The Viscum album Gene Space database

Abstract

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).

Keywords: Complexome profiling; Illumina sequencing; PacBio sequencing; complex I; database development; mitochondria; oxidative phosphorylation (OXPHOS); supercomplex.

Figure 1

Number of transcripts according to assignment to phyla. Assignment of transcripts obtained by Illumina Sequencing was based on the best blast hit method on the UniProt/Swiss-Prot and UniProt/TrEMBL databases. Number of transcripts of summer and winter samples were displayed separately. Phyla considered are Viridiplantae (green algae and the land plants (embryophytes)) and fungi (as yeasts, molds and mushrooms). Transcripts resembling those of other phyla (e.g.: metazoan, bacteria etc.) were grouped under “other”. Transcripts not significantly resembling known sequences were labeled “unknown”. The Venn diagram was created by BioVenn (Hulsen et al., 2008).

Figure 2

Transcript levels within the V. album summer and winter samples according to their assignment of phyla. Transcript levels are given in log10 Transcripts Per Million (TPM). The line within the box shows the median for each dataset. The upper end of the box is the “upper quartile”, which is the median of the upper half of the dataset. The lower quartile (lower end of the box) is the median of the lower half of the dataset. The end of the vertical lines indicate the lowest and highest values of the dataset. The diagram was created by instant clue (Nolte et al., 2018).

Figure 3

Comparison of the completeness of the VaGs I database (Schröder et al., 2022b) and the new VaGs II database as revealed by ‘Benchmarking Universal Single Copy Orthologs’ (BUSCO) analysis (Seppey et al., 2019).

Figure 4

The Viscum album Gene space database at https://viscumalbum.pflanzenproteomik.de/.

Figure 5

Number of identified proteins or peptides in a V. album mitochondrial complexome dataset upon data evaluation with TAIR, VaGs I and VaGs II. TAIR10: Arabidopsis protein database provided by The Arabidopsis thaliana Information resource (TAIR), Version 10 (https://www.arabidopsis.org/). The mitochondrial complexome dataset is derived from Senkler et al., 2018. The dataset was evaluated by TAIR10 (as in Senkler et al., 2018), VaGs I (Schröder et al., 2022b) and VaGs II (this study).

Figure 6

Identified subunits of complexes III and IV of the respiratory chain from V. album in a mitochondrial complexome dataset (Senkler et al., 2018) upon analysis using the VaGs II database. Mitochondrial proteins were separated by Blue native (BN) PAGE and stained by Coomassie-blue (horizontal gel lane in the center of the Figure). The gel lane was dissected into 54 slices, which all were subjected to label-free quantitative shotgun mass spectrometry. Abundance profiles of individual proteins along the gel lane are visualized as a heatmap (figure part above the gel lane; the columns correspond to the 54 gel slices; the rows to the abundance profiles of individual proteins; normalized (max) abundance profiles are given; see scale to the left of the heatmap) and aligned according to similarity (using the NOVA software package version 0.5.8). Only profiles of the subunits of respiratory complexes III and IV are shown (for complete dataset see Supplementary Data S1 and ComplexomeMap at https://complexomemap.de/75). Protein identities are given to the right of the heatmap. Summed-up abundance profiles (given as ‘intensity based absolute quantification’ (iBAQ) values) of the subunits of the two protein complexes as well as assemblies of the complexes (supercomplexes III₂IV₁ and III₂IV₂) are given in the diagram (figure part below the gel lane; y-axis: iBAQ score; x-axis: gel slices 1-54). Results of the original evaluation (Senkler et al., 2018) are shown in Supplementary Figure S1 ).