Nucleotide sequence of a cDNA clone from broccoli with high identity with the PSST subunit of NADH:ubiquinone oxidoreductase.

NADHxbiquinone oxidoreductase (complex I) is the first enzyme in the electron transport chain of mitochondrial respiration and catalyzes electron transfer from NADH to ubiquinone by a large number of redox groups. In bovine heart mitochondria the membrane-bound enzyme is composed of at least 40 subunits, 33 of which are nuclear encoded and 7 of which originate in the mitochondria. The estimated protein mass is 880,000 (Walker et al., 1992). Until recently the plant enzyme has been studied little. Animals have two oxidation systems, whereas plants possess two or three additional NAD(P)H oxidoreductases (Douce and Neuburger, 1989). Complex I has now been purified from broad bean and partially characterized, and the N-terminal sequence has been obtained for 16 of the subunits (Leterme and Boutry, 1993). In addition to the mitochondrial location for complex I, there is evidence of a chloroplast-encoded NAD(P)H oxidoreductase. Eleven subunits of complex I have homologs in chloroplast genomes, and transcripts of three of these chloroplast genes (NDH-H, NDH-I, NDH-K) are detected in plastids, and the protein of one is located on the thylakoid membrane (Nixon et al., 1989; Arizmendi et al., 1992; Berger et al., 1993). The function of this as-yet-uncharacterized NAD(P)H oxidoreductase may involve the regulation of electron flow and ATP and NADPH production (Berger et al., 1993). A cDNA library from Brassica napus senescing leaves was differentially screened against a library constructed from green leaves. One of the clones isolated, LSC8, hybridized to a mRNA of 1.1 kb (Buchanan-Wollaston, 1994). In the work presented here the LSC8 clone was used to probe a cDNA library constructed from a poly(Af) mRNA from Bmssica oleraceae florets held at 20°C in darkness for 48 h after harvest. A homolog to LSC8 was isolated from the broccoli library and is called pbroc4 (Table I). The pbroc4 cDNA is 911 bp long, and starting at the first Met the open reading frame encodes a protein of 214 amino acids. The