Thermal stress accelerates Arabidopsis thaliana mutation rate

Abstract

Mutations are the source of both genetic diversity and mutational load. However, the effects of increasing environmental temperature on plant mutation rates and relative impact on specific mutational classes (e.g., insertion/deletion [indel] vs. single nucleotide variant [SNV]) are unknown. This topic is important because of the poorly defined effects of anthropogenic global temperature rise on biological systems. Here, we show the impact of temperature increase on Arabidopsis thaliana mutation, studying whole genome profiles of mutation accumulation (MA) lineages grown for 11 successive generations at 29°C. Whereas growth of A. thaliana at standard temperature (ST; 23°C) is associated with a mutation rate of 7 × 10^-9 base substitutions per site per generation, growth at stressful high temperature (HT; 29°C) is highly mutagenic, increasing the mutation rate to 12 × 10^-9 SNV frequency is approximately two- to threefold higher at HT than at ST, and HT-growth causes an ∼19- to 23-fold increase in indel frequency, resulting in a disproportionate increase in indels (vs. SNVs). Most HT-induced indels are 1-2 bp in size and particularly affect homopolymeric or dinucleotide A or T stretch regions of the genome. HT-induced indels occur disproportionately in nucleosome-free regions, suggesting that much HT-induced mutational damage occurs during cell-cycle phases when genomic DNA is packaged into nucleosomes. We conclude that stressful experimental temperature increases accelerate plant mutation rates and particularly accelerate the rate of indel mutation. Increasing environmental temperatures are thus likely to have significant mutagenic consequences for plants growing in the wild and may, in particular, add detrimentally to mutational load.

Figure 1.

Stressful HT growth increases A. thaliana mutation rates. (A) Plants grown for 28 d or 58 d in cold (CT; 16°C), standard (ST; 23°C), or high temperature (HT; 29°C) conditions. HT-grown plants display characteristic high temperature developmental and stress responses. (B) Mutation rates (per genome per generation) for transitions, transversions, and indels accumulating in CT, ST (Jiang et al. 2014), and HT MA lines. (C) Overview of the 15 mutations detected at G6 in five independent CT MA lines. Deletions are 1 bp in size; SNVs are single-nucleotide variants (single-nucleotide substitutions). (D) Overview of the 52 mutations detected at G10 in nine ST MA lines from three independent lineages (Jiang et al. 2014). Deletions are 1–66 bp in size; insertion is 2 bp in size. (E) Overview of the 230 mutations (Supplemental Table 2) detected at G11 in six independent HT MA lines. Deletions are 1–22 bp in size; insertions are 1–5 bp in size. (F) SNV and indel mutation rates in ST (Jiang et al. 2014) and HT compared with those in salinity stress (Saline-ST) (Jiang et al. 2014). Error bars (B,F) indicate SEM from five (CT data), nine (ST data) (Jiang et al. 2014), nine (Saline-ST data) (Jiang et al. 2014), or six (HT data) biological replicates.

Figure 2.

HT growth increases SNV frequency and maintains GC-to-AT bias. (A) SNV spectrum and Ti/Tv ratio in ST (Ossowski et al. 2010) and HT conditions. (B) Relative percentage of transitions versus transversions in ST (Ossowski et al. 2010) versus HT-grown MA lines. Error bars (A,B) indicate SEM from five (ST data) (Ossowski et al. 2010) or six (HT data) biological replicates.

Figure 3.

Genome-wide distribution of SNVs in HT, ST, and MMR-deficient MA lines. Black bars show the relative distribution of Arabidopsis thaliana TAIR10 reference genome annotation categories (expressed as % of total genome). (CDS) Coding DNA sequence, (UTRs) untranslated regions, (TE) transposable element, (Other) noncoding RNAs and pseudogenes. The remaining bars show relative distribution (%) of SNVs in those annotation categories in: (red) HT MA lines (N = 92); (green) ST MA lines (N = 98) (Ossowski et al. 2010) and N = 44 (Jiang et al. 2014) (averaged for each category); and (blue) MMR-deficient MA lines (N = 4048) (Belfield et al. 2018).

Figure 4.

Most HT-induced indels are of 1- to 2-bp length. Length distributions (in bp) of HT-grown MA (G11) deletions (A) and insertions (B). (C) Comparison of the frequencies of the different classes of HT-induced 1- and 2-bp indels. For example, 40 A, 32 T, 4 C, and 1 C single base deletions were detected.

Figure 5.

HT-induced indels cluster in homopolymeric/microsatellite stretches and cause extreme indel/SNV bias. (A) A histogram showing the frequency (left y-axis) of 1-bp A or T indels found in homopolymeric repeat regions of different lengths (x-axis: values normalized with respect to the numbers of each length category in TAIR10, numbers as indicated by the red diamond-marked line [right y-axis]) (see also Supplemental Fig. 5A,B). The black dotted line indicates a moving average trendline for indel frequency. (B) Genomic distribution of indels in HT MA lines. (CDS) Coding DNA sequence, (UTR) untranslated region, (TE) transposable element, (Other) pseudogenes and noncoding RNAs. (C) Indel/SNV ratio comparisons; data from unicellular eukaryotes (red bars), multicellular eukaryotes (green bars), and eubacteria (blue bars). Organisms were grown at relative high temperature (HT), standard temperature (ST), or alternative cold temperature (CT), as indicated. The studies shown are: Plasmodium falciparum – ST (Hamilton et al. 2017); Arabidopsis thaliana – HT (this study); A. thaliana - MMR – ST (Belfield et al. 2018); A. thaliana - FN – ST (Belfield et al. 2012); A. thaliana - Saline – ST (Jiang et al. 2014); Oryza sativa – ST (Yang et al. 2015); Escherichia coli – ST (Chu et al. 2018); E. coli – CT (28°C) (Chu et al. 2018); E. coli – CT (25°C) (Chu et al. 2018); A. thaliana A – ST (Ossowski et al. 2010); A. thaliana B – ST (Jiang et al. 2014); Saccharomyces cerevisiae – HT (Huang et al. 2018); Caenorhabditis elegans – ST (Meier et al. 2014); S. cerevisiae – ST (Liu and Zhang 2019); Homo sapiens – ST (Besenbacher et al. 2016); and Daphnia pulex – ST (Keith et al. 2016). Error bars (A,B) indicate SEM from six HT biological replicates.

Figure 6.

HT-induced indels are disproportionally located in nucleosome-free DNA. Nucleosome profiling of indel and SNV mutations in ST-grown (Ossowski et al. 2010; Jiang et al. 2014) and HT-grown MA lines. (Predicted) Random expectation nucleosomal-associated versus nonnucleosomal distributions, as based on the distributions of MNase-seq bases (Supplemental Table S4A,B); (Observed) actual distribution of indels and SNVs in ST (Ossowski et al. 2010; Jiang et al. 2014) and HT conditions Supplemental Table S5A–D).