Structure and Regulation of Ferredoxin‐Dependent Glutamase Synthase from Arabidopsis Thaliana
Authors: Suzuki Akira , Rothstein Steven 
Published: July 2004

Ferredoxin (Fd)‐dependent glutamate synthase is present in green leaves, etiolated leaves, shoots and roots of Arabidopsis thaliana (ecotype Columbia). In photosynthetic green leaves and shoots, Fd‐dependent glutamate synthase accounts for more than 96% of the total glutamate synthase activity in vitro with the remaining activity derived from an enzyme that uses NADH as the electron donor. In etiolated leaves and roots, Fd‐dependent glutamate synthase is 3–4‐fold less active than in green leaves, but represents 70–85% of the total glutamate synthase activity in these tissues. Fd‐dependent glutamate synthase is detected as a single peptide of 165kDa on a western blot of green leaf and shoot tissues, and this Fd‐dependent glutamate synthase polypeptide is 3–4‐fold less abundant in etiolated leaves and roots. In these non‐photosynthetic tissues, there is a higher activity of NADH‐dependent glutamate synthase. The A. thaliana gltS mutant (strain CS254) contains only 1.7% and 17.5% of the wild‐type Fd‐dependent glutamate synthase activity in leaves and roots, respectively. Western blots indicate that the Fd‐dependent glutamate synthase peptide of 165kDa is absent from leaves and roots of the gltS mutant. In contrast, NADH‐dependent glutamate synthase activity in leaves and roots is unaffected. During illumination of wild‐type dark‐grown leaves for 72h, the levels of Fd‐dependent glutamate synthase protein and its activity increased threefold to levels equivalent to those in green leaves. In contrast, NADH‐dependent glutamate synthase activity decreases twofold during illumination. The complete nucleotide sequence of the complementary DNA for A. thaliana Fd‐dependent glutamate synthase has been determined. Analysis of the amino acid sequence deduced from the complete cDNA sequence (5178bp) has revealed that A. thaliana Fd‐dependent glutamate synthase is synthesized as a 1648‐amino‐acid precursor protein (180090Da) which consists of a 131‐amino‐acid transit peptide (14603Da) and a 1517‐amino‐acid mature peptide (165487Da). The A. thaliana Fd‐dependent glutamate synthase has a high similarity to maize Fd‐dependent glutamate synthase (839%) and to the analogous region of NADH‐dependent glutamate synthase (42%) and NADPH‐dependent glutamate synthases (40–43%) from different organisms. The A. thaliana Fd‐dependent glutamate synthase contains the purF‐type glutamine‐amido‐transfer domain as well as flavin and iron‐sulfur‐cluster‐binding domains. The deduced primary structures of A. thaliaina Fd‐dependent glutamate synthase and of glutamate synthases from other organisms indicate that Fd‐dependent glutamate synthase may have evolved from bacterial NADPH‐dependent glutamate synthase. The cDNA hybridized to RNA of about 5.3 kb from different tissues of A. thaliana. A high steady‐state level of Fd‐dependent glutamate synthase mRNA is found in photosynthetic green leaves and shoots, and roots contain less mRNA for Fd‐dependent glutamate synthase. In the gltS mutant, there are twofold and fourfold lower levels of Fd‐dependent glutamate synthase mRNA in leaves and roots, respectively, relative to those in wild‐type A. thaliana. Under continuous illumination of dark‐grown leaves, the Fd‐dependent glutamate synthase mRNA is induced twofold to a level equivalent to that in green leaves.