Klaus Zetsche

Cytoplasmic male sterility in sunflower is correlated with the co-transcription of a new open reading frame with the atpA gene

SummaryThe organization and expression of the mitochondrial (mt) genome of fertile, male-sterile and restored lines of Helianthus annuus and of H. petiolaris were compared to identify alterations which might lead to cytoplasmic male sterility (CMS). The mtDNAs of fertile and male-sterile lines differ by an 11 kb inversion and a 5 kb insertion. The rearrangements seem to be the result of recombination events within an inverted repeat of 261 bp. Detectable alterations in the transcript pattern of the rearranged mtDNA regions are restricted to the atpA locus. The male-sterile line CMSBaso shows three additional transcripts of the atpA locus of about 2500, 1200 and 250 nucleotides which are not detectable in Baso. However, the coding sequences of the atpA gene are entirely identical in the fertile line Baso and the male-sterile line CMSBaso. But a new open reading frame (orfH522) of 522 nucleotides is co-transcribed with the atpA gene as an additional larger transcript of about 2500 nucleotides in CMSBaso. orfH522 is also included in a second additional transcript of about 1200 nucleotides. The predicted translation product of orfH522 might play a role in CMS in sunflower.

Rubisco genes indicate a close phylogenetic relation between the plastids of Chromophyta and Rhodophyta

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 Φ. 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.

A mitochondrial 16 kDa protein is associated with cytoplasmic male sterility in sunflower

Cytoplasmic male-sterile lines CMS89 and CMSBaso of sunflower (Helianthus annuus) differ from the fertile lines HA89 and Baso in a mitochondrial DNA sequence in the vicinity of theatpA gene. In addition, the transcriptional pattern of theatpA gene is changed in male-sterile lines compared to fertile ones. Besides one main transcript in the fertile lines, the male-sterile lines additionally show larger transcripts. Investigation of Baso and CMSBaso revealed that the two fertility-restored lines of CMS89 have the same transcripts as CMSBaso or a combination of CMSBaso and CMS89. Comparing the mitochondrialin organello translation products we observed a unique 16 kDa protein, which is expressed in male-sterile lines carrying theH. petiolaris cytoplasm but is not detectable in fertile lines withH. annuus cytoplasm. The 16 kDa protein can also be observed in restored lines but not inH. petiolaris. As the expression of the 16 kDa polypeptide seems to be linked to the interspecific cross betweenH. petiolaris andH. annuus it may play a role in CMS. By different criteria such as molecular mass, isoelectric point and peptide fingerprinting the α subunit of the F1-ATPase of male-sterile and fertile lines is very similar if not identical.

The genes of both subunits of ribulose-1,5-bisphosphate carboxylase constitute an operon on the plastome of a red alga

SummaryPlastid (pt) DNA from the red alga Porphyridium aerugineum was purified by CsCI 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 by 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.

A structural, functional and molecular analysis of plastids of the holoparasites Cuscuta reflexa and Cuscuta europaea.

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 14CO2 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 14CO2 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.

Structure of the rubisco operon from the unicellular red alga Cyanidium caldarium: evidence for a polyphyletic origin of the plastids.

SummaryThe genes for both subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (Rubisco) were located on the plastid DNA (ptDNA) of the unicellular red algaCyanidium caldarium. Both genes are organized together in an operon. The sequence homology of both genes to the corresponding genes from the unicellular red algaPorphyridium 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 torbcS genes from cryptophytes and chromophytes. Thus, homologies in therbcS gene indicate a close phylogenetic relationship between rhodoplasts and the plastids of Chromophyta.

Functional loss of all ndh genes in an otherwise relatively unaltered plastid genome of the holoparasitic flowering plant Cuscuta reflexa

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.

Structure of the rubisco operon from the multicellular red alga Antithamnion spec.

SummaryIn the multicellular red alga Antithamnion spec. both rubisco genes (rbcL and rbcS) are encoded on the plastid DNA (ptDNA). Both genes are separated by a short A/T-rich spacer of 100 bp and are cotranscribed into an mRNA of approximately 2.7 kb. These findings are in extensive agreement with those obtained from two unicellular red algae (Porphyridium aerugineum and Cyanidium caldarium). The large subunit (LSU) of rubisco shows an amino acid homology of 82–87% with the LSUs from the two unicellular red algae and only about 55% to LSUs from green algae, higher plants and two cyanobacteria. The small subunit (SSU) of rubisco is more similar to those from the unicellular red algae and two algae which are members of the Chromophyta (about 60% homology) than to cyanobacterial and higher plant proteins (27–36% homology). These data indicate that rhodoplasts originated independently from the chloroplast line. The plastids of chromophytes and rhodophytes appear to be closely related.

Organization and sequence of photosynthetic genes from the plastid genome of the holoparasitic flowering plant Cuscuta reflexa.

SummaryWe have cloned and sequenced an area of about 6 kb of the plastid DNA (ptDNA) from the holoparasitic plant Cuscuta refexa. This region contains (in the following order) genes for the cytochrome b6/f-complex subunit V (petG), tRNAVal (trnV), tRNAMet (trnM), the ε and β-subunit of the chloroplast ATP-synthase (atpE and atpB) and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; rbcL). In addition we identified other photosynthesis-related genes (atpA, petB, psaA, psbA, psbB, psbC, and psbD) in C. refexa by heterologous hybridization. The gene arrangement of the sequenced area is, except for the petG gene, the same as in ptDNAs of other higher plants (e.g. Nicotiana tabacum). Sequence homologies between the Cuscuta genes and corresponding genes from higher plants are in the range of 90%. The only significant difference is that the rbcL gene of C. refexa encodes a polypeptide which is 18–23 amino acids longer than in other higher plants. This is remarkable since C. refexa has lost its ability to grow photoautotrophically. The transcript level of the rbcL gene, however, is strongly reduced as compared to tobacco. These findings are compatible with results from Western blotting analysis, where no Rubisco large subunit was detectable, and with the lack of Rubisco activity in crude extracts of C. ref lexa.

In-vitro synthesis of phycobiliproteids and ribulose-1,5-bisphosphate carboxylase by non-poly-adenylated-RNA of Cyanidium caldarium and Porphyridium aerugineum.

Polyadenylated and nonpolyadenylated mRNa from the red algae Cyanidium caldarium Geitler and Porphyridium aerugineum Geitler were translated in a cell-free system. The α-and β-subunits of phycocyanin and allophycocyanin and also both subunits of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase) were identified with the help of specific antibodies as translation products of non-polyadenylated mRNA. Both subunits of RuBPCase were synthesized with molecular weights in the range of the mature forms. This is in contrast to the findings with green algae and higher plants where the small subunits of RuBPCase are always made by polyadenylated mRNA in the form of a larger precursor molecule. We discuss our findings with regard to the systematic position of the algae used and the evolution of plastids.