Overexpression of cytosolic glutamine synthetase. Relation to nitrogen, light, and photorespiration

Abstract

In plants, ammonium released during photorespiration exceeds primary nitrogen assimilation by as much as 10-fold. Analysis of photorespiratory mutants indicates that photorespiratory ammonium released in mitochondria is reassimilated in the chloroplast by a chloroplastic isoenzyme of glutamine synthetase (GS2), the predominant GS isoform in leaves of Solanaceous species including tobacco (Nicotiana tabacum). By contrast, cytosolic GS1 is expressed in the vasculature of several species including tobacco. Here, we report the effects on growth and photorespiration of overexpressing a cytosolic GS1 isoenzyme in leaf mesophyll cells of tobacco. The plants, which ectopically overexpress cytosolic GS1 in leaves, display a light-dependent improved growth phenotype under nitrogen-limiting and nitrogen-non-limiting conditions. Improved growth was evidenced by increases in fresh weight, dry weight, and leaf soluble protein. Because the improved growth phenotype was dependent on light, this suggested that the ectopic expression of cytosolic GS1 in leaves may act via photosynthetic/photorespiratory process. The ectopic overexpression of cytosolic GS1 in tobacco leaves resulted in a 6- to 7-fold decrease in levels of free ammonium in leaves. Thus, the overexpression of cytosolic GS1 in leaf mesophyll cells seems to provide an alternate route to chloroplastic GS2 for the assimilation of photorespiratory ammonium. The cytosolic GS1 transgenic plants also exhibit an increase in the CO(2) photorespiratory burst and an increase in levels of photorespiratory intermediates, suggesting changes in photorespiration. Because the GS1 transgenic plants have an unaltered CO(2) compensation point, this may reflect an accompanying increase in photosynthetic capacity. Together, these results provide new insights into the possible mechanisms responsible for the improved growth phenotype of cytosolic GS1 overexpressing plants. Our studies provide further support for the notion that the ectopic overexpression of genes for cytosolic GS1 can potentially be used to affect increases in nitrogen use efficiency in transgenic crop plants.

Figure 1

GS expression profiles in leaves of 35S-GS transgenic tobacco plants. A, GS mRNA was detected by hybridization with full-length cDNA probes for pea cytosolic GS1 (lanes 1–3), pea cytosolic GS3A (lanes 4 and 5), and pea chloroplastic GS2 (lanes 6–8). B, Western-blot analysis with a mixture of antibodies to bean (Phaseolus vulgaris) cytosolic GS1 and tobacco chloroplastic GS2 (Hirel et al., 1984; Lara et al., 1984; Tingey et al., 1988). C, Non-denaturing gel and GS activity stain showing GS holoenzymes A, B, and C. GS holoenzyme A (*) is a nonnative GS isoenzyme detected only in CytGS3A-TR plants. CytGS1-TR and CytGS3A-TR lines contain normal levels of native chloroplastic GS2 (band B). D, Non-denaturing gel and GS activity stain showing a side-by-side comparison between CytGS3A-TR (lane 6) and CytGS1-TR (lane 5) leaf extracts. The cytosolic GS1 holoenzyme (band C), which is detected in leaves of CytGS1-TR plants but not in the control plants, corresponds to the native root-specific tobacco cytosolic GS1 holoenzyme (lanes 4 and 6). Controls: lanes 1 and 2, pea chloroplast and root extracts; lanes 3 and 4, tobacco chloroplast and root extracts. E, Subunit composition of GS holoenzymes. GS holoenzymes A*, B, and C, respectively, were excised from preparative native gels, denatured, separated by PAGE, and detected by western-blot analysis. Crude leaf extract of untransformed tobacco (lane 1), GS holoenzyme A* from CytGS3A-TR (lane 2), GS holoenzyme band B isolated from isolated chloroplasts from untransformed tobacco (lane 3), and GS holoenzyme C from CytGS1-TR (lane 4).

Figure 2

Qualitative and quantitative growth phenotype of GS transgenic plants. A, Plants from the control line (SR1–6) and the cosuppressed chloroplastic GS2 (ChlGS2-TR) line are shown next to three independent lines of cytosolic GS1 overexpressors: CytGS1-TR1 (1), CytGS1-TR2 (2), and CytGS1-TR3 (3). The same ameliorated growth phenotype was also observed in another independent CytGS1-TR line, CytGS1-TR4 (not shown). B through D, Growth analysis of cytosolic GS1 overexpressor lines (●) CytGS1-TR1 (1), CytGS1-TR2 (2), and CytGS1-TR3 (3). Also represented are the control tobacco line (SR1–6, □) and the cosuppressed chloroplastic GS2 line (ChlGS2-TR, ▪). The growth assays were performed in 19 plants for the CytGS1-TR or ChlGS2-TR lines and 10 plants for the SR1–6 line. All plants were analyzed individually for total plant fresh weight (B), dry weight (C), and soluble protein (D) as a function of total leaf GS specific activity (Shapiro and Stadtman, 1971). The plants were grown and assayed as described in “Materials and Methods.”

Figure 3

Qualitative growth phenotype of soil-grown GS transgenic plants. Control line (SR1–6; A), CytGS1-TR1 (B), CytGS1-TR2 (C), and CytGS1-TR3 (D) were germinated and grown for 28 d in soil as described in “Materials and Methods.”

Figure 4

Effect of light on growth of GS transgenic plants grown under different nitrogen regimes. Plants were incubated in a normal day/night cycle either under high light (moderate PFD, 200 μmol cm⁻² s⁻¹) or low light (low PFD, 50 μmol cm⁻² s⁻¹) and subirrigated with ammonium-free/nitrate-free liquid Murashige and Skoog medium containing 0× nitrogen (no nitrogen supplementation), 0.1× nitrogen (4 mm nitrate/2 mm ammonium), or 1× nitrogen (40 mm nitrate/20 mm ammonium). A, Qualitative growth phenotype. B, Fresh weight (n = 4, mean ± se) from plants in A. The plants for this experiment were grown as described in “Materials and Methods.”

Figure 5

Levels of photorespiration correlate with GS expression in transgenic plants. Detached leaves of the cosuppressed chloroplastic GS2 line (ChlGS2-TR1, □), the control tobacco line (SR1–6, ▪), and a cytosolic GS1 overexpressor line (CytGS1-TR1, ●) were initially illuminated (1,000 μmol cm⁻² s⁻¹) for 1 h and subsequently exposed to dark by blocking the light source for a period of 2 min. The composition of the gas entering the chamber was 79 μL CO₂ L⁻¹ (PPM), 21% (v/v) O₂, and balanced nitrogen. Total gas flow was approximately 1 L min⁻¹. The temperature was kept at 28°C to 29°C for dark and light conditions. The rate of CO₂ exchange was measured at 12-s intervals. The measurements were done in two individual plants from each transgenic line analyzed. A representative result is shown.

Figure 6

Correlation between the levels of ammonium and expression of GS in tobacco transgenic plants. The plants were incubated under moderate light (moderate PFD, 200 μmol cm⁻² s⁻¹) subirrigated with 0.5× Hoagland for 20 to 30 d. Ammonium was determined from leaf extracts of the tobacco transgenic lines as indicated. Results are in nanograms of NH₄⁺ per microgram of protein ± se, n = 3 individual plants.