In the plastids of Cuscuta reflexa the number of thylakoids is strongly reduced compared with true chloroplasts and no typical grana are visible. The plastids of Cuscuta europaea lack thylakoids and the stroma is filled with either starch grains or lipid droplets. In Cuscuta reflexa both chlorophylls are present in low concentrations, while in C. europaea chlorophylls are totally absent. Light slightly stimulates the incorporation of (14)CO2 in C. reflexa. This is in accordance with a low activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which was measured in extracts of this parasite. No stimulation of (14)CO2 incorporation by light was observed in C. europaea. Instead, a relatively strong incorporation was measured in darkness. Malate and aspartate were the main products of this incorporation. In agreement with these results, no Rubisco activity was detected in C. europaea but there was a moderate activity of phosphoenolpyruvate-carboxylase. The presence of the genes for both subunits of Rubisco (rbcL, rbcS) and of the gene which codes for the 32-kDa protein of photosystem II (psbA) was established for C. europaea by hybridization experiments. In both species only very small amounts of transcripts of these genes were detected.
Plastid (pt) DNA from the red alga Porphyridium aerugineum was purified by CsCl gradient centrifugation. An EcoRI library of the ptDNA was screened with a gene probe specific for the gene encoding the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco EC 4.1.1.39) from spinach. A 5.8 kb EcoRI clone containing the LSU gene (rbcL) was isolated and the DNA sequence of the Porphyridium rbcL gene and its flanking regions was determined. An open reading frame was found 130 bp downstream from the rbcL gene that shows homology to genes coding for the small subunit of Rubisco (rbcS) from higher plants and cyanobacteria. Both genes (rbcL + rbcS) are cotranscribed. Comparison of rbcL and rbcS sequences from Porphyridium, higher plants and cyanobacteria seems to reveal a remarkable evolutionary distance between the plastids of the red algae (rhodoplasts), chloroplasts and cyanobacteria.
The genes for both subunits of Rubisco (rbcL, rbcS) are located on the plastome of the brown alga Ectocarpus siliculosus (Chromophyta, Phaeophyceae). The organization of these genes in the form of an operon was similar to that found in rhodoplasts, cyanobacteria and the plastids of Cryptomonas phi. Sequence analysis of the complete operon revealed a high degree of homology and great structural similarities to corresponding genes from two red algae. In contrast, sequence homology to Rubisco genes from chloroplasts and cyanobacteria was much lower. This clearly indicated a close phylogenetic relationship between the plastids of Rhodophyta and Chromophyta which seem to have evolved independently from the chloroplasts (polyphyletic origin). Our data suggest that the plastids of Chromophyta and Cryptophyta have originated from endosymbiotic unicellular red algae. Surprisingly, red and brown algal Rubiscos show a significantly higher degree of homology to that from a hydrogen bacterium than to those from cyanobacteria.
The genes for both subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (Rubisco) were located on the plastid DNA (ptDNA) of the unicellular red alga Cyanidium caldarium. Both genes are organized together in an operon. The sequence homology of both genes to the corresponding genes from the unicellular red alga Porphyridium aerugineum is remarkably high, whereas homology to Rubisco genes from chloroplasts and two recent cyanobacteria is significantly lower. These data provide strong evidence for a polyphyletic origin of chloroplasts and rhodoplasts. In addition the genes for the small subunit of Rubisco (rbcS) from red algae show about 60% homology to rbcS genes from cryptophytes and chromophytes. Thus, homologies in the rbcS gene indicate a close phylogenetic relationship between rhodoplasts and the plastids of Chromophyta.
We have cloned and sequenced an area of about 9.0 kb of the plastid DNA (ptDNA) from the holoparasitic flowering plant Cuscuta reflexa to investigate the evolutionary response of plastid genes to a reduced selective pressure. The region contains genes for the 16S rRNA, a subunit of a plastid NAD(P)H dehydrogenase (ndhB), three transfer RNAs (trnA, trnI, trnV) as well as the gene coding for the ribosomal protein S7 (rps7). While the other genes are strongly conserved in C. reflexa, the ndhB gene is a pseudogene due to many frameshift mutations. In addition we used heterologous gene probes to identify the other ndh genes encoded by the plastid genome in higher plants. No hybridization signals could be obtained, suggesting that these genes are either lost or strongly altered in the ptDNA of C. reflexa. Together with evidence of deleted genes in the ptDNA of C. reflexa, the plastid genome can be grouped into four classes reflecting a different evolutionary rate in each case. The phylogenetic position of Cuscuta and the significance of ndh genes in the plastid genome of higher plants are discussed.
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