The energy biology of European Mistletoe (Viscum album)

Abstract

The hemiparasitic European mistletoe (Viscum album) is known for its extraordinary way of life. Not only its huge genome of about 90 Gbp is noticeable, but also the absence of mitochondrial complex I of the Oxidative Phosphorylation system. Since a large genome indicates a high energy demand during cellular division, absence of complex I, which strongly contributes to the proton gradient across the inner mitochondrial membrane and thus to ATP production, is to be considered remarkable. How can V. album accomplish its energy metabolism? This is the central research question of this thesis. To this end, the transcriptome of V. album was first sequenced to provide the basis for efficient proteome analysis. RNA was isolated from mistletoe leaves, flowers, and stems harvested in summer and winter. The RNA was next transcribed into cDNA and sequenced as a pooled sample via the PacBio sequencing strategy. The resulting initial Viscum album Gene Space (VaGs) database showed 78% completeness based on Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis. To further develop this database, additional Illumina sequencing of the individual samples (summer and winter) was performed. The resulting Viscum album Gene Space database II (VaGsII) has a completeness of 93% and contains sequences of 90,039 transcripts. Based on these sequences, a GC content of 50% could be calculated. This is an unusually high GC content, as in other dicotyledonous plants the GC content usually ranges between 43-45 %. Due to the resulting enhanced stability of the DNA, an increased energy requirement must also be anticipated for DNA replication and transcription. In addition to the absence of the mitochondrial genes encoding subunits of complex I, the absence of almost all nuclear genes encoding complex I subunits could be shown. Furthermore, by re-evaluating an existing complexome dataset of V. album mitochondria using the new VaGs II database, more than 1,000 additional mitochondrial proteins could be identified with respect to the original evaluation. Besides the mitochondria, also the chloroplasts were examined in more detail to determine their contribution to the energy metabolism of V. album cells through photosynthesis and photophosphorylation. In the course of this examination, a complete absence of the NDH complex (NADH dehydrogenase- like complex, chloroplast pendant of mitochondrial complex I), which contributes to cyclic electron transport around photosystem I, was proven on the proteome level. In addition, PGR5 and PGRL1, two proteins which were shown to be alternatively involved in cyclic electron transport around photosystem I, were found to be of reduced abundance in V. album compared to the model plant Arabidopsis thaliana. Abundance of the chloroplast ATP synthase complex is comparable to A. thaliana; however, its stability clearly is increased in V. album. Also, the photosystem II is of similar abundance in A. thaliana and V. album, in contrast to the photosystem I, which is of comparatively low abundance in V. album. It can be concluded that both, linear and cyclic electron transport and thus ATP synthesis by photophosphorylation are comparatively low in V. album. In summary, it can be concluded that: 1. V. album has an even higher energy demand than previously thought due to its high GC content. 2. ATP production in mitochondria and chloroplasts is limited due to the absence or reduced abundance of some of the involved proteins and protein complexes. How sufficient amounts of ATP are provided in V. album cells is therefore still not entirely clear. It is hypothesized that the slow growth and reduced cell division rate of V. album might reduce its energy demand. In addition, sugar compounds transported in the host xylem in spring may be a source of energy for V. album. This may also explain the strong growth rate of V. album in spring. Further research is needed to understand the way of life of this very particular plant.