Molecular characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice leaves

Abstract.

A full-length cDNA (designated rcaII) encoding the Rubisco activase (RCA) of rice (Oryza sativa L.) has been cloned from a cDNA library constructed with mRNA from green leaves. Sequence analysis resulted in a reading frame of 432 amino acids with a calculated molecular mass of 47.9 kDa and an estimated isoelectric point of 5.97. The deduced amino acid sequence showed 74–89% identity with other Rubisco activases from higher plants. Two highly conserved motifs were identified. Southern blot analysis suggested the presence of a single rca gene in the rice genome. The accumulation of leaf rca mRNA was found to be regulated by an oscillating circadian rhythm, in rice plants grown in a light-dark photoperiod. To purify the rice RCA protein, total soluble protein from rice green leaves was fractionated by ammonium sulfate precipitation, followed by preparative gel electrophoresis. Two polypeptides, designated RCAI and RCAII, were isolated by two-dimensional gel electrophoresis and further confirmed by N-terminal sequencing. The polyclonal antibodies prepared against rice RCAI and RCAII were found to cross-react with two RCA polypeptides present in leaf extracts of spinach and tobacco. Furthermore, two different 3′ ends of rca mRNA were detected by reverse transcription-polymerase chain reaction analysis. These cDNA fragments and the related genomic DNA fragment were cloned and sequenced. The sequence of rcaI is almost identical to the corresponding sequence of rcaII, except for its having 33 additional amino acids at the C-terminal portion. It can be concluded that a novel alternative splicing mechanism for a common rca mRNA precursor near the 3′ end exists in rice plants.