RESUMO
Isotope effects, studied with precision isotope ratio mass spectrometry, have been used to locate critical steps in the H metabolism of plants. By manipulating the growth conditions of versatile microalgae, the discrimination of H isotopes between water in the growth medium and the organically bonded H in carbohydrates from these microalgae was -100 to -120 per thousand and was regulated by both the light and the dark reactions of photosynthesis. Photosynthetic electron transport discriminated against the heavy isotope of H and formed a pool of reductant available for biosynthesis that was enriched in the light isotope. Growth in red or white light activated phosphoglyceric acid reduction and H isotope discrimination, when H was fixed into organic matter. An additional fractionation of -30 to -60 per thousand occurred during the biosynthesis of proteins and lipids and was associated with glycolysis. This fractionation paralleled the isotope effect seen in carbohydrate metabolism, indicating that H metabolism in photosynthesis was coupled with that in dark biosynthetic reactions via the pool of reductant, probably NADPH.
RESUMO
Stable-isotope discrimination factors (D) for the uptake of oxygen during respiration by a variety of plant materials were determined by measuring (18)O enrichment in a closed system. Baker's yeast (Saccharomyces cerevisiae Meyer) and mitochondrial preparations from baker's yeast and from castor bean (Ricinus communis L.) endosperm, all of which are fully sensitive to cyanide, discriminated againt (18)O by about 16-18. Whole Medicago sativa L. seedlings, isolated intact Asparagus sprengeri Regel mesophyll cells, and spadix mitochondria of Eastern skunk cabbage (Symplocarpus foetidus L.) had higher Ds of about 20-22. These materials all had some capacity for the cyanide-resistant alternative respiration pathway and in the presence of cyanide discriminated by about 24-26. When treated with salicylhydroxamic acid or tetraethylthiuram disulfide, which inhibit the alternative pathway, discrimination was about 17-19. Where respiration was limited by oxygen diffusion (slices of thermogenic tissues from S. foetidus and Sauromatum gutfatum Schott), fractionation was much reduced and the difference between the two respiratory pathways was masked. Isotope discrimination by soybean lipoxygenase (EC 1.13.11.12) supplied with linoleic acid was much lower than by respiration. Where diffusion is not a problem, the D value obtained in the absence of inhibitor can be used to estimate the partitioning of electron transport between the two pathways at steady-state by linear interpolation between the Ds characteristic of cyanide-resistant and cyanide-sensitive respiration.