European mistletoe (Viscum album) is a hemiparasitic flowering plant that is known for its very special life cycle and extraordinary biochemical properties. Particularly, V. album has an unusual mode of cellular respiration that takes place in the absence of mitochondrial complex I. However, insights into the molecular biology of V. album so far are very limited. Since the genome of V. album is extremely large (estimated 600 times larger than the genome of the model plant Arabidopsis thaliana) it has not been sequenced up to now. We here report sequencing of the V. album gene space (defined as the space including and surrounding genic regions, encompassing coding as well as 5 0 and 3 0 non-coding regions). mRNA fractions were isolated from different V. album organs harvested in summer or winter and were analyzed via single-molecule real-time sequencing. We determined sequences of 39 092 distinct open reading frames encoding 32 064 V. album proteins (designated V. album protein space). Our data give new insights into the metabolism and molecular biology of V. album, including the biosynthesis of lectins and viscotoxins. The benefits of the V. album gene space information are demonstrated by re-evaluating mass spectrometry-based data of the V. album mitochondrial proteome, which previously had been evaluated using the A. thaliana genome sequence. Our re-examination allowed the additional identification of nearly 200 mitochondrial proteins, including four proteins related to complex I, which all have a secondary function not related to respiratory electron transport. The V. album gene space sequences are available at the NCBI.
European mistletoe (Viscum album) is known for its special mode of cellular respiration. It lacks the mitochondrial NADH dehydrogenase complex (complex I of the respiratory chain) and has restricted capacities to generate mitochondrial adenosine triphosphate (ATP). Here, we present an investigation of the V. album energy metabolism taking place in chloroplasts. Thylakoids were purified from young V. album leaves, and membrane bound protein complexes were characterized by Blue native polyacrylamide gel electrophoresis as well as by the complexome profiling approach. Proteins were systematically identified by label-free quantitative shotgun proteomics. We identified > 1800 distinct proteins (accessible at https://complexomemap.de/va_leaves), including nearly 100 proteins forming part of the protein complexes involved in the light-dependent part of photosynthesis. The photosynthesis apparatus of V. album has distinct features: (i) comparatively low amounts of photosystem I; (ii) absence of the NDH complex (the chloroplast pendant of mitochondrial complex I involved in cyclic electron transport around photosystem I); (iii) reduced levels of the proton gradient regulation 5 (PGR5) and proton gradient regulation 5-like 1 (PGRL1) proteins, which offer an alternative route for cyclic electron transport around photosystem I; (iv) comparable amounts of photosystem II and the chloroplast ATP synthase complex to other seed plants. Our data suggest a restricted capacity for chloroplast ATP biosynthesis by the photophosphorylation process. This is in addition to the limited ATP supply by the mitochondria. We propose a view on mistletoe's mode of life, according to which its metabolism relies to a greater extent on energy-rich compounds provided by the host trees.
The hemiparasitic flowering plant Viscum album (European mistletoe) is known for its very special life cycle, extraordinary biochemical properties, and extremely large genome. The size of its genome is estimated to be 30 times larger than the human genome and 600 times larger than the genome of the model plant Arabidopsis thaliana. To achieve insights into the Gene Space of the genome, which is defined as the space including and surrounding protein-coding regions, a transcriptome project based on PacBio sequencing has recently been conducted. A database resulting from this project contains sequences of 39,092 different open reading frames encoding 32,064 distinct proteins. Based on ‘Benchmarking Universal Single-Copy Orthologs’ (BUSCO) analysis, the completeness of the database was estimated to be in the range of 78%. To further develop this database, we performed a transcriptome project of V. album organs harvested in summer and winter based on Illumina sequencing. Data from both sequencing strategies were combined. The new V. album Gene Space database II (VaGs II) contains 90,039 sequences and has a completeness of 93% as revealed by BUSCO analysis. Sequences from other organisms, particularly fungi, which are known to colonize mistletoe leaves, have been removed. To evaluate the quality of the new database, proteome data of a mitochondrial fraction of V. album were re-analyzed. Compared to the original evaluation published five years ago, nearly 1000 additional proteins could be identified in the mitochondrial fraction, providing new insights into the Oxidative Phosphorylation System of V. album. The VaGs II database is available at https://viscumalbum.pflanzenproteomik.de/. Furthermore, all V. album sequences have been uploaded at the European Nucleotide Archive (ENA).
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