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Retrotransposons have played an important role in the evolution of host genomes1,2. Their impact is mainly deduced from the composition of DNA sequences that have been fixed over evolutionary time2. Such studies provide important ‘snapshots’ reflecting the historical activities of transposons but do not predict current transposition potential. We previously reported sequence-independent retrotransposon trapping (SIRT) as a method that, by identification of extrachromosomal linear DNA (eclDNA), revealed the presence of active long terminal repeat (LTR) retrotransposons in Arabidopsis3. However, SIRT cannot be applied to large and transposon-rich genomes, as found in crop plants. We have developed an alternative approach named ALE-seq (amplification of LTR of eclDNAs followed by sequencing) for such situations. ALE-seq reveals sequences of 5′ LTRs of eclDNAs after two-step amplification: in vitro transcription and subsequent reverse transcription. Using ALE-seq in rice, we detected eclDNAs for a novel Copiafamily LTR retrotransposon, Go-on, which is activated by heat stress. Sequencing of rice accessions revealed that Go-onhas preferentially accumulated in Oryza sativassp. indicarice grown at higher temperatures. Furthermore, ALE-seq applied to tomato fruits identified a developmentally regulated Gypsyfamily of retrotransposons. A bioinformatic pipeline adapted for ALE-seq data analyses is used for the direct and reference-free annotation of new, active retroelements. This pipeline allows assessment of LTR retrotransposon activities in organisms for which genomic sequences and/or reference genomes are either unavailable or of low quality.

Heat-imposed crop failure is often attributed to reduced thermotolerance of floral tissues; however, the underlying mechanism remains unknown. Here, we demonstrate that m6A RNA methylation increases in Arabidopsisflowers and negatively regulates gene expression variability. Stochastic gene expression provides flexibility to cope with environmental stresses. We find that reduced transcriptional fluctuation is associated with compromised activation of heat-responsive genes. Moreover, disruption of an RNA demethylase AtALKBH10B leads to lower gene expression variability, suppression of heat-activated genes, and strong reduction of plant fertility. Our work proposes a novel role for RNA methylation in the bet-hedging strategy of heat stress response.