Regulation of Maize Leaf Nitrate Reductase Activity Involves Both Gene Expression and Protein Phosphorylation

Abstract

Nitrate reductase (NR; EC 1.6.6.1) activity increased at the beginning of the photoperiod in mature green maize (rea mays l.) leaves as a result of increased enzyme protein level and protein dephosphorylation. In vitro experiments suggested that phosphorylation of maize leaf NR affected sensitivity to Mgz* inhibition, as shown previously in spinach. When excised leaves were fed 32P-labeled inorganic phosphate, NR was phosphorylated on seryl residues in both the light and dark. Tryptic peptide mapping of NR labeled in vivo indicated three major 3ZP-phosphopeptide fragments, and labeling of all three was reduced when leaves were illuminated. Maize leaf NR mRNA levels that were low at the end of the dark period peaked within 2 h in the light and decreased thereafter, and NR activity generally remained high. It appears that light signals, rather than an endogenous rhythm, account primarily for diurnal variations in NR mRNA levels. Overall, regulation of NR activity in mature maize leaves in response to light signals appears to involve control of gene expression, enzyme protein synthesis, and reversible protein phosphorylation.

Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris

“…No obvious correlation was detected between levels of NR mRNA and NR activity (Galangau et al, 1988). A similar phenomenon was also found in maize leaves in which NR mRNA levels were low at the end of the dark period, peaked within 2 h in the light and decreased thereafter, whereas NR activity generally remained high (Huber et al, 1994). Apparently, the positive feed-forward induction of NR genes by sugars, and the negative feedback by nitrogen compound(s) from nitrate assimilation results in the diurnal oscillation of NR mRNA.…”

“…One of the unusual characters of authentic Ricinus NR was its insensitivity to preincubation with ATP (Kandlbinder et al, 2000), which contrasted sharply with the situation in spinach and other plants (Kaiser and Spill, 1991;Huber et al, 1994). It has been shown previously that under continuous darkness for up to 6 days, spinach NR protein was almost completely degraded.…”

“…Additionally, CO 2 enrichment markedly elevated nitrate reductase activity in young, but not old, tobacco plants, presumably because growth rates were much greater in the younger plants and they needed the extra nitrogen provided by this phenomenon to maintain their heightened level of development (Geiger et al, 1998). The demonstration that light is an effective regulator of NR at the post-translational level by reversible protein phosphorylation (Kaiser et al, 1992;Huber et al, 1994), has been a major breakthrough in our knowledge on NR regulation. Thus, more details on this aspect are given below.…”

“…It has been shown that NR is phosphorylated on multiple sites (Huber et al, 1992;LaBrie and Crawford, 1994;Huber et al, 1994); therefore, the highly conserved Ser residues in this region could be sites for NR phosphorylation. Sequence comparison of the Moco domain revealed that Ricinus NR possesses those highly conserved Ser residues (Ser211,253,258,316,357,387 and 430) as Arabidopsis NR2 does.…”

Developmental regulation of photosynthate distribution in leaves of rice

“…It has been proposed that the distribution of photosynthetically fixed carbon (C) between amino acids and saccharides is regulated by the relative 2 activities of sucrose phosphate synthase (SPS) and phosphoenolpyruvate carboxylase (PEPC; Champigny and Foyer 1992, Foyer et al 1994, Huber et al 1994). In C 3 -plants, PEPC acts to replenish TCA cycle intermediates which serve as source material for the synthesis of amino acids (Stitt 1999).…”