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. 2003:2:e0010.
doi: 10.1199/tab.0010. Epub 2003 Sep 30.

Primary N-assimilation into Amino Acids in Arabidopsis

Gloria M Coruzzi  1
Affiliations
Free PMC article

Primary N-assimilation into Amino Acids in Arabidopsis

Gloria M Coruzzi. Arabidopsis Book. 2003.
Free PMC article
No abstract available

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Figures

Figure 1.
Figure 1.
Nitrogen Assimilatory Pathway
Figure 2.
Figure 2.
Amino Acid Levels in Light-Grown and Dark-Adapted Wild-Type Arabidopsis Plants. Amino acids levels in Arabidopsis plants grown in light (empty boxes) or subsequently dark adapted for 24 hr (filled boxes). The standard three letter code is used for all amino acids. (A) Average total free amino acid content. Each sample represents the average of three different plants (two leaves/plant). Gaba, γ-amino butyric acid. (B) Average free amino acid content in phloem exudates of three independent plants (one leaf/plant). (C) Average free amino acid content of xylem sap collected from cut hypocotyls of three independent plants. Data are from Schultz (1994). From: Hon-Ming Lam, Karen Coschigano, Carolyn Schultz, Rosana Melo-Oliveira, Gabrielle Tjaden, Igor Oliveira, Nora Ngai, Ming-Hsiun Hsieh, and Gloria Coruzzi (1995). Use of Arabidopsis Mutants and Genes to Study Amide Amino Acid Biosynthesis. Plant Cell 7, 887–898.
Figure 3.
Figure 3.
C:N Regulation of GS mRNA levels and GS Activity. (A) Amino acids and carbon reciprocally regulate GS mRNA accumulation and enzyme levels. Arabidopsis plants were grown semihydroponically and dark-adapted. The dark-adapted Arabidopsis plants were transferred in the dark to fresh low-nitrogen MS medium with no carbon supplementation (Con, lane 1), to fresh low-nitrogen MS medium with 3% Suc (lane 2), or to fresh low-nitrogen MS medium with 3% Suc in addition of either 10 mM Asp (lane 3), 10 mM Asn (lane 4), 10 mM Glu (lane 5), or 10 mM Gln (lane 6). After transfer, the plants were further incubated for 12 h in the dark. (B) Same as A except that an aliquot of each sample was collected for determination of total GS activity. A representative experiment of two repetitions is shown (results are GS activity per milligram of total protein ± SE of three independent determinations). From: Igor C. Oliveira and Gloria M. Coruzzi (1999). Carbon and Amino Acid Reciprocally Modulate the Expression of Glutamine Synthetase in Arabidopsis. Plant Physiology 121, 301–309.
Figure 4.
Figure 4.
Photorespiratory Phenotype of gls Mutants Defective in Fd-GOGAT Activity. In (A) – (E), Arabidopsis plants were grown in air; in (F) – (J), plants were grown in 2% CO2 Genotypes: (A) and (F) Arabidopsis wild-type Columbia. (B) and (G) Photorespiratory gls mutant CS103. (C) and (H) NA60. (D) and (I) CS37. (E) and (J) CS340. From: Karen T. Coschigano, Rosana Melo-Oliveira, Jackie Lam, and Gloria Coruzzi (1998). Arabidopsis gls Mutants and Distinct Fd-GOGAT Genes: Implications for Photorespiration and Primary Nitrogen Assimilation. Plant Cell 10, 741–752.
Figure 5.
Figure 5.
Levels of mRNA for Fd-GOGAT genes GLU1 & GLU2 in leaves vs. roots. GLU1 is the major mRNA in leaves, while GLU2 predominates in roots. Control RNA is tubulin. From: Karen T. Coschigano, Rosana Melo-Oliveira, Jackie Lam, and Gloria Coruzzi (1998). Arabidopsis gls Mutants and Distinct Fd-GOGAT Genes: Implications for Photorespiration and Primary Nitrogen Assimilation. Plant Cell 10, 741–752.
Figure 6.
Figure 6.
A gls Mutant Displays a Defect in the Assimilation of Inorganic Nitrogen under Nonphotorespiratory Conditions. (A) and (B) Plants grown in air and plants in 1% CO2, respectively. Wild-type Columbia (WT) and a gls mutant (NA60) were grown for 12 days on nitrogen-free MS media supplemented with either low levels of inorganic nitrogen (2 mM ammonium and 4 mM nitrate; open bars) or high levels of inorganic nitrogen (20 mM ammonium and 40 mM nitrate; hatched bars). Mean values of total chlorophyll measurements ± SE are shown. (C) Two-way ANOVA of (1) the wild type (WT) versus a gls mutant in air and CO2, (2) low versus high nitrogen conditions in air and CO2, and (3) interactions of 1 and 2 as given above. Statistically significant results are indicated by an asterisk. From: Karen T. Coschigano, Rosana Melo-Oliveira, Jackie Lam, and Gloria Coruzzi (1998). Arabidopsis gls Mutants and Distinct Fd-GOGAT Genes: Implications for Photorespiration and Primary Nitrogen Assimilation. Plant Cell 10, 741–752.
Figure 7.
Figure 7.
A Model for the Non-Redundant Roles of GOGAT Isoenzymes in Leaves and Roots. Model based on analysis of Arabidopsis GOGAT mutants in Fd-GOGAT (gls) or NADH-GOGAT (glt1-T) (Somerville and Ogren, 1980), (Coschigano et al., 1998), (Lancien et al., 2002). Fd-GOGAT in leaves encoded by Glu1 is primarily responsible for assimilation of photorespiratory ammonia while NADH-GOGAT is responsible for glutamate synthesis in roots. From: Muriel Lancien, Melinda Martin, Ming-Hsiun Hsieh, Tom Leustek, Howard Goodman and Gloria M. Coruzzi (2002). Arabidopsis glt1-T mutant defines a role for NADH-GOGAT in the non-photorespiratory ammonium assimilatory pathway. Plant Journal 29(3), 347–358.
Figure 8.
Figure 8.
GDH Activity in Wild-Type Arabidopsis and gdh 1-1 Mutant. Crude leaf protein extracts were made from rosette leaves of 21-day-old Arabidopsis plants, separated by electrophoresis on a nondenaturing polyacrylamide gel, and stained for GDH activity. Lanes 1 and 7, extract of wild-type Arabidopsis (Columbia). The seven holoenzymes result from the formation of two homohexamers (GDH1 and GDH2), and five heterohexamers of GDH are indicated on the right (GDH1/GDH2). M3 individuals from a selfed gdh 1-1mutant display only the GDH2 homohexamer (lanes 2–6). From: Rosana Melo-Oliveira, Igor Cunha Oliveira, and Gloria M. Coruzzi (1996). Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation. Proc. Natl. Acad. Sci. 93, 4718–4723.
Figure 9.
Figure 9.
Growth Phenotype of the gdh 1-1 Mutant. Growth of wild-type Arabidopsis versus the gdh 1-1 mutant seedlings was measured in a vertical root length assay. Wild-type (wt) and M3 seeds of the gdh 1-1 mutant were sown side-by-side on ammonia-free/nitrate-free MS media containing vitamins and 3% sucrose supplemented with either: (A) no organic nitrogen (0 mM ammonia, 0 mM nitrate); (B) intermediate levels of inorganic nitrogen (2 mM ammonia, 4 mM nitrate); or (C) high levels of inorganic nitrogen (20 mM ammonia, 40 mM nitrate). (D) Plants grown on MS media supplemented with 3% sucrose containing high levels of inorganic nitrogen (20 mM ammonia, 40 mM nitrate) without the vitamin supplement (*). Plates were incubated vertically for 12 days and grown under a normal day/night cycle. From: Rosana Melo-Oliveira, Igor Cunha Oliveira, and Gloria M. Coruzzi (1996). Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation. Proc. Natl. Acad. Sci. 93, 4718–4723.
Figure 10.
Figure 10.
ASP Genes and Major AAT Isoenzymes in Arabidopsis. Five ASP genes encoding AAT isoenzymes: ASP1 encodes mitochondrial AAT1, ASP2 encodes cytosolic AAT2, ASP5 encodes chloroplastic AAT3, ASP3 encodes a putative peroxisomal AAT, and ASP4 encodes a minor cytosolic AAT.
Figure 11.
Figure 11.
AspAT Activity Gels of Wild-Type, Single and Double aat Mutants. AspAT activity gel of seven different F2 individuals from the cross aat2-1/aat2-1 X aat3-2/aat3-2 showing individuals which are homozygous wild-type for both cytosolic and chloroplastic activity (lanes 1 and 5); homozygous mutant for cytosolic AAT2 but wild-type for chloroplastic AAT3 (lanes 2 and 6); homozygous mutant for both cytosolic and chloroplastic AspAT (lanes 3 and 7); and wild-type for cytosolic AAT2 but homozygous mutants for chloroplastic AAT3 (lane 4). The genotypes of the F2 individuals shown are as follows: AAT2-1/AAT2-1, AAT3-2/AAT3-2 (lanes 1 and 5); aat2-1/aat2-1, AAT3-2/AAT3-2 (lanes 2 and 6); aat2-1/aat2-1, aat3-2/aat3-2 (lanes 3 and 7); AAT2-1/AAT2-1, aat3-2/aat3-2 (lane 4). The amount of rubisco protein was determined by Coomassie staining of the gel after activity staining. From: Schultz CJ (1994) A molecular and genetic dissection of the aspartate aminotransferase isoenzymes of Arabidopsis thaliana. A Ph.D. thesis. New York University, New York, NY
Figure 12.
Figure 12.
aat2-2 mutants have specific reductions in levels of aspartate in light-grown plants and asparagine in dark-adapted plants. The relative proportions of aspartate and asparagine in the phloem exudates from wild-type Columbia (Col) and aat2-2 mutant plants grown in light (unshaded boxes) or dark adapted (shaded boxes). Each sample is the average of a single leaf from three representative plants. Plants were grown in soil in a normal day/night cycle (16 hr light/8 hr dark) for 3 wk and either light adapted (unshaded box) or dark adapted (shaded box) for 24 hr. Error bars represent the standard error of the mean. From: Carolyn J. Schultz, Meier Hsu, Barbara Miesak and Gloria Coruzzi (1998). Arabidopsis Mutants Define an in Vivo Role for Isoenzymes of Aspartate Aminotransferase in Plant Nitrogen Assimilation. Genetics 149; 491–499.
Figure 13.
Figure 13.
Relative levels of ASN1 and ASN2 mRNA in dark-adapted versus light-treated plants. Sixteen-day-old Arabidopsis seedlings were grown on tissue culture plates (MS plus 3% sucrose) and dark-adapted (D, lane 1) or light-treated (L, lane 2) for 48 h. Ten µg of total RNA was subjected to Northern blot analysis and hybridized with ASN1 or ASN2. ASN1 and ASN2 gene-specific probes were approximately the same specific activity and X-ray exposure times were identical. 18S rRNA was detected on a replicate blot as a loading control. Following normalization to the corresponding rRNA, the highest level of mRNA (ASN1, lane 1) was set at 1.0. The bar graph (b) represents the relative levels of ASN1 and ASN2 transcripts in (a). From: Hon-Ming Lam, Ming-Hsiun Hsieh and Gloria Coruzzi (1998). Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana. Plant Journal 16(3), 345–353.
Figure 14.
Figure 14.
N-assimilation into Amino Acids is regulated by light. Cytosolic AAT2 controls the synthesis of aspartate in the light, which is converted to asparagine in the dark. A model is depicted for the metabolic flow of nitrogen assimilation into the nitrogen-transport amino acids glutamate, glutamine, aspartate, and asparagine in the light and dark. In the light, inorganic nitrogen is assimilated initially into glutamate and glutamine by the combined actions of the plastid enzymes: chloroplastic glutamine synthetase (GS2, encoded by GLN2), and ferredoxin-dependent glutamate synthase (Fd-GOGAT, encoded by GLU1; Oliveira et al. 1997; Coschigano et al. 1998). The conversion of glutamate into aspartate in the light is controlled by cytosolic AAT2. In the dark, this pool of aspartate is converted into asparagine by asparagine synthetase (ASN1) (Lam et al. 1994, 1995). From: Carolyn J. Schultz, Meier Hsu, Barbara Miesak and Gloria Coruzzi (1998). Arabidopsis Mutants Define an in Vivo Role for Isoenzymes of Aspartate Aminotransferase in Plant Nitrogen Assimilation. Genetics 149; 491–499.
Figure 15.
Figure 15.
C:N Regulation of GLN2/ASN1 mRNA levels. Light and metabolites cause a reciprocal effect on Arabidopsis GLN2 and ASN1 gene expression. The mRNA levels of ASN1 are repressed to nearly undetectable levels in light-grown plants (lane 8) and are strongly enhanced in dark-adapted plants (lane 9). By contrast, the low, undetectable levels of GLN2 mRNA from plants grown in the dark (lane 2) are highly elevated in light-grown plants (lane 1). Sucrose can partially mimic the effects of light by causing the induction of GLN2 and repressing the expression of ASN1 mRNA in the dark (lane 10). In the dark, the light-mimicking effects of sucrose can be antagonized by treatment with amino acids (AA) (GLN2, lane 4 to 7 and ASN1, lanes 11 to 13) with each AA affecting the expression of GLN2 and ASN1 to different extents. The differential effects of the AA on both GLN2 and ASN1 mRNA levels may be explained by the possibility that each AA exerts its effects through different but partially overlapping pathways. However, one cannot rule out differences due to rate of uptake or metabolism. PHY, phytochrome; GLN2, Arabidopsis GS2 (glutamate synthetase) gene; ASN1, Arabidopsis gene AS1 (asparagine synthetase) gene. From: I.C. Oliveira, E. Brenner, J. Chiu, M.-H. Hsieh, A. Kouranov, H.-M. Lam, M.J.Shin and G. Coruzzi (2001). Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway. Brazillian Journal of Medical and Biological Research. 34: 567–575.
Figure 16.
Figure 16.
Multiple Input Regulation of N-assimilation genes. A simplified scheme depicting reciprocal regulation of GLN2 & ASN1 by Light, Carbon and Nitrogen. Also depicted are NR (nitrate reductase) and NiR (nitrite reductase).
Figure 17.
Figure 17.
Boolean Circuits for Interaction of Light and Carbon (light grown). Boolean circuits model ASN1, ASN2 and GLN2 regulation by light and carbon in light-grown plants. A, B, and C Boolean circuits based on 16 experiments. A. GLN2 regulation by white, blue, red or far-red light when compared against a base-case of no light, no carbon or no light, carbon. B. ASN2 regulation by white, blue, red or far-red light when compared against a base-case of no light, no carbon or no light, carbon. C. ASN1 regulation by white, blue, red or far-red light when compared against a base-case of no light, no carbon or no light, carbon. The inputs are white light (WL), blue light low fluence (BLF), blue light high fluence (BHF), red light low fluence (RLF), red light high fluence (RHF), far-red light low fluence (FRLF), far-red light high fluence (FRHF). Low fluence is 2µµmol m−2 s−1, high fluence is 100µµmol m−2 s−1. The arrow or barred lines indicate the function of the inputs as either inductive or repressive. Double arrows or double bars denote super-induction or super-repression, respectively. For a Boolean OR, if any one of the inputs is active, the output will also be active. Differences in the input for Boolean circuits when comparing ‘absence of carbon’ to ‘presence of carbon’ are shown as boxed inputs. From: Thum KE, Shasha DE, Lejay L, Coruzzi G (2003) Light- and Carbon Signaling Pathways. Modeling Circuits of Interactions. Plant Physiology 132: 440–452.
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