Two isoforms of Rubisco activase in cotton, the products of separate genes not alternative splicing

In several plant species, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase consists of two isoforms that are produced by alternative splicing of a pre-mRNA. Two forms of activase corresponding to the longer, redox-regulated α and the shorter, β forms were detected immunologically in cotton (Gossypium hirsutum L.) leaves, but their N-termini differed in 4 of 14 residues. The cDNAs for the α and β forms of cotton activase diverged throughout the translated and 3′-untranslated regions, including variations that accounted for the differences in N-terminal amino acid sequence. Analysis of genomic DNA confirmed that separate genes encoded the α and β forms of cotton activase. Separate activase genes were also detected in diploid species of cotton containing the different progenitor genomes of the cultivated allotetraploid, indicating that the occurrence of separate α- and β-form genes in cotton predates the merger of the diploid genomes. The deduced amino acid sequences of the two forms of cotton activase exhibited 84% identity and both forms were active after expression in Escherichia coli. The recombinant α and β forms exhibited similar affinities for ATP and only minor differences in thermotolerance, but their ATPase specific activities differed. The results show for the first time a plant species with two forms of activase that are structurally and functionally equivalent to the alternatively spliced α and β forms in other plants, but that are encoded by separate genes. That cotton still expresses both forms of activase, even without alternative splicing, suggests that each form has a required function in photosynthesis.