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.