Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, Cuscuta reflexa and Cuscuta gronovii

Abstract

Background: The holoparasitic plant genus Cuscuta comprises species with photosynthetic capacity and functional chloroplasts as well as achlorophyllous and intermediate forms with restricted photosynthetic activity and degenerated chloroplasts. Previous data indicated significant differences with respect to the plastid genome coding capacity in different Cuscuta species that could correlate with their photosynthetic activity. In order to shed light on the molecular changes accompanying the parasitic lifestyle, we sequenced the plastid chromosomes of the two species Cuscuta reflexa and Cuscuta gronovii. Both species are capable of performing photosynthesis, albeit with varying efficiencies. Together with the plastid genome of Epifagus virginiana, an achlorophyllous parasitic plant whose plastid genome has been sequenced, these species represent a series of progression towards total dependency on the host plant, ranging from reduced levels of photosynthesis in C. reflexa to a restricted photosynthetic activity and degenerated chloroplasts in C. gronovii to an achlorophyllous state in E. virginiana.

Results: The newly sequenced plastid genomes of C. reflexa and C. gronovii reveal that the chromosome structures are generally very similar to that of non-parasitic plants, although a number of species-specific insertions, deletions (indels) and sequence inversions were identified. However, we observed a gradual adaptation of the plastid genome to the different degrees of parasitism. The changes are particularly evident in C. gronovii and include (a) the parallel losses of genes for the subunits of the plastid-encoded RNA polymerase and the corresponding promoters from the plastid genome, (b) the first documented loss of the gene for a putative splicing factor, MatK, from the plastid genome and (c) a significant reduction of RNA editing.

Conclusion: Overall, the comparative genomic analysis of plastid DNA from parasitic plants indicates a bias towards a simplification of the plastid gene expression machinery as a consequence of an increasing dependency on the host plant. A tentative assignment of the successive events in the adaptation of the plastid genomes to parasitism can be inferred from the current data set. This includes (1) a loss of non-coding regions in photosynthetic Cuscuta species that has resulted in a condensation of the plastid genome, (2) the simplification of plastid gene expression in species with largely impaired photosynthetic capacity and (3) the deletion of a significant part of the genetic information, including the information for the photosynthetic apparatus, in non-photosynthetic parasitic plants.

Figure 1

Gene Maps of the plastid chromosomes of Cuscuta reflexa and Cuscuta gronovii. Genes shown on the right hand side are transcribed top down and genes on the left hand side bottom up. The large single copy region (LSC) and the small single copy region (SSC) are separated by two inverted repeats (IR_A and IR_B). Asterisks indicate intron containing genes. Pseudogenes are marked by Ψ. Dashed lines indicate the inverted regions between C. reflexa and C. gronovii.

Figure 2

Comparison of promoter sequences of five PEP promoters in Nicotiana tabacum, Cuscuta reflexa and Cuscuta gronovii. Double lines indicate the consensus motifs of the -10 and -35 boxes typical of plastid PEP promoters. Other conserved regions are marked with a single black line. The distance in nucleotides between the transcription start (indicated by a rightward arrow) and the translation start (ATG) is given. Black dots represent residues that are identical to the nucleotides of N. tabacum shown at the top.

Figure 3

Splicing of the intron 2 of clpP in Cuscuta gronovii. A: PCR (DNA) and RT-PCR (cDNA) products of clpP overlapping the intron 2 B: DNA and cDNA sequences of the region around the exon/intron 2 boundaries of clpP

Figure 4

Sequencing chromatogram excerpts of the editing sites in Cuscuta. The uppercase letter indicates the editing site or the conserved amino acid at the DNA level; for partial editing sites two chromatograms are shown in photosynthetic active tissue (top) and in pale tissue (bottom).