZYME STRUCTURE AND ASSEMBLY

TOP INTRODUCTION REGULATION OF THE NITRATE... INSIGHTS INTO ENZYME STRUCTURE... THE NO3 TRANSPORT SYSTEM... LONG-RANGE SIGNALING IN PLANTS "OME"-OPHOBIA? CONCLUDING REMARKS

Despite the massive interest in NR, only one structure, that of the unusual periplasmic, dissimilatory NR from a sulfate-reducing bacterium, has so far been published. High-level expression of other NRs, especially the plant enzyme, in a form that can be purified and crystallized, remains a challenge. Therefore, of particular interest was progress reported by Campbell (The Nitrate Elimination Company, Lake Linden, MI). A simplified expression system using Pichia pastoris now offers special promise for the production of a soluble form of the Arabidopsis NR2 enzyme that, as a first step, might be suitable for biosensor applications. More structures of NRs are certainly required, presenting a challenge for the next NAMGA meeting. Meanwhile, we were tantalized by the elegant and highly plausible "three-dimensional model of a five-domain NR" based on structural data from overexpressed fragments of the enzyme accumulated over many years and presented at this meeting by Campbell.

Several speakers presented crystal structures of their proteins. These included elegant structural studies at atomic resolution of the Alcaligenes xylosoxidans blue copper nitrite reductase and its mutants (Hasnain, De Montfort University, Leicester, UK), of the apo-ModE transcription factor, required for molydopterin synthesis by enteric bacteria (Boxer, University of Dundee, UK), and of the free and DNA-bound forms of NarL, the NO₃ response regulator from E. coli (Gunsalus, University of California, Los Angeles).

An inevitable recurring theme of the meeting was the discovery from genome sequencing projects that many processes believed to be simple are in fact quite complex, involving more proteins than previously anticipated. Particular interest in the last five years has focused on the periplasmic NRs (Nap) found in gram-negative bacteria. These enzymes fulfill different physiological roles in different types of bacteria, and multiple structural components that differ between bacteria of different physiological types appear to correlate, at last in part, with the physiology of the strain studied. Moreno-Vivián (University of Córdoba) proposed that the enigmatic NapF in Rhodobacter sphaeroides is involved in the posttranslational assembly of a functional NapA. The insertion of the prosthetic multicopper center into the denitrification enzyme, nitrous oxide reductase of Pseudomonas stutzeri, requires a posttranslational maturation complex encoded by the nosDFYLtatE operon (tatE encodes a component of the twin-Arg targeting pathway for the secretion of partially folded redox proteins across the bacteria membrane). Zumft (University of Karlsruhe, Germany) reviewed the progress in understanding how this process is regulated, and the roles of these components in copper acquisition and assembly. Many proteins are also involved in the posttranslational assembly of c-type cytochromes such as those involved in periplasmic NO₃ reduction by bacteria. Ferguson (University of Oxford) reported that the Cys-X-X-Cys-His motif in a c-type cytochrome can form a disulfide bond that must be reduced before thio-ether bond formation can occur.

The molybdenum cofactor (Moco) is an essential component of NRs and other molybdoenzymes. Schwarz (University of Braunschweig, Germany) reported on the proteins CNX1 in plants and Gephyrin in humans involved in catalyzing the final step of Moco biosynthesis, whose structure-function relationship is becoming understood. Moco maturation of molybdoenzymes by the Moco-sulfurase abscisic acid 3 (ABA3) as a posttranslational substitution of a Mo-bound oxygen with a sulfur was addressed by Mendel (University of Braunschweig). In Chlamydomonas, Moco is bound to a small carrier protein known as MocoCP. Ataya (University of Córdoba) reported the cloning of a cDNA for MocoCP from Chlamydomonas using microsequenced peptides as the starting point. Antibodies raised against the recombinant MocoCP were used to demonstrate the likely existence of related proteins in higher plants. The precise function of MocoCPs is unknown, but there is speculation that it might act as a Moco storage protein with a role in recycling Moco.

	THE NO3 TRANSPORT SYSTEM IN PLANTS: YET MORE COMPLEXITY

TOP INTRODUCTION REGULATION OF THE NITRATE... INSIGHTS INTO ENZYME STRUCTURE... THE NO3 TRANSPORT SYSTEM... LONG-RANGE SIGNALING IN PLANTS "OME"-OPHOBIA? CONCLUDING REMARKS

Multigene Families: Redundancy or
Diversity of Function?

Chlamydomonas, the model organism for NO₃ transport research in plants, has five known NO₃/NO₂ transport systems, comprising four plasma membrane influx systems with differing substrate affinities and one plastidic NO₂ uptake system. The number of structural genes so far implicated in these transport systems is six. It is perhaps not surprising that the genetics of NO₃ transport in a multicellular organism like a higher plant would prove to be even more complex. The Arabidopsis genome contains seven members of the NRT2 NO₃ transporter gene family (AtNRT2.1-AtNRT2.7), plus at least two NO₃ transporters from an unrelated family (AtNRT1.1 and AtNRT1.2). Krapp (Institut National de la Recherche Agronomique, Versailles, France) and Glass (University of British Columbia, Vancouver, Canada) reported their progress in trying to elucidate the physiological roles of the different NRT1 and NRT2 genes from knockout mutants, promoter-reporter gene fusions, and analysis of mRNA expression patterns throughout the plant. Previous studies from these laboratories have established AtNRT2.1 as a major structural gene encoding the inducible high-affinity NO₃ influx system in roots. Recent work discussed here made clear the diverse regulatory properties and spatial patterns of expression displayed by different AtNRT2 genes. For example, although AtNRT2.1 and AtNRT2.2 are induced by NO₃ in the classical manner, other members of the family are unaffected by the N supply, or are even repressed by NO₃; several of the genes are expressed in the shoot and one is expressed in mature pollen grains. Krapp reported that two AtNRT2 genes are very strongly derepressed in a mutant (atnrt2a) that has the AtNRT2.1 and AtNRT2.2 genes deleted, yet the mutant still has a major defect in the inducible high-affinity NO₃ influx system. This suggests that these other AtNRT2 genes may not contribute to high-affinity NO₃ influx. There is still no information on the subcellular localization of the different NRT2 family members, leaving open the possibility that some may have roles in intracellular NO₃ transport.

It may have been of some comfort to the plant biologists at the meeting to note that gene duplication and its attendant complications are not something restricted to higher organisms. Enteric bacteria have been revealed through genome sequencing projects to have three separate NRs that enable them to reduce NO₃ rapidly to NH₄⁺ during anaerobic growth. Cole (University of Birmingham, UK) reported that three transport proteins, three NR complexes involving multiple components, and two NO₂ reduction pathways were needed to enable fermentative bacteria to survive anaerobically when NO₃ is abundant, when NO₃ is scarce, or when all nutrients are depleted.

One Gene, More Than One Function

Gene duplication is not the only route to complexity. The Arabidopsis AtNRT1.1 (CHL1) gene encodes an unusual dual-affinity component of the root NO₃ influx system that is NO₃ inducible and auxin regulated. Crawford (University of California, San Diego) reported the latest twist in the AtNRT1.1 story. Finding that reporter gene fusions with the AtNRT1.1 promoter were strongly expressed in stomatal guard cells of mature leaves (as well as in roots and in young shoot tissues), he and his collaborators investigated what role this NO₃ transporter might play in stomatal function. They found that NO₃ could substitute for Cl in light-induced stomatal opening, that atnrt1.1 mutants were defective in this response (when NO₃ was the available anion), and that the mutant guard cells were defective in NO₃ accumulation. When grown on NO₃, atnrt1.1 mutants had reduced stomatal apertures and increased drought tolerance. Thus, AtNRT1.1 emerges as fascinating example of a transporter with diverse functions, not only contributing to high- and low-affinity NO₃ uptake by the root, but also with a role in the control of stomatal opening.

One Transporter, More Than One Subunit?

Miller (Rothamsted Research, Harpenden, UK) reported another example illustrating the value of Chlamydomonas as a model for NO₃ transport research. It is known that the NRT2.1 and NRT2.2 genes that encode the high-affinity NO₃ transport Systems I and II in Chlamydomonas require the product of a third gene (NAR2) for their functional expression. NAR2 encodes a small (28-kD) protein with one to two transmembrane domains, but NAR2 homologs are restricted to the plant kingdom and their sequences give little clue to their function. Miller's group has isolated three full-length NAR2 cDNAs from barley (Hordeum vulgare) and used a Xenopus oocyte expression system to show that mRNA from one of these (HvNAR2.3) was able to reconstitute high-affinity NO₃ transport activity when coinjected with mRNA for the otherwise inactive HvNRT2.1 protein. It is still unclear whether NAR2 is a second subunit of this NO₃ transporter or has another role such as in the translocation of NRT2 to the plasma membrane. Other members of the NAR2 family may have different functions: Paneque (Consejo Superior de Investigaciones Cientificas, Madrid) reported that a member of the Arabidopsis NAR2 gene family (WR3.1) is expressed most strongly in hydatodes and stipules, is wound inducible and NO₃ inducible, and is partly localized in the nuclear envelope.

	LONG-RANGE SIGNALING IN PLANTS

TOP INTRODUCTION REGULATION OF THE NITRATE... INSIGHTS INTO ENZYME STRUCTURE... THE NO3 TRANSPORT SYSTEM... LONG-RANGE SIGNALING IN PLANTS "OME"-OPHOBIA? CONCLUDING REMARKS

Signals Going Up...

For many plant species, NH₄⁺ on its own is not the preferred N source, and NH₄⁺-fed plants grow more slowly than NO₃-fed plants and have altered patterns of development. However, the negative effect of NH₄⁺ on vegetative growth is not without a potential commercial advantage because the shift to reproductive growth can lead to increased harvest indices and better fruit quality. Lips (Ben Gurion University of the Negev, Israel) discussed a model for how NO₃ and NH₄⁺ may regulate the balance between vegetative and reproductive growth. The model is based on observations that NO₃ promotes cytokinin production in the root and NH₄⁺ promotes ABA production. Thus, a switch between NO₃ and NH₄⁺ nutrition alters the cytokinin/ABA balance in the xylem sap, providing a mechanism for long-distance signaling from root to shoot. Walch-Liu (Hohenheim) discussed the close link between NO₃ supply to the roots and increased rates of leaf expansion, which are correlated with increased cytokinin fluxes from root to shoot. Latest results showed that addition of NO₃ to NH₄⁺-grown plants transiently induced daily oscillations in leaf growth rates that were directly correlated with oscillations in the concentration of cytokinins (but not NO₃) in the xylem sap.

How might NO₃ regulate cytokinin production in the root? A potential control point in cytokinin biosynthesis is in the step catalyzed by adenylate isopentenyltransferase (IPT). Sakakibara (RIKEN, Japan) reported the analysis of a family of seven Arabidopsis IPT genes and identified two root-expressed genes (AtIPT3 and AtIPT5) that responded positively to increased NO₃ availability. This raises the exciting possibility that one or both of these IPT genes plays a pivotal role in the process of converting an external NO₃ signal into a long-range cytokinin signal.

... and Signals Coming Down

Demand for N in the shoot has long been known as an important regulator of NO₃ uptake activity in the root, but how is this demand communicated from the top to the bottom of the plant? One possible signal that should be positively correlated with the shoot's demand for N is the phloem-mediated flux of sugars arriving in the root. Gojon (Institut National de la Recherche Agronomique, Montpellier) reported that external application of Suc to Arabidopsis roots stimulated not only the AtNRT1.1 and AtNRT2.1 NO₃ transporter genes, but also a range of other nutrient transporter genes. There was a strong correlation between those genes whose root expression was diurnally regulated and those that were up-regulated by Suc. This would support the idea that the rate of sugar export from the shoot plays an important role in integrating nutrient uptake in the root with the requirements of the shoot. However, more specific signals related to the demand for particular nutrients are also likely to exist. In an attempt to gain some insight into these signals, Gojon and colleagues are using an Arabidopsis split root system in combination with Serial Analysis of Gene Expression to identify genes responsive to long-range signals related to changes in the N status of the shoot. In a collection of 40,000 gene tags representing 5,000 different genes, they found 85 genes whose expression levels were altered in one-half of the root system when the rest of the root was deprived of NO₃ for 24 h.

	"OME"-OPHOBIA?

TOP INTRODUCTION REGULATION OF THE NITRATE... INSIGHTS INTO ENZYME STRUCTURE... THE NO3 TRANSPORT SYSTEM... LONG-RANGE SIGNALING IN PLANTS "OME"-OPHOBIA? CONCLUDING REMARKS

Although the impact of genomic sequencing projects on NO₃ assimilation research permeated the whole meeting, there was a marked scarcity of talks describing the application of downstream "-omic" technologies. One notable exception was the presentation of Lejay (New York University, New York) who reported progress toward a systems approach to understanding C:N signaling in Arabidopsis using transcriptomics. She also described the development of a powerful bioinformatics tool called "PathExplore" that can be used to query microarray data to determine how genes common to particular metabolic pathways are regulated. Valuable input for such an analysis is likely to come from microarray experiments such as those reported by Boivin (Institut National de la Recherche Agronomique, d'Evry, France) in which the responses of the transcriptome to N starvation and resupply are being analyzed. Metabolomics are being used to try to understand the phenotype of a plant mutant defective in the plastidic form of Gln synthase (Marquez, University of Seville).

Transcription profiling was not the only "whole genome" technique discussed in the context of uncovering N regulatory circuits. Hirel (Institut National de la Recherche Agronomique, Versailles, France) and Yamaya (Tohoku University, Sendai, Japan) are using quantitative trait loci mapping in maize (Zea mays) and rice (Oryza sativa), respectively, to identify genetic determinants of N-use efficiency. Hirel pointed out the potential that this approach holds for identifying the master regulatory genes that orchestrate the plant's responses to changing N nutrition.

	CONCLUDING REMARKS

TOP INTRODUCTION REGULATION OF THE NITRATE... INSIGHTS INTO ENZYME STRUCTURE... THE NO3 TRANSPORT SYSTEM... LONG-RANGE SIGNALING IN PLANTS "OME"-OPHOBIA? CONCLUDING REMARKS

This latest meeting in the NAMGA series reminded us yet again how valuable it can be to exchange ideas, approaches, and results across the great divide that so often keeps plant biologists apart from their colleagues working on analogous problems in other kingdoms. Clear homologies exist between the proteins that catalyze the NO₃ assimilatory pathways in the different kingdoms. This gives ample scope for plant biologists to benefit from the structural and functional insights emerging from the more amenable microbial systems. It is curious that the nuts and bolts of NO₃ regulation seem not to have been similarly conserved across the kingdoms. Did the mechanisms for NO₃ induction and feedback repression of the pathway evolve independently in bacteria, fungi, and plants? Until we know more about how plant NO₃ assimilation is regulated, we cannot be sure. However, with the rapid advances now possible in the postgenomics era, it seems safe to predict that by the time NAMGA2006 comes around, at least some of the key factors that regulate NO₃ assimilation in plants will have been identified. The comparisons that can then be made between NO₃ regulatory circuits in diverse phylogenetic groups will no doubt contribute to another fruitful and memorable meeting in the NAMGA series.