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)

Mobilization of the reserve beta -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 mu mol photons(.)m(-2.)s(-1)) with C-14-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 mu mol photons-m(-2.)s(-1)) with additions of 40 muM NH4+. In the C-14 pulse phase with high light and N depletion, beta -1,3-glucan accumulated and accounted for 85% of incorporated C-14. In the subsequent C-14 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 beta -1,3-glucan (dark, 26%; low light, 19%). Biochemical fractionation of organic C-14 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 muM NH4+ under inorganic carbon limitation. Added NH4+ was assimilated at an N-specific rate of 0.023 h(-1), and cellular beta -1,3-glucan decreased by 15% within 6 h. Hence, beta -1,3-glucan was mobilized during NH4+ assimilation, even though inorganic carbon was modifying the metabolic rates. The results provide new evidence of beta -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.