Molecular characteristics and expression patterns of Rubisco activase, novel alternative splicing variants in a heterophyllous aquatic plant, Sagittaria graminea

Two full-length cDNAs (SGrca1 and SGrca2) encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) were cloned from a heterophyllous aquatic plant, Sagittaria graminea, using Rapid-Amplification of cDNA Ends (RACE). SGrca1 contains a 1,320 bp open reading frame encoding a protein of 440 amino acids, and SGrca2 is exactly identical to SGrca1 except for 330 bp missing in the middle of SGrca1. Sequence analysis of cDNA and genomic DNA indicated both two cDNAs were generated from a common gene via alternative splicing. The deduced amino acid sequence encoded by SGrca1 showed 75-82% identity with other RCAs from higher plants and showed high homology in three highly conserved motifs associated with ATP-binding sites. RT-PCR analysis suggested both SGrca1 and SGrca2 were expressed in green tissues. During a 14 h light/10 h dark photoperiod, both aerial and submerged leaves exhibited the similar expression pattern of SGrca1 and SGrca2 with SGrca1 as the dominant form, but the accumulation of both SGrca1 and SGrca2 mRNA was significantly inhibited in the submerged leaves. Western blot analysis showed that both SGrca1 and SGrca2 had their translation products, the 43 kDa form and the 31 kDa form expressing in leaves. Interestingly, the aerial leaves expressed higher amount of the 43 kDa form compared with the 31 kDa form, while it was reversed in the submerged leaves. The results demonstrated that both environments regulated the RCA gene expression at both transcriptional and posttranscriptional level. In addition, co-immunoprecipitation assay revealed that the isolated Rubisco-RCA complex contained both the 43 and 31 kDa forms, and the proportion of the 31 kDa form was obviously enhanced in the submerged leaves. The results indicated that both the 43 kDa and 31 kDa forms were involved in Rubisco and RCA interaction and the increased incorporation of the 31 kDa form was associated with submerged photosynthetic environment.