3.2. Water environmental increases RNA methylation
N6-methyladenosine (m6A) RNA modification is a prevalent and dynamic modification in eukaryotic RNA, playing a crucial role in various physiological aspects of living organisms, including growth, development, and stress responses . The m6A modification is involved in the regulation of mRNA stability, alternative splicing, translation, export, and maturation of microRNA, which can influence the plant’s ability to adapt to environmental changes . In plants, m6A RNA modification has been linked to abiotic stress responses, such as salt and osmotic stress, drought, cold and UV radiation . For example, inArabidopsis thalliana the m6A modification has been important for salt stress tolerance . In the context of amphibious plants like analyzed Riccia fluitans , which exhibit remarkable adaptability to fluctuating aquatic and terrestrial environments, m6A RNA modification could potentially play a role in their fast adaptation to changing environments.
Information on 2 190 probable aquatic methylation sites and 464 terrestrial methylation sites was revealed by analysis of raw Nanopore signals. Identifying 173 sites from 126 transcripts as significant in the water form (Supporting Information S1: Table 9) and 27 from 24 transcripts as significant in the land form (Supporting Information S1: Table 10) was based on the previously mentioned sites. The 16 methylation biases shared both forms (Figure 3A and 3B). The CL.22551.1 transcript coded cytochrome-c oxidase/electron carrier was the most methylated transcript in the aquatic form, with five significant methylation sites. In the terrestrial form, the most frequently significantly methylated transcripts were CL.33843.1 encoded ribosomal protein S11 family protein, CL.6664.1 encoded papain family cysteine protease and CL.8794.1 translated 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein, each with two significant sites. Among the detected methylation sites in the aquatic form, CL.33844.1, encoded ribosomal protein S4 (RPS4A) family protein, exhibited the highest probability of methylation (0.97). Whereas, in the terrestrial form, CL.303.1 (Ribosomal protein S14p/S29e family protein) showed the highest methylation probability of approximately 0.9. Methylation was most frequently detected in the GAACT motif in both forms of Riccia fluitans (Figure 3C). Transcripts with significant methylation sites in the aquatic form were involved in the following gene ontology processes (FDR < 0.05): aerobic (GO:0019646) and cellular respiration (GO:0045333) (Figure 3D and Supporting Information S1: Table 11), while transcripts methylated frequently in the land form were involved in the chloroplast envelope (GO:0009941) and located within plastoglobules (GO:0010287) (Figure 3E and Supporting Information S1: Table 12). An overlap was identified between aquatic methylation positions and unique DEGs identified by Illumina technology CL.28438 (Gamma vacuolar processing enzyme), CL.28820 (Low temperature and salt responsive protein family), CL.3354 (Disease resistance-responsive family protein), CL.19054 (Peroxidase superfamily protein), and Nanopore technology CL.8117 (Chitinase family protein). Notably, the CL.2289 (Unknown) gene was shared between the methods. Similarly, terrestrial methylation positions showed overlap with Illumina DEGs and Nanopore DEGs. The unknown CL.3752 (Unknown) gene was identified as DEGs only in Nanopore sequencing technology. Other common elements, including genes CL.19794 (Unknown), CL.21493 (Unknown), CL.2593 (Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family protein), CL.31915 (Carbonic anhydrase 2), were found to be relevant for both sequencing methods (Supporting Information S2: Figure 6). Additional, transcript encoded Cytochrome P450 superfamily protein, which is DETs in short-read analysis, also revealed significant methylation modification in water environment (Supporting Information S1: Figure 7). Three methylations of transcript CL.6664.1 were detected in aquatic Riccia and two other epitranscriptome events of the same transcript in the land form. Papain family cysteine proteases are involved in the response to abiotic stress. Zang et al. showed that transgenic Arabidopsis overexpressing the gene encoding a papain family cysteine protease exhibited stronger drought tolerance under water-stressed conditions than the wild type, suggesting that the gene plays a role in mediating dehydration tolerance . The sweet potato papain family cysteine proteases 2 gene was involved in the response to darkness. In addition, the same gene in Arabidopsisincreased resistance to drought and salt stress . On the other hand, overexpression of the sweet potato papain family cysteine proteases 3 gene in Arabidopsis conferred sensitivity to drought stress . Despite differences in methylated sites between water and land forms (Figure 3A), the expression on gene complexes involved in m6A methylation processes is similar (Supporting Information S1: Table 13). Both environmental forms of R. fluitans did not differ in expression of homologs identified in A. thaliana as writers (MTA, MTB), readers (YTH) or erasers (ALKBH9B, ALKBH10B), which can be explained by high abundance modified transcripts in mRNA.