Reciprocal cybrids reveal how organellar genomes affect plant phenotypes

Abstract

Assessment of the impact of variation in chloroplast and mitochondrial DNA (collectively termed the plasmotype) on plant phenotypes is challenging due to the difficulty in separating their effect from nuclear-derived variation (the nucleotype). Haploid-inducer lines can be used as efficient plasmotype donors to generate new plasmotype–nucleotype combinations (cybrids)1. We generated a panel comprising all possible cybrids of seven Arabidopsis thaliana accessions and extensively phenotyped these lines for 1,859 phenotypes under both stable and fluctuating conditions. We show that natural variation in the plasmotype results in both additive and epistatic effects across all phenotypic categories. Plasmotypes that induce more additive phenotypic changes also cause more epistatic effects, suggesting a possible common basis for both additive and epistatic effects. On average, epistatic interactions explained twice as much of the variance in phenotypes as additive plasmotype effects. The impact of plasmotypic variation was also more pronounced under fluctuating and stressful environmental conditions. Thus, the phenotypic impact of variation in plasmotypes is the outcome of multi-level nucleotype–plasmotype–environment interactions and, as such, the plasmotype is likely to serve as a reservoir of variation that is predominantly exposed under certain conditions. The production of cybrids using haploid inducers is a rapid and precise method for assessment of the phenotypic effects of natural variation in organellar genomes. It will facilitate efficient screening of unique nucleotype–plasmotype combinations to both improve our understanding of natural variation in these combinations and identify favourable combinations to enhance plant performance.

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Fig. 1: Generation of a cybrid test panel.
Fig. 2: Significant plasmotype-induced effects in 92 phenotypes.
Fig. 3: Plasmotype changes result in cytonuclear epistasis and also, in the case of cybrids with the Ely and Bur plasmotype, in additive effects.
Fig. 4: The fraction of explained genetic variation for photosynthesis phenotypes differs depending on light conditions.

Data availability

Sequencing and transcriptome data are available through the European Nucleotide Archive with the primary accession codes PRJEB29654 and PRJEB35324. The raw datasets are available through Dryad at https://doi.org/10.5061/dryad.cz8w9gj05. The analysed datasets that support our findings are available as Supplementary Data. The associated raw data for Figs. 3 and 4 are provided in Supplementary Data 1, and the raw data for Fig. 2 are provided in Supplementary Data 2. Source data for Figs. 3 and Fig. 4 and Extended Data Figs. 1, 3, 4 and 79 are provided with the paper. The germplasm generated in this project will be available via the European Arabidopsis Stock Centre (www.arabidopsis.info).

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Acknowledgements

H. Blankestijn, J. van de Belt, D. Oberste-Lehn, E. Schijlen, C. Hanhart, J. ter Riele and S. Schop (Wageningen University & Research) are acknowledged for help with experiments; J. Klasen (Max Planck Institute for Plant Breeding Research), A. Languillaume and R. van Bezouw (Wageningen University & Research) for statistical advice; and D. Aanen (Wageningen University & Research) for helpful discussions. This work was, in part, supported by the Netherlands Organization for Scientific Research through ALW-TTI Green Genetics (P.J.F.) and ALWGS.2016.012 (T.P.J.M.T). The European Molecular Biology Organization supported this work through grant no. ALTF 679-2013 (E.W.), and the European Community through the Marie-Curie Initial Training Network ‘COMREC’ project no. 606956 funded under FP7-PEOPLE (V.C.-B.). ZonMw Enabling Technology Hotels and the Consortium for Improving Plant Yield Enabling Technology Hotels provided funds for the metabolomics, RNA-seq and seed phenotyping. Work at Michigan State University for DEPI phenotyping was supported by the US Department of Energy, Chemical Sciences, Geosciences, and Biosciences Division, Basic Energy Sciences, Office of Science at the US Department of Energy (through grant no. DE-FG02–91ER20021).

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Authors

Contributions

P.J.F. and E.W. conceived and designed the study. T.P.J.M.T. designed and performed the statistical analysis with help from P.J.F., W.K. and F.v.E. P.J.F., T.P.J.M.T., E.K., F.F.M.B., L.A.J.W., V.C.-B., J.v.A., J.M.G. and L.S. performed experiments. P.J.F., T.P.J.M.T., K.S., P.K., E.S., J.A.H., S.K.S., R.W., W.L., R.M., F.v.E. and E.W. analysed data. D.M.K., J.J.B.K., M.K., J.H. and M.G.M.A. contributed to the interpretation of results. P.J.F., T.P.J.M.T. and E.W. wrote the paper with substantial contributions from M.K., J.H. and M.G.M.A. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Pádraic J. Flood or Tom P. J. M. Theeuwen or Erik Wijnker.

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Extended data

Extended Data Fig. 1 Coverage plots reveal a duplication on chromosome 2 in two cybrid lines.

Extended Data Fig. 1: Coverage plots reveal a duplication on chromosome 2 in two cybrid lines. This coverage plot shows the normalized read coverage at the lower end of the long arm of chromosome 2 for wild-type Bur (Bur-WT) and six cybrids with the Bur nucleotype (genotypes indicated as NucleotypePlasmotype). The coverage plot reveals the presence of a spontaneous nuclear DNA duplication in two cybrid lines (BurBur and BurC24), presumably derived from their wild-type Bur progenitor. These lines were excluded from all further analyses. Source data

Extended Data Fig. 2

Neighbor joining (NJ) trees based on SNPs and INDELs for nucleus (a), chloroplast (b) and mitochondria (c) for the seven Arabidopsis thaliana accessions.

Extended Data Fig. 3 Correlation of plasmotype effects amongst the subset of 92 phenotypes.

For more information on the 92 phenotypes see Supplementary Tables 2 and 4. Dark blue has a correlation of 1 and dark red a correlation of -1. Correlation represents the Pearson correlation coefficient. Source data

Extended Data Fig. 4 Scatterplots showing the correlation between the number of plasmotype additive and plasmotype epistatic effects.

a, Shows the correlation for additive and epistatic effects for all plasmotypes (including the Ely plasmotype; n = 7 plasmotypes) in every comparison, averaged over the nucleotypes, and counted over the 92 phenotypes. b, Shows the same correlation when excluding the Ely plasmotype (n = 6 plasmotypes). R is the Pearson correlation coefficient, the P value is based on a two-sided t-test and the shaded area shows the 95% confidence interval around the regression line. Source data

Extended Data Fig. 5 Cytoplasmic male sterility in ElySha.

a, An ElySha plant was polinated on three open flowers using Ely wild-type pollen, which produced elongated siliques (indicated with red arrows), scale bar is 1 cm. b, Shows an anther and pollen of ElySha, stained using Alexander stain. Pollen viability was assessed in 250 pollen per flower (n = 3 flowers). Note the presence of a high percentage (45%) of greenish, almost colourless aborted pollen. Pollen with a red colour in this line are not able to fertilize ovules, as deduced from the male sterile phenotype of ElySha (as shown in panel a), scale bar is 500 μm. c, Anther and pollen of Ely wildtype. Note that all pollen have a dark red colour, suggesting high viability. Ely wildtype is able to fertilize ElySha (as shown in panel a), scale bar is 500 μm.

Extended Data Fig. 6 Changes in gene expression between cybrids with a Ler nucleus (panel a), an Ely nucleus (panel b) and changes they have in common (panel c).

Cybrid genotypes are indicated as NucleotypePlasmotype. The triangle in panel a shows cybrid comparisons with a Ler nuclear background (for plasmotypes Ely, Ler and Bur) and panel b shows cybrid comparisons with an Ely nuclear background for the same plasmotypes. Significantly differentially expressed (DE) genes between cybrid comparisons are indicated with black numbers. These DE genes are subdivided in upregulated genes (green numbers in superscript) and downregulated genes (red numbers in subscript), following the direction of the arrows between cybrids (that is the change from an Ely to a Ler plasmotype in a Ler nuclear background resulted in 726 DE genes, of which 426 were upregulated and 300 were downregulated). The green triangle in panel c shows what differentially expressed genes the comparisons in panels a and b have in common. For example, the Ler and Ely nuclear backgrounds show a common response of 78 DE genes when the Ely plasmotype is changed for a Ler plasmotype. The absence of one of the comparisons in this triangle is due to the absence of shared DE genes. The common effect of changing an Ely plasmotype for either Bur of Ler was derived by assessing what DE genes are similar along the axes in the green triangle c. These 78 and 150 genes have 40 shared DE genes (see Supplementary Table 6). For the raw data see Supplementary Data 3.

Extended Data Fig. 7 The fraction of explained genetic variation (H2) for changes in photosynthesis phenotypes (ΦPSII, ΦNPQ, ΦNO, NPQ, qE, qI) in response to light conditions.

a, Shows the fraction of H2 for epistatic interactions (nucleotype x plasmotype). b, Shows H2 for plasmotype additive effects. c, Shows the light intensity for five consecutive days with growth under: steady light (day 1); in- and decreasing light intensity (day 2); fluctuating in- and decreasing light intensity (day 3); steady light (day 4) and fluctuating in- and decreasing light intensity (day 5). Days are separated by nights (shaded areas). The first three days of this experiment are identical to the light conditions of the experiment shown in Fig. 3. Source data

Extended Data Fig. 8 KCN sensitive O2 consumption of mitochondria in seedlings of wild-type accessions.

Mitochondrial ATP-synthesis proceeds mainly through the phosphoryl ating cytochrome (KCN sensitive) pathway. KCN sensitive O2 consumption by Bur does not differ significantly from C24, Col and Ws-4. Error bars represent the standard error of the mean (n = minimally 6 biologically independent samples). Letters indicate significant differences using the Tukey posthoc test, with an α = 0.05 threshold. Source data

Extended Data Fig. 9 Coverage plots reveal no unique deletions in Bur chloroplasts or mitochondria.

Normalized read depth for the chloroplast (a) and mitochondrial (b) genome sequences were calculated in a sliding window of 1-kb. Because we observe no unique deletions or duplications in the Bur plasmotype that might be causal to the phenotypic effects observed in cybrids with the Bur plasmotype. Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Supplementary Tables 1–8.

Reporting Summary

Supplementary Data 1

Supplementary Data 1. Summary and test statistics for all 1,859 phenotypes. Table 1 shows the least squared means for all phenotypes per cybrid; these are calculated via the models as specified in Supplementary Table 4. Table 2 shows the fraction of explained variance for nucleotype, plasmotype and interaction separately, including cybrids with the Ely plasmotype. We show the variance components for all terms in the model. Using the variance components of the genetic components, we calculated the broad-sense heritability (H2). We used a H2 threshold of 0.05 for phenotypes to be included in summary and test statistics. The fraction of H2 for all three genetic components is also provided. Table 3 shows the same data as given in Table 2, but excluding cybrids with the Ely plasmotype. Table 4 shows significant plasmotype additive effects, with Hochberg’s P value correction. Letters indicate significance or not. Table 5 shows significant plasmotype epistatic effects. This was performed for every plasmotype within each nucleotype (with Hochberg’s P value correction), as well as a comparison between self-cybrids and other cybrids within each nucleotype (with Dunnett’s P value correction).

Supplementary Data 2

Supplementary Data 2. Summary and test statistics of 92 phenotypes, with easy-to-use tables provided to search for significant differences among all separate phenotypes. Table 1 shows the significant additive effects for each pairwise comparison, with a text box to explain the interpretation of the data presented. Table 1 contains the underlying data used for generation of Table 1a. Table 2 shows the significant difference for every pairwise comparison within a nucleotype—that is, epistatic effects. A text box is provided to explain interpretation of the data. Table 2 contains the underlying data used for generation of Table 1b. The remaining tables show the summary and test statistics for the 92 phenotypes, as in Supplementary Data 1.

Supplementary Data 3

Supplementary Data 3. Differential expression overview from the RNA-seq experiment for the six cybrid comparisons. Tables 1–6 show pairwise comparisons of cybrid lines, indicated as ‘nucleotype–plasmotype versus nucleotype–plasmotype’. For every nuclear gene detected, the differential expression data are given with the adjusted P value cut-off set at α = 0.05 (in yellow). The summary statistics for this are given in Supplementary Table 5. Table 7 shows Gene Ontology enrichment for five cybrid comparisons. For one comparison (LerBur versus LerLer) we detected only three significantly expressed genes for which Gene Ontology enrichment yielded no results. This table also shows Gene Ontology enrichment for genes that differentially changed expression when the Ely plasmotype was replaced by Ler or Bur in a Ler or Ely nuclear background (indicated as ‘Ely main’).

Supplementary Data 4

Supplementary Data 4. Predicted impact of SNPs and INDELs on the chloroplastic and mitochondrial genomes of all seven accessions used. For every variant the reference allele, based on TAIR10.1, is given next to the alternative allele. We indicate, for all seven accessions, whether they share the reference (0/0) or alternative allele (1/1), and used SnpEff to predict the impact. The changes are ranked as ‘Low’, ‘Modifier’, ‘Moderate’ or ‘High’ based on the location in respect to a gene, and these predicted amino acid change. In our interpretation we used the ‘Moderate-’ and ‘High’-impact variants. The genes affected, as well as nucleotide and amino acid change, are provided.

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Flood, P.J., Theeuwen, T.P.J.M., Schneeberger, K. et al. Reciprocal cybrids reveal how organellar genomes affect plant phenotypes. Nat. Plants 6, 13–21 (2020). https://doi.org/10.1038/s41477-019-0575-9

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