Ammonia Regulation of Carbon Metabolism in Photosynthesizing Leaf Discs

Abstract

The over-all process of nitrate reduction and ammonia incorporation into amino acids in higher plant leaves (along with the role of light in that process) has been a subject of recent research (3,10,12). Given the production of amino acids as well as sucrose during photosynthesis (16), it is of interest to determine whether leaf regulatory mechanisms involve ammonia. The intracellular NH4+ level could influence leaf carbon metabolism during photosynthesis with respect to sucrose synthesis versus the amino acid synthesis necessary for protein production and leaf growth. Kinetic studies of "4C-labeled compounds formed in the unicellular green alga Chlorella pyrenoidosa during photosynthesis with "4CO2 have indicated that ammonia brings about increased amino acid synthesis in part due to stimulation of pyruvate kinase (8). We have now investigated whether a similar regulatory mechanism is active in the leaves of higher plants. The specific plant chosen for investigation was alfalfa because of its potential as a source of leaf protein for direct human consumption (5).In our experiments we have used the techniques of kinetic tracer analysis of steady-state photosynthesis (2,19 MATERIALS AND METHODS Plant Material. Alfalfa (Medicago sativa L., var. El Unico) seeds were planted in 12 cm of vermiculite. The plants were grown at 3,000 ft-c and 15 C with a 9-hr light period and a 15-hr dark period. Plants were fertilized with modified Hoagland solution. Second through fourth unfolded leaves (numbered from the plant apex; not fully expanded) were excised after 1 hr of light from 6-week-old plants. The leaves were placed on a rubber sheet and 1-cm-diameter leaf discs were cut out with a cork borer.Photosynthetic Carbon Fixation. The closed steady-state gas circulation system described previously was employed (19). Leaf discs were exposed to CO2 in 4.5-cm-diameter flasks made from ground-glass joints and having transparent upper and lower surfaces. Each flask was fitted with gas inlet and outlet tubes, a serum stopper (allowing for addition of reagents during experiments), and five glass leaf disc holders (serving to prevent leaf overlap). Immediately after being cut, the leaf discs were floated on 4 ml of pH 7.4 buffer (0.05 M K2HPO4, pH adjusted with HCl) in the disc exposure flasks. Twelve flasks were utilized; each contained five discs comprising a single sample. The flasks were placed in a temperature-regulated shaking device (previously described for chloroplast experiments) (7) and attached to the closed gas circulation system through manifolds. Gas flow was commenced with 0.04% CO2 in air (20% 02); flask temperature was 27 C. After 12 min of preincubation in the dark, the lights were turned on and ammonium chloride in the pH 7.4 buffer was injected into six of the flasks to give a final concentration of 5 mm NH4Cl. Photosynthesis with 12CO2 was observed for 17 min. The unlabeled CO2 in air was then replaced by 0.038% "4CO2 (30.2 ,uCi/Amol) in air (20% 02). Samples of control and ammoniumtreated leaf discs were remove...

Monovalent Cation Activation of Plant Pyruvate Dehydrogenase Kinase

“…Such an effect would be consistent with the increased carbon flux through the Krebs cycle observed in response to exogenously supplied NH4+ (Platt et al, 1977;Larsen et al, 1981). However, NH4+ in the range of 1 to 10 mM did not inhibit PDH kinase in the presence of saturating (10 mM) K+ (data not shown).…”

Nonlinear Time-Course Uptake of Carbon and Ammonium by Marine Phytoplankton

“…8) the low molecular weight pools were significantly more labile during the dark period when high levels of NH: were present, reflecting a significant modification in the metabolism of the contained phytoplankton prior to this time. In recent photosynthesis studies (e. g. Kanazawa et al, 1970;Bassham, 1971 ;Hipkin and Syrett, 1977;Platt et al, 1977;Mohamed and Gnanam, 1979) increased levels of NH: have been shown to lead to modifications of cellular metabolism in response to both enhanced availability of nitrogen and enzyme regulation. Typical influences include increased synthesis of amino acids and protein relative to the production of carbohydrate precursors and polymers.…”

Short‐term exposure to atmospheric ammonia does not affect low‐temperature hardening of winter wheat

“…At the same time, ammonia exposure resulted in a much lower water-soluble sugar content than in the control plants during low-temperature acclimation, which again indicates that ammonia taken up by the leaves was metabolized (Platt et al., 1977;Champigny et al, 1992;Lea et al, 1992).…”

“…Apparently ammonia w-as taken up by the lea\'es, as has been demonstrated in leaves of Italian ryegrass {Lolivm multifiorum) and poplar {Populus euramericana) (Lockyer & Whitehead,1 986 ;Van Hove et al,1 991) and was subsequently metabolized, as evident from the higher biomass. Ammonia can be assimilated to glutamate by the glutamine/glutamate synthase cycle at the expense of carbohydrates (Platt, Plaut & Bassham, 1977;Champigny et al, 1992;Lea, Blackwell & Joy. 1992).…”