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Role of light in the in vivo and in vitro synthesis of spinach thioredoxin f
Author(s) -
Carrasco José L.,
Chueca Ana,
Sahrawy Mariam,
Hermoso Rosario,
Lázaro Juan J.,
Gorgé Julio López
Publication year - 1992
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1992.tb04659.x
Subject(s) - spinach , spinacia , chloroplast , in vivo , organelle , in vitro , biology , thioredoxin , greening , antiserum , chenopodiaceae , biochemistry , microbiology and biotechnology , biophysics , enzyme , ecology , antibody , immunology , gene
Upon continuous illumination of dark‐grown spinach ( Spinacia oleracea L. cv. Winter Giant) seedlings, the thioredoxin f (Td f) content (ELISA) showed a steep rise, which can be evaluated after 3 and 36 h illumination as 3 times and 10 times the dark value, respectively. These figures correspond to 0.03% and 0.1% of total soluble protein, which means a higher biosynthetic rate for Td f compared to the average of total proteins in the earlier steps of plant development. After 40‐50 h light the Td f level reached its highest value which remained stable for an additional 40 h and then decreased. Pulse‐chase in vivo experiments with [ 35 S]‐methionine also showed this sharp increase of Td f in the dark‐light transition. From the pattern of decay of [ 35 S]‐labelled Td f, a half‐life of 7 h was determined for this chloroplast protein. In vitro translation experiments with poly(A)‐mRNA isolated from illuminated young spinach seedlings, coupled to a wheat‐germ synthesizing system, showed the appearance of a labelled fraction of ca 19 kDa molecular mass, recognizable by a specific Td f antiserum. When intact spinach chloroplasts were added to the translation assay medium, and then illuminated, the 19 kDa band disappeared, with a parallel increase of an internalized 13 kDa labelled polypeptide, also recognized by the Td f antiserum. These results are good evidence for a nuclear‐coded synthesis of a Td f precursor, which travels through the chloroplast envelope, leaving the functional protein inside the organelle after the loss of a 6 kDa transit peptide.