Ribulose-1,5-bisphosphate carboxylase/oxygenase activase cDNAs from Nicotiana tabacum.

Rubisco-the key regulatory enzyme in photosynthetic carbon assimilation-is activated in vivo by the chloroplast enzyme Rubisco activase (Portis, 1990). Previous studies in which Rubisco antisense DNA mutants of tobacco were used have revealed that under some circumstances Rubisco activase can play a central role in regulating the photosynthetic process (Rodermel et al., 1988; Quick et al., 1991a, 1991b; Stitt et al., 1991). To assess better the impact of activase on photosynthesis, we wish to generate and characterize activase antisense DNA mutants of tobacco. A prerequisite for these experiments is that a highly expressed Rubisco activase gene (Rca) sequence from tobacco be cloned and sequenced. Higher plant Rca sequences have been previously reported in Arabidopsis (Wemeke and Ogren, 1989; Werneke et al., 1989), spinach (Wemeke et al., 1988, 1989), and barley (Rundle and Zielinski, 1991). Screening of two Nicotiana tabacum cDNA libraries yielded a total of 20 Rca cDNA clones (Table I). These clones fel1 into three classes. One class is represented by pJQ4, which is a near full-length cDNA (1656 bp), and the other two classes are represented by pJQll and pTAl.l, which are partia1 cDNAs (897 and 1012 bp, respectively). The coding regions of pTAl.1 and pJQ4 are greater than 99% similar to one another at both the nucleotide and amino acid sequence levels, and the 3’ flanking regions of these cDNAs are 96% similar to one another. In contrast, pJQ11 is the most divergent of the three cDNAs. Although the amino acid sequence of the coding region of this clone is 96% similar to pTAl.l and pJQ4, the coding and flanking regions of this cDNA bear only 90 and 76% nucleotide similarity, respectively, to the corresponding sequences from the other two clones. Overall, the three tobacco Rca cDNAs code for polypeptides that are from 85 to 94% similar to other higher plant activase proteins. The data in this report indicate that Rca is a member of a small multigene family in N. tabacum. This finding is consistent with genomic Southem hybridization experiments, which have revealed that the N. tabacum genome may contain from three to five Rca gene family members (data not shown). This is the highest number of Rca copies found in any plant genome to date (Wemeke et al., 1988; Rundle and Zielinski, 1991).