Genes encoding photosynthetic enzymes experience increased rates of mutation in holoparasitic plants due to the relaxation of functional constraints, resulting in gene deletion, pseudogene formation, or unfavorable amino acid substitutions. The chloroplast gene rbcL encodes the large subunit of the holoenzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxidase). In this study, the evolution of rbcL was examined in parasitic sister genera, Hyobanche L. which demonstrates rbcL pseudogene formation, and Harveya Hook. which retains a seemingly functional form of the gene. The overall rate of evolution of rbcL is greater in Hyobanche than Harveya, but a comparison of synonymous/non-synonymous substitution rates indicates that rbcL has evolved under greater functional constraint in Harveya than in Hyobanche. Two hypotheses explain this phenomenon: A) Rubisco retains function in Harveya, in which case Harveya may be a cryptic hemiparasite. B) The loss of Rubisco functionality has occurred via a different pathway in Harveya than in Hyobanche, perhaps at the level of transcription or translation. Conservation of the rbcL nucleotide sequence in Harveya may indicate a later origin of the holoparasitic habit than in the sister lineage, Hyobanche. To differentiate between these hypotheses, gene expression of rbcL is examined at three levels. Nucleotide sequences of the 5' and 3' untranslated regions which flank rbcL were examined as these have known transcriptional and translational function, RT-PCR was used to detect rbcL transcripts in tissues, and western blot was utilized to assay for the presence of Rubisco.

Key words: Harveya, Hyobanche, Orobanchaceae, parasitic plants, rbcL, Rubisco