MOBILIZATION OF β-1,3-GLUCAN AND BIOSYNTHESIS OF AMINO ACIDS INDUCED BY NH4+ ADDITION TO N-LIMITED CELLS OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE) |
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Authors: | Espen Granum and Sverre M Myklestad |
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Institution: | Department of Biotechnology, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway |
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Abstract: | Mobilization of the reserve β-1,3-glucan (chrysolaminaran) in N-limited cells of the marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) was investigated. The diatom was grown in pH-regulated batch cultures with a 14:10-h light:dark cycle until N depletion. In a pulse-chase experiment, the cells were first incubated in high light (200 μmol photons·m − 2·s − 1) with 14C-bicarbonate until dissolved inorganic carbon was exhausted. Unlabeled bicarbonate (1 mM) was then added, and the cells were incubated in the dark and subsequently in low light (20 μmol photons·m − 2·s − 1) with additions of 40 μM NH4 + . In the 14C pulse phase with high light and N depletion, β-1,3-glucan accumulated and accounted for 85% of incorporated 14C. In the subsequent 14C chase phases, added NH4 + was assimilated at an N-specific rate of 0.11 h − 1 in both the dark and low light, and in both cases it caused a significant mobilization of β-1,3-glucan (dark, 26%; low light, 19%). Biochemical fractionation of organic 14C showed that free amino acids were most rapidly labeled in the early stage of NH4 + assimilation, whereas proteins and polysaccharides were labeled more rapidly after 1.2 h. Analysis of the cellular free amino acids strongly indicated that de novo biosynthesis was occurring, with a Gln:Glu ratio increasing from 0.4 to 10 within 1.2 h. After the NH4 + was exhausted, the cellular pools of glucan and amino acids became constant or slowly decreased. In another experiment, N-limited cells were first incubated in high light until dissolved inorganic carbon was exhausted and were further incubated in high light with 150 μM NH4 + under inorganic carbon limitation. Added NH4 + was assimilated at an N-specific rate of 0.023 h − 1, and cellular β-1,3-glucan decreased by 15% within 6 h. Hence, β-1,3-glucan was mobilized during NH4 + assimilation, even though inorganic carbon was modifying the metabolic rates. The results provide new evidence of β-1,3-glucan supplying essential precursors for biosynthesis of amino acids and other components in S. costatum in both the dark and subsaturating light and even saturating light under inorganic carbon limitation. |
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Keywords: | ammonium assimilation biochemical fractionation free amino acids β-1 3-d-glucan mobilization nitrogen limitation polysaccharides Skeletonema costatum |
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