Adaptive combinatorial design to explore large experimental spaces: approach and validation.

Systems biology requires mathematical tools not only to analyse large genomic datasets, but also to explore large experimental spaces in a systematic yet economical way. We demonstrate that two-factor combinatorial design (CD), shown to be useful in software testing, can be used to design a small set of experiments that would allow biologists to explore larger experimental spaces. Further, the results of an initial set of experiments can be used to seed further 'Adaptive' CD experimental designs. As a proof of principle, we demonstrate the usefulness of this Adaptive CD approach by analysing data from the effects of six binary inputs on the regulation of genes in the N-assimilation pathway of Arabidopsis. This CD approach identified the more important regulatory signals previously discovered by traditional experiments using far fewer experiments, and also identified examples of input interactions previously unknown. Tests using simulated data show that Adaptive CD suffers from fewer false positives than traditional experimental designs in determining decisive inputs, and succeeds far more often than traditional or random experimental designs in determining when genes are regulated by input interactions. We conclude that Adaptive CD offers an economical framework for discovering dominant inputs and interactions that affect different aspects of genomic outputs and organismal responses.

Carbon and nitrogen sensing and signaling in plants: emerging 'matrix effects'.

Plants, like other organisms, have developed mechanisms that allow them to sense and respond to changes in levels of carbon and nitrogen metabolites. These mechanisms, in turn, regulate the expression of genes and the activities of proteins involved in C and N transport and metabolism, allowing plants to optimize the use of energy resources. Recent studies, which have involved molecular-genetic, genomic, and cell biological approaches, have begun to uncover the signals and components of C:N sensing and signaling mechanisms in plants. For sugar sensing, analysis of Arabidopsis mutants has revealed intersections with hormone and nitrogen signaling. For nitrogen sensing/signaling, recent progress has identified transcriptional and posttranslational mechanisms of regulation. In all, a complex picture is emerging in which C:N signaling systems are subject to a 'matrix effect' in which downstream responses are dependent upon cell-type, developmental, metabolic, and/or environmental conditions.

Arabidopsis mutants resistant to S(+)-beta-methyl-alpha, beta-diaminopropionic acid, a cycad-derived glutamate receptor agonist.

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that are the predominant neuroreceptors in the mammalian brain. Genes with high sequence similarity to animal iGluRs have been identified in Arabidopsis. To understand the role of Arabidopsis glutamate receptor-like (AtGLR) genes in plants, we have taken a pharmacological approach by examining the effects of BMAA [S(+)-beta-methyl-alpha, beta-diaminopropionic acid], a cycad-derived iGluR agonist, on Arabidopsis morphogenesis. When applied to Arabidopsis seedlings, BMAA caused a 2- to 3-fold increase in hypocotyl elongation and inhibited cotyledon opening during early seedling development. The effect of BMAA on hypocotyl elongation is light specific. Furthermore, BMAA effects on early morphogenesis of Arabidopsis can be reversed by the simultaneous application of glutamate, the native iGluR agonist in animals. To determine the targets of BMAA action in Arabidopsis, a genetic screen was devised to isolate Arabidopsis mutants with a BMAA insensitive morphology (bim). When grown in the light on BMAA, bim mutants exhibited short hypocotyls compared with wild type. bim mutants were grouped into three classes based on their morphology when grown in the dark in the absence of BMAA. Class-I bim mutants have a normal, etiolated morphology, similar to wild-type plants. Class-II bim mutants have shorter hypocotyls and closed cotyledons when grown in the dark. Class-III bim mutants have short hypocotyls and open cotyledons when grown in the dark, resembling the previously characterized constitutively photomorphogenic mutants (cop, det, fus, and shy). Further analysis of the bim mutants should help define whether plant-derived iGluR agonists target glutamate receptor signaling pathways in plants.

Carbon and amino acids reciprocally modulate the expression of glutamine synthetase in Arabidopsis.

In bacteria and yeast, glutamine synthetase (GS) expression is tightly regulated by the metabolic status of the cell, both at the transcriptional and posttranscriptional levels. We discuss the relative contributions of light and metabolic cues on the regulation of members of the GS gene family (chloroplastic GS2 and cytosolic GS1) in Arabidopsis. These studies reveal that the dramatic induction of mRNA for chloroplastic GS2 by light is mediated in part by phytochrome and in part by light-induced changes in sucrose (Suc) levels. In contrast, the modest induction of mRNA for cytosolic GS1 by light is primarily mediated by changes in the levels of carbon metabolites. Suc induction of mRNA for GS2 and GS1 occurs in a time- and dose-dependent manner. Suc-induced changes in GS mRNA levels were also observed at the level of GS enzyme activity. In contrast, amino acids were shown to antagonize the Suc induction of GS, both at the level of mRNA accumulation and that of enzyme activity. For GS2, the gene whose expression was the most dramatically regulated by metabolites, we used a GS2 promoter-beta-glucuronidase fusion to demonstrate that transcriptional control is involved in this metabolic regulation. Our results suggest that the metabolic regulation of GS expression in plants is controlled by the relative abundance of carbon skeletons versus amino acids. This would allow nitrogen assimilation into glutamine to proceed (or not) according to the metabolic status and biosynthetic needs of the plant. This type of GS gene regulation is reminiscent of the nitrogen regulatory system in bacteria, and suggests an evolutionary link between metabolic sensing and signaling in bacteria and plants.

Arabidopsis mutants define an in vivo role for isoenzymes of aspartate aminotransferase in plant nitrogen assimilation.

Arabidopsis contains five isoenzymes of aspartate aminotransferase (AspAT) localized to the cytosol, chloroplast, mitochondria, or peroxisomes. To define the in vivo function of individual isoenzymes, we screened for Arabidopsis mutants deficient in either of the two major isoenzymes, cytosolic AAT2 or chloroplastic AAT3, using a native gel activity assay. In a screen of 8,000 M2 seedlings, three independent mutants deficient in cytosolic AAT2 (aat2) and two independent mutants deficient in chloroplastic AAT3 (aat3) were isolated. Mapping of aat2 and aat3 mutations and the five AspAT genes (ASP1-ASP5) established associations as follows: the mutation affecting aat2 maps with and cosegregates with ASP2, one of two expressed genes for cytosolic AspAT; the mutation affecting aat3 maps to the same location as the ASP5 gene encoding chloroplastic AspAT. Phenotypic analysis of the aat2 and aat3 mutants revealed a dramatic aspartate-related phenotype in one of the mutants deficient in cytosolic AAT2. The aat2-2 mutant displays an 80% reduction in levels of aspartate transported in the phloem of light-grown plants, and a 50% reduction in levels of asparagine transported in dark-adapted plants. These results indicate that cytosolic AAT2 is the major isoenzyme controlling aspartate synthesized for nitrogen transport in the light, and that this aspartate pool is converted to asparagine when plants are dark adapted.

Arabidopsis gls mutants and distinct Fd-GOGAT genes. Implications for photorespiration and primary nitrogen assimilation.

Ferredoxin-dependent glutamate synthase (Fd-GOGAT) plays a major role in photorespiration in Arabidopsis, as has been determined by the characterization of mutants deficient in Fd-GOGAT enzyme activity (gls). Despite genetic evidence for a single Fd-GOGAT locus and gene, we discovered that Arabidopsis contains two expressed genes for Fd-GOGAT (GLU1 and GLU2). Physical and genetic mapping of the gls1 locus and GLU genes indicates that GLU1 is linked to the gls1 locus, whereas GLU2 maps to a different chromosome. Contrasting patterns of GLU1 and GLU2 expression explain why a mutation in only one of the two genes for Fd-GOGAT leads to a photorespiratory phenotype in the gls1 mutants. GLU1 mRNA was expressed at the highest levels in leaves, and its mRNA levels were specifically induced by light or sucrose. In contrast, GLU2 mRNA was expressed at lower constitutive levels in leaves and preferentially accumulated in roots. Although these results suggest a major role for GLU1 in photorespiration, the sucrose induction of GLU1 mRNA in leaves also suggests a role in primary nitrogen assimilation. This possibility is supported by the finding that chlorophyll levels of a gls mutant are significantly lower than those of the wild type when grown under conditions that suppress photorespiration. Both the mutant analysis and gene regulation studies suggest that GLU1 plays a major role in photorespiration and also plays a role in primary nitrogen assimilation in leaves, whereas the GLU2 gene may play a major role in primary nitrogen assimilation in roots.

THE MOLECULAR-GENETICS OF NITROGEN ASSIMILATION INTO AMINO ACIDS IN HIGHER PLANTS.

Nitrogen assimilation is a vital process controlling plant growth and development. Inorganic nitrogen is assimilated into the amino acids glutamine, glutamate, asparagine, and aspartate, which serve as important nitrogen carriers in plants. The enzymes glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), aspartate aminotransferase (AspAT), and asparagine synthetase (AS) are responsible for the biosynthesis of these nitrogen-carrying amino acids. Biochemical studies have revealed the existence of multiple isoenzymes for each of these enzymes. Recent molecular analyses demonstrate that each enzyme is encoded by a gene family wherein individual members encode distinct isoenzymes that are differentially regulated by environmental stimuli, metabolic control, developmental control, and tissue/cell-type specificity. We review the recent progress in using molecular-genetic approaches to delineate the regulatory mechanisms controlling nitrogen assimilation into amino acids and to define the physiological role of each isoenzyme involved in this metabolic pathway.

Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation.

Glutamate dehydrogenase (GDH) is ubiquitous to all organisms, yet its role in higher plants remains enigmatic. To better understand the role of GDH in plant nitrogen metabolism, we have characterized an Arabidopsis mutant (gdh1-1) defective in one of two GDH gene products and have studied GDH1 gene expression. GDH1 mRNA accumulates to highest levels in dark-adapted or sucrose-starved plants, and light or sucrose treatment each repress GDH1 mRNA accumulation. These results suggest that the GDH1 gene product functions in the direction of glutamate catabolism under carbon-limiting conditions. Low levels of GDH1 mRNA present in leaves of light-grown plants can be induced by exogenously supplied ammonia. Under such conditions of carbon and ammonia excess, GDH1 may function in the direction of glutamate biosynthesis. The Arabidopsis gdh-deficient mutant allele gdh1-1 cosegregates with the GDH1 gene and behaves as a recessive mutation. The gdh1-1 mutant displays a conditional phenotype in that seedling growth is specifically retarded on media containing exogenously supplied inorganic nitrogen. These results suggest that GDH1 plays a nonredundant role in ammonia assimilation under conditions of inorganic nitrogen excess. This notion is further supported by the fact that the levels of mRNA for GDH1 and chloroplastic glutamine synthetase (GS2) are reciprocally regulated by light.

Molecular evolution of duplicate copies of genes encoding cytosolic glutamine synthetase in Pisum sativum.

Here, we describe two nearly identical expressed genes for cytosolic glutamine synthetase (GS3A and GS3B) in Pisum sativum L. RFLP mapping data indicates that the GS3A and GS3B genes are separate loci located on different chromosomes. DNA sequencing of the GS3A and GS3B genes revealed that the coding regions are 99% identical with only simple nucleotide substitutions resulting in three amino acid differences. Surprisingly, the non-coding regions (5' non-coding leader, the 11 introns, and 3' non-coding tail) all showed a high degree of identity (96%). In these non-coding regions, 25% of the observed differences between the GS3A and GS3B genes were deletions or duplications. The single difference in the 3' non-coding regions of the GS3A and GS3B genes was a 25 bp duplication of an AU-rich element in the GS3B gene. As the GS3B mRNA accumulates to lower levels than the GS3A gene, we tested whether this sequence which resembles an mRNA instability determinant functioned as such in the context of the GS mRNA. Using the GS3B 3' tail as part of a chimeric gene in transgenic plants, we showed that this AU-rich sequence has little effect on transgene mRNA levels. To determine whether the GS3A/GS3B genes represent a recent duplication, we examined GS3-like genes in genomic DNA of ancient relatives of P. sativum. We observed that several members of the Viceae each contain two genomic DNA fragments homologous to the GS3B gene, suggesting that this is an ancient duplication event. Gene conversion has been invoked as a possible mechanism for maintaining the high level of nucleotide similarity found between GS3A and GS3B genes. Possible evolutionary reasons for the maintenance of these 'twin' GS genes in pea, and the general duplication of genes for cytosolic GS in all plant species are discussed.

Cis elements and trans-acting factors affecting regulation of a nonphotosynthetic light-regulated gene for chloroplast glutamine synthetase.

The glutamine synthetase (GS) gene family in pea (Pisum sativum) consists of four nuclear genes encoding distinct isoenzymes. Molecular studies have show that the GS2 gene encoding chloroplast-localized GS is expected in specific cell types and is regulated by diverse factors such as light and photorespiration. Here, we present the nucleotide sequence of the pea GS2 gene promoter. To identify the elements involved in regulation of GS2 expression, GS2 promoter-deletion analyses were performed using GS2-GUS fusions in tobacco (Nicotiana tabacum). This analysis revealed that the GS2 transit peptide is not required for mesophyll cell-specific expression of beta-glucuronidase (GUS). GUS activity was induced 2- to 4-fold in light-grown versus etiolated T1 seedlings. However, high levels of GUS activity were observed in etiolated seedlings. This observation demonstrated that regulation of expression of GS2, a nonphotosynthetic light-regulated gene, involves additional factors. A 323-bp GS2 promoter sequence is sufficient to confer light regulation to the GUS reporter gene in leaves of mature transgenic tobacco. Light-regulated expression of this pea gene promoter is observed in both tobacco and Arabidopsis, suggesting that the regulatory elements are conserved. Gel-shift analysis detected DNA-protein complexes formed with potential transcription elements within this short, light-responsive GS2 promoter fragment.

The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments.

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana. Four classes of cDNAs representing four distinct AspAT genes (ASP1-ASP4) have been cloned from Arabidopsis. Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1-AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis. These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.

Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana.

Here, we characterize a cDNA encoding a glutamine-dependent asparagine synthetase (ASN1) from Arabidopsis thaliana and assess the effects of metabolic regulation on ASN1 mRNA levels. Sequence analysis shows that the predicted ASN1 peptide contains a purF-type glutamine-binding domain. Southern blot experiments and cDNA clone analysis suggest that ASN1 is the only gene encoding glutamine-dependent asparagine synthetase in A. thaliana. The ASN1 gene is expressed predominantly in shoot tissues, where light has a negative effect on its mRNA accumulation. This negative effect of light on ASN1 mRNA levels was shown to be mediated, at least in part, via the photoreceptor phytochrome. We also investigated whether light-induced changes in nitrogen to carbon ratios might exert a metabolic regulation of the ASN1 mRNA accumulation. These experiments demonstrated that the accumulation of ASN1 mRNA in dark-grown plants is strongly repressed by the presence of exogenous sucrose. Moreover, this sucrose repression of ASN1 expression can be partially rescued by supplementation with exogenous amino acids such as asparagine, glutamine, and glutamate. These findings suggest that the expression of the ASN1 gene is under the metabolic control of the nitrogen to carbon ratio in cells. This is consistent with the fact that asparagine, synthesized by the ASN1 gene product, is a favored compound for nitrogen storage and nitrogen transport in dark-grown plants. We have put forth a working model suggesting that when nitrogen to carbon ratios are high, the gene product of ASN1 functions to re-direct the flow of nitrogen into asparagine, which acts as a shunt for storage and/or long-distance transport of nitrogen.

A novel AT-rich DNA binding protein that combines an HMG I-like DNA binding domain with a putative transcription domain.

There is growing evidence that AT-rich promoter elements play a role in transcription of plant genes. For the promoter of the nuclear gene for chloroplast glutamine synthetase from pea (GS2), the deletion of a 33-bp AT-rich sequence (box 1 native) from the 5' end of a GS2 promoter-beta-glucuronidase (GUS) fusion resulted in a 10-fold reduction in GUS activity. The box 1 native element was used in gel shift analysis and two distinct complexes were detected. One complex is related to the low-mobility complex reported previously for AT-rich elements from several other plant promoters. A multimer of the box 1 sequence was used to isolate a cDNA encoding an AT-rich DNA binding protein (ATBP-1). ATBP-1 is not a high-mobility group protein, but it is a novel protein that combines a high-mobility group I/Y-like DNA binding domain with a glutamine-rich putative transcriptional domain.

Ectopic Overexpression of Asparagine Synthetase in Transgenic Tobacco.

Here, we monitor the effects of ectopic overexpression of genes for pea asparagine synthetase (AS1) in transgenic tobacco (Nicotiana tabacum). The AS genes of pea and tobacco are normally expressed only during the dark phase of the diurnal growth cycle and specifically in phloem cells. A hybrid gene was constructed in which a pea AS1 cDNA was fused to the cauliflower mosaic virus 35S promoter. The 35S-AS1 gene was therefore ectopically expressed in all cell types in transgenic tobacco and constitutively expressed at high levels in both the light and the dark. Northern analysis demonstrated that the 35S-AS1 transgene was constitutively expressed at high levels in leaves of several independent transformants. Furthermore, amino acid analysis revealed a 10- to 100-fold increase in free asparagine in leaves of transgenic 35S-AS1 plants (construct z127) compared with controls. Plant growth analyses showed increases (although statistically insignificant) in growth phenotype during the vegetative stage of growth in 35S-AS1 transgenic lines. The 35S-AS1 construct was further modified by deletion of the glutamine-binding domain of the enzyme (gln[delta]AS1; construct z167). By analogy to animal AS, we reasoned that inhibition of glutamine-dependent AS activity might enhance the ammonia-dependent AS activity. The 3- to 19-fold increase in asparagine levels in the transgenic plants expressing gln[delta]AS1 compared with wild type suggests that the novel AS holoenzyme present in the transgenic plants (gln[delta]AS1 homodimer) has enhanced ammonia-dependent activity. These data indicate that manipulation of AS expression in transgenic plants causes an increase in nitrogen assimilation into asparagine, which in turn produces effects on plant growth and asparagine biosynthesis.

A promoter sequence involved in cell-specific expression of the pea glutamine synthetase GS3A gene in organs of transgenic tobacco and alfalfa.

The DNA sequence of the pea cytosolic glutamine synthetase GS3A gene promoter has been determined and the start of transcription mapped using S1 nuclease. The full-length promoter and a series of 5' deletions were fused to beta-glucuronidase (GUS) and introduced into transgenic tobacco and alfalfa. In transgenic tobacco the GS3A promoter directed GUS expression in the phloem cells of the vasculature in leaves, stems and roots. GUS expression was also detected in the vasculature of cotyledons and the root tips of germinating T1 seedlings. The promoter conferred a similar expression pattern in transgenic alfalfa, and expression was also observed in root nodules. Nodule expression was located in nodule primordia, as well as the meristem, symbiotic zone, and vasculature of mature nodules. The promoter was found to be active even when deleted to -132 relative to the start of transcription. DNA mobility-shift analysis identified a protein present in nuclear and whole-cell plant extracts which bound to a 17 bp DNA element contained within the minimal -132 promoter required for expression.

Cell-specific expression in transgenic plants reveals nonoverlapping roles for chloroplast and cytosolic glutamine synthetase.

Chloroplast and cytosolic isoforms of glutamine synthetase (GS; EC 6.3.1.2) are encoded by separate nuclear genes in plants. Here we report that the promoters for chloroplast GS2 and cytosolic GS3A of Pisum sativum confer nonoverlapping, cell-specific expression patterns on the beta-glucuronidase (GUS) reporter gene in transgenic tobacco. The promoter for chloroplast GS2 directs GUS expression within photosynthetic cell types (e.g., palisade parenchymal cells of the leaf blade, chlorenchymal cells of the midrib and stem, and photosynthetic cells of tobacco cotyledons). The promoter for chloroplast GS2 retains the ability to confer light-regulated gene expression in the heterologous transgenic tobacco system in a manner analogous to the light-regulated expression of the cognate gene for chloroplast GS2 in pea. These expression patterns reflect the physiological role of the chloroplast GS2 isoform in the assimilation of ammonia generated by nitrite reduction and photorespiration. In contrast, the promoter for cytosolic GS3A directs expression of GUS specifically within the phloem elements in all organs of mature plants. This phloem-specific expression pattern suggests that the cytosolic GS3A isoenzyme functions to generate glutamine for intercellular nitrogen transport. In germinating seedlings, the intense expression of the cytosolic GS3A-GUS transgene in the vasculature of cotyledons reveals a role for cytosolic GS in the mobilization of seed storage reserves. The distinct, cell-specific patterns of expression conferred by the promoters for chloroplast GS2 and cytosolic GS3A indicate that the corresponding GS isoforms perform separate metabolic functions.