EXPRESSION OF FERREDOXIN-DEPENDENT GLUTAMATE SYNTHASE IN DARK-GROWN PINE-SEEDLINGS

Pine seedlings are able to accumulate chlorophylls and develop green p lastids in a light-independent manner. In this work, we have character ized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pin us pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163-168 kDa that is con fined to the chloroplast stroma. Polyclonal antibodies generated again st the purified enzyme were used to immunoscreen a lambda gt11 express ion library from Scots pine (Pinus sylvestris) seedlings and partial c DNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (5 50 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross- reactivity and comparative amino sequence analysis revealed that Fd-GO GAT is a well conserved protein in higher plants. Western blot analyse s showed that protein was expressed in chloroplast-containing pine tis sues and this expression pattern was not affected by exogenously suppl ied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulat ed in substantial amounts in dark-grown pine seedlings. The presence o f a functional Fd-GOGAT may be important to provide the required gluta mate for the biosynthesis of nitrogen compounds during chloroplast bio genesis in the dark.