Isolation of cDNA clones coding for spinach nitrite reductase: complete sequence and nitrate induction

Area of Science:

• Plant Molecular Biology
• Biochemistry

Background:

• Plants primarily use soil nitrate as their main nitrogen source.
• Nitrate assimilation involves two enzymatic steps: nitrate reductase and nitrite reductase.
• Nitrite reductase converts nitrite to ammonia, a crucial step in amino acid synthesis.

Purpose of the Study:

• To determine the complete primary sequence of the spinach nitrite reductase precursor protein.
• To investigate the role of the N-terminal extension in protein localization.

Main Methods:

• Isolation of cDNA clones from a nitrate-induced library.
• Use of oligonucleotide probes and antibodies for cDNA screening.
• Deduction of the precursor protein's primary sequence from cloned cDNAs.

Main Results:

• The spinach nitrite reductase precursor protein is 594 amino acids long.
• A 32-amino acid N-terminal extension likely functions as a transit peptide.
• cDNA hybridizes to a 2.3 kb RNA, with increased levels upon nitrate induction.

Conclusions:

• The deduced sequence provides insights into nitrite reductase structure and function.
• The transit peptide directs the nuclear-encoded protein to the chloroplast.
• Nitrate availability regulates nitrite reductase gene expression at the RNA level.