Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco

Abstract

Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.

Figure 1.

Proposed diagram of the photorespiratory nitrogen cycle involving respiratory Glu metabolism with release of ammonium and 2-oxoglutarate between three subcellular compartments. Ammonium can be produced during photorespiration and oxidative deamination of Glu by GDH and assimilated by the action of the GS-GOGAT pathway. The stoichiometry of the cycle is not included. GDC, Gly decarboxylase multienzyme complex (EC 1.4.4.2/2.1.2.10); GGAT, Glu:glyoxylate aminotransferase (EC 2.6.1.4); SHMT, Ser hydroxymethyltransferase (EC 2.1.2.1); SGAT, Ser:glyoxylate aminotransferase (EC 2.6.1.45); CH₂-THF, N⁵,N¹⁰-methylene tetrahydrofolate; G3P, glycerate-3-P; OH-pyruvate, hydroxypyruvate; RuBP, ribulose 1,5-bisphoshate.

Figure 2.

Changes in the levels of total ammonium in young and old leaves either in the light or in the dark. Young and old leaf discs from 10-week-old tobacco plants were floated on incubation buffer for 1 h in the dark. Leaf discs were dipped into the solutions without (A) or with (B) 1 mm MSO, quickly removed, and washed with water (time 0). Leaf discs were further incubated in the light or in the dark and harvested at 5, 10, 20, 30, 45, 60, 120, and 240 min. Ammonium content was expressed as nmol (mg DW)⁻¹. Values represent the means of analysis on leaf discs from five independent plants. DW, Dry weight.

Figure 3.

Changes in the levels of [¹⁵N]ammonium in young and old leaves either in the light (A) or in the dark (B). Experiments were carried out under identical conditions to those described in the legend of Figure 2. Leaf discs were harvested at 5, 10, 20, 30, 45, 60, 120, and 240 min. [¹⁵N]Ammonium levels were expressed as nmol (mg DW)⁻¹. Values represent means of analysis on leaf discs from five independent plants. DW, Dry weight.

Figure 4.

Kinetic analysis of [¹⁵N]ammonium incorporation into amides and amino acids in the light. Young and old leaf discs from 10-week-old tobacco plants were floated on incubation buffer for 1 h in the dark. After addition of [¹⁵N]ammonium (time 0), samples were incubated in the light and harvested at 5, 10, 20, 30, 45, 60, and 120 min from the solution without (A–D) or with (E–H) AZA (GOGAT inhibitor). ¹⁵N labeling in amide and amino nitrogen was determined by a GC-MS analyzer. Values represent the means of analysis on leaf discs from five independent plants.

Figure 5.

Kinetic analysis of [¹⁵N]ammonium incorporation into amides and amino acids in the dark. After addition of [¹⁵N]ammonium (time 0), samples were incubated in the dark and harvested at 5, 10, 20, 30, 45, 60, and 120 min from the solution without (A–D) or with (E–H) AZA. Values represent the means of analysis on leaf discs from five independent plants from discs.

Figure 6.

Scheme of ammonium assimilation into amides and amino acids. The first step of [¹⁵N]ammonium entry is into the Gln-amide group by GS. Then GOGAT transfers the ¹⁵N amide group of Gln to the 2-oxoglutarate position yielding one [2-¹⁵N]Glu and one Glu. AS catalyzes Asn formation from Asp using either the Gln-amide group or ammonium. AAT, Asp aminotransferase (EC 2.6.1.1); GGAT, Glu:glyoxylate aminotransferase; AZA, GOGAT inhibitor; MSO, GS inhibitor.

Figure 7.

Changes in the level of Fd-Glu synthase in young and old tissues from two 10-week-old transgenic tobacco lines, harboring the promoter of GLU1 encoding Arabidopsis Fd-GOGAT, fused to the GUS reporter gene (GLU1∷GUS*1 and GLU1∷GUS*2). A, Western-blot analysis of Fd-GOGAT. The enzyme protein amounts were compared in the mesophyll, vein, and petiole, and expressed as percent relative to the values in the young leaves. The Fd-GOGAT protein of 165 kD was shown with the prestained markers including a myosin (233 kD). B, In vivo activity of the promoter of GLU1 (Arabidopsis Fd-GOGAT) fused to the GUS reporter gene. GUS activity was assayed in the young and old leaves of the transgenic line GLU1∷GUS*1. Bar = 50 μm. mes, Mesophyll. C, Relative quantitative RT-PCR analysis of the Fd-GOGAT mRNA levels. The mRNA levels were compared in the mesophyll and vein and expressed as percent relative to the values in the young leaves.

Figure 8.

Immunocytochemical localization of Fd-Glu synthase in tobacco leaves. A, Leaf mesophyll section. B, Control leaf mesophyll section treated with nonimmune serum as the primary antibody. C, Leaf vascular section. D, Transmission of the leaf vascular section corresponding to C. chl, Chloroplast; pp, palisade parenchyma; sp, spongy parenchyma; x, xylem.