Comprehensive Analysis of Small RNA Modifications in Arabidopsis thaliana and Their Dynamics During Seed Germination

<b>Background</b>: Small RNA, defined as RNA molecules of less than 200 nucleotides in length, play pivotal regulatory roles in plant growth, development, and environmental stress responses. However, research on modifications in plant small RNA remains limited. <b>Methods</b>: In this study, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection of 41 RNA modifications, facilitating the systematic qualification and quantification of modifications in plant small RNA. <b>Results</b>: We identified a total of nine modifications, among which <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine (m^6,6A) is a newly identified modification in plant small RNA. Furthermore, we conducted a quantitative analysis of these modifications in <i>Arabidopsis thaliana</i> during the germination process and observed significant dynamic changes in their abundance from 1 to 5 days post-germination. Notably, the trends in the contents of these modifications exhibited a strong correlation with the reported gene expression levels of the relevant modifying enzymes and demodifying enzymes, suggesting that these modifications may play essential roles during seed germination and are tightly regulated by the genes of the corresponding enzymes. <b>Conclusions</b>: The discovery of these modifications in plant small RNA, coupled with the dynamic changes in their levels during germination, holds great promise for a further understanding of the physiological functions of small RNA modifications and their associated regulatory mechanisms in plant seed germination.